научная статья по теме SIMULTANEOUS DETERMINATION OF ALISKIREN, AMLODIPINE AND HYDROCHLOROTHIAZIDE IN SPIKED HUMAN PLASMA AND URINE BY HIGH PERFORMANCE LIQUID CHROMATOGRAPHY Химия

Текст научной статьи на тему «SIMULTANEOUS DETERMINATION OF ALISKIREN, AMLODIPINE AND HYDROCHLOROTHIAZIDE IN SPIKED HUMAN PLASMA AND URINE BY HIGH PERFORMANCE LIQUID CHROMATOGRAPHY»

ЖУРНАЛ АНАЛИТИЧЕСКОЙ ХИМИИ, 2015, том 70, № 4, с. 426-433

ОРИГИНАЛЬНЫЕ СТАТЬИ

УДК 543

SIMULTANEOUS DETERMINATION OF ALISKIREN, AMLODIPINE AND HYDROCHLOROTHIAZIDE IN SPIKED HUMAN PLASMA AND URINE BY HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

© 2015 Zeynep Aydogmu§

Istanbul University, Faculty of Pharmacy, Department of Analytical Chemistry 34116, Beyazit, Istanbul, Turkey E-mail: zaydogmus@yahoo.com Received 26.04.2013; in final form 10.10.2014

A reversed-phase (RP) liquid chromatographic technique for the simultaneous determination of aliskiren (ALS), amlodipine (AMD) and hydrochlorothiazide (HCT) in spiked human plasma and urine was presented. The method employs a gradient elution using 10 mM orthophosphoric acid containing 0.1% triethy-lamine (pH 2.5, v/v) and acetonitrile and an RP-C18 column (4.6 mm x 250 mm, 5 p.m, Phenomenex) at 1 mL/min of flow rate, with a UV photo diode array detector at 271 nm. The linear ranges were 0.01—10 p.g/mL in plasma and 0.05—10 p.g/mL in urine for both ALS and AMD. The linearty of HCT was in the range of 0.0125—2.5 p.g/mL in plasma and urine. Correlation coefficients (r2) were higher than 0.9983 for all of the analytes, indicating good linear relationship. The method validation was performed with respect to linearity, recovery, accuracy, precision and stability. The developed method could be applied in the routine clinical analysis.

Keywords: aliskiren, amlodipine, hydrochlorothiazide, HPLC, biological fluids. DOI: 10.7868/S0044450215040210

Aliskiren hemifumarate (Scheme) is the direct renin enzyme inhibitor clinically used in treatment of hypertension either alone or in combination with other antihypertensive agents [1]. After administration, ALS is rapid-

ly absorbed with peak plasma concentration occurring after 1—3 h. Its bioavailability is about 2.6% and it is eliminated unchanged primarily by the biliary-fecal route (91%) and renal excretion is only about 0.6% [2].

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Aliskiren

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ch3 Amlodipine

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Hydrochlorothiazide

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Amlodipine besylate is a dihydropyridine calcium and angina pectoris. Pharmacokinetic studies have channel blocker clinically used to treat hypertension demonstrated that AMD is well absorbed orally, with

peak plasma concentrations reaching within 6—12 h after oral administration. Mean bioavailability of AMD is about 64%. Plasma protein binding of amlo-dipine in humans was 98% [3]. Amlodipine is metabolised by both the renal and biliary routes to inactive metabolites with 10% of a dose as unchanged compound and excretion metabolites is about 60% in the urine [4].

Hydrochlorothiazide is a thiazide class of diuretic. HCT used in the treatment of moderate hypertension alone or widely in combination with different antihy-pertensive agents [5].

Literature review revealed that there are some analytical techniques for quantification and pharmacoki-netic studies of ALS including micro-radioimmu-noassay [6], spectrofluorimetric [7], spectrophotometry [8], HPLC-tandem mass (HPLC-MS-MS)

[9], micellar electrokinetic chromatography (MEKC)

[10] and HPLC [11]. As for AMD, several techniques have been published concerning its determination either in pharmaceutical preparations and biological fluids, or in the mixture with other drugs. These include HPLC—MS—MS using solid phase extraction [12], spectrophotometric methods [13], gas chromatography with mass spectrometry (GC—MS) [14], enzyme associated with immunosorbent method [15], high performance-thin layer chromatography [16], voltammetry [17], capillary electrophoresis (CE) [18], isotachophoresis-capillary zone electrophoresis [19], flow injection method [20], electrospray ionization LC-MS-MS [21] and CE [22]. Also, different methods have been reported for the determination of HCT either in pure form and in biological fluids or in mixture with antihypertensive drugs like chemiluminom-etry [23, 24], electrochemical method [25], GC-MS [26], CE and capillary electrochromatography [27].

Among reported techniques for the simultaneous assay of these drugs in the biological fluids and/or pharmaceutical preparations, HPLC with UV detector was the most common technique since it has high selectivity, provides good separation and does not usually need derivatization. Numerous HPLC-UV methods have been reported for the simultaneous determination of ALS with the combination of HCT and other drugs [28-30]. For AMD either with HCT or with other drugs, HPLC-UV methods [31-37] have been reported. Similarly, some HPLC-UV methods for determination of HCT combined with the different drugs [38, 39] have been described.

