Simultaneous determination of alpha-, beta- and gamma-hydroxybutyric acids in micro-pulverized human hair by GC-MS: Method development, validation and application

An analytical method was developed and validated for the simultaneous determination of alpha, beta and gamma-hydroxybutyric acids (AHB, BHB and GHB, respectively) in human hair. Hair samples (10 mg) were pulverized and submitted to extraction using methanol (1 mL) for 10 min. The extract was centrifuged, filtered, and evaporated to dryness at 40 °C under a gentle N2 flow, dissolved in ethyl acetate (0.050 mL) and derivatized using 0.050 mL of BSTFA containing 1% TMCS, at °C for 40 min. The derivatives were determined by GC/MS. Aliquots of natural hair (“blank“) samples after water extraction (3 steps) were pulverized, spiked with AHB, BHB, GHB or d6-GHB and used for method validation. Figures of merit showed the method’s feasibility. The LOQ values were 1 ng mg−1 for BHB and AHB and 0.6 ng mg−1 for GHB, and mean recoveries were 54%, 69% and 75% for AHB, BHB and GHB respectively. Precision and bias were better than 15% and 20% respectively. Calibration curves (LOQ to 20 ng mg−1 for AHB and BHB and LOQ to 16 ng mg−1 for GHB) obtained in 5 days were pooled for statistical analysis, but the data were heteroscedastic, as demonstrated by the White test. Therefore, weighted linear regression and data transformations were applied and the best results were obtained using the Box-Cox transformation [c′ = (cλ-1)/λ] for GHB and log transformation for AHB and BHB. Authentic hair samples were collected from the posterior head vertex of 23 volunteers (16 females and 7 males). Four of them declared having type-2 diabetes (T2D). Only female volunteers reported hair treatments such as dyeing, flat ironing, straightening and/or bleaching. Dyeing appeared not to affect GHB levels in hair, but no data were found in the literature about other cosmetic treatments. GHB levels varied from