New Analytical Method for the Determination of
Metronidazole in Human Plasma: Application to
Bioequivalence Study
Essam Ezzeldin1,2* and Toqa M. El-Nahhas1,3
1Drug Bioavailability Center, National
Organization for Drug Control and Research (NODCAR),
Cairo, Egypt, 2Drug Bioavailability
Laboratory, College of Pharmacy, King Saud University,
Saudi Arabia, 3Clinical Pharmacology
Department, Queen Marry and Westfield College of London,
University College of London, EC1M 6BQ.
*For correspondence:
Email:
ezzeldin24@hotmail.com Tel:
+996597449358
Received: 17
December
2011 Revised
accepted: 6 July 2012
Tropical
Journal of Pharmaceutical Research, October 2012;
11(5): 799-805
http://dx.doi.org/10.4314/tjpr.v11i5.14
Abstract
Purpose: To develop new sensitive, accurate, rapid and
reproducible high performance liquid chromatography (HPLC)
method to determine metronidazole levels in human plasma
and to apply the method in a bioequivalence study.
Methods: Metronidazole was extracted from human plasma
through one step of protein precipitation by methanol
using carbamazepine as internal standard (IS). After
centrifugation of the plasma sample, the supernatant
layer was separated and injected into HPLC system using
Eclipse XDB-phenyl column. The mobile phase consisted of
phosphate buffer (pH 4.5): acetonitrile (95:5, v/v). The
UV detector was set at 320 nm. The bioavailability of
the test metronidazole product (Brand A) was compared to
a commercial metronidazole brand as reference product in
24 healthy volunteers who received a single dose
equivalent to 500 mg of the test and reference products
in a randomized balanced two-way cross-over design
separated by two-week wash-out period.
Results: Mean standard calibration curves of metronidazole
over the concentration range of 0.05 – 30 μg/ml were
linear. No significant differences were found based on
analysis of variance of the pharmacokinetics parameters
required for the assessment of bioequivalence of test
and reference formulations. The mean value and 90 %CI of
test/reference ratios for the derived parameters were: Cmax,
9.64 vs. 8.38 (0.93 – 1.10), AUC0-24, 124.6
vs.122.3 μg.h/mL (0.973 – 1.051) and AUC0-∞,
140.9 vs. 128.4 h/mL (1.15 – 1.23).
Conclusion: The test metronidazole product was bioequivalent
to the reference. The method is suitable for
bioequivalence and pharmacokinetic studies in humans
with a low limit of quantification of 0.05 µg/ml.
Keywords: Metronidazole, Bioequivalence, HPLC,
Pharmacokinetics, Human plasma.
