Rapid Determination of Two Kinds of Metabolites in Urine by Acetonitrile Precipitation-Ultra High Performance Liquid Chromatography-Tandem Mass Spectrometry
A method for rapid,simultaneous determination of two metabolites of benzene,trans,trans-muconic acid (t,t-MA) and S-phenylmercapturic acid (S-PMA),in human urine by ultra high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was established. Urine sample was acidified and protein was removed by acetonitrile precipitation. The supernatant was concentrated by nitrogen blowing and redissolved by solvent,and then analyzed by UPLC-MS/MS. The compounds were separated on a BEH C18 column with acidic ammonium formate solution and methanol solution as mobile phases for gradient elution and then quantified in multiple reaction monitoring (MRM) mode by internal standard method. Combined with the 96-well sample collection plate as a pre-treatment vessel,large quantities of samples could be rapidly analyzed. The linear ranges for detection of t,t-MA and S-PMA were 20-2000μg/L and 0.5-20μg/L,respectively,and the correlation coefficients were 0.9998. The limits of detection were 1.2 and 0.06μg/L,respectively. The recoveries were 90.6％-103.4％ and 87.3％-101.7％,and the relative standard deviations (RSDs) were in range of 1.5％-3.5％ and 1.8％-4.2％,respectively. The method was applied to detection of 41 urine samples of employees exposed to benzene. The detection rates of t,t-MA and S-PMA were 95％ and 78％,respectively. Compared with solid phase extraction method,this method showed many advantages such as simple operation,rapidity and high efficiency,high sensitivity and accuracy,which was suitable for rapid detection of t,t-MA and S-PMA in large quantities of urine samples.
Acetonitrile precipitationUltra performance liquid chromatography-tandem mass spectrometryUrineTrans,trans-muconic acidS-Phenylmercapturic acid
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