TY - JOUR
AU1 - Huang,, Dan
AU2 - Zhao,, Xinqing
AU3 - Liu,, Xiuxiu
AU4 - Chao,, Ruobing
AB - Abstract A convenient and accurate high-performance liquid chromatography coupled with evaporative light scattering detection (HPLC–ELSD) method using solid phase extraction (SPE) was established for quantification of five aminoalcohol-diterpenoid alkaloids (ADAs), including mesaconine, aconine, hypaconine, fuziline and neoline, in the lateral roots of Aconitum carmichaeli (Fuzi) for the first time. The Fuzi extractive was purified using strong cation-exchange SPE. The chromatographic separation was performed on a Gemini C18 column (150 × 4.60 mm, 5 μm) with the mobile phase of methanol–water–diethylamine (48:52:0.01, v/v/v), adjusted to pH 10.2 with acetic acid. The detector was Alltech ELSD 2000ES (drift tube temperature: 90°C; gas flow-rate: 2.3 L/min). Five ADAs in Fuzi were well separated. The calibration curves showed good linearity (r > 0.9990) in the range of 0.0125–0.3750 mg/mL for each alkaloid. The recoveries were in the range of 95.1–105.7%, with relative standard deviations < 5.0%. This method is accurate, specific and repeatable for the determination of five ADAs in different Fuzi samples, which can be applied to the quality control of Fuzi. Introduction The lateral root of Aconitum carmichaeli (Fuzi) is widely used as an important ingredient in traditional Chinese medicines, such as Sini Tang, Fuzi Lizhong Wan and Fuzi Tang. Fuzi, originally growing in Sichuan province of China, is also cultivated for clinical use in other Eastern Asian countries, such as Japan and Korea. Multiple biological activities were reported in literatures (1, 2) including cardiotonic effect, anti-arrhythmic activity, anti-inflammatory, cartilage protection, abirritation, sedation and hypothermia. Fuzi can be classified as black slices, white slices and salted aconite daughter roots according to the different processing methods (3). Diterpenoid alkaloids (DAs), as the main components in Fuzi, can be divided into diester-, monoester- and aminoalcohol- groups (DDAs, MDAs and ADAs, respectively) in terms of the esterification at C8 and C14. In the past few decades, researchers have intensively focused on the chemical and pharmacological properties of DDAs and MDAs, which have been the criteria to assess the quality of Fuzi in compendial methods (3). Meanwhile, various analytical methods such as HPLC-UV, CE, LC–MS, IL-ATPS-HPLC have been developed for determination of DDAs and MDAs (4–12). However, researchers previously paid little attention to the aminoalcohol-diterpenoid alkaloids (ADAs) mainly because (i) their pharmacological activities remained unclear; and (ii) no UV absorption of ADAs led to a little difficult for detection. In the study of bioactive components, Ye et al. reported the presence of DDAs, MDAs and ADAs in rat blood after intravenous administration of a mixture of DDAs (13), which manifested that the ADAs were likely the active ingredients. In our previous study, ADAs such as mesaconine, hypaconine and beiwutinine showed strong cardiotonic effect, which demonstrated that the cardioactive substances in Fuzi were ADAs (14, 15). Moreover, Liu et al. reported that hypaconine was the main anti-acute pancreatitis component (16); Suzuki et al. reported that neoline was promising agent to alleviate oxaliplatin-induced neuropathic pain (17). Above studies indicate that ADAs are important effective components in Fuzi. Regarding analytical methods, ADAs currently can be detected by mass spectrometry (MS) detector. The reported literatures mainly focused on the structural characterization of DAs (including ADAs) in Fuzi by HPLC or UPLC coupled with various tandem MS detectors (11, 18–23). Our group used electrospray ionization time-of-flight MS to study the fragmentation patterns of ADAs (24). In the aspect of quantitative determination, only one literature in our previous work developed an LC–MS-MS method for determination of 13 ADAs in Fuzi (25). However, MS detector is difficult to be widely applied to routine quality control at present, and it is necessary to develop a more convenient, low-cost and accurate method for the quantitative determination of ADAs, thereby providing a more feasible solution for the quality control of Fuzi. Evaporative light scattering detection (ELSD) coupled with HPLC has been extensively used to detect compounds without UV absorption for routine drug quality control (26). In the previous established LC–MS-MS method (25), MS detection is specific and selective, which can be used to identify and quantify analogous compounds with similar retention times. However, ELSD is a non-selective detector, which is quite different from MS detector, and which requires more strict demands on chromatographic separation. ADAs are exactly a kind of structural analogs, so finding a proper chromatographic condition is very critical to achieving complete separation. In our study, an HPLC–ELSD method was established to determine five main ADAs in Fuzi. The separation conditions for the five alkaloids were investigated. A strong cation-exchange solid phase extraction (SPE) was still used in the sample preparation for its obvious purifying effect showed in our previous work. The five ADAs were well separated in the optimized chromatographic condition. The whole analytical method was validated according to ICH guideline and the results manifested that it was accurate, specific and repeatable. This method was successfully applied to determine five ADAs in Fuzi from different origins and with different processing methods. Experimental Chemicals and reagents Standards of mesaconine, aconine and hypaconine, were provided by the laboratory of Prof. Fengpeng Wang, West China School of Pharmacy, Sichuan University. Their structures were elucidated based on high-resolution mass spectrometry, 1H-NMR and 13C-NMR spectroscopic methods. Fuziline and neoline were purchased from Chengdu Must Bio-technology (Chengdu, China). The purities of all standards were >98%, and the chemical structures were shown in Figure 1. Figure 1. Open in new tabDownload slide Chemical structures of five ADAs. Figure 1. Open in new tabDownload slide Chemical structures of five ADAs. Methanol and acetic acid of HPLC grade were from Sigma-Aldrich (St. Louis, USA) and Kelong Chemicals (Chengdu, China), respectively. The water for HPLC analysis was Cestbon purified water (Chengdu, China). All other reagents were of analytical grade. The SPE partridge (ProElut PXC 150 mg) was obtained from Dikma Technologies (Beijing, China) Plant samples Eight batches of Fuzi from different places of origin and with different processing methods, including white slices (place of origin from Jiangyou, Hanzhong, Anxian and Yunnan), black slices (from Jiangyou and Anxian) and salted aconite daughter roots (from Jiangyou and Yunnan), were purchased from Hehuachi Chinese herbal medicine market (Chengdu, China). One batch of black slices (places of origin unknown) was purchased from Yumintang drugstore. All the above nine batches of Fuzi were processed from the lateral roots of A. carmichaeli, and identified by Dr Shu Wang, West China School of Pharmacy, Sichuan University. Two batches of crude Fuzi from Jiangyou and Xichang, which were not processed, were provided and identified by Prof. Fengpeng Wang. Instrumentation and chromatographic conditions The HPLC system was a SHIMADZU LC-10ATvp series apparatus (Japan), coupled with an ELSD detector (ELSD 2000ES, Alltech, USA). The HPLC separation was performed on a Gemini C18 column (150 × 4.60 mm, 5 μm, Phenomenex, USA) with the mobile phase of methanol–water–diethylamine (48:52:0.01, v/v/v), adjusted to pH 10.2 with acetic acid. The flow rate was 1.0 mL/min, and the column-temperature was set at 30°C. The parameters for ELSD were set as follows: gas flow-rate, 2.3 L/min; drift tube temperature, 90°C; gain, 1; impactor, OFF. Preparation of the mixed standard solution Five ADAs were accurately