δ13C analysis of amino acids in human hair using trimethylsilyl derivatives and gas chromatography/combustion/isotope ratio mass spectrometry

RATIONALE To provide a simple one-step derivatization procedure for the analysis of a wide variety of amino acids in human hair by gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS). N,O-Bis(trimethylsilyl)trifluoroacetamide (BSTFA) derivatization is already widely used outside the IRMS community, is applicable to a variety of functional groups, and provides products that are common entries in mass spectral databases, thus simplifying compound identification. METHODS Method optimization and validation were performed on a mixture of ten standard amino acids found abundantly in human hair. The method was then applied to the analysis of scalp hair from six human subjects. The hair was washed, hydrolyzed with 6 M HCl, derivatized using BSTFA in acetonitrile and analyzed using gas chromatography (GC) with concurrent quadrupole and isotope ratio mass spectrometry (IRMS) detectors. RESULTS The reproducibility for the 13C measurements, including the derivatization procedure and GC/C/IRMS analysis, on a day-to-day comparison was between 0.19 parts per thousand and 0.35 parts per thousand (SD, N=12), with an average standard deviation of 0.26 parts per thousand. Because trimethylsilylation adds 3N carbon atoms (where N=# reactive protons) to each amino acid, the 13C values for amino acid derivatives were corrected using a mass balance correction and the measured kinetic isotope effect (KIE). The KIE values ranged from 0.984 to 1.020. CONCLUSIONS The procedure gave consistent 13C values with precision similar to other derivatization methods for the range of sample sizes studied: 50-1000 mu g of each amino acid. The method gave 13C values consistent with the known literature values when applied to the analysis of amino acids in human hair. Copyright (c) 2013 John Wiley & Sons, Ltd.