Pharmacokinetic analysis
BLOOD
Blood samples were taken from patients before NMP and at one, two, four, eight and 24 h post dose. Plasma samples (20 μl) were prepared by adding five volumes of methanol containing deuterated internal standard. The samples were vortexed then centrifuged, and the supernatant was transferred to a vial and injected onto the LCMSMS. The LCMSMS system consisted of a Shimadzu UHPLC with a SCIEX 6500QTrap, a KinetexC18 (3 × 50 mm, 2.6 μm) column and using a gradient of 0.1% formic acid and acetonitrile. The initial conditions were 0.5 ml/min and 2% acetonitrile; this was held for 1 min then increased to 20% acetonitrile at 2 min then increased to 70% acetonitrile and held for 0.5 min. Equilibration time was 0.5 min. MRMs for NMP were 100 → 58, 100 → 69, 100 → 41 and for the internal standard d9NMP 109 → 62, 100 → 61, 100 → 46. PK analysis was performed using Pkanalix (version 2021R1. Antony, France: Lixoft SAS, 2021).