weighed and dissolved in methanol–water (1:1, v/v) to prepare the 0.5 mg/mL mixed stock solution. The mixed standard solution was prepared by diluting the mixed stock solution with methanol–water (1:1, v/v) to 0.05 mg/mL. Sample preparation Five gram of Fuzi powder was accurately weighed and soaked with 50 mL of ethanol–water–aqueous ammonia (50:46:4, v/v/v) for 2 h, and sonicated for 30 min followed by filtration. The extracts were evaporated under reduced pressure at 50°C. The residue was dissolved with 0.1 mol/L HCl, then filtrated with filter paper, and rinsed with 0.1 mol/L HCl to get aqueous acidic solution of Fuzi. The SPE was operated as previously reported (25) with some modifications. Briefly, the Fuzi acidic solution was loaded on the pretreated SPE column, and 30 mL of water and 20 mL of methanol–water (1:1, v/v) were then used to wash the SPE column sequentially. The alkaloids was eluted by 20 mL of aqueous ammonia–methanol (8:92, v/v) and collected, and the eluant was evaporated under reduced pressure at 50°C to dryness. About 10 mL methanol–water (1:1, v/v) was precisely added into the residue to dissolve as the sample solution. Method validation Several parameters, i.e. specificity, linearity, limit of quantitation (LOQ), accuracy, injection precision, repeatability and stability of sample solution, were validated according to ICH guideline to ensure the reliability of the established method. The specificity was assessed by investigating the interference between the solvent, the five determined alkaloids and the Fuzi sample, which was manifested in the chromatograms of the blank solvent, mixed standard solution and sample solution. The linearity of five ADAs was investigated by constructing the calibration curves. The mixed working solutions of five concentration levels from 0.0125 to 0.3750 mg/mL for five alkaloids were prepared by diluting the mixed stock solution step by step with methanol–water (1:1, v/v). The calibration curves were constructed by plotting the logarithmic value of the peak area versus logarithmic value of the concentration. Linear regression analysis was calculated by least-squares regression method. To assess LOQ, the mixed standard solution was gradually diluted to an appropriate concentration with methanol–water (1:1, v/v). The LOQs were determined based on the S/N of ~10. Injection precision was evaluated by analyzing 15 μL of mixed standard solution for five replicates consecutively. Variations were expressed by relative standard deviations (RSDs) of peak areas. Six independent assays of black slices powder were performed at the same time to investigate the method repeatability. About 20 μL of each test sample solution was measured for HPLC analysis, and the peak areas were obtained to calculate the contents and the corresponding RSDs by accompanying calibration curve. The stability of sample solution under room temperature was examined by analyzing the same sample solution at 0, 2, 4, 6 and 8 h. The content of each alkaloid was calculated by accompanying calibration curve, and variations were expressed by RSDs. The accuracy was evaluated by the standard-added recovery experiment. White slices powder, in which the contents of five alkaloids were known, was spiked with the mixed standard solution at high, intermediate and low concentration levels; each level was performed in triplicate and prepared using the established method. The recovery was calculated by measured value compared with the nominal values. Results and discussions Optimization of the extraction The polarity and acid-base property of extraction solvent may affect the permeability to the plant cell wall and the dissolution of the determined alkaloids. Hence, different extraction solvents, i.e. different concentrations of ethanol aqueous solutions added with hydrochloric acid or aqueous ammonia, were investigated. The extraction effects were shown in Table S (Supplementary Material). Differences in the extraction efficiency of