Method for the Determination of Aconitum Alkaloids in Dietary Supplements and Raw Materials by Reversed-Phase Liquid Chromatography with

Ultraviolet Detection and Confirmation by Tandem Mass Spectrometry: Single-Laboratory Validation Wai-Tong Tang, Sui-Kay Wong, Tin-Yau Law, Kwok-Chu Pang, Della Sin, and Yin-King Tam Government Laboratory, 88 Chung Hau St, Homantin, Hong Kong SAR, China Corresponding authors e-mail: wttang@govtlab.gov.hk See other articles in PMC that cite the published article.

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Abstract A study of single-laboratory validation (SLV) of a reversed-phase liquid Chromatography (RPLC) method was conducted for the determination of diester-diterpene Aconitum alkaloids, viz., aconitine, mesaconitine, and hypaconitine, in a variety of dietary supplements, including singlearid multiple-ingredient dry powder extracts, pills, capsules, and raw materials. The Aconitum alkaloids in the samples were extracted by diethyl ether in the presence of ammonia. After cleanup with solid-phase extraction to remove the matrix interferences, the alkaloids were determined by RP-LC with UV detection at 235 nm, and the results were confirmed by tandem mass Spectrometry. The linear responses for aconitine, mesaconitine, and hypaconitine based on the present LC system ranged from 0.5 to 200 g/mL. Relative standard deviations of 2.0 to 6.9% were obtained from duplicate analysis of 6 test materials of different matrixes for the 3 Aconitum alkaloids performed by 2 analysts on 5 different days. The recoveries determined for supplements and raw materials spiked with 3 Aconitum alkaloids at levels of 2.510 g/g were in the range of 8699%. In view of the attainment of satisfactory results for accuracy, precision, and recovery in the SLV study, it is recommended that the method validation process proceed to a collaborative study.

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The diester-diterpene Aconitum alkaloids, viz., aconitine, hypaconitine, and mesaconitine, are highly toxic compounds commonly present in the aconite roots such as Radix 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 of 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 contents, and hence 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. This study is of particular importance not only to the public but also to dietary supplement manufacturers for setting quality standards and regulatory agencies for monitoring the safety in the use of dietary supplements containing aconite roots.

In this single-laboratory validation (SLV) study, the evaluation of the method was based on the use of 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, the details of which are given in Table 1. Table 1 Aconite root products and negative controls for single-laboratory validation of the determination of Aconitum alkaloids

The composition of dietary supplements is usually very complex and usually poses difficulties in their determinations due to significant matrix interferences. The present method utilizes a 2-step cleanup procedure, involving initial liquidliquid extraction followed by solid-phase extraction (SPE) to remove interferences prior to liquid chromatographic (LC) separation using C8 columns (3). In this cleanup procedure, the 3 Aconitum alkaloids of interest (aconitine, hypaconitine, and mesaconitine; Figure 1) are extracted from the samples with diethyl ether in the presence of ammonia. Matrix interferences of the samples, including neutral, acidic, and basic polar compounds, are sequentially removed using OASIS MCX SPE cartridges exhibiting a dual-mode [reversed-phase (RP) and ion-exchange] retention mechanism. The 3 alkaloids in the final extract are separated by RP-LC with UV detection at 235 nm. The results obtained are confirmed by tandem mass spectrometry (MS/MS). Figure 1 Molecular structures of aconitine, mesaconitine, and hypaconitine.

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Experimental Apparatus a. LC-UV system.Agilent (Wilmington, DE) 1100 Series LC system with a quaternary pump, autosampler, degasser, column heater, variable wavelength detector, and ChemStation for data acquisition and processing. b. LC/MS/MS system.Perkin-Elmer (Shelton, CT) SCIEX API-300 triple-quadruple mass spectrometer equipped with turbo ion spray source and an Agilent 1100 Series HPLC system consisting of a binary pump, autosampler, degasser, column heater, control unit, and data acquisition and processing unit provided by Applied Biosystems (Foster City, CA). c. LC column (for LC-UV system).Zorbax Eclipse XDB-C8, 5 m, 4.6 150 mm with guard cartridge (Agilent). d. LC column (for LC/MS/MS system).Xterra MS C8, 3.5 m, 2.1 150 mm (Waters Corp., Milford, MA). e. ASPEC XL4 SPE system.Gilson Inc. (Middleton, WI).

f. g. h. i. j. k. l. m. n. o. p. q. r. s.

