44. Method Development to Measure Phthalate in Human Urine Specimen using LC-MS/MS
Poster: Tue 6:30-7:30PM
Rana Zahedi
California Department of Public Health
Rana Zahedi, Robert Ramage, Paramjit Behniwal, Frank Barley, Jianwen She

Biochemistry Section, Environmental Health Laboratory Branch
California Department of Public Health, Richmond, CA 94804
There is a wide variety of consumer products that contain phthalate or are covered with phthalate-containing plastic packaging. They are primarily used as plasticizer (substances added to plastics to increase their flexibility, transparency, durability, and longevity) to soften polyvinyl chloride. Diethyl phthalate was reported as an ingredient in 67 cosmetic formulations at concentrations ranging from ¡Ü0.1 % to 50%. As of 2004 manufacturers produced about 363 thousand metric tons of phthalates each year. Phthalates are easily released into the environment because there is no covalent bond between them and plastics in which they are mixed. As plastics age and break down, the release of phthalates accelerates. People are commonly exposed to phthalates, and most Americans tested by the Centers for Disease Control and Prevention have metabolites of multiple phthalates in their urine.

Some of the phthalates and their metabolites are shown to be developmental toxicants in the rodents due to different modes of action such as hypospadias and delay testes descent or vaginal pouch development, to that end they are discussed as endocrine disrupting compounds. They have also shown to have weak carcinogenic and teratogenic effects in animals. Due to their high volume use and potential toxicity to human, they are the high priority chemicals to monitor in Californians in California Environmental Contaminants Biomonitoring Program (CECBP).

We are in the process of developing and validating a method to measure phthalates in urine samples isotope dilution HPLC-tandem mass spectrometry (MS/MS). The analytical procedure involves an enzymatic deconjugation, solid-phase extraction, separation with high performance liquid chromatography, and detection by MS/MS. The MS/MS is operated in negative electrospray ionization mode. Multiple Reaction Monitoring (MRM) transitions were used for the quantitative determination of phthalate metabolites. The analytical challenges we faced included laboratory background contamination for some metabolites and instability of mBzP. Introducing a trapping column and careful sample preparation are necessary for reproducible measurement. Our detection limit for phthalate analytes are 0.5 ppb for mCPP, 2ppb for mEP, and 4ppb for mBP and mBzP. The coefficient of variation (CV) for each analyte from 8 replicates is smaller than 20%.