UHPLC-PDA Method for Simultaneous Determination of Sartans Antihypertensive Drugs

Hamid Khan

Rungta Institute of Pharmaceutical Sciences, Bhilai, Chhattisgarh, India

*Corresponding Author E-mail: khanhamid770@gmail.com

ABSTRACT:

In the presented work the Ultra-high performance Liquid Chromatographic-Photodiode Array Detection (UHPLC-PDA) method has been developed and validated for Simultaneous determination of Sartans Antihypertensive Drugs such as Losartan, Telmisartan, Valsartan, Candesartan, Irbesartan and Olmesartan. The chromatographic separation was achieved on Acquity UPLC^TM BEH C₁₈(100.0 × 2.1 mm, 1.7µm) column using isocratic mobile phase consisting of acetonitrile-2mM ammonium acetate (50:50, v/v) at a flow rate of 0.25 mL/min. The Detection of all the drugs was carried out by PDA detector at 245 nm. The method was applied for routine quality control analysis and pharmacokinetic study of these drugs in human plasma.

KEYWORDS: UPLC-PDA, Antihypertensive Drugs