| Summary: | We have developed and validated a novel, sensitive, selective and reproducible reversed-phase high-performance liquid chromatography method coupled with electrospray ionization mass spectrometry (HPLC–ESI-MS/MS) for the simultaneous quantitation of ceftriaxone (CEF), metronidazole (MET) and hydroxymetronidazole (MET-OH) from only 50 μL of human plasma, and unbound CEF from 25 μL plasma ultra-filtrate to evaluate the effect of protein binding. Cefuroxime axetil (CEFU) was used as an internal standard (IS). The analytes were extracted by a protein precipitation procedure with acetonitrile and separated on a reversed-phase Polaris 5 C18-Analytical column using a mobile phase composed of acetonitrile containing 0.1% (v/v) formic acid and 10 mM aqueous ammonium formate pH 2.5, delivered at a flow-rate of 300 μL/min. Multiple reaction monitoring was performed in the positive ion mode using the transitions m/z555.1→m/z396.0 (CEF), m/z172.2→m/z 128.2 (MET), m/z188.0→m/z125.9 (MET-OH) and m/z528.1→m/z 364.0 (CEFU) to quantify the drugs. Calibration curves in spiked plasma and ultra-filtrate were linear (r ≥ 0.9948) from 0.4–300 μg/mL for CEF, 0.05–50 μg/mL for MET and 0.02 – 30 μg/mL for MET-OH. The intra- and inter- assay precisions were less than 9% and the mean extraction recoveries were 94.0% (CEF), 98.2% (MET), 99.6% (MET-OH) and 104.6% (CEF in ultra-filtrate); the recoveries for the IS were 93.8% (in plasma) and 97.6% (in ultra-filtrate). The validated method was successfully applied to a pharmacokinetic study of CEF, MET and MET-OH in hospitalized children with complicated severe acute malnutrition following an oral administration of MET and intravenous administration of CEF over the course of 72 hours.
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