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Original Research Article
Development and Validation of
Reverse Phase High Performance Chromatography Method for
Determination of Olanzapine in Microsample Rat Plasma:
Application to Preclinical Pharmacokinetic Study
Fahad Pervaiz*, Mahmood Ahmad,
Muhammad Usman Minhas and Muhammad Sohail
Faculty of Pharmacy and
Alternative Medicines, The Islamia University of
Bahawalpur, 63100, Bahawalpur, Pakistan
*For correspondence:
Email: fahad_bwp@yahoo.com;
Tel: +92-3216805365; Fax: +92-62925556
Received: 7 October 2014
Revised accepted: 21 December 2014
Tropical Journal of Pharmaceutical Research,
January 2015;
14(1): 141-147
http://dx.doi.org/10.4314/tjpr.v14i1.20
Abstract
Purpose: To develop a sensitive and
validated reverse phase-high performance liquid
chromatographic (RP-HPLC) method for quantification of
olanzapine in micro-sample of rat plasma using UV
detection.
Methods: A single oral dose of
olanzapine (7 mg/kg) was given to overnight fasted rats
(n = 6). Rat plasma samples containing the drug were
extracted by liquid-liquid extraction using a
combination of dichloromethane: n-hexane (80:20). A
reverse phase chromatographic column C18 hypersil-BDS
was used for chromatographic separation with a mobile
phase consisting of 50 mM phosphate buffer pH 5.5,
acetonitrile and methanol (50:30:20, v/v/v) pumped at a
flow rate of 1.2 ml/min. Olanzapine was measured using
ultraviolet (UV) detection at 214 nm. The method was
validated for precision and accuracy.
Results: Separation of compounds of
interest was not affected by endogenous interference.
Good linearity within the concentration range of 1 - 500
ng/ml in rat plasma was obtained with coefficient of
regression (r2) of 0.9986. Liquid-liquid
extraction produced comparable recovery to solid phase
extraction. Retention time of olanzapine and internal
standard (fluoxetine) was 5.0 and 13.4 min,
respectively. Lowest limit of quantification (LLOQ) was
1 ng/ml while inter-day and intra-day precision was <
12.5 and 5.1 %, respectively. Accuracy of the method was
between 94 and 105 % and the variation of results
between two analysts was not significant (p = 0.626).
Mean maximum plasma concentration (Cmax) of olanzapine
was 412.7 ng/ml, time to attain maximum plasma
concentration (tmax) was 1 h and half life (t1/2)
was 2.54 h.
Conclusion: The proposed method has
been successfully validated for precision and accuracy
that are within the limits of U.S. Food and Drug
Administration (FDA)�s guidance for bioanalyitcal assay
validation. The method was successfully applied to
preclinical pharmacokinetic analysis of olanzapine in
rats.
Keywords: Olanzapine, Antipsychotic,
Pharmacokinetics, Rat, Plasma, Bioanalytical assay |