Quality Control of the Fuzi Lizhong Pill Through Simultaneous Determination of 16 Major Bioactive Constituents by RRLC–MS-MS

Quality Control of the Fuzi Lizhong Pill Through Simultaneous Determination of 16 Major Bioactive... Abstract Fuzi Lizhong pill (FLP) is used to treat gastritis, and the monarch drug of it is Aconiti Lateralis Radix Praeparata (Fuzi, aconite roots) which is a toxic herbal medicine. To better control the safety and quality of FLP, an effective method to analyze the contents of 16 toxic and bioactive components using rapid resolution liquid chromatography–tandem triple-quadrupole mass spectrometer was established. The 16 constituents included aconine, mesaconine, hypaconitine, benzoylaconine, benzoylmesaconine, benzoylhypaconine, adenosine, liquiritin, liquiritigenin, glycyrrhizic acid, isoliquiritigenin, 6-gingerol, atractylenolide III, atractylenolide I, atractylenolide II and glycyrrhetic acid. Ideal separation was performed using gradient elution in 13 min by optimized conditions. All the isomerides were isolated to baseline. The improved method with a polarity switch in contiguous time segments could analyze the five types of components, including polar and nonpolar compounds, without decreasing sensitivity. The proposed method was fully validated. The results revealed that contents of six alkaloids from Fuzi were significantly different among the samples. Using the established method and multivariate statistical method, the quality consistency of two dosage forms of FLP from different companies were analyzed. The optimized method could be used for the quality control of FLP and investigate index compound variation between two dosage forms. Introduction Chinese patent medicines (Zhong-Cheng-Yao in Chinese, CPMs) are the representative form of traditional Chinese medicines, which have gained increasing popularity for their health care and improving the body’s immunity against diseases around the world over the past decades (1). Moreover, several prescriptions of CPMs derived from traditional classical prescriptions, which have been applied in clinic for centuries, have been demonstrated to be effective in modern pharmacological studies and clinical trials (2–4). For example, the Fuzi Lizhong pill (FLP), which was initially recorded in “Shanghan Lun” 1800 years ago, is one of the popular traditional Chinese patent medicines used to treat such conditions as gastritis, enteritis, diarrhea and hypothermia with indigestion (2, 5). FLP has obvious anti-inflammatory and analgesic effects (6, 7). It consists of five herbal materials and Aconiti Lateralis Radix Praeparata (Fuzi, aconite roots) is the monarch drug in the prescription that exists the major therapeutic effects following the TCM theory. However, Fuzi is a toxic herbal medicine. Processing, one of the characteristics of the Chinese herbal medicine, could greatly reduce the toxicity of Fuzi by hydrolyzing the toxic diester diterpene alkaloids mainly including aconitine, mesaconitine and hypaconitine (8). Nevertheless, poison and side effects sometimes still occur in clinic (9, 10). Thus, it is urgent and essential to determinate the toxic components in FLP to guarantee its clinical safety. As the main active and toxic compounds in Fuzi, alkaloids could perform the cardiotonic action and reinforce the immune system by improve the peritoneal macrophage Ia expression (11). Among them, aconitine (AC), mesaconitine (MA), hypaconitine (HA), benzoylaconine (BA), benzoylmesaconine (BMA) and benzoylhypaconine (BHA) are the main toxic and active important markers in chemical analysis of Fuzi (8). Their contents are extensively qualified in Chinese Pharmacopeia resulting from that they are possessed of toxicity and simultaneously pharmacological activities. Moreover, it is well-known that the active components in herbal materials of CPMs are multiple, and the curative effects of CPMs are principally based on the synergic effects of these ingredients (12). Except Fuzi, FLP also consists of other four herbal materials: Codonopsis Radix (Dangshen), Atractylodis Macrocephalae Rhizoma (Baizhu), Glycyrrhizae Radix et Rhizoma (Gancao) and Zingiberis Rhizoma (Ganjiang) (2). The flavones and triterpenes from Gancao have a wide range of biological and physiological activities, such as anti-inflammatory (13), anti-proliferative (14) and anti-diabetic (15). Liquiritigenin (LQG), liquiritin (LQR), glycyrrhizic acid (GIA), isoliquiritigenin (OLQG) and glycyrrhetic acid (GEA) are the main markers in quality control of Gancao. The lactones from Baizhu are important for their anti-inflammatory, antioxidant and modulating gastrointestinal function (16, 17). Atractylenolide I (ALI), atractylenolide II (ALII) and atractylenolide III (ALIII) are main chemical markers in quality analysis of Baizhu. Adenosine (AD) and 6-gingerol (GE) from other included herbal materials are also important due to their antioxidant, anti-inflammatory and xanthine oxidase inhibitory effects (18, 19). Chromatographic fingerprints have identified several compounds in the FLP (the dosage form is not described), where differences were found among 12 batches of samples from four companies (20). While, whether the contents of main active components exist difference in different dosage forms of FLP and what may cause the variations are still unclear. Therefore, it is imperative to qualify control of FLP by these active components in order to minimize the variability and ensure its repeatable therapeutic effects. With the rapid development of advanced technology and precise instrument, many qualitative and quantitative methods have been devised regarding the quality evaluation of CPMs, such as rapid resolution liquid chromatography/tandem quadrupole time-of-flight mass spectrometer and rapid resolution liquid chromatography/tandem triple-quadrupole mass spectrometer (21–24). In contrast to HPLC method, rapid resolution liquid chromatography (RRLC) exhibits a higher peak capacity, increased sensitivity, better resolution and a greater rate of analysis. RRLC coupled to a triple quadrupole tandem mass spectrometer (TQ-MS-MS) has advantages of high selectivity and sensitivity through the multiple reaction monitoring (MRM) scan mode, in which baseline chromatographic separation of selected constituents is unnecessary (8). Moreover, the application of small-diameter particle columns in this method could guarantee rapid separation (8, 25). This method could quantify target constituents in complex entities at trace amounts, which implies its good potential in simultaneously determining multiple target constituents in CPMs. Despite the great advantages of RRLC–TQ-MS-MS in terms of quantification, analyzing various types of components with the same polarity is still a problem, particularly in guaranteeing sensitivity. In this study, an improved method with polarity switching in contiguous time segments achieved the goal of analyzing six types of 16 components, including alkaloids, adenosines, lactones, flavonoids, triterpenes and phenolic compounds, the major chemical components in herbal materials of FLP, without decreasing sensitivity. Based on the sample data, two dosage forms FLP samples, big honey pill (BHP) and the concentrated pill (CP), from 10 companies were determined. Moreover, OPLS-DA, heat map and ROC curve were used in the quality evaluation of the variation of samples. It is suggested that the established method would be applicable for quality evaluation of other constituents in CPMs. Experimental Chemicals, solvents and materials A total of 49 batches of FLPs were collected from eleven pharmaceutical companies in China (Table SI). The reference standards of AD, LQR, AC, MA, HA, GE, GIA and GEA were obtained from the Chinese National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China); BMA, BA, BHA, LQG and OLQG were purchased from Chengdu Must Biotechnology Co., Ltd. (Chengdu, China); ALI, ALII and ALIII were provided by Shanghai Tiyo Biotechnology Co., Ltd. (Shanghai, China). The purities of all the standards were no <95%. All of the analytes are summarized in Table SII, and their structures are shown in Figure 1. Figure 1. View largeDownload slide Structures of the 16 major constituents in the FLP. Adenosine (AD, 1), liquiritin (LQR, 2), benzoylmesaconine (BMA, 3), benzoylaconine (BA, 4), benzoylhypaconine (BHA, 5), liquiritigenin (LQG, 6), mesaconine (MA, 7), aconine (AC, 8), hypaconitine (HA, 9), glycyrrhizic acid (GIA, 10), isoliquiritigenin (OLQG, 11), 6-gingerol (GE, 12), atractylenolide III (ALIII, 13), atractylenolide I (ALI, 14), atractylenolide II (ALII, 15), glycyrrhetic acid (GEA, 16). Figure 1. View largeDownload slide Structures of the 16 major constituents in the FLP. Adenosine (AD, 1), liquiritin (LQR, 2), benzoylmesaconine (BMA, 3), benzoylaconine (BA, 4), benzoylhypaconine (BHA, 5), liquiritigenin (LQG, 6), mesaconine (MA, 7), aconine (AC, 8), hypaconitine (HA, 9), glycyrrhizic acid (GIA, 10), isoliquiritigenin (OLQG, 11), 6-gingerol (GE, 12), atractylenolide III (ALIII, 13), atractylenolide I (ALI, 14), atractylenolide II (ALII, 15), glycyrrhetic acid (GEA, 16). HPLC-grade acetonitrile and methanol was purchased from Fisher Scientific (Fair Lawn, NJ, USA), and MS grade formic acid was provided by MREDA Technology Inc. (USA). Deionized water was redistilled. All other chemicals and solvents were of analytical grade. Sample preparation and standard solutions For the sample preparation, the BHP samples were cut into pieces, whereas the CP samples were ground into a fine powder. The 0.2 g of BHP samples and 0.050 g of CP samples were accurately weighed and ultrasonically extracted with 50 mL methanol for 60 min in a conical flask, cooled to room temperature and then replenished to their original weight with methanol. The obtained solution was filtered through a 0.22-μm syringe filter. The first portion of the filter liquor was abandoned, and the subsequent 1–2 μL was directly injected into the HPLC instruments analysis. Stock solutions were obtained from the procedure, in which each reference standard was accurately weighed and dissolved with methanol. Specific amounts of the 16 reference standard solutions were diverted into a 10-mL volumetric flask to obtain the mixed stock solution. And the concentration of standard of AD, LQR, BMA, BA, BHA, LQG, MA, AC, HA, GIA, OLQG, GE, ALIII, ALI, ALII and GEA in the mixed stock solution were 60.00, 5,040, 462.0, 88.40, 324.0, 680.0, 29.12, 7.385, 92.40,13,027, 115.2, 5,952, 408.0, 496.0, 399.6 and 130.0 ng/mL, respectively. A set of standard solutions were prepared by appropriate dilution of the mixed stock solution with methanol to obtain the calibration curve. Chromatographic and mass spectrometric conditions All data were acquired using an RRLC system (1,260 series liquid chromatography, Agilent Technologies, Inc., USA) coupled with an electrospray MS (6410B Triple Quadrupole Mass Spectrometer, Agilent Technologies, Inc., USA; accuracy of 0.1 Da and resolution of 0.7 Da within the range of 1–1,000 Da). The separation was performed on a Thermo Accucore RP-MS column (100 mm × 2.1 mm; 2.6 μm particle size). The mobile phase consisted of (A) water containing 0.1% formic acid and (B) acetonitrile containing 0.1% formic acid. The linear gradient conditions were as follows: 0–3 min, 15–55% B; 3–9 min, 55–55%; 9–12 min, 55–80%; 12–12.01 min, 80–100%; 12.01–13 min, 100–100%. The flow rate was 0.3 mL/min. The column temperature was set at 30°C. Typical RRLC chromatograms of detected chemicals in the FLP are shown in Figure 2. Figure 2. View largeDownload slide Typical chromatograms of RRLC–MS-MS for a (A) sample and (B) standard mixture. The peak number is the same as the compound number summarized in Figure 1. This figure is available in black and white in print and in color at JCS online. Figure 2. View largeDownload slide Typical chromatograms of RRLC–MS-MS for a (A) sample and (B) standard mixture. The peak number is the same as the compound number summarized in Figure 1. This figure is available in black and white in print and in color at JCS online. The conditions of MS analysis were as follows: LQR was detected in negative ion mode, whereas the rest of the constituents were detected in positive ion mode; the drying gas N2 flow rate was 11 L/min at a gas temperature 300°C, and the pressure of the nebulizer was 45 psi. The capillary voltage was set to 4,000 V. MRM was employed for quantification. The fragmentor voltage, collision energy values and dwell time of each ion pair were optimized to obtain the highest abundance. Method validation The quantitative analysis was an external calibration method, and the linearity calibration curves were constructed by six different concentrations of 16 components, where all of them were prepared by serial dilutions with methanol. Each concentration was analyzed in triplicate, and the calibration curves were established by plotting the peak areas with the concentrations of each component. For the limit of detection (LOD) and limit of quantification (LOQ), namely, the sensitivity of the method was measured at a signal-to-noise ratio of 3 and 10, respectively, as the criteria. The intra-day precision was evaluated by detection of the mixed standard solution at six times within 1 day, whereas the inter-day precision was determined by repeated analysis of the mixed standard solution in 3 consecutive days. To confirm repeatability, five replicates of the same samples were prepared according to the procedures described above. Regarding the stability, it was examined by testing the sample every 2 h for 24 h continuously. The standard addition method was applied in the recovery test of this method. Three different concentration levels (approximately equivalent to 0.8, 1.0 and 1.2 times of the concentration of the matrix) of the references standards were added into the sample in triplicate. The total amount of each analyte was calculated from the corresponding calibration curve, and the average recoveries were determined and calculated. Quantification of FLP samples Forty-nine batches of two dosage forms of FLP samples from 11 companies were analyzed by the established method as described above, and each sample was analyzed three times. The quantification of the 16 standard compounds in the FLP samples was performed using their individual calibration curves. Chemometric data analysis The data of the two dosage forms of the FLP samples were calculated and analyzed by Simca-P 13.0 software (Umetrics, Umeå, Sweden), which included a multivariate statistical analysis, such as OPLS-DA. The receiver operating characteristic (ROC) curve and heat map generation was performed by a web-based tool called MetaboloAnalyst 3.0 (http://www.metaboanalyst.ca/). The areas (AUC) under ROC were constructed to evaluate the accuracy of differential markers in distinguishing dosage form. In addition, Student’s t test was used to calculate the statistical significance between two groups by SPSS 22.0, and the P value of Student’s t test was set to 0.05 for this study. Results Method validation To verify the reliability of the results, the established method was validated with respect to linearity, precision, sensitivity, stability and accuracy for the two dosage forms. The results in Table I show that full calibration curves of the 16 compounds were obtained with relatively wide concentration ranges, where the correlation coefficients of all the calibration curves were >0.9997. A portion of the BHP samples were diluted 5-fold due to their high contents to ensure that the detection value satisfied the linear ranges. For different compounds, the LOD ranged from 0.007 to 8.349 ng/mL, and the LOQ ranged from 0.024 to 27.83 ng/mL. The RSDs of intra- and inter-day precision were in the range of 2.04–3.00% and 2.76–3.99%, respectively. The stability was <4.42%. Recoveries of the 16 compounds ranged from 90.84 to 106.7% with all of the RSDs <7.65% (Table SIII), which indicates that the optimized method is accurate. Table I. Calibration Curves, LOD, LOQ, Precision and Repeatability for 16 Compounds in FLP No.  Compound  Calibration curve  r  Linear range (ng/mL)  LOD (ng/mL)  LOQ (ng/mL)  Intra-day (RSD,%) (n = 6)  Inter-day (RSD,%) (n = 6)  Repeatability (RSD,%) (n = 5)  1  AD  y = 3,998x + 6.105  0.9998  1.200–60.00  0.145  0.483  2.42  3.03  3.10  2  LQR  y = 845.1x + 16.68  0.9998  100.8–5,040  0.785  2.615  2.48  3.39  3.32  3  BMA  y = 19,238x + 56.66  0.9998  1.848–462.0  0.128  0.427  2.86  3.90  3.69  4  BA  y = 24,844x + 17.11  0.9998  0.3536–88.40  0.082  0.274  2.68  3.34  3.98  5  BHA  y = 24,181x + 68.04  0.9998  1.296–324.0  0.018  0.058  2.63  3.56  3.20  6  LQG  y = 9,009x + 189.1  0.9998  2.720–680.0  0.814  2.713  2.84  3.23  2.97  7  MA  y = 12,767x + 2.703  0.9999  0.1165–29.12  0.035  0.115  2.21  3.02  3.43  8  AC  y = 19,072x + 0.8950  0.9999  0.02954–7.385  0.007  0.024  3.00  3.87  3.77  9  HA  y = 22,114x + 10.13  0.9998  0.3696–92.40  0.022  0.072  2.40  3.35  3.09  10  GIA  y = 2,048x + 1,119  0.9998  52.11–13,027  0.372  1.238  2.08  2.76  3.13  11  OLQG  y = 30,198x + 64.96  0.9998  0.3840–115.2  0.105  0.350  2.16  2.88  3.85  12  GE  y = 5.544x + 2.660  0.9997  29.76–5,952  8.349  27.83  2.86  3.99  4.42  13  ALIII  y = 2,813x + 19.49  0.9998  1.632–408.0  0.435  1.450  2.68  3.19  2.57  14  ALI  y = 3,923x + 9.701  0.9998  1.984–496.0  0.388  1.292  2.04  3.50  3.65  15  ALII  y = 2,041x + 6.923  0.9999  1.598–399.6  0.425  1.417  2.75  3.83  3.83  16  GEA  y = 5,718x − 1.576  0.9999  0.5200–130.0  0.135  0.452  2.21  3.42  3.07  No.  Compound  Calibration curve  r  Linear range (ng/mL)  LOD (ng/mL)  LOQ (ng/mL)  Intra-day (RSD,%) (n = 6)  Inter-day (RSD,%) (n = 6)  Repeatability (RSD,%) (n = 5)  1  AD  y = 3,998x + 6.105  0.9998  1.200–60.00  0.145  0.483  2.42  3.03  3.10  2  LQR  y = 845.1x + 16.68  0.9998  100.8–5,040  0.785  2.615  2.48  3.39  3.32  3  BMA  y = 19,238x + 56.66  0.9998  1.848–462.0  0.128  0.427  2.86  3.90  3.69  4  BA  y = 24,844x + 17.11  0.9998  0.3536–88.40  0.082  0.274  2.68  3.34  3.98  5  BHA  y = 24,181x + 68.04  0.9998  1.296–324.0  0.018  0.058  2.63  3.56  3.20  6  LQG  y = 9,009x + 189.1  0.9998  2.720–680.0  0.814  2.713  2.84  3.23  2.97  7  MA  y = 12,767x + 2.703  0.9999  0.1165–29.12  0.035  0.115  2.21  3.02  3.43  8  AC  y = 19,072x + 0.8950  0.9999  0.02954–7.385  0.007  0.024  3.00  3.87  3.77  9  HA  y = 22,114x + 10.13  0.9998  0.3696–92.40  0.022  0.072  2.40  3.35  3.09  10  GIA  y = 2,048x + 1,119  0.9998  52.11–13,027  0.372  1.238  2.08  2.76  3.13  11  OLQG  y = 30,198x + 64.96  0.9998  0.3840–115.2  0.105  0.350  2.16  2.88  3.85  12  GE  y = 5.544x + 2.660  0.9997  29.76–5,952  8.349  27.83  2.86  3.99  4.42  13  ALIII  y = 2,813x + 19.49  0.9998  1.632–408.0  0.435  1.450  2.68  3.19  2.57  14  ALI  y = 3,923x + 9.701  0.9998  1.984–496.0  0.388  1.292  2.04  3.50  3.65  15  ALII  y = 2,041x + 6.923  0.9999  1.598–399.6  0.425  1.417  2.75  3.83  3.83  16  GEA  y = 5,718x − 1.576  0.9999  0.5200–130.0  0.135  0.452  2.21  3.42  3.07  Table I. Calibration Curves, LOD, LOQ, Precision and Repeatability for 16 Compounds in FLP No.  Compound  Calibration curve  r  Linear range (ng/mL)  LOD (ng/mL)  LOQ (ng/mL)  Intra-day (RSD,%) (n = 6)  Inter-day (RSD,%) (n = 6)  Repeatability (RSD,%) (n = 5)  1  AD  y = 3,998x + 6.105  0.9998  1.200–60.00  0.145  0.483  2.42  3.03  3.10  2  LQR  y = 845.1x + 16.68  0.9998  100.8–5,040  0.785  2.615  2.48  3.39  3.32  3  BMA  y = 19,238x + 56.66  0.9998  1.848–462.0  0.128  0.427  2.86  3.90  3.69  4  BA  y = 24,844x + 17.11  0.9998  0.3536–88.40  0.082  0.274  2.68  3.34  3.98  5  BHA  y = 24,181x + 68.04  0.9998  1.296–324.0  0.018  0.058  2.63  3.56  3.20  6  LQG  y = 9,009x + 189.1  0.9998  2.720–680.0  0.814  2.713  2.84  3.23  2.97  7  MA  y = 12,767x + 2.703  0.9999  0.1165–29.12  0.035  0.115  2.21  3.02  3.43  8  AC  y = 19,072x + 0.8950  0.9999  0.02954–7.385  0.007  0.024  3.00  3.87  3.77  9  HA  y = 22,114x + 10.13  0.9998  0.3696–92.40  0.022  0.072  2.40  3.35  3.09  10  GIA  y = 2,048x + 1,119  0.9998  52.11–13,027  0.372  1.238  2.08  2.76  3.13  11  OLQG  y = 30,198x + 64.96  0.9998  0.3840–115.2  0.105  0.350  2.16  2.88  3.85  12  GE  y = 5.544x + 2.660  0.9997  29.76–5,952  8.349  27.83  2.86  3.99  4.42  13  ALIII  y = 2,813x + 19.49  0.9998  1.632–408.0  0.435  1.450  2.68  3.19  2.57  14  ALI  y = 3,923x + 9.701  0.9998  1.984–496.0  0.388  1.292  2.04  3.50  3.65  15  ALII  y = 2,041x + 6.923  0.9999  1.598–399.6  0.425  1.417  2.75  3.83  3.83  16  GEA  y = 5,718x − 1.576  0.9999  0.5200–130.0  0.135  0.452  2.21  3.42  3.07  No.  Compound  Calibration curve  r  Linear range (ng/mL)  LOD (ng/mL)  LOQ (ng/mL)  Intra-day (RSD,%) (n = 6)  Inter-day (RSD,%) (n = 6)  Repeatability (RSD,%) (n = 5)  1  AD  y = 3,998x + 6.105  0.9998  1.200–60.00  0.145  0.483  2.42  3.03  3.10  2  LQR  y = 845.1x + 16.68  0.9998  100.8–5,040  0.785  2.615  2.48  3.39  3.32  3  BMA  y = 19,238x + 56.66  0.9998  1.848–462.0  0.128  0.427  2.86  3.90  3.69  4  BA  y = 24,844x + 17.11  0.9998  0.3536–88.40  0.082  0.274  2.68  3.34  3.98  5  BHA  y = 24,181x + 68.04  0.9998  1.296–324.0  0.018  0.058  2.63  3.56  3.20  6  LQG  y = 9,009x + 189.1  0.9998  2.720–680.0  0.814  2.713  2.84  3.23  2.97  7  MA  y = 12,767x + 2.703  0.9999  0.1165–29.12  0.035  0.115  2.21  3.02  3.43  8  AC  y = 19,072x + 0.8950  0.9999  0.02954–7.385  0.007  0.024  3.00  3.87  3.77  9  HA  y = 22,114x + 10.13  0.9998  0.3696–92.40  0.022  0.072  2.40  3.35  3.09  10  GIA  y = 2,048x + 1,119  0.9998  52.11–13,027  0.372  1.238  2.08  2.76  3.13  11  OLQG  y = 30,198x + 64.96  0.9998  0.3840–115.2  0.105  0.350  2.16  2.88  3.85  12  GE  y = 5.544x + 2.660  0.9997  29.76–5,952  8.349  27.83  2.86  3.99  4.42  13  ALIII  y = 2,813x + 19.49  0.9998  1.632–408.0  0.435  1.450  2.68  3.19  2.57  14  ALI  y = 3,923x + 9.701  0.9998  1.984–496.0  0.388  1.292  2.04  3.50  3.65  15  ALII  y = 2,041x + 6.923  0.9999  1.598–399.6  0.425  1.417  2.75  3.83  3.83  16  GEA  y = 5,718x − 1.576  0.9999  0.5200–130.0  0.135  0.452  2.21  3.42  3.07  Quantitative analysis of samples The established RRLC–MS-MS method was successfully applied to determine the content of the 16 components in the 49 batches of commercial FLP samples (Table II). To ensure the comparability of results, the concentration of each component was at the same level as in material drugs in all samples. Total contents of monoester-diterpenoid alkaloids (MDAs), including BMA, BA and BHA, from Fuzi in BHP and CP samples were in the range of 0.573–240.67 and 37.076–605.73 μg/g, respectively, while total diester-diterpenoid alkaloids (DDAs) (including MA, AC and HA) in BHP and CP samples were in the range of 0.046–44.933 and 0.357–20.48 μg/g. It could be easily found that all samples contained a high concentration of LQR in Gancao except sample No. 39 and its presence is mandatory for every company to meet the Chinese patent medicine standard. Among the 16 analytes, GIA was the compound with the highest content because it was the component with the highest content in Gancao (26). Besides, it has been found that the contents of GE in Ganjiang were 280.7–3,867 μg/g in all BHP samples, while they were very lower and even couldn’t be detected in CP samples. The low contents of GE in CP may derived by the different pharmaceutical process of Ganjiang in CP and BHP. In CP, Ganjiang was extracted by 70% ethanol, while it was herb power in BHP. The extracting solvent had a significant influence on the content of GE according to other literature reports (27). Similarly, contents of the compounds ALIII, ALII, ALI in Baizhu were much higher in BHP samples than those in CP samples. Besides, it is indicated that there was significant difference among products of batch-to-batch or from different manufactures even in the same dosage form, as the literature reported that the variation in contents of the constituents could be derived from the quality of raw herbal material (28). Table II. Contents of the 16 Compounds in 49 Batches of Samples Dosage form  Batch no.  