Determination of Organophosphate Esters and Metabolites in Serum and Urine by Ultra-High Performance Liquid Chromatography-Tandem Mass Spectrometry

A new method was developed for simultaneous detection of total 19 kinds of organophosphate esters (OPEs) and their diester metabolites (di-OPEs) in human serum (1.0 mL) and urine (1.5 mL) with low volume of samples. The target compounds were determined using ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) after acetonitrile liquid-liquid extraction combined with purification using an ENVI-18 solid-phase extraction (SPE) column. OPEs and di-OPEs were separated using a Shim-pack GIST C 18 column (100 mmx2.1 mm, 2 mu m) with a Shim-pack GIST-HP (G) C (18) guard column. An electrospray ionization source (ESI) was employed in mass spectrometry analysis, with positive/negative ion mode using the multiple reaction monitoring (MRM). All target compounds were separated within 15 min, and exhibited good linear relationships in the concentration range of 2-100 ng/mL, with correlation coefficients (R-2) above 0.994. The method detection limits (MDL) in serum ranged from 0.001 to 0.178 ng/mL and the MDL in urine ranged from 0.001 to 0.119 ng/mL. The recoveries of the analytes spiked in serum and urine matrices at two concentration levels were 30.5%-126.8%, with the relative standard deviations (RSDs) ranged from 1% to 23%. In addition, paired serum and urine samples from 11 patients were analyzed. For all samples tested, the internal standards of OPEs exhibited recoveries between 61% and 114%, whereas the internal standards for di-OPEs had recoveries ranging from 43% to 103%. OPEs and di-OPEs exhibited high detection frequencies in 22 serum and urine samples. Triethyl phosphate (TEP), tributyl phosphate (TBP), tris(2-ethylhexyl) phosphate (TEHP), tris(2-butoxyethyl) phosphate (TBEP), tris(1-chloro-2-propyl) phosphate (TCIPP), triphenyl phosphate (TPHP), tri-m-tolyl-phosphate (TMTP) and 2-ethylhexyl diphenyl phosphate (EHDPP) were universally detected in all serum samples. TCIPP was identified at the highest concentrations (median 0.548 ng/mL) in serum samples. In urine samples, the detection frequency for 12 kinds of target compounds reached 100%. Notably, TBP emerged as the predominant OPE in urine, demonstrating a median concentration of 0.506 ng/mL. Regarding di-OPEs, bis(2-chloroethyl) phosphate (BCEP) and bis(2-butoxyethyl) hydrogen phosphate (BBOEP) were the most abundant in urine, with median concentrations of 6.404 and 2.136 ng/mL, respectively. The total concentrations of OPEs and di-OPEs in serum and urine were 1.580-3.843 ng/mL and 5.149-17.537 ng/mL, respectively. These results not only confirmed the effectiveness of the method in detection of OPEs and di-OPEs in biological matrices, but also revealed the widespread presence of OPE compounds in human body and pointed to potential exposure risks.