Quantification of metronidazole in human plasma using a highly sensitive and rugged LC–MS/MS method for a bioequivalence study

Abstract

A highly sensitive, selective and rugged method has been described for the quantification of metronidazole (MTZ) in human plasma by liquid chromatography-tandem mass spectrometry using metronidazole-d4 as the internal standard (IS). The analyte and the IS were extracted from 100 μL plasma by liquid-liquid extraction. The clear samples obtained were chromatographed on an ACE C (100 × 4.6 mm, 5 μm) column using acetonitrile and 10.0 mm ammonium formate in water, pH 4.00 (80:20, v/v) as the mobile phase. A triple quadrupole mass spectrometer system equipped with turbo ion spray source and operated in multiple reaction monitoring mode was used for the detection and quantification of MTZ. The calibration range was established from 0.01 to 10.0 μg/mL. The results of validation testing for precision and accuracy, selectivity, matrix effects, recovery and stability complied with current bioanalytical guidelines. A run time of 3.0 min permitted analysis of more than 300 samples in a day. The method was applied to a bioequivalence study with 250 mg MTZ tablet formulation in 24 healthy Indian males.

Quantification of metronidazole in healthy subjects' feces using an LC–MS/MS method

“…Thus, zidovudine, a general internal standard, was used in their work. In the present work, four potential analytical columns, namely Symmetry C 18 , ACE Phenyl, Cosmosil C 18 and Alltima C 18 , having identical dimensions (100 × 4.6 mm, 5 μm), were tested to achieve adequate retention, short analysis time, symmetric peak shape and acceptable response for MTZ based on our previous work (Vanol et al, ). Separation was tried using various combinations of acetonitrile–methanol along with additives like formic acid–acetic acid (0.1%) and ammonium formate–ammonium acetate (5–10 m m ).…”

“…Numerous methods are available in literature to determine MTZ alone (Bolton & Culshaw, ; Cox, Allender, & Yarbrough, ; de Freitas Silva et al, ; Emami, Ghassami, & Hamishehkar, ; Galmier et al, ; Ilomuanya et al, ; Jeffery et al, ; Krook et al, ; Kubodera, Tokumura, & Machida, ; Mustapha, Odunola, Garba, & Obodozie, ; Silva et al, ; Vanol, Sanyal, Shah, & Shrivastav, ; Wibawa, Shaw, & Barrett, ), together with its hydroxy metabolite (Klimowicz, Bielecka‐Grzela, & Tomaszewska, ) and with other drugs like ranitidine (Nascimento, Oliveira, & Macêdo, ) and spiramycin I (Sagan et al, ) in a variety of biological matrices. Analytical techniques such as liquid chromatography with UV (Bolton & Culshaw, ; Cox et al, ; Emami et al, ; Galmier et al, ; Klimowicz et al, ; Krook et al, ; Kubodera et al, ; Mustapha et al, ; Wibawa et al, ), photodiode array (Nascimento et al, ) or mass spectrometry (de Freitas Silva et al, ; Ilomuanya et al, ; Jeffery et al, ; Sagan et al, ; Silva et al, ; Vanol et al, ) detection have been used for the quantification of MTZ in these matrices. The salient features of these methods are assessed in our previous report (Vanol et al, ).…”

“…Analytical techniques such as liquid chromatography with UV (Bolton & Culshaw, ; Cox et al, ; Emami et al, ; Galmier et al, ; Klimowicz et al, ; Krook et al, ; Kubodera et al, ; Mustapha et al, ; Wibawa et al, ), photodiode array (Nascimento et al, ) or mass spectrometry (de Freitas Silva et al, ; Ilomuanya et al, ; Jeffery et al, ; Sagan et al, ; Silva et al, ; Vanol et al, ) detection have been used for the quantification of MTZ in these matrices. The salient features of these methods are assessed in our previous report (Vanol et al, ). However, there are very few methods which deal with the analysis of fecal MTZ (Bolton & Culshaw, ; Jeffery et al, ; Krook et al, ).…”