Contents of each compound (μg/g relevant herbal material)  AD  LQR  BMA  BA  BHA  LQG  MA  AC  Big honeyed-pill  1  26.00 ± 0.437  2,095 ± 23.38  18.55 ± 0.821  3.478 ± 0.051  4.335 ± 0.080  62.12 ± 0.790  0.130 ± 0.005  0.057 ± 0.002  2  21.77 ± 0.574  1,659 ± 4.632  81.57 ± 0.228  8.989 ± 0.159  10.96 ± 0.121  207.8 ± 2.806  7.458 ± 0.005  1.601 ± 0.042  3  15.88 ± 0.168  1,581 ± 27.51  77.00 ± 1.379  7.934 ± 0.168  10.54 ± 0.180  188.3 ± 1.129  6.490 ± 0.253  1.515 ± 0.046  4  31.37 ± 0.194  2,067 ± 20.22  54.91 ± 0.591  6.704 ± 0.240  12.63 ± 0.468  78.47 ± 2.381  2.628 ± 0.176  0.529 ± 0.014  5  15.34 ± 0.225  1,744 ± 46.48  81.10 ± 1.676  8.863 ± 0.213  10.69 ± 0.283  201.9 ± 1.563  6.580 ± 0.090  1.562 ± 0.062  6  20.81 ± 0.321  1,577 ± 27.20  102.5 ± 1.018  9.725 ± 0.381  11.16 ± 0.403  259.2 ± 1.079  11.89 ± 0.173  2.403 ± 0.037  7  23.30 ± 0.314  2,996 ± 25.53  136.2 ± 1.042  14.49 ± 0.484  22.88 ± 0.523  56.66 ± 2.431  11.62 ± 0.183  2.253 ± 0.025  8  34.23 ± 0.641  2,998 ± 29.53  84.61 ± 1.325  12.07 ± 0.931  23.91 ± 0.515  56.18 ± 1.934  3.131 ± 0.293  0.724 ± 0.041  9  34.52 ± 0.376  3,105 ± 21.42  94.33 ± 1.016  14.46 ± 0.231  22.14 ± 0.356  70.53 ± 1.221  1.065 ± 0.923  0.474 ± 0.056  10  1.847 ± 0.072  1,796 ± 17.55  6.584 ± 0.316  0.889 ± 0.033  1.795 ± 0.063  108.0 ± 4.831  0.053 ± 0.407  0.034 ± 0.002  11  12.16 ± 0.134  2,676 ± 21.53  1.944 ± 0.413  0.089 ± 0.045  0.008 ± 0.001  95.19 ± 1.842  0.016 ± 0.002  0.009 ± 0.000  12  8.128 ± 0.274  2,686 ± 19.37  0.316 ± 0.041  0.062 ± 0.031  0.195 ± 0.034  69.59 ± 1.780  0.021 ± 0.008  0.012 ± 0.000  13  3.171 ± 0.384  2,807 ± 27.53  3.590 ± 0.319  0.490 ± 0.016  1.524 ± 0.051  97.58 ± 2.014  0.080 ± 0.007  0.038 ± 0.013  14  0.163 ± 0.064  3,921 ± 25.48  10.82 ± 1.452  1.343 ± 0.084  0.886 ± 0.093  150.5 ± 3.858  0.031 ± 0.008  0.022 ± 0.001  15  3.365 ± 0.092  5,659 ± 25.84  46.13 ± 0.924  7.494 ± 0.153  10.40 ± 0.073  147.7 ± 3.239  0.143 ± 0.019  0.172 ± 0.014  16  3.230 ± 0.124  1,557 ± 30.28  102.5 ± 1.099  11.38 ± 0.275  17.34 ± 0.373  68.32 ± 0.285  4.996 ± 0.002  1.109 ± 0.042  17  2.105 ± 0.072  1,875 ± 9.186  81.72 ± 1.818  9.724 ± 0.096  16.29 ± 0.340  172.9 ± 1.233  4.575 ± 0.068  1.226 ± 0.049  18  2.369 ± 0.072  1,503 ± 7.465  97.21 ± 0.560  10.73 ± 0.143  17.39 ± 0.348  68.39 ± 1.862  4.569 ± 0.097  1.201 ± 0.025  19  5.704 ± 0.143  1,776 ± 24.02  90.73 ± 1.636  9.930 ± 0.128  15.60 ± 0.228  43.65 ± 0.922  5.058 ± 0.084  1.310 ± 0.025  20  9.860 ± 0.235  2,317 ± 22.51  63.12 ± 1.782  12.81 ± 1.024  28.27 ± 0.142  55.21 ± 1.523  1.600 ± 0.083  0.779 ± 0.044  21  4.073 ± 0.327  2,155 ± 17.42  113.5 ± 3.515  12.48 ± 0.931  20.00 ± 0.201  52.07 ± 1.664  4.015 ± 0.081  1.264 ± 0.053  22  18.53 ± 0.415  1,392 ± 18.20  44.27 ± 1.833  0.311 ± 0.020  11.58 ± 0.272  96.95 ± 2.323  0.246 ± 0.160  0.171 ± 0.003  23  19.81 ± 0.327  1,509 ± 62.60  37.37 ± 4.046  0.409 ± 0.025  11.26 ± 0.403  141.5 ± 2.064  0.306 ± 0.008  0.173 ± 0.003  24  18.74 ± 0.579  1,432 ± 42.79  31.40 ± 0.455  0.186 ± 0.001  9.628 ± 0.052  111.0 ± 2.425  0.293 ± 0.005  0.177 ± 0.003  25  31.22 ± 0.632  2,046 ± 20.51  54.42 ± 2.013  4.112 ± 0.146  5.142 ± 0.109  82.90 ± 3.028  10.01 ± 0.098  1.844 ± 0.028  26  13.29 ± 0.234  1,722 ± 21.58  198.3 ± 8.421  20.96 ± 1.023  21.41 ± 0.113  106.9 ± 3.741  4.287 ± 0.122  1.389 ± 0.084  27  16.57 ± 0.134  1,706 ± 19.52  92.45 ± 1.951  11.29 ± 1.184  16.60 ± 0.142  64.92 ± 1.447  1.794 ± 0.053  0.516 ± 0.032    RSD/%  73.3  41.8  69.3  76.1  63.9  52.7  106.1  88.2  Concentrated pill  28  26.06 ± 1.056  1,386 ± 38.24  133.3 ± 4.227  30.83 ± 0.531  107.3 ± 4.381  84.83 ± 1.935  0.062 ± 0.239  0.055 ± 0.001  29  19.36 ± 0.483  546.0 ± 6.419  102.9 ± 3.848  22.36 ± 0.475  93.01 ± 0.948  54.72 ± 2.849  0.102 ± 0.001  0.060 ± 0.001  30  21.63 ± 0.394  1,170 ± 26.72  117.8 ± 1.050  29.02 ± 0.991  94.39 ± 1.083  91.24 ± 0.992  0.088 ± 0.002  0.073 ± 0.001  31  22.01 ± 0.676  563.8 ± 19.10  88.64 ± 0.255  21.26 ± 0.554  106.5 ± 4.126  74.07 ± 3.309  0.142 ± 0.003  0.123 ± 0.003  32  33.98 ± 0.374  4,084 ± 28.83  319.1 ± 2.941  68.63 ± 1.251  218.0 ± 3.416  118.2 ± 2.041  0.185 ± 0.003  0.111 ± 0.007  33  37.35 ± 0.452  2,602 ± 26.31  304.6 ± 2.055  64.68 ± 0.491  198.5 ± 2.955  98.19 ± 1.453  0.176 ± 0.004  0.192 ± 0.008  34  7.166 ± 0.341  575.9 ± 3.932  45.81 ± 0.166  10.55 ± 0.124  24.23 ± 0.941  91.37 ± 2.450  0.058 ± 0.004  0.055 ± 0.001  35  9.863 ± 0.293  1,200 ± 14.11  96.86 ± 3.031  16.28 ± 0.283  44.73 ± 1.651  23.20 ± 1.533  0.173 ± 0.010  0.064 ± 0.003  36  15.47 ± 0.473  2,010 ± 17.82  99.95 ± 2.951  23.12 ± 0.341  51.60 ± 2.042  51.19 ± 2.951  0.197 ± 0.011  0.174 ± 0.005  37  13.47 ± 0.417  528.0 ± 10.30  58.71 ± 1.809  12.31 ± 0.152  39.60 ± 0.652  35.12 ± 1.240  0.036 ± 0.001  0.042 ± 0.000  38  10.71 ± 0.154  438.6 ± 1.613  64.72 ± 1.155  12.22 ± 0.272  38.79 ± 0.551  34.86 ± 0.358  0.069 ± 0.001  0.045 ± 0.001  39  12.40 ± 0.229  172.9 ± 2.009  35.98 ± 0.576  8.178 ± 0.115  31.10 ± 0.397  0.958 ± 0.032  0.055 ± 0.001  0.025 ± 0.000  40  20.49 ± 0.245  401.9 ± 8.352  114.4 ± 4.014  24.95 ± 0.398  64.94 ± 1.432  24.68 ± 1.542  0.003 ± 0.000  0.011 ± 0.000  41  7.184 ± 0.197  903.5 ± 5.530  26.36 ± 0.468  4.497 ± 0.195  9.323 ± 0.360  65.92 ± 2.121  0.080 ± 0.000  0.028 ± 0.001  42  17.92 ± 0.831  3,069 ± 10.51  67.96 ± 1.031  11.16 ± 0.312  20.06 ± 0.966  77.29 ± 3.133  0.170 ± 0.004  0.075 ± 0.000  43  14.43 ± 0.624  3,206 ± 28.41  70.98 ± 0.941  11.98 ± 0.284  19.60 ± 0.455  85.53 ± 2.414  0.090 ± 0.002  0.046 ± 0.000  44  8.560 ± 0.532  2,137 ± 15.32  87.56 ± 1.252  11.84 ± 0.184  29.41 ± 1.053  282.9 ± 3.988  0.683 ± 0.010  0.231 ± 0.001  45  4.262 ± 0.135  1,023 ± 11.42  44.59 ± 0.836  5.705 ± 0.167  18.82 ± 0.533  139.1 ± 3.556  0.560 ± 0.009  0.170 ± 0.000  46  16.02 ± 0.142  698.7 ± 2.889  16.33 ± 0.125  8.276 ± 0.084  12.47 ± 0.250  34.48 ± 0.509  1.409 ± 0.002  0.531 ± 0.000  47  2.909 ± 0.068  1,000 ± 3.059  175.3 ± 0.537  32.29 ± 0.337  79.26 ± 0.240  68.99 ± 0.691  0.217 ± 0.049  0.140 ± 0.000  48  23.65 ± 0.523  2,174 ± 16.82  252.7 ± 1.934  58.30 ± 0.253  129.4 ± 0.675  17.17 ± 0.886  1.102 ± 0.088  0.680 ± 0.001  49  7.264 ± 0.834  1,966 ± 12.44  253.2 ± 0.894  43.46 ± 0.384  115.6 ± 1.143  110.0 ± 3.452  0.895 ± 0.014  0.675 ± 0.003    RSD/%  56.6  73.7  75.7  78.4  82.6  77.2  129.2  121.8  Dosage form  Batch no.  Contents of each compound (μg/g relevant herbal material)  AD  LQR  BMA  BA  BHA  LQG  MA  AC  Big honeyed-pill  1  26.00 ± 0.437  2,095 ± 23.38  18.55 ± 0.821  3.478 ± 0.051  4.335 ± 0.080  62.12 ± 0.790  0.130 ± 0.005  0.057 ± 0.002  2  21.77 ± 0.574  1,659 ± 4.632  81.57 ± 0.228  8.989 ± 0.159  10.96 ± 0.121  207.8 ± 2.806  7.458 ± 0.005  1.601 ± 0.042  3  15.88 ± 0.168  1,581 ± 27.51  77.00 ± 1.379  7.934 ± 0.168  10.54 ± 0.180  188.3 ± 1.129  6.490 ± 0.253  1.515 ± 0.046  4  31.37 ± 0.194  2,067 ± 20.22  54.91 ± 0.591  6.704 ± 0.240  12.63 ± 0.468  78.47 ± 2.381  2.628 ± 0.176  0.529 ± 0.014  5  15.34 ± 0.225  1,744 ± 46.48  81.10 ± 1.676  8.863 ± 0.213  10.69 ± 0.283  201.9 ± 1.563  6.580 ± 0.090  1.562 ± 0.062  6  20.81 ± 0.321  1,577 ± 27.20  102.5 ± 1.018  9.725 ± 0.381  11.16 ± 0.403  259.2 ± 1.079  11.89 ± 0.173  2.403 ± 0.037  7  23.30 ± 0.314  2,996 ± 25.53  136.2 ± 1.042  14.49 ± 0.484  22.88 ± 0.523  56.66 ± 2.431  11.62 ± 0.183  2.253 ± 0.025  8  34.23 ± 0.641  2,998 ± 29.53  84.61 ± 1.325  12.07 ± 0.931  23.91 ± 0.515  56.18 ± 1.934  3.131 ± 0.293  0.724 ± 0.041  9  34.52 ± 0.376  3,105 ± 21.42  94.33 ± 1.016  14.46 ± 0.231  22.14 ± 0.356  70.53 ± 1.221  1.065 ± 0.923  0.474 ± 0.056  10  1.847 ± 0.072  1,796 ± 17.55  6.584 ± 0.316  0.889 ± 0.033  1.795 ± 0.063  108.0 ± 4.831  0.053 ± 0.407  0.034 ± 0.002  11  12.16 ± 0.134  2,676 ± 21.53  1.944 ± 0.413  0.089 ± 0.045  0.008 ± 0.001  95.19 ± 1.842  0.016 ± 0.002  0.009 ± 0.000  12  8.128 ± 0.274  2,686 ± 19.37  0.316 ± 0.041  0.062 ± 0.031  0.195 ± 0.034  69.59 ± 1.780  0.021 ± 0.008  0.012 ± 0.000  13  3.171 ± 0.384  2,807 ± 27.53  3.590 ± 0.319  0.490 ± 0.016  1.524 ± 0.051  97.58 ± 2.014  0.080 ± 0.007  0.038 ± 0.013  14  0.163 ± 0.064  3,921 ± 25.48  10.82 ± 1.452  1.343 ± 0.084  0.886 ± 0.093  150.5 ± 3.858  0.031 ± 0.008  0.022 ± 0.001  15  3.365 ± 0.092  5,659 ± 25.84  46.13 ± 0.924  7.494 ± 0.153  10.40 ± 0.073  147.7 ± 3.239  0.143 ± 0.019  0.172 ± 0.014  16  3.230 ± 0.124  1,557 ± 30.28  102.5 ± 1.099  11.38 ± 0.275  17.34 ± 0.373  68.32 ± 0.285  4.996 ± 0.002  1.109 ± 0.042  17  2.105 ± 0.072  1,875 ± 9.186  81.72 ± 1.818  9.724 ± 0.096  16.29 ± 0.340  172.9 ± 1.233  4.575 ± 0.068  1.226 ± 0.049  18  2.369 ± 0.072  1,503 ± 7.465  97.21 ± 0.560  10.73 ± 0.143  17.39 ± 0.348  68.39 ± 1.862  4.569 ± 0.097  1.201 ± 0.025  19  5.704 ± 0.143  1,776 ± 24.02  90.73 ± 1.636  9.930 ± 0.128  15.60 ± 0.228  43.65 ± 0.922  5.058 ± 0.084  1.310 ± 0.025  20  9.860 ± 0.235  2,317 ± 22.51  63.12 ± 1.782  12.81 ± 1.024  28.27 ± 0.142  55.21 ± 1.523  1.600 ± 0.083  0.779 ± 0.044  21  4.073 ± 0.327  2,155 ± 17.42  113.5 ± 3.515  12.48 ± 0.931  20.00 ± 0.201  52.07 ± 1.664  4.015 ± 0.081  1.264 ± 0.053  22  18.53 ± 0.415  1,392 ± 18.20  44.27 ± 1.833  0.311 ± 0.020  11.58 ± 0.272  96.95 ± 2.323  0.246 ± 0.160  0.171 ± 0.003  23  19.81 ± 0.327  1,509 ± 62.60  37.37 ± 4.046  0.409 ± 0.025  11.26 ± 0.403  141.5 ± 2.064  0.306 ± 0.008  0.173 ± 0.003  24  18.74 ± 0.579  1,432 ± 42.79  31.40 ± 0.455  0.186 ± 0.001  9.628 ± 0.052  111.0 ± 2.425  0.293 ± 0.005  0.177 ± 0.003  25  31.22 ± 0.632  2,046 ± 20.51  54.42 ± 2.013  4.112 ± 0.146  5.142 ± 0.109  82.90 ± 3.028  10.01 ± 0.098  1.844 ± 0.028  26  13.29 ± 0.234  1,722 ± 21.58  198.3 ± 8.421  20.96 ± 1.023  21.41 ± 0.113  106.9 ± 3.741  4.287 ± 0.122  1.389 ± 0.084  27  16.57 ± 0.134  1,706 ± 19.52  92.45 ± 1.951  11.29 ± 1.184  16.60 ± 0.142  64.92 ± 1.447  1.794 ± 0.053  0.516 ± 0.032    RSD/%  73.3  41.8  69.3  76.1  63.9  52.7  106.1  88.2  Concentrated pill  28  26.06 ± 1.056  1,386 ± 38.24  133.3 ± 4.227  30.83 ± 0.531  107.3 ± 4.381  84.83 ± 1.935  0.062 ± 0.239  0.055 ± 0.001  29  19.36 ± 0.483  546.0 ± 6.419  102.9 ± 3.848  22.36 ± 0.475  93.01 ± 0.948  54.72 ± 2.849  0.102 ± 0.001  0.060 ± 0.001  30  21.63 ± 0.394  1,170 ± 26.72  117.8 ± 1.050  29.02 ± 0.991  94.39 ± 1.083  91.24 ± 0.992  0.088 ± 0.002  0.073 ± 0.001  31  22.01 ± 0.676  563.8 ± 19.10  88.64 ± 0.255  21.26 ± 0.554  106.5 ± 4.126  74.07 ± 3.309  0.142 ± 0.003  0.123 ± 0.003  32  33.98 ± 0.374  4,084 ± 28.83  319.1 ± 2.941  68.63 ± 1.251  218.0 ± 3.416  118.2 ± 2.041  0.185 ± 0.003  0.111 ± 0.007  33  37.35 ± 0.452  2,602 ± 26.31  304.6 ± 2.055  64.68 ± 0.491  198.5 ± 2.955  98.19 ± 1.453  0.176 ± 0.004  0.192 ± 0.008  34  7.166 ± 0.341  575.9 ± 3.932  45.81 ± 0.166  10.55 ± 0.124  24.23 ± 0.941  91.37 ± 2.450  0.058 ± 0.004  0.055 ± 0.001  35  9.863 ± 0.293  1,200 ± 14.11  96.86 ± 3.031  16.28 ± 0.283  44.73 ± 1.651  23.20 ± 1.533  0.173 ± 0.010  0.064 ± 0.003  36  15.47 ± 0.473  2,010 ± 17.82  99.95 ± 2.951  23.12 ± 0.341  51.60 ± 2.042  51.19 ± 2.951  0.197 ± 0.011  0.174 ± 0.005  37  13.47 ± 0.417  528.0 ± 10.30  58.71 ± 1.809  12.31 ± 0.152  39.60 ± 0.652  35.12 ± 1.240  0.036 ± 0.001  0.042 ± 0.000  38  10.71 ± 0.154  438.6 ± 1.613  64.72 ± 1.155  12.22 ± 0.272  38.79 ± 0.551  34.86 ± 0.358  0.069 ± 0.001  0.045 ± 0.001  39  12.40 ± 0.229  172.9 ± 2.009  35.98 ± 0.576  8.178 ± 0.115  31.10 ± 0.397  0.958 ± 0.032  0.055 ± 0.001  0.025 ± 0.000  40  20.49 ± 0.245  401.9 ± 8.352  114.4 ± 4.014  24.95 ± 0.398  64.94 ± 1.432  24.68 ± 1.542  0.003 ± 0.000  0.011 ± 0.000  41  7.184 ± 0.197  903.5 ± 5.530  26.36 ± 0.468  4.497 ± 0.195  9.323 ± 0.360  65.92 ± 2.121  0.080 ± 0.000  0.028 ± 0.001  42  17.92 ± 0.831  3,069 ± 10.51  67.96 ± 1.031  11.16 ± 0.312  20.06 ± 0.966  77.29 ± 3.133  0.170 ± 0.004  0.075 ± 0.000  43  14.43 ± 0.624  3,206 ± 28.41  70.98 ± 0.941  11.98 ± 0.284  19.60 ± 0.455  85.53 ± 2.414  0.090 ± 0.002  0.046 ± 0.000  44  8.560 ± 0.532  2,137 ± 15.32  87.56 ± 1.252  11.84 ± 0.184  29.41 ± 1.053  282.9 ± 3.988  0.683 ± 0.010  0.231 ± 0.001  45  4.262 ± 0.135  1,023 ± 11.42  44.59 ± 0.836  5.705 ± 0.167  18.82 ± 0.533  139.1 ± 3.556  0.560 ± 0.009  0.170 ± 0.000  46  16.02 ± 0.142  698.7 ± 2.889  16.33 ± 0.125  8.276 ± 0.084  12.47 ± 0.250  34.48 ± 0.509  1.409 ± 0.002  0.531 ± 0.000  47  2.909 ± 0.068  1,000 ± 3.059  175.3 ± 0.537  32.29 ± 0.337  79.26 ± 0.240  68.99 ± 0.691  0.217 ± 0.049  0.140 ± 0.000  48  23.65 ± 0.523  2,174 ± 16.82  252.7 ± 1.934  58.30 ± 0.253  129.4 ± 0.675  17.17 ± 0.886  1.102 ± 0.088  0.680 ± 0.001  49  7.264 ± 0.834  1,966 ± 12.44  253.2 ± 0.894  43.46 ± 0.384  115.6 ± 1.143  110.0 ± 3.452  0.895 ± 0.014  0.675 ± 0.003    RSD/%  56.6  73.7  75.7  78.4  82.6  77.2  129.2  121.8  Dosage form  Batch no.  Contents of each compound (μg/g material drugs)  HA  GIA  OLQG  GE  ALIII  ALI  ALII  GEA  Big honeyed-pill  1  1.452 ± 0.021  2,650 ± 43.32  11.30 ± 0.463  1,595 ± 54.68  42.46 ± 1.757  45.63 ± 1.223  60.20 ± 0.534  2.659 ± 0.053  2  22.99 ± 0.469  3,012 ± 90.64  26.27 ± 0.319  2,031 ± 53.48  172.0 ± 1.676  182.6 ± 3.168  158.0 ± 1.022  6.260 ± 0.142  3  20.77 ± 0.346  3,202 ± 137.4  24.64 ± 0.340  1,464 ± 50.18  141.9 ± 3.387  151.1 ± 1.446  126.1 ± 2.015  5.337 ± 0.204  4  11.87 ± 0.313  3,914 ± 78.75  15.01 ± 0.051  2,341 ± 109.5  100.8 ± 1.155  97.61 ± 1.023  114.4 ± 1.472  2.624 ± 0.046  5  21.61 ± 0.087  3,735 ± 93.67  26.73 ± 0.293  1,217 ± 51.34  129.0 ± 1.124  126.9 ± 0.593  113.5 ± 3.811  6.583 ± 0.068  6  30.64 ± 0.610  3,285 ± 63.06  33.21 ± 1.256  872.9 ± 2.160  94.88 ± 3.543  125.3 ± 1.032  139.0 ± 2.415  8.481 ± 0.248  7  28.42 ± 0.435  3,183 ± 54.76  4.228 ± 0.863  2,554 ± 46.76  81.52 ± 2.657  158.4 ± 2.907  131.6 ± 2.098  0.927 ± 0.097  8  13.29 ± 0.521  4,383 ± 66.87  4.845 ± 0.956  3,112 ± 64.38  89.50 ± 1.543  122.1 ± 2.065  109.0 ± 2.218  1.716 ± 0.087  9  9.252 ± 0.367  4,533 ± 57.43  5.375 ± 0.675  2,545 ± 58.62  89.85 ± 2.989  115.7 ± 1.648  92.76 ± 3.112  1.252 ± 0.057  10  1.178 ± 0.016  4,633 ± 34.81  38.83 ± 0.802  780.8 ± 1.079  106.2 ± 3.058  140.8 ± 3.498  96.39 ± 1.544  21.81 ± 0.884  11  0.021 ± 0.000  4,879 ± 48.67  33.17 ± 0.586  683.2 ± 20.76  222.1 ± 1.677  322.3 ± 2.767  200.1 ± 2.056  35.57 ± 1.076  12  0.021 ± 0.000  4,684 ± 87.43  19.76 ± 0.437  865.9 ± 16.44  245.8 ± 2.452  403.0 ± 2.234  247.1 ± 2.675  9.749 ± 0.078  13  1.150 ± 0.129  5,149 ± 76.44  28.21 ± 0.532  644.1 ± 23.98  99.78 ± 1.777  181.1 ± 0.986  79.67 ± 0.887  23.63 ± 0.254  14  0.695 ± 0.034  8,127 ± 48.61  19.56 ± 0.338  509.9 ± 21.61  248.2 ± 3.665  160.4 ± 1.065  202.0 ± 1.965  7.088 ± 0.334  15  1.680 ± 0.384  10814 ± 98.43  34.65 ± 0.674  600.5 ± 19.833  159.4 ± 2.787  133.1 ± 1.254  113.1 ± 2.137  23.47 ± 0.546  16  26.26 ± 0.718  3,980 ± 121.8  9.190 ± 0.224  1,934 ± 52.95  116.9 ± 0.699  181.6 ± 1.233  156.3 ± 0.929  32.89 ± 1.045  17  24.56 ± 0.323  5,442 ± 139.1  25.94 ± 0.748  2,276 ± 92.71  58.32 ± 1.480  96.40 ± 0.257  87.48 ± 1.436  43.10 ± 1.332  18  24.69 ± 0.378  2,682 ± 88.74  8.464 ± 0.150  2,031 ± 57.87  112.7 ± 2.215  186.4 ± 5.955  159.9 ± 3.114  32.39 ± 0.622  19  24.62 ± 0.462  2,966 ± 20.79  7.386 ± 0.184  2,031 ± 49.40  78.49 ± 1.840  135.0 ± 0.424  99.51 ± 2.247  29.82 ± 0.493  20  31.96 ± 0.532  2,800 ± 47.53  3.054 ± 0.205  3,867 ± 56.63  86.73 ± 2.096  221.0 ± 2.665  121.5 ± 1.877  23.93 ± 0.376  21  22.28 ± 0.787  3,736 ± 25.47  3.564 ± 0.397  3,771 ± 68.42  113.3 ± 2.136  222.1 ± 1.772  163.2 ± 2.870  20.47 ± 0.265  22  3.552 ± 0.122  4,402 ± 42.62  36.27 ± 0.447  875.2 ± 2.344  75.00 ± 1.335  82.91 ± 0.415  80.02 ± 1.082  9.714 ± 0.252  23  2.989 ± 0.033  4,397 ± 55.22  46.85 ± 1.037  687.3 ± 0.791  114.8 ± 4.771  86.91 ± 2.792  90.56 ± 11.312  13.97 ± 0.460  24  3.253 ± 0.052  4,430 ± 76.95  32.60 ± 1.153  781.2 ± 2.178  80.94 ± 1.116  92.23 ± 1.593  88.38 ± 1.604  12.65 ± 0.412  25  18.84 ± 0.452  3,613 ± 17.53  6.957 ± 0.654  2,892 ± 10.75  87.89 ± 2.343  116.0 ± 2.076  106.1 ± 2.122  3.381 ± 0.008  26  28.22 ± 0.445  3,175 ± 35.58  8.621 ± 0.788  2,716 ± 21.09  125.1 ± 2.766  178.1 ± 1.996  121.5 ± 1.942  2.513 ± 0.006  27  12.73 ± 0.782  2,448 ± 17.37  4.007 ± 0.965  280.7 ± 30.07  66.75 ± 2.087  94.44 ± 0.879  78.52 ± 1.209  3.072 ± 0.005    RSD/%  78.8  41.5  69.0  60.8  45.3  48.5  35.2  87.5  Concentrated pill  28  0.924 ± 0.002  1,987 ± 51.50  27.14 ± 0.749  N.D.a  16.45 ± 0.658  3.691 ± 0.074  1.300 ± 0.013  10.68 ± 0.401  29  1.378 ± 0.008  1,850 ± 44.50  33.03 ± 1.046  15.93 ± 0.026  15.28 ± 0.471  4.238 ± 0.179  0.705 ± 0.007  9.821 ± 0.208  30  1.342 ± 0.016  2,282 ± 44.85  54.09 ± 0.672  15.68 ± 0.026  17.49 ± 0.239  6.309 ± 0.086  0.596 ± 0.007  10.70 ± 0.464  31  4.264 ± 0.063  2,384 ± 95.77  46.24 ± 0.842  6.316 ± 0.025  12.05 ± 0.540  3.564 ± 0.178  0.257 ± 0.001  10.10 ± 0.238  32  2.219 ± 0.012  6,741 ± 67.33  21.35 ± 0.531  87.60 ± 1.035  41.86 ± 0.561  21.18 ± 0.093  3.951 ± 0.061  11.56 ± 0.051  33  3.330 ± 0.067  6,161 ± 52.88  18.12 ± 0.312  93.14 ± 1.237  41.22 ± 0.611  25.33 ± 0.123  3.089 ± 0.061  11.74 ± 0.086  34  1.035 ± 0.007  1,311 ± 25.10  43.31 ± 1.231  1.817 ± 0.009  13.82 ± 0.549  20.57 ± 0.856  0.844 ± 0.024  5.012 ± 0.135  35  1.579 ± 0.023  3,786 ± 46.83  6.253 ± 2.577  91.79 ± 1.535  68.57 ± 0.882  103.0 ± 0.562  75.39 ± 0.006  5.097 ± 0.026  36  3.796 ± 0.076  1,633 ± 32.66  6.986 ± 0.566  99.59 ± 2.044  64.18 ± 0.436  98.36 ± 1.053  59.14 ± 0.016  4.091 ± 0.003  37  1.092 ± 0.003  2,049 ± 10.92  23.10 ± 0.431  1.592 ± 0.005  12.94 ± 0.477  2.233 ± 0.015  0.260 ± 0.007  7.134 ± 0.059  38  0.983 ± 0.005  1,973 ± 27.67  23.11 ± 0.173  N.D.a  9.764 ± 0.460  2.137 ± 0.002  0.379 ± 0.007  4.280 ± 0.090  39  0.577 ± 0.018  547.3 ± 23.18  3.369 ± 0.078  N.D.a  8.834 ± 0.304  1.632 ± 0.005  0.260 ± 0.001  4.180 ± 0.058  40  0.343 ± 0.008  128.7 ± 9.531  3.595 ± 0.035  N.D.a  43.33 ± 0.531  14.96 ± 0.012  5.494 ± 0.059  4.521 ± 0.008  41  0.600 ± 0.009  3,872 ± 146.7  55.58 ± 0.473  N.D.a  15.11 ± 0.609  3.484 ± 0.075  1.068 ± 0.013  32.43 ± 0.662  42  1.328 ± 0.033  8,413 ± 125.3  22.99 ± 0.661  N.D.a  44.01 ± 0.844  22.65 ± 0.073  4.964 ± 0.065  39.81 ± 0.035  43  1.450 ± 0.037  9,473 ± 94.31  25.12 ± 0.775  N.D.a  50.67 ± 0.398  36.23 ± 0.015  8.063 ± 0.013  40.35 ± 0.035  44  4.840 ± 0.103  2,959 ± 36.23  84.89 ± 1.356  N.D.a  39.93 ± 0.448  21.87 ± 0.064  5.293 ± 0.058  46.86 ± 0.051  45  5.688 ± 0.112  1,244 ± 17.43  40.77 ± 1.532  N.D. a  16.71 ± 0.562  7.114 ± 0.058  0.653 ± 0.043  25.32 ± 0.044  46  6.687 ± 0.137  2,788 ± 68.26  32.95 ± 0.654  N.D.a  9.299 ± 0.282  1.661 ± 0.001  0.250 ± 0.002  18.49 ± 0.681  47  6.842 ± 0.118  2,415 ± 38.86  24.43 ± 0.229  N.D.a  40.50 ± 0.871  59.27 ± 1.662  45.81 ± 0.998  2.432 ± 0.006  48  17.86 ± 0.271  1,567 ± 14.52  4.972 ± 0.733  159.9 ± 11.53  32.73 ± 0.511  57.32 ± 0.056  37.04 ± 0.051  4.278 ± 0.023  49  18.91 ± 0.176  4,410 ± 17.44  11.94 ± 0.561  48.97 ± 1.442  55.94 ± 0.823  21.06 ± 0.061  5.299 ± 0.066  4.577 ± 0.002    RSD/%  128.5  77.5  72.6  164.7  62.9  121.7  182.6  95.6  Dosage form  Batch no.  Contents of each compound (μg/g material drugs)  HA  GIA  OLQG  GE  ALIII  ALI  ALII  GEA  Big honeyed-pill  1  1.452 ± 0.021  2,650 ± 43.32  11.30 ± 0.463  1,595 ± 54.68  42.46 ± 1.757  45.63 ± 1.223  60.20 ± 0.534  2.659 ± 0.053  2  22.99 ± 0.469  3,012 ± 90.64  26.27 ± 0.319  2,031 ± 53.48  172.0 ± 1.676  182.6 ± 3.168  158.0 ± 1.022  6.260 ± 0.142  3  20.77 ± 0.346  3,202 ± 137.4  24.64 ± 0.340  1,464 ± 50.18  141.9 ± 3.387  151.1 ± 1.446  126.1 ± 2.015  5.337 ± 0.204  4  11.87 ± 0.313  3,914 ± 78.75  15.01 ± 0.051  2,341 ± 109.5  100.8 ± 1.155  97.61 ± 1.023  114.4 ± 1.472  2.624 ± 0.046  5  21.61 ± 0.087  3,735 ± 93.67  26.73 ± 0.293  1,217 ± 51.34  129.0 ± 1.124  126.9 ± 0.593  113.5 ± 3.811  6.583 ± 0.068  6  30.64 ± 0.610  3,285 ± 63.06  33.21 ± 1.256  872.9 ± 2.160  94.88 ± 3.543  125.3 ± 1.032  139.0 ± 2.415  8.481 ± 0.248  7  28.42 ± 0.435  3,183 ± 54.76  4.228 ± 0.863  2,554 ± 46.76  81.52 ± 2.657  158.4 ± 2.907  131.6 ± 2.098  0.927 ± 0.097  8  13.29 ± 0.521  4,383 ± 66.87  4.845 ± 0.956  3,112 ± 64.38  89.50 ± 1.543  122.1 ± 2.065  109.0 ± 2.218  1.716 ± 0.087  9  9.252 ± 0.367  4,533 ± 57.43  5.375 ± 0.675  2,545 ± 58.62  89.85 ± 2.989  115.7 ± 1.648  92.76 ± 3.112  1.252 ± 0.057  10  1.178 ± 0.016  4,633 ± 34.81  38.83 ± 0.802  780.8 ± 1.079  106.2 ± 3.058  140.8 ± 3.498  96.39 ± 1.544  21.81 ± 0.884  11  0.021 ± 0.000  4,879 ± 48.67  33.17 ± 0.586  683.2 ± 20.76  222.1 ± 1.677  322.3 ± 2.767  200.1 ± 2.056  35.57 ± 1.076  12  0.021 ± 0.000  4,684 ± 87.43  19.76 ± 0.437  865.9 ± 16.44  245.8 ± 2.452  403.0 ± 2.234  247.1 ± 2.675  9.749 ± 0.078  13  1.150 ± 0.129  5,149 ± 76.44  28.21 ± 0.532  644.1 ± 23.98  99.78 ± 1.777  181.1 ± 0.986  79.67 ± 0.887  23.63 ± 0.254  14  0.695 ± 0.034  8,127 ± 48.61  19.56 ± 0.338  509.9 ± 21.61  248.2 ± 3.665  160.4 ± 1.065  202.0 ± 1.965  7.088 ± 0.334  15  1.680 ± 0.384  10814 ± 98.43  34.65 ± 0.674  600.5 ± 19.833  159.4 ± 2.787  133.1 ± 1.254  113.1 ± 2.137  23.47 ± 0.546  16  26.26 ± 0.718  3,980 ± 121.8  9.190 ± 0.224  1,934 ± 52.95  116.9 ± 0.699  181.6 ± 1.233  156.3 ± 0.929  32.89 ± 1.045  17  24.56 ± 0.323  5,442 ± 139.1  25.94 ± 0.748  2,276 ± 92.71  58.32 ± 1.480  96.40 ± 0.257  87.48 ± 1.436  43.10 ± 1.332  18  24.69 ± 0.378  2,682 ± 88.74  8.464 ± 0.150  2,031 ± 57.87  112.7 ± 2.215  186.4 ± 5.955  159.9 ± 3.114  32.39 ± 0.622  19  24.62 ± 0.462  2,966 ± 20.79  7.386 ± 0.184  2,031 ± 49.40  78.49 ± 1.840  135.0 ± 0.424  99.51 ± 2.247  29.82 ± 0.493  20  31.96 ± 0.532  2,800 ± 47.53  3.054 ± 0.205  3,867 ± 56.63  86.73 ± 2.096  221.0 ± 2.665  121.5 ± 1.877  23.93 ± 0.376  21  22.28 ± 0.787  3,736 ± 25.47  3.564 ± 0.397  3,771 ± 68.42  113.3 ± 2.136  222.1 ± 1.772  163.2 ± 2.870  20.47 ± 0.265  22  3.552 ± 0.122  4,402 ± 42.62  36.27 ± 0.447  875.2 ± 2.344  75.00 ± 1.335  82.91 ± 0.415  80.02 ± 1.082  9.714 ± 0.252  23  2.989 ± 0.033  4,397 ± 55.22  46.85 ± 1.037  687.3 ± 0.791  114.8 ± 4.771  86.91 ± 2.792  90.56 ± 11.312  13.97 ± 0.460  24  3.253 ± 0.052  4,430 ± 76.95  32.60 ± 1.153  781.2 ± 2.178  80.94 ± 1.116  92.23 ± 1.593  88.38 ± 1.604  12.65 ± 0.412  25  18.84 ± 0.452  3,613 ± 17.53  6.957 ± 0.654  2,892 ± 10.75  87.89 ± 2.343  116.0 ± 2.076  106.1 ± 2.122  3.381 ± 0.008  26  28.22 ± 0.445  3,175 ± 35.58  8.621 ± 0.788  2,716 ± 21.09  125.1 ± 2.766  178.1 ± 1.996  121.5 ± 1.942  2.513 ± 0.006  27  12.73 ± 0.782  2,448 ± 17.37  4.007 ± 0.965  280.7 ± 30.07  66.75 ± 2.087  94.44 ± 0.879  78.52 ± 1.209  3.072 ± 0.005    RSD/%  78.8  41.5  69.0  60.8  45.3  48.5  35.2  87.5  Concentrated pill  28  0.924 ± 0.002  1,987 ± 51.50  27.14 ± 0.749  N.D.a  16.45 ± 0.658  3.691 ± 0.074  1.300 ± 0.013  10.68 ± 0.401  29  1.378 ± 0.008  1,850 ± 44.50  33.03 ± 1.046  15.93 ± 0.026  15.28 ± 0.471  4.238 ± 0.179  0.705 ± 0.007  9.821 ± 0.208  30  1.342 ± 0.016  2,282 ± 44.85  54.09 ± 0.672  15.68 ± 0.026  17.49 ± 0.239  6.309 ± 0.086  0.596 ± 0.007  10.70 ± 0.464  31  4.264 ± 0.063  2,384 ± 95.77  46.24 ± 0.842  6.316 ± 0.025  12.05 ± 0.540  3.564 ± 0.178  0.257 ± 0.001  10.10 ± 0.238  32  2.219 ± 0.012  6,741 ± 67.33  21.35 ± 0.531  87.60 ± 1.035  41.86 ± 0.561  21.18 ± 0.093  3.951 ± 0.061  11.56 ± 0.051  33  3.330 ± 0.067  6,161 ± 52.88  18.12 ± 0.312  93.14 ± 1.237  41.22 ± 0.611  25.33 ± 0.123  3.089 ± 0.061  11.74 ± 0.086  34  1.035 ± 0.007  1,311 ± 25.10  43.31 ± 1.231  1.817 ± 0.009  13.82 ± 0.549  20.57 ± 0.856  0.844 ± 0.024  5.012 ± 0.135  35  1.579 ± 0.023  3,786 ± 46.83  6.253 ± 2.577  91.79 ± 1.535  68.57 ± 0.882  103.0 ± 0.562  75.39 ± 0.006  5.097 ± 0.026  36  3.796 ± 0.076  1,633 ± 32.66  6.986 ± 0.566  99.59 ± 2.044  64.18 ± 0.436  98.36 ± 1.053  59.14 ± 0.016  4.091 ± 0.003  37  1.092 ± 0.003  2,049 ± 10.92  23.10 ± 0.431  1.592 ± 0.005  12.94 ± 0.477  2.233 ± 0.015  0.260 ± 0.007  7.134 ± 0.059  38  0.983 ± 0.005  1,973 ± 27.67  23.11 ± 0.173  N.D.a  9.764 ± 0.460  2.137 ± 0.002  0.379 ± 0.007  4.280 ± 0.090  39  0.577 ± 0.018  547.3 ± 23.18  3.369 ± 0.078  N.D.a  8.834 ± 0.304  1.632 ± 0.005  0.260 ± 0.001  4.180 ± 0.058  40  0.343 ± 0.008  128.7 ± 9.531  3.595 ± 0.035  N.D.a  43.33 ± 0.531  14.96 ± 0.012  5.494 ± 0.059  4.521 ± 0.008  41  0.600 ± 0.009  3,872 ± 146.7  55.58 ± 0.473  N.D.a  15.11 ± 0.609  3.484 ± 0.075  1.068 ± 0.013  32.43 ± 0.662  42  1.328 ± 0.033  8,413 ± 125.3  22.99 ± 0.661  N.D.a  44.01 ± 0.844  22.65 ± 0.073  4.964 ± 0.065  39.81 ± 0.035  43  1.450 ± 0.037  9,473 ± 94.31  25.12 ± 0.775  N.D.a  50.67 ± 0.398  36.23 ± 0.015  8.063 ± 0.013  40.35 ± 0.035  44  4.840 ± 0.103  2,959 ± 36.23  84.89 ± 1.356  N.D.a  39.93 ± 0.448  21.87 ± 0.064  5.293 ± 0.058  46.86 ± 0.051  45  5.688 ± 0.112  1,244 ± 17.43  40.77 ± 1.532  N.D. a  16.71 ± 0.562  7.114 ± 0.058  0.653 ± 0.043  25.32 ± 0.044  46  6.687 ± 0.137  2,788 ± 68.26  32.95 ± 0.654  N.D.a  9.299 ± 0.282  1.661 ± 0.001  0.250 ± 0.002  18.49 ± 0.681  47  6.842 ± 0.118  2,415 ± 38.86  24.43 ± 0.229  N.D.a  40.50 ± 0.871  59.27 ± 1.662  45.81 ± 0.998  2.432 ± 0.006  48  17.86 ± 0.271  1,567 ± 14.52  4.972 ± 0.733  159.9 ± 11.53  32.73 ± 0.511  57.32 ± 0.056  37.04 ± 0.051  4.278 ± 0.023  49  18.91 ± 0.176  4,410 ± 17.44  11.94 ± 0.561  48.97 ± 1.442  55.94 ± 0.823  21.06 ± 0.061  5.299 ± 0.066  4.577 ± 0.002    RSD/%  128.5  77.5  72.6  164.7  62.9  121.7  182.6  95.6  aUndetectable. Table II. Contents of the 16 Compounds in 49 Batches of Samples Dosage form  Batch no.  