The cooked meat carcinogens 2-amino-1-methyl-6-phenylimidazo[4 5 to 250 or 500 at a scan speed of 500 amu/s using the same acquisition parameters as above. 6 12 16 20 40 60 or 100 pg whereas the unlabeled IQx-8-COOH standard was added at 0 30 60 80 100 200 300 or 500 pg/mL of urine. The calibration curves of PhIP metabolites had been designed with [2H3C]-PhIP-N2-Gl [2H3C]-PhIP-N3-Gl [2H3C]-HON-PhIP-N2-Gl and [2H3C]-HON-PhIP-N3-Gl added at a set focus of 1000 pg/mL of urine and each unlabeled analyte was added at concentrations of 0 60 120 160 200 400 600 or 1000 pg/mL of urine. The urine examples had been prepared by Ciproxifan SPE (vide supra). The calibration curves for every urine sample had been run double on two different times Ciproxifan to possess six data factors per calibrant level. The calibration data had been suited to a direct line using the normal Ciproxifan least-squares technique with identical weightings. The functionality from Ciproxifan the analytical technique was executed on urine examples from three different topics gathered over 12 hrs pursuing consumption of prepared meat. The topics drank orange juice as the drink. Urine (1 mL) underwent the SPE handling conditions defined above. The within- and between-day precisions for MeIQx PhIP and their metabolites had been computed in quadruplicate as defined 28 with urine examples. The test planning and data analyses had been performed on four different times over a period amount of 1 month. RESULTS AND DISCUSSION UPLC-ESI/MS/MS Analysis of of MeIQx 8 IQx-8-COOH PhIP PhIP-N2-Gl PhIP-N3-Gl HON-PhIP-N2-Gl and HON-PhIP-N3-Gl in the Urine of Omnivores The chemical structures of PhIP MeIQx and their metabolites are shown in Figure 1. A mixed-mode reverse phase cation exchange resin enrichment procedure developed in our lab 21 is a rapid and high throughput method for the simultaneous isolation of PhIP MeIQx and Ciproxifan six of their metabolites from urine. The metabolism of both HAAs was extensive: Less than 9% of the dose was eliminated in urine as unaltered MeIQx and <1% was eliminated as unaltered PhIP.21 We estimated that 60 to 85% of the ingested dose of MeIQx was eliminated in urine of subjects as a combination of unaltered MeIQx and cytochrome P450 1A2 derived metabolites 8 and IQx-8-COOH (Figure 1) within 12 h of consumption of cooked beef. The rate of elimination of PhIP and its N-glucuronidated metabolites was slower than that for MeIQx and its metabolites and up to 35% of the ingested dose of PhIP was eliminated within 12 h of consumption of cooked beef;21 PhIP and its N-glucuronidated metabolites account for 60-82% of the ingested dose that was eliminated in urine within 24 h.29 The capillary HPLC-ESI/MS/MS analytical method was sensitive. The LOQ values of the unaltered HAAs and metabolites were 50 parts per trillion (ppt) or less when 100 μL equivalent of urine was assayed on column.21 However the duty cycle time was protracted at 57 min: Calcrl this duty cycle included a 41 min gradient for analyte separation two min column washing cycle and a 14 min equilibration time (Supporting Information Figure S-1). We sought to determine if the duty cycle time could be significantly shortened by UPLC without sacrificing resolution diminished sensitivity or reproducibility in the quantification of the urinary analytes. The duty cycle time of the UPLC-ESI/MS/MS assay could Ciproxifan be decreased to 19 min; the duty cycle includes a 13 min gradient for separation and analysis of the analytes a 2 min column wash at 100% B followed by a 4 min equilibration time. The UPLC-ESI/MS/MS chromatograms of MeIQx PhIP and their metabolites in urine of a subject collected before and after consumption of meat are depicted in Figure 2. The subject drank black tea with the meal. The SRM traces of parent compounds all of the analytes and the internal standards in the urine extract are well resolved from isobaric inteferences. A steeper gradient could not be employed to further reduce the time of analysis because of an isobaric interferent detected at tR 7.9 min monitored using the same transition as.