Kim Annink

200 Chapter 9 and hypoxanthine. A dilution of the supernatant with mobile phase was performed for the determination of uric acid before injection into the chromatographic system. The chromatographic separation was performed on an Astec ® CHIROBIOTIC ® V HPLC column using isocratic elution. An API 4000 tandem mass spectrometer equipped with a turbo ion spray probe operating in the negative multiple reaction monitoring mode was used for quantification. The assay was validated according the FDA and EMA guidelines. The lower limit of quantification (LLOQ) was 0.1 mg/L for allopurinol and 0.0934 mg/L for oxypurinol. Pharmacokinetic modelling A population PK model was developed for allopurinol and oxypurinol using nonlinear mixed effect modeling (NONMEM 7.3, Icon Development Solutions). Pirana (version 2.9.9) was used as graphical user interface for NONMEM (31). R (version 3.4.3) was used for data handling and visualization. The estimated pharmacokinetic parameters were: volume of distribution (Vd) and total body clearance (CL) for allopurinol and oxypurinol. The fraction of allopurinol converted to oxypurinol in neonates was unknown. Therefore, oxypurinol parameter estimates were relative to formation fraction (fm), i.e. Vd/fm and CL/fm. Birth weight (BW), normalized to BW of 3.5kg, was used as a description of body size and was related to PK parameters using allometric relationships (32). The exponent defining the relationship of BW and CL was fixed to 0.75 and the exponent defining the relationship of BW and Vd was fixed to 1. Measurements below the LLOQ were included. Values were set to LLOQ/2, which was 0.05 mg/L for allopurinol and 0.0467 mg/L for oxypurinol (33). Interindividual variability (IIV) was incorporated using a proportional model and was tested for CL and Vd for both allopurinol and oxypurinol. A combination of a proportional and additive error model was used to describe residual unexplained variability. Additive errors were fixed to 0.05 for allopurinol and 0.0467 for oxypurinol to account for the measurements below LLOQ (33). Separate error models were used for allopurinol and oxypurinol. Parameter precision was accessed with sampling importance resampling (SIR) (34). Model adequacy was evaluated through both statistical procedures, goodness-of-fit (GOF) plots and physiological plausibility. Statistical significance was evaluated by the change in objective function value (OFV), which is equal to minus two the log-likelihood. A decrease in OFV of