five alkaloids were observed when using 30%, 50% and 80% ethanol solutions, and 50% ethanol solution was chosen as the extraction solvent based on the total extraction amount. The results also showed that the extraction amount of each alkaloid in alkaline solvent was higher than in acidic solvent (except fuziline) at the same ethanol concentration level. Thus, the basic condition was more suitable for the extraction. Finally, the solvent of ethanol–water–aqueous ammonia (50:46:4, v/v/v) was selected for extraction. Methanol was once attempted as solvent of sample solution. However, the chromatographic peak shapes obviously changed and broadened, and the separation efficiencies were unsatisfied, which was likely due to the large polarity differences between the mobile phase and the sample solvent. Therefore, methanol–water (1:1, v/v) was used as the solvent of sample solution. Optimization of HPLC conditions The optimization of mobile phase was carried out in the pilot test. Series of acidic or alkaline mobile phase were examined. The mixture of methanol–water–trifluoroacetic acid (TFA) was initially investigated. In this condition, TFA was used as an ion pair reagent to form complexes with alkaloids. Our results showed that the five ADAs in mixed standard solution were in good separation but could not be separated with other components in the sample solution when the mobile phase was methanol–water–TFA (20:80:0.1, v/v/v). Under basic conditions, the retention of ADAs was increased by the inhibition of ionization of alkaloids, while the retention of other components in the sample solution decreased thus resulting in less sample disturbance. Therefore, the basic mobile phase is more suitable for the analysis of alkaloids. Different volatile alkaline modifiers including aqueous ammonia, diethylamine and triethylamine were examined. It was found that peaks broadened and alkaloids could not be well separated with other components in sample solution when aqueous ammonia or triethylamine were added, only the diethylamine benefited the separation. The pH values of mobile phase from 9.8 to 10.8 were investigated to study the influence of pH on separation. The results showed that pH value had great influence on the retention of hypaconine; a good separation of five alkaloids was obtained at the pH of 10.2, which meant an alkali-resistant column was necessarily required. Gemini C18 column and other alkali-resistant columns were compared, and separation efficiency of the five alkaloids on Gemini C18 column was obviously better than that on other columns. Finally, methanol–water–diethylamine (48:52:0.01, v/v/v, adjusted to pH 10.2 with acetic acid) and Gemini C18 column were found to give the best separation of all alkaloids and other components. In this established chromatographic condition, 11 ADAs, including beiwutinine, mesaconine, 8-methoxymesaconine, aconine, hypaconine, 8-methoxyhypaconine, isotalatizidine, fuziline, neoline and karakoline mentioned in literature (25) and 8-methoxyaconine, could be well separated (Figure S1 in Supplementary Material). The other three alkaloids, i.e. talatizamine and chasmanine could not be eluted under this condition due to their strong retention; 3-deoxyaconine was eluted after karakoline but did not go on the test due to the lack of standards. Finally, only five alkaloids were selected to conduct method validation and determination based on the contents in Fuzi, biological activities and the availability of standards. Optimization of ELSD parameters Drift tube temperature and nebulizing gas flow-rate are important parameters for ELSD. These parameters were optimized by evaluating the ratio of S/N. The impacts of evaporator tube temperature (85–100°C) and gas flow-rate (2.0–2.5 L/min) were further investigated and optimized. Accordingly, the optimized parameters of ELSD were 90°C for drift tube temperature and 2.3 L/min for gas flow-rate. Validation of the developed method The chromatograms of mixed standard solution and sample solution were shown in Figure 2, and the chromatogram of blank solvent was in Figure S2 (Supplementary Material). It is indicated that five ADAs were in good separation without interference of blank solvent and other components in Fuzi under this chromatographic condition. Figure 2. Open in new tabDownload slide Chromatograms of mixed standard solution (A) and sample solution (B) (Jiangyou salted aconite daughter root). 