Validation of a sensitive bioanalytical method for metronidazole extraction from human plasma.

Metronidazole (MTZ) is a broad-spectrum antibiotic with numerous routes of administration, including topical. Topical application of MTZ gel or cream results in very low systemic absorption, resulting in the need for a sensitive extraction method to quantify plasma concentrations. Currently published methods are not suitable for analysis of plasma concentrations after topical application, as undetectable MTZ concentrations commonly occur. We validated a simple extraction method for MTZ recovery from plasma and quantified it using an LC-MS/MS analytical method. Methods: Plasma samples were spiked with MTZ (0.5 - 5 ng/mL) and internal standard (tinidazole, 2 ng/mL). MTZ was extracted by liquid-liquid extraction using ethyl acetate and acetonitrile mixture (4:1) as the extraction solvent. A quadrupole mass spectrometer interfaced with an Acquity H-Class HPLC was used to quantify MTZ concentrations in positive ion mode. A Kinetix C18 analytical column (150 mm × 4.6 mm i. d., 5 μm particle size) was used for separation. The plasma extraction method was validated for various parameters, including % recovery, precision, accuracy, and stability. Results: The extraction method demonstrated high MTZ recovery, ranging from 93.7 - 97.5%. The calibration curve prepared using MTZ samples extracted from plasma (0.5 - 5 ng/mL) had excellent linearity with R