Kombucha is a sweetened tea beverage fermented by bacterial and yeast cultures. Sweeteners such as glucose, sucrose, fructose and others are converted by yeasts into ethanol, then by Acetobacter and other bacterial species into a weak acetic acid solution that is diluted, flavoured and packaged into glass or aluminum cans for consumer consumption. Naturally, fermented Kombucha contains 0 to 3% alcohol-by-volume (ABV). However, Kombucha containing ethanol is concerning for pregnant women and young children for whom low levels of ethanol consumption (1% ABV are regulated as liquor. This study assessed ethanol concentrations in Kombucha collected from processors and purchased at retail venues in BC. Ethanol values were compared to the place of manufacture (country or province) and place of purchase (grocery stores, restaurants, farmers markets, recreational centres, and processors). Ethanol (n = 684) levels were measured using a headspace gas chromatography mass spectrometry (HS-GCMS) method with a detection limit of 0.0002% ABV for ethanol. Overall, teas contained mean and median ethanol of 0.77% and 0.62% ABV, respectively, ranging from non-detectable up to 3.62% ABV. Four Kombucha teas (0.6%) made by BC processors tested over 3% ABV, and 31.5% of samples contained ethanol that exceeded the BC regulatory limits for non-alcoholic beverages of 1% ABV. Kombucha manufactured in BC had significantly higher mean ethanol values (1.16% ABV) in comparison to all other places of manufacture. Similarly, mean ethanol tea values obtained from BC processors (1.2% ABV) and restaurants (1.01% ABV) were significantly higher than those obtained at other retail venues. This study demonstrates the potential for alcohol harm to at-risk populations consuming Kombucha teas sold in BC.

Kombucha is a fermented beverage made with tea, sugar, and a symbiotic colony of bacteria and yeast that is usually marketed as a non-alcoholic beverage. Products must contain <0.5% and <1.1% alcohol by volume in the United States and Canada respectively to be classified as non-alcoholic products. Prior studies have found that Kombucha beverages can become very acidic and may contain levels of alcohol above 1% which can be a potential health risk to children and the developing fetus during pregnancy. Given the public safety concerns and legal requirements associated with the level of alcohol within Kombucha beverages, there is a need for accurate and reliable methods. Herein we describe the validation of a sensitive, rapid, and simple Headspace Gas Chromatographic method with mass spectrometric detection for determining ethanol in Kombucha. Method performance characteristics measured included linearity, accuracy, precision, limit of detection (LOD), and limit of quantification (LOQ) as per AOAC International guideline Appendix K Part 1. Performance was evaluated against the AOAC Standard Method Performance Requirements 2016.001 for determination of ethanol in Kombucha. The linear dynamic range for this method was confirmed over the range of 0.025 to 2.47% ABV. The LOD and LOQ were determined to be 0.0002% and 0.002% ABV, respectively. With a spike recovery of 102% for accuracy and precision of RSD_r ≤ 4% the method met the SMPR requirements within the analytical range. The results of this validation study demonstrated the method is fit for the purpose of quantifying ethanol in Kombucha and is suitable for rapid and easy integration by laboratories to ensure that regulatory requirements are met.

Background: The leaves of Mitragyna speciosa, commonly called kratom, have been consumed for centuries for their energizing and analgesic properties. Kratom has gained popularity in North America, with an estimated 16 million American consumers. Recently, a new product has emerged, labeled “kratom extract” but purportedly composed of high concentrations of 7-hydroxymitragynine, an oxidation product of mitragynine, the primary alkaloid found in kratom. Objective: 7-hydroxymitragynine, a potent mu-agonist, may pose a serious public health risk. Commercial products labeled as containing 7-hydroxymitragynine were evaluated to determine whether the constituent was present and in what concentration. Method: The concentrations of 7-hydroxymitragynine and mitragynine were determined by a previously validated HPLC-DAD method, AOAC Official MethodSM 2017.14. The chromatographic profiles of products were also compared to authentic kratom leaf. Results: All products were described on their label as containing “kratom extracts.” The concentration of mitragynine ranged from 2.0 to 6.0 mg/g dry weight, substantially lower than typically found in kratom leaves or extracts. 7-hydroxymitragynine was determined at 22–75 mg/g dry weight, 5–28% higher than the label claims. The chromatographic profiles of all products were inconsistent with kratom leaf, having exceptional levels of 7-hydroxymitragynine, lacking the other major naturally occurring alkaloids, and exhibiting peaks not observed in leaf. Conclusions: The products were verified to contain very high levels of 7-hydroxymitragynine, achievable only by synthetic means. These products differ significantly from authentic native kratom leaf and are not kratom extracts, as labeled, but rather synthetic derivatives that constitute unapproved new drugs being unlawfully marketed. There is an urgent need to differentiate kratom, which has been safely consumed for centuries, from 7-hydroxymitragynine, which may pose a significant risk to public health. Highlights: Traditional kratom leaf is distinguished from products found to contain high concentrations of 7-hydroxymitragynine, only achievable by synthetic means. These unapproved new drugs contained other unidentified constituents, also not present in the native kratom leaf. The presence of concentrated 7-hydroxymitragynine products in the market masquerading as kratom is misleading, unlawful, and potentially dangerous to the public.

