title: Determination of five di-(2-ethylhexyl)phthalate metabolites in urine by UPLC-MS/MS, markers of blood transfusion misuse in sports.
abstract: Di-(2-ethylhexyl)phthalate (DEHP) is the most commonly used plasticizer for polyvinyl chloride, which is found in a large variety of products, including most of the bags used for blood storage because of its protective role on erythrocytes survival. DEHP metabolites have been recently proposed as markers of the misuse of blood transfusion in athletes. In this study, a method to quantify the main five DEHP metabolites in urine has been developed: mono-(2-ethylhexyl)phthalate (MEHP), mono-(2-ethyl-5-hydroxyhexyl)phthalate (MEHHP), mono-(2-ethyl-5-oxohexyl)phthalate (MEOHP), mono-(2-ethyl-5-carboxypentyl)phthalate (5cx-MEPP), and mono-(2-carboxymethylhexyl)phthalate (2cx-MMHP). The method involved an enzymatic hydrolysis with β-glucuronidase from Escherichia coli followed by an acidic extraction with ethyl acetate. The hydrolysed extracts were analysed by ultraperformance liquid chromatography tandem mass spectrometry. Isotope labelled MEHP, MEOHP and 5cx-MEPP were used as internal standards. Analysis of all the metabolites was achieved in a total run time of 10min, using a C(18) column and a mobile phase containing deionized water and acetonitrile with formic acid, with gradient elution at a flow-rate of 0.6mLmin(-1). Detection of the compounds was performed by multiple reaction monitoring, using electrospray ionization in positive and negative ion modes. The method was validated for quantitative purposes. Extraction recoveries were greater than 90% and the limits of quantitation ranged from 1.2 to 2.6ngmL(-1). Intra-day precisions were better than 8% for all metabolites while inter-assay precisions were better than 12%. Concentrations of DEHP metabolites were measured in a control group (n=30, subjects reflecting the common environmental DEHP exposure), and in sportsmen (n=464), to evaluate population distribution exposure to DEHP. Additionally, threshold concentrations indicating outliers of common exposure for DEHP metabolites are proposed.
authors.affiliation: Grup de Recerca en Bioanàlisi i Serveis Analítics, Programa de Recerca en Neurociències, Institut Hospital del Mar d'Investigacions Mèdiques, Barcelona, Spain.
authors.forename: Núria
authors.initials: N
authors.lastname: Monfort
authors.forename: Rosa
authors.initials: R
authors.lastname: Ventura
authors.forename: Georgina
authors.initials: G
authors.lastname: Balcells
authors.forename: Jordi
authors.initials: J
authors.lastname: Segura
mesh_terms: D000328:Adult; D001803:Blood Transfusion; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004051:Diethylhexyl Phthalate / Q000652:urine*; D004300:Doping in Sports; D005260:Female; D006801:Humans; D016018:Least-Squares Analysis; D008297:Male; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D021241:Spectrometry, Mass, Electrospray Ionization; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D004051:Diethylhexyl Phthalate