5. AOACSPDSMethods-2018AwardsV3

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12  B rown & Y u : J ournal of AOAC I nternational V ol . 96, N o . 1, 2013

DIETARY SUPPLEMENTS

Determination of Ginsenoside Content in Panax ginseng C.A. Meyer and Panax quinquefolius L. Root Materials and Finished Products by High-Performance Liquid Chromatography with Ultraviolet Absorbance Detection: Interlaboratory Study P aula N. B rown and R onan Y u British Columbia Institute of Technology, Centre for Applied Research and Innovation, Natural Health & Food Products Research Group, 3700 Willingdon Ave, Burnaby, British Columbia, Canada V5G 3H2 Collaborators : T. Cain, G. Huie, C.D. Jin, J.N. Kababick, G. Leong, K. LeVanseler, S. Lunetta, Y.C. Ma, K. Reif, B. Schaneberg, C. Shevchuk, R. Smith, D. Sullivan, N. Wijewickreme, A. Windust

G inseng, a perennial species from the Araliaceae family, is recognized as having adaptogenic and stimulating effects on the human body. There are a total of 11 ginseng species, but those most commonly found in dietary supplements are North American ginseng ( Panax quinquefolius L.) and Asian ginseng ( P. ginseng C.A. Meyer). The majority of the active secondary metabolites are found in the roots of the ginseng plant. Ginseng has long been used as a prophylactic treatment consumed in teas, soups, and drinks. Furthermore, the roots are processed into extracts, such as powders or tinctures, and are sold as dietary supplements worldwide. The major secondary metabolites found in ginseng roots are steroidal-type molecules known as ginsenosides, which are a class of triterpene saponins that occur naturally in P. ginseng C.A.Meyer (Asian ginseng) and P. quinquefolius (North American ginseng). The two main classes of ginsenosides are protopanaxadiols (Rb 1 , Rc, Rb 2 , and Rd) and protopanaxatriols (Rg 1 and Re), which are usually found in their neutral forms (1); however, acidic moieties of these ginsenosides exist ( m -Rb 1 , m -Rc, m -Rb 2 , and m -Rd; 2–4). Analytical quantification of the total ginsenosides in a given root sample can be challenging because of these malonyl compounds, since they can be variably hydrolyzed to their neutral forms without any pretreatment during extraction with an organic solvent (4–6). This can contribute to the variability of ginsenoside content reported in a given root sample when analyzed using different extraction protocols (4, 5). There are published methods that use a hydrolysis step to mitigate this situation by completely converting the acidic ginsenosides to their neutral forms by either applying heat or by using an acidic or basic hydrolysis reaction (4–6). The use of basic hydrolysis is considered a more favorable route in hydrolyzing the ginsenosides since excessive heating has been shown to cause degradation while acidic hydrolysis produces unwanted side products and artifacts (5–7). Similarly, there are several analytical methods that are designed to separate and quantify these ginsenosides that utilize GC (7, 8), LC, and LC/MS/MS (9–14). None of these methods, however, had undergone a comprehensive validation study to assess accuracy and reliability in the quantitative determination of ginsenosides until recently (15, 16). A single-laboratory validation (SLV) for the determination of ginsenosides in P. ginseng and P. quinquefolius ginseng raw

An interlaboratory study was conducted on an HPLC method with UV absorbance detection, previously validated using AOAC single-laboratory validation guidelines, for the determination of the six major ginsenosides (Rg 1 , Re, Rb 1 , Rc, Rb 2 , and Rd) in Panax ginseng C.A. Meyer and Panax quinquefolius L. root materials, extracts, and finished products. Fourteen participating laboratories analyzed five test materials ( P. ginseng whole root, P. ginseng powdered extract, P. quinquefolius whole root, P. quinquefolius powdered extract, and P. ginseng powdered extract spiked in a matrix blank) as blind duplicates, and two test materials ( P. ginseng powdered whole root tablet and P. quinquefolius powdered extract hard-filled capsule) as single samples. Due to the variability of the ginsenosides (low level concentration of Rb 2 in P. quinquefolius raw materials and in P. ginseng spiked matrix blanks, and the possibility of incomplete hydrolysis of the finished products during processing), it was deemed more applicable to analyze total ginsenosides rather than individual ones. Outliers were evaluated and omitted using the Cochran’s test and single and double Grubbs’ tests. The reproducibility RSD (RSD R ) for the blind duplicate samples ranged from 4.38 to 5.39%, with reproducibility Horwitz Ratio (HorRat R ) values ranging from 1.5 to 1.9. For the single replicate samples, the data sets were evaluated solely by their repeatability HorRat (HorRat r ), which were 2.9 and 3.5 for the capsule and tablet samples, respectively. Based on these results, the method is recommended for AOAC Official First Action for the determination of total ginsenosides in P. ginseng and P. quinquefolius root materials and powdered extracts.

Received April 6, 2012. Accepted by AP June 21, 2012. Corresponding author’s e-mail: paula_brown@bcit.ca DOI: 10.5740/jaoacint.12-153