Background: Three aconitine poisoning events occurred in two Canadian provinces in 2022: one in British Columbia (BC) and two in Ontario (ON). Aconitine is a potent alkaloid found in several species of the plant Aconitum, containing cardiotoxins and neurotoxins. It is used in traditional Chinese medicine (TCM) for pain management, and in powdered form, Aconitum is similar in appearance to sand ginger (Kaempferia galanga), which can lead to poisonings from misidentification and mislabeling. Methods: Aconitine poisoning is rare in Canada; here, we compare communications, collaborations, laboratory testing options and actions during investigations. Results: Fourteen cases occurred from the consumption of sand ginger: in BC (n = 2), purchased at an Asian health food store; in ON (n = 11), Kaempferia galanga powder (KGP) spices were used to prepare meals at a restaurant, and in one ON case, KGP was purchased. Traceback found product imported from China contained aconitine levels ranging from 1304 to 5500 ppm. Later investigations revealed mislabeling of Aconitum as KGP from the same imported lot (January 2020). Plant DNA testing found no KGP in any spice packets, including lots not linked to illness, suggestive of adulteration. Conclusion: Method development for aconitine in BC led to an improved response time for testing in ON. BC and ON updated outbreak response protocols and communications.

Introduction: The U-Dream line of products are marketed as natural health product sleep aids in Canada and as dietary supplements in the United States. Several user reviews of the product mention concerning side effects not typically associated with the listed herbal ingredients stated on the product label. Based on these concerns an analytical study was undertaken to determine if the products contained any undeclared pharmaceuticals. Methods: Product was screened by high resolution mass spectrometry (HRMS) for known pharmaceuticals with sedative and sleep promoting properties. Based on the mass spectral screens an unknown compound was isolated for characterization by nuclear magnetic resonance (NMR) and presence confirmed by Enzymelinked Immunosorbent Assay (ELISA). Results: The mass spectral analyses indicated the presence of an undeclared analogue of the pharmaceutical drug zopiclone within the product lot tested. NMR characterization confirmed the compound to be a brominated analogue of zopiclone and a commercial zopiclone/eszopiclone ELISA kit tested positive. Discussion: The undeclared compound was found to be an analogue of zopiclone whereby the chlorine atom was substituted with bromine. Given the results of the ELISA assay and the structural similarity to zopiclone it is likely the compound exhibits biological activity. Of considerable concern is not only the potential of the unknown compound to exhibit pharmacological activity, but the lack of a safety profile by which the risk can be properly assessed. Conclusions: The natural health product regulations provide a framework for high quality, safe and efficacious products to access the market. It is the responsibility of the manufacturer to assure traceability and transparency in their supply chain and establish verifiable compliance with GMP. This study illustrates the importance of careful evaluation of analytical data in order to detect undeclared adulterants and highlights the need for an active monitoring and surveillance system for potentially high-risk products.

Water kefir is a fermented beverage using water, sugar and cultured microorganism grains as the primary ingredients. Ethanol may be present at varying levels within the final product due to the fermentation process so it is vital to have a validated method to meet regulatory, quality and safety requirements. This study describes using water kefir as a matrix for the evaluation of the previously validated method employing headspace gas chromatography mass spectrometry (HS-GCMS) detection for ethanol in Kombucha. The study objective is to demonstrate the method originally using kombucha is also fit for the analysis of water kefir. This method will also evaluate the determination of methanol within the water kefir samples. The matrix extension study was performed as per the AOAC guidance documents outlined in Appendix K: Guidelines for Dietary Supplements and Botanicals utilizing HS-GCMS for ethanol determination [1]. Ethanol determination in each water kefir sample is quantified against an external standard calibration curve. The same instrumentation is used for methanol characterization. RSD_r and HorRat values obtained for from the study demonstrated acceptable precision with RSD_r values of 1.03% to 6.68% and HorRat values determined to be between 0.23-1.52 for ethanol determination within kefir samples. Similarly, acceptable values of RSD_r ranging from 1.45% to 3.39% and HorRat ranging from 0.25 to 0.49 were observed with methanol determination. For methanol determination, the LOD and LOQ determined for this method in this study to be 16 and 21 ppm respectively. The methanol spike recovery study gave overall recoveries ranging from 89-91% demonstrating acceptable method accuracy. The results of this study demonstrate the previously validated HS-GCMS method for ethanol determination in kombucha can also be used to quantify ethanol in water kefir samples. The method is also suitable for the determination of methanol within water kefir samples.