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Abstract

METHOD VALIDATION OF HIGH PERFORMANCE LIQUID CHROMATOGRAPHY MASS SPECTROMETRIC METHOD FOR THE ESTIMATION OF LOTEPREDNOL IN AQUEOUS HUMOUR USING LOTEPREDNOL ETABONATE D5 AS INTERNAL STANDARD

Malini Sharma* and Umesh B. A. 

ABSTRACT

A novel, simple, specific, sensitive and reproducible liquid chromatography mass Spectrometric (LC/MS-MS) assay method has been developed and validated for estimation of Loteprednol in aqueous humour biological matrix. The LC/MS-MS method includes use of Loteprednol D5 as an internal standard (IS). Samples were extracted using liquid-liquid extraction methodology. The chromatographic analysis was performed using API 3200 and PC based data system with Analyst 1.4.2 software. Chromatographic separation was achieved on BDS Hypersil C-18, 150 ×4.6mm, 5μm column using isocratic mobile phase composition with (0.1% Formic acid in Methanol and Milli-Q water 1:1 V/V) at a flow rate of 0.8 ml/min at 40ºC column oven temperature, without splitter over a total run time of 5 min. Method validation was performed as per USFDA guidelines and the results met the acceptance criteria. The calibration curve was linear over a concentration range of 2.05 to 203.35 ng/ml for Loteprednol using Loteprednol-D5 as internal standard (r2 ≥ 0.99). Inter-batch quality control samples accuracy for Loteprednol ranged from 94.95 to 102.77% with inter-batch precision values of 1.81% to 5.70% during the course of validation, demonstrating acceptable assay accuracy and precision. The validated LCMS-MS method was successfully applied for assay and stability determination of clinical study samples.

Keywords: Loteprednol; LCMS-MS; Aqueous humour, Method validation; Clinical study.


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