1, mesaconine (3.10 min); 2, aconine (4.36 min); 3, hypaconine (6.89 min); 4, fuziline (11.42 min); 5, neoline (16.49 min). Figure 2. Open in new tabDownload slide Chromatograms of mixed standard solution (A) and sample solution (B) (Jiangyou salted aconite daughter root). 1, mesaconine (3.10 min); 2, aconine (4.36 min); 3, hypaconine (6.89 min); 4, fuziline (11.42 min); 5, neoline (16.49 min). The results of the linearity, LOQ, injection precision, repeatability and stability were shown in Table I. Satisfactory regression equations and correlation coefficients were obtained for these five alkaloids. The values of LOQ indicated that the sensitivity of the established method could quantitatively determinate ~0.01–0.02 mg for each alkaloid per gram of Fuzi powder. The RSDs of injection precision showed that the variations of sample injecting and detection of ELSD remained low. Meanwhile, this method exhibited good repeatability. The sample solution could remain stable within 8 h. The established method had a recovery of 95–105% for the five alkaloids (Table II), displaying a satisfactory accuracy. Table I. The linearity, LOQ, Repeatability, Injection Precision and Stability of the Method Alkaloids . Linear range (mg/mL) . Linear equation . Correlation coefficient . LOQ (μg) . Repeatability . Injection precision (RSD/%) . Stability (RSD/%) . Content (mg/g) . RSD/% . Mesaconine 0.01265–0.3795 y = 1.2793x + 7.5883 0.9995 0.127 0.1700 1.4 1.4 3.4 Aconine 0.01270–0.3810 y = 1.2536x + 7.4015 0.9995 0.127 0.0509 4.2 1.4 4.3 Hypaconine 0.01260–0.3780 y = 1.1546x + 7.2991 0.9999 0.126 0.0259 4.1 0.9 3.7 Fuziline 0.01285–0.3855 y = 1.2243x + 7.3722 0.9998 0.257 0.0754 2.0 1.2 2.1 Neoline 0.01185–0.3550 y = 1.2592x + 7.3424 0.9992 0.237 0.1250 4.1 1.5 4.4 Alkaloids . Linear range (mg/mL) . Linear equation . Correlation coefficient . LOQ (μg) . Repeatability . Injection precision (RSD/%) . Stability (RSD/%) . Content (mg/g) . RSD/% . Mesaconine 0.01265–0.3795 y = 1.2793x + 7.5883 0.9995 0.127 0.1700 1.4 1.4 3.4 Aconine 0.01270–0.3810 y = 1.2536x + 7.4015 0.9995 0.127 0.0509 4.2 1.4 4.3 Hypaconine 0.01260–0.3780 y = 1.1546x + 7.2991 0.9999 0.126 0.0259 4.1 0.9 3.7 Fuziline 0.01285–0.3855 y = 1.2243x + 7.3722 0.9998 0.257 0.0754 2.0 1.2 2.1 Neoline 0.01185–0.3550 y = 1.2592x + 7.3424 0.9992 0.237 0.1250 4.1 1.5 4.4 Table I. The linearity, LOQ, Repeatability, Injection Precision and Stability of the Method Alkaloids . Linear range (mg/mL) . Linear equation . Correlation coefficient . LOQ (μg) . Repeatability . Injection precision (RSD/%) . Stability (RSD/%) . Content (mg/g) . RSD/% . Mesaconine 0.01265–0.3795 y = 1.2793x + 7.5883 0.9995 0.127 0.1700 1.4 1.4 3.4 Aconine 0.01270–0.3810 y = 1.2536x + 7.4015 0.9995 0.127 0.0509 4.2 1.4 4.3 Hypaconine 0.01260–0.3780 y = 1.1546x + 7.2991 0.9999 0.126 0.0259 4.1 0.9 3.7 Fuziline 0.01285–0.3855 y = 1.2243x + 7.3722 0.9998 0.257 0.0754 2.0 1.2 2.1 Neoline 0.01185–0.3550 y = 1.2592x + 7.3424 0.9992 0.237 0.1250 4.1 1.5 4.4 Alkaloids . Linear range (mg/mL) . Linear equation . Correlation coefficient . LOQ (μg) . Repeatability . Injection precision (RSD/%) . Stability (RSD/%) . Content (mg/g) . RSD/% . Mesaconine 0.01265–0.3795 y = 1.2793x + 7.5883 0.9995 0.127 0.1700 1.4 1.4 3.4 Aconine 0.01270–0.3810 y = 1.2536x + 7.4015 0.9995 0.127 0.0509 4.2 1.4 4.3 Hypaconine 0.01260–0.3780 y = 1.1546x + 7.2991 0.9999 0.126 0.0259 4.1 0.9 3.7 Fuziline 0.01285–0.3855 y = 1.2243x + 7.3722 0.9998 0.257 0.0754 2.0 1.2 2.1 Neoline 0.01185–0.3550 y = 1.2592x + 7.3424 0.9992 0.237 0.1250 4.1 