Determination of thiazolidine-4-carboxylates in urine by chloroformate derivatization and gas chromatography-electron impact mass spectrometry

The derivatization method of thiazolidine-4-carboxylic acid (TZCA) and methyl-thiazolidine-4-carboxylic acid (Me-TZCA) in urine with alcohol/chloroformate was achieved. TZCA and Me-TZCA were derivatized in one step in urine with ethyl chloroformate in 1 min at room temperature. The derivatives of TZCA and Me-TZCA had very good chromatographic properties and offered very sensitive response for gas chromatography-electron impact ionization-mass spectrometry (GC-EI-MS). On the basis of derivatization, the method for simultaneous determination of TZCA and Me-TZCA in human urine was developed. Deuterated Me-TZCA (Me-TZCA-d(4)) was synthesized as the internal standard (IS) for the analysis of urine samples. TZCA and Me-TZCA were derivatized and extracted from urine at pH 9.5 with toluene, and then the dried extract was dissolved with 100 microl ethyl acetate and injected in GC/MS system. The recoveries of TZCA and Me-TZCA were about 102 and 103%, respectively, at the concentration of 0.05 mg/l. The method detection limits (MDL) were 1.0 and 0.5 microg/l, respectively, for TZCA and Me-TZCA in 1 ml human urine. The coefficients of variation of TZCA and Me-TZCA were less than 6% at the concentrations of 0.05 and 0.2 mg/l, respectively. To assess the formation of TZCA during inhalation with formaldehyde (FA) (about 3.1 and 38.1 ppm FA in air), urine samples from rats were taken during 3 days after initiation of treatment. The mean amount of TZCA determined was 0.07 mg/l in control group and 0.18 mg/l during treatment with 3.1 ppm. The TZCA levels increased up to about 1.01 mg/l during treatment with 38.1 ppm. It is planned to study whether urinary TZCA can be used as an indicator in the biological monitoring of exposure to FA.