การพัฒนาและตรวจสอบความถูกต้องของวิธีโครมาโทกราฟีของเหลวสมรรถนะสูงเพื่อใช้วิเคราะห์ปริมาณพรีกาบาลินในพลาสมามนุษย์ Development and Validation of an High Performance Liquid Chromatography Method for Determination of Pregabalin in Human Plasma

Abstract

Objective: To develop and validate method for quantitative determination ofpregabalin in human plasma by high performance liquid chromatographyequipped with fluorescence detector. Method: Sample preparation requiredplasma protein precipitation using acetonitrile. The supernatant was thenaliquoted and pre-column derivatized with 4-chloro-7-nitrobenzofurazan(NBD-Cl). The optimum derivatization conditions established were 2.24mg/ml of NBD-Cl, pH 11 and 80 °C. After 15 min, the reaction wasterminated using 0.02 M HCl. The chromatographic separation was carriedout on a Phenomenex C18 column. Mobile phase, a mixture of KH2PO4buffer (10 mM; pH 2.5) and acetonitrile (50:50, v/v), was eluted at the flowrate of 1.2 ml/min. The fluorescent derivatives were monitored at theexcitation and emission wavelengths of 460 and 558 nm, respectively.Results: The obtained derivatives were found to be stable (> 96%)remaining for at least 48 hours at 20 °C. The developed assay forpregabalin was linear over a range of 20 – 10,000 ng/ml in plasma (R2 >0.9999). The lower limit of quantification (LLOQ) was 20 ng/ml when 20 μlinjection volume was applied. The average percentage coefficient ofvariation (% CV) and % recovery at LLOQ level were within 5.19% and101.86%, respectively. Conclusion: The proposed method, proved to besensitive, selective, precise and accurate, meets the standards ofbioanalysis method validation accepted by the United States Food andDrug Administration (US FDA) guideline 2013 and the European MedicinesAgency (EMEA) guideline 2011.Keywords: Pregabalin, Gabapentin, Human plasma, HPLC, NBD-Cl