Abstract
Objective: A simple, selective and sensitive reverse-phase high-performance liquid chromatography (RP-HPLC) method to estimate repaglinide (REP) in rabbit plasma using rabeprazole (RAB) as an internal standard was developed and validated for various qualifications.
 Methods: The chromatographic separation was performed on C18 (2) analytical column (5 μ, 250×4.6 mm) using acetonitrile: 0.05% trifluoroacetic acid in water (55:45, v/v) as mobile phase at the flow rate of 1 ml/min. Validation of the analytical method was performed as per ICH guidelines.
 Results: The retention times of REP and RAB were found at ~4.3 and 5.1 min respectively, with adequate system suitability parameters (theoretical plates ≥3619, tailing factor ≤1.38, resolution factor 2.37). The method has linearity over a concentration range of 10 to 1000 ng/ml (r2=0.9987). The results of accuracy (≥98.17%), intra-, inter-day precision (≤2.9%), recovery (101.21±2.09%) and process efficiency (99.77±3.74%) found satisfactory with no matrix effect. The analyte in samples were found stable up to 6 h, 3 freeze-thaw cycles and not more than 2 mo corresponding to bench-top, short and long term stability studies respectively.
 Conclusion: The developed RP-HPLC method for estimation of REP in rabbit plasma was developed. The method was found to be rapid, cost-effective and accurate to estimate the REP from the sample matrix. The method can be a most useful tool for in vivo study of REP in the rabbit.
Highlights
Repaglinide (REP) is a novel carbomoxyl methyl benzoic acid derivative, known as 2-ethoxy-4-[2-[[3-methyl-1-[2(lpiperidinyl)-phenyl] butyl] amino]-2-oxoethyl]-benzoic acid [1]
The chromatographic separation and elution of analyte was done by mobile phase consisting of ACN:0.05% Trifluoroacetic acid (TFA) in water (55:45, v/v) in reverse phase analytical column (250×4.6 mm; 5 μ particle size)
A) was compared with the chromatogram developed for rabbit plasma spiked with REP and internal standard (IS)
Summary
Repaglinide (REP) is a novel carbomoxyl methyl benzoic acid derivative, known as 2-ethoxy-4-[2-[[3-methyl-1-[2(lpiperidinyl)-phenyl] butyl] amino]-2-oxoethyl]-benzoic acid (fig. 1) [1]. Gradient elution techniques for concurrent estimation of REP with other anti-diabetic agents from the biological matrix on HPLC using UV detection were reported [8, 9]. HPLC-tandem mass spectrometry (LC/MS/MS) methods were established to assess the REP from plasma and urine samples of human [10, 11], monkey [12], rat [13] and equine [14]. All the above methods reported for REP estimation from biological matrix need of expensive instruments for gradient elution and for eluate detection, restrict the application of them in common research laboratories equipped with limited resources
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