Ultra-performance liquid chromatographic assay coupled with two-dimensional separation for spectrometric determination of urinary S-phenylmercapturic acid

Abstract

A gradient ultra-performance liquid chromatographic (UPLC) assay coupled with the two dimensional separation and diode array detection method was developed to determine S-phenylmercapturic acid (SPMA), a specific urinary metabolite of benzene. Gradient separation was performed on two C18 core-shell columns (2.6 and 1.7 μm, 50 × 2.1 mm i.d.) maintained at 40 and 20 °C. Among various solvents evaluated, dichloromethane which offered >85% recovery rates of both SPMA and d-benzylmercapturic acid (internal standard) was used for extraction. The yield of free-SPMA was maximized by hydrolyzing 200 μL of urine with 20 μL of 6 M HCl prior to dichloromethane extraction. The limit of detection was 1 μg L-1(s/n = 5) and the limit of quantification was 2 μg L-1. Using our method, a correlation of 0.99 was obtained with 10 specimens (range, 1.7 to 182 μg L-1) from the German External Quality Assessment Scheme. Among the 75 non-occupational exposure individuals, 61 (81%) had SPMA levels below the LOQ. Among the 26 male petroleum workers, 5 who were exposed to