Contents of each compound (μg/g relevant herbal material)  AD  LQR  BMA  BA  BHA  LQG  MA  AC  Big honeyed-pill  1  26.00 ± 0.437  2,095 ± 23.38  18.55 ± 0.821  3.478 ± 0.051  4.335 ± 0.080  62.12 ± 0.790  0.130 ± 0.005  0.057 ± 0.002  2  21.77 ± 0.574  1,659 ± 4.632  81.57 ± 0.228  8.989 ± 0.159  10.96 ± 0.121  207.8 ± 2.806  7.458 ± 0.005  1.601 ± 0.042  3  15.88 ± 0.168  1,581 ± 27.51  77.00 ± 1.379  7.934 ± 0.168  10.54 ± 0.180  188.3 ± 1.129  6.490 ± 0.253  1.515 ± 0.046  4  31.37 ± 0.194  2,067 ± 20.22  54.91 ± 0.591  6.704 ± 0.240  12.63 ± 0.468  78.47 ± 2.381  2.628 ± 0.176  0.529 ± 0.014  5  15.34 ± 0.225  1,744 ± 46.48  81.10 ± 1.676  8.863 ± 0.213  10.69 ± 0.283  201.9 ± 1.563  6.580 ± 0.090  1.562 ± 0.062  6  20.81 ± 0.321  1,577 ± 27.20  102.5 ± 1.018  9.725 ± 0.381  11.16 ± 0.403  259.2 ± 1.079  11.89 ± 0.173  2.403 ± 0.037  7  23.30 ± 0.314  2,996 ± 25.53  136.2 ± 1.042  14.49 ± 0.484  22.88 ± 0.523  56.66 ± 2.431  11.62 ± 0.183  2.253 ± 0.025  8  34.23 ± 0.641  2,998 ± 29.53  84.61 ± 1.325  12.07 ± 0.931  23.91 ± 0.515  56.18 ± 1.934  3.131 ± 0.293  0.724 ± 0.041  9  34.52 ± 0.376  3,105 ± 21.42  94.33 ± 1.016  14.46 ± 0.231  22.14 ± 0.356  70.53 ± 1.221  1.065 ± 0.923  0.474 ± 0.056  10  1.847 ± 0.072  1,796 ± 17.55  6.584 ± 0.316  0.889 ± 0.033  1.795 ± 0.063  108.0 ± 4.831  0.053 ± 0.407  0.034 ± 0.002  11  12.16 ± 0.134  2,676 ± 21.53  1.944 ± 0.413  0.089 ± 0.045  0.008 ± 0.001  95.19 ± 1.842  0.016 ± 0.002  0.009 ± 0.000  12  8.128 ± 0.274  2,686 ± 19.37  0.316 ± 0.041  0.062 ± 0.031  0.195 ± 0.034  69.59 ± 1.780  0.021 ± 0.008  0.012 ± 0.000  13  3.171 ± 0.384  2,807 ± 27.53  3.590 ± 0.319  0.490 ± 0.016  1.524 ± 0.051  97.58 ± 2.014  0.080 ± 0.007  0.038 ± 0.013  14  0.163 ± 0.064  3,921 ± 25.48  10.82 ± 1.452  1.343 ± 0.084  0.886 ± 0.093  150.5 ± 3.858  0.031 ± 0.008  0.022 ± 0.001  15  3.365 ± 0.092  5,659 ± 25.84  46.13 ± 0.924  7.494 ± 0.153  10.40 ± 0.073  147.7 ± 3.239  0.143 ± 0.019  0.172 ± 0.014  16  3.230 ± 0.124  1,557 ± 30.28  102.5 ± 1.099  11.38 ± 0.275  17.34 ± 0.373  68.32 ± 0.285  4.996 ± 0.002  1.109 ± 0.042  17  2.105 ± 0.072  1,875 ± 9.186  81.72 ± 1.818  9.724 ± 0.096  16.29 ± 0.340  172.9 ± 1.233  4.575 ± 0.068  1.226 ± 0.049  18  2.369 ± 0.072  1,503 ± 7.465  97.21 ± 0.560  10.73 ± 0.143  17.39 ± 0.348  68.39 ± 1.862  4.569 ± 0.097  1.201 ± 0.025  19  5.704 ± 0.143  1,776 ± 24.02  90.73 ± 1.636  9.930 ± 0.128  15.60 ± 0.228  43.65 ± 0.922  5.058 ± 0.084  1.310 ± 0.025  20  9.860 ± 0.235  2,317 ± 22.51  63.12 ± 1.782  12.81 ± 1.024  28.27 ± 0.142  55.21 ± 1.523  1.600 ± 0.083  0.779 ± 0.044  21  4.073 ± 0.327  2,155 ± 17.42  113.5 ± 3.515  12.48 ± 0.931  20.00 ± 0.201  52.07 ± 1.664  4.015 ± 0.081  1.264 ± 0.053  22  18.53 ± 0.415  1,392 ± 18.20  44.27 ± 1.833  0.311 ± 0.020  11.58 ± 0.272  96.95 ± 2.323  0.246 ± 0.160  0.171 ± 0.003  23  19.81 ± 0.327  1,509 ± 62.60  37.37 ± 4.046  0.409 ± 0.025  11.26 ± 0.403  141.5 ± 2.064  0.306 ± 0.008  0.173 ± 0.003  24  18.74 ± 0.579  1,432 ± 42.79  31.40 ± 0.455  0.186 ± 0.001  9.628 ± 0.052  111.0 ± 2.425  0.293 ± 0.005  0.177 ± 0.003  25  31.22 ± 0.632  2,046 ± 20.51  54.42 ± 2.013  4.112 ± 0.146  5.142 ± 0.109  82.90 ± 3.028  10.01 ± 0.098  1.844 ± 0.028  26  13.29 ± 0.234  1,722 ± 21.58  198.3 ± 8.421  20.96 ± 1.023  21.41 ± 0.113  106.9 ± 3.741  4.287 ± 0.122  1.389 ± 0.084  27  16.57 ± 0.134  1,706 ± 19.52  92.45 ± 1.951  11.29 ± 1.184  16.60 ± 0.142  64.92 ± 1.447  1.794 ± 0.053  0.516 ± 0.032    RSD/%  73.3  41.8  69.3  76.1  63.9  52.7  106.1  88.2  Concentrated pill  28  26.06 ± 1.056  1,386 ± 38.24  133.3 ± 4.227  30.83 ± 0.531  107.3 ± 4.381  84.83 ± 1.935  0.062 ± 0.239  0.055 ± 0.001  29  19.36 ± 0.483  546.0 ± 6.419  102.9 ± 3.848  22.36 ± 0.475  93.01 ± 0.948  54.72 ± 2.849  0.102 ± 0.001  0.060 ± 0.001  30  21.63 ± 0.394  1,170 ± 26.72  117.8 ± 1.050  29.02 ± 0.991  94.39 ± 1.083  91.24 ± 0.992  0.088 ± 0.002  0.073 ± 0.001  31  22.01 ± 0.676  563.8 ± 19.10  88.64 ± 0.255  21.26 ± 0.554  106.5 ± 4.126  74.07 ± 3.309  0.142 ± 0.003  0.123 ± 0.003  32  33.98 ± 0.374  4,084 ± 28.83  319.1 ± 2.941  68.63 ± 1.251  218.0 ± 3.416  118.2 ± 2.041  0.185 ± 0.003  0.111 ± 0.007  33  37.35 ± 0.452  2,602 ± 26.31  304.6 ± 2.055  64.68 ± 0.491  198.5 ± 2.955  98.19 ± 1.453  0.176 ± 0.004  0.192 ± 0.008  34  7.166 ± 0.341  575.9 ± 3.932  45.81 ± 0.166  10.55 ± 0.124  24.23 ± 0.941  91.37 ± 2.450  0.058 ± 0.004  0.055 ± 0.001  35  9.863 ± 0.293  1,200 ± 14.11  96.86 ± 3.031  16.28 ± 0.283  44.73 ± 1.651  23.20 ± 1.533  0.173 ± 0.010  0.064 ± 0.003  36  15.47 ± 0.473  2,010 ± 17.82  99.95 ± 2.951  23.12 ± 0.341  51.60 ± 2.042  51.19 ± 2.951  0.197 ± 0.011  0.174 ± 0.005  37  13.47 ± 0.417  528.0 ± 10.30  58.71 ± 1.809  12.31 ± 0.152  39.60 ± 0.652  35.12 ± 1.240  0.036 ± 0.001  0.042 ± 0.000  38  10.71 ± 0.154  438.6 ± 1.613  64.72 ± 1.155  12.22 ± 0.272  38.79 ± 0.551  34.86 ± 0.358  0.069 ± 0.001  0.045 ± 0.001  39  12.40 ± 0.229  172.9 ± 2.009  35.98 ± 0.576  8.178 ± 0.115  31.10 ± 0.397  0.958 ± 0.032  0.055 ± 0.001  0.025 ± 0.000  40  20.49 ± 0.245  401.9 ± 8.352  114.4 ± 4.014  24.95 ± 0.398  64.94 ± 1.432  24.68 ± 1.542  0.003 ± 0.000  0.011 ± 0.000  41  7.184 ± 0.197  903.5 ± 5.530  26.36 ± 0.468  4.497 ± 0.195  9.323 ± 0.360  65.92 ± 2.121  0.080 ± 0.000  0.028 ± 0.001  42  17.92 ± 0.831  3,069 ± 10.51  67.96 ± 1.031  11.16 ± 0.312  20.06 ± 0.966  77.29 ± 3.133  0.170 ± 0.004  0.075 ± 0.000  43  14.43 ± 0.624  3,206 ± 28.41  70.98 ± 0.941  11.98 ± 0.284  19.60 ± 0.455  85.53 ± 2.414  0.090 ± 0.002  0.046 ± 0.000  44  8.560 ± 0.532  2,137 ± 15.32  87.56 ± 1.252  11.84 ± 0.184  29.41 ± 1.053  282.9 ± 3.988  0.683 ± 0.010  0.231 ± 0.001  45  4.262 ± 0.135  1,023 ± 11.42  44.59 ± 0.836  5.705 ± 0.167  18.82 ± 0.533  139.1 ± 3.556  0.560 ± 0.009  0.170 ± 0.000  46  16.02 ± 0.142  698.7 ± 2.889  16.33 ± 0.125  8.276 ± 0.084  12.47 ± 0.250  34.48 ± 0.509  1.409 ± 0.002  0.531 ± 0.000  47  2.909 ± 0.068  1,000 ± 3.059  175.3 ± 0.537  32.29 ± 0.337  79.26 ± 0.240  68.99 ± 0.691  0.217 ± 0.049  0.140 ± 0.000  48  23.65 ± 0.523  2,174 ± 16.82  252.7 ± 1.934  58.30 ± 0.253  129.4 ± 0.675  17.17 ± 0.886  1.102 ± 0.088  0.680 ± 0.001  49  7.264 ± 0.834  1,966 ± 12.44  253.2 ± 0.894  43.46 ± 0.384  115.6 ± 1.143  110.0 ± 3.452  0.895 ± 0.014  0.675 ± 0.003    RSD/%  56.6  73.7  75.7  78.4  82.6  77.2  129.2  121.8  Dosage form  Batch no.  Contents of each compound (μg/g relevant herbal material)  AD  LQR  BMA  BA  BHA  LQG  MA  AC  Big honeyed-pill  1  26.00 ± 0.437  2,095 ± 23.38  18.55 ± 0.821  3.478 ± 0.051  4.335 ± 0.080  62.12 ± 0.790  0.130 ± 0.005  0.057 ± 0.002  2  21.77 ± 0.574  1,659 ± 4.632  81.57 ± 0.228  8.989 ± 0.159  10.96 ± 0.121  207.8 ± 2.806  7.458 ± 0.005  1.601 ± 0.042  3  15.88 ± 0.168  1,581 ± 27.51  77.00 ± 1.379  7.934 ± 0.168  10.54 ± 0.180  188.3 ± 1.129  6.490 ± 0.253  1.515 ± 0.046  4  31.37 ± 0.194  2,067 ± 20.22  54.91 ± 0.591  6.704 ± 0.240  12.63 ± 0.468  78.47 ± 2.381  2.628 ± 0.176  0.529 ± 0.014  5  15.34 ± 0.225  1,744 ± 46.48  81.10 ± 1.676  8.863 ± 0.213  10.69 ± 0.283  201.9 ± 1.563  6.580 ± 0.090  1.562 ± 0.062  6  20.81 ± 0.321  1,577 ± 27.20  102.5 ± 1.018  9.725 ± 0.381  11.16 ± 0.403  259.2 ± 1.079  11.89 ± 0.173  2.403 ± 0.037  7  23.30 ± 0.314  2,996 ± 25.53  136.2 ± 1.042  14.49 ± 0.484  22.88 ± 0.523  56.66 ± 2.431  11.62 ± 0.183  2.253 ± 0.025  8  34.23 ± 0.641  2,998 ± 29.53  84.61 ± 1.325  12.07 ± 0.931  23.91 ± 0.515  56.18 ± 1.934  3.131 ± 0.293  0.724 ± 0.041  9  34.52 ± 0.376  3,105 ± 21.42  94.33 ± 1.016  14.46 ± 0.231  22.14 ± 0.356  70.53 ± 1.221  1.065 ± 0.923  0.474 ± 0.056  10  1.847 ± 0.072  1,796 ± 17.55  6.584 ± 0.316  0.889 ± 0.033  1.795 ± 0.063  108.0 ± 4.831  0.053 ± 0.407  0.034 ± 0.002  11  12.16 ± 0.134  2,676 ± 21.53  1.944 ± 0.413  0.089 ± 0.045  0.008 ± 0.001  95.19 ± 1.842  0.016 ± 0.002  0.009 ± 0.000  12  8.128 ± 0.274  2,686 ± 19.37  0.316 ± 0.041  0.062 ± 0.031  0.195 ± 0.034  69.59 ± 1.780  0.021 ± 0.008  0.012 ± 0.000  13  3.171 ± 0.384  2,807 ± 27.53  3.590 ± 0.319  0.490 ± 0.016  1.524 ± 0.051  97.58 ± 2.014  0.080 ± 0.007  0.038 ± 0.013  14  0.163 ± 0.064  3,921 ± 25.48  10.82 ± 1.452  1.343 ± 0.084  0.886 ± 0.093  150.5 ± 3.858  0.031 ± 0.008  0.022 ± 0.001  15  3.365 ± 0.092  5,659 ± 25.84  46.13 ± 0.924  7.494 ± 0.153  10.40 ± 0.073  147.7 ± 3.239  0.143 ± 0.019  0.172 ± 0.014  16  3.230 ± 0.124  1,557 ± 30.28  102.5 ± 1.099  11.38 ± 0.275  17.34 ± 0.373  68.32 ± 0.285  4.996 ± 0.002  1.109 ± 0.042  17  2.105 ± 0.072  1,875 ± 9.186  81.72 ± 1.818  9.724 ± 0.096  16.29 ± 0.340  172.9 ± 1.233  4.575 ± 0.068  1.226 ± 0.049  18  2.369 ± 0.072  1,503 ± 7.465  97.21 ± 0.560  10.73 ± 0.143  17.39 ± 0.348  68.39 ± 1.862  4.569 ± 0.097  1.201 ± 0.025  19  5.704 ± 0.143  1,776 ± 24.02  90.73 ± 1.636  9.930 ± 0.128  15.60 ± 0.228  43.65 ± 0.922  5.058 ± 0.084  1.310 ± 0.025  20  9.860 ± 0.235  2,317 ± 22.51  63.12 ± 1.782  12.81 ± 1.024  28.27 ± 0.142  55.21 ± 1.523  1.600 ± 0.083  0.779 ± 0.044  21  4.073 ± 0.327  2,155 ± 17.42  113.5 ± 3.515  12.48 ± 0.931  20.00 ± 0.201  52.07 ± 1.664  4.015 ± 0.081  1.264 ± 0.053  22  18.53 ± 0.415  1,392 ± 18.20  44.27 ± 1.833  0.311 ± 0.020  11.58 ± 0.272  96.95 ± 2.323  0.246 ± 0.160  0.171 ± 0.003  23  19.81 ± 0.327  1,509 ± 62.60  37.37 ± 4.046  0.409 ± 0.025  11.26 ± 0.403  141.5 ± 2.064  0.306 ± 0.008  0.173 ± 0.003  24  18.74 ± 0.579  1,432 ± 42.79  31.40 ± 0.455  0.186 ± 0.001  9.628 ± 0.052  111.0 ± 2.425  0.293 ± 0.005  0.177 ± 0.003  25  31.22 ± 0.632  2,046 ± 20.51  54.42 ± 2.013  4.112 ± 0.146  5.142 ± 0.109  82.90 ± 3.028  10.01 ± 0.098  1.844 ± 0.028  26  13.29 ± 0.234  1,722 ± 21.58  198.3 ± 8.421  20.96 ± 1.023  21.41 ± 0.113  106.9 ± 3.741  4.287 ± 0.122  1.389 ± 0.084  27  16.57 ± 0.134  1,706 ± 19.52  92.45 ± 1.951  11.29 ± 1.184  16.60 ± 0.142  64.92 ± 1.447  1.794 ± 0.053  0.516 ± 0.032    RSD/%  73.3  41.8  69.3  76.1  63.9  52.7  106.1  88.2  Concentrated pill  28  26.06 ± 1.056  1,386 ± 38.24  133.3 ± 4.227  30.83 ± 0.531  107.3 ± 4.381  84.83 ± 1.935  0.062 ± 0.239  0.055 ± 0.001  29  19.36 ± 0.483  546.0 ± 6.419  102.9 ± 3.848  22.36 ± 0.475  93.01 ± 0.948  54.72 ± 2.849  0.102 ± 0.001  0.060 ± 0.001  30  21.63 ± 0.394  1,170 ± 26.72  117.8 ± 1.050  29.02 ± 0.991  94.39 ± 1.083  91.24 ± 0.992  0.088 ± 0.002  0.073 ± 0.001  31  22.01 ± 0.676  563.8 ± 19.10  88.64 ± 0.255  21.26 ± 0.554  106.5 ± 4.126  74.07 ± 3.309  0.142 ± 0.003  0.123 ± 0.003  32  33.98 ± 0.374  4,084 ± 28.83  319.1 ± 2.941  68.63 ± 1.251  218.0 ± 3.416  118.2 ± 2.041  0.185 ± 0.003  0.111 ± 0.007  33  37.35 ± 0.452  2,602 ± 26.31  304.6 ± 2.055  64.68 ± 0.491  198.5 ± 2.955  98.19 ± 1.453  0.176 ± 0.004  0.192 ± 0.008  34  7.166 ± 0.341  575.9 ± 3.932  45.81 ± 0.166  10.55 ± 0.124  24.23 ± 0.941  91.37 ± 2.450  0.058 ± 0.004  0.055 ± 0.001  35  9.863 ± 0.293  1,200 ± 14.11  96.86 ± 3.031  16.28 ± 0.283  44.73 ± 1.651  23.20 ± 1.533  0.173 ± 0.010  0.064 ± 0.003  36  15.47 ± 0.473  2,010 ± 17.82  99.95 ± 2.951  23.12 ± 0.341  51.60 ± 2.042  51.19 ± 2.951  0.197 ± 0.011  0.174 ± 0.005  37  13.47 ± 0.417  528.0 ± 10.30  58.71 ± 1.809  12.31 ± 0.152  39.60 ± 0.652  35.12 ± 1.240  0.036 ± 0.001  0.042 ± 0.000  38  10.71 ± 0.154  438.6 ± 1.613  64.72 ± 1.155  12.22 ± 0.272  38.79 ± 0.551  34.86 ± 0.358  0.069 ± 0.001  0.045 ± 0.001  39  12.40 ± 0.229  172.9 ± 2.009  35.98 ± 0.576  8.178 ± 0.115  31.10 ± 0.397  0.958 ± 0.032  0.055 ± 0.001  0.025 ± 0.000  40  20.49 ± 0.245  401.9 ± 8.352  114.4 ± 4.014  24.95 ± 0.398  64.94 ± 1.432  24.68 ± 1.542  0.003 ± 0.000  0.011 ± 0.000  41  7.184 ± 0.197  903.5 ± 5.530  26.36 ± 0.468  4.497 ± 0.195  9.323 ± 0.360  65.92 ± 2.121  0.080 ± 0.000  0.028 ± 0.001  42  17.92 ± 0.831  3,069 ± 10.51  67.96 ± 1.031  11.16 ± 0.312  20.06 ± 0.966  77.29 ± 3.133  0.170 ± 0.004  0.075 ± 0.000  43  14.43 ± 0.624  3,206 ± 28.41  70.98 ± 0.941  11.98 ± 0.284  19.60 ± 0.455  85.53 ± 2.414  0.090 ± 0.002  0.046 ± 0.000  44  8.560 ± 0.532  2,137 ± 15.32  87.56 ± 1.252  11.84 ± 0.184  29.41 ± 1.053  282.9 ± 3.988  0.683 ± 0.010  0.231 ± 0.001  45  4.262 ± 0.135  1,023 ± 11.42  44.59 ± 0.836  5.705 ± 0.167  18.82 ± 0.533  139.1 ± 3.556  0.560 ± 0.009  0.170 ± 0.000  46  16.02 ± 0.142  698.7 ± 2.889  16.33 ± 0.125  8.276 ± 0.084  12.47 ± 0.250  34.48 ± 0.509  1.409 ± 0.002  0.531 ± 0.000  47  2.909 ± 0.068  1,000 ± 3.059  175.3 ± 0.537  32.29 ± 0.337  79.26 ± 0.240  68.99 ± 0.691  0.217 ± 0.049  0.140 ± 0.000  48  23.65 ± 0.523  2,174 ± 16.82  252.7 ± 1.934  58.30 ± 0.253  129.4 ± 0.675  17.17 ± 0.886  1.102 ± 0.088  0.680 ± 0.001  49  7.264 ± 0.834  1,966 ± 12.44  253.2 ± 0.894  43.46 ± 0.384  115.6 ± 1.143  110.0 ± 3.452  0.895 ± 0.014  0.675 ± 0.003    RSD/%  56.6  73.7  75.7  78.4  82.6  77.2  129.2  121.8  Dosage form  Batch no.  Contents of each compound (μg/g material drugs)  HA  GIA  OLQG  GE  ALIII  ALI  ALII  GEA  Big honeyed-pill  1  1.452 ± 0.021  2,650 ± 43.32  11.30 ± 0.463  1,595 ± 54.68  42.46 ± 1.757  45.63 ± 1.223  60.20 ± 0.534  2.659 ± 0.053  2  22.99 ± 0.469  3,012 ± 90.64  26.27 ± 0.319  2,031 ± 53.48  172.0 ± 1.676  182.6 ± 3.168  158.0 ± 1.022  6.260 ± 0.142  3  20.77 ± 0.346  3,202 ± 137.4  24.64 ± 0.340  1,464 ± 50.18  141.9 ± 3.387  151.1 ± 1.446  126.1 ± 2.015  5.337 ± 0.204  4  11.87 ± 0.313  3,914 ± 78.75  15.01 ± 0.051  2,341 ± 109.5  100.8 ± 1.155  97.61 ± 1.023  114.4 ± 1.472  2.624 ± 0.046  5  21.61 ± 0.087  3,735 ± 93.67  26.73 ± 0.293  1,217 ± 51.34  129.0 ± 1.124  126.9 ± 0.593  113.5 ± 3.811  6.583 ± 0.068  6  30.64 ± 0.610  3,285 ± 63.06  33.21 ± 1.256  872.9 ± 2.160  94.88 ± 3.543  125.3 ± 1.032  139.0 ± 2.415  8.481 ± 0.248  7  28.42 ± 0.435  3,183 ± 54.76  4.228 ± 0.863  2,554 ± 46.76  81.52 ± 2.657  158.4 ± 2.907  131.6 ± 2.098  0.927 ± 0.097  8  13.29 ± 0.521  4,383 ± 66.87  4.845 ± 0.956  3,112 ± 64.38  89.50 ± 1.543  122.1 ± 2.065  109.0 ± 2.218  1.716 ± 0.087  9  9.252 ± 0.367  4,533 ± 57.43  5.375 ± 0.675  2,545 ± 58.62  89.85 ± 2.989  115.7 ± 1.648  92.76 ± 3.112  1.252 ± 0.057  10  1.178 ± 0.016  4,633 ± 34.81  38.83 ± 0.802  780.8 ± 1.079  106.2 ± 3.058  140.8 ± 3.498  96.39 ± 1.544  21.81 ± 0.884  11  0.021 ± 0.000  4,879 ± 48.67  33.17 ± 0.586  683.2 ± 20.76  222.1 ± 1.677  322.3 ± 2.767  200.1 ± 2.056  35.57 ± 1.076  12  0.021 ± 0.000  4,684 ± 87.43  19.76 ± 0.437  865.9 ± 16.44  245.8 ± 2.452  403.0 ± 2.234  247.1 ± 2.675  9.749 ± 0.078  13  1.150 ± 0.129  5,149 ± 76.44  28.21 ± 0.532  644.1 ± 23.98  99.78 ± 1.777  181.1 ± 0.986  79.67 ± 0.887  23.63 ± 0.254  14  0.695 ± 0.034  8,127 ± 48.61  19.56 ± 0.338  509.9 ± 21.61  248.2 ± 3.665  160.4 ± 1.065  202.0 ± 1.965  7.088 ± 0.334  15  1.680 ± 0.384  10814 ± 98.43  34.65 ± 0.674  600.5 ± 19.833  159.4 ± 2.787  133.1 ± 1.254  113.1 ± 2.137  23.47 ± 0.546  16  26.26 ± 0.718  3,980 ± 121.8  9.190 ± 0.224  1,934 ± 52.95  116.9 ± 0.699  181.6 ± 1.233  156.3 ± 0.929  32.89 ± 1.045  17  24.56 ± 0.323  5,442 ± 139.1  25.94 ± 0.748  2,276 ± 92.71  58.32 ± 1.480  96.40 ± 0.257  87.48 ± 1.436  43.10 ± 1.332  18  24.69 ± 0.378  2,682 ± 88.74  8.464 ± 0.150  2,031 ± 57.87  112.7 ± 2.215  186.4 ± 5.955  159.9 ± 3.114  32.39 ± 0.622  19  24.62 ± 0.462  2,966 ± 20.79  7.386 ± 0.184  2,031 ± 49.40  78.49 ± 1.840  135.0 ± 0.424  99.51 ± 2.247  29.82 ± 0.493  20  31.96 ± 0.532  2,800 ± 47.53  3.054 ± 0.205  3,867 ± 56.63  86.73 ± 2.096  221.0 ± 2.665  121.5 ± 1.877  23.93 ± 0.376  21  22.28 ± 0.787  3,736 ± 25.47  3.564 ± 0.397  3,771 ± 68.42  113.3 ± 2.136  222.1 ± 1.772  163.2 ± 2.870  20.47 ± 0.265  22  3.552 ± 0.122  4,402 ± 42.62  36.27 ± 0.447  875.2 ± 2.344  75.00 ± 1.335  82.91 ± 0.415  80.02 ± 1.082  9.714 ± 0.252  23  2.989 ± 0.033  4,397 ± 55.22  46.85 ± 1.037  687.3 ± 0.791  114.8 ± 4.771  86.91 ± 2.792  90.56 ± 11.312  13.97 ± 0.460  24  3.253 ± 0.052  4,430 ± 76.95  32.60 ± 1.153  781.2 ± 2.178  80.94 ± 1.116  92.23 ± 1.593  88.38 ± 1.604  12.65 ± 0.412  25  18.84 ± 0.452  3,613 ± 17.53  6.957 ± 0.654  2,892 ± 10.75  87.89 ± 2.343  116.0 ± 2.076  106.1 ± 2.122  3.381 ± 0.008  26  28.22 ± 0.445  3,175 ± 35.58  8.621 ± 0.788  2,716 ± 21.09  125.1 ± 2.766  178.1 ± 1.996  121.5 ± 1.942  2.513 ± 0.006  27  12.73 ± 0.782  2,448 ± 17.37  4.007 ± 0.965  280.7 ± 30.07  66.75 ± 2.087  94.44 ± 0.879  78.52 ± 1.209  3.072 ± 0.005    RSD/%  78.8  41.5  69.0  60.8  45.3  48.5  35.2  87.5  Concentrated pill  28  0.924 ± 0.002  1,987 ± 51.50  27.14 ± 0.749  N.D.a  16.45 ± 0.658  3.691 ± 0.074  1.300 ± 0.013  10.68 ± 0.401  29  1.378 ± 0.008  1,850 ± 44.50  33.03 ± 1.046  15.93 ± 0.026  15.28 ± 0.471  4.238 ± 0.179  0.705 ± 0.007  9.821 ± 0.208  30  1.342 ± 0.016  2,282 ± 44.85  54.09 ± 0.672  15.68 ± 0.026  17.49 ± 0.239  6.309 ± 0.086  0.596 ± 0.007  10.70 ± 0.464  31  4.264 ± 0.063  2,384 ± 95.77  46.24 ± 0.842  6.316 ± 0.025  12.05 ± 0.540  3.564 ± 0.178  0.257 ± 0.001  10.10 ± 0.238  32  2.219 ± 0.012  6,741 ± 67.33  21.35 ± 0.531  87.60 ± 1.035  41.86 ± 0.561  21.18 ± 0.093  3.951 ± 0.061  11.56 ± 0.051  33  3.330 ± 0.067  6,161 ± 52.88  18.12 ± 0.312  93.14 ± 1.237  41.22 ± 0.611  25.33 ± 0.123  3.089 ± 0.061  11.74 ± 0.086  34  1.035 ± 0.007  1,311 ± 25.10  43.31 ± 1.231  1.817 ± 0.009  13.82 ± 0.549  20.57 ± 0.856  0.844 ± 0.024  5.012 ± 0.135  35  1.579 ± 0.023  3,786 ± 46.83  6.253 ± 2.577  91.79 ± 1.535  68.57 ± 0.882  103.0 ± 0.562  75.39 ± 0.006  5.097 ± 0.026  36  3.796 ± 0.076  1,633 ± 32.66  6.986 ± 0.566  99.59 ± 2.044  64.18 ± 0.436  98.36 ± 1.053  59.14 ± 0.016  4.091 ± 0.003  37  1.092 ± 0.003  2,049 ± 10.92  23.10 ± 0.431  1.592 ± 0.005  12.94 ± 0.477  2.233 ± 0.015  0.260 ± 0.007  7.134 ± 0.059  38  0.983 ± 0.005  1,973 ± 27.67  23.11 ± 0.173  N.D.a  9.764 ± 0.460  2.137 ± 0.002  0.379 ± 0.007  4.280 ± 0.090  39  0.577 ± 0.018  547.3 ± 23.18  3.369 ± 0.078  N.D.a  8.834 ± 0.304  1.632 ± 0.005  0.260 ± 0.001  4.180 ± 0.058  40  0.343 ± 0.008  128.7 ± 9.531  3.595 ± 0.035  N.D.a  43.33 ± 0.531  14.96 ± 0.012  5.494 ± 0.059  4.521 ± 0.008  41  0.600 ± 0.009  3,872 ± 146.7  55.58 ± 0.473  N.D.a  15.11 ± 0.609  3.484 ± 0.075  1.068 ± 0.013  32.43 ± 0.662  42  1.328 ± 0.033  8,413 ± 125.3  22.99 ± 0.661  N.D.a  44.01 ± 0.844  22.65 ± 0.073  4.964 ± 0.065  39.81 ± 0.035  43  1.450 ± 0.037  9,473 ± 94.31  25.12 ± 0.775  N.D.a  50.67 ± 0.398  36.23 ± 0.015  8.063 ± 0.013  40.35 ± 0.035  44  4.840 ± 0.103  2,959 ± 36.23  84.89 ± 1.356  N.D.a  39.93 ± 0.448  21.87 ± 0.064  5.293 ± 0.058  46.86 ± 0.051  45  5.688 ± 0.112  1,244 ± 17.43  40.77 ± 1.532  N.D. a  16.71 ± 0.562  7.114 ± 0.058  0.653 ± 0.043  25.32 ± 0.044  46  6.687 ± 0.137  2,788 ± 68.26  32.95 ± 0.654  N.D.a  9.299 ± 0.282  1.661 ± 0.001  0.250 ± 0.002  18.49 ± 0.681  47  6.842 ± 0.118  2,415 ± 38.86  24.43 ± 0.229  N.D.a  40.50 ± 0.871  59.27 ± 1.662  45.81 ± 0.998  2.432 ± 0.006  48  17.86 ± 0.271  1,567 ± 14.52  4.972 ± 0.733  159.9 ± 11.53  32.73 ± 0.511  57.32 ± 0.056  37.04 ± 0.051  4.278 ± 0.023  49  18.91 ± 0.176  4,410 ± 17.44  11.94 ± 0.561  48.97 ± 1.442  55.94 ± 0.823  21.06 ± 0.061  5.299 ± 0.066  4.577 ± 0.002    RSD/%  128.5  77.5  72.6  164.7  62.9  121.7  182.6  95.6  Dosage form  Batch no.  Contents of each compound (μg/g material drugs)  HA  GIA  OLQG  GE  ALIII  ALI  ALII  GEA  Big honeyed-pill  1  1.452 ± 0.021  2,650 ± 43.32  11.30 ± 0.463  1,595 ± 54.68  42.46 ± 1.757  45.63 ± 1.223  60.20 ± 0.534  2.659 ± 0.053  2  22.99 ± 0.469  3,012 ± 90.64  26.27 ± 0.319  2,031 ± 53.48  172.0 ± 1.676  182.6 ± 3.168  158.0 ± 1.022  6.260 ± 0.142  3  20.77 ± 0.346  3,202 ± 137.4  24.64 ± 0.340  1,464 ± 50.18  141.9 ± 3.387  151.1 ± 1.446  126.1 ± 2.015  5.337 ± 0.204  4  11.87 ± 0.313  3,914 ± 78.75  15.01 ± 0.051  2,341 ± 109.5  100.8 ± 1.155  97.61 ± 1.023  114.4 ± 1.472  2.624 ± 0.046  5  21.61 ± 0.087  3,735 ± 93.67  26.73 ± 0.293  1,217 ± 51.34  129.0 ± 1.124  126.9 ± 0.593  113.5 ± 3.811  6.583 ± 0.068  6  30.64 ± 0.610  3,285 ± 63.06  33.21 ± 1.256  872.9 ± 2.160  94.88 ± 3.543  125.3 ± 1.032  139.0 ± 2.415  8.481 ± 0.248  7  28.42 ± 0.435  3,183 ± 54.76  4.228 ± 0.863  2,554 ± 46.76  81.52 ± 2.657  158.4 ± 2.907  131.6 ± 2.098  0.927 ± 0.097  8  13.29 ± 0.521  4,383 ± 66.87  4.845 ± 0.956  3,112 ± 64.38  89.50 ± 1.543  122.1 ± 2.065  109.0 ± 2.218  1.716 ± 0.087  9  9.252 ± 0.367  4,533 ± 57.43  5.375 ± 0.675  2,545 ± 58.62  89.85 ± 2.989  115.7 ± 1.648  92.76 ± 3.112  1.252 ± 0.057  10  1.178 ± 0.016  4,633 ± 34.81  38.83 ± 0.802  780.8 ± 1.079  106.2 ± 3.058  140.8 ± 3.498  96.39 ± 1.544  21.81 ± 0.884  11  0.021 ± 0.000  4,879 ± 48.67  33.17 ± 0.586  683.2 ± 20.76  222.1 ± 1.677  322.3 ± 2.767  200.1 ± 2.056  35.57 ± 1.076  12  0.021 ± 0.000  4,684 ± 87.43  19.76 ± 0.437  865.9 ± 16.44  245.8 ± 2.452  403.0 ± 2.234  247.1 ± 2.675  9.749 ± 0.078  13  1.150 ± 0.129  5,149 ± 76.44  28.21 ± 0.532  644.1 ± 23.98  99.78 ± 1.777  181.1 ± 0.986  79.67 ± 0.887  23.63 ± 0.254  14  0.695 ± 0.034  8,127 ± 48.61  19.56 ± 0.338  509.9 ± 21.61  248.2 ± 3.665  160.4 ± 1.065  202.0 ± 1.965  7.088 ± 0.334  15  1.680 ± 0.384  10814 ± 98.43  34.65 ± 0.674  600.5 ± 19.833  159.4 ± 2.787  133.1 ± 1.254  113.1 ± 2.137  23.47 ± 0.546  16  26.26 ± 0.718  3,980 ± 121.8  9.190 ± 0.224  1,934 ± 52.95  116.9 ± 0.699  181.6 ± 1.233  156.3 ± 0.929  32.89 ± 1.045  17  24.56 ± 0.323  5,442 ± 139.1  25.94 ± 0.748  2,276 ± 92.71  58.32 ± 1.480  96.40 ± 0.257  87.48 ± 1.436  43.10 ± 1.332  18  24.69 ± 0.378  2,682 ± 88.74  8.464 ± 0.150  2,031 ± 57.87  112.7 ± 2.215  186.4 ± 5.955  159.9 ± 3.114  32.39 ± 0.622  19  24.62 ± 0.462  2,966 ± 20.79  7.386 ± 0.184  2,031 ± 49.40  78.49 ± 1.840  135.0 ± 0.424  99.51 ± 2.247  29.82 ± 0.493  20  31.96 ± 0.532  2,800 ± 47.53  3.054 ± 0.205  3,867 ± 56.63  86.73 ± 2.096  221.0 ± 2.665  121.5 ± 1.877  23.93 ± 0.376  21  22.28 ± 0.787  3,736 ± 25.47  3.564 ± 0.397  3,771 ± 68.42  113.3 ± 2.136  222.1 ± 1.772  163.2 ± 2.870  20.47 ± 0.265  22  3.552 ± 0.122  4,402 ± 42.62  36.27 ± 0.447  875.2 ± 2.344  75.00 ± 1.335  82.91 ± 0.415  80.02 ± 1.082  9.714 ± 0.252  23  2.989 ± 0.033  4,397 ± 55.22  46.85 ± 1.037  687.3 ± 0.791  114.8 ± 4.771  86.91 ± 2.792  90.56 ± 11.312  13.97 ± 0.460  24  3.253 ± 0.052  4,430 ± 76.95  32.60 ± 1.153  781.2 ± 2.178  80.94 ± 1.116  92.23 ± 1.593  88.38 ± 1.604  12.65 ± 0.412  25  18.84 ± 0.452  3,613 ± 17.53  6.957 ± 0.654  2,892 ± 10.75  87.89 ± 2.343  116.0 ± 2.076  106.1 ± 2.122  3.381 ± 0.008  26  28.22 ± 0.445  3,175 ± 35.58  8.621 ± 0.788  2,716 ± 21.09  125.1 ± 2.766  178.1 ± 1.996  121.5 ± 1.942  2.513 ± 0.006  27  12.73 ± 0.782  2,448 ± 17.37  4.007 ± 0.965  280.7 ± 30.07  66.75 ± 2.087  94.44 ± 0.879  78.52 ± 1.209  3.072 ± 0.005    RSD/%  78.8  41.5  69.0  60.8  45.3  48.5  35.2  87.5  Concentrated pill  28  0.924 ± 0.002  1,987 ± 51.50  27.14 ± 0.749  N.D.a  16.45 ± 0.658  3.691 ± 0.074  1.300 ± 0.013  10.68 ± 0.401  29  1.378 ± 0.008  1,850 ± 44.50  33.03 ± 1.046  15.93 ± 0.026  15.28 ± 0.471  4.238 ± 0.179  0.705 ± 0.007  9.821 ± 0.208  30  1.342 ± 0.016  2,282 ± 44.85  54.09 ± 0.672  15.68 ± 0.026  17.49 ± 0.239  6.309 ± 0.086  0.596 ± 0.007  10.70 ± 0.464  31  4.264 ± 0.063  2,384 ± 95.77  46.24 ± 0.842  6.316 ± 0.025  12.05 ± 0.540  3.564 ± 0.178  0.257 ± 0.001  10.10 ± 0.238  32  2.219 ± 0.012  6,741 ± 67.33  21.35 ± 0.531  87.60 ± 1.035  41.86 ± 0.561  21.18 ± 0.093  3.951 ± 0.061  11.56 ± 0.051  33  3.330 ± 0.067  6,161 ± 52.88  18.12 ± 0.312  93.14 ± 1.237  41.22 ± 0.611  25.33 ± 0.123  3.089 ± 0.061  11.74 ± 0.086  34  1.035 ± 0.007  1,311 ± 25.10  43.31 ± 1.231  1.817 ± 0.009  13.82 ± 0.549  20.57 ± 0.856  0.844 ± 0.024  5.012 ± 0.135  35  1.579 ± 0.023  3,786 ± 46.83  6.253 ± 2.577  91.79 ± 1.535  68.57 ± 0.882  103.0 ± 0.562  75.39 ± 0.006  5.097 ± 0.026  36  3.796 ± 0.076  1,633 ± 32.66  6.986 ± 0.566  99.59 ± 2.044  64.18 ± 0.436  98.36 ± 1.053  59.14 ± 0.016  4.091 ± 0.003  37  1.092 ± 0.003  2,049 ± 10.92  23.10 ± 0.431  1.592 ± 0.005  12.94 ± 0.477  2.233 ± 0.015  0.260 ± 0.007  7.134 ± 0.059  38  0.983 ± 0.005  1,973 ± 27.67  23.11 ± 0.173  N.D.a  9.764 ± 0.460  2.137 ± 0.002  0.379 ± 0.007  4.280 ± 0.090  39  0.577 ± 0.018  547.3 ± 23.18  3.369 ± 0.078  N.D.a  8.834 ± 0.304  1.632 ± 0.005  0.260 ± 0.001  4.180 ± 0.058  40  0.343 ± 0.008  128.7 ± 9.531  3.595 ± 0.035  N.D.a  43.33 ± 0.531  14.96 ± 0.012  5.494 ± 0.059  4.521 ± 0.008  41  0.600 ± 0.009  3,872 ± 146.7  55.58 ± 0.473  N.D.a  15.11 ± 0.609  3.484 ± 0.075  1.068 ± 0.013  32.43 ± 0.662  42  1.328 ± 0.033  8,413 ± 125.3  22.99 ± 0.661  N.D.a  44.01 ± 0.844  22.65 ± 0.073  4.964 ± 0.065  39.81 ± 0.035  43  1.450 ± 0.037  9,473 ± 94.31  25.12 ± 0.775  N.D.a  50.67 ± 0.398  36.23 ± 0.015  8.063 ± 0.013  40.35 ± 0.035  44  4.840 ± 0.103  2,959 ± 36.23  84.89 ± 1.356  N.D.a  39.93 ± 0.448  21.87 ± 0.064  5.293 ± 0.058  46.86 ± 0.051  45  5.688 ± 0.112  1,244 ± 17.43  40.77 ± 1.532  N.D. a  16.71 ± 0.562  7.114 ± 0.058  0.653 ± 0.043  25.32 ± 0.044  46  6.687 ± 0.137  2,788 ± 68.26  32.95 ± 0.654  N.D.a  9.299 ± 0.282  1.661 ± 0.001  0.250 ± 0.002  18.49 ± 0.681  47  6.842 ± 0.118  2,415 ± 38.86  24.43 ± 0.229  N.D.a  40.50 ± 0.871  59.27 ± 1.662  45.81 ± 0.998  2.432 ± 0.006  48  17.86 ± 0.271  1,567 ± 14.52  4.972 ± 0.733  159.9 ± 11.53  32.73 ± 0.511  57.32 ± 0.056  37.04 ± 0.051  4.278 ± 0.023  49  18.91 ± 0.176  4,410 ± 17.44  11.94 ± 0.561  48.97 ± 1.442  55.94 ± 0.823  21.06 ± 0.061  5.299 ± 0.066  4.577 ± 0.002    RSD/%  128.5  77.5  72.6  164.7  62.9  121.7  182.6  95.6  aUndetectable. OPLS-DA of the samples To better evaluate the quality consistency of the samples in different dosage forms, the content data of the 16 constituents in all samples were treated by the software SIMCA (Version 13.0). OPLS-DA method is a supervised statistical model, which was carried out to optimize separation between different groups of samples. To maximize class discrimination and component variation, the OPLS model was performed, and then the contents of the 16 analytes (49 × 16) were imported to the SIMCA for OPLS-DA analysis. An OPLS-DA model with predictive ability Q2(Y) of 76.7% was obtained. As shown in Figure 3A, a separation tendency between BHP and CP samples was established. In order to determine the predictive ability of the classifier models, 31 batches of the samples were selected as the calibration set to construct OPLS-DA models and the remaining 18 batches of samples were the prediction set. The predicted samples with a Y value above 0.6 will be recognized as a member of the BHP. The total accuracy, classification accuracy of BHP and CP in the prediction set were 94, 100 and 89%, respectively. Thus, the use of OPLS-DA to perform FLP classification would make it possible to evaluate the quality consistency of CPM. Figure 3. View largeDownload slide Multivariate statistical analysis of the tested samples in the two BHP and CP dosage forms. (A) OPLS-DA score plot of BHP and CP; (B) column diagram of the contents of the most relevant components related to the differences between the BHP and CP. BHP represents the big honey pill, and CP represents concentrated pills. This figure is available in black and white in print and in color at JCS online. Figure 3. View largeDownload slide Multivariate statistical analysis of the tested samples in the two BHP and CP dosage forms. (A) OPLS-DA score plot of BHP and CP; (B) column diagram of the contents of the most relevant components related to the differences between the BHP and CP. BHP represents the big honey pill, and CP represents concentrated pills. This figure is available in black and white in print and in color at JCS online. The VIP plot, displaying the VIP values of all of the variables, was used to assist in finding the most relevant variables which contributed to distinguishing between BHP and CP group. ALIII, ALII, ALI and GE were the most relevant. As shown in Figure 3B, the contents of ALIII, ALII, ALI and GE in the BHP samples were significantly higher than those in the CP samples, which may indicate the disparity between the pharmaceutical process of the two dosage forms. ROC of the selected analytes The potential markers that significantly contributed to the discrimination of different dosage forms were identified by using ROC curve analysis in MetaboAnalyst 3.0 (Figure 4). Heat map was constructed for unsupervised clustering using contents of the selected analytes as variables. As shown in Figure 5, the heat map showed a clear clustering for each group, which showed direct variation of each differential marker. Figure 4. View largeDownload slide (A) Distinction accuracy evaluation using ROC curves of selected analytes between BHP and CP samples. (AUC > 0.85); (B) ROC curve curve-based model evaluation (AUC = 1). This figure is available in black and white in print and in color at JCS online. Figure 4. View largeDownload slide (A) Distinction accuracy evaluation using ROC curves of selected analytes between BHP and CP samples. (AUC > 0.85); (B) ROC curve curve-based model evaluation (AUC = 1). This figure is available in black and white in print and in color at JCS online. Figure 5. View largeDownload slide Heat map of selected analytes between BHP and CP groups from contents. The color of each section is proportional to the significance of change of metabolites (red, up-regulated; blue, downregulated). The abbreviation is the same as the compound summarized in Figure 1. This figure is available in black and white in print and in color at JCS online. Figure 5. View largeDownload slide Heat map of selected analytes between BHP and CP groups from contents. The color of each section is proportional to the significance of change of metabolites (red, up-regulated; blue, downregulated). The abbreviation is the same as the compound summarized in Figure 1. This figure is available in black and white in print and in color at JCS online. As a result, four markers with the areas under the ROC curves ranging from 0.85 to 1 (Figure 4A), were considered to show the greatest distinction accuracy. Subsequently, ROC curve-based model was established, and the AUC value of the established model is 1 (Figure 4B), which showed a good ability for discriminating CP samples from the BHP samples. Thus, those four markers (GE, ALI, ALII, ALIII) (Figure 4A) can be defined as potential markers associated with dosage form. Discussion Optimization of sample preparation The solvent (including water, 50% methanol, 70% methanol, methanol first methanol and then subsequently ammonia water), volume of solvent (25, 50, 75 mL) and extraction time (15, 30, 60, 90 min) were investigated for the two dosage forms. Note that compared with refluxing, ultrasonic extraction is simple, reproducible and effective. To assess the extraction efficiency, a normalization method was used based on the fact that the contents of the 16 components have wide ranges, e.g., the highest content was 1,000-fold greater than the lowest content; thus, it was impossible to evaluate the extraction efficiency when their absolute peak areas were integrated together because the effect of the low content constituents would be overlooked. Therefore, the normalization arithmetic method was applied to dismiss the content variance; the peak area of each analyte was divided by the square root of the sum of squares obtained with all runs of every single investigation factor (every single integral extraction experiment was independent of other runs); then, despite the wide content ranges, the cumulative data from each run would represent its extraction efficiency. Eventually, the 60-min procedure with 50 mL/70% methanol by ultrasonic extraction was chosen because it could attain a greater response of the selected analytes. Optimization of the RRLC condition The 16 analytes were selected to obtain the optimum elution conditions because they belong to different chemical classes and cover a wide range of polarities. Various mobile phases, such as 0.05, 0.1 and 0.2% aqueous formic acid and acetic acid, were tested in this study. The best peak shape and resolution were obtained from a mixture of acetonitrile containing 0.1% formic acid and 0.1% formic acid water solution. Indeed, the addition of 0.1% formic acid in both of the mobile phases not only dramatically enhanced the abundance of [M + H]+ ions but also eliminated the peak tailing of the target compounds in the negative ion mode. In addition, liquid chromatographic conditions, such as flow rate and column temperature, were investigated. Because there was no requirement for baseline chromatographic separation of target analytes using the MRM scan mode and time segments mode, the run time was minimized to 13 min. Optimization of MS conditions After the MS condition for the transitions was optimized, it was found that all analytes demonstrated a better response in positive ion mode except LQR. To simultaneously detect all analytes, a polarity switch is necessary. Unfortunately, the frequent switching of polarity in an identical segment would reduce the sensitivity and accuracy of the quantification. A method with two optimized time segments (polarity switch in different segments) was devised to obtain a better response value. As shown in Table III, a polarity switch in different segments will not reduce the accuracy and sensitivity of quantification using the optimized MS instrumentation (namely by maintaining the same polarity in one segment). Given that LQR could only obtain a good response in negative ion mode, it was necessary to switch the polarity in different segments to analyze the analytes. The procedure of optimizing the MRM transitions for all the target analytes was as follows: initially, the precursor and product ions of the constituents were determined by standard solutions in scan and product ion mode, respectively. According to this, the fragmentor energy and collision energy parameters were further optimized based on the relative richest abundance of precursor and product ions (Table IV). Table III. The LOD and LOQ Data of Different Method No.  Ionization mode  Time segment  Compound  LOD (ng/mL)  LOQ (ng/mL)  1  ESI+  0–5  AD  0.120  0.399  2  ESI−  0–5  LQR  0.798  2.661  3  ESI±  0–5  AD  1.149  3.831    ESI±  0–5  LQR  6.205  20.68  4  ESI+  0–3.5  AD  0.145  0.483    ESI−  3.5–5  LQR  0.784  2.615  No.  Ionization mode  Time segment  Compound  LOD (ng/mL)  LOQ (ng/mL)  1  ESI+  0–5  AD  0.120  0.399  2  ESI−  0–5  LQR  0.798  2.661  3  ESI±  0–5  AD  1.149  3.831    ESI±  0–5  LQR  6.205  20.68  4  ESI+  0–3.5  AD  0.145  0.483    ESI−  3.5–5  LQR  0.784  2.615  Table III. The LOD and LOQ Data of Different Method No.  Ionization mode  Time segment  Compound  LOD (ng/mL)  LOQ (ng/mL)  1  ESI+  0–5  AD  0.120  0.399  2  ESI−  0–5  LQR  0.798  2.661  3  ESI±  0–5  AD  1.149  3.831    ESI±  0–5  LQR  6.205  20.68  4  ESI+  0–3.5  AD  0.145  0.483    ESI−  3.5–5  LQR  0.784  2.615  No.  Ionization mode  Time segment  Compound  LOD (ng/mL)  LOQ (ng/mL)  1  ESI+  0–5  AD  0.120  0.399  2  ESI−  0–5  LQR  0.798  2.661  3  ESI±  0–5  AD  1.149  3.831    ESI±  0–5  LQR  6.205  20.68  4  ESI+  0–3.5  AD  0.145  0.483    ESI−  3.5–5  LQR  0.784  2.615  Table IV. The Information for MRM Parameters No.  Compounds (abbreviations)  Ionization mode  Precursor ion  Product ion  Fragmentor  CE (eV)  Retention time (min)  Time segments: 0–3.5 min  1  Adenosine (AD)  ESI+  268.1  136a  80  50  0.831      119  80  15    Time segments: 3.5–4.8 min  2  Liquiritin (LQR)  ESI-  417  255.2a  100  10  4.087      135  100  10    Time segments: 4.8–6.6 min  3  Benzoylmesaconine (BMA)  ESI+  590.3  105a  160  45  5.283      540.3  160  45    4  Benzoylaconine (BA)  ESI+  604.3  105a  95  50  5.541      554.3  95  50    5  Benzoylhypaconine (BHA)  ESI+  574.3  542.3a  115  40  5.665      105  115  40    6  Liquiritigenin (LQG)  ESI+  257.1  137a  105  20  5.994      147  105  20    7  Mesaconine (MA)  ESI+  632.2  572.3a  100  45  6.052      354.1  100  45    8  Aconine (AC)  ESI+  646.3  586.3a  105  45  6.287      368.2  105  45    9  Hypaconitine (HA)  ESI+  616.3  556.3a  115  40  6.292      338.2  115  40    10  Glycyrrhizic acid (GIA)  ESI+  823.5  453.4a  160  10  6.468      647.2  160  10    Time segments: 6.6–13 min  11  Isoliquiritigenin (OLQG)  ESI+  257.1  137a  105  20  6.735      147  105  20    12  6-Gingerol (GE)  ESI+  317.2  217.3a  145  16  7.542  13  Atractylenolide III (ALIII)  ESI+  249.2  231.1a  55  12  8.107      163.2  55  12    14  Atractylenolide I (ALI)  ESI+  233.2  151.1a  95  10  10.047      215.3  95  10    15  Atractylenolide II (ALII)  ESI+  231.1  185a  85  16  12.014      157.1  85  16    16  Glycyrrhetic acid (GEA)  ESI+  471.4  317.2a  45  20  12.372      263.1  45  20    No.  Compounds (abbreviations)  Ionization mode  Precursor ion  Product ion  Fragmentor  CE (eV)  Retention time (min)  Time segments: 0–3.5 min  1  Adenosine (AD)  ESI+  268.1  136a  80  50  0.831      119  80  15    Time segments: 3.5–4.8 min  2  Liquiritin (LQR)  ESI-  417  255.2a  100  10  4.087      135  100  10    Time segments: 4.8–6.6 min  3  Benzoylmesaconine (BMA)  ESI+  590.3  105a  160  45  5.283      540.3  160  45    4  Benzoylaconine (BA)  ESI+  604.3  105a  95  50  5.541      554.3  95  50    5  Benzoylhypaconine (BHA)  ESI+  574.3  542.3a  115  40  5.665      105  115  40    6  Liquiritigenin (LQG)  ESI+  257.1  137a  105  20  5.994      147  105  20    7  Mesaconine (MA)  ESI+  632.2  572.3a  100  45  6.052      354.1  100  45    8  Aconine (AC)  ESI+  646.3  586.3a  105  45  6.287      368.2  105  45    9  Hypaconitine (HA)  ESI+  616.3  556.3a  115  40  6.292      338.2  115  40    10  Glycyrrhizic acid (GIA)  ESI+  823.5  453.4a  160  10  6.468      647.2  160  10    Time segments: 6.6–13 min  11  Isoliquiritigenin (OLQG)  ESI+  257.1  137a  105  20  6.735      147  105  20    12  6-Gingerol (GE)  ESI+  317.2  217.3a  145  16  7.542  13  Atractylenolide III (ALIII)  ESI+  249.2  231.1a  55  12  8.107      163.2  55  12    14  Atractylenolide I (ALI)  ESI+  233.2  151.1a  95  10  10.047      215.3  95  10    15  Atractylenolide II (ALII)  ESI+  231.1  185a  85  16  12.014      157.1  85  16    16  Glycyrrhetic acid (GEA)  ESI+  471.4  317.2a  45  20  12.372      263.1  45  20    aQuantitative transition. Table IV. The Information for MRM Parameters No.  Compounds (abbreviations)  Ionization mode  Precursor ion  Product ion  Fragmentor  CE (eV)  Retention time (min)  Time segments: 0–3.5 min  1  Adenosine (AD)  ESI+  268.1  136a  80  50  0.831      119  80  15    Time segments: 3.5–4.8 min  2  Liquiritin (LQR)  ESI-  417  255.2a  100  10  4.087      135  100  10    Time segments: 4.8–6.6 min  3  Benzoylmesaconine (BMA)  ESI+  590.3  105a  160  45  5.283      540.3  160  45    4  Benzoylaconine (BA)  ESI+  604.3  105a  95  50  5.541      554.3  95  50    5  Benzoylhypaconine (BHA)  ESI+  574.3  542.3a  115  40  5.665      105  115  40    6  Liquiritigenin (LQG)  ESI+  257.1  137a  105  20  5.994      147  105  20    7  Mesaconine (MA)  ESI+  632.2  572.3a  100  45  6.052      354.1  100  45    8  Aconine (AC)  ESI+  646.3  586.3a  105  45  6.287      368.2  105  45    9  Hypaconitine (HA)  ESI+  616.3  556.3a  115  40  6.292      338.2  115  40    10  Glycyrrhizic acid (GIA)  ESI+  823.5  453.4a  160  10  6.468      647.2  160  10    Time segments: 6.6–13 min  11  Isoliquiritigenin (OLQG)  ESI+  257.1  137a  105  20  6.735      147  105  20    12  6-Gingerol (GE)  ESI+  317.2  217.3a  145  16  7.542  13  Atractylenolide III (ALIII)  ESI+  249.2  231.1a  55  12  8.107      163.2  55  12    14  Atractylenolide I (ALI)  ESI+  233.2  151.1a  95  10  10.047      215.3  95  10    15  Atractylenolide II (ALII)  ESI+  231.1  185a  85  16  12.014      157.1  85  16    16  Glycyrrhetic acid (GEA)  ESI+  471.4  317.2a  45  20  12.372      263.1  45  20    No.  Compounds (abbreviations)  Ionization mode  Precursor ion  Product ion  Fragmentor  CE (eV)  Retention time (min)  Time segments: 0–3.5 min  1  Adenosine (AD)  ESI+  268.1  136a  80  50  0.831      119  80  15    Time segments: 3.5–4.8 min  2  Liquiritin (LQR)  ESI-  417  255.2a  100  10  4.087      135  100  10    Time segments: 4.8–6.6 min  3  Benzoylmesaconine (BMA)  ESI+  590.3  105a  160  45  5.283      540.3  160  45    4  Benzoylaconine (BA)  ESI+  604.3  105a  95  50  5.541      554.3  95  50    5  Benzoylhypaconine (BHA)  ESI+  574.3  542.3a  115  40  5.665      105  115  40    6  Liquiritigenin (LQG)  ESI+  257.1  137a  105  20  5.994      147  105  20    7  Mesaconine (MA)  ESI+  632.2  572.3a  100  45  6.052      354.1  100  45    8  Aconine (AC)  ESI+  646.3  586.3a  105  45  6.287      368.2  105  45    9  Hypaconitine (HA)  ESI+  616.3  556.3a  115  40  6.292      338.2  115  40    10  Glycyrrhizic acid (GIA)  ESI+  823.5  453.4a  160  10  6.468      647.2  160  10    Time segments: 6.6–13 min  11  Isoliquiritigenin (OLQG)  ESI+  257.1  137a  105  20  6.735      147  105  20    12  6-Gingerol (GE)  ESI+  317.2  217.3a  145  16  7.542  13  Atractylenolide III (ALIII)  ESI+  249.2  231.1a  55  12  8.107      163.2  55  12    14  Atractylenolide I (ALI)  ESI+  233.2  151.1a  95  10  10.047      215.3  95  10    15  Atractylenolide II (ALII)  ESI+  231.1  185a  85  16  12.014      157.1  85  16    16  Glycyrrhetic acid (GEA)  ESI+  471.4  317.2a  45  20  12.372      263.1  45  20    aQuantitative transition. Quantitative analysis of samples So far, over 200 alkaloids have been isolated and identified from Fuzi (29, 30), and they were classified into four major groups, nonester alkaloids, MDAs, DDAs and lipo alkaloids. Among the alkaloids in Fuzi, DDAs and MDAs have been investigated to possess many effects, such as antinociceptive, antiarrhythmic and anti-epileptic properties due to a blockade of the voltage-dependent Na+ channel (31). As a toxic herbal medicine, the toxicity of Fuzi mainly derives from DDAs including AC, MA and HA, etc. The processing methods in traditional Chinese medicine are mandatory to reduce the toxicity of Fuzi by decomposing DDAs to the less toxic MDAs or even unesterified compounds, but in the meantime, their pharmacological activities may alter to weaken or even wear off (32). Therefore, an appropriate quality control method capable of ensuring the safety and efficacy is absolutely necessary. In Chinese pharmacopoeia 2015, it is mandatory that the total amount of BA, BMA and BHA in Fuzi should be over 0.01%, and the total contents of AC, MA and HA in Fuzi should be lower than 0.01%. In pharmaceutical process of BHP and CP samples, Fuzi were both added as crude herb powder. Table II revealed that contents of six alkaloids from Fuzi were obviously different among all the samples. A point diagram (Figure 6) was performed to distribute the qualified and non-qualified Fuzi in the FLP samples following the lecture (24) It is obvious that the total contents of AC, MA and HA in fourteen batches was over 0.01%, named non-qualified, which is insecurity as their toxicity. The total contents of BA, BMA and BHA in five batches was below 0.01%, named non-qualified, which results in a decrease in curative effect of FLP, and even no effect. So, it is highly recommended that the determination of those six alkaloids in the proprietary Chinese medicines containing Fuzi must be done as a routine measurement, so as to provide a safe application to patients in clinics, and good manufacture practices (33). The data reveal that this method could be successfully applied to CPMs, such as BHP, with the prerequisite that the herbal medicines in them are only powdered, whereas for CPMs in CP form, this method should be further improved after carefully taking the constituents into account, particularly the unstable constituents, which may thermally decompose during the processes of extraction, concentration and desiccation. Figure 6. View largeDownload slide Point diagram of the distribution of the qualified and non-qualified TCMs in the FLP samples. x-axis: BHP and CP represent big honey pills and concentrated pills, respectively; Fuzi represent Aconiti Lateralis Radix Praeparata, respectively. The analytes in the brackets are required by Chinese Pharmacopeia. MDA represents monoester-diterpenoid alkaloids (includes BA, BMA, BHA), and DDA represents diester-diterpenoid alkaloids (includes AC, MA, HA); the y-axis is the content of the analyte shown in percentage and is the quantitative standard for the Chinese Pharmacopeia. The direction of the arrow represents the content requirement by the Chinese Pharmacopeia. Figure 6. View largeDownload slide Point diagram of the distribution of the qualified and non-qualified TCMs in the FLP samples. x-axis: BHP and CP represent big honey pills and concentrated pills, respectively; Fuzi represent Aconiti Lateralis Radix Praeparata, respectively. The analytes in the brackets are required by Chinese Pharmacopeia. MDA represents monoester-diterpenoid alkaloids (includes BA, BMA, BHA), and DDA represents diester-diterpenoid alkaloids (includes AC, MA, HA); the y-axis is the content of the analyte shown in percentage and is the quantitative standard for the Chinese Pharmacopeia. The direction of the arrow represents the content requirement by the Chinese Pharmacopeia. OPLS-DA of the sample The results (Figure 3) suggest that OPLS-DA might be an effective method to evaluate the quality consistency of CPM. These observations also demonstrate that the content variations of the 16 components between the two dosage forms were larger than those between samples in the same dosage form. To further compare the two dosage forms on manufacturing procedures: Dangshen, Baizhu, Gancao and Ganjiang in CP are extracted by 70% alcohol or water, which is in contrast to all herbal medicines in the BHP form are only powdered. Furthermore, it should be noted that the most relevant four variables, i.e., ALIII, ALII, ALI and GE, are heat- or sunlight-unstable compounds (34, 35); the instability of these components implies that their contents decrease significantly during CP sample production. The results suggest that more attention should be paid to those unstable constituents in the quality control of CPMs. ROC of the selected analytes ROC analysis is a useful tool for evaluating the accuracy of a statistical model (eg, logistic regression, linear discriminate analysis). The sensitivity and specificity trade-offs were calculated for each selected marker using the area under the ROC curve (AUC), which is a summary measure that essentially averages distinction accuracy. The clear clustering in heat map for each group was in agreement with the OPLS-DA results. The 16 analytes selected in this paper were not merely selected based on suitable therapeutic, bioactive, characteristic, main, synergistic, correlative, toxic and analytical properties. Through this study, the results suggest that the problem in the quality control of multiple components of FLPs is urgent and important. Furthermore, the content variation of major active components between two dosage forms of FLP is an issue of great concern. The primary reason for the variations between the two dosage forms was the fact that the unstable components decreased significantly during the CP manufacturing process, and the reason for variations among different companies may be derived from the quality of herbal material with different properties, such as origins, sources and processing methods. Additionally, the differences in the manufacturing procedures and scales among the companies may be other possible reasons. Because only the chemical variation between two dosage forms is discussed, whether the chemical variation results in a variation in the therapeutic effect should be questioned and further explored. Conclusion A convenient and reliable approach using RRLC–MS was developed to quantitatively analyze the 16 components in FLP. The establishment of pattern recognition using OPLS-DA method and heat map demonstrated content variations between the two dosage forms. Through this approach, the correct classification and the differentiation of samples were obtained; moreover, by VIP plots and ROC curves, markers with large variations that were related to different dosage forms or quality variation of herbal materials could be easily found. The quality of the crude material herb could be qualified and evaluated as well. The method in this work could be used as an alternative approach in the quality evaluation of CPMs. Supplementary data Supplementary material is available at Journal of Chromatographic Science online. Funding Major State Basic Research Development Program of China, 973 Program (Project No. 2012CB518401), the National Natural Science Foundation of China (Project No. 81470177) and the Central Research Institutes of Basic Research and Public Service Special Operations (Project No. YZ-1542). Conflict of interest statement The authors declare that have no competing interests. References 1 Jiang, W.Y.; Therapeutic wisdom in traditional Chinese medicine: a perspective from modern science; Trends in Pharmacological Sciences , ( 2005); 26( 11): 558– 563. 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For Permissions, please email: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Chromatographic Science Oxford University Press

