Development and validation of a method for simultaneous analysis of hair underivatized amino acids and damage...

The forensic and medical fields are seeing growing interest in the amino acid and damage biomarker composition of hair, in order to identify adulteration of drug hair testing and for diagnostic purposes. Therefore, there is an increased demand for quick and accurate analytical methods. This study presents the first liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay for the simultaneous quantification of hair amino acids and four damage biomarkers, which also implements an isotopic dilution strategy to improve recovery and precision of the acid hydrolysis-sensitive analytes. The applied strategy enabled a recovery of the hydrolysis-sensitive amino acids between 83 and 120% (vs. 33-77%, without isotopic dilution) for two different protein standards, and a precision with a relative standard deviation (RSD) between 1.3 and 7.5% (vs. 9.0-29.4%, without isotopic dilution). All 21 analytes could be measured without interferences by matrix and sample components, thus demonstrating satisfactory selectivity of the method. For spiked samples of hair hydrolyzate, recovery was between 88 and 120%, whereas precision and intermediate precision were below 10.1%. The high sensitivity of the method made it possible to reduce sample preparation to a 10000-fold dilution of the raw hydrolyzate. The wide linear range displayed by the method allowed the simultaneous quantification of minor (0.3 μmol/g of hair) and major (up to 1000 μmol/g of hair) components of the biological fiber. This method was successfully applied to the analysis of real hair samples submitted to six different treatments. Statistical data analysis by means of t-test and principal component analysis (PCA) showed a clear discrimination of the treated from the untreated hair samples and of the different treatments. Since these hair treatments can interfere with hair drug testing, the method possesses the ability of identifying hair samples with potential for attempted drug test evasion. In addition, lanthionine emerged as a new biomarker for heat damaged hair.