Original Article QUANTITATION OF AMLODIPINE IN HUMAN PLASMA BY LCMS/MS ASSAY SYED N. ALVI, RAJAA F. HUSSEIN, SALEH AL-DGITHER, MUHAMMAD M. HAMMAMI Clinical Studies and Empirical Ethics Department, King Faisal Specialist Hospital and Research Center, P O Box # 3354, MBC-03, Riyadh 11211, Kingdom of Saudi Arabia Email: muhammad@kfshrc.edu.sa Received: 27 Apr 2016 Revised and Accepted: 20 June 2016 ABSTRACT Objective: To develop and validate a simple, precise, and rapid liquid chromatographic-tandem mass spectrometric (LC-MS/MS) method for quantification of amlodipine in human plasma. Methods: Chromatographic analysis was performed on Atlantis dC18 column (2.1 x 100 mm, 3 µm) with a mobile phase consisting of acetonitrile and 10 mM formic acid (80:20, v: v) that was delivered at a flow rate of 0.3 ml/min. The eluents were monitored using electrospray ionization in the positive ion mode set at transition 409 → 238.4 and 254.3 → 43.9 for amlodipine and tizanidine hydrochloride (IS), respectively. The method was validated for linearity, accuracy, precision, and recovery as per US-FDA guidelines. Results: The retention times of amlodipine and tizanidine (IS) were 1.26 and 1.22 respectively. The relationship between amlodipine concentration and peak height ratio of amlodipine to the IS was linear (R 2 Conclusion: The proposed method is simple, precise, and accurate for rapid measurement of amlodipine level using 0.5 ml human plasma. Further, the assay was successfully applied to determine amlodipine level in human plasma samples obtained from a healthy volunteer. ≥ 0.9868) in the range of 0.2–20 ng/ml, and the intra-and inter-day coefficient of variations and bias were ≤14.4% and ≤13.6% and ≤13.7% and ≤11.2%, respectively. Keywords: Amlodipine, Tizanidine, Human plasma, LC-MS/MS © 2016 The Authors. Published by Innovare Academic Sciences Pvt Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/ ) INTRODUCTION Amlodipine (CAS: 88150-42-9), a derivative of dihydropyridine, is widely used in the treatment of hypertension. It lowers blood pressure by inhibiting the influx of calcium ions Various analytical methods have been reported for quantification of amlodipine in human plasma. They include thin-layer chromate- graphy (TLC) [4], gas chromatography equipped with electron capture detection (GC-ECD) [5, 6], high-performance thin-layer chroma- tography (HP-TLC) [7], high performance liquid chromate-graphy (HPLC) with ultra-violet (UV) detection [8, 9], fluorimeteric detection [10, 11]or electrochemical detection [12], and liquid chromatography- tandem mass spectrometry (LCMS-MS) [13-15]. In general, HPLC with UV detection is the preferred method for quantification of analytes that have strong absorbance in the UV range. Because amlodipine has low absorbance in UV range, most reported assays used either pre- column derivatization with 4-chloro-7-nitrobenzofurazan or LCMS/MS. There is limited data on the stability of amlodipine in processed and unprocessed human plasma [1]. Its absolute bioavailability is in the range of 60-65%, and it has a peak plasma concentration of 6-12 ng/ml within 8-10 h after the ingestion of a 10 mg therapeutic dosage [2, 3]. The present manuscript describes a precise and rapid LCMS/MS assay for quantitative determination of amlodipine in human plasma using tizanidine as an internal standard. The method involves simple liquid/liquid extraction, using 500 µl human plasma. The validated method was used to determine the stability of amlodipine under various clinical laboratory conditions, particularly in unprocessed human plasma samples for more than one year and has been successfully used to determine amlodipine level in human plasma samples obtained from a healthy volunteer. [9, 14]. MATERIALS AND METHODS Chemicals and reagents All chemicals were of analytical grade unless stated otherwise. Amlodipine USP reference standard and tizanidine hydrochloride (IS) were purchased from Sigma-Aldrich Co., St. Louis, MO, USA. Acetonitrile, methanol, dichloromethane, formic acid and tert. butyl methyl ether (HPLC grade) were purchased from Fisher Scientific, NJ. USA. Water for HPLC was prepared by reverse osmosis and further purified by using synergy water purification system (Millipore, Bedford, MA, USA). Drug-free human plasma was obtained from the blood bank of King Faisal Specialist Hospital and Research Centre (KFSHRC) Riyadh, Saudi Arabia. The study was approved by the Research Ethics Committee of KFSHRC, under Research Advisory Council (RAC# 2101100). Instrument and chromatographic conditions LC-MS/MS analysis was performed on Waters Alliance HPLC 2695 Separation module, consisting of quaternary pump, autosampler, column thermostat, and Micromass Quattro micro API bench-top triple quadruple mass spectrometer, interfaced with Z-spray electro- spray ionization probe. Data acquisition and analysis were performed using MassLynx 4.0 software with Quan Lynx program (Waters Associates Inc, Milford, MA, USA). Analysis was performed on a reversed phase Atlantis dC18 (2.1 X 100 mm, 3 µm) column equipped with Symmetry C 18 (3.9 x 20 mm, 5 µm) guard column. The mobile phase, composed of acetonitrile and 10 mM formic acid (80:20, v: v), was filtered through a 0.22 µm membrane filter (Millipore Corporation, Bedford, MA, USA), degassed, and delivered at a flow rate of 0.3 ml/min. MassLynx software working under Microsoft Window XP professional environment was used to control the instruments, data acquisition, peak integration, peak smoothing, and signal-to-noise ratio measurements. The electrospray ionization source was operated in the positive-ion mode at a capillary voltage of 4.0 kV and a cone voltage of 10 V. Nitrogen was used as nebulizing and desolvation gas at a flow rate of 60 and 600 L/hr, respectively. Argon was used as the collision gas at a pressure of 1.28 x 10 -3 mbar. The optimum collision energy for amlodipine and tizanidine hydrochloride (internal standard, IS) was 25 eV. The ion source and the desolvation temperatures were maintained at 105 and 350 ̊C, respectively. The product ion transitions response were recorded at m/z 409.8 → International Journal of Pharmacy and Pharmaceutical Sciences ISSN- 0975-1491 Vol 8, Issue 8, 2016