Negative chemical ionization gas chromatography/mass spectrometry to quantify urinary 3-methylhistidine: Application to burn injury

A rapid method for measuring 3-methylhistidine (3MH) in rat and human urine with higher sensitivity and precision than any previously reported method is described using internal standard [1-13C]3MH (M + 1) and negative chemical ionization (NCI) gas chromatography/mass spectrometry (GC/MS). Internal standard [1-13C]3MH (M + 1) was added to rat and human urine samples, hydrolyzed, and absorbed onto cation exchange columns. The column eluent was dried and derivatized for GC/MS analysis. Quantification of 3MH levels was accomplished by monitoring the m/z 204 fragment. The m/z 204 fragment was chosen due to the fragment’s abundance and stability as determined by analysis of [methyl-2H3, 18O2]3MH (M + 7) and [methyl-13C]3MH (M + 1) fragmentation patterns under NCI conditions. This method shows excellent linearity (0.9989) over the range studied (0–0.5 mol), high recovery (95.9%), and low coefficient of variation (4.7%). The described method is sensitive enough to detect 6.8 pmol amount of urinary 3MH with a precision of 9.1%. The in vivo utility of this method to quantify urinary 3MH was tested in a burn injury rat model and on urine specimens from pediatric burn patients. Data obtained from the urine of burn-injured rats and pediatric burn patients match previously reported trends and validate the in vivo utility of this method.