Quality Control of the Fuzi Lizhong Pill Through Simultaneous Determination of 16 Major Bioactive Constituents by RRLC–MS-MS

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Oxford University Press
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© The Author(s) 2018. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com
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0021-9665
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1945-239X
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10.1093/chromsci/bmy029
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Abstract

Abstract Fuzi Lizhong pill (FLP) is used to treat gastritis, and the monarch drug of it is Aconiti Lateralis Radix Praeparata (Fuzi, aconite roots) which is a toxic herbal medicine. To better control the safety and quality of FLP, an effective method to analyze the contents of 16 toxic and bioactive components using rapid resolution liquid chromatography–tandem triple-quadrupole mass spectrometer was established. The 16 constituents included aconine, mesaconine, hypaconitine, benzoylaconine, benzoylmesaconine, benzoylhypaconine, adenosine, liquiritin, liquiritigenin, glycyrrhizic acid, isoliquiritigenin, 6-gingerol, atractylenolide III, atractylenolide I, atractylenolide II and glycyrrhetic acid. Ideal separation was performed using gradient elution in 13 min by optimized conditions. All the isomerides were isolated to baseline. The improved method with a polarity switch in contiguous time segments could analyze the five types of components, including polar and nonpolar compounds, without decreasing sensitivity. The proposed method was fully validated. The results revealed that contents of six alkaloids from Fuzi were significantly different among the samples. Using the established method and multivariate statistical method, the quality consistency of two dosage forms of FLP from different companies were analyzed. The optimized method could be used for the quality control of FLP and investigate index compound variation between two dosage forms. Introduction Chinese patent medicines (Zhong-Cheng-Yao in Chinese, CPMs) are the representative form of traditional Chinese medicines, which have gained increasing popularity for their health care and improving the body’s immunity against diseases around the world over the past decades (1). Moreover, several prescriptions of CPMs derived from traditional classical prescriptions, which have been applied in clinic for centuries, have been demonstrated to be effective in modern pharmacological studies and clinical trials (2–4). For example, the Fuzi Lizhong pill (FLP), which was initially recorded in “Shanghan Lun” 1800 years ago, is one of the popular traditional Chinese patent medicines used to treat such conditions as gastritis, enteritis, diarrhea and hypothermia with indigestion (2, 5). FLP has obvious anti-inflammatory and analgesic effects (6, 7). It consists of five herbal materials and Aconiti Lateralis Radix Praeparata (Fuzi, aconite roots) is the monarch drug in the prescription that exists the major therapeutic effects following the TCM theory. However, Fuzi is a toxic herbal medicine. Processing, one of the characteristics of the Chinese herbal medicine, could greatly reduce the toxicity of Fuzi by hydrolyzing the toxic diester diterpene alkaloids mainly including aconitine, mesaconitine and hypaconitine (8). Nevertheless, poison and side effects sometimes still occur in clinic (9, 10). Thus, it is urgent and essential to determinate the toxic components in FLP to guarantee its clinical safety. As the main active and toxic compounds in Fuzi, alkaloids could perform the cardiotonic action and reinforce the immune system by improve the peritoneal macrophage Ia expression (11). Among them, aconitine (AC), mesaconitine (MA), hypaconitine (HA), benzoylaconine (BA), benzoylmesaconine (BMA) and benzoylhypaconine (BHA) are the main toxic and active important markers in chemical analysis of Fuzi (8). Their contents are extensively qualified in Chinese Pharmacopeia resulting from that they are possessed of toxicity and simultaneously pharmacological activities. Moreover, it is well-known that the active components in herbal materials of CPMs are multiple, and the curative effects of CPMs are principally based on the synergic effects of these ingredients (12). Except Fuzi, FLP also consists of other four herbal materials: Codonopsis Radix (Dangshen), Atractylodis Macrocephalae Rhizoma (Baizhu), Glycyrrhizae Radix et Rhizoma (Gancao) and Zingiberis Rhizoma (Ganjiang) (2). The flavones and triterpenes from Gancao have a wide range of biological and physiological activities, such as anti-inflammatory (13), anti-proliferative (14) and anti-diabetic (15). Liquiritigenin (LQG), liquiritin (LQR), glycyrrhizic acid (GIA), isoliquiritigenin (OLQG) and glycyrrhetic acid (GEA) are the main markers in quality control of Gancao. The lactones from Baizhu are important for their anti-inflammatory, antioxidant and modulating gastrointestinal function (16, 17). Atractylenolide I (ALI), atractylenolide II (ALII) and atractylenolide III (ALIII) are main chemical markers in quality analysis of Baizhu. Adenosine (AD) and 6-gingerol (GE) from other included herbal materials are also important due to their antioxidant, anti-inflammatory and xanthine oxidase inhibitory effects (18, 19). Chromatographic fingerprints have identified several compounds in the FLP (the dosage form is not described), where differences were found among 12 batches of samples from four companies (20). While, whether the contents of main active components exist difference in different dosage forms of FLP and what may cause the variations are still unclear. Therefore, it is imperative to qualify control of FLP by these active components in order to minimize the variability and ensure its repeatable therapeutic effects. With the rapid development of advanced technology and precise instrument, many qualitative and quantitative methods have been devised regarding the quality evaluation of CPMs, such as rapid resolution liquid chromatography/tandem quadrupole time-of-flight mass spectrometer and rapid resolution liquid chromatography/tandem triple-quadrupole mass spectrometer (21–24). In contrast to HPLC method, rapid resolution liquid chromatography (RRLC) exhibits a higher peak capacity, increased sensitivity, better resolution and a greater rate of analysis. RRLC coupled to a triple quadrupole tandem mass spectrometer (TQ-MS-MS) has advantages of high selectivity and sensitivity through the multiple reaction monitoring (MRM) scan mode, in which baseline chromatographic separation of selected constituents is unnecessary (8). Moreover, the application of small-diameter particle columns in this method could guarantee rapid separation (8, 25). This method could quantify target constituents in complex entities at trace amounts, which implies its good potential in simultaneously determining multiple target constituents in CPMs. Despite the great advantages of RRLC–TQ-MS-MS in terms of quantification, analyzing various types of components with the same polarity is still a problem, particularly in guaranteeing sensitivity. In this study, an improved method with polarity switching in contiguous time segments achieved the goal of analyzing six types of 16 components, including alkaloids, adenosines, lactones, flavonoids, triterpenes and phenolic compounds, the major chemical components in herbal materials of FLP, without decreasing sensitivity. Based on the sample data, two dosage forms FLP samples, big honey pill (BHP) and the concentrated pill (CP), from 10 companies were determined. Moreover, OPLS-DA, heat map and ROC curve were used in the quality evaluation of the variation of samples. It is suggested that the established method would be applicable for quality evaluation of other constituents in CPMs. Experimental Chemicals, solvents and materials A total of 49 batches of FLPs were collected from eleven pharmaceutical companies in China (Table SI). The reference standards of AD, LQR, AC, MA, HA, GE, GIA and GEA were obtained from the Chinese National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China); BMA, BA, BHA, LQG and OLQG were purchased from Chengdu Must Biotechnology Co., Ltd. (Chengdu, China); ALI, ALII and ALIII were provided by Shanghai Tiyo Biotechnology Co., Ltd. (Shanghai, China). The purities of all the standards were no <95%. All of the analytes are summarized in Table SII, and their structures are shown in Figure 1. Figure 1. View largeDownload slide Structures of the 16 major constituents in the FLP. Adenosine (AD, 1), liquiritin (LQR, 2), benzoylmesaconine (BMA, 3), benzoylaconine (BA, 4), benzoylhypaconine (BHA, 5), liquiritigenin (LQG, 6), mesaconine (MA, 7), aconine (AC, 8), hypaconitine (HA, 9), glycyrrhizic acid (GIA, 10), isoliquiritigenin (OLQG, 11), 6-gingerol (GE, 12), atractylenolide III (ALIII, 13), atractylenolide I (ALI, 14), atractylenolide II (ALII, 15), glycyrrhetic acid (GEA, 16). Figure 1. View largeDownload slide Structures of the 16 major constituents in the FLP. Adenosine (AD, 1), liquiritin (LQR, 2), benzoylmesaconine (BMA, 3), benzoylaconine (BA, 4), benzoylhypaconine (BHA, 5), liquiritigenin (LQG, 6), mesaconine (MA, 7), aconine (AC, 8), hypaconitine (HA, 9), glycyrrhizic acid (GIA, 10), isoliquiritigenin (OLQG, 11), 6-gingerol (GE, 12), atractylenolide III (ALIII, 13), atractylenolide I (ALI, 14), atractylenolide II (ALII, 15), glycyrrhetic acid (GEA, 16). HPLC-grade acetonitrile and methanol was purchased from Fisher Scientific (Fair Lawn, NJ, USA), and MS grade formic acid was provided by MREDA Technology Inc. (USA). Deionized water was redistilled. All other chemicals and solvents were of analytical grade. Sample preparation and standard solutions For the sample preparation, the BHP samples were cut into pieces, whereas the CP samples were ground into a fine powder. The 0.2 g of BHP samples and 0.050 g of CP samples were accurately weighed and ultrasonically extracted with 50 mL methanol for 60 min in a conical flask, cooled to room temperature and then replenished to their original weight with methanol. The obtained solution was filtered through a 0.22-μm syringe filter. The first portion of the filter liquor was abandoned, and the subsequent 1–2 μL was directly injected into the HPLC instruments analysis. Stock solutions were obtained from the procedure, in which each reference standard was accurately weighed and dissolved with methanol. Specific amounts of the 16 reference standard solutions were diverted into a 10-mL volumetric flask to obtain the mixed stock solution. And the concentration of standard of AD, LQR, BMA, BA, BHA, LQG, MA, AC, HA, GIA, OLQG, GE, ALIII, ALI, ALII and GEA in the mixed stock solution were 60.00, 5,040, 462.0, 88.40, 324.0, 680.0, 29.12, 7.385, 92.40,13,027, 115.2, 5,952, 408.0, 496.0, 399.6 and 130.0 ng/mL, respectively. A set of standard solutions were prepared by appropriate dilution of the mixed stock solution with methanol to obtain the calibration curve. Chromatographic and mass spectrometric conditions All data were acquired using an RRLC system (1,260 series liquid chromatography, Agilent Technologies, Inc., USA) coupled with an electrospray MS (6410B Triple Quadrupole Mass Spectrometer, Agilent Technologies, Inc., USA; accuracy of 0.1 Da and resolution of 0.7 Da within the range of 1–1,000 Da). The separation was performed on a Thermo Accucore RP-MS column (100 mm × 2.1 mm; 2.6 μm particle size). The mobile phase consisted of (A) water containing 0.1% formic acid and (B) acetonitrile containing 0.1% formic acid. The linear gradient conditions were as follows: 0–3 min, 15–55% B; 3–9 min, 55–55%; 9–12 min, 55–80%; 12–12.01 min, 80–100%; 12.01–13 min, 100–100%. The flow rate was 0.3 mL/min. The column temperature was set at 30°C. Typical RRLC chromatograms of detected chemicals in the FLP are shown in Figure 2. Figure 2. View largeDownload slide Typical chromatograms of RRLC–MS-MS for a (A) sample and (B) standard mixture. The peak number is the same as the compound number summarized in Figure 1. This figure is available in black and white in print and in color at JCS online. Figure 2. View largeDownload slide Typical chromatograms of RRLC–MS-MS for a (A) sample and (B) standard mixture. The peak number is the same as the compound number summarized in Figure 1. This figure is available in black and white in print and in color at JCS online. The conditions of MS analysis were as follows: LQR was detected in negative ion mode, whereas the rest of the constituents were detected in positive ion mode; the drying gas N2 flow rate was 11 L/min at a gas temperature 300°C, and the pressure of the nebulizer was 45 psi. The capillary voltage was set to 4,000 V. MRM was employed for quantification. The fragmentor voltage, collision energy values and dwell time of each ion pair were optimized to obtain the highest abundance. Method validation The quantitative analysis was an external calibration method, and the linearity calibration curves were constructed by six different concentrations of 16 components, where all of them were prepared by serial dilutions with methanol. Each concentration was analyzed in triplicate, and the calibration curves were established by plotting the peak areas with the concentrations of each component. For the limit of detection (LOD) and limit of quantification (LOQ), namely, the sensitivity of the method was measured at a signal-to-noise ratio of 3 and 10, respectively, as the criteria. The intra-day precision was evaluated by detection of the mixed standard solution at six times within 1 day, whereas the inter-day precision was determined by repeated analysis of the mixed standard solution in 3 consecutive days. To confirm repeatability, five replicates of the same samples were prepared according to the procedures described above. Regarding the stability, it was examined by testing the sample every 2 h for 24 h continuously. The standard addition method was applied in the recovery test of this method. Three different concentration levels (approximately equivalent to 0.8, 1.0 and 1.2 times of the concentration of the matrix) of the references standards were added into the sample in triplicate. The total amount of each analyte was calculated from the corresponding calibration curve, and the average recoveries were determined and calculated. Quantification of FLP samples Forty-nine batches of two dosage forms of FLP samples from 11 companies were analyzed by the established method as described above, and each sample was analyzed three times. The quantification of the 16 standard compounds in the FLP samples was performed using their individual calibration curves. Chemometric data analysis The data of the two dosage forms of the FLP samples were calculated and analyzed by Simca-P 13.0 software (Umetrics, Umeå, Sweden), which included a multivariate statistical analysis, such as OPLS-DA. The receiver operating characteristic (ROC) curve and heat map generation was performed by a web-based tool called MetaboloAnalyst 3.0 (http://www.metaboanalyst.ca/). The areas (AUC) under ROC were constructed to evaluate the accuracy of differential markers in distinguishing dosage form. In addition, Student’s t test was used to calculate the statistical significance between two groups by SPSS 22.0, and the P value of Student’s t test was set to 0.05 for this study. Results Method validation To verify the reliability of the results, the established method was validated with respect to linearity, precision, sensitivity, stability and accuracy for the two dosage forms. The results in Table I show that full calibration curves of the 16 compounds were obtained with relatively wide concentration ranges, where the correlation coefficients of all the calibration curves were >0.9997. A portion of the BHP samples were diluted 5-fold due to their high contents to ensure that the detection value satisfied the linear ranges. For different compounds, the LOD ranged from 0.007 to 8.349 ng/mL, and the LOQ ranged from 0.024 to 27.83 ng/mL. The RSDs of intra- and inter-day precision were in the range of 2.04–3.00% and 2.76–3.99%, respectively. The stability was <4.42%. Recoveries of the 16 compounds ranged from 90.84 to 106.7% with all of the RSDs <7.65% (Table SIII), which indicates that the optimized method is accurate. Table I. Calibration Curves, LOD, LOQ, Precision and Repeatability for 16 Compounds in FLP No.  Compound  Calibration curve  r  Linear range (ng/mL)  LOD (ng/mL)  LOQ (ng/mL)  Intra-day (RSD,%) (n = 6)  Inter-day (RSD,%) (n = 6)  Repeatability (RSD,%) (n = 5)  1  AD  y = 3,998x + 6.105  0.9998  1.200–60.00  0.145  0.483  2.42  3.03  3.10  2  LQR  y = 845.1x + 16.68  0.9998  100.8–5,040  0.785  2.615  2.48  3.39  3.32  3  BMA  y = 19,238x + 56.66  0.9998  1.848–462.0  0.128  0.427  2.86  3.90  3.69  4  BA  y = 24,844x + 17.11  0.9998  0.3536–88.40  0.082  0.274  2.68  3.34  3.98  5  BHA  y = 24,181x + 68.04  0.9998  1.296–324.0  0.018  0.058  2.63  3.56  3.20  6  LQG  y = 9,009x + 189.1  0.9998  2.720–680.0  0.814  2.713  2.84  3.23  2.97  7  MA  y = 12,767x + 2.703  0.9999  0.1165–29.12  0.035  0.115  2.21  3.02  3.43  8  AC  y = 19,072x + 0.8950  0.9999  0.02954–7.385  0.007  0.024  3.00  3.87  3.77  9  HA  y = 22,114x + 10.13  0.9998  0.3696–92.40  0.022  0.072  2.40  3.35  3.09  10  GIA  y = 2,048x + 1,119  0.9998  52.11–13,027  0.372  1.238  2.08  2.76  3.13  11  OLQG  y = 30,198x + 64.96  0.9998  0.3840–115.2  0.105  0.350  2.16  2.88  3.85  12  GE  y = 5.544x + 2.660  0.9997  29.76–5,952  8.349  27.83  2.86  3.99  4.42  13  ALIII  y = 2,813x + 19.49  0.9998  1.632–408.0  0.435  1.450  2.68  3.19  2.57  14  ALI  y = 3,923x + 9.701  0.9998  1.984–496.0  0.388  1.292  2.04  3.50  3.65  15  ALII  y = 2,041x + 6.923  0.9999  1.598–399.6  0.425  1.417  2.75  3.83  3.83  16  GEA  y = 5,718x − 1.576  0.9999  0.5200–130.0  0.135  0.452  2.21  3.42  3.07  No.  Compound  Calibration curve  r  Linear range (ng/mL)  LOD (ng/mL)  LOQ (ng/mL)  Intra-day (RSD,%) (n = 6)  Inter-day (RSD,%) (n = 6)  Repeatability (RSD,%) (n = 5)  1  AD  y = 3,998x + 6.105  0.9998  1.200–60.00  0.145  0.483  2.42  3.03  3.10  2  LQR  y = 845.1x + 16.68  0.9998  100.8–5,040  0.785  2.615  2.48  3.39  3.32  3  BMA  y = 19,238x + 56.66  0.9998  1.848–462.0  0.128  0.427  2.86  3.90  3.69  4  BA  y = 24,844x + 17.11  0.9998  0.3536–88.40  0.082  0.274  2.68  3.34  3.98  5  BHA  y = 24,181x + 68.04  0.9998  1.296–324.0  0.018  0.058  2.63  3.56  3.20  6  LQG  y = 9,009x + 189.1  0.9998  2.720–680.0  0.814  2.713  2.84  3.23  2.97  7  MA  y = 12,767x + 2.703  0.9999  0.1165–29.12  0.035  0.115  2.21  3.02  3.43  8  AC  y = 19,072x + 0.8950  0.9999  0.02954–7.385  0.007  0.024  3.00  3.87  3.77  9  HA  y = 22,114x + 10.13  0.9998  0.3696–92.40  0.022  0.072  2.40  3.35  3.09  10  GIA  y = 2,048x + 1,119  0.9998  52.11–13,027  0.372  1.238  2.08  2.76  3.13  11  OLQG  y = 30,198x + 64.96  0.9998  0.3840–115.2  0.105  0.350  2.16  2.88  3.85  12  GE  y = 5.544x + 2.660  0.9997  29.76–5,952  8.349  27.83  2.86  3.99  4.42  13  ALIII  y = 2,813x + 19.49  0.9998  1.632–408.0  0.435  1.450  2.68  3.19  2.57  14  ALI  y = 3,923x + 9.701  0.9998  1.984–496.0  0.388  1.292  2.04  3.50  3.65  15  ALII  y = 2,041x + 6.923  0.9999  1.598–399.6  0.425  1.417  2.75  3.83  3.83  16  GEA  y = 5,718x − 1.576  0.9999  0.5200–130.0  0.135  0.452  2.21  3.42  3.07  Table I. Calibration Curves, LOD, LOQ, Precision and Repeatability for 16 Compounds in FLP No.  Compound  Calibration curve  r  Linear range (ng/mL)  LOD (ng/mL)  LOQ (ng/mL)  Intra-day (RSD,%) (n = 6)  Inter-day (RSD,%) (n = 6)  Repeatability (RSD,%) (n = 5)  1  AD  y = 3,998x + 6.105  0.9998  1.200–60.00  0.145  0.483  2.42  3.03  3.10  2  LQR  y = 845.1x + 16.68  0.9998  100.8–5,040  0.785  2.615  2.48  3.39  3.32  3  BMA  y = 19,238x + 56.66  0.9998  1.848–462.0  0.128  0.427  2.86  3.90  3.69  4  BA  y = 24,844x + 17.11  0.9998  0.3536–88.40  0.082  0.274  2.68  3.34  3.98  5  BHA  y = 24,181x + 68.04  0.9998  1.296–324.0  0.018  0.058  2.63  3.56  3.20  6  LQG  y = 9,009x + 189.1  0.9998  2.720–680.0  0.814  2.713  2.84  3.23  2.97  7  MA  y = 12,767x + 2.703  0.9999  0.1165–29.12  0.035  0.115  2.21  3.02  3.43  8  AC  y = 19,072x + 0.8950  0.9999  0.02954–7.385  0.007  0.024  3.00  3.87  3.77  9  HA  y = 22,114x + 10.13  0.9998  0.3696–92.40  0.022  0.072  2.40  3.35  3.09  10  GIA  y = 2,048x + 1,119  0.9998  52.11–13,027  0.372  1.238  2.08  2.76  3.13  11  OLQG  y = 30,198x + 64.96  0.9998  0.3840–115.2  0.105  0.350  2.16  2.88  3.85  12  GE  y = 5.544x + 2.660  0.9997  29.76–5,952  8.349  27.83  2.86  3.99  4.42  13  ALIII  y = 2,813x + 19.49  0.9998  1.632–408.0  0.435  1.450  2.68  3.19  2.57  14  ALI  y = 3,923x + 9.701  0.9998  1.984–496.0  0.388  1.292  2.04  3.50  3.65  15  ALII  y = 2,041x + 6.923  0.9999  1.598–399.6  0.425  1.417  2.75  3.83  3.83  16  GEA  y = 5,718x − 1.576  0.9999  0.5200–130.0  0.135  0.452  2.21  3.42  3.07  No.  Compound  Calibration curve  r  Linear range (ng/mL)  LOD (ng/mL)  LOQ (ng/mL)  Intra-day (RSD,%) (n = 6)  Inter-day (RSD,%) (n = 6)  Repeatability (RSD,%) (n = 5)  1  AD  y = 3,998x + 6.105  0.9998  1.200–60.00  0.145  0.483  2.42  3.03  3.10  2  LQR  y = 845.1x + 16.68  0.9998  100.8–5,040  0.785  2.615  2.48  3.39  3.32  3  BMA  y = 19,238x + 56.66  0.9998  1.848–462.0  0.128  0.427  2.86  3.90  3.69  4  BA  y = 24,844x + 17.11  0.9998  0.3536–88.40  0.082  0.274  2.68  3.34  3.98  5  BHA  y = 24,181x + 68.04  0.9998  1.296–324.0  0.018  0.058  2.63  3.56  3.20  6  LQG  y = 9,009x + 189.1  0.9998  2.720–680.0  0.814  2.713  2.84  3.23  2.97  7  MA  y = 12,767x + 2.703  0.9999  0.1165–29.12  0.035  0.115  2.21  3.02  3.43  8  AC  y = 19,072x + 0.8950  0.9999  0.02954–7.385  0.007  0.024  3.00  3.87  3.77  9  HA  y = 22,114x + 10.13  0.9998  0.3696–92.40  0.022  0.072  2.40  3.35  3.09  10  GIA  y = 2,048x + 1,119  0.9998  52.11–13,027  0.372  1.238  2.08  2.76  3.13  11  OLQG  y = 30,198x + 64.96  0.9998  0.3840–115.2  0.105  0.350  2.16  2.88  3.85  12  GE  y = 5.544x + 2.660  0.9997  29.76–5,952  8.349  27.83  2.86  3.99  4.42  13  ALIII  y = 2,813x + 19.49  0.9998  1.632–408.0  0.435  1.450  2.68  3.19  2.57  14  ALI  y = 3,923x + 9.701  0.9998  1.984–496.0  0.388  1.292  2.04  3.50  3.65  15  ALII  y = 2,041x + 6.923  0.9999  1.598–399.6  0.425  1.417  2.75  3.83  3.83  16  GEA  y = 5,718x − 1.576  0.9999  0.5200–130.0  0.135  0.452  2.21  3.42  3.07  Quantitative analysis of samples The established RRLC–MS-MS method was successfully applied to determine the content of the 16 components in the 49 batches of commercial FLP samples (Table II). To ensure the comparability of results, the concentration of each component was at the same level as in material drugs in all samples. Total contents of monoester-diterpenoid alkaloids (MDAs), including BMA, BA and BHA, from Fuzi in BHP and CP samples were in the range of 0.573–240.67 and 37.076–605.73 μg/g, respectively, while total diester-diterpenoid alkaloids (DDAs) (including MA, AC and HA) in BHP and CP samples were in the range of 0.046–44.933 and 0.357–20.48 μg/g. It could be easily found that all samples contained a high concentration of LQR in Gancao except sample No. 39 and its presence is mandatory for every company to meet the Chinese patent medicine standard. Among the 16 analytes, GIA was the compound with the highest content because it was the component with the highest content in Gancao (26). Besides, it has been found that the contents of GE in Ganjiang were 280.7–3,867 μg/g in all BHP samples, while they were very lower and even couldn’t be detected in CP samples. The low contents of GE in CP may derived by the different pharmaceutical process of Ganjiang in CP and BHP. In CP, Ganjiang was extracted by 70% ethanol, while it was herb power in BHP. The extracting solvent had a significant influence on the content of GE according to other literature reports (27). Similarly, contents of the compounds ALIII, ALII, ALI in Baizhu were much higher in BHP samples than those in CP samples. Besides, it is indicated that there was significant difference among products of batch-to-batch or from different manufactures even in the same dosage form, as the literature reported that the variation in contents of the constituents could be derived from the quality of raw herbal material (28). Table II. Contents of the 16 Compounds in 49 Batches of Samples Dosage form  Batch no.  