Research article Received: 18 September 2013,

Revised: 25 April 2014,

Accepted: 24 May 2014

Published online in Wiley Online Library

(wileyonlinelibrary.com) DOI 10.1002/bmc.3279

Development and validation of a HPLC-PDA bioanalytical method for the simultaneous estimation of Aliskiren and Amlodipine in human plasma Sai Sandeep Mannemalaa,b* and Janaki Sankarachari Krishnan Nagarajana ABSTRACT: A simple, unique and selective HPLC-PDA method was developed and validated for the simultaneous estimation of aliskiren (ALS) and amlodipine (AML) in human plasma. Extraction of the sample was accomplished by protein precipitation. Plasma proteins were precipitated by employing acetonitrile containing hydrochlorothiazide as internal standard. The compounds were analyzed by HPLC by using PDA detector on a Hibar C18 (250 × 4.6 mm) column with a mobile phase comprising acetonitrile and phosphate buffer (pH 4.2 and 25 mM; 60:40 v/v) with a flow rate of 0.8 mL/min. Different sample pretreatment techniques were evaluated but protein precipitation was found to be satisfactory, offering good recovery values of 97.11–98.45% for ALS and 97.5–99.12% for AML. The within-day precisions for ALS were 96.66, 99.16 and 99.41% at 90, 240 and 480 ng/mL, respectively, and for AML they were 97.27, 99.54 and 99.31% at 3.3, 8.8 and 17.6 ng/mL, respectively. The between-day precisions for ALS were 96.66, 99.16 and 99.41% at 90, 240 and 480 ng/mL, respectively and the between-day precisions for AML were 98.18, 99.20 and 99.40% at 3.3, 8.8 and 17.6 ng/mL, respectively. The limit of quantitation was 30 and 1.0 ng/mL for ALS and AML respectively. Different constituents of plasma proteins did not interfere with the absolute recovery of ALS and AML. Copyright © 2014 John Wiley & Sons, Ltd. Keywords: liquid chromatography; aliskiren; amlodipine; bioanalytical method; validation

Introduction Hypertension, also known as high or raised blood pressure, is a global public health issue. It contributes to the burden of heart disease, stroke, kidney failure and premature mortality and disability. It mostly affects populations in low and middle income countries where health systems are weak. Hypertension is responsible for at least 45% of deaths according to the World Health Organization (Lloyd-Jones et al., 2009; WHO, 2013). Inappropriate diet and other bad habits like alcohol and tobacco consumption are some of the factors closely related to the risk of suffering from hypertension. Other important risk factors are dyslipidemia and diabetes. The pathologies associated with suffering from some of these conditions simultaneously are known as ‘Metabolic Syndrome’ (Alberti et al., 2005; Grundy et al., 2004). To counteract hypertension either new drug molecules or combinations of them are released from time to time. The monitoring of the plasma concentrations of these drugs is essential for bioequivalence studies and therapeutic drug monitoring. Therefore the simultaneous determination of these drugs is necessary. The complexity of the chosen biological matrix and the low expected concentrations of some analytes require the development of sensitive and selective methods for the analysis. Aliskiren (ALS), (2S,4S,5S,7S)-N-(2-carbamoyl-2-methylpropyl)5-amino-4-hydroxy-2,7-diisopropyl-8-[4-methoxy-3-(3-methoxypropoxy)phenyl]-octanamide (Fig. 1a) is a direct rennin inhibitor; amlodipine (AML), 2-[(2-amino ethoxy)-methyl]-4-(2-chloro phenyl)-

Biomed. Chromatogr. 2014

1,4-dihydro-6-methyl-3,5-pyridine dicarboxylic acid 3-ethyl-5-methyl ester (Fig. 1b) is a long-acting calcium channel blocker; and hydrochlorothiazide (HCTZ), 6-chloro-1,1-dioxo-3,4-dihydro2H-1,2,4-benzothiadiazine-7-sulfonamide (Fig. 1c) belongs to thiazide class of diuretics. Aliskiren lowers the blood pressure by decreasing plasma renin activity by binding to the S3bp site of renin, which essential for its activity. Binding to this pocket prevents the conversion of angiotensinogen to angiotensin I, which lowers blood pressure (Rahuel et al., 2000). Amlodipine is a peripheral arterial vasodilator that acts directly on vascular smooth muscle to cause a reduction in peripheral vascular resistance and reduction in blood pressure. The clinical significance of combined treatment of aliskiren and amlodipine arise from the actions of these two agents on different, but complementary mechanisms that regulate blood pressure, calcium channelmediated vasoconstriction and renin–angiotensin–aldosterone

