Tamara van Donge

Personalized use of ibuprofen in preterm neonates 113 6 for each enantiomer of the calibration curve is 50, 25, 12.5, 5, 2.5, 0.5, 0.25, 0.05 and 0.025 µg/ mL . The internal standard was prepared by dissolving 5 mg of (R/S)-ibuprofen-d3 in 5 mL of methanol and subsequent dilution by a factor of 200 with methanol/water (50/50) resulting in a final concentration of 5 µg/mL (R/S)-ibuprofen or 2.5 µg/mL for each enantiomer. For quantification, calibration curves were performed in triplicates referring each enantiomer to the corresponding deuterated standard. For quality controls, adult plasma (2 * 2mL; 4ml in total) was spiked with 20 µL and 200 µL of (R/S)-ibuprofen (diluted certified ibuprofen solution; 100 µg/mL) and aliquoted in 100 µL equivalents. The final QC concentration was 0.5 µg/mL (QC low ) and 5 µg/mL (QC high ). Samples were stored at -80°C. Sample preparation Blood samples (200 μL) were collected in lithium heparin tubes and plasma was obtained by centrifugation at 3000 rpm for 15 minutes within two hours after blood collection. The plasma samples were stored at -80°C until analysis. The used sample preparation procedure was adopted from a validated method published by Nakov et al. (2015). The samples were analysed at the McMaster Regional Centre for Mass Spectrometry. Briefly, frozen samples and quality controls were thawed prior analysis. A mixture of 50 μL of plasma samples, 50 μL deuterated internal standard and 50 μL hydrochloric acid (0.15N) were vortexed for 1 min. To extract ibuprofen, ethyl acetate (1000 μL) was added, shaken for 10 min and centrifuged (5 min, 10,000 rpm). The supernatant was dried under nitrogen at 37°C. All samples were reconstituted with 100 μL methanol/H₂0 (80/20) containing 0.1% acetic acid. Method validation The retention time for the signal of R-Ibuprofen and S-ibuprofen were 5.96 and 6.94 minutes and were sufficiently separated to allow the quantification of the enantiomers (Fig.1). For the assessment of the selectivity, blank plasma samples were taken before the study. The results show that all Ibuprofen levels were below the lowest standard (n=15, <0.025 µg/mL). Linearity of the method was shown for a range from 0.025 to 50 µg/mL for each both enantiomers. The nine-point calibration curve was fitted by 1/x weighted, linear least-square regression resulting in the following regression equations: SR = 0.294 * C – 0.001 (r=0.9982) for R-ibuprofen and SR = 0.299 * C – 0.001 (r=0.9981) for S-ibuprofen with SR =signal ratio and C = analyte concentration. The lowest limit of detection (LLOQ) was 0.01 µg/mL. To test the repeatability of the analytical device, a single random sample was injected 10 times (Table S1). The coefficient of variation was 1.5 and 1.2 % for R-ibuprofen and S-ibuprofen, respectively. The overall precision and accuracy of the method was tested using quality control samples. The results of the inter- and intra-assay samples (n=10, each) are depicted in Table 2.