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An interlaboratory study was conducted to evaluate a method for the determination of 3 Aconitum alkaloids, viz., aconitine, mesaconitine, and hypaconitine, in raw botanical material and dietary supplements. The alkaloids were extracted with diethyl ether in the presence of ammonia. After cleanup by solid-phase extraction to remove matrix interferences, the alkaloids were determined by reversed-phase liquid chromatography (LC)/UV detection at 235 nm with confirmation by LC/tandem mass spectrometry (MS/MS). A total of 14 blind duplicates were successfully analyzed by 12 collaborators. For repeatability, the relative standard deviation (RSDr) values ranged from 1.9 to 16.7%, and for reproducibility, the RSDR values ranged from 6.5 to 33%. The HorRat values were all <2 with only one exception at 2.3. All collaborating laboratories had calibration curves with correlation coefficients of >0.998. In addition, 6 collaborators performed the confirmation and were able to verify the identities of the alkaloids by using LC/MS/MS.
The diester-diterpene Aconitum alkaloids, viz., aconitine, hypaconitine, and mesaconitine, are highly toxic compounds commonly present in aconite roots such as R. aconiti (Chuanwu), R. aconiti kusnezoffii (Caowu), and R. aconiti lateralis (Fuzi). Many dietary supplements for enhancing sexual ability and circulation, restoring health, and relieving pain contain processed aconite roots and thus the Aconitum alkaloids. Proper processing by heating, steaming, and soaking the aconite roots can hydrolyze the highly toxic diester-diterpene Aconitum alkaloids to compounds of much lower toxicity, e.g., benzoylaconine, benzoylmesaconine, and benzoylhypaconine (1). Pharmacological studies indicated that the diester-diterpene Aconitum alkaloids have the same or similar anti-inflammatory and analgesic actions as their hydrolyzed analogs (2). The absence of standardized methods for processing aconite roots has resulted in drastic variation in the alkaloid content, and thus in the safety, of the supplement products containing aconite roots. Intoxication cases arising from consumption of improperly processed aconite roots have been reported in many countries.
Because of this significant public health impact, a single-laboratory validation (SLV) study was conducted for the determination of the 3 Aconitum alkaloids in 6 representative matrixes of aconite root products, including processed raw material (Fuzi), single-ingredient dry powder extract, multi-ingredient dry powder extract, pills, and capsules found in the marketplace (3). This method will facilitate the accurate determination of the quality of botanicals and dietary supplements with respect to the 3 Aconitum alkaloids. In addition, the use of this method may allow dietary supplement manufacturers to set quality standards, and regulatory agencies to monitor safety in the use of dietary supplements containing aconite roots.
The liquid chromatography/UV detection (LC/UV) phase of this study was conducted with 7 materials as blind duplicates. Some of the materials contained known concentrations of naturally occurring or purposely added (formulated or fortified) aconitine, mesaconitine, and hypaconitine. Two of the materials were negative controls. In addition, each participant was supplied with sufficient quantities of reference standards to conduct the study. Random identification numbers were assigned to each of the blind duplicate materials. A practice sample of dietary supplement material of known concentration was provided for participants. Also, a blank of botanical raw material was provided for the recovery study. These samples were used to ensure that each laboratory could successfully follow the method and to optimize each participant’s instruments and chromatography before the collaborative study samples were analyzed.
Thirteen laboratories participated in this study and received collaborative study materials. From these 13 laboratories, 12 sets of LC/UV data and 6 sets of confirmation data were generated for this collaborative study. The locations of the 13 laboratories were as follows: 4 in the United States, 4 in mainland China, 1 in the United Kingdom, 1 in Korea, and 3 in Hong Kong.
Liquid Chromatography with UV Detection First Action 2008
See Table 2008.11A for the results of the interlaboratory study supporting acceptance of the method.
