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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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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