br Total NNN and total NNAL in urine A

Total NNN and total NNAL in urine A bioanalytical assay was developed to quantitate NNAL and NNN in human urine. A 9 point standard calibrator line was established in ultrapure water across the NNAL (5.00–1000pg/mL) and NNN (0.200–40.0pg/mL) analytical ranges. Calibration standards, quality control samples and clinical samples were supplemented with stable label internal standards (d4-NNAL and d4-NNN). The target analytes were retained through a solid phase extraction process. The extraction solvent was dried under a stream of nitrogen gas. The extracts were reconstituted in a polar organic solvent and injected onto an LC-MS/MS system for detection. Positive ions were monitored in multiple reaction monitoring mode. The validation testing included precision and accuracy testing with 6 replicates of quality control samples prepared at 4 concentrations (NNAL <18.2% C.V., <3.0% bias; NNN <24.6% C.V., <3.5% bias), recovery (NNAL 88.0–85.0%, NNN 64.0–66.0%), multiple-lot quantitation, stock/sub-stock solution buy GSK256066 at −20°C in polypropylene, long-term clinical sample stability (807 days at −20°C; 318 days (LLOQ QC) and 296 days (dilution QC) at −20°C), freeze/thaw stability (6 cycles at ambient temperature for NNAL, 6 cycles at ambient temperature for NNN), short-term stability (24h at ambient temperature for NNAL, 24h at ambient temperature for NNN), and post-preparative stability (126h for NNAL, 179h for NNN). All data from the method validation was reviewed by an independent quality assurance unit as per Celerion standard operating procedures.
Total 3-hydroxy benzo[a]pyrene in urine A bioanalytical assay was developed to quantitate 3-hydroxybenzo[a]pyrene in human urine. An eight point standard calibrator line was established across the 3-hydroxybenzo[a]pyrene (25.0–600fg/mL) analytical range and was validated. Calibration standards, quality control samples and clinical samples were supplemented with stable label internal standard (13C6-3-hydroxybenzo[a]pyrene). All samples were treated with β-glucuronidase to convert the glucuronide metabolite to the aglycone which was measured. The target analyte was retained through a solid-phase extraction process. The eluents were dried under a stream of nitrogen gas. The extracts were reconstituted with a polar organic solvent and were injected onto an LC-MS/MS system for detection. Positive ions were monitored in multiple reaction monitoring mode. The validation testing included precision and accuracy testing with 6 replicates of quality control samples prepared at 5 concentrations (<15.8% C.V., <2.8% bias), recovery (91–104%), multiple-lot quantitation, stock/sub-stock solution stability at −20°C in silanized glass containers, long-term clinical sample stability stored at −20°C (570 days), freeze/thaw stability (6 cycles under UV-shielded light), short-term stability (55hours under UV-shielded at ambient temperature), and post-preparative stability (156h). All data from the method validations were reviewed by an independent quality assurance unit as per Celerion standard operating procedures.
Caffeine and paraxanthine in plasma A bioanalytical assay was developed to quantitate caffeine and paraxanthine in human plasma. A 9 point standard calibrator line was established in human plasma (heparin) across the caffeine and paraxanthine (20.0–5000ng/mL) analytical ranges. Calibration standards, quality control samples and clinical samples were supplemented with stable label internal standards (d9-caffeine and d3-paraxanthine). The target analytes were retained through a liquid-liquid extraction process. The extraction solvent was dried under a stream of nitrogen gas. The extracts were reconstituted in a polar organic solvent and injected onto an LC-MS/MS system for detection. Positive ions were monitored in multiple reaction monitoring mode. The validation testing included precision and accuracy testing with 6 replicates of quality control samples prepared at 4 concentrations (caffeine <7.9% C.V., <3.6% bias; paraxanthine <6.5% C.V., <−0.6% bias), recovery (caffeine 81.0–87.0%, paraxanthine 59.0–63.0%), multiple-lot quantitation, stock/sub-stock solution stability at −20°C in polypropylene, long-term clinical sample stability (545 days at −20°C), freeze/thaw stability (6 cycles at ambient temperature and in an ice water bath), short-term stability (24h at ambient temperature and in an ice water bath), and post-preparative stability (162h). All data from the method validation was reviewed by an independent quality assurance unit as per Celerion standard operating procedures.