Oasis MCX extraction cartridges.3 cc, 60 mg (Waters). Grinder, blender, or mill. Analytical balance.Capable of weighing to 0.1 mg. Volumetric flasks.Various sizes. Ultrasonic bath. Platform shaker. Centrifuge tube.50 mL. Centrifuge. Nitrogen evaporator.N-EVAP 112 (Organomation Associates Inc., Berlin, MA). Autopipets.250 and 1000 L. Droppers.About 2 mL. Membrane filters.0.45 m. LC vials.12 32 mm, 2 mL amber glass with screw caps. Filter papers.Whatman (Florham Park, NJ) 541, 90 m pore size or equivalent.

Reagents a. Mobile phase A (for LC-UV).20 mM triethylamine (TEA; adjusted to pH 3 with phosphoric acid)methanol (95 + 5). b. Mobile phase B (for LC/MS/MS).20 mM ammonium formate in 0.1% formic acid. c. Methanol.HPLC grade. d. Acetonitrile (ACN).HPLC grade. e. Acetic acid.AR grade. f. 0.1% acetic acid.Dilute 1 mL acetic acid to 1 L with water. g. Acetonitrile0.1% acetic acid (1 + 1).Mix 500 mL acetonitrile with 500 mL of 0.1% acetic acid. h. Water.Distilled and deionized. i. Diethyl ether.AR grade. j. TEA.AR grade. k. Concentrated ammonia solution (ca 25% NH3).AR grade. l. 10% ammonia solution.Dilute 400 mL concentrated ammonia solution to 1 L with water. m. 5% ammonia solution.Dilute 200 mL concentrated ammonia solution to 1 L with water. n. SPE washing solution.Mix 70 mL of 5% ammonia solution with 30 mL methanol. o. SPE elution solution.Mix 5 mL of 5% ammonia solution with 95 mL methanol. p. Aconitine.Purity >98% (Wako Chemicals USA, Inc., Richmond, VA). q. Hypaconitine.Purity >98% (Wako). r. Mesaconitine.Purity >98% (Wako). Stock Standard Solutions (a) Aconitine stock standard solution (1000 mg/L) About but no less than 10 mg aconitine was weighed accurately into a 10 mL volumetric flask. Six mL acetonitrile0.1% acetic acid (1 + 1) was added to dissolve the solid. Additional solvent was added to dilute to the mark. (b) Mesaconitine stock standard solution (1000 mg/L)

About but no less than 10 mg mesaconitine was weighed accurately into a 10 mL volumetric flask. Six mL acetonitrile0.1% acetic acid (1 + 1) was added to dissolve the solid. Additional solvent was added to dilute to the mark. (c) Hypaconitine stock standard solution (1000 mg/L) About but no less than 10 mg hypaconitine was weighed into a 10 mL volumetric flask. Six mL acetonitrile0.1% acetic acid (1 + 1) was added to dissolve the solid. Additional solvent was added to dilute to the mark. (d) Mixed intermediate standard solution (ca 100 mg/L) One mL each of the stock standard solutions was pipetted into a 10 mL volumetric flask, and acetonitrile0.1% acetic acid (1 + 1) was added to dilute to the mark. Serial dilution was performed to obtain the required concentration, if required. (e) Working standard solutions (120 mg/L) No less than 5 working standard solutions within this range according to Table 2 were freshly prepared by dilution of the mixed intermediate standard solution with acetonitrile0.1% acetic acid (1 + 1). Table 2 Preparation of standard (std) solutions

Validation Samples Six samples comprising 1 raw material and 5 dietary supplements containing aconite roots or their extracts in the form of single-ingredient dry powder extract, multi-ingredient dry powder extract, pills, or capsules were chosen to represent products commonly available on the market. The product names, descriptions, forms, and label claims are given in Table 1. Test Samples Preparation (a) Raw material (processed Radix aconiti lateralis) Ca 50 g of the sample were ground using a blender to produce a homogeneous powder capable of passing through a 150 m sieve. The contents were transferred into an amber wide-mouth bottle with a screw cap and then mixed thoroughly. (b) Dry powder extracts (single- and multi-ingredient dry powder extracts) The original test sample was ground, and the powder was passed through a 150 m sieve, if necessary. About 30 g of the powder was transferred to an amber wide-mouth bottle with a screw cap and then mixed thoroughly. (c) Pills Ca 30 g of randomly selected pills were powdered using a blender, and the powder was passed through a 150 m sieve. The powdered contents were transferred into an amber wide-mouth bottle with a screw cap and mixed thoroughly. (d) Capsules Ca 100 capsules were randomly selected. The shells were removed, and their contents were powdered and passed through a 150 m sieve, if necessary. The powdered contents were transferred into an amber wide-mouth bottle with a screw cap and then mixed thoroughly. Sample Treatment (a) Extraction of samples