* Correspondence to: Sai Sandeep M, Department of Pharmaceutical Analysis, JSS University, Udhagamandalam, Tamilnadu, India. Email: [email protected] a

Department of Pharmaceutical Analysis, JSS University, Udhagamandalam, Tamilnadu, India

b

Department of Pharmacy, Annamalai University, Annamalai Nagar, Tamilnadu, India Abbreviations used: ALS, aliskiren; AML, amlodipine; HCTZ, hydrochlorothiazide.

Copyright © 2014 John Wiley & Sons, Ltd.

S. S. M and J. S. K. Nagarajan Instrumentation and analysis conditions

Figure 1. Chemical structures of (a) aliskiren; (b) amlodipine; and (c) hydrochlorothiazide.

system-mediated effects on vascular tone and sodium excretion (Goldenberg, 2010). Many UV and HPLC methods for quantitative and qualitative analysis of these drugs have been reported individually for ALS and AML (Anandakumar and Jayamariappan, 2011; Bhatia et al., 2009; Chitlange et al., 2008; Dongre et al., 2008; Feng et al., 2002; Jothieswari et al., 2010; Kumar, 2010; Mohammadi et al., 2007; Nalwade et al., 2011; Patil et al., 2009; Rathee et al., 2010; Salve et al., 2009; Wrasse-Sangoi et al., 2011). However, an intensive literature search revealed the bioanalytical methods reported for determination of ALS (Adireddy et al., 2013; Aydoğmuş et al., 2012; Burckhardt et al., 2013; Limoges et al., 2008; Waldmeier et al., 2007), AML (Bahrami and Mirzaeei, 2004; Bhatt et al., 2007; Ma et al., 2007; Tatar and Atmaca, 2001; Zarghi et al., 2005), a combination of AML and HCTZ (El-Gizawy et al., 2012; Sharma and Pancholi, 2012) and a combination of ALS and HCTZ (Belal et al., 2013). To the best of our knowledge there is no bioanalytical method available for the simultaneous determination of ALS and AML. Although a recent method was developed by Kumara Swamy et al. (2012) for the simultaneous analysis of ALS, AML and HCTZ in bulk and pharmaceutical formulations, the method suffers from limitations like lack of sensitivity, and also the retention time of the first eluting analyte (HCTZ) may not be acceptable for its accurate quantitation, especially in biological matrices. Furthermore, the developed method was aimed at determining these drugs in biological matrices for bioequivalence studies and therapeutic drug monitoring. In this work, a simple, fast and sensitive HPLC-PDA method has been established for the simultaneous analysis of ALS and AML in human plasma. The suitability of the method is demonstrated by validation, carried out by following the guidelines proposed by the US Food and Drug Administration (2001).

Experimental Chemicals and reagents Working standards of ALS (98.9% pure), AML (99.3% pure) and HCTZ (98.2% pure) were procured from Drugs Testing Laboratory (Tamil nadu, India). Acetonitrile of HPLC grade was purchased from Sigma Aldrich (Bangalore, India). di-potassium ortho-phosphate and ortho-phosphoric acid of AR grade were purchased from SD Fine Chemicals (Mumbai, India). Milli-Q water was prepared from Milli-Q systems (Bangalore, India).

The LC system used for analysis consists of a Shimadzu LC-10 AT VP solvent delivery module with an SPD M-10AVP photodiode array detector. Data acquisition was performed using Shimadzu Class VP software. Chromatographic analysis was carried out on a Hibar C18 column (250 × 4.6 mm; Merck, Inc.) using a mobile phase comprising di-potassium ortho-phosphate (25 mM, pH 4.2) and acetonitrile (60:40 v/v) at a flow rate of 0.8mL/min, and the sample pretreatment was carried out by protein precipitation. All eluents were filtered through 0.45 μm membrane filter (Gelman, India) and degassed ultrasonically for 15 min before use. A sample volume of 20 μL was injected and the detector was set at 232 nm for determination of all of the analytes.

