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

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.

Keywords: LC-MS/MS; ceftriaxone; complicated severe acute malnutrition; metronidazole; protein binding; ultrafiltration.

Conflict of interest statement

Competing interests: No competing interests were disclosed. No writing assistance was utilized in the production of this manuscript.

Figures

Figure 1.

Chemical structures of ceftriaxone (A), metronidazole (B), hydroxymetronidazole (D) and cefuroxime axetil, IS (C).

Figure 2A.. Representative chromatograms from extracted zero sample (with IS only), cefuroxime (RT 3.71 min).

Figure 2B.. Representative chromatograms of ceftriaxone (RT 2.59 min), metronidazole (RT 2.67 min), hydroxymetronidazole (RT 2.69 min), and cefuroxime (IS) (RT 3.71 min) from extracted spiked plasma at LLOQ.

Figure 2C.. Representative chromatograms of ceftriaxone (RT 2.59 min), metronidazole (RT 2.67 min), hydroxymetronidazole (RT 2.69 min) and cefuroxime (IS) (RT 3.71 min) from extracted spiked plasma at ULOQ.

Figure 3.

Example concentration-time data of each of the four blood samples (baseline and 3 post first dose), where ceftriaxone, metronidazole and hydroxymetronidazole were quantified. In 2 samples, unbound ceftriaxone was also quantified. This example shows a patient who has clearly taken at least one previous dose of metronidazole prior to study enrolment.

Figure 4A.. Representative chromatograms from processed plasma study sample at baseline before drug administration with undetectable levels of the drugs.

Figure 4B.. Representative chromatograms of ceftriaxone 266.27µg/mL (RT 2.59 min), metronidazole 2.54µg/mL (RT 2.67 min), hydroxymetronidazole 0.13µg/mL (RT 2.69 min) and cefuroxime (IS) (RT 3.71 min) from processed plasma study sample at 5 min after administering ceftriaxone IV.

Figure 4C.. Representative chromatograms of ceftriaxone 74.39µg/mL (RT 2.59 min), metronidazole 1.99µg/mL (RT 2.67 min), hydroxymetronidazole 0.66µg/mL (RT 2.69 min) and cefuroxime (IS) (RT 3.71 min) from processed plasma study sample at 30 min after administering ceftriaxone IV.