Asian Journal of Research in Chemistry

Srinivas Reddy ¹, Jibin V. Joseph ¹, Aravind Kumar ¹, Arindam Mukhopadhyay ¹, Saral Thangam ¹

Affiliations
1 Norwich Clinical Services Pvt. Ltd., 147/F, 8th Main, 3rd Block, Koramangala, Bangalore– 560034

Abstract

After extraction from plasma by liquid - liquid extraction method, Felodipine and FelodipineD5, (IS), were separated on a Reverse phase chromatography using the mobile phase mixture of ammonium acetate and methanol at a flow rate of 1.0 ml/min. The analytes were detected in API 4000 Mass spectrometer in the positive atmospheric pressure chemical Ionization (APCI) mode with multiple reactions monitoring (MRM). The MRM transitions were monitored by following m/z for parent ion 384.0&daughter ion 338.0 (Felodipine), and m/z 389.1&daughter 338.1 (Felodipine D5, IS). A linear calibration plot of Felodipine was achieved in the concentration ranges of 0.105 ng/ml to 20.000 ng/ml. Mean recovery was 69.4%.This method was fully validated for specificity, precision, accuracy, reproducibility and other criteria as per regulations.

Keywords

Felodipine, Liquid liquid extraction, LCMS/MS, Human plasma, Validation

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Anil Kumar Meher ¹, P. Anandan ¹, Arindam Mukhopadhyay ¹

Affiliations
1 Bioanalytical Laboratories, Lotus Clinical Research Academy Pvt. Ltd., 582, KCA Enclave, Koramangala 8th Block, Bangalore – 560 095, IN

Abstract

A rapid and sensitive method for quantification of the antihypertensive drug, isradipine in human plasma using LCMS/MS has been developed. Protein was precipitated from the plasma by 20% trichloroacetic acid. It was then made alkaline with 1M sodium hydroxide. Isradipine and the internal standard (IS), Nimodipine, were extracted by a simple liquid -liquid extraction method using extraction solvent mixture (n-Hexane: t-butyl methyl ether-70:30, v/v). The extract was then separated by a reverse phase HPLC on a XTerra MSC18 column (100mm x 3.0 mm, 5μm) using solvent mixture 42:58, v/v ( Mobile phase 1: 0.1%, v/v, Formic Acid; Mobile phase 2: Acetonitrile: Methanol- 75:25, v/v). Flow rate was 0.5 ml/min without splitter and the column oven temperature was 35°C. The analytes were then detected by monitoring the transitions m/z 372.1 → m/z 312.0 for isradipine and 419.0 → 343.0 for nimodipine using API 3000 LCMS/MS system (Applied Biosystems) with Turbo Ion Spray interface. A good linear standard curve was obtained within the range of 0.051ng/ml - 20.448ng/ml (r > 0.9973). The lower limit of quantitation (LLOQ) was 0.051 ng/ml. The overall accuracy and precision were 94.28% and 6.19%, respectively. No significant degradation for isradipine in human plasma was observed at room temperature (7h) or when subjected to freeze thaw procedures (3 cycles). No significant metabolic compounds were found to interfere with the analysis. The results of method validation offer the acceptable performance for the specificity, selectivity, sensitivity, linearity, accuracy, precision and stability. This method can be applied for quantitation of isradipine in dosed human plasma samples.

Keywords

Isradipine, Nimodipine, LCMS/MS, Validation, Human Plasma.

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