Although several chromatographic methods have been described for the simultaneous determination of ALS, AMD and HCT in combination with each other or with other drugs, only two RP-HPLC methods [40, 41] for these three drugs in pharmaceutical dosage forms have been published very recently. These reported methods are not sensitive enough to determine these drugs in biological fluids. In the study by Alagar et al. [40], linear ranges were found between 1-

6 ^g/mL for ALS and AMD and 2-12 ^g/mL for HCT. In the study by Swamy et. al. [41], linear ranges (^g/mL) were reported between 12.5-62.5 for HCT, 5-25 for AMD and 150-750 for ALS. Also, one pharmacokinetic study [1] was published previously (linear ranges were reported between 24-400 ng/mL for three drugs). However, up to now, there is no HPLC technique for simultaneous determination of ALS, AMD and HCT in plasma and urine. Therefore, there is a need to develop simple, more sensitive, rapid and validated methods that may be used in quality control and clinical laboratories. For this purpose, a RP-HPLC method has been developed for the determination of the three drugs in mixture in human plasma and urine samples.

EXPERIMENTAL

Chemicals and materials. ALS, AMD and HCT

were kindly supplied from Novartis, Eczacibas i pharmaceuticals, and Abdi Ibrahim pharmaceuticals (all from Istanbul, Turkey), respectively. All solvents and chemicals were HPLC-grade from Merck (Germany). A WTW pH 526 digital pH meter was used in the pH measurements. An aquaMAXTM-ultra water purification system (Young-lin instrument, Korea) was used for water purification. Drug free human plasma was purchased from Blood Center of Cerrahpasa Medical Faculty (Istanbul, Turkey), kept in freezer (-20°C), and thawed in warm water before use.

Instrumentation and chromatographic conditions.

Analysis was carried out on Thermo Separation system (San Jose, CA). The system with the following parts: controller SN 4000, pump P 4000 and autosampler AS 3000, fitted with 20 ^L sample loop, and photodiode array detector UV 6000 LP. Data acquisition was performed with ChromQuest 5.0 software. Separations were carried out with C18-column (250 mm x 4.6 mm i.d., 5 ^m, Phenomenex Luna). A guard column with the same packing material was used. 10 mM phosphoric acid-0.1% triethylamine (solution A) and acetonitrile (solution B) were used as mobile phase in gradient mode with 1 mL/min flow rate. The used elution conditions were: initial A-B (90 : 10), in 12 min A-B (35 : 65), back to initial conditions in 4 min, and equilibration for 2 min. The mobile phase was filtered through a Milli-pore vacuum filter system. A UV photo diode array detector set at 271 nm was used to monitor eluents.

Solutions and quality control samples. Stock solutions of the studied drugs at 1.0 mg/mL (for ALS and AMD concentration expressed as base compound) were prepared separately in acetonitrile. The preparations of working standard solutions were made by appropriate dilutions in acetonitrile. Under these conditions the stock solutions remained stable at least two weeks.

Procedure for the determination of ALS, AMD and HCT in human plasma. Aliquots of 0.250 mL of drug

free plasma samples were transferred into tubes and added standard analytes solutions at six different concentration levels to obtain a final concentration (in the three mixtures) ranges of 0.01—10 |g/mL for ALS and AMD, and 0.0125-2.5 |g/mL for HCT. Plasma proteins precipitated with 1.0 mL acetonitrile and then centrifuged at 4500 rpm for 20 min. The supernatant of these samples was evaporated to dryness under nitrogen at 40°C. The residue was redissolved with 1.0 mL mobile phase, and loaded into autosampler vials before injecting 20 |L into the HPLC system. A blank experiment was analyzed with drug-free plasma under the same conditions.

Procedure for the determination of ALS, AMD and HCT in spiked human urine. Aliquots of 0.250 mL of urine spiked with six different concentrations of standard analytes solutions to give a final concentration of mixture ranges of 0.05-10 |g/mL (for ALS and AMD) and 0.0125-2.5 |g/mL (for HCT) in mobile phase and injected into HPLC system. A blank experiment was carried out simultaneously.

Method validation. Linearity. To evaluate of linearity, calibration curves (five replicate analyses) were prepared using a set of six calibrations points, prepared in the ranges from 0.01 to 10 |g/mL in plasma and from 0.05 to 10 |g/mL in urine for both ALS and AMD. The linearity of HCT in samples was over the range of 0.0125 |g/mL. To construct the calibration curves, the peak areas were plotted against the concentration of the analytes under the optimized conditions. Alternatively, the corresponding regression equations were derived.

Precision and accuracy. The intra- and inter-day accuracy and precision were validated by investigated samples spiked with the quality control (QC) samples at three concentrations (0.05, 1.0 and 10.0 |g/mL for ALS and AMD, and 0.02, 0.1 and 2.5 |g/mL for HCT). Determinations were carried out with five replicates within same day for intra-day and on five separate days for inter-day precision.

Recovery. The absolute recovery was calculated by comparing the recovery of results obtained by extracted spiked human plasma and urine samples with the unextra

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