The Aconitum alkaloids are extracted from the various matrixes with diethyl ether in the presence of ammonia, the extract is cleaned up by solid-phase extraction (SPE), and the analytes (aconitine, mesaconitine, and hypaconitine) are quantitated by liquid chromatography/UV detection (LC/UV) at 235 nm. Identities are confirmed by liquid chromatography/tandem mass spectrometry (LC/MS/MS).
(Note: The 3 Aconitum alkaloids are susceptible to hydrolysis in methanol, especially under alkaline conditions. The half-life of aconitine, hypaconitine, and mesaconitine in methanol with 5% ammonia is about 4–5 days. However, there were no signs of degradation of the alkaloids when stored in acetonitrile or acidic media even for a period of 6 months.)
Establish a calibration curve of peak area of each analyte in the working standard solutions versus concentration of analyte Determine the concentration of each analyte in the sample extract solution from the calibration curve If the concentration of an analyte in the sample extract exceeds the calibration range make appropriate dilution and repeat the determination Calculate the concentration of each analyte in the sample, C, in mg/kg, by using the following equation:
where A = concentration of the analyte found in the sample solution mg/L; W = weight of sample used g; V = final volume of the sample solution, mL; D = dilution factor if any; and 5/4 = factor that accounted for the portion of extract taken for the cleanup.
When an Aconitum alkaloid is detected at ≥1 mg/kg as determined by LC/UV, the identity is confirmed by LC/MS/MS. For confirmation, the intensity of the respective multireaction monitoring ion pair signal should be ≥400 cps (Table 2008.11D).
References: J. AOAC Int. 89, 1496 (2006); 92, 111 (2009).
Thirteen laboratories participated in the collaborative study. One laboratory was not able to finish the study because of lack of time. The remaining laboratories were able to submit data before the submission deadline.
Results, in mg aconitine, mesaconitine, and hypaconitine/kg product, for each of the 7 blind replicates are presented in Table 1. Test samples were given codes before shipment to the collaborators and then decoded when the results were returned. To allow the collaborators to check their competence in conducting the analysis, the procedure and blank botanical material were provided for them to conduct a recovery study before the analysis of the test materials. According to the data received, 11 laboratories were able to obtain average recoveries for the 3 Aconitum alkaloids that were very close to or within the acceptable range of 80–100%, whereas the recoveries obtained by the remaining laboratory (Laboratory L) were only about 35%. This laboratory had discussed the problem with the organizing laboratory, but it still failed to identify the causes for the poor recovery. It was also noted that most of the results of Laboratory L were inconsistent with those of the other collaborators. Therefore, it was decided not to include the results of Laboratory L in the evaluation of the performance characteristics for this study.
Laboratory B reported that samples A1 and C1 were lost during the sample preparation. As a result, no data were provided for these 2 samples. Laboratory H observed that, during the sample extraction for samples A1 and A2, sample at the bottom of the centrifuge tube lumped together and was not able to be wetted by the extraction solvent. Because this observation implied that the extraction for these 2 samples might not be complete, the respective data entries were thus not included in the statistical analysis. Judging from the data, it was suspected that Laboratory I and Laboratory J might have mixed up some of their results. For example, Laboratory I may have mixed up the results for samples C1 and C2 with those for samples F1 and F2, and Laboratory J may have mixed up the results for samples D2, E2, and F2. The organizing laboratory raised this issue with both laboratories but the issue was not resolved by either laboratory. In order not to affect the evaluation of the performance characteristics for this study, the data entries in question were not included in the statistical analysis. In addition, Laboratory E reported that mesaconitine was detected in samples G1 and G2, and hypaconitine, in samples F1 and F2, which were the dietary supplement negative control replicates. These results might be due to contamination or to carry-over peaks in the LC/UV analysis.
