(670f) A Parsimonious and Accurate Method for Determination of Phthalates and Dinch Metabolites in Human Samples, Using High Performance Liquid Chromatography (HPLC–MS/MS)

Phthalates and their substitute Hexamoll® DINCH®, are a group of plasticizers with a production volume of millions of tons per year. They are widely used in the manufacture of plastics, to make them soft and flexible, and in personal care products. They can be found in everyday products such as soaps, suntan lotion, soft plastic toys, plastic bottles, raincoats, shoes and food packaging. Due to their endocrine-disrupting properties, some phthalates have been assigned use restrictions since the late 1990s. There is a societal concern due to their toxicity to reproduction and presence in biological matrices of humans with Greenpeace having conducted several studies addressing phthalates in consumer products and the potential health effects emerging from its endocrine-disrupting effects in the early 2000s.

For this study, we analyzed phthalate metabolites urine samples from a pilot study of 20 volunteers. The volunteers cover an age range between 5 and 45 years. Three of the volunteers were children under 10 years, ten volunteers were between 20-30 years old, five between 30-40 years old and 2 older than 40 years old. Eleven were male, and 9 were female. The urine samples were collected in the morning before eating anything. All the volunteers provide their last day description of physical activities and also their last meal description. The morning urine samples, after determination of their full volume, were aliquoted into polypropylene tubes or glassware vials and stored at temperatures −80◦C, prior to sample preparation and analysis.

In the morning urine samples of the pilot study, 14 phthalate metabolites and 2 Hexamoll® DINCH® metabolites were determined by on-line high performance liquid chromatography coupled to tandem mass spectrometry (HPLC–MS/MS) using internal isotope-labelled standards according to the new method that we have develop in our lab. Our analytical method can lead to the quantification of 14 phthalates metabolites (MEP, MBzP, MiBP, MnBP, MCHP, MnPeP, MEHP, 5OH-MEHP, 5oxo-MEHP, 5cx-MEHP, MnOP, OH-MiNP, cx-MiNP, and OH-MiDP) and 2 Hexamoll® DINCH® metabolites (OH-MINCH and cx-MINCH) with very low limits of detection and quantification (0.2 μg/L).

In brief, for sample preparation, aliquots of 900μL of the urine samples, calibration samples and quality control samples were added to 372 μL of ammonium acetate buffer (1 M; pH = 6.0–6.4), 18 μL Glucuronidase K12 from E.coli, arylsulfatase-free (Roche Diagnostics Mannheim, Germany; diluted 1:1 with ammonium acetate buffer) and 67.5μL internal standard solution (of the isotope labelled analogues). Samples were incubated in a water bath for 3 h at 37◦C for enzymatic hydrolyses of the conjugates. After incubation,33.8 μL of acetic acid were added to samples to adjust the pH value. Samples were frozen for at least 3 hrs at −20◦C to precipitate proteins. After that, samples were thawed at room temperature and transferred into a 1.8 ml glass vial for instrumental analysis. For liquid chromatography 500 μL of the above solution were injected into a Thermo Scientific Accela LC Systems. Sample clean-up and enrichment was performed on the Spark Holland On-Line SPE system. Chromatographic separation was performed on a Target-Core Phthalates column C18, 100 x2.1mm, 2.6um. The solvents used were LC-MS grade 100% water with 0.1% acetic acid (solvent A) and 100% acetonitrile with acetic acid with 0.1% acetic acid (solvent B). The 6-port valve was switched to direct the analytical gradient in backflush mode through the clean-up column onto the analytical column. At 18 min the 6port valve was changed back into starting position. The total runtime for one sample was 26 min. Mass spectrometric detection and quantification was performed on a Thermo TSQ Quantum Ultra tandem MS mass spectrometer used in negative ionization mode (ESI-). For each analyte, two ion transitions were recorded. One was used for quantification (quantifier) and the other one for verification (qualifier). For the labelled internal standards, one ion transition was recorded. The mass spectrometer was operated in scheduled multiple reaction monitoring (sMRM) mode. The respective ion transitions are given in Table 2. The precision of the method was determined by analyzing a low and high concentration control material pooled from native urine samples in each analytical batch. Metabolite concentrations and conjugation status thus represented the characteristics of general population urine samples. The accuracy of the method was determined by spiking standard solutions to native urine samples. The limits of quantification (LOQ) of the individual phthalate metabolites were derived based on a signal-to-noise ratio. Accuracy and international comparability of the analytical procedure were ensured and verified by the successful participation in the External Quality Assessment Scheme (EQUAS) of the European HBM4EU project on human-biomonitoring and by obtaining certificates of the RIKILT –Wageningen University & Research Organiser of the Phthalates exercise on behalf of the HBM4EU Quality Assurance Unit offered for the metabolites, MEP, MBzP, MiBP, MnBP, MEHP, 5OH-MEHP, 5oxo-MEHP, 5cx-MEPP, MnOP, OH-MiNP, cx-MiNP, OH-MINCH and cx-MINCH in human urine to the Health and Exposome Research Centre (HERACLES), Center for Transdisciplinary Research and Innovation (KEDEK), Aristotle University of Thessaloniki.

In sum, the presented human biomonitoring method of phthalates and Hexamoll® DINCH® is characterized by higher sensitive in comparison to the existing ones in the literature thus results in reliable results of 16 urinary biomarkers of exposure to plasticizers faster than before (in only one analytical run).