Tandem derivatization combined with salting-out assisted liquid–liquid microextraction for determination of biothiols in urine by gas chromatography–mass spectrometry

• Published in: Journal of Chromatography A 2017-11, Vol.1524, p.29-36
• Main Authors: Tsai, Chia-Ju, Liao, Fang-Yi, Weng, Jing-Ru, Feng, Chia-Hsien
• Format: Article
• Language: English
• Subjects: Cysteine; Gas chromatography–mass spectrometry; Homocysteine; Polar organic compound; Salting-out assisted liquid–liquid microextraction; Tandem microwave-assisted derivatization
• ISSN: 0021-9673
• DOI: 10.1016/j.chroma.2017.09.069

•Tandem derivatization of biothiols with multi-polar groups using Br-TFB was achieved.•Excess derivatizing reagent can be recovered and used again by SALLME.•This analytical method enhanced the detectable signal for biothiol analysis by GC–MS in urine.•A fast and environment-friendly derivatization and microextraction technique was developed. Detection of polar organic compounds (POCs) using gas chromatography (GC) is not straightforward due to high polarity, hydrophilicity, and low volatility of POCs. In this study, we report a tandem microwave-assisted derivatization method combined with salting-out assisted liquid–liquid microextraction (SALLME) to modify successively the polar groups of POCs in protic and aprotic solvents. Biothiols (cysteine and homocysteine) served as a proof of concept for this method because they possess three polar groups (thiol, amine, and carboxyl); the derivatizing reagent was 3,4,5-trifluorobenzyl bromide (Br-TFB) for alkylation. The solubility of the POCs in the protic or aprotic reaction medium affected the number of TFB molecules attached. Using the tandem derivatization with Br-TFB, the thiol and amine groups of biothiols were alkylated in the protic system, and the carboxylic groups of biothiols were alkylated in the aprotic system. The developed method was then successfully applied to measure biothiols in human urine. Because of the complex urine matrix and the lack of urine samples without endogenous biothiols, the standard addition method was utilized to avoid the matrix effect, check the recovery, and calculate the initial biothiol content in the urine. Regarding the linearity of the standard addition curves, the coefficient of determination was >0.996, and the linear regression showed satisfactory reproducibility with a relative standard deviation