Single-Lab Validation for Determination of Kavalactones and Flavokavains in Piper methysticum (Kava)

Abstract

Piper methysticum (Kava) is a plant whose roots are used in the preparation of traditional beverages with spiritual, medicinal, and social importance for the Pacific Islanders. Kava is also sold as a herbal supplement or recreational beverage consumed for its mild inebriating effect in Europe and North America. With an ongoing interest in the safety and quality of kava products, it is necessary to develop a validated method for determination of kava chemical composition to ensure confidence in quality assessment. Thus, an high-performance liquid chromatography with ultraviolet detection (HPLC-UV) method was developed, optimized, and validated for determining six major kavalactones and three flavokavains in kava raw materials and finished products based on AOAC single-laboratory validation guidelines. This is the first fully validated analytical method for measuring kavalactones and flavokavains in a single run. The separation of the analytes was achieved in 10 min with an Agilent Poroshell C18 column using gradient separation. The sample was extracted with methanol first and then acetone. The signals were detected at 240 nm and 355 nm. The limit of quantification was under 1.2 µg/mL (0.3 mg/g) for kavalactones and under 0.35 µg/mL (0.01 mg/g) for flavokavains. The Horwitz ratio values described ranged from 0.3 to 1.82. The spike recovery experiments showed an accuracy between 92 and 105% for all analytes. The results of the study demonstrate that the method is fit for the purpose of determining methysticin, dihydromethysticin, kavain, dihydrokavain, yangonin, desmethoxyyangonin, flavokavain A, flavokavain B, and flavokavain C in kava raw material and finished products (dry-filled capsule, liquid phytocaps, and tincture).

Key words

kava - Piper methysticum - kavalactones - flavokavains - HPLC-UV - single-laboratory validation - Piperaceae - quality control

• References

• 1 Lebot V. Kava (Piper methysticum Forst. f.): the Polynesian Dispersal of an Oceanian Plant. In: Cox PA, Banack SA. eds. Islands, Plants, and Polynesians: An Introduction to Polynesian Ethnobotany. Portland: Dioscorides Press; 1991
• 2 Teschke R, Lebot V. Proposal for a kava quality standardization code. Food Chem Toxicol 2011; 49: 2503-2516
• 3 Food and Agriculture Organization of the United Nations. Kava: a review of the safety of traditional and recreational beverage consumption. Available at: http://www.fao.org/3/a-i5770e.pdf Accessed August 17, 2017
• 4 Drug Enforcement Administration. Kava. Available at: https://www.deadiversion.usdoj.gov/drug_chem_info/kava.pdf Accessed August 17, 2017
• 5 Sarris J, LaPorte E, Schweitzer I. Kava: a comprehensive review of efficacy, safety, and psychopharmacology. Aust N Z J Psychiatry 2011; 45: 27-35
• 6 Lebot V, Do TKT, Legendre L. Detection of flavokavins (A, B, C) in cultivars of kava (Piper methysticum) using high performance thin layer chromatography (HPTLC). Food Chem 2014; 151: 554-560
• 7 U.S. Food and Drug Administration. Consumer advisory: kava-containing dietary supplements may be associated with severe liver injury. Available at: http://www.thebody.com/content/art13885.html Accessed June 21, 2018
• 8 World Health Organization. Assessments of the risk of hepatotoxicity with kava products. Geneva: WHO Press; 2007
• 9 Ross-van Dopr CIJM. Besluit van 23 april 2003, houdende wijziging van het Warenwetbesluit Kruidenpreparaten (verbod op Kava Kava in kruidenpreparaten). Staatsblad van het Konnikrijik der Nederlanden 2003; 181: 1-4
• 10 Kuchta K, Schmidt M, Nahrstedt A. German kava ban lifted by court: the alleged hepatotoxicity of kava (Piper methyticum) as a case of ill-defined herbal drug identity, lacking quality control, and misguided regulatory politics. Planta Med 2015; 81: 1647-1653
• 11 Wang J, Qu WY, Jun SJ, Bittenbender HC, Li QX. Rapid determination of six kavalactones in kava root and rhizome samples using Fourier transform infrared spectroscopy and multivariate analysis in comparison with gas chromatography. Anal Methods 2010; 2: 492-498
• 12 Murauer A, Ganzera M. Quantitative determination of lactones in Piper methysticum (kava-kava) by supercritical fluid chromatography. Planta Med 2017; 83: 1053-1057
• 13 Meissner O, Häberlein H. HPLC analysis of flavokavins and kavapyrones from Piper methysticum Forst. J Chromatogr B 2005; 826: 46-49
• 14 Jhoo JW, Freeman JP, Heinze TM, Moody JD, Schnackenberg LK, Beger RD, Dragull K, Tang CS, Ang CYW. In vitro cytotoxicity of nonpolar constituents from different parts of kava plant (Piper methysticum). J Agric Food Chem 2006; 54: 3157-3162
• 15 Zhou P, Gross S, Liu JH, Yu BY, Feng LL, Nolta J, Sharma V, Piwnica-Worms D, Qiu SX. Flavokawain B, the hepatotoxic constituent from kava root, induces GSH-sensitive oxidative stress through modulation of IKK/NF-κB and MAPK signaling pathways. FASEB J 2010; 24: 4722-4732
• 16 Teschke R, Qiu SX, Lebot V. Herbal hepatotoxicity by kava: update on pipermethystine, flavokavain B, and mould hepatotoxins as primarily assumed culprits. Dig Liver Dis 2011; 43: 676-681
• 17 AOAC International. Appendix K: Guidelines for Dietary Supplements and Botanicals – Part I AOAC Guidelines for Single-Laboratory Validation of Chemical Methods for Dietary Supplements and Botanicals. AOAC Official Methods of Analysis. Maryland: AOAC International; 2013
• 18 Environmental Protection Agency. Guidelines establishing test procedures for the analysis of pollutants; procedures for detection and quantification, 40 CFR pt. 136, Appendix D, rev. 1.11.2002. Available at: https://www.epa.gov/sites/production/files/2015-06/documents/40cfr136_03.pdf Accessed June 21, 2018