Contents of each compound (μg/g relevant herbal material)  AD  LQR  BMA  BA  BHA  LQG  MA  AC  Big honeyed-pill  1  26.00 ± 0.437  2,095 ± 23.38  18.55 ± 0.821  3.478 ± 0.051  4.335 ± 0.080  62.12 ± 0.790  0.130 ± 0.005  0.057 ± 0.002  2  21.77 ± 0.574  1,659 ± 4.632  81.57 ± 0.228  8.989 ± 0.159  10.96 ± 0.121  207.8 ± 2.806  7.458 ± 0.005  1.601 ± 0.042  3  15.88 ± 0.168  1,581 ± 27.51  77.00 ± 1.379  7.934 ± 0.168  10.54 ± 0.180  188.3 ± 1.129  6.490 ± 0.253  1.515 ± 0.046  4  31.37 ± 0.194  2,067 ± 20.22  54.91 ± 0.591  6.704 ± 0.240  12.63 ± 0.468  78.47 ± 2.381  2.628 ± 0.176  0.529 ± 0.014  5  15.34 ± 0.225  1,744 ± 46.48  81.10 ± 1.676  8.863 ± 0.213  10.69 ± 0.283  201.9 ± 1.563  6.580 ± 0.090  1.562 ± 0.062  6  20.81 ± 0.321  1,577 ± 27.20  102.5 ± 1.018  9.725 ± 0.381  11.16 ± 0.403  259.2 ± 1.079  11.89 ± 0.173  2.403 ± 0.037  7  23.30 ± 0.314  2,996 ± 25.53  136.2 ± 1.042  14.49 ± 0.484  22.88 ± 0.523  56.66 ± 2.431  11.62 ± 0.183  2.253 ± 0.025  8  34.23 ± 0.641  2,998 ± 29.53  84.61 ± 1.325  12.07 ± 0.931  23.91 ± 0.515  56.18 ± 1.934  3.131 ± 0.293  0.724 ± 0.041  9  34.52 ± 0.376  3,105 ± 21.42  94.33 ± 1.016  14.46 ± 0.231  22.14 ± 0.356  70.53 ± 1.221  1.065 ± 0.923  0.474 ± 0.056  10  1.847 ± 0.072  1,796 ± 17.55  6.584 ± 0.316  0.889 ± 0.033  1.795 ± 0.063  108.0 ± 4.831  0.053 ± 0.407  0.034 ± 0.002  11  12.16 ± 0.134  2,676 ± 21.53  1.944 ± 0.413  0.089 ± 0.045  0.008 ± 0.001  95.19 ± 1.842  0.016 ± 0.002  0.009 ± 0.000  12  8.128 ± 0.274  2,686 ± 19.37  0.316 ± 0.041  0.062 ± 0.031  0.195 ± 0.034  69.59 ± 1.780  0.021 ± 0.008  0.012 ± 0.000  13  3.171 ± 0.384  2,807 ± 27.53  3.590 ± 0.319  0.490 ± 0.016  1.524 ± 0.051  97.58 ± 2.014  0.080 ± 0.007  0.038 ± 0.013  14  0.163 ± 0.064  3,921 ± 25.48  10.82 ± 1.452  1.343 ± 0.084  0.886 ± 0.093  150.5 ± 3.858  0.031 ± 0.008  0.022 ± 0.001  15  3.365 ± 0.092  5,659 ± 25.84  46.13 ± 0.924  7.494 ± 0.153  10.40 ± 0.073  147.7 ± 3.239  0.143 ± 0.019  0.172 ± 0.014  16  3.230 ± 0.124  1,557 ± 30.28  102.5 ± 1.099  11.38 ± 0.275  17.34 ± 0.373  68.32 ± 0.285  4.996 ± 0.002  1.109 ± 0.042  17  2.105 ± 0.072  1,875 ± 9.186  81.72 ± 1.818  9.724 ± 0.096  16.29 ± 0.340  172.9 ± 1.233  4.575 ± 0.068  1.226 ± 0.049  18  2.369 ± 0.072  1,503 ± 7.465  97.21 ± 0.560  10.73 ± 0.143  17.39 ± 0.348  68.39 ± 1.862  4.569 ± 0.097  1.201 ± 0.025  19  5.704 ± 0.143  1,776 ± 24.02  90.73 ± 1.636  9.930 ± 0.128  15.60 ± 0.228  43.65 ± 0.922  5.058 ± 0.084  1.310 ± 0.025  20  9.860 ± 0.235  2,317 ± 22.51  63.12 ± 1.782  12.81 ± 1.024  28.27 ± 0.142  55.21 ± 1.523  1.600 ± 0.083  0.779 ± 0.044  21  4.073 ± 0.327  2,155 ± 17.42  113.5 ± 3.515  12.48 ± 0.931  20.00 ± 0.201  52.07 ± 1.664  4.015 ± 0.081  1.264 ± 0.053  22  18.53 ± 0.415  1,392 ± 18.20  44.27 ± 1.833  0.311 ± 0.020  11.58 ± 0.272  96.95 ± 2.323  0.246 ± 0.160  0.171 ± 0.003  23  19.81 ± 0.327  1,509 ± 62.60  37.37 ± 4.046  0.409 ± 0.025  11.26 ± 0.403  141.5 ± 2.064  0.306 ± 0.008  0.173 ± 0.003  24  18.74 ± 0.579  1,432 ± 42.79  31.40 ± 0.455  0.186 ± 0.001  9.628 ± 0.052  111.0 ± 2.425  0.293 ± 0.005  0.177 ± 0.003  25  31.22 ± 0.632  2,046 ± 20.51  54.42 ± 2.013  4.112 ± 0.146  5.142 ± 0.109  82.90 ± 3.028  10.01 ± 0.098  1.844 ± 0.028  26  13.29 ± 0.234  1,722 ± 21.58  198.3 ± 8.421  20.96 ± 1.023  21.41 ± 0.113  106.9 ± 3.741  4.287 ± 0.122  1.389 ± 0.084  27  16.57 ± 0.134  1,706 ± 19.52  92.45 ± 1.951  11.29 ± 1.184  16.60 ± 0.142  64.92 ± 1.447  1.794 ± 0.053  0.516 ± 0.032    RSD/%  73.3  41.8  69.3  76.1  63.9  52.7  106.1  88.2  Concentrated pill  28  26.06 ± 1.056  1,386 ± 38.24  133.3 ± 4.227  30.83 ± 0.531  107.3 ± 4.381  84.83 ± 1.935  0.062 ± 0.239  0.055 ± 0.001  29  19.36 ± 0.483  546.0 ± 6.419  102.9 ± 3.848  22.36 ± 0.475  93.01 ± 0.948  54.72 ± 2.849  0.102 ± 0.001  0.060 ± 0.001  30  21.63 ± 0.394  1,170 ± 26.72  117.8 ± 1.050  29.02 ± 0.991  94.39 ± 1.083  91.24 ± 0.992  0.088 ± 0.002  0.073 ± 0.001  31  22.01 ± 0.676  563.8 ± 19.10  88.64 ± 0.255  21.26 ± 0.554  106.5 ± 4.126  74.07 ± 3.309  0.142 ± 0.003  0.123 ± 0.003  32  33.98 ± 0.374  4,084 ± 28.83  319.1 ± 2.941  68.63 ± 1.251  218.0 ± 3.416  118.2 ± 2.041  0.185 ± 0.003  0.111 ± 0.007  33  37.35 ± 0.452  2,602 ± 26.31  304.6 ± 2.055  64.68 ± 0.491  198.5 ± 2.955  98.19 ± 1.453  0.176 ± 0.004  0.192 ± 0.008  34  7.166 ± 0.341  575.9 ± 3.932  45.81 ± 0.166  10.55 ± 0.124  24.23 ± 0.941  91.37 ± 2.450  0.058 ± 0.004  0.055 ± 0.001  35  9.863 ± 0.293  1,200 ± 14.11  96.86 ± 3.031  16.28 ± 0.283  44.73 ± 1.651  23.20 ± 1.533  0.173 ± 0.010  0.064 ± 0.003  36  15.47 ± 0.473  2,010 ± 17.82  99.95 ± 2.951  23.12 ± 0.341  51.60 ± 2.042  51.19 ± 2.951  0.197 ± 0.011  0.174 ± 0.005  37  13.47 ± 0.417  528.0 ± 10.30  58.71 ± 1.809  12.31 ± 0.152  39.60 ± 0.652  35.12 ± 1.240  0.036 ± 0.001  0.042 ± 0.000  38  10.71 ± 0.154  438.6 ± 1.613  64.72 ± 1.155  12.22 ± 0.272  38.79 ± 0.551  34.86 ± 0.358  0.069 ± 0.001  0.045 ± 0.001  39  12.40 ± 0.229  172.9 ± 2.009  35.98 ± 0.576  8.178 ± 0.115  31.10 ± 0.397  0.958 ± 0.032  0.055 ± 0.001  0.025 ± 0.000  40  20.49 ± 0.245  401.9 ± 8.352  114.4 ± 4.014  24.95 ± 0.398  64.94 ± 1.432  24.68 ± 1.542  0.003 ± 0.000  0.011 ± 0.000  41  7.184 ± 0.197  903.5 ± 5.530  26.36 ± 0.468  4.497 ± 0.195  9.323 ± 0.360  65.92 ± 2.121  0.080 ± 0.000  0.028 ± 0.001  42  17.92 ± 0.831  3,069 ± 10.51  67.96 ± 1.031  11.16 ± 0.312  20.06 ± 0.966  77.29 ± 3.133  0.170 ± 0.004  0.075 ± 0.000  43  14.43 ± 0.624  3,206 ± 28.41  70.98 ± 0.941  11.98 ± 0.284  19.60 ± 0.455  85.53 ± 2.414  0.090 ± 0.002  0.046 ± 0.000  44  8.560 ± 0.532  2,137 ± 15.32  87.56 ± 1.252  11.84 ± 0.184  29.41 ± 1.053  282.9 ± 3.988  0.683 ± 0.010  0.231 ± 0.001  45  4.262 ± 0.135  1,023 ± 11.42  44.59 ± 0.836  5.705 ± 0.167  18.82 ± 0.533  139.1 ± 3.556  0.560 ± 0.009  0.170 ± 0.000  46  16.02 ± 0.142  698.7 ± 2.889  16.33 ± 0.125  8.276 ± 0.084  12.47 ± 0.250  34.48 ± 0.509  1.409 ± 0.002  0.531 ± 0.000  47  2.909 ± 0.068  1,000 ± 3.059  175.3 ± 0.537  32.29 ± 0.337  79.26 ± 0.240  68.99 ± 0.691  0.217 ± 0.049  0.140 ± 0.000  48  23.65 ± 0.523  2,174 ± 16.82  252.7 ± 1.934  58.30 ± 0.253  129.4 ± 0.675  17.17 ± 0.886  1.102 ± 0.088  0.680 ± 0.001  49  7.264 ± 0.834  1,966 ± 12.44  253.2 ± 0.894  43.46 ± 0.384  115.6 ± 1.143  110.0 ± 3.452  0.895 ± 0.014  0.675 ± 0.003    RSD/%  56.6  73.7  75.7  78.4  82.6  77.2  129.2  121.8  Dosage form  Batch no.  Contents of each compound (μg/g relevant herbal material)  AD  LQR  BMA  BA  BHA  LQG  MA  AC  Big honeyed-pill  1  26.00 ± 0.437  2,095 ± 23.38  18.55 ± 0.821  3.478 ± 0.051  4.335 ± 0.080  62.12 ± 0.790  0.130 ± 0.005  0.057 ± 0.002  2  21.77 ± 0.574  1,659 ± 4.632  81.57 ± 0.228  8.989 ± 0.159  10.96 ± 0.121  207.8 ± 2.806  7.458 ± 0.005  1.601 ± 0.042  3  15.88 ± 0.168  1,581 ± 27.51  77.00 ± 1.379  7.934 ± 0.168  10.54 ± 0.180  188.3 ± 1.129  6.490 ± 0.253  1.515 ± 0.046  4  31.37 ± 0.194  2,067 ± 20.22  54.91 ± 0.591  6.704 ± 0.240  12.63 ± 0.468  78.47 ± 2.381  2.628 ± 0.176  0.529 ± 0.014  5  15.34 ± 0.225  1,744 ± 46.48  81.10 ± 1.676  8.863 ± 0.213  10.69 ± 0.283  201.9 ± 1.563  6.580 ± 0.090  1.562 ± 0.062  6  20.81 ± 0.321  1,577 ± 27.20  102.5 ± 1.018  9.725 ± 0.381  11.16 ± 0.403  259.2 ± 1.079  11.89 ± 0.173  2.403 ± 0.037  7  23.30 ± 0.314  2,996 ± 25.53  136.2 ± 1.042  14.49 ± 0.484  22.88 ± 0.523  56.66 ± 2.431  11.62 ± 0.183  2.253 ± 0.025  8  34.23 ± 0.641  2,998 ± 29.53  84.61 ± 1.325  12.07 ± 0.931  23.91 ± 0.515  56.18 ± 1.934  3.131 ± 0.293  0.724 ± 0.041  9  34.52 ± 0.376  3,105 ± 21.42  94.33 ± 1.016  14.46 ± 0.231  22.14 ± 0.356  70.53 ± 1.221  1.065 ± 0.923  0.474 ± 0.056  10  1.847 ± 0.072  1,796 ± 17.55  6.584 ± 0.316  0.889 ± 0.033  1.795 ± 0.063  108.0 ± 4.831  0.053 ± 0.407  0.034 ± 0.002  11  12.16 ± 0.134  2,676 ± 21.53  1.944 ± 0.413  0.089 ± 0.045  0.008 ± 0.001  95.19 ± 1.842  0.016 ± 0.002  0.009 ± 0.000  12  8.128 ± 0.274  2,686 ± 19.37  0.316 ± 0.041  0.062 ± 0.031  0.195 ± 0.034  69.59 ± 1.780  0.021 ± 0.008  0.012 ± 0.000  13  3.171 ± 0.384  2,807 ± 27.53  3.590 ± 0.319  0.490 ± 0.016  1.524 ± 0.051  97.58 ± 2.014  0.080 ± 0.007  0.038 ± 0.013  14  0.163 ± 0.064  3,921 ± 25.48  10.82 ± 1.452  1.343 ± 0.084  0.886 ± 0.093  150.5 ± 3.858  0.031 ± 0.008  0.022 ± 0.001  15  3.365 ± 0.092  5,659 ± 25.84  46.13 ± 0.924  7.494 ± 0.153  10.40 ± 0.073  147.7 ± 3.239  0.143 ± 0.019  0.172 ± 0.014  16  3.230 ± 0.124  1,557 ± 30.28  102.5 ± 1.099  11.38 ± 0.275  17.34 ± 0.373  68.32 ± 0.285  4.996 ± 0.002  1.109 ± 0.042  17  2.105 ± 0.072  1,875 ± 9.186  81.72 ± 1.818  9.724 ± 0.096  16.29 ± 0.340  172.9 ± 1.233  4.575 ± 0.068  1.226 ± 0.049  18  2.369 ± 0.072  1,503 ± 7.465  97.21 ± 0.560  10.73 ± 0.143  17.39 ± 0.348  68.39 ± 1.862  4.569 ± 0.097  1.201 ± 0.025  19  5.704 ± 0.143  1,776 ± 24.02  90.73 ± 1.636  9.930 ± 0.128  15.60 ± 0.228  43.65 ± 0.922  5.058 ± 0.084  1.310 ± 0.025  20  9.860 ± 0.235  2,317 ± 22.51  63.12 ± 1.782  12.81 ± 1.024  28.27 ± 0.142  55.21 ± 1.523  1.600 ± 0.083  0.779 ± 0.044  21  4.073 ± 0.327  2,155 ± 17.42  113.5 ± 3.515  12.48 ± 0.931  20.00 ± 0.201  52.07 ± 1.664  4.015 ± 0.081  1.264 ± 0.053  22  18.53 ± 0.415  1,392 ± 18.20  44.27 ± 1.833  0.311 ± 0.020  11.58 ± 0.272  96.95 ± 2.323  0.246 ± 0.160  0.171 ± 0.003  23  19.81 ± 0.327  1,509 ± 62.60  37.37 ± 4.046  0.409 ± 0.025  11.26 ± 0.403  141.5 ± 2.064  0.306 ± 0.008  0.173 ± 0.003  24  18.74 ± 0.579  1,432 ± 42.79  31.40 ± 0.455  0.186 ± 0.001  9.628 ± 0.052  111.0 ± 2.425  0.293 ± 0.005  0.177 ± 0.003  25  31.22 ± 0.632  2,046 ± 20.51  54.42 ± 2.013  4.112 ± 0.146  5.142 ± 0.109  82.90 ± 3.028  10.01 ± 0.098  1.844 ± 0.028  26  13.29 ± 0.234  1,722 ± 21.58  198.3 ± 8.421  20.96 ± 1.023  21.41 ± 0.113  106.9 ± 3.741  4.287 ± 0.122  1.389 ± 0.084  27  16.57 ± 0.134  1,706 ± 19.52  92.45 ± 1.951  11.29 ± 1.184  16.60 ± 0.142  64.92 ± 1.447  1.794 ± 0.053  0.516 ± 0.032    RSD/%  73.3  41.8  69.3  76.1  63.9  52.7  106.1  88.2  Concentrated pill  28  26.06 ± 1.056  1,386 ± 38.24  133.3 ± 4.227  30.83 ± 0.531  107.3 ± 4.381  84.83 ± 1.935  0.062 ± 0.239  0.055 ± 0.001  29  19.36 ± 0.483  546.0 ± 6.419  102.9 ± 3.848  22.36 ± 0.475  93.01 ± 0.948  54.72 ± 2.849  0.102 ± 0.001  0.060 ± 0.001  30  21.63 ± 0.394  1,170 ± 26.72  117.8 ± 1.050  29.02 ± 0.991  94.39 ± 1.083  91.24 ± 0.992  0.088 ± 0.002  0.073 ± 0.001  31  22.01 ± 0.676  563.8 ± 19.10  88.64 ± 0.255  21.26 ± 0.554  106.5 ± 4.126  74.07 ± 3.309  0.142 ± 0.003  0.123 ± 0.003  32  33.98 ± 0.374  4,084 ± 28.83  319.1 ± 2.941  68.63 ± 1.251  218.0 ± 3.416  118.2 ± 2.041  0.185 ± 0.003  0.111 ± 0.007  33  37.35 ± 0.452  2,602 ± 26.31  304.6 ± 2.055  64.68 ± 0.491  198.5 ± 2.955  98.19 ± 1.453  0.176 ± 0.004  0.192 ± 0.008  34  7.166 ± 0.341  575.9 ± 3.932  45.81 ± 0.166  10.55 ± 0.124  24.23 ± 0.941  91.37 ± 2.450  0.058 ± 0.004  0.055 ± 0.001  35  9.863 ± 0.293  1,200 ± 14.11  96.86 ± 3.031  16.28 ± 0.283  44.73 ± 1.651  23.20 ± 1.533  0.173 ± 0.010  0.064 ± 0.003  36  15.47 ± 0.473  2,010 ± 17.82  99.95 ± 2.951  23.12 ± 0.341  51.60 ± 2.042  51.19 ± 2.951  0.197 ± 0.011  0.174 ± 0.005  37  13.47 ± 0.417  528.0 ± 10.30  58.71 ± 1.809  12.31 ± 0.152  39.60 ± 0.652  35.12 ± 1.240  0.036 ± 0.001  0.042 ± 0.000  38  10.71 ± 0.154  438.6 ± 1.613  64.72 ± 1.155  12.22 ± 0.272  38.79 ± 0.551  34.86 ± 0.358  0.069 ± 0.001  0.045 ± 0.001  39  12.40 ± 0.229  172.9 ± 2.009  35.98 ± 0.576  8.178 ± 0.115  31.10 ± 0.397  0.958 ± 0.032  0.055 ± 0.001  0.025 ± 0.000  40  20.49 ± 0.245  401.9 ± 8.352  114.4 ± 4.014  24.95 ± 0.398  64.94 ± 1.432  24.68 ± 1.542  0.003 ± 0.000  0.011 ± 0.000  41  7.184 ± 0.197  903.5 ± 5.530  26.36 ± 0.468  4.497 ± 0.195  9.323 ± 0.360  65.92 ± 2.121  0.080 ± 0.000  0.028 ± 0.001  42  17.92 ± 0.831  3,069 ± 10.51  67.96 ± 1.031  11.16 ± 0.312  20.06 ± 0.966  77.29 ± 3.133  0.170 ± 0.004  0.075 ± 0.000  43  14.43 ± 0.624  3,206 ± 28.41  70.98 ± 0.941  11.98 ± 0.284  19.60 ± 0.455  85.53 ± 2.414  0.090 ± 0.002  0.046 ± 0.000  44  8.560 ± 0.532  2,137 ± 15.32  87.56 ± 1.252  11.84 ± 0.184  29.41 ± 1.053  282.9 ± 3.988  0.683 ± 0.010  0.231 ± 0.001  45  4.262 ± 0.135  1,023 ± 11.42  44.59 ± 0.836  5.705 ± 0.167  18.82 ± 0.533  139.1 ± 3.556  0.560 ± 0.009  0.170 ± 0.000  46  16.02 ± 0.142  698.7 ± 2.889  16.33 ± 0.125  8.276 ± 0.084  12.47 ± 0.250  34.48 ± 0.509  1.409 ± 0.002  0.531 ± 0.000  47  2.909 ± 0.068  1,000 ± 3.059  175.3 ± 0.537  32.29 ± 0.337  79.26 ± 0.240  68.99 ± 0.691  0.217 ± 0.049  0.140 ± 0.000  48  23.65 ± 0.523  2,174 ± 16.82  252.7 ± 1.934  58.30 ± 0.253  129.4 ± 0.675  17.17 ± 0.886  1.102 ± 0.088  0.680 ± 0.001  49  7.264 ± 0.834  1,966 ± 12.44  253.2 ± 0.894  43.46 ± 0.384  115.6 ± 1.143  110.0 ± 3.452  0.895 ± 0.014  0.675 ± 0.003    RSD/%  56.6  73.7  75.7  78.4  82.6  77.2  129.2  121.8  Dosage form  Batch no.  Contents of each compound (μg/g material drugs)  HA  GIA  OLQG  GE  ALIII  ALI  ALII  GEA  Big honeyed-pill  1  1.452 ± 0.021  2,650 ± 43.32  11.30 ± 0.463  1,595 ± 54.68  42.46 ± 1.757  45.63 ± 1.223  60.20 ± 0.534  2.659 ± 0.053  2  22.99 ± 0.469  3,012 ± 90.64  26.27 ± 0.319  2,031 ± 53.48  172.0 ± 1.676  182.6 ± 3.168  158.0 ± 1.022  6.260 ± 0.142  3  20.77 ± 0.346  3,202 ± 137.4  24.64 ± 0.340  1,464 ± 50.18  141.9 ± 3.387  151.1 ± 1.446  126.1 ± 2.015  5.337 ± 0.204  4  11.87 ± 0.313  3,914 ± 78.75  15.01 ± 0.051  2,341 ± 109.5  100.8 ± 1.155  97.61 ± 1.023  114.4 ± 1.472  2.624 ± 0.046  5  21.61 ± 0.087  3,735 ± 93.67  26.73 ± 0.293  1,217 ± 51.34  129.0 ± 1.124  126.9 ± 0.593  113.5 ± 3.811  6.583 ± 0.068  6  30.64 ± 0.610  3,285 ± 63.06  33.21 ± 1.256  872.9 ± 2.160  94.88 ± 3.543  125.3 ± 1.032  139.0 ± 2.415  8.481 ± 0.248  7  28.42 ± 0.435  3,183 ± 54.76  4.228 ± 0.863  2,554 ± 46.76  81.52 ± 2.657  158.4 ± 2.907  131.6 ± 2.098  0.927 ± 0.097  8  13.29 ± 0.521  4,383 ± 66.87  4.845 ± 0.956  3,112 ± 64.38  89.50 ± 1.543  122.1 ± 2.065  109.0 ± 2.218  1.716 ± 0.087  9  9.252 ± 0.367  4,533 ± 57.43  5.375 ± 0.675  2,545 ± 58.62  89.85 ± 2.989  115.7 ± 1.648  92.76 ± 3.112  1.252 ± 0.057  10  1.178 ± 0.016  4,633 ± 34.81  38.83 ± 0.802  780.8 ± 1.079  106.2 ± 3.058  140.8 ± 3.498  96.39 ± 1.544  21.81 ± 0.884  11  0.021 ± 0.000  4,879 ± 48.67  33.17 ± 0.586  683.2 ± 20.76  222.1 ± 1.677  322.3 ± 2.767  200.1 ± 2.056  35.57 ± 1.076  12  0.021 ± 0.000  4,684 ± 87.43  19.76 ± 0.437  865.9 ± 16.44  245.8 ± 2.452  403.0 ± 2.234  247.1 ± 2.675  9.749 ± 0.078  13  1.150 ± 0.129  5,149 ± 76.44  28.21 ± 0.532  644.1 ± 23.98  99.78 ± 1.777  181.1 ± 0.986  79.67 ± 0.887  23.63 ± 0.254  14  0.695 ± 0.034  8,127 ± 48.61  19.56 ± 0.338  509.9 ± 21.61  248.2 ± 3.665  160.4 ± 1.065  202.0 ± 1.965  7.088 ± 0.334  15  1.680 ± 0.384  10814 ± 98.43  34.65 ± 0.674  600.5 ± 19.833  159.4 ± 2.787  133.1 ± 1.254  113.1 ± 2.137  23.47 ± 0.546  16  26.26 ± 0.718  3,980 ± 121.8  9.190 ± 0.224  1,934 ± 52.95  116.9 ± 0.699  181.6 ± 1.233  156.3 ± 0.929  32.89 ± 1.045  17  24.56 ± 0.323  5,442 ± 139.1  25.94 ± 0.748  2,276 ± 92.71  58.32 ± 1.480  96.40 ± 0.257  87.48 ± 1.436  43.10 ± 1.332  18  24.69 ± 0.378  2,682 ± 88.74  8.464 ± 0.150  2,031 ± 57.87  112.7 ± 2.215  186.4 ± 5.955  159.9 ± 3.114  32.39 ± 0.622  19  24.62 ± 0.462  2,966 ± 20.79  7.386 ± 0.184  2,031 ± 49.40  78.49 ± 1.840  135.0 ± 0.424  99.51 ± 2.247  29.82 ± 0.493  20  31.96 ± 0.532  2,800 ± 47.53  3.054 ± 0.205  3,867 ± 56.63  86.73 ± 2.096  221.0 ± 2.665  121.5 ± 1.877  23.93 ± 0.376  21  22.28 ± 0.787  3,736 ± 25.47  3.564 ± 0.397  3,771 ± 68.42  113.3 ± 2.136  222.1 ± 1.772  163.2 ± 2.870  20.47 ± 0.265  22  3.552 ± 0.122  4,402 ± 42.62  36.27 ± 0.447  875.2 ± 2.344  75.00 ± 1.335  82.91 ± 0.415  80.02 ± 1.082  9.714 ± 0.252  23  2.989 ± 0.033  4,397 ± 55.22  46.85 ± 1.037  687.3 ± 0.791  114.8 ± 4.771  86.91 ± 2.792  90.56 ± 11.312  13.97 ± 0.460  24  3.253 ± 0.052  4,430 ± 76.95  32.60 ± 1.153  781.2 ± 2.178  80.94 ± 1.116  92.23 ± 1.593  88.38 ± 1.604  12.65 ± 0.412  25  18.84 ± 0.452  3,613 ± 17.53  6.957 ± 0.654  2,892 ± 10.75  87.89 ± 2.343  116.0 ± 2.076  106.1 ± 2.122  3.381 ± 0.008  26  28.22 ± 0.445  3,175 ± 35.58  8.621 ± 0.788  2,716 ± 21.09  125.1 ± 2.766  178.1 ± 1.996  121.5 ± 1.942  2.513 ± 0.006  27  12.73 ± 0.782  2,448 ± 17.37  4.007 ± 0.965  280.7 ± 30.07  66.75 ± 2.087  94.44 ± 0.879  78.52 ± 1.209  3.072 ± 0.005    RSD/%  78.8  41.5  69.0  60.8  45.3  48.5  35.2  87.5  Concentrated pill  28  0.924 ± 0.002  1,987 ± 51.50  27.14 ± 0.749  N.D.a  16.45 ± 0.658  3.691 ± 0.074  1.300 ± 0.013  10.68 ± 0.401  29  1.378 ± 0.008  1,850 ± 44.50  33.03 ± 1.046  15.93 ± 0.026  15.28 ± 0.471  4.238 ± 0.179  0.705 ± 0.007  9.821 ± 0.208  30  1.342 ± 0.016  2,282 ± 44.85  54.09 ± 0.672  15.68 ± 0.026  17.49 ± 0.239  6.309 ± 0.086  0.596 ± 0.007  10.70 ± 0.464  31  4.264 ± 0.063  2,384 ± 95.77  46.24 ± 0.842  6.316 ± 0.025  12.05 ± 0.540  3.564 ± 0.178  0.257 ± 0.001  10.10 ± 0.238  32  2.219 ± 0.012  6,741 ± 67.33  21.35 ± 0.531  87.60 ± 1.035  41.86 ± 0.561  21.18 ± 0.093  3.951 ± 0.061  11.56 ± 0.051  33  3.330 ± 0.067  6,161 ± 52.88  18.12 ± 0.312  93.14 ± 1.237  41.22 ± 0.611  25.33 ± 0.123  3.089 ± 0.061  11.74 ± 0.086  34  1.035 ± 0.007  1,311 ± 25.10  43.31 ± 1.231  1.817 ± 0.009  13.82 ± 0.549  20.57 ± 0.856  0.844 ± 0.024  5.012 ± 0.135  35  1.579 ± 0.023  3,786 ± 46.83  6.253 ± 2.577  91.79 ± 1.535  68.57 ± 0.882  103.0 ± 0.562  75.39 ± 0.006  5.097 ± 0.026  36  3.796 ± 0.076  1,633 ± 32.66  6.986 ± 0.566  99.59 ± 2.044  64.18 ± 0.436  98.36 ± 1.053  59.14 ± 0.016  4.091 ± 0.003  37  1.092 ± 0.003  2,049 ± 10.92  23.10 ± 0.431  1.592 ± 0.005  12.94 ± 0.477  2.233 ± 0.015  0.260 ± 0.007  7.134 ± 0.059  38  0.983 ± 0.005  1,973 ± 27.67  23.11 ± 0.173  N.D.a  9.764 ± 0.460  2.137 ± 0.002  0.379 ± 0.007  4.280 ± 0.090  39  0.577 ± 0.018  547.3 ± 23.18  3.369 ± 0.078  N.D.a  8.834 ± 0.304  1.632 ± 0.005  0.260 ± 0.001  4.180 ± 0.058  40  0.343 ± 0.008  128.7 ± 9.531  3.595 ± 0.035  N.D.a  43.33 ± 0.531  14.96 ± 0.012  5.494 ± 0.059  4.521 ± 0.008  41  0.600 ± 0.009  3,872 ± 146.7  55.58 ± 0.473  N.D.a  15.11 ± 0.609  3.484 ± 0.075  1.068 ± 0.013  32.43 ± 0.662  42  1.328 ± 0.033  8,413 ± 125.3  22.99 ± 0.661  N.D.a  44.01 ± 0.844  22.65 ± 0.073  4.964 ± 0.065  39.81 ± 0.035  43  1.450 ± 0.037  9,473 ± 94.31  25.12 ± 0.775  N.D.a  50.67 ± 0.398  36.23 ± 0.015  8.063 ± 0.013  40.35 ± 0.035  44  4.840 ± 0.103  2,959 ± 36.23  84.89 ± 1.356  N.D.a  39.93 ± 0.448  21.87 ± 0.064  5.293 ± 0.058  46.86 ± 0.051  45  5.688 ± 0.112  1,244 ± 17.43  40.77 ± 1.532  N.D. a  16.71 ± 0.562  7.114 ± 0.058  0.653 ± 0.043  25.32 ± 0.044  46  6.687 ± 0.137  2,788 ± 68.26  32.95 ± 0.654  N.D.a  9.299 ± 0.282  1.661 ± 0.001  0.250 ± 0.002  18.49 ± 0.681  47  6.842 ± 0.118  2,415 ± 38.86  24.43 ± 0.229  N.D.a  40.50 ± 0.871  59.27 ± 1.662  45.81 ± 0.998  2.432 ± 0.006  48  17.86 ± 0.271  1,567 ± 14.52  4.972 ± 0.733  159.9 ± 11.53  32.73 ± 0.511  57.32 ± 0.056  37.04 ± 0.051  4.278 ± 0.023  49  18.91 ± 0.176  4,410 ± 17.44  11.94 ± 0.561  48.97 ± 1.442  55.94 ± 0.823  21.06 ± 0.061  5.299 ± 0.066  4.577 ± 0.002    RSD/%  128.5  77.5  72.6  164.7  62.9  121.7  182.6  95.6  Dosage form  Batch no.  Contents of each compound (μg/g material drugs)  HA  GIA  OLQG  GE  ALIII  ALI  ALII  GEA  Big honeyed-pill  1  1.452 ± 0.021  2,650 ± 43.32  11.30 ± 0.463  1,595 ± 54.68  42.46 ± 1.757  45.63 ± 1.223  60.20 ± 0.534  2.659 ± 0.053  2  22.99 ± 0.469  3,012 ± 90.64  26.27 ± 0.319  2,031 ± 53.48  172.0 ± 1.676  182.6 ± 3.168  158.0 ± 1.022  6.260 ± 0.142  3  20.77 ± 0.346  3,202 ± 137.4  24.64 ± 0.340  1,464 ± 50.18  141.9 ± 3.387  151.1 ± 1.446  126.1 ± 2.015  5.337 ± 0.204  4  11.87 ± 0.313  3,914 ± 78.75  15.01 ± 0.051  2,341 ± 109.5  100.8 ± 1.155  97.61 ± 1.023  114.4 ± 1.472  2.624 ± 0.046  5  21.61 ± 0.087  3,735 ± 93.67  26.73 ± 0.293  1,217 ± 51.34  129.0 ± 1.124  126.9 ± 0.593  113.5 ± 3.811  6.583 ± 0.068  6  30.64 ± 0.610  3,285 ± 63.06  33.21 ± 1.256  872.9 ± 2.160  94.88 ± 3.543  125.3 ± 1.032  139.0 ± 2.415  8.481 ± 0.248  7  28.42 ± 0.435  3,183 ± 54.76  4.228 ± 0.863  2,554 ± 46.76  81.52 ± 2.657  158.4 ± 2.907  131.6 ± 2.098  0.927 ± 0.097  8  13.29 ± 0.521  4,383 ± 66.87  4.845 ± 0.956  3,112 ± 64.38  89.50 ± 1.543  122.1 ± 2.065  109.0 ± 2.218  1.716 ± 0.087  9  9.252 ± 0.367  4,533 ± 57.43  5.375 ± 0.675  2,545 ± 58.62  89.85 ± 2.989  115.7 ± 1.648  92.76 ± 3.112  1.252 ± 0.057  10  1.178 ± 0.016  4,633 ± 34.81  38.83 ± 0.802  780.8 ± 1.079  106.2 ± 3.058  140.8 ± 3.498  96.39 ± 1.544  21.81 ± 0.884  11  0.021 ± 0.000  4,879 ± 48.67  33.17 ± 0.586  683.2 ± 20.76  222.1 ± 1.677  322.3 ± 2.767  200.1 ± 2.056  35.57 ± 1.076  12  0.021 ± 0.000  4,684 ± 87.43  19.76 ± 0.437  865.9 ± 16.44  245.8 ± 2.452  403.0 ± 2.234  247.1 ± 2.675  9.749 ± 0.078  13  1.150 ± 0.129  5,149 ± 76.44  28.21 ± 0.532  644.1 ± 23.98  99.78 ± 1.777  181.1 ± 0.986  79.67 ± 0.887  23.63 ± 0.254  14  0.695 ± 0.034  8,127 ± 48.61  19.56 ± 0.338  509.9 ± 21.61  248.2 ± 3.665  160.4 ± 1.065  202.0 ± 1.965  7.088 ± 0.334  15  1.680 ± 0.384  10814 ± 98.43  34.65 ± 0.674  600.5 ± 19.833  159.4 ± 2.787  133.1 ± 1.254  113.1 ± 2.137  23.47 ± 0.546  16  26.26 ± 0.718  3,980 ± 121.8  9.190 ± 0.224  1,934 ± 52.95  116.9 ± 0.699  181.6 ± 1.233  156.3 ± 0.929  32.89 ± 1.045  17  24.56 ± 0.323  5,442 ± 139.1  25.94 ± 0.748  2,276 ± 92.71  58.32 ± 1.480  96.40 ± 0.257  87.48 ± 1.436  43.10 ± 1.332  18  24.69 ± 0.378  2,682 ± 88.74  8.464 ± 0.150  2,031 ± 57.87  112.7 ± 2.215  186.4 ± 5.955  159.9 ± 3.114  32.39 ± 0.622  19  24.62 ± 0.462  2,966 ± 20.79  7.386 ± 0.184  2,031 ± 49.40  78.49 ± 1.840  135.0 ± 0.424  99.51 ± 2.247  29.82 ± 0.493  20  31.96 ± 0.532  2,800 ± 47.53  3.054 ± 0.205  3,867 ± 56.63  86.73 ± 2.096  221.0 ± 2.665  121.5 ± 1.877  23.93 ± 0.376  21  22.28 ± 0.787  3,736 ± 25.47  3.564 ± 0.397  3,771 ± 68.42  113.3 ± 2.136  222.1 ± 1.772  163.2 ± 2.870  20.47 ± 0.265  22  3.552 ± 0.122  4,402 ± 42.62  36.27 ± 0.447  875.2 ± 2.344  75.00 ± 1.335  82.91 ± 0.415  80.02 ± 1.082  9.714 ± 0.252  23  2.989 ± 0.033  4,397 ± 55.22  46.85 ± 1.037  687.3 ± 0.791  114.8 ± 4.771  86.91 ± 2.792  90.56 ± 11.312  13.97 ± 0.460  24  3.253 ± 0.052  4,430 ± 76.95  32.60 ± 1.153  781.2 ± 2.178  80.94 ± 1.116  92.23 ± 1.593  88.38 ± 1.604  12.65 ± 0.412  25  18.84 ± 0.452  3,613 ± 17.53  6.957 ± 0.654  2,892 ± 10.75  87.89 ± 2.343  116.0 ± 2.076  106.1 ± 2.122  3.381 ± 0.008  26  28.22 ± 0.445  3,175 ± 35.58  8.621 ± 0.788  2,716 ± 21.09  125.1 ± 2.766  178.1 ± 1.996  121.5 ± 1.942  2.513 ± 0.006  27  12.73 ± 0.782  2,448 ± 17.37  4.007 ± 0.965  280.7 ± 30.07  66.75 ± 2.087  94.44 ± 0.879  78.52 ± 1.209  3.072 ± 0.005    RSD/%  78.8  41.5  69.0  60.8  45.3  48.5  35.2  87.5  Concentrated pill  28  0.924 ± 0.002  1,987 ± 51.50  27.14 ± 0.749  N.D.a  16.45 ± 0.658  3.691 ± 0.074  1.300 ± 0.013  10.68 ± 0.401  29  1.378 ± 0.008  1,850 ± 44.50  33.03 ± 1.046  15.93 ± 0.026  15.28 ± 0.471  4.238 ± 0.179  0.705 ± 0.007  9.821 ± 0.208  30  1.342 ± 0.016  2,282 ± 44.85  54.09 ± 0.672  15.68 ± 0.026  17.49 ± 0.239  6.309 ± 0.086  0.596 ± 0.007  10.70 ± 0.464  31  4.264 ± 0.063  2,384 ± 95.77  46.24 ± 0.842  6.316 ± 0.025  12.05 ± 0.540  3.564 ± 0.178  0.257 ± 0.001  10.10 ± 0.238  32  2.219 ± 0.012  6,741 ± 67.33  21.35 ± 0.531  87.60 ± 1.035  41.86 ± 0.561  21.18 ± 0.093  3.951 ± 0.061  11.56 ± 0.051  33  3.330 ± 0.067  6,161 ± 52.88  18.12 ± 0.312  93.14 ± 1.237  41.22 ± 0.611  25.33 ± 0.123  3.089 ± 0.061  11.74 ± 0.086  34  1.035 ± 0.007  1,311 ± 25.10  43.31 ± 1.231  1.817 ± 0.009  13.82 ± 0.549  20.57 ± 0.856  0.844 ± 0.024  5.012 ± 0.135  35  1.579 ± 0.023  3,786 ± 46.83  6.253 ± 2.577  91.79 ± 1.535  68.57 ± 0.882  103.0 ± 0.562  75.39 ± 0.006  5.097 ± 0.026  36  3.796 ± 0.076  1,633 ± 32.66  6.986 ± 0.566  99.59 ± 2.044  64.18 ± 0.436  98.36 ± 1.053  59.14 ± 0.016  4.091 ± 0.003  37  1.092 ± 0.003  2,049 ± 10.92  23.10 ± 0.431  1.592 ± 0.005  12.94 ± 0.477  2.233 ± 0.015  0.260 ± 0.007  7.134 ± 0.059  38  0.983 ± 0.005  1,973 ± 27.67  23.11 ± 0.173  N.D.a  9.764 ± 0.460  2.137 ± 0.002  0.379 ± 0.007  4.280 ± 0.090  39  0.577 ± 0.018  547.3 ± 23.18  3.369 ± 0.078  N.D.a  8.834 ± 0.304  1.632 ± 0.005  0.260 ± 0.001  4.180 ± 0.058  40  0.343 ± 0.008  128.7 ± 9.531  3.595 ± 0.035  N.D.a  43.33 ± 0.531  14.96 ± 0.012  5.494 ± 0.059  4.521 ± 0.008  41  0.600 ± 0.009  3,872 ± 146.7  55.58 ± 0.473  N.D.a  15.11 ± 0.609  3.484 ± 0.075  1.068 ± 0.013  32.43 ± 0.662  42  1.328 ± 0.033  8,413 ± 125.3  22.99 ± 0.661  N.D.a  44.01 ± 0.844  22.65 ± 0.073  4.964 ± 0.065  39.81 ± 0.035  43  1.450 ± 0.037  9,473 ± 94.31  25.12 ± 0.775  N.D.a  50.67 ± 0.398  36.23 ± 0.015  8.063 ± 0.013  40.35 ± 0.035  44  4.840 ± 0.103  2,959 ± 36.23  84.89 ± 1.356  N.D.a  39.93 ± 0.448  21.87 ± 0.064  5.293 ± 0.058  46.86 ± 0.051  45  5.688 ± 0.112  1,244 ± 17.43  40.77 ± 1.532  N.D. a  16.71 ± 0.562  7.114 ± 0.058  0.653 ± 0.043  25.32 ± 0.044  46  6.687 ± 0.137  2,788 ± 68.26  32.95 ± 0.654  N.D.a  9.299 ± 0.282  1.661 ± 0.001  0.250 ± 0.002  18.49 ± 0.681  47  6.842 ± 0.118  2,415 ± 38.86  24.43 ± 0.229  N.D.a  40.50 ± 0.871  59.27 ± 1.662  45.81 ± 0.998  2.432 ± 0.006  48  17.86 ± 0.271  1,567 ± 14.52  4.972 ± 0.733  159.9 ± 11.53  32.73 ± 0.511  57.32 ± 0.056  37.04 ± 0.051  4.278 ± 0.023  49  18.91 ± 0.176  4,410 ± 17.44  11.94 ± 0.561  48.97 ± 1.442  55.94 ± 0.823  21.06 ± 0.061  5.299 ± 0.066  4.577 ± 0.002    RSD/%  128.5  77.5  72.6  164.7  62.9  121.7  182.6  95.6  aUndetectable. Table II. Contents of the 16 Compounds in 49 Batches of Samples Dosage form  Batch no.  