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

Martin Ongas,James A Berkley,Bernhards Ogutu,Joseph Waichungo,Joseph Standing,Karin Kipper

doi:10.12688/wellcomeopenres.11728.2

Abstract

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 2 ≥ 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.

Highlights

Serious infections are common in children, especially those with severe acute malnutrition (SAM) admitted sick to hospitals, with over 50% of patients estimated to be infected at any one time[1,2]
Empiric antibiotics are routinely given[4,5,6,7], it is not clear whether the currently recommended regimen is the most effective in the context of increasing antimicrobial resistance (AMR), and whether expected therapeutic levels are achieved in this group of patients
Like many β-lactam antibiotics, CEF is more susceptible to chemical and biological degradation due to its labile β-lactam ring[32,33]

Summary

Introduction

Serious infections are common in children, especially those with severe acute malnutrition (SAM) admitted sick to hospitals, with over 50% of patients estimated to be infected at any one time[1,2]. Empiric antibiotics are routinely given[4,5,6,7], it is not clear whether the currently recommended regimen is the most effective in the context of increasing antimicrobial resistance (AMR), and whether expected therapeutic levels are achieved in this group of patients. A quantitative determination of MET and CEF in plasma is essential in order to evaluate the pharmacokinetics of these co-administrated antibiotics (Figure 1). O’Keefe et al.[11] evaluated the activity of the metronidazole metabolites against anaerobic bacteria; the LC-UV method was limited in quantifying lower levels of the metabolites in a biological matrix due to its low sensitivity and poor selectivity. Silva et al.[12] developed an HPLC-MS-MS method for the quantitation of metronidazole in plasma. The method required large sample volumes and complex sample preparation steps, with large volumes of extraction solvents

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