1.5 4.4 Table II. Results of Recovery Test for the Five Alkaloids (n = 3) Alkaloids . Added (mg) . Determined (mg) . Recovery/% . Average recovery/% . RSD/% . Mesaconine 0.2024 0.2261 111.7 105.7 4.9 0.2530 0.2602 102.8 0.3036 0.3112 102.5 Aconine 0.2032 0.2041 100.4 98.6 3.0 0.2540 0.2545 100.2 0.3038 0.2890 95.1 Hypaconine 0.2016 0.2054 101.9 103.0 0.9 0.2520 0.2606 103.4 0.3024 0.3137 103.7 Fuziline 0.2056 0.1960 95.3 95.1 1.4 0.2570 0.2476 96.3 0.3084 0.2891 93.7 Neoline 0.1896 0.1960 103.4 102.6 0.7 0.2370 0.2419 102.1 0.2844 0.2911 102.4 Alkaloids . Added (mg) . Determined (mg) . Recovery/% . Average recovery/% . RSD/% . Mesaconine 0.2024 0.2261 111.7 105.7 4.9 0.2530 0.2602 102.8 0.3036 0.3112 102.5 Aconine 0.2032 0.2041 100.4 98.6 3.0 0.2540 0.2545 100.2 0.3038 0.2890 95.1 Hypaconine 0.2016 0.2054 101.9 103.0 0.9 0.2520 0.2606 103.4 0.3024 0.3137 103.7 Fuziline 0.2056 0.1960 95.3 95.1 1.4 0.2570 0.2476 96.3 0.3084 0.2891 93.7 Neoline 0.1896 0.1960 103.4 102.6 0.7 0.2370 0.2419 102.1 0.2844 0.2911 102.4 Table II. Results of Recovery Test for the Five Alkaloids (n = 3) Alkaloids . Added (mg) . Determined (mg) . Recovery/% . Average recovery/% . RSD/% . Mesaconine 0.2024 0.2261 111.7 105.7 4.9 0.2530 0.2602 102.8 0.3036 0.3112 102.5 Aconine 0.2032 0.2041 100.4 98.6 3.0 0.2540 0.2545 100.2 0.3038 0.2890 95.1 Hypaconine 0.2016 0.2054 101.9 103.0 0.9 0.2520 0.2606 103.4 0.3024 0.3137 103.7 Fuziline 0.2056 0.1960 95.3 95.1 1.4 0.2570 0.2476 96.3 0.3084 0.2891 93.7 Neoline 0.1896 0.1960 103.4 102.6 0.7 0.2370 0.2419 102.1 0.2844 0.2911 102.4 Alkaloids . Added (mg) . Determined (mg) . Recovery/% . Average recovery/% . RSD/% . Mesaconine 0.2024 0.2261 111.7 105.7 4.9 0.2530 0.2602 102.8 0.3036 0.3112 102.5 Aconine 0.2032 0.2041 100.4 98.6 3.0 0.2540 0.2545 100.2 0.3038 0.2890 95.1 Hypaconine 0.2016 0.2054 101.9 103.0 0.9 0.2520 0.2606 103.4 0.3024 0.3137 103.7 Fuziline 0.2056 0.1960 95.3 95.1 1.4 0.2570 0.2476 96.3 0.3084 0.2891 93.7 Neoline 0.1896 0.1960 103.4 102.6 0.7 0.2370 0.2419 102.1 0.2844 0.2911 102.4 Sample determination An accompanying calibration curve is needed for the calculation of content of each alkaloid due to the exponential response of ELSD. About 10, 15 and 20 μL of mixed standard solution were measured for HPLC analysis, and the calibration curve was obtained by plotting the logarithmic values of peak areas and logarithmic values of concentration for each alkaloid. Then, 20 μL of sample solution was injected for HPLC analysis, the peak areas were recorded and the content of each alkaloid was calculated according to the accompanying calibration curve. The contents of five ADAs in the 11 batches of Fuzi from different origins and different processing methods were determined by the established method, and the results were listed in Table III. The results showed that the contents of the five alkaloids in Fuzi from different regions with different processing method. The contents of mesaconine and fuziline in the tested 11 batches were higher than that of the other alkaloids. The salted aconite daughter roots and crude Fuzi contained lower amounts of the five alkaloids than white slices and black slices probably due to different processing methods and varying places of origin. Table III. Determination of the Five Alkaloids in 11 Batches of Fuzi (n = 4) Material . Location . Mean contents (mg/g) . Mesaconine . Aconine . Hypaconine . Fuziline . Neoline . White slices Jiangyou 0.2407 0.0338 0.0202 0.1488 0.0297 White slices Anxian 0.1408 0.0251 0.0183 0.1527 - White slices Yunnan 0.1602 0.0247 0.0301 0.1548 0.0477 White slices Hanzhong 0.1703 0.0509 0.0259 0.0754 0.1249 Black slices Jiangyou 0.2411 0.0400 0.0227 0.1589 0.0440 Black slices Anxian 0.1703 0.0546 0.0248 0.1094 0.1356 Black slices Unknown 0.1700 0.0509 0.0259 0.0754 0.1250 Salted aconite daughter root Jiangyou 0.0699 0.0221 0.0200 0.0250 0.0305 Salted aconite daughter root Yunnan 0.0342 0.0130 - 0.1112 0.0371 Crude Fuzi Jiangyou 0.0492 0.0249 0.0366 - 0.0700 Crude