Method Validation of High Performance Liquid Chromatography Mass Spectrometric Method for the Estimation of Cis and Trans isomers of Phytonadione in Human Plasma using D–labeledTrans Phytonadione as an Internal Standard

Sheeba Manoj Nair; Malini Sharma; Dr. Denish Karia

Volume : IV, Issue : VIII, August - 2015

Method Validation of High Performance Liquid Chromatography Mass Spectrometric Method for the Estimation of Cis and Trans isomers of Phytonadione in Human Plasma using D–labeledTrans Phytonadione as an Internal Standard

Sheeba Manoj Nair, Malini Sharma, Dr. Denish Karia

Abstract :

A novel, simple, specific, sensitive and reproducible liquid chromatography mass Spectrometric (LCMS–MS) assay method has been developed and validated for estimation of Cis and Trans isomers of Phytonadione. The LCMS–MS method includes use of D–labeledtransphytonadione as an internal standard (IS). The chromatographic analysis was performed using API 4000, Applied biosystems Liquid Chromatography Mass Spectrometer system equipped with MS detector and PC based data system with Analyst 1.6.1 supplied by Lab India. Chromatographic separation was achieved on C30 column, 250×4.6mm, 200A, 5� column maintained at 40ºC temperature using isocratic mobile phase composition with (0.1% Formic acid in Methanol V/V) at a flow rate of 0.8 ml/min, without splitter with a total run time of 18 min. Method validation was performed as per USFDA guidelines and the results met the acceptance criteria. The caliation curve was linear over a concentration range of 0.650 to 40.596 ng/ml for Cis Phytonadione and 0.652 to 81.461 ng/ml for Trans Phytonadione using (D–labeledTrans–Phytonadione) as internal standard (r2 ≥ 0.99). Precicion& accuracy batches analysed on 3 different day�s revealed accuracy of Inter–batch caliation standard accuracy for ranged from 93.69% to 103.11% for Cis Phytonadione and 98.19% to 101.60% for Trans Phytonadione with inter–batch precision values of 1.41% to 3.96% for Cis Phytonadione and 1.24% to 3.00% for Trans Phytonadione during the course of validation, demonstrating acceptable assay linearity. The validated LCMS–MS method was successfully applied for assay and stability determination of clinical study samples.