Ca 1 g powdered sample was accurately weighed in a 50 mL centrifuge tube. One mL 10% ammonia solution and 25 mL diethyl ether were added. The tube was shaken on a platform shaker for 1 h at a speed of 300 rpm. The sample was centrifuged at 4000 rpm for 10 min to settle the solid. The diethyl ether was decanted into another centrifuge tube. The sample extraction was repeated twice, each with 10 mL diethyl ether, and the mixture was shaken for 30 and 10 min, respectively. The extracts were combined and evaporated to dryness at 40C under a stream of nitrogen. The residue was redissolved in 5 mL acetonitrile0.1% acetic acid (1 + 1). The solution was ready for SPE cleanup after passing through a 0.45 m membrane filter. (b) MCX cartridge SPE The SPE cartridge was conditioned with 1 mL methanol and 1 mL water. After loading 4 mL sample solution onto the SPE cartridge, it was washed sequentially with 1 mL 0.1% acetic acid, 1 mL methanol, 1 mL water, and 1 mL SPE washing solution. The cartridge was dried, and the analytes were eluted with 2 mL SPE elution solution. The solvent was evaporated to dryness at ca 40C under a stream of nitrogen. The residue was reconstituted in 1 mL acetonitrile0.1% acetic acid (1 + 1). The solution was ready for chromatographic analysis after passing through a 0.45 m membrane filter. Instrument Operating Conditions (a) LC-UV Column temperature, 25C; flow rate, 1.0 mL/min; injection volume, 10 L; detection wavelength, 235 nm; run time, 45 min; post-run, 10 min; and gradient program as in Table 3. Table 3 Gradient program for the separation of aconitine, hypaconitine, and mesaconitine

(b) LC/MS/MS MS settings: ionization mode, electrospray positive ionization, curtain gas, 8; nebulizer gas, 15; ionspray voltage, 5000 V, and temperature, 350C. Other MS parameters are given in Table 4. The flow rate of the mobile phase was set at 200 L/min. Table 4 Mass spectrometric parameters

Calibration (a) Calibration curve Five-point (excluding zero) calibration graphs were established for each of the 3 analytes from freshly prepared standard solutions. The calibration graphs were checked by injection of check standard solutions at the beginning, middle, and end of the analytical run. (b) Linearity The relationship between the peak area response and concentration was determined using linear regression. The correlation coefficient (r) for each analyte must be >0.999. Calculations

A calibration graph of peak area of analytes in working standard solutions against the concentration of analytes was established. The concentration of analytes in sample extract solution was determined from the calibration graph. The concentration of analytes in the sample, C in g/g, was calculated with the following equation:

where A = concentration of the analyte found in the sample solution, g/mL; W = weight of sample used, g; V = final volume of the sample solution, mL; and D = dilution factor, if any. Determination of Limit of Detection (LOD) and Limit of Quantitation (LOQ) At least 7 replicates of blank samples (negative controls) spiked with aconitine, mesaconitine, and hypaconitine at levels close to the detection limit were prepared, and the standard deviations () were determined. The LOD and LOQ were calculated as follows:

The LOD and LOQ were verified by inspecting the appropriate chromatograms. Accuracy The low, intermediate, and high spike recoveries were determined by spiking, in triplicate, negative controls AC-C-1, AC-C-2, and AC-C-3 with the aconitine, hypaconitine, and mesaconitine reference standards at levels of ca 2.5, 5, and 10 g/g, respectively. The solvent in the spiking solution was evaporated to dryness prior to the addition of sample because Aconitum alkaloids were found soluble in polar solvents such as acetonitrile and methanol, which would affect the yield of the extraction. A known amount of reference standard solution was pipetted into a centrifuge tube, and the solvent was evaporated to dryness at 40C under a stream of nitrogen. The extraction, SPE cleanup, chromatographic separation, and analysis were then performed after adding the negative controls. Precision Duplicate sample preparations of 6 samples in 5 days were performed by 2 different analysts. The within-day precision (Sw), between-day precision (Sb), and the total precision (St) were calculated as follows:

Ruggedness Testing A ruggedness trial for 7 factors potentially affecting the quantitative results, including weight of sample, volume of extraction solvent, polarity of extraction solvent, extraction time, shaking rate, strength of ammonia for extraction, and volume of SPE elution solvent was designed. Test sample AC-S-2 was prepared in duplicate for each experiment. The results of ruggedness testing are presented in Table 5.

Table 5 Youden ruggedness trial design and calculation

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Results and Discussion Optimization Optimization of the method in terms of sample extraction, SPE cleanup procedures, and column separation conditions (composition of the mobile phase and choice of columns) has been studied. In the present method, Aconitum alkaloids in the samples were converted to the free base form in the presence of ammonia and extracted into diethyl ether. Shaking the sample with a platform shaker at 300 rpm increases the partition efficiency of the analytes between the aqueous and organic layers and, thus, the yield. Matrix interferences were removed using SPE with OASIS MCX employing a dual-mode (RP and ion-exchange) retention mechanism prior to LC-UV determination. The use of other SPE systems was studied and proved to yield either none or much lower recoveries. Compared to other columns, the Eclipse XDB column, in which the stationary phase materials are extra densely bonded and double end-capped, provide better peak shape for basic compounds such as Aconitum alkaloids. Comparison of Eclipse XDB C8 columns from 3 different batches gave retention times of 30.81, 33.73, and 36.29 min, respectively, for mesaconitine, hypaconitine, and aconitine that were very consistent [relative standard deviation (RSD) 1.8, 2.0, and 2.2%, respectively], and the interferences were well separated from the analytes of interest. The effect of the concentration of TEA on the separation of the analytes was studied, and the use of 20 mM TEA is recommended. The addition of 5% methanol in the mobile phase is also recommended to prevent precipitation of TEA upon prolonged storage. LOD and LOQ The LOD and LOQ for the LC-UV method based on 1 g sample are 0.2 and 0.6 g/g, respectively. However, for ease of computation, a higher LOQ of 1 g/g was used in this study. Stability of the Analytes 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 was found to be about 45 days (Figure 2). However, there were no signs of degradation of the alkaloids when being stored in acetonitrile or acidic media, even for a period of 6 months. Figure 2 Stability of (A) aconitine, (B) mesaconitine, and (C) hypaconitine in a methanol 5% ammonia (95 + 50) mixture.

Linearity and Working Range The peak area responses for the 3 Aconitum alkaloids were found linear from 0.05 to 100 g/mL with r-values > 0.9999. Standard Curve Precision The r-values for the 3 Aconitum alkaloids for a 5-point calibration graph (approximately 1 to 20 /mL) was >0.9999 (Figure 3), and the slopes of 5 replicates of the calibration graph covering the analytical range for each Aconitum alkaloid standard varied by no more than 1.5% in terms of RSD over a period of 2 weeks. Figure 3 Calibration graphs for (A) aconitine, (B) hypaconitine, and (C) mesaconitine.

Youden Ruggedness Trial Results Given in Figure 4 is the graphical presentation of results from a Youden ruggedness trial. Of the 7 factors tested (high and low parameters for sample weight, volume of extraction solvent, polarity of extraction solvent composing ether and dichloromethaneether mixture, extraction time, shaking rate, strength of ammonia used, and volume of SPE elution solvent), the selection of extraction solvent has the greatest impact on the yield of Aconitum alkaloids. The neutral Aconitum alkaloids were found to be more soluble in diethyl ether than in dichloromethane. The change from pure diethyl ether to a 1 + 1 mixture of dichloromethanediethyl ether resulted in a cleaner background but lower yield. Weight of sample (Factors A/a) and extraction time (Factors D/d) affected the results to a lesser extent. On the other hand, it should be noted that the use of a smaller amount of sample resulted in amounts of alkaloids close to the LOQ and, thus, greater variation in recoveries. On the other hand, the longer the extraction time under alkaline conditions, the more susceptible the alkaloids were to hydrolysis, thus lowering recoveries. Slight changes in the remaining factors were least likely to affect the quantitation results. Figure 4 Results of Youden ruggedness test.

