An electrospray ionization tandem mass spectrometry based system with an online dual‐loop cleanup device for simultaneous quantitation of urinary benzene exposure biomarkers trans,trans‐muconic acid and S‐phenylmercapturic acid

Abstract

An electrospray ionization tandem mass spectrometry (ESI-MS/MS) system with an online dual-loop cleanup device was developed for simultaneous quantitation of the urinary benzene exposure biomarkers trans,trans-muconic acid (ttMA) and S-phenylmercapturic acid (SPMA). The cleanup device was constructed from an autosampler, two electrically operated two-position switching valves, a reversed-phase C18 trap cartridge, a 200-microL loop, and two solvent-delivery pumps. The device was interfaced directly with a triple-quadrupole mass spectrometer and fully controlled by computer software and hardware. Because isotope dilution by introducing 13C-labeled ttMA and SPMA as internal standards was employed, the precision of the analytical system was high (for ttMA, intra- and inter-day CV values ranged from 3.82-4.53%; for SPMA, 2.13-7.06%). The calibration curves obtained using human urine spiked with ttMA were linear from 15.6-4000 microg/L (R = 0.9998) and SPMA at concentrations from 0.78-200 microg/L (R = 0.9993). The method detection limit (MDL) for SPMA was 0.23 microg/L. The MDL of ttMA could not be determined accurately because of unavailability of an appropriate blank urine matrix, but was estimated to be lower than 7.43 microg/L. Without tedious manual sample cleanup procedures the analytical system is fully automated and is therefore useful for high-throughput simultaneous determination of urinary ttMA and SPMA. The sample throughput is roughly 100 samples per day. With the selectivity and the sensitivity provided by MS/MS detection, the analytical system can be used for large-scale monitoring of environmental or occupational exposure of humans to benzene.

Validation of an HPLC–MS/MS method for the simultaneous determination of phenylmercapturic acid, benzylmercapturic acid and o-methylbenzyl mercapturic acid in urine as biomarkers of exposure to benzene, toluene and xylenes

“…Several LC-MS published methods reported slightly lower LOQ values for PMA or BMA in urine. However, these values were obtained by analysis of a pool of urine and not from different urine samples [13,[15][16][17][18][19]. Due to the unavailability of a urine sample completely free of BMA, the determination of the LOQ for BMA presented some problems.…”

“…In particular, previously published methods for the determination of MAs in urine did not test matrix effect and ion suppression [13,[15][16][17][18][19][20]. Due to the complexity of biological fluids and to the high inter-subject variability of urine in particular, assessment of matrix effects in different batches of urine has been included as part of the validation procedure.…”

“…Among published methods, HPLC/tandem mass spectrometry (MS/MS) is currently considered as the first choice for MAs determination in human urine due to its high specificity and sensitivity. Several HPLC-MS/MS methods have been reported for the determination of PMA and BMA in urine [13,[15][16][17][18][19][20] and Gonzalez-Reche et al [21] described a method for the quantification of dimethylphenyl mercapturic acid (a MA derived from the metabolism of o-xylene) in urine as a biomarker of exposure to xylenes. However, the metabolism of alkyl aromatics mainly occurs at the side chain [22][23][24], producing, in the case of xylene, a higher amount of MBMA.…”

Validation of an HPLC/MS/MS method with isotopic dilution for quantitative determination of trans,trans-muconic acid in urine samples of workers exposed to low benzene concentrations

“…Almost all today's HPLC methods use strong anionic exchange cartridges for sample cleaning and reverse-phase column separation, with UV detection at 259 nm [2][3][4][5][6]. New methods have been proposed, that still use SPE for urine clean-up and HPLC separation, but instead of UV they employ electrospray tandem mass spectrometry (MS) detection, a technique widely applied for the determination of biomarkers on account of its higher sensitivity and specificity [7][8][9][10][11].…”

“…The matrix effect changes the instrumental response of the analyte when it is dissolved in urine compared to pure solvent, lowering the accuracy in quantitative analysis; this can be overcome by using an isotopic labeled internal standard. Some methods have been validated for quantitative analysis with isotopic dilution using as internal standard a 13 C 6 labeled t,t-MA, biosynthetically prepared by injecting 13 C 6 benzene into rats [7,8] or synthetized starting from 13 C 6 labeled adipic acid [9]. These solutions, however, are not applicable in all laboratories that do routine biological monitoring of workers.…”