Liquid chromatography–tandem mass spectrometry for the simultaneous quantitation of ceftriaxone, metronidazole and hydroxymetronidazole in plasma from seriously ill, severely malnourished children

Abstract

<ns3:p> 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 <ns3:italic>m/z</ns3:italic> 555.1→ <ns3:italic>m/z</ns3:italic> 396.0 (CEF), <ns3:italic>m/z</ns3:italic> 172.2→ <ns3:italic>m/z</ns3:italic> 128.2 (MET), <ns3:italic>m/z</ns3:italic> 188.0→ <ns3:italic>m/z</ns3:italic> 125.9 (MET-OH) and <ns3:italic>m/z</ns3:italic> 528.1→ <ns3:italic>m/z</ns3:italic> 364.0 (CEFU) to quantify the drugs. Calibration curves in spiked plasma and ultra-filtrate were linear ( <ns3:italic> r <ns3:sup>2</ns3:sup> </ns3:italic> ≥ 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. </ns3:p>