Stock solutions, calibration standards and quality control samples The standard stock solutions of ALS, AML and HCTZ (IS) at 1 mg/mL were prepared by dissolving appropriate amounts of each compound in acetonitrile. The calibration standards and the QC samples were prepared by spiking blank plasma with stock solution. Calibration standards of range the 30.0–600.0 ng/mL (30.0, 60.0, 90.0, 180.0, 240.0, 360.0, 480.0 and 600.0 ng/mL) for ALS and 1.1–22 ng/mL (1.1, 2.2, 3.3, 6.6, 8.8, 13.2, 17.6 and 22 ng/ mL) for AML were prepared and made up the volume with blank plasma, transferred into different 2 mL Eppendorf centrifuge tubes and stored at 70 ± 2°C until further processing. Quality control (QC) samples were prepared independently in the same matrix (blank human plasma).

Plasma sample preparation At the time of analysis, the samples were removed from the deep freezer and kept at room temperature and allowed to thaw. A 100 μL aliquot of HCTZ (I.S) solution was added to 200 μL of plasma containing the drug sample. The final sample was vortex-mixed and extracted with acetonitrile. The clear supernatant was obtained and evaporated under stream of nitrogen, the residue obtained was reconstituted with 250 μL mobile phase and a 20 μL aliquot of the reconstituted extract was injected for analysis.

Method validation The method validation was performed according to the US Food and Drug Administration (2001) guidelines for bioanalytical method validation. The validation parameters like selectivity, specificity, linearity, limits of quantification and detection, precision, accuracy, recovery and stability were addressed. Selectivity and specificity. For selectivity six lots of blank plasma without analytes were processed as per procedure and screened for any traces of interference from endogenous compounds (matrix effects) at the retention times of ALS, AML and IS. Lots that are free from potential interference were used for further analysis. Interference from other commonly co-prescribed drugs for combined cardiovascular therapy such as aspirin, ibuprofen, valsartan, telmisartan, enalapril, clopidiogrel, paracetamol and ramipril at a concentration of 1 μg/mL was also tested. For specificity LLOQ (lower limit of quantification) concentrations of analytes were spiked into blank plasma free from interferences and processed as per procedure, then analyzed for CV (coefficient of variation) for recovery.

Plasma sampling

Linearity. The linearity of the bioanalytical method was established in the range of 30–600 ng/mL for ALS and 1.1–22 μg/mL for AML using calibration curves prepared on five separate days (n = 5) with spiked plasma calibration standards at six different concentrations levels. Calibration curves were prepared by plotting the peak-area ratios (analyte peak area/IS peak area) vs concentration, and fitted to the equation y = mx + c.

Blank human plasma (drug-free) containing heparin sodium (anticoagulant) was collected from healthy volunteers and was stored at 20°C until use.

Limits of detection and quantification. The limit of detection (LOD), defined as the lowest concentration that can be distinguished from the

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Biomed. Chromatogr. 2014

Simultaneous estimation of aliskiren and amlodipine in human plasma noise level, was determined by analyzing plasma samples with known concentrations of ALS and AML after successive dilutions, and it was established by measuring signal-to-noise ratios at 5:1 by measuring signals of known low concentrations of ALS and AML and comparing them with those of blank plasma samples that could be reliably detected. The limit of quantification (LOQ) was defined as the lowest concentration of the analyte that could be measured with precision (expressed as CV) and accuracy under the stated experimental conditions. The LOQ was evaluated by comparing the signal-to-noise ratios at 10:1 by measuring signals of known low concentrations of ALS and AML with those of blank plasma samples which were prepared in five replicates (n = 5). Precision and accuracy. Intra and interday precision and accuracy were assessed using QC samples analyzed in five replicates (n = 5) at three different concentration levels representative of the entire range of the calibration curves (low, medium and high QC samples). The concentrations tested were 90, 240 and 480 ng/mL for ALS and 3.3, 8.8 and 17.6 ng/mL for AML. The acceptance criterion for precision was CV ≤15% (or 20% for LOQ), and that for accuracy was a bias value within

Development and validation of a HPLC-PDA bioanalytical method for the simultaneous estimation of Aliskiren and Amlodipine in human plasma.

A simple, unique and selective HPLC-PDA method was developed and validated for the simultaneous estimation of aliskiren (ALS) and amlodipine (AML) in ...
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