Cyclohexanone and Metabolite Assays

Blood samples were collected from neonates at 3 points: preoperatively (prior to skin incision), postoperatively (immediately after CPB), and 12 hours postoperatively. Blood samples were held on ice until the serum was isolated, then aliquoted, and stored at −80 °C until assayed. To determine serum cyclohexanone and its metabolite concentrations, samples were spiked with isotopically labeled standards (Sigma-Aldrich) prior to acetonitrile extraction. All samples were analyzed within 48 hours of processing. Analysis of the samples was conducted on a 7010 GC-triple quadrupole mass spectrometer (Agilent) operating in electron-impact ionization mode (70eV source energy, 225 °C) and fitted with a Stabilwax analytical column (30 m × 250-μm diameter × 0.25-μm film thickness, 5-m integrated guard column, Restek #10623-124). Ultra high purity helium was used as a carrier gas, and ultra high purity nitrogen was used as a collision gas. Multiple reaction monitoring transitions for each target compound had been previously determined empirically by analysis of authentic standards. Peak integration and reporting was conducted using MassHunter Quantitative Analysis software version B.08.00 (Agilent). Retention times for all peaks were within 4 seconds of the expected values obtained from authentic standards. Quantitation was performed via an area ratio of cyclohexanone or its breakdown products to the internal standard area. All target compounds demonstrated a linear response across the concentration range of 1 pg to 1 ng on-column based on a dilution series of authentic standards. The percent coefficient of variability from the assay of a pooled plasma sample including 15 samples was cyclohexanone, 1.8%; cis 1,2 cyclohexanediol, 7.9%; trans 1,2 cyclohexanediol, 10.7%; 1,3 cyclohexanediol, 13.5%; and trans 1,4 cyclohexanediol, 15.2%. Samples that showed poor peak shape for the internal standard (apex >6 seconds from expected value) or showed chromatographic abnormalities were excluded from further analysis.