Fuzi Xichang 0.0582 0.0190 0.0281 0.1189 0.0519 Material . Location . Mean contents (mg/g) . Mesaconine . Aconine . Hypaconine . Fuziline . Neoline . White slices Jiangyou 0.2407 0.0338 0.0202 0.1488 0.0297 White slices Anxian 0.1408 0.0251 0.0183 0.1527 - White slices Yunnan 0.1602 0.0247 0.0301 0.1548 0.0477 White slices Hanzhong 0.1703 0.0509 0.0259 0.0754 0.1249 Black slices Jiangyou 0.2411 0.0400 0.0227 0.1589 0.0440 Black slices Anxian 0.1703 0.0546 0.0248 0.1094 0.1356 Black slices Unknown 0.1700 0.0509 0.0259 0.0754 0.1250 Salted aconite daughter root Jiangyou 0.0699 0.0221 0.0200 0.0250 0.0305 Salted aconite daughter root Yunnan 0.0342 0.0130 - 0.1112 0.0371 Crude Fuzi Jiangyou 0.0492 0.0249 0.0366 - 0.0700 Crude Fuzi Xichang 0.0582 0.0190 0.0281 0.1189 0.0519 -, not detected. Table III. Determination of the Five Alkaloids in 11 Batches of Fuzi (n = 4) Material . Location . Mean contents (mg/g) . Mesaconine . Aconine . Hypaconine . Fuziline . Neoline . White slices Jiangyou 0.2407 0.0338 0.0202 0.1488 0.0297 White slices Anxian 0.1408 0.0251 0.0183 0.1527 - White slices Yunnan 0.1602 0.0247 0.0301 0.1548 0.0477 White slices Hanzhong 0.1703 0.0509 0.0259 0.0754 0.1249 Black slices Jiangyou 0.2411 0.0400 0.0227 0.1589 0.0440 Black slices Anxian 0.1703 0.0546 0.0248 0.1094 0.1356 Black slices Unknown 0.1700 0.0509 0.0259 0.0754 0.1250 Salted aconite daughter root Jiangyou 0.0699 0.0221 0.0200 0.0250 0.0305 Salted aconite daughter root Yunnan 0.0342 0.0130 - 0.1112 0.0371 Crude Fuzi Jiangyou 0.0492 0.0249 0.0366 - 0.0700 Crude Fuzi Xichang 0.0582 0.0190 0.0281 0.1189 0.0519 Material . Location . Mean contents (mg/g) . Mesaconine . Aconine . Hypaconine . Fuziline . Neoline . White slices Jiangyou 0.2407 0.0338 0.0202 0.1488 0.0297 White slices Anxian 0.1408 0.0251 0.0183 0.1527 - White slices Yunnan 0.1602 0.0247 0.0301 0.1548 0.0477 White slices Hanzhong 0.1703 0.0509 0.0259 0.0754 0.1249 Black slices Jiangyou 0.2411 0.0400 0.0227 0.1589 0.0440 Black slices Anxian 0.1703 0.0546 0.0248 0.1094 0.1356 Black slices Unknown 0.1700 0.0509 0.0259 0.0754 0.1250 Salted aconite daughter root Jiangyou 0.0699 0.0221 0.0200 0.0250 0.0305 Salted aconite daughter root Yunnan 0.0342 0.0130 - 0.1112 0.0371 Crude Fuzi Jiangyou 0.0492 0.0249 0.0366 - 0.0700 Crude Fuzi Xichang 0.0582 0.0190 0.0281 0.1189 0.0519 -, not detected. Conclusion At present, the quality control of Fuzi is performed by monitoring DDAs and MDAs (3), and the application of analytical methods in quantitative assessments of Fuzi has also focused on DDAs and MDAs (4–12). ADAs have been well proved as important effective components in Fuzi. Because of no UV absorption, MS (25) or ELSD detector must be used for the application of chromatographic science in quantitative determination of ADAs. In this paper, an accurate, reliable and repeatable HPLC–ELSD method was established to determinate mesaconine, aconine, hypaconine, fuziline and neoline in Fuzi for the first time, which was proven convenient and low-cost for the routine quality control of Fuzi by evaluating ADAs. In fact, the established chromatographic condition could separate a dozen ADAs, showing the powerful separation efficiency of chromatographic science. This established method may further be combined with the present quantitative method for DDAs and MDAs to comprehensively evaluate the quality of Fuzi. Overall, this work represents a meaningful progress of routine quality control of Fuzi. Supplementary data Supplementary data are available at Journal of Chromatographic Science online. 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TI - Determination of Five Aminoalcohol-diterpenoid Alkaloids in the Lateral Root of Aconitum carmichaeli by HPLC–ELSD with SPE
JF - Journal of Chromatographic Science
DO - 10.1093/chromsci/bmx059
DA - 2017-10-01
UR - https://www.deepdyve.com/lp/oxford-university-press/determination-of-five-aminoalcohol-diterpenoid-alkaloids-in-the-NbCsKTMLqh
SP - 940
VL - 55
IS - 9
DP - DeepDyve
ER -