Comparison of Results Obtained from LC-UV and LC/MS/MS The results of analysis of all samples under study for 3 Aconitum alkaloids by LC-UV and LC/MS/MS methods are summarized in Table 6. The ratios of average amount of hypaconitine and mesaconitine obtained by the LC-UV and LC/MS/MS methods were found to range from 0.94 to 1.09. All the samples under study were found to contain aconitine below the LOQ (i.e., 1 g/g), and the results were confirmed by LC/MS/MS. The close agreement of results obtained by the LC-UV and LC/MS/MS methods indicated that interferences were well separated from the analytes of interest. Table 6 Comparison of results obtained by the LC/UV and LC/MS/MS techniques

Precision Precision experiments were performed on 6 matrixes (AC-S-1 to AC-S-6), with duplicate sample preparations over a consecutive 5-day period by 2 different analysts. Sw, Sb, and St values were determined and are given in Tables 7 and and88 for hypaconitine and mesaconitine, respectively. Table 7 Repeatability test data for determination of hypaconitine

Table 8 Repeatability test data for determination of mesaconitine

For all materials tested in duplicate over a 5-day period, the repeatability (RSDr) ranged from 1.98 to 3.72% (Table 7) and 3.57 to 6.94% (Table 8) for hypaconitine and mesaconitine, respectively. All calculated HorRat scores were within the limits for performance acceptability, ranging from 0.35 to 0.61 and 0.46 to 0.91 for hypaconitine and mesaconitine respectively, indicating satisfactory precision of the method.

Because all samples were found to contain aconitine at levels lower than the LOQ of 1 g/g, the corresponding precision could not be evaluated. The precision was estimated with reference to the negative control samples being spiked with aconitine. Triplicate analyses on each of the 3 control samples spiked with reference standards at levels of about 2.5, 5, and 10 g/g were conducted over 3 different days. Satisfactory precision of the method for aconitine was also demonstrated by its RSDr of 2.68 to 4.82% and the HorRat score of 0.47 to 0.68 (Table 9). Table 9 Repeatability test data for determination of aconitine (data obtained from spiked samples)

Accuracy Results from Negative Control Recovery Experiments Negative control recovery was determined by spiking all 3 reference compounds in triplicate into 3 blank matrixes at 3 concentrations (low at approximately 50%, intermediate at 100%, and high at 200% of the normal concentrations encountered, about 5 g/g). Negative Control 1 is a ginseng sample similar to aconite roots, viz., R. aconiti (Chuanwu), R. aconiti kusnezoffii (Caowu), and R. aconiti lateralis (Fuzi). Negative Control 2 is a multiingredient powder extract dietary supplement similar to AC-S-2. Negative Control 3 is a dietary supplement capsule for enhancing circulation and relieving pain, similar to sample AC-S-6. The results of recovery studies are summarized in Tables 1012. The recoveries for the 3 Aconitum alkaloids ranged from 86 to 94%, 90 to 97%, and 91 to 99% for low, intermediate, and high spike levels, respectively, indicating satisfactory performance. Table 10 Accuracy results from negative control recovery experiments consisting of triplicate sample preparations at 3 spike levels (50, 100, and 200%) by LC-UV: results for AC-C-1a

Table 12 Accuracy results from negative control recovery experiments consisting of triplicate sample preparations at 3 spike levels (50, 100, and 200%) by LC-UV: results for AC-C-3a

Representative LC-UVand LC/MS/MS Chromatograms Representative LC-UV and LC/MS/MS chromatograms for reference standards and samples are given in Figures 5 and and66. Figure 5 Representative LC-UV chromatograms of (A) Aconitum alkaloid stanards (10 g/mL), (B) sample AC-S-1, (C) sample AC-S-2, and (D) sample AC-S-3.

Representative LC-UV chromatograms of (E) sample AC-S-4, (F) sample AC-S5, and (G) sample AC-S-6.

Figure 6 Representative LC/MS/MS chromatograms. Extracted ion chromatogram of 0.8 /mL mixed standard (A) MRM 646/586 for aconitine and (B) MRM 616/556 for hypaconitine. Representative LC/MS/MS chromatograms. Extracted ion chromatogram of 0.8 /mL (more ...)