Table 2Phthalate compounds tested.The methodology for overnight delivery determining parent phthalates in serum and sweat was as follows. Samples (serum and sweat) were weighed into glass tubes (ca 1g) and 1mL of acetonitrile was added in order to precipitate serum and plasma proteins. The resulting mixture was serially extracted twice with 5mL portions of hexane:dichloromethane (8:1, v/v) using sonication as per Colon et al., [55]. The resulting extracts were combined and concentrated to 200 microliters. Analysis was performed using gas chromatography/selected ion-monitoring mass spectrometry. Ions monitored include: dimethylphthalate (DMP), m/z 194/163; diethylphthalate (DEP), m/z 177/222; dibutylphthalate (DBP), m/z 223/278/205; benzylbutylphthalate (BBP), m/z 206/238; dicyclohexylphthalate (DCHP), m/z 249/330; diethylhexylphthalate (DEHP), m/z 279/390; disonylphthalate (DiNP), m/z 293/418.

Prior to analysis all extracts were diluted 1:4 with hexane. Quantitation was performed using external standard calibration. Quality control was measured by analyzing method blanks, analyzing water fortified with the analytes of interest, as well as calf serum samples. The recovery of the phthalates from fortified water was 87�C108% with a relative standard deviation of 1.9 to 9.0%. The relative percent difference for calf serum was 0.7 to 12% with the exception of DMP which was 23%. Instrument detection limits were determined to be 8ng/g. Serum, sweat, and urine were analyzed for phthalate metabolites following the general procedures established by the US Centers for Disease Control and Prevention [80, 81].

Briefly, 1.0g of serum, sweat, or urine was fortified with 10 nanograms of isotopically-labelled phthalate metabolites, 20 micrograms of 4-methylumbelliferone glucuronide, 20-micrograms of labeled 4-methylumbelliferone, 500 microliters of ammonium acetate buffer (pH 6.5), and 10 microliters of ��-glucuronidase (Escherichia coli K12, Roche Biomedical). The samples were mixed and incubated at 37��C for 90 minutes to allow for the deglucuronidation of the phthalate metabolites. Following enzymatic hydrolysis, an aliquot (20uL) was removed and analyzed for 4-methylumbilliferone GSK-3 to determine enzymatic hydrolysis efficiency. The remainder was removed and loaded onto a Zymark Rapid Trace Station for automated solid phase extraction (SPE). The 60 milligram/3mL Oasis-HLB cartridges was conditioned with HPLC-grade methanol (2mL) and 0.1M formic acid (1mL). The samples were diluted with 5mL of 0.1M formic acid and loaded onto the SPE cartridge at a rate of 0.5mL/min.