Contents of each compound (μg/g relevant herbal material)  AD  LQR  BMA  BA  BHA  LQG  MA  AC  Big honeyed-pill  1  26.00 ± 0.437  2,095 ± 23.38  18.55 ± 0.821  3.478 ± 0.051  4.335 ± 0.080  62.12 ± 0.790  0.130 ± 0.005  0.057 ± 0.002  2  21.77 ± 0.574  1,659 ± 4.632  81.57 ± 0.228  8.989 ± 0.159  10.96 ± 0.121  207.8 ± 2.806  7.458 ± 0.005  1.601 ± 0.042  3  15.88 ± 0.168  1,581 ± 27.51  77.00 ± 1.379  7.934 ± 0.168  10.54 ± 0.180  188.3 ± 1.129  6.490 ± 0.253  1.515 ± 0.046  4  31.37 ± 0.194  2,067 ± 20.22  54.91 ± 0.591  6.704 ± 0.240  12.63 ± 0.468  78.47 ± 2.381  2.628 ± 0.176  0.529 ± 0.014  5  15.34 ± 0.225  1,744 ± 46.48  81.10 ± 1.676  8.863 ± 0.213  10.69 ± 0.283  201.9 ± 1.563  6.580 ± 0.090  1.562 ± 0.062  6  20.81 ± 0.321  1,577 ± 27.20  102.5 ± 1.018  9.725 ± 0.381  11.16 ± 0.403  259.2 ± 1.079  11.89 ± 0.173  2.403 ± 0.037  7  23.30 ± 0.314  2,996 ± 25.53  136.2 ± 1.042  14.49 ± 0.484  22.88 ± 0.523  56.66 ± 2.431  11.62 ± 0.183  2.253 ± 0.025  8  34.23 ± 0.641  2,998 ± 29.53  84.61 ± 1.325  12.07 ± 0.931  23.91 ± 0.515  56.18 ± 1.934  3.131 ± 0.293  0.724 ± 0.041  9  34.52 ± 0.376  3,105 ± 21.42  94.33 ± 1.016  14.46 ± 0.231  22.14 ± 0.356  70.53 ± 1.221  1.065 ± 0.923  0.474 ± 0.056  10  1.847 ± 0.072  1,796 ± 17.55  6.584 ± 0.316  0.889 ± 0.033  1.795 ± 0.063  108.0 ± 4.831  0.053 ± 0.407  0.034 ± 0.002  11  12.16 ± 0.134  2,676 ± 21.53  1.944 ± 0.413  0.089 ± 0.045  0.008 ± 0.001  95.19 ± 1.842  0.016 ± 0.002  0.009 ± 0.000  12  8.128 ± 0.274  2,686 ± 19.37  0.316 ± 0.041  0.062 ± 0.031  0.195 ± 0.034  69.59 ± 1.780  0.021 ± 0.008  0.012 ± 0.000  13  3.171 ± 0.384  2,807 ± 27.53  3.590 ± 0.319  0.490 ± 0.016  1.524 ± 0.051  97.58 ± 2.014  0.080 ± 0.007  0.038 ± 0.013  14  0.163 ± 0.064  3,921 ± 25.48  10.82 ± 1.452  1.343 ± 0.084  0.886 ± 0.093  150.5 ± 3.858  0.031 ± 0.008  0.022 ± 0.001  15  3.365 ± 0.092  5,659 ± 25.84  46.13 ± 0.924  7.494 ± 0.153  10.40 ± 0.073  147.7 ± 3.239  0.143 ± 0.019  0.172 ± 0.014  16  3.230 ± 0.124  1,557 ± 30.28  102.5 ± 1.099  11.38 ± 0.275  17.34 ± 0.373  68.32 ± 0.285  4.996 ± 0.002  1.109 ± 0.042  17  2.105 ± 0.072  1,875 ± 9.186  81.72 ± 1.818  9.724 ± 0.096  16.29 ± 0.340  172.9 ± 1.233  4.575 ± 0.068  1.226 ± 0.049  18  2.369 ± 0.072  1,503 ± 7.465  97.21 ± 0.560  10.73 ± 0.143  17.39 ± 0.348  68.39 ± 1.862  4.569 ± 0.097  1.201 ± 0.025  19  5.704 ± 0.143  1,776 ± 24.02  90.73 ± 1.636  9.930 ± 0.128  15.60 ± 0.228  43.65 ± 0.922  5.058 ± 0.084  1.310 ± 0.025  20  9.860 ± 0.235  2,317 ± 22.51  63.12 ± 1.782  12.81 ± 1.024  28.27 ± 0.142  55.21 ± 1.523  1.600 ± 0.083  0.779 ± 0.044  21  4.073 ± 0.327  2,155 ± 17.42  113.5 ± 3.515  12.48 ± 0.931  20.00 ± 0.201  52.07 ± 1.664  4.015 ± 0.081  1.264 ± 0.053  22  18.53 ± 0.415  1,392 ± 18.20  44.27 ± 1.833  0.311 ± 0.020  11.58 ± 0.272  96.95 ± 2.323  0.246 ± 0.160  0.171 ± 0.003  23  19.81 ± 0.327  1,509 ± 62.60  37.37 ± 4.046  0.409 ± 0.025  11.26 ± 0.403  141.5 ± 2.064  0.306 ± 0.008  0.173 ± 0.003  24  18.74 ± 0.579  1,432 ± 42.79  31.40 ± 0.455  0.186 ± 0.001  9.628 ± 0.052  111.0 ± 2.425  0.293 ± 0.005  0.177 ± 0.003  25  31.22 ± 0.632  2,046 ± 20.51  54.42 ± 2.013  4.112 ± 0.146  5.142 ± 0.109  82.90 ± 3.028  10.01 ± 0.098  1.844 ± 0.028  26  13.29 ± 0.234  1,722 ± 21.58  198.3 ± 8.421  20.96 ± 1.023  21.41 ± 0.113  106.9 ± 3.741  4.287 ± 0.122  1.389 ± 0.084  27  16.57 ± 0.134  1,706 ± 19.52  92.45 ± 1.951  11.29 ± 1.184  16.60 ± 0.142  64.92 ± 1.447  1.794 ± 0.053  0.516 ± 0.032    RSD/%  73.3  41.8  69.3  76.1  63.9  52.7  106.1  88.2  Concentrated pill  28  26.06 ± 1.056  1,386 ± 38.24  133.3 ± 4.227  30.83 ± 0.531  107.3 ± 4.381  84.83 ± 1.935  0.062 ± 0.239  0.055 ± 0.001  29  19.36 ± 0.483  546.0 ± 6.419  102.9 ± 3.848  22.36 ± 0.475  93.01 ± 0.948  54.72 ± 2.849  0.102 ± 0.001  0.060 ± 0.001  30  21.63 ± 0.394  1,170 ± 26.72  117.8 ± 1.050  29.02 ± 0.991  94.39 ± 1.083  91.24 ± 0.992  0.088 ± 0.002  0.073 ± 0.001  31  22.01 ± 0.676  563.8 ± 19.10  88.64 ± 0.255  21.26 ± 0.554  106.5 ± 4.126  74.07 ± 3.309  0.142 ± 0.003  0.123 ± 0.003  32  33.98 ± 0.374  4,084 ± 28.83  319.1 ± 2.941  68.63 ± 1.251  218.0 ± 3.416  118.2 ± 2.041  0.185 ± 0.003  0.111 ± 0.007  33  37.35 ± 0.452  2,602 ± 26.31  304.6 ± 2.055  64.68 ± 0.491  198.5 ± 2.955  98.19 ± 1.453  0.176 ± 0.004  0.192 ± 0.008  34  7.166 ± 0.341  575.9 ± 3.932  45.81 ± 0.166  10.55 ± 0.124  24.23 ± 0.941  91.37 ± 2.450  0.058 ± 0.004  0.055 ± 0.001  35  9.863 ± 0.293  1,200 ± 14.11  96.86 ± 3.031  16.28 ± 0.283  44.73 ± 1.651  23.20 ± 1.533  0.173 ± 0.010  0.064 ± 0.003  36  15.47 ± 0.473  2,010 ± 17.82  99.95 ± 2.951  23.12 ± 0.341  51.60 ± 2.042  51.19 ± 2.951  0.197 ± 0.011  0.174 ± 0.005  37  13.47 ± 0.417  528.0 ± 10.30  58.71 ± 1.809  12.31 ± 0.152  39.60 ± 0.652  35.12 ± 1.240  0.036 ± 0.001  0.042 ± 0.000  38  10.71 ± 0.154  438.6 ± 1.613  64.72 ± 1.155  12.22 ± 0.272  38.79 ± 0.551  34.86 ± 0.358  0.069 ± 0.001  0.045 ± 0.001  39  12.40 ± 0.229  172.9 ± 2.009  35.98 ± 0.576  8.178 ± 0.115  31.10 ± 0.397  0.958 ± 0.032  0.055 ± 0.001  0.025 ± 0.000  40  20.49 ± 0.245  401.9 ± 8.352  114.4 ± 4.014  24.95 ± 0.398  64.94 ± 1.432  24.68 ± 1.542  0.003 ± 0.000  0.011 ± 0.000  41  7.184 ± 0.197  903.5 ± 5.530  26.36 ± 0.468  4.497 ± 0.195  9.323 ± 0.360  65.92 ± 2.121  0.080 ± 0.000  0.028 ± 0.001  42  17.92 ± 0.831  3,069 ± 10.51  67.96 ± 1.031  11.16 ± 0.312  20.06 ± 0.966  77.29 ± 3.133  0.170 ± 0.004  0.075 ± 0.000  43  14.43 ± 0.624  3,206 ± 28.41  70.98 ± 0.941  11.98 ± 0.284  19.60 ± 0.455  85.53 ± 2.414  0.090 ± 0.002  0.046 ± 0.000  44  8.560 ± 0.532  2,137 ± 15.32  87.56 ± 1.252  11.84 ± 0.184  29.41 ± 1.053  282.9 ± 3.988  0.683 ± 0.010  0.231 ± 0.001  45  4.262 ± 0.135  1,023 ± 11.42  44.59 ± 0.836  5.705 ± 0.167  18.82 ± 0.533  139.1 ± 3.556  0.560 ± 0.009  0.170 ± 0.000  46  16.02 ± 0.142  698.7 ± 2.889  16.33 ± 0.125  8.276 ± 0.084  12.47 ± 0.250  34.48 ± 0.509  1.409 ± 0.002  0.531 ± 0.000  47  2.909 ± 0.068  1,000 ± 3.059  175.3 ± 0.537  32.29 ± 0.337  79.26 ± 0.240  68.99 ± 0.691  0.217 ± 0.049  0.140 ± 0.000  48  23.65 ± 0.523  2,174 ± 16.82  252.7 ± 1.934  58.30 ± 0.253  129.4 ± 0.675  17.17 ± 0.886  1.102 ± 0.088  0.680 ± 0.001  49  7.264 ± 0.834  1,966 ± 12.44  253.2 ± 0.894  43.46 ± 0.384  115.6 ± 1.143  110.0 ± 3.452  0.895 ± 0.014  0.675 ± 0.003    RSD/%  56.6  73.7  75.7  78.4  82.6  77.2  129.2  121.8  Dosage form  Batch no.  Contents of each compound (μg/g relevant herbal material)  AD  LQR  BMA  BA  BHA  LQG  MA  AC  Big honeyed-pill  1  26.00 ± 0.437  2,095 ± 23.38  18.55 ± 0.821  3.478 ± 0.051  4.335 ± 0.080  62.12 ± 0.790  0.130 ± 0.005  0.057 ± 0.002  2  21.77 ± 0.574  1,659 ± 4.632  81.57 ± 0.228  8.989 ± 0.159  10.96 ± 0.121  207.8 ± 2.806  7.458 ± 0.005  1.601 ± 0.042  3  15.88 ± 0.168  1,581 ± 27.51  77.00 ± 1.379  7.934 ± 0.168  10.54 ± 0.180  188.3 ± 1.129  6.490 ± 0.253  1.515 ± 0.046  4  31.37 ± 0.194  2,067 ± 20.22  54.91 ± 0.591  6.704 ± 0.240  12.63 ± 0.468  78.47 ± 2.381  2.628 ± 0.176  0.529 ± 0.014  5  15.34 ± 0.225  1,744 ± 46.48  81.10 ± 1.676  8.863 ± 0.213  10.69 ± 0.283  201.9 ± 1.563  6.580 ± 0.090  1.562 ± 0.062  6  20.81 ± 0.321  1,577 ± 27.20  102.5 ± 1.018  9.725 ± 0.381  11.16 ± 0.403  259.2 ± 1.079  11.89 ± 0.173  2.403 ± 0.037  7  23.30 ± 0.314  2,996 ± 25.53  136.2 ± 1.042  14.49 ± 0.484  22.88 ± 0.523  56.66 ± 2.431  11.62 ± 0.183  2.253 ± 0.025  8  34.23 ± 0.641  2,998 ± 29.53  84.61 ± 1.325  12.07 ± 0.931  23.91 ± 0.515  56.18 ± 1.934  3.131 ± 0.293  0.724 ± 0.041  9  34.52 ± 0.376  3,105 ± 21.42  94.33 ± 1.016  14.46 ± 0.231  22.14 ± 0.356  70.53 ± 1.221  1.065 ± 0.923  0.474 ± 0.056  10  1.847 ± 0.072  1,796 ± 17.55  6.584 ± 0.316  0.889 ± 0.033  1.795 ± 0.063  108.0 ± 4.831  0.053 ± 0.407  0.034 ± 0.002  11  12.16 ± 0.134  2,676 ± 21.53  1.944 ± 0.413  0.089 ± 0.045  0.008 ± 0.001  95.19 ± 1.842  0.016 ± 0.002  0.009 ± 0.000  12  8.128 ± 0.274  2,686 ± 19.37  0.316 ± 0.041  0.062 ± 0.031  0.195 ± 0.034  69.59 ± 1.780  0.021 ± 0.008  0.012 ± 0.000  13  3.171 ± 0.384  2,807 ± 27.53  3.590 ± 0.319  0.490 ± 0.016  1.524 ± 0.051  97.58 ± 2.014  0.080 ± 0.007  0.038 ± 0.013  14  0.163 ± 0.064  3,921 ± 25.48  10.82 ± 1.452  1.343 ± 0.084  0.886 ± 0.093  150.5 ± 3.858  0.031 ± 0.008  0.022 ± 0.001  15  3.365 ± 0.092  5,659 ± 25.84  46.13 ± 0.924  7.494 ± 0.153  10.40 ± 0.073  147.7 ± 3.239  0.143 ± 0.019  0.172 ± 0.014  16  3.230 ± 0.124  1,557 ± 30.28  102.5 ± 1.099  11.38 ± 0.275  17.34 ± 0.373  68.32 ± 0.285  4.996 ± 0.002  1.109 ± 0.042  17  2.105 ± 0.072  1,875 ± 9.186  81.72 ± 1.818  9.724 ± 0.096  16.29 ± 0.340  172.9 ± 1.233  4.575 ± 0.068  1.226 ± 0.049  18  2.369 ± 0.072  1,503 ± 7.465  97.21 ± 0.560  10.73 ± 0.143  17.39 ± 0.348  68.39 ± 1.862  4.569 ± 0.097  1.201 ± 0.025  19  5.704 ± 0.143  1,776 ± 24.02  90.73 ± 1.636  9.930 ± 0.128  15.60 ± 0.228  43.65 ± 0.922  5.058 ± 0.084  1.310 ± 0.025  20  9.860 ± 0.235  2,317 ± 22.51  63.12 ± 1.782  12.81 ± 1.024  28.27 ± 0.142  55.21 ± 1.523  1.600 ± 0.083  0.779 ± 0.044  21  4.073 ± 0.327  2,155 ± 17.42  113.5 ± 3.515  12.48 ± 0.931  20.00 ± 0.201  52.07 ± 1.664  4.015 ± 0.081  1.264 ± 0.053  22  18.53 ± 0.415  1,392 ± 18.20  44.27 ± 1.833  0.311 ± 0.020  11.58 ± 0.272  96.95 ± 2.323  0.246 ± 0.160  0.171 ± 0.003  23  19.81 ± 0.327  1,509 ± 62.60  37.37 ± 4.046  0.409 ± 0.025  11.26 ± 0.403  141.5 ± 2.064  0.306 ± 0.008  0.173 ± 0.003  24  18.74 ± 0.579  1,432 ± 42.79  31.40 ± 0.455  0.186 ± 0.001  9.628 ± 0.052  111.0 ± 2.425  0.293 ± 0.005  0.177 ± 0.003  25  31.22 ± 0.632  2,046 ± 20.51  54.42 ± 2.013  4.112 ± 0.146  5.142 ± 0.109  82.90 ± 3.028  10.01 ± 0.098  1.844 ± 0.028  26  13.29 ± 0.234  1,722 ± 21.58  198.3 ± 8.421  20.96 ± 1.023  21.41 ± 0.113  106.9 ± 3.741  4.287 ± 0.122  1.389 ± 0.084  27  16.57 ± 0.134  1,706 ± 19.52  92.45 ± 1.951  11.29 ± 1.184  16.60 ± 0.142  64.92 ± 1.447  1.794 ± 0.053  0.516 ± 0.032    RSD/%  73.3  41.8  69.3  76.1  63.9  52.7  106.1  88.2  Concentrated pill  28  26.06 ± 1.056  1,386 ± 38.24  133.3 ± 4.227  30.83 ± 0.531  107.3 ± 4.381  84.83 ± 1.935  0.062 ± 0.239  0.055 ± 0.001  29  19.36 ± 0.483  546.0 ± 6.419  102.9 ± 3.848  22.36 ± 0.475  93.01 ± 0.948  54.72 ± 2.849  0.102 ± 0.001  0.060 ± 0.001  30  21.63 ± 0.394  1,170 ± 26.72  117.8 ± 1.050  29.02 ± 0.991  94.39 ± 1.083  91.24 ± 0.992  0.088 ± 0.002  0.073 ± 0.001  31  22.01 ± 0.676  563.8 ± 19.10  88.64 ± 0.255  21.26 ± 0.554  106.5 ± 4.126  74.07 ± 3.309  0.142 ± 0.003  0.123 ± 0.003  32  33.98 ± 0.374  4,084 ± 28.83  319.1 ± 2.941  68.63 ± 1.251  218.0 ± 3.416  118.2 ± 2.041  0.185 ± 0.003  0.111 ± 0.007  33  37.35 ± 0.452  2,602 ± 26.31  304.6 ± 2.055  64.68 ± 0.491  198.5 ± 2.955  98.19 ± 1.453  0.176 ± 0.004  0.192 ± 0.008  34  7.166 ± 0.341  575.9 ± 3.932  45.81 ± 0.166  10.55 ± 0.124  24.23 ± 0.941  91.37 ± 2.450  0.058 ± 0.004  0.055 ± 0.001  35  9.863 ± 0.293  1,200 ± 14.11  96.86 ± 3.031  16.28 ± 0.283  44.73 ± 1.651  23.20 ± 1.533  0.173 ± 0.010  0.064 ± 0.003  36  15.47 ± 0.473  2,010 ± 17.82  99.95 ± 2.951  23.12 ± 0.341  51.60 ± 2.042  51.19 ± 2.951  0.197 ± 0.011  0.174 ± 0.005  37  13.47 ± 0.417  528.0 ± 10.30  58.71 ± 1.809  12.31 ± 0.152  39.60 ± 0.652  35.12 ± 1.240  0.036 ± 0.001  0.042 ± 0.000  38  10.71 ± 0.154  438.6 ± 1.613  64.72 ± 1.155  12.22 ± 0.272  38.79 ± 0.551  34.86 ± 0.358  0.069 ± 0.001  0.045 ± 0.001  39  12.40 ± 0.229  172.9 ± 2.009  35.98 ± 0.576  8.178 ± 0.115  31.10 ± 0.397  0.958 ± 0.032  0.055 ± 0.001  0.025 ± 0.000  40  20.49 ± 0.245  401.9 ± 8.352  114.4 ± 4.014  24.95 ± 0.398  64.94 ± 1.432  24.68 ± 1.542  0.003 ± 0.000  0.011 ± 0.000  41  7.184 ± 0.197  903.5 ± 5.530  26.36 ± 0.468  4.497 ± 0.195  9.323 ± 0.360  65.92 ± 2.121  0.080 ± 0.000  0.028 ± 0.001  42  17.92 ± 0.831  3,069 ± 10.51  67.96 ± 1.031  11.16 ± 0.312  20.06 ± 0.966  77.29 ± 3.133  0.170 ± 0.004  0.075 ± 0.000  43  14.43 ± 0.624  3,206 ± 28.41  70.98 ± 0.941  11.98 ± 0.284  19.60 ± 0.455  85.53 ± 2.414  0.090 ± 0.002  0.046 ± 0.000  44  8.560 ± 0.532  2,137 ± 15.32  87.56 ± 1.252  11.84 ± 0.184  29.41 ± 1.053  282.9 ± 3.988  0.683 ± 0.010  0.231 ± 0.001  45  4.262 ± 0.135  1,023 ± 11.42  44.59 ± 0.836  5.705 ± 0.167  18.82 ± 0.533  139.1 ± 3.556  0.560 ± 0.009  0.170 ± 0.000  46  16.02 ± 0.142  698.7 ± 2.889  16.33 ± 0.125  8.276 ± 0.084  12.47 ± 0.250  34.48 ± 0.509  1.409 ± 0.002  0.531 ± 0.000  47  2.909 ± 0.068  1,000 ± 3.059  175.3 ± 0.537  32.29 ± 0.337  79.26 ± 0.240  68.99 ± 0.691  0.217 ± 0.049  0.140 ± 0.000  48  23.65 ± 0.523  2,174 ± 16.82  252.7 ± 1.934  58.30 ± 0.253  129.4 ± 0.675  17.17 ± 0.886  1.102 ± 0.088  0.680 ± 0.001  49  7.264 ± 0.834  1,966 ± 12.44  253.2 ± 0.894  43.46 ± 0.384  115.6 ± 1.143  110.0 ± 3.452  0.895 ± 0.014  0.675 ± 0.003    RSD/%  56.6  73.7  75.7  78.4  82.6  77.2  129.2  121.8  Dosage form  Batch no.  Contents of each compound (μg/g material drugs)  HA  GIA  OLQG  GE  ALIII  ALI  ALII  GEA  Big honeyed-pill  1  1.452 ± 0.021  2,650 ± 43.32  11.30 ± 0.463  1,595 ± 54.68  42.46 ± 1.757  45.63 ± 1.223  60.20 ± 0.534  2.659 ± 0.053  2  22.99 ± 0.469  3,012 ± 90.64  26.27 ± 0.319  2,031 ± 53.48  172.0 ± 1.676  182.6 ± 3.168  158.0 ± 1.022  6.260 ± 0.142  3  20.77 ± 0.346  3,202 ± 137.4  24.64 ± 0.340  1,464 ± 50.18  141.9 ± 3.387  151.1 ± 1.446  126.1 ± 2.015  5.337 ± 0.204  4  11.87 ± 0.313  3,914 ± 78.75  15.01 ± 0.051  2,341 ± 109.5  100.8 ± 1.155  97.61 ± 1.023  114.4 ± 1.472  2.624 ± 0.046  5  21.61 ± 0.087  3,735 ± 93.67  26.73 ± 0.293  1,217 ± 51.34  129.0 ± 1.124  126.9 ± 0.593  113.5 ± 3.811  6.583 ± 0.068  6  30.64 ± 0.610  3,285 ± 63.06  33.21 ± 1.256  872.9 ± 2.160  94.88 ± 3.543  125.3 ± 1.032  139.0 ± 2.415  8.481 ± 0.248  7  28.42 ± 0.435  3,183 ± 54.76  4.228 ± 0.863  2,554 ± 46.76  81.52 ± 2.657  158.4 ± 2.907  131.6 ± 2.098  0.927 ± 0.097  8  13.29 ± 0.521  4,383 ± 66.87  4.845 ± 0.956  3,112 ± 64.38  89.50 ± 1.543  122.1 ± 2.065  109.0 ± 2.218  1.716 ± 0.087  9  9.252 ± 0.367  4,533 ± 57.43  5.375 ± 0.675  2,545 ± 58.62  89.85 ± 2.989  115.7 ± 1.648  92.76 ± 3.112  1.252 ± 0.057  10  1.178 ± 0.016  4,633 ± 34.81  38.83 ± 0.802  780.8 ± 1.079  106.2 ± 3.058  140.8 ± 3.498  96.39 ± 1.544  21.81 ± 0.884  11  0.021 ± 0.000  4,879 ± 48.67  33.17 ± 0.586  683.2 ± 20.76  222.1 ± 1.677  322.3 ± 2.767  200.1 ± 2.056  35.57 ± 1.076  12  0.021 ± 0.000  4,684 ± 87.43  19.76 ± 0.437  865.9 ± 16.44  245.8 ± 2.452  403.0 ± 2.234  247.1 ± 2.675  9.749 ± 0.078  13  1.150 ± 0.129  5,149 ± 76.44  28.21 ± 0.532  644.1 ± 23.98  99.78 ± 1.777  181.1 ± 0.986  79.67 ± 0.887  23.63 ± 0.254  14  0.695 ± 0.034  8,127 ± 48.61  19.56 ± 0.338  509.9 ± 21.61  248.2 ± 3.665  160.4 ± 1.065  202.0 ± 1.965  7.088 ± 0.334  15  1.680 ± 0.384  10814 ± 98.43  34.65 ± 0.674  600.5 ± 19.833  159.4 ± 2.787  133.1 ± 1.254  113.1 ± 2.137  23.47 ± 0.546  16  26.26 ± 0.718  3,980 ± 121.8  9.190 ± 0.224  1,934 ± 52.95  116.9 ± 0.699  181.6 ± 1.233  156.3 ± 0.929  32.89 ± 1.045  17  24.56 ± 0.323  5,442 ± 139.1  25.94 ± 0.748  2,276 ± 92.71  58.32 ± 1.480  96.40 ± 0.257  87.48 ± 1.436  43.10 ± 1.332  18  24.69 ± 0.378  2,682 ± 88.74  8.464 ± 0.150  2,031 ± 57.87  112.7 ± 2.215  186.4 ± 5.955  159.9 ± 3.114  32.39 ± 0.622  19  24.62 ± 0.462  2,966 ± 20.79  7.386 ± 0.184  2,031 ± 49.40  78.49 ± 1.840  135.0 ± 0.424  99.51 ± 2.247  29.82 ± 0.493  20  31.96 ± 0.532  2,800 ± 47.53  3.054 ± 0.205  3,867 ± 56.63  86.73 ± 2.096  221.0 ± 2.665  121.5 ± 1.877  23.93 ± 0.376  21  22.28 ± 0.787  3,736 ± 25.47  3.564 ± 0.397  3,771 ± 68.42  113.3 ± 2.136  222.1 ± 1.772  163.2 ± 2.870  20.47 ± 0.265  22  3.552 ± 0.122  4,402 ± 42.62  36.27 ± 0.447  875.2 ± 2.344  75.00 ± 1.335  82.91 ± 0.415  80.02 ± 1.082  9.714 ± 0.252  23  2.989 ± 0.033  4,397 ± 55.22  46.85 ± 1.037  687.3 ± 0.791  114.8 ± 4.771  86.91 ± 2.792  90.56 ± 11.312  13.97 ± 0.460  24  3.253 ± 0.052  4,430 ± 76.95  32.60 ± 1.153  781.2 ± 2.178  80.94 ± 1.116  92.23 ± 1.593  88.38 ± 1.604  12.65 ± 0.412  25  18.84 ± 0.452  3,613 ± 17.53  6.957 ± 0.654  2,892 ± 10.75  87.89 ± 2.343  116.0 ± 2.076  106.1 ± 2.122  3.381 ± 0.008  26  28.22 ± 0.445  3,175 ± 35.58  8.621 ± 0.788  2,716 ± 21.09  125.1 ± 2.766  178.1 ± 1.996  121.5 ± 1.942  2.513 ± 0.006  27  12.73 ± 0.782  2,448 ± 17.37  4.007 ± 0.965  280.7 ± 30.07  66.75 ± 2.087  94.44 ± 0.879  78.52 ± 1.209  3.072 ± 0.005    RSD/%  78.8  41.5  69.0  60.8  45.3  48.5  35.2  87.5  Concentrated pill  28  0.924 ± 0.002  1,987 ± 51.50  27.14 ± 0.749  N.D.a  16.45 ± 0.658  3.691 ± 0.074  1.300 ± 0.013  10.68 ± 0.401  29  1.378 ± 0.008  1,850 ± 44.50  33.03 ± 1.046  15.93 ± 0.026  15.28 ± 0.471  4.238 ± 0.179  0.705 ± 0.007  9.821 ± 0.208  30  1.342 ± 0.016  2,282 ± 44.85  54.09 ± 0.672  15.68 ± 0.026  17.49 ± 0.239  6.309 ± 0.086  0.596 ± 0.007  10.70 ± 0.464  31  4.264 ± 0.063  2,384 ± 95.77  46.24 ± 0.842  6.316 ± 0.025  12.05 ± 0.540  3.564 ± 0.178  0.257 ± 0.001  10.10 ± 0.238  32  2.219 ± 0.012  6,741 ± 67.33  21.35 ± 0.531  87.60 ± 1.035  41.86 ± 0.561  21.18 ± 0.093  3.951 ± 0.061  11.56 ± 0.051  33  3.330 ± 0.067  6,161 ± 52.88  18.12 ± 0.312  93.14 ± 1.237  41.22 ± 0.611  25.33 ± 0.123  3.089 ± 0.061  11.74 ± 0.086  34  1.035 ± 0.007  1,311 ± 25.10  43.31 ± 1.231  1.817 ± 0.009  13.82 ± 0.549  20.57 ± 0.856  0.844 ± 0.024  5.012 ± 0.135  35  1.579 ± 0.023  3,786 ± 46.83  6.253 ± 2.577  91.79 ± 1.535  68.57 ± 0.882  103.0 ± 0.562  75.39 ± 0.006  5.097 ± 0.026  36  3.796 ± 0.076  1,633 ± 32.66  6.986 ± 0.566  99.59 ± 2.044  64.18 ± 0.436  98.36 ± 1.053  59.14 ± 0.016  4.091 ± 0.003  37  1.092 ± 0.003  2,049 ± 10.92  23.10 ± 0.431  1.592 ± 0.005  12.94 ± 0.477  2.233 ± 0.015  0.260 ± 0.007  7.134 ± 0.059  38  0.983 ± 0.005  1,973 ± 27.67  23.11 ± 0.173  N.D.a  9.764 ± 0.460  2.137 ± 0.002  0.379 ± 0.007  4.280 ± 0.090  39  0.577 ± 0.018  547.3 ± 23.18  3.369 ± 0.078  N.D.a  8.834 ± 0.304  1.632 ± 0.005  0.260 ± 0.001  4.180 ± 0.058  40  0.343 ± 0.008  128.7 ± 9.531  3.595 ± 0.035  N.D.a  43.33 ± 0.531  14.96 ± 0.012  5.494 ± 0.059  4.521 ± 0.008  41  0.600 ± 0.009  3,872 ± 146.7  55.58 ± 0.473  N.D.a  15.11 ± 0.609  3.484 ± 0.075  1.068 ± 0.013  32.43 ± 0.662  42  1.328 ± 0.033  8,413 ± 125.3  22.99 ± 0.661  N.D.a  44.01 ± 0.844  22.65 ± 0.073  4.964 ± 0.065  39.81 ± 0.035  43  1.450 ± 0.037  9,473 ± 94.31  25.12 ± 0.775  N.D.a  50.67 ± 0.398  36.23 ± 0.015  8.063 ± 0.013  40.35 ± 0.035  44  4.840 ± 0.103  2,959 ± 36.23  84.89 ± 1.356  N.D.a  39.93 ± 0.448  21.87 ± 0.064  5.293 ± 0.058  46.86 ± 0.051  45  5.688 ± 0.112  1,244 ± 17.43  40.77 ± 1.532  N.D. a  16.71 ± 0.562  7.114 ± 0.058  0.653 ± 0.043  25.32 ± 0.044  46  6.687 ± 0.137  2,788 ± 68.26  32.95 ± 0.654  N.D.a  9.299 ± 0.282  1.661 ± 0.001  0.250 ± 0.002  18.49 ± 0.681  47  6.842 ± 0.118  2,415 ± 38.86  24.43 ± 0.229  N.D.a  40.50 ± 0.871  59.27 ± 1.662  45.81 ± 0.998  2.432 ± 0.006  48  17.86 ± 0.271  1,567 ± 14.52  4.972 ± 0.733  159.9 ± 11.53  32.73 ± 0.511  57.32 ± 0.056  37.04 ± 0.051  4.278 ± 0.023  49  18.91 ± 0.176  4,410 ± 17.44  11.94 ± 0.561  48.97 ± 1.442  55.94 ± 0.823  21.06 ± 0.061  5.299 ± 0.066  4.577 ± 0.002    RSD/%  128.5  77.5  72.6  164.7  62.9  121.7  182.6  95.6  Dosage form  Batch no.  Contents of each compound (μg/g material drugs)  HA  GIA  OLQG  GE  ALIII  ALI  ALII  GEA  Big honeyed-pill  1  1.452 ± 0.021  2,650 ± 43.32  11.30 ± 0.463  1,595 ± 54.68  42.46 ± 1.757  45.63 ± 1.223  60.20 ± 0.534  2.659 ± 0.053  2  22.99 ± 0.469  3,012 ± 90.64  26.27 ± 0.319  2,031 ± 53.48  172.0 ± 1.676  182.6 ± 3.168  158.0 ± 1.022  6.260 ± 0.142  3  20.77 ± 0.346  3,202 ± 137.4  24.64 ± 0.340  1,464 ± 50.18  141.9 ± 3.387  151.1 ± 1.446  126.1 ± 2.015  5.337 ± 0.204  4  11.87 ± 0.313  3,914 ± 78.75  15.01 ± 0.051  2,341 ± 109.5  100.8 ± 1.155  97.61 ± 1.023  114.4 ± 1.472  2.624 ± 0.046  5  21.61 ± 0.087  3,735 ± 93.67  26.73 ± 0.293  1,217 ± 51.34  129.0 ± 1.124  126.9 ± 0.593  113.5 ± 3.811  6.583 ± 0.068  6  30.64 ± 0.610  3,285 ± 63.06  33.21 ± 1.256  872.9 ± 2.160  94.88 ± 3.543  125.3 ± 1.032  139.0 ± 2.415  8.481 ± 0.248  7  28.42 ± 0.435  3,183 ± 54.76  4.228 ± 0.863  2,554 ± 46.76  81.52 ± 2.657  158.4 ± 2.907  131.6 ± 2.098  0.927 ± 0.097  8  13.29 ± 0.521  4,383 ± 66.87  4.845 ± 0.956  3,112 ± 64.38  89.50 ± 1.543  122.1 ± 2.065  109.0 ± 2.218  1.716 ± 0.087  9  9.252 ± 0.367  4,533 ± 57.43  5.375 ± 0.675  2,545 ± 58.62  89.85 ± 2.989  115.7 ± 1.648  92.76 ± 3.112  1.252 ± 0.057  10  1.178 ± 0.016  4,633 ± 34.81  38.83 ± 0.802  780.8 ± 1.079  106.2 ± 3.058  140.8 ± 3.498  96.39 ± 1.544  21.81 ± 0.884  11  0.021 ± 0.000  4,879 ± 48.67  33.17 ± 0.586  683.2 ± 20.76  222.1 ± 1.677  322.3 ± 2.767  200.1 ± 2.056  35.57 ± 1.076  12  0.021 ± 0.000  4,684 ± 87.43  19.76 ± 0.437  865.9 ± 16.44  245.8 ± 2.452  403.0 ± 2.234  247.1 ± 2.675  9.749 ± 0.078  13  1.150 ± 0.129  5,149 ± 76.44  28.21 ± 0.532  644.1 ± 23.98  99.78 ± 1.777  181.1 ± 0.986  79.67 ± 0.887  23.63 ± 0.254  14  0.695 ± 0.034  8,127 ± 48.61  19.56 ± 0.338  509.9 ± 21.61  248.2 ± 3.665  160.4 ± 1.065  202.0 ± 1.965  7.088 ± 0.334  15  1.680 ± 0.384  10814 ± 98.43  34.65 ± 0.674  600.5 ± 19.833  159.4 ± 2.787  133.1 ± 1.254  113.1 ± 2.137  23.47 ± 0.546  16  26.26 ± 0.718  3,980 ± 121.8  9.190 ± 0.224  1,934 ± 52.95  116.9 ± 0.699  181.6 ± 1.233  156.3 ± 0.929  32.89 ± 1.045  17  24.56 ± 0.323  5,442 ± 139.1  25.94 ± 0.748  2,276 ± 92.71  58.32 ± 1.480  96.40 ± 0.257  87.48 ± 1.436  43.10 ± 1.332  18  24.69 ± 0.378  2,682 ± 88.74  8.464 ± 0.150  2,031 ± 57.87  112.7 ± 2.215  186.4 ± 5.955  159.9 ± 3.114  32.39 ± 0.622  19  24.62 ± 0.462  2,966 ± 20.79  7.386 ± 0.184  2,031 ± 49.40  78.49 ± 1.840  135.0 ± 0.424  99.51 ± 2.247  29.82 ± 0.493  20  31.96 ± 0.532  2,800 ± 47.53  3.054 ± 0.205  3,867 ± 56.63  86.73 ± 2.096  221.0 ± 2.665  121.5 ± 1.877  23.93 ± 0.376  21  22.28 ± 0.787  3,736 ± 25.47  3.564 ± 0.397  3,771 ± 68.42  113.3 ± 2.136  222.1 ± 1.772  163.2 ± 2.870  20.47 ± 0.265  22  3.552 ± 0.122  4,402 ± 42.62  36.27 ± 0.447  875.2 ± 2.344  75.00 ± 1.335  82.91 ± 0.415  80.02 ± 1.082  9.714 ± 0.252  23  2.989 ± 0.033  4,397 ± 55.22  46.85 ± 1.037  687.3 ± 0.791  114.8 ± 4.771  86.91 ± 2.792  90.56 ± 11.312  13.97 ± 0.460  24  3.253 ± 0.052  4,430 ± 76.95  32.60 ± 1.153  781.2 ± 2.178  80.94 ± 1.116  92.23 ± 1.593  88.38 ± 1.604  12.65 ± 0.412  25  18.84 ± 0.452  3,613 ± 17.53  6.957 ± 0.654  2,892 ± 10.75  87.89 ± 2.343  116.0 ± 2.076  106.1 ± 2.122  3.381 ± 0.008  26  28.22 ± 0.445  3,175 ± 35.58  8.621 ± 0.788  2,716 ± 21.09  125.1 ± 2.766  178.1 ± 1.996  121.5 ± 1.942  2.513 ± 0.006  27  12.73 ± 0.782  2,448 ± 17.37  4.007 ± 0.965  280.7 ± 30.07  66.75 ± 2.087  94.44 ± 0.879  78.52 ± 1.209  3.072 ± 0.005    RSD/%  78.8  41.5  69.0  60.8  45.3  48.5  35.2  87.5  Concentrated pill  28  0.924 ± 0.002  1,987 ± 51.50  27.14 ± 0.749  N.D.a  16.45 ± 0.658  3.691 ± 0.074  1.300 ± 0.013  10.68 ± 0.401  29  1.378 ± 0.008  1,850 ± 44.50  33.03 ± 1.046  15.93 ± 0.026  15.28 ± 0.471  4.238 ± 0.179  0.705 ± 0.007  9.821 ± 0.208  30  1.342 ± 0.016  2,282 ± 44.85  54.09 ± 0.672  15.68 ± 0.026  17.49 ± 0.239  6.309 ± 0.086  0.596 ± 0.007  10.70 ± 0.464  31  4.264 ± 0.063  2,384 ± 95.77  46.24 ± 0.842  6.316 ± 0.025  12.05 ± 0.540  3.564 ± 0.178  0.257 ± 0.001  10.10 ± 0.238  32  2.219 ± 0.012  6,741 ± 67.33  21.35 ± 0.531  87.60 ± 1.035  41.86 ± 0.561  21.18 ± 0.093  3.951 ± 0.061  11.56 ± 0.051  33  3.330 ± 0.067  6,161 ± 52.88  18.12 ± 0.312  93.14 ± 1.237  41.22 ± 0.611  25.33 ± 0.123  3.089 ± 0.061  11.74 ± 0.086  34  1.035 ± 0.007  1,311 ± 25.10  43.31 ± 1.231  1.817 ± 0.009  13.82 ± 0.549  20.57 ± 0.856  0.844 ± 0.024  5.012 ± 0.135  35  1.579 ± 0.023  3,786 ± 46.83  6.253 ± 2.577  91.79 ± 1.535  68.57 ± 0.882  103.0 ± 0.562  75.39 ± 0.006  5.097 ± 0.026  36  3.796 ± 0.076  1,633 ± 32.66  6.986 ± 0.566  99.59 ± 2.044  64.18 ± 0.436  98.36 ± 1.053  59.14 ± 0.016  4.091 ± 0.003  37  1.092 ± 0.003  2,049 ± 10.92  23.10 ± 0.431  1.592 ± 0.005  12.94 ± 0.477  2.233 ± 