The distribution of amino acids in hair can divulge information regarding the health (e.g., diabetes) and provide a means for detecting the history of the disease by segmentation of the hair as well as attributes of an individual (e.g., sex and age). Therefore, an nonenzymatic method of hair digestion and profiling is required. In addition to optimizing and validating a method for measuring the distribution of amino acids in human hair, a robust and comprehensive approach to objectively compare the most effective means of extracting and manipulating chromatographic data to obtain the best limits of detection, linearity, and sensitivity are provided.
Data comparisons were made by operating the mass spectrometer in a mode that rapidly switches between total ion current (TIC) and selected ion monitoring (SIM) modes during each sample injection. In this way, any external confounding factors were negated that may otherwise influence the comparison of the linearity and sensitivity between the two modes of operation. The use of SIM, peak areas, and an internal standard provided significantly better sensitivity and limits of detection than using peak heights, TICs, or no internal standard.
The sample preparation steps included protein acid hydrolysis using hydrochloric acid and trimethylsilyl (TMS) derivatization using N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA). The optimal derivatization conditions were acetonitrile as reaction solvent, temperature of 100°C, and a reaction time of 30 min.
The method was validated by measuring the amino acid content of myoglobin. This validation was accurate for nine of the fourteen amino acids found in myoglobin and gave detection limits in the range of 0.04–0.1 μmol/L, quantitation limits in the range of 0.1–0.5 μmol/L, recoveries between 80% and 110%, and linear models with coefficients of determination (R2) greater than 0.99 in the tested range from 1 to 300 μmol/L. The remaining five amino acids of myoglobin were deleteriously affected by acid hydrolysis.