Testing for betel nut alkaloids in hair using UHPLC-MS/MS and UHPLC-Q-ToF-MS

Abstract

Simultaneous identification and quantification of 4 betel nut alkaloids (arecoline, arecaidine, guvacoline and guvacine), in the hair of papuans abusers. Betel nut or areca nut, also known as “buai” in Papua New Guinea is the fruit of Areca catechu palm. This nut is highly consumed by the local population and represents a large part of the internal commercial activity of the island. The betel nut is mixed with lime powder made of shell or coral and stick mustard, which is a kind of bean also called “daka”. This mixture, of a typical red color, is chewed all along the day and finally spat in the street by the residents. Two major compounds are presents in betel nut: arecoline and guvacoline. In presence of lime (alkaline conditions), these alkaloids are hydrolyzed into arecaidine and guvacine respectively. These 4 substances have stimulating, anorectic and mildly euphoric effects. Some side effects are associated with this addiction, and the use of betel nut is the principal cause of mouth and esophagus cancer in Papua New Guinea. As part of the study on dietary habits, phenotypic and genetic diversity of papuans residents, our laboratory was asked to analyze the active substances in hair to document exposure. Hair samples were collected from 19 adult subjects (males = 11; females = 8) by some of the authors and sent to the laboratory for analysis. The 4 alkaloids of betel nut have very similar chemical structures. In order to identify accurately the drugs, two methods were developed. First, the compounds were identified using an ultra-high-performance liquid chromatography system coupled to time-of-flight mass spectrometry (Waters XEVO ® G2-XS qTof). Then, they were quantified by an ultra-high-performance liquid chromatography system coupled to tandem mass spectrometry (Waters XEVO ® TQD). Hair samples were twice decontaminated with dichloromethane and cut into very small segments using scissors. Given the curvy nature of all the specimens, there was no attempt to do segmentation. Then, 20 mg were incubated in methanol during 2 h 30 in an ultrasound bath. After cooling, the methanol was evaporated to dryness in presence of 20 μL octanol to prevent volatilization. Linearity was observed for concentration ranging from LOQ to 20 ng/mg with a correlation coefficient R 2 of 0.9994, 0.9999, 0.9999 and 0.9981 for arecoline, arecaidine, guvacine and guvacoline respectively. Measured concentrations were in the range: 60 pg/mg to 18 ng/mg for arecoline ( n = 19), 14 pg/mg to 2.5 ng/mg for guvacoline ( n = 11), 63 pg/mg to 3.8 ng/mg for arecaidine ( n = 11) and 100 pg/mg to 3.2 ng/mg for guvacine ( n = 6). There was no correlation between concentrations of arecoline and arecaidine (ratio arecaidine/arecoline from 0.01 to 0.18) and guvacoline and guvacine (ratio guvacine/guvacoline from 0.06 to 3.50). However, the identification of these substances in hair is a good marker of consumption of betel nut, and allows to document a local practice which remains difficult to evaluate just by questioning.