Determination of S‐phenylmercapturic acid in human urine using an automated sample extraction and fast liquid chromatography‐tandem mass spectrometric method

Abstract

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.

Methodological issues in the biological monitoring of urinary benzene and S-phenylmercapturic acid at low exposure levels

“…The levels of SPMA found were also in good agreement with those obtained using similar analytical conditions in nonoccupationally exposed subjects, which were 0.12 g/L in non-smokers and 1.31 g/L in smokers (overall range <0.05-3.33 g/L) [15], and below 0.5 g/L in non-smokers and 0.7-9.5 g/L in smokers [16]. Conversely, the SPMA concentrations reported in the present study were much lower than those reported in the above cited investigation [3], where median concentrations ranging from 4.1 g/L to 13.7 g/L were reported.…”

“…Particularly, a CV of 10.4% for intra-day precision and 12.2% for inter-day precision, and accuracy in the range of 88-125% were reported at 0.43 g/L of SPMA by Schettgen et al [15], and withinbatch precision of 4.6% and accuracy of about 108% were reported Table 3 SPMA: within-and between-run precision and accuracy, assessed using low, medium and high level quality controls. by Li et al [16]. These better results may be due to higher linearity in the response of the instruments used, i.e.…”

Fast determination of urinary S-phenylmercapturic acid (S-PMA) and S-benzylmercapturic acid (S-BMA) by column-switching liquid chromatography–tandem mass spectrometry

“…Methods published so far for the determination of urinary mercapturic acids of benzene and toluene involve mainly LC-MS-MS [26,27,32,33,34], LC-MS [35] or GC-MS after derivatisation of the mercapturic acids with carcinogenic diazomethane [15,31]. However, most of these methods determine only one metabolite [15,23,27,28,31,32,33,35].…”

(2-Methoxyethoxy)acetic acid: a urinary biomarker of exposure for jet fuel JP-8

“…Urinary 1-and 2-naphthols have previously been used as biomarkers for JP-8 exposure (Chao et al 2006;Serdar et al 2003Serdar et al , 2004. Metabolites of the aromatic compounds benzene and toluene have also been used to assess SD standard deviation, Max maximum detected concentration, Min minimum concentration or 0.07 lg/ml, LOD limit of detection (0.1 lg/ml), samples less than the LOD are coded as 0.07 lg/ml = LOD divided by the square root of 2 (see the text for a full explanation) exposure (Li et al 2006;Maestri et al 2005;Manini et al 2006;Marchese et al 2004). However, these compounds are encountered in the general environment through household products, cigarette smoke, fuels, and other products or factors outside the work environment.…”