Objective To achieve high throughput determination of metabolites of phthalates in human urine, a simultaneous determination of 12 phthalates metabolites in human urine using ultra high performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method was established by optimizing a solid phase extraction process of a previous study.
Methods An aliquot of 2 mL human urine was hydrolyzed with β-glucuronidase, followed by enrichment and purification with a strong anion-exchange solid-phase extraction cartridge. The target analytes were eluted by 2% formic acid in methanol solution and directly injected for analysis without evaporation or reconstitution. The separation of target analytes was conducted on an ACQUITY UPLC BEH Phenyl column (100 mm×2.1 mm, 1.7 μm) with gradient elution by 0.1% acetic acid aqueous solution and 0.1% acetic acid in acetonitrile. Twelve phthalates metabolites were simultaneously determined under multiple reaction monitoring mode using negative electrospray ionization. The concentrations of 12 target analytes were quantified by isotope dilution analysis.
Results The 12 phthalates metabolites showed good linearities in the range of 1.5-200 ng/mL for MiBP, 2.5-300 ng/mL for MnBP, or LOQ-100 ng/mL for other 10 target analytes, all with correlation coefficients of >0.998. The recoveries of 12 target analytes at low, moderate, and high spiked levels ranged 86.4%-106%. The intra-and inert-day precisions were 1.2%-5.8% and 5.9%-12%, respectively. Standard reference material was used to evaluate method accuracy. The relative error between the measured values and the reference values ranged from 2.0%-11%.
Conclusion Compared with the previous method, UPLC-MS/MS method is superior in convenience and efficiency. It is suitable for high throughput determination of the 12 phthalates metabolites in human urine.
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