Quantification of free and total bisphenol A and bisphenol B in human urine by dispersive liquid–liquid microextraction (DLLME) and heart-cutting multidimensional gas chromatography–mass spectrometry (MD–GC/MS)

Abstract

A novel method combining dispersive liquid–liquid microextraction (DLLME) and heart-cutting multidimensional gas chromatography coupled to mass spectrometry was developed for the determination of free and total bisphenol A (BPA) and bisphenol B (BPB) in human urine samples. The DLLME procedure combines extraction, derivatization and concentration of the analytes into one step. Several important variables influencing the extraction efficiency and selectivity such as nature and volume of extractive and dispersive solvents as well as the amount of acetylating reagent were investigated. The temperature and time to hydrolyze BPA and BPB conjugates with a β-glucuronidase and sulfatase enzyme preparation were also studied. Under the optimized conditions good efficiency extraction (71–93%) and acceptable total DLLME yields (56–77%) were obtained for both analytes. Matrix-matched calibration curves were linear with correlation coefficients higher than 0.996 in the range level 0.1–5 μg/l, and the relative standard deviations (%RSD) were lower than 20% ( n = 6). The limits of detection were 0.03 and 0.05 μg/l for BPA and BPB, respectively. The applicability of the proposed method for determining urinary free and total BPA and BPB was assessed by analyzing the human urine of a group of 20 volunteers. Free BPA was detected in 45% of the sample whereas total BPA was detected in 85% of the samples at concentrations ranging between 0.39 and 4.99 μg/l. BPB was detected in conjugated form in two samples.