Table 2008.11A presents statistical summaries of the results from Laboratories A–K. Statistical analysis to determine repeatability and reproducibility was performed by using the AOAC INTERNATIONAL Statistical Program (Version 2.0) for Blind Replicates. The values for repeatability standard deviation (Sr), reproducibility standard deviation (SR), RSDr, reproducibility relative standard deviation (RSDR), and number of statistical outliers are presented. HorRat values are also presented and are calculated as RSDR (observed)/RSDR(predicted), where the RSDR(predicted) is calculated by using the following equation:
where C is the measured analyte concentration in decimal mass units (4). The Cochran, Grubbs, and Double Grubbs tests were used to remove statistical outliers where appropriate. Data from laboratories reporting values for individual alkaloids as greater-than or less-than values were not included in the statistical analysis.
The collaborators were able to follow the method with very few difficulties. Five laboratories reported difficulties in mixing the entire sample inside the centrifuge tube with the 1 mL 10% ammonium hydroxide solution before the extraction. One collaborator suggested using 2 mL 5% ammonium hydroxide solution instead. Another laboratory performed the extraction by using a 50 mL conical flask instead of the centrifuge tube.
The method performed well in the collaborative study for both detection and confirmation of the 3 Aconitum alkaloids. For repeatability, the RSDr values ranged from 1.9 to 16.7% and for reproducibility, the RSDR values ranged from 6.5 to 33%. Five materials had acceptable HorRat values except for the aconitine determined in the processed R. aconiti, for which the HorRat value was 2.37. For the 2 negative control materials, the HorRat values were not applicable. The number of results identified as outliers and disregarded was ≤4 out of 21 or 22 for all samples except for hypaconitine determined in the spiked dietary supplement negative control for which 2 pairs of Cochran and 1 pair of Grubbs outliers were identified. The number of outlier laboratories identified, excluding those suspected of having results mixed up with those of other samples, was <2 out of 11, except for the determination of hypaconitine in the spiked dietary supplement negative control, for which 3 laboratories were identified as outliers.
In addition, all collaborating laboratories had correlation coefficients of >0.998 for the calibration curves generated. Among the participating laboratories, 6 verified the identity of the Aconitum alkaloids found in the samples by using LC/MS/MS. The reason that the other laboratories failed to do so might be the unavailability of the required equipment.
On the basis of the results of this collaborative study, it is recommended that the method be adopted Official First Action for the determination of Aconitum alkaloids in dietary supplements and raw botanical materials.
We thank T.L. Ting, Government Laboratory, for his encouragement and support throughout the course of this study. We would also like to thank the following collaborators for their participation in this study:
Darryl Sullivan, Covance Laboratories, Madison, WI
Brian T. Schaneberg, ChromaDex Co., Boulder, CO
Shen Ji, Shanghai Institute for Drug Control, People’s Republic of China
Kelvin S.Y. Leung, Baptist University, Hong Kong SAR, China
C.T. Che, Chinese University of Hong Kong, Hong Kong SAR, China
Alan Richards, Scientific Services, Durham, UK
Jong-Hwan Kim, Korean Food and Drug Administration, Korea
Sidney Sudberg, Alkemists Pharmaceticals, Costa Mesa, CA
Wendi Wang, Advanced Botanical Consulting & Testing, Inc., Tustin, CA
Hong-Zhu Guo, Beijing Institute for Drug Control, People’s Republic of China
Li-Xin Zhang, Chengdu Institute for Drug Control, People’s Republic of China
Feng-Yi Zheng, Guangdong Institute for Drug Control, People’s Republic of China
Collaborators: C.T. Che; H.-Z. Guo; S. Ji; J.-H. Kim; K.S.Y. Leung; C.K. Lo; A. Richards; B.T. Schaneberg; S. Sudberg; D. Sullivan; W. Wang; Y.C. Wong; L.-X. Zhang; F.-Y. Zheng
The recommendation was approved by the Methods Committee on Dietary Supplements as First Action. See “Official Methods Program Actions,” (2008) Inside Laboratory Management, November/December issue.