Journal de bioanalyse et de biomédecine

Determination of S-amlodipine in Human Plasma Using Tizanidine as Internal Standard by Lc/Ms/Ms Method

Abstract

Rajesh Dhiman, Vijaya Durga and Konsam Hamlet Meetei

High performance liquid chromatographic tandom mass spectrometric method for the estimation of S-amlodipine in human plasma has been developed and validated using Tizanidine as internal standard. Sample process was accomplished by Liquid-Liquid Extraction technique. The processed sample was chromatographed and analysed on Phenomenex, Lux 3u Cellulose-2 (150 × 4.6) mm column using mobile phase [0.1% formic acid in water and Acetonitrile (50:50% v/v)]. S-amlodipine was chromatographed and analysed by MS detector. Amlodipine has two basic moiety (one primary and one secondary amino) so it shows pKa=9.45 with two ester linkages and one ether linkage, so it is highly lipid soluble (logP=2.22) whereas Tizanidine has two secondary amino and three tertiary amino groups, so it shows pKa=7.49. Primary amine –NH2 is aliphatic in nature in amlodipine and secondary amine –NH is in ring so it has less action of basicity. Tizanidine has secondary amine –NH is in chain which predominates the basicity (pKa=7.49) and other secondary and tertiary amines are in ring so they show less action with formic acid/ACN mobile phase. Formic acid ionizes (primary amino) –NH2 part, then (secondary amino) –NH part and then (tertiary amino) =N part. So amlodipine elutes 1st then tizanidine comes out. LogP of amlodipine is 2.22 and for tizanidine it is 1.4, so due to Formic acid-water/CAN mobile phase Amlodipine has higher retention time than tizanidine and comparison Rt. The analytical method described is valid the determination of S-amlodipine (over a range of 0.77 ng/ml to 50.98 ng/ml) using Tizanidine as internal standard in human plasma. Signals from the detector were captured in a computer and processed using Analyst software. This validation report provides results of various validation parameters.