Fast determination of mazindol in hair sample using UPLC-MS/MS: An isotopic identification approach

Abstract

Accidents on Brazilian highways kill more than 40 thousand people every year, not considering those who are injured and/or disabled by them. Since the implementation of federal law 13103/2015 to control the use of drugs by truck drivers, where the control is done by analysis of the substance in hair by a retroactive minimum period of 90 days, more than 100.000 people have been detected with a presence of one or more prohibited drugs in their hair samples. The Mazindol (cutoff of 0.5 ng/mg) is on of fourteen prohibited substances in the list and the second most stimulant in use. Mazindol is a sympathomimetic agonist causing central nervous system stimulation, acting together at peripheral alpha and beta-receptors. However, a serious side effect is the alteration of perception, which can be associated with accidents involving roads. In Brazil, the National Sanitary Agency (ANVISA) banned the production and marketing of this substance throughout the national territory in 2011, as the drug did not present proven effectiveness in its main indication: the treatment of obesity. After six years, in June of 2017 the federal law by the Brazilian congress (13.454/2017) authorizes once again the production and marketing of mazindol, amphetamine, sibutramine and femproporex. Nowadays, the majority of Brazilian laboratories use the mass spectrometry technique as a screening and confirmatory test on their routine analyses. The present study presents the use of isotopic separation by UPLC-MS/MS to confirm the presence of mazindol in a hair sample. Hair samples collected from real cases were obtained from authorized patients for the study. The method was validated and accredited to ISO/IEC 17025 standards for the specificity, sensitivity, linearity, accuracy, precision, recovery, and stability using 10 mg of hair. LOD and LOQ were 0.25 ng/mg. The chromatography run time was 2.2 min. The hair samples were washed and then extracted with organic solvent in the presence of deuterated analogues followed by an incubation under 60 °C for 8 h Reverse phase separation was performed with the Acquity BEH C18 chromatographic column (50 × 2.1 mm × 1.7 μm). Mazindol has chlorine atom in its chemical structure; it's possible to perform an isotopic separation and identification as the approach to confirm mazindol in hair. Considering the chlorine isotope has 35 Cl and 37 Cl in 1/3 proportion, the mass spectrometry was set to determine 285.1 and 287.1 m/z with transition 44 m/z. Instead of evaluating one parent ion and one product ion, we evaluated two parent ions (isotopic) and the same product ion. Due the mazindol presents interference in the most intense fragment in a fast-chromatographic analysis, the fragment 285.2 > 44 m/z has no interference in real samples. For mazindol quantitation, the transition 285.1 > 44 m/z can be used and for confirmation criteria an isotope transition of mazindol 287.1 > 44 m/z can be used, thus increasing the reliability of the generated data without interference. The method was applied in a two real sample and mazindol was determined and confirmed by the present method, 1.18 ng/mg and 2.18 ng/mg. The isotope separation and identification by tandem triple quads is a useful way to confirm chemical structure when there is no formation or there is a no abundant formation of ion for quality analysis and confirmation the presence of substance using a fat chromatographic separation.