0.015  0.260 ± 0.007  7.134 ± 0.059  38  0.983 ± 0.005  1,973 ± 27.67  23.11 ± 0.173  N.D.a  9.764 ± 0.460  2.137 ± 0.002  0.379 ± 0.007  4.280 ± 0.090  39  0.577 ± 0.018  547.3 ± 23.18  3.369 ± 0.078  N.D.a  8.834 ± 0.304  1.632 ± 0.005  0.260 ± 0.001  4.180 ± 0.058  40  0.343 ± 0.008  128.7 ± 9.531  3.595 ± 0.035  N.D.a  43.33 ± 0.531  14.96 ± 0.012  5.494 ± 0.059  4.521 ± 0.008  41  0.600 ± 0.009  3,872 ± 146.7  55.58 ± 0.473  N.D.a  15.11 ± 0.609  3.484 ± 0.075  1.068 ± 0.013  32.43 ± 0.662  42  1.328 ± 0.033  8,413 ± 125.3  22.99 ± 0.661  N.D.a  44.01 ± 0.844  22.65 ± 0.073  4.964 ± 0.065  39.81 ± 0.035  43  1.450 ± 0.037  9,473 ± 94.31  25.12 ± 0.775  N.D.a  50.67 ± 0.398  36.23 ± 0.015  8.063 ± 0.013  40.35 ± 0.035  44  4.840 ± 0.103  2,959 ± 36.23  84.89 ± 1.356  N.D.a  39.93 ± 0.448  21.87 ± 0.064  5.293 ± 0.058  46.86 ± 0.051  45  5.688 ± 0.112  1,244 ± 17.43  40.77 ± 1.532  N.D. a  16.71 ± 0.562  7.114 ± 0.058  0.653 ± 0.043  25.32 ± 0.044  46  6.687 ± 0.137  2,788 ± 68.26  32.95 ± 0.654  N.D.a  9.299 ± 0.282  1.661 ± 0.001  0.250 ± 0.002  18.49 ± 0.681  47  6.842 ± 0.118  2,415 ± 38.86  24.43 ± 0.229  N.D.a  40.50 ± 0.871  59.27 ± 1.662  45.81 ± 0.998  2.432 ± 0.006  48  17.86 ± 0.271  1,567 ± 14.52  4.972 ± 0.733  159.9 ± 11.53  32.73 ± 0.511  57.32 ± 0.056  37.04 ± 0.051  4.278 ± 0.023  49  18.91 ± 0.176  4,410 ± 17.44  11.94 ± 0.561  48.97 ± 1.442  55.94 ± 0.823  21.06 ± 0.061  5.299 ± 0.066  4.577 ± 0.002    RSD/%  128.5  77.5  72.6  164.7  62.9  121.7  182.6  95.6  aUndetectable. OPLS-DA of the samples To better evaluate the quality consistency of the samples in different dosage forms, the content data of the 16 constituents in all samples were treated by the software SIMCA (Version 13.0). OPLS-DA method is a supervised statistical model, which was carried out to optimize separation between different groups of samples. To maximize class discrimination and component variation, the OPLS model was performed, and then the contents of the 16 analytes (49 × 16) were imported to the SIMCA for OPLS-DA analysis. An OPLS-DA model with predictive ability Q2(Y) of 76.7% was obtained. As shown in Figure 3A, a separation tendency between BHP and CP samples was established. In order to determine the predictive ability of the classifier models, 31 batches of the samples were selected as the calibration set to construct OPLS-DA models and the remaining 18 batches of samples were the prediction set. The predicted samples with a Y value above 0.6 will be recognized as a member of the BHP. The total accuracy, classification accuracy of BHP and CP in the prediction set were 94, 100 and 89%, respectively. Thus, the use of OPLS-DA to perform FLP classification would make it possible to evaluate the quality consistency of CPM. Figure 3. View largeDownload slide Multivariate statistical analysis of the tested samples in the two BHP and CP dosage forms. (A) OPLS-DA score plot of BHP and CP; (B) column diagram of the contents of the most relevant components related to the differences between the BHP and CP. BHP represents the big honey pill, and CP represents concentrated pills. This figure is available in black and white in print and in color at JCS online. Figure 3. View largeDownload slide Multivariate statistical analysis of the tested samples in the two BHP and CP dosage forms. (A) OPLS-DA score plot of BHP and CP; (B) column diagram of the contents of the most relevant components related to the differences between the BHP and CP. BHP represents the big honey pill, and CP represents concentrated pills. This figure is available in black and white in print and in color at JCS online. The VIP plot, displaying the VIP values of all of the variables, was used to assist in finding the most relevant variables which contributed to distinguishing between BHP and CP group. ALIII, ALII, ALI and GE were the most relevant. As shown in Figure 3B, the contents of ALIII, ALII, ALI and GE in the BHP samples were significantly higher than those in the CP samples, which may indicate the disparity between the pharmaceutical process of the two dosage forms. ROC of the selected analytes The potential markers that significantly contributed to the discrimination of different dosage forms were identified by using ROC curve analysis in MetaboAnalyst 3.0 (Figure 4). Heat map was constructed for unsupervised clustering using contents of the selected analytes as variables. As shown in Figure 5, the heat map showed a clear clustering for each group, which showed direct variation of each differential marker. Figure 4. View largeDownload slide (A) Distinction accuracy evaluation using ROC curves of selected analytes between BHP and CP samples. (AUC > 0.85); (B) ROC curve curve-based model evaluation (AUC = 1). This figure is available in black and white in print and in color at JCS online. Figure 4. View largeDownload slide (A) Distinction accuracy evaluation using ROC curves of selected analytes between BHP and CP samples. (AUC > 0.85); (B) ROC curve curve-based model evaluation (AUC = 1). This figure is available in black and white in print and in color at JCS online. Figure 5. View largeDownload slide Heat map of selected analytes between BHP and CP groups from contents. The color of each section is proportional to the significance of change of metabolites (red, up-regulated; blue, downregulated). The abbreviation is the same as the compound summarized in Figure 1. This figure is available in black and white in print and in color at JCS online. Figure 5. View largeDownload slide Heat map of selected analytes between BHP and CP groups from contents. The color of each section is proportional to the significance of change of metabolites (red, up-regulated; blue, downregulated). The abbreviation is the same as the compound summarized in Figure 1. This figure is available in black and white in print and in color at JCS online. As a result, four markers with the areas under the ROC curves ranging from 0.85 to 1 (Figure 4A), were considered to show the greatest distinction accuracy. Subsequently, ROC curve-based model was established, and the AUC value of the established model is 1 (Figure 4B), which showed a good ability for discriminating CP samples from the BHP samples. Thus, those four markers (GE, ALI, ALII, ALIII) (Figure 4A) can be defined as potential markers associated with dosage form. Discussion Optimization of sample preparation The solvent (including water, 50% methanol, 70% methanol, methanol first methanol and then subsequently ammonia water), volume of solvent (25, 50, 75 mL) and extraction time (15, 30, 60, 90 min) were investigated for the two dosage forms. Note that compared with refluxing, ultrasonic extraction is simple, reproducible and effective. To assess the extraction efficiency, a normalization method was used based on the fact that the contents of the 16 components have wide ranges, e.g., the highest content was 1,000-fold greater than the lowest content; thus, it was impossible to evaluate the extraction efficiency when their absolute peak areas were integrated together because the effect of the low content constituents would be overlooked. Therefore, the normalization arithmetic method was applied to dismiss the content variance; the peak area of each analyte was divided by the square root of the sum of squares obtained with all runs of every single investigation factor (every single integral extraction experiment was independent of other runs); then, despite the wide content ranges, the cumulative data from each run would represent its extraction efficiency. Eventually, the 60-min procedure with 50 mL/70% methanol by ultrasonic extraction was chosen because it could attain a greater response of the selected analytes. Optimization of the RRLC condition The 16 analytes were selected to obtain the optimum elution conditions because they belong to different chemical classes and cover a wide range of polarities. Various mobile phases, such as 0.05, 0.1 and 0.2% aqueous formic acid and acetic acid, were tested in this study. The best peak shape and resolution were obtained from a mixture of acetonitrile containing 0.1% formic acid and 0.1% formic acid water solution. Indeed, the addition of 0.1% formic acid in both of the mobile phases not only dramatically enhanced the abundance of [M + H]+ ions but also eliminated the peak tailing of the target compounds in the negative ion mode. In addition, liquid chromatographic conditions, such as flow rate and column temperature, were investigated. Because there was no requirement for baseline chromatographic separation of target analytes using the MRM scan mode and time segments mode, the run time was minimized to 13 min. Optimization of MS conditions After the MS condition for the transitions was optimized, it was found that all analytes demonstrated a better response in positive ion mode except LQR. To simultaneously detect all analytes, a polarity switch is necessary. Unfortunately, the frequent switching of polarity in an identical segment would reduce the sensitivity and accuracy of the quantification. A method with two optimized time segments (polarity switch in different segments) was devised to obtain a better response value. As shown in Table III, a polarity switch in different segments will not reduce the accuracy and sensitivity of quantification using the optimized MS instrumentation (namely by maintaining the same polarity in one segment). Given that LQR could only obtain a good response in negative ion mode, it was necessary to switch the polarity in different segments to analyze the analytes. The procedure of optimizing the MRM transitions for all the target analytes was as follows: initially, the precursor and product ions of the constituents were determined by standard solutions in scan and product ion mode, respectively. According to this, the fragmentor energy and collision energy parameters were further optimized based on the relative richest abundance of precursor and product ions (Table IV). Table III. The LOD and LOQ Data of Different Method No.  Ionization mode  Time segment  Compound  LOD (ng/mL)  LOQ (ng/mL)  1  ESI+  0–5  AD  0.120  0.399  2  ESI−  0–5  LQR  0.798  2.661  3  ESI±  0–5  AD  1.149  3.831    ESI±  0–5  LQR  6.205  20.68  4  ESI+  0–3.5  AD  0.145  0.483    ESI−  3.5–5  LQR  0.784  2.615  No.  Ionization mode  Time segment  Compound  LOD (ng/mL)  LOQ (ng/mL)  1  ESI+  0–5  AD  0.120  0.399  2  ESI−  0–5  LQR  0.798  2.661  3  ESI±  0–5  AD  1.149  3.831    ESI±  0–5  LQR  6.205  20.68  4  ESI+  0–3.5  AD  0.145  0.483    ESI−  3.5–5  LQR  0.784  2.615  Table III. The LOD and LOQ Data of Different Method No.  Ionization mode  Time segment  Compound  LOD (ng/mL)  LOQ (ng/mL)  1  ESI+  0–5  AD  0.120  0.399  2  ESI−  0–5  LQR  0.798  2.661  3  ESI±  0–5  AD  1.149  3.831    ESI±  0–5  LQR  6.205  20.68  4  ESI+  0–3.5  AD  0.145  0.483    ESI−  3.5–5  LQR  0.784  2.615  No.  Ionization mode  Time segment  Compound  LOD (ng/mL)  LOQ (ng/mL)  1  ESI+  0–5  AD  0.120  0.399  2  ESI−  0–5  LQR  0.798  2.661  3  ESI±  0–5  AD  1.149  3.831    ESI±  0–5  LQR  6.205  20.68  4  ESI+  0–3.5  AD  0.145  0.483    ESI−  3.5–5  LQR  0.784  2.615  Table IV. The Information for MRM Parameters No.  Compounds (abbreviations)  Ionization mode  Precursor ion  Product ion  Fragmentor  CE (eV)  Retention time (min)  Time segments: 0–3.5 min  1  Adenosine (AD)  ESI+  268.1  136a  80  50  0.831      119  80  15    Time segments: 3.5–4.8 min  2  Liquiritin (LQR)  ESI-  417  255.2a  100  10  4.087      135  100  10    Time segments: 4.8–6.6 min  3  Benzoylmesaconine (BMA)  ESI+  590.3  105a  160  45  5.283      540.3  160  45    4  Benzoylaconine (BA)  ESI+  604.3  105a  95  50  5.541      554.3  95  50    5  Benzoylhypaconine (BHA)  ESI+  574.3  542.3a  115  40  5.665      105  115  40    6  Liquiritigenin (LQG)  ESI+  257.1  137a  105  20  5.994      147  105  20    7  Mesaconine (MA)  ESI+  632.2  572.3a  100  45  6.052      354.1  100  45    8  Aconine (AC)  ESI+  646.3  586.3a  105  45  6.287      368.2  105  45    9  Hypaconitine (HA)  ESI+  616.3  556.3a  115  40  6.292      338.2  115  40    10  Glycyrrhizic acid (GIA)  ESI+  823.5  453.4a  160  10  6.468      647.2  160  10    Time segments: 6.6–13 min  11  Isoliquiritigenin (OLQG)  ESI+  257.1  137a  105  20  6.735      147  105  20    12  6-Gingerol (GE)  ESI+  317.2  217.3a  145  16  7.542  13  Atractylenolide III (ALIII)  ESI+  249.2  231.1a  55  12  8.107      163.2  55  12    14  Atractylenolide I (ALI)  ESI+  233.2  151.1a  95  10  10.047      215.3  95  10    15  Atractylenolide II (ALII)  ESI+  231.1  185a  85  16  12.014      157.1  85  16    16  Glycyrrhetic acid (GEA)  ESI+  471.4  317.2a  45  20  12.372      263.1  45  20    No.  Compounds (abbreviations)  Ionization mode  Precursor ion  Product ion  Fragmentor  CE (eV)  Retention time (min)  Time segments: 0–3.5 min  1  Adenosine (AD)  ESI+  268.1  136a  80  50  0.831      119  80  15    Time segments: 3.5–4.8 min  2  Liquiritin (LQR)  ESI-  417  255.2a  100  10  4.087      135  100  10    Time segments: 4.8–6.6 min  3  Benzoylmesaconine (BMA)  ESI+  590.3  105a  160  45  5.283      540.3  160  45    4  Benzoylaconine (BA)  ESI+  604.3  105a  95  50  5.541      554.3  95  50    5  Benzoylhypaconine (BHA)  ESI+  574.3  542.3a  115  40  5.665      105  115  40    6  Liquiritigenin (LQG)  ESI+  257.1  137a  105  20  5.994      147  105  20    7  Mesaconine (MA)  ESI+  632.2  572.3a  100  45  6.052      354.1  100  45    8  Aconine (AC)  ESI+  646.3  586.3a  105  45  6.287      368.2  105  45    9  Hypaconitine (HA)  ESI+  616.3  556.3a  115  40  6.292      338.2  115  40    10  Glycyrrhizic acid (GIA)  ESI+  823.5  453.4a  160  10  6.468      647.2  160  10    Time segments: 6.6–13 min  11  Isoliquiritigenin (OLQG)  ESI+  257.1  137a  105  20  6.735      147  105  20    12  6-Gingerol (GE)  ESI+  317.2  217.3a  145  16  7.542  13  Atractylenolide III (ALIII)  ESI+  249.2  231.1a  55  12  8.107      163.2  55  12    14  Atractylenolide I (ALI)  ESI+  233.2  151.1a  95  10  10.047      215.3  95  10    15  Atractylenolide II (ALII)  ESI+  231.1  185a  85  16  12.014      157.1  85  16    16  Glycyrrhetic acid (GEA)  ESI+  471.4  317.2a  45  20  12.372      263.1  45  20    aQuantitative transition. Table IV. The Information for MRM Parameters No.  Compounds (abbreviations)  Ionization mode  Precursor ion  Product ion  Fragmentor  CE (eV)  Retention time (min)  Time segments: 0–3.5 min  1  Adenosine (AD)  ESI+  268.1  136a  80  50  0.831      119  80  15    Time segments: 3.5–4.8 min  2  Liquiritin (LQR)  ESI-  417  255.2a  100  10  4.087      135  100  10    Time segments: 4.8–6.6 min  3  Benzoylmesaconine (BMA)  ESI+  590.3  105a  160  45  5.283      540.3  160  45    4  Benzoylaconine (BA)  ESI+  604.3  105a  95  50  5.541      554.3  95  50    5  Benzoylhypaconine (BHA)  ESI+  574.3  542.3a  115  40  5.665      105  115  40    6  Liquiritigenin (LQG)  ESI+  257.1  137a  105  20  5.994      147  105  20    7  Mesaconine (MA)  ESI+  632.2  572.3a  100  45  6.052      354.1  100  45    8  Aconine (AC)  ESI+  646.3  586.3a  105  45  6.287      368.2  105  45    9  Hypaconitine (HA)  ESI+  616.3  556.3a  115  40  6.292      338.2  115  40    10  Glycyrrhizic acid (GIA)  ESI+  823.5  453.4a  160  10  6.468      647.2  160  10    Time segments: 6.6–13 min  11  Isoliquiritigenin (OLQG)  ESI+  257.1  137a  105  20  6.735      147  105  20    12  6-Gingerol (GE)  ESI+  317.2  217.3a  145  16  7.542  13  Atractylenolide III (ALIII)  ESI+  249.2  231.1a  55  12  8.107      163.2  55  12    14  Atractylenolide I (ALI)  ESI+  233.2  151.1a  95  10  10.047      215.3  95  10    15  Atractylenolide II (ALII)  ESI+  231.1  185a  85  16  12.014      157.1  85  16    16  Glycyrrhetic acid (GEA)  ESI+  471.4  317.2a  45  20  12.372      263.1  45  20    No.  Compounds (abbreviations)  Ionization mode  Precursor ion  Product ion  Fragmentor  CE (eV)  Retention time (min)  Time segments: 0–3.5 min  1  Adenosine (AD)  ESI+  268.1  136a  80  50  0.831      119  80  15    Time segments: 3.5–4.8 min  2  Liquiritin (LQR)  ESI-  417  255.2a  100  10  4.087      135  100  10    Time segments: 4.8–6.6 min  3  Benzoylmesaconine (BMA)  ESI+  590.3  105a  160  45  5.283      540.3  160  45    4  Benzoylaconine (BA)  ESI+  604.3  105a  95  50  5.541      554.3  95  50    5  Benzoylhypaconine (BHA)  ESI+  574.3  542.3a  115  40  5.665      105  115  40    6  Liquiritigenin (LQG)  ESI+  257.1  137a  105  20  5.994      147  105  20    7  Mesaconine (MA)  ESI+  632.2  572.3a  100  45  6.052      354.1  100  45    8  Aconine (AC)  ESI+  646.3  586.3a  105  45  6.287      368.2  105  45    9  Hypaconitine (HA)  ESI+  616.3  556.3a  115  40  6.292      338.2  115  40    10  Glycyrrhizic acid (GIA)  ESI+  823.5  453.4a  160  10  6.468      647.2  160  10    Time segments: 6.6–13 min  11  Isoliquiritigenin (OLQG)  ESI+  257.1  137a  105  20  6.735      147  105  20    12  6-Gingerol (GE)  ESI+  317.2  217.3a  145  16  7.542  13  Atractylenolide III (ALIII)  ESI+  249.2  231.1a  55  12  8.107      163.2  55  12    14  Atractylenolide I (ALI)  ESI+  233.2  151.1a  95  10  10.047      215.3  95  10    15  Atractylenolide II (ALII)  ESI+  231.1  185a  85  16  12.014      157.1  85  16    16  Glycyrrhetic acid (GEA)  ESI+  471.4  317.2a  45  20  12.372      263.1  45  20    aQuantitative transition. Quantitative analysis of samples So far, over 200 alkaloids have been isolated and identified from Fuzi (29, 30), and they were classified into four major groups, nonester alkaloids, MDAs, DDAs and lipo alkaloids. Among the alkaloids in Fuzi, DDAs and MDAs have been investigated to possess many effects, such as antinociceptive, antiarrhythmic and anti-epileptic properties due to a blockade of the voltage-dependent Na+ channel (31). As a toxic herbal medicine, the toxicity of Fuzi mainly derives from DDAs including AC, MA and HA, etc. The processing methods in traditional Chinese medicine are mandatory to reduce the toxicity of Fuzi by decomposing DDAs to the less toxic MDAs or even unesterified compounds, but in the meantime, their pharmacological activities may alter to weaken or even wear off (32). Therefore, an appropriate quality control method capable of ensuring the safety and efficacy is absolutely necessary. In Chinese pharmacopoeia 2015, it is mandatory that the total amount of BA, BMA and BHA in Fuzi should be over 0.01%, and the total contents of AC, MA and HA in Fuzi should be lower than 0.01%. In pharmaceutical process of BHP and CP samples, Fuzi were both added as crude herb powder. Table II revealed that contents of six alkaloids from Fuzi were obviously different among all the samples. A point diagram (Figure 6) was performed to distribute the qualified and non-qualified Fuzi in the FLP samples following the lecture (24) It is obvious that the total contents of AC, MA and HA in fourteen batches was over 0.01%, named non-qualified, which is insecurity as their toxicity. The total contents of BA, BMA and BHA in five batches was below 0.01%, named non-qualified, which results in a decrease in curative effect of FLP, and even no effect. So, it is highly recommended that the determination of those six alkaloids in the proprietary Chinese medicines containing Fuzi must be done as a routine measurement, so as to provide a safe application to patients in clinics, and good manufacture practices (33). The data reveal that this method could be successfully applied to CPMs, such as BHP, with the prerequisite that the herbal medicines in them are only powdered, whereas for CPMs in CP form, this method should be further improved after carefully taking the constituents into account, particularly the unstable constituents, which may thermally decompose during the processes of extraction, concentration and desiccation. Figure 6. View largeDownload slide Point diagram of the distribution of the qualified and non-qualified TCMs in the FLP samples. x-axis: BHP and CP represent big honey pills and concentrated pills, respectively; Fuzi represent Aconiti Lateralis Radix Praeparata, respectively. The analytes in the brackets are required by Chinese Pharmacopeia. MDA represents monoester-diterpenoid alkaloids (includes BA, BMA, BHA), and DDA represents diester-diterpenoid alkaloids (includes AC, MA, HA); the y-axis is the content of the analyte shown in percentage and is the quantitative standard for the Chinese Pharmacopeia. The direction of the arrow represents the content requirement by the Chinese Pharmacopeia. Figure 6. View largeDownload slide Point diagram of the distribution of the qualified and non-qualified TCMs in the FLP samples. x-axis: BHP and CP represent big honey pills and concentrated pills, respectively; Fuzi represent Aconiti Lateralis Radix Praeparata, respectively. The analytes in the brackets are required by Chinese Pharmacopeia. MDA represents monoester-diterpenoid alkaloids (includes BA, BMA, BHA), and DDA represents diester-diterpenoid alkaloids (includes AC, MA, HA); the y-axis is the content of the analyte shown in percentage and is the quantitative standard for the Chinese Pharmacopeia. The direction of the arrow represents the content requirement by the Chinese Pharmacopeia. OPLS-DA of the sample The results (Figure 3) suggest that OPLS-DA might be an effective method to evaluate the quality consistency of CPM. These observations also demonstrate that the content variations of the 16 components between the two dosage forms were larger than those between samples in the same dosage form. To further compare the two dosage forms on manufacturing procedures: Dangshen, Baizhu, Gancao and Ganjiang in CP are extracted by 70% alcohol or water, which is in contrast to all herbal medicines in the BHP form are only powdered. Furthermore, it should be noted that the most relevant four variables, i.e., ALIII, ALII, ALI and GE, are heat- or sunlight-unstable compounds (34, 35); the instability of these components implies that their contents decrease significantly during CP sample production. The results suggest that more attention should be paid to those unstable constituents in the quality control of CPMs. ROC of the selected analytes ROC analysis is a useful tool for evaluating the accuracy of a statistical model (eg, logistic regression, linear discriminate analysis). The sensitivity and specificity trade-offs were calculated for each selected marker using the area under the ROC curve (AUC), which is a summary measure that essentially averages distinction accuracy. The clear clustering in heat map for each group was in agreement with the OPLS-DA results. The 16 analytes selected in this paper were not merely selected based on suitable therapeutic, bioactive, characteristic, main, synergistic, correlative, toxic and analytical properties. Through this study, the results suggest that the problem in the quality control of multiple components of FLPs is urgent and important. Furthermore, the content variation of major active components between two dosage forms of FLP is an issue of great concern. The primary reason for the variations between the two dosage forms was the fact that the unstable components decreased significantly during the CP manufacturing process, and the reason for variations among different companies may be derived from the quality of herbal material with different properties, such as origins, sources and processing methods. Additionally, the differences in the manufacturing procedures and scales among the companies may be other possible reasons. Because only the chemical variation between two dosage forms is discussed, whether the chemical variation results in a variation in the therapeutic effect should be questioned and further explored. Conclusion A convenient and reliable approach using RRLC–MS was developed to quantitatively analyze the 16 components in FLP. The establishment of pattern recognition using OPLS-DA method and heat map demonstrated content variations between the two dosage forms. Through this approach, the correct classification and the differentiation of samples were obtained; moreover, by VIP plots and ROC curves, markers with large variations that were related to different dosage forms or quality variation of herbal materials could be easily found. The quality of the crude material herb could be qualified and evaluated as well. 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Published: Apr 4, 2018

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