Application of ethyl chloroformate derivatization for gas chromatography-mass spectrometry based metabonomic profiling.

A new combined gas chromatography and mass spectrometry (GC-MS) method has been developed suitable for the urine sample treatment in aqueous phase with ethyl chloroformate (ECF) derivatization agents. The method has been extensively optimized and validated over a broad range of different compounds and urine samples. Analysis of test metabolite derivatives, containing spiked standards, or rat urine exhibited acceptable linearity, satisfactory intra-batch precision (repeatability) and stability, relative standard deviations (R.S.D.) less than 10 and 15% within 48 h, respectively. The quantification limits were 150-300 pg on column for most metabolites. Recovery of several representative compounds, at different concentrations, ranged from 70 to 120%, with R.S.D. better than 10% for rat urine. We were able to generally eliminate potentially confounding variables such as medium complexity, different urea concentrations, and/or derivatization procedure variability. Metabonomic profiling of 1,2-dimethylhydrazine (DMH)-induced precancerous colon rat urine using GC-MS with ECF derivatization was performed to evaluate the proposed method. The analytical variation of the method was smaller than the biological variation in the rat urine samples, proving the suitability of the method to analyze differences in the metabonome of a living system with perturbed metabolic network. Thus, the proposed GC-MS analytical method is reliable to analyze a large variety of metabolites and can be used to investigate human pathology including disease onset, progression, and mortality.

Analysis of methanol or formic acid in body fluids by headspace solid-phase microextraction and capillary gas chromatography.

Methanol and its metabolite formic acid have been found extractable from human whole blood and urine by headspace solid-phase microextraction (SPME) with a Carboxen/polydimethylsiloxane fiber. The headspace SPME for formic acid was carried out after derivatization to methyl formate under acidic conditions. The determinations of both compounds were made by using acetonitrile as internal standard (IS) and capillary gas chromatography (GC) with flame ionization detection. The headspace SPME-GC gave sharp peaks for methanol, methyl formate and I.S.; and low background noises for whole blood and urine samples. Extraction efficiencies were 0.25-1.05% of methanol and 0.38-0.84% formic acid for whole blood and urine. The calibration curves for methanol and formic acid showed excellent linearity in the range of 1.56 to 800 and 1.56 to 500 microg/0.5 ml of whole blood or urine, respectively. The detection limits were 0.1-0.5 microg/0.5 ml for methanol and 0.6 microg/0.5 ml for formic acid for both body fluids. The within-day relative standard deviations in terms of extraction efficiency for both compounds in whole blood and urine samples were not greater than 9.8%. By using the established SPME method, methanol and formic acid were successfully separated and determined in rat blood after oral administration of methanol.

Rapid GC-MS confirmation of urinary amphetamine and methamphetamine as their propylchloroformate derivatives.

Amphetamine and methamphetamine are extracted from 200 microL of urine into an organic solvent containing propylchloroformate. The amines immediately react to form propylcarbamate derivatives. The aqueous phase is removed, and a portion of the organic phase is analyzed by gas chromatography-mass spectrometry. Either the deuterated analogues or N-propylamphetamine internal standards give acceptable results. For day-to-day precision, coefficients of variation in the range of 6-10% are found. The method is linear to 10,000 ng/mL. Limits of quantitation and detection are 50 and 5 ng/mL, respectively, when using N-propylamphetamine as the internal standard. Extracts are stable at room temperature for at least 60 days. There is no interference from other amphetamine drugs.