Liu Chang-Cai, Liu Shi-Lei, Xi Hai-Ling, Yu Hui-Lan, Zhou Shi-Kun, Huang Gui-Lan, Liang Long-Hui, Liu Jing-Quan
Laboratory of Analytical Chemistry, Research Institute of Chemical Defence, Beijing 102205, China; State Key Laboratory of NBC Protection for Civilian, Beijing 102205, China.
Laboratory of Analytical Chemistry, Research Institute of Chemical Defence, Beijing 102205, China; State Key Laboratory of NBC Protection for Civilian, Beijing 102205, China.
J Chromatogr A. 2017 Apr 7;1492:41-48. doi: 10.1016/j.chroma.2017.02.056. Epub 2017 Feb 27.
Four HD urinary metabolites including hydrolysis metabolite thiodiglycol (TDG), glutathione-derived metabolite 1,1'-sulfonylbis[2-S-(N-acetylcysteinyl)ethane] (SBSNAE), as well as the β-lyase metabolites 1,1'-sulfonylbis[2-(methylsulfinyl)ethane] (SBMSE) and 1-methylsulfinyl-2-[2-(methylthio) ethylsulfonyl]ethane (MSMTESE) are considered as important biomarkers for short-term retrospective detection of HD exposure. In this study, a single method for simultaneous quantification of the four HD metabolites in urine samples was developed using ultra high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). The four urinary metabolites were simultaneously extracted from urinary samples using a solid phase extraction (SPE) method with high extraction recoveries for all four metabolites varied in the range of 71.1-103% followed by UHPLC-MS/MS analysis. The SPE is simple and high effective only requiring 0.1mL of urinary samples and 0.5h time consuming. The problem of previous co-elution of TDG and SBSNAE in UHPLC was well solved, and complete separation of TDG, SBSNAE, SBMSE and MSMTESE from SPE-processed urine matrix was obtained to increase specificity and sensitivity. A full method validation was performed for each analyte in urine matrix. The linear range of calibration curves for the four analytes were respectively from 0.50-500ngmL for TDG and SBSNAE, 0.05-500ngmL for SBMSE and MSMTESE with coefficient of determination value (R) ≥0.990. The limit of detection was 0.25ngmL for TDG and SBSNAE, 0.01ngmL for SBMSE and MSMTESE spiked in normal urine. The intra/inter-day precision for each analyte at three QC levels had relative standard deviation (%RSD) of ≤10.3%, and the intra/inter-day accuracy ranged between 88.0-108%. This developed method allows for simultaneous and trace measurement of four HD urinary metabolites within one single determination with the lowest usage amount of urine samples over all previous methods This study provides a useful tool for early diagnosis and monitoring of HD poisoning for medical treatment with high confidence, avoiding the need for application of several analysis methods.
四种HD尿液代谢物，包括水解代谢物硫代二甘醇（TDG）、谷胱甘肽衍生代谢物1,1'-磺酰基双[2-S-（N-乙酰半胱氨酰）乙烷]（SBSNAE），以及β-裂解酶代谢物1,1'-磺酰基双[2-（甲基亚磺酰基）乙烷]（SBMSE）和1-甲基亚磺酰基-2-[2-（甲硫基）乙基磺酰基]乙烷（MSMTESE），被认为是HD暴露短期回顾性检测的重要生物标志物。在本研究中，使用超高效液相色谱-串联质谱法（UHPLC-MS/MS）开发了一种同时定量尿液样本中四种HD代谢物的单一方法。采用固相萃取（SPE）方法从尿液样本中同时提取四种尿液代谢物，所有四种代谢物的提取回收率都很高，在71.1%-103%范围内，随后进行UHPLC-MS/MS分析。SPE简单高效，仅需0.1mL尿液样本，耗时0.5小时。很好地解决了UHPLC中先前TDG和SBSNAE共洗脱的问题，实现了TDG、SBSNAE、SBMSE和MSMTESE与SPE处理后的尿液基质的完全分离，以提高特异性和灵敏度。对尿液基质中的每种分析物进行了全面的方法验证。四种分析物校准曲线的线性范围分别为：TDG和SBSNAE为0.50-500ng/mL，SBMSE和MSMTESE为0.05-500ng/mL，测定系数值（R）≥0.990。在正常尿液中加标的TDG和SBSNAE的检测限为0.25ng/mL，SBMSE和MSMTESE的检测限为0.01ng/mL。每种分析物在三个质量控制水平下的日内/日间精密度的相对标准偏差（%RSD）≤10.3%，日内/日间准确度在88.0%-108%之间。这种开发的方法允许在一次测定中同时对四种HD尿液代谢物进行痕量测量，与之前所有方法相比尿液样本用量最少。本研究为HD中毒的早期诊断和监测提供了一种有用的工具，用于高可信度的医疗治疗，避免了应用多种分析方法的需要。
