Determination of S-phenylmercapturic acid in human urine using an automated sample extraction and fast liquid chromatography-tandem mass spectrometric method.
S-phenylmercapturic acid is widely accepted as a specific biomarker for the evaluation of benzene exposure. Here, we describe a fast, specific and sensitive high-performance liquid achromatography coupled with tandem mass spectrometry (LC-MS/MS) method that has been developed and validated for the determination of S-phenylmercapturic acid in human urine. Isotope-labeled S-phenylmercapturic acid-d5 was used as internal standard to improve the method ruggedness. The fully automated solid-phase extraction method on a 96-well Oasis MAX (mix-mode anion exchange) plate was employed to clean up the urine samples before analysis. The rapid LC-MS/MS analysis of extracted samples was achieved on a Genesis C18 column with a run time of only 3 min. Negative electrospray ionization with multiple reaction monitoring (ESI-MRM) mode was used to detect S-phenylmercapturic acid (m/z 238 --> 109) and S-phenylmercapturic acid -d5 (m/z 243 --> 114). The method fulfils all the standard requirements of method validation. The calibration curve was linear within the concentration range 0.400-200 ng/mL. The method performed accurately and precisely in validation with <7.5% relative error and <6.5% relative standard deviation of quality control samples. The method efficacy was also verified by the analysis of urine samples from 12 smokers and 12 non-smokers. With the fully automated sample cleanup procedure and the fast LC-MS/MS analysis, a sample analysis throughput of 384 samples per day could be achieved.
Li
Yinghe
Y
Covance Laboratories Inc., Madison, WI 53704, USA. yinghe.li@covance.com
Li
Austin C
AC
Shi
Haihong
H
Junga
Heiko
H
Jiang
Xiangyu
X
Naidong
Weng
W
Lauterbach
John H
JH
D000111:Acetylcysteine / Q000031:analogs & derivatives* / Q000652:urine; D001331:Automation; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D005260:Female; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D012015:Reference Standards; D012680:Sensitivity and Specificity
C053003:S-phenyl-N-acetylcysteine; D000111:Acetylcysteine