Logo image
Back
Validation of a sensitive bioanalytical method for metronidazole extraction from human plasma
Journal article   Open access

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

Krishna Kumar Patel, Valeria Cota and Nicole K. Brogden
Journal of Pharmaceutical and Biomedical Analysis Open, Vol.3, 100025
06/2024
DOI: 10.1016/j.jpbao.2024.100025
PMCID: PMC11583922
PMID: 39582498
url
https://doi.org/10.1016/j.jpbao.2024.100025View
Published (Version of record) Open Access

Abstract

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 – 5ng/mL) and internal standard (tinidazole, 2ng/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 (150mm × 4.6mm 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 – 5ng/mL) had excellent linearity with R2 = 0.999. The extracted samples also showed higher autosampler and freeze-thaw stability over a 72-hr period. The mean intra- and inter-day accuracy and precision of the extraction assay ranged from 97 to 101.6%. and 2.7 – 4.8% RSD, respectively. The assay was highly efficient, with a limit of quantification (0.53 ± 0.04ng/mL) lower than previously published methods (≥5ng/mL). The extraction method was successfully validated using LC-MS/MS and can be used to extract and detect trace amounts of MTZ in plasma after topical application.
 bioanalytical assay extraction human plasma LC-MS/MS metronidazole validation

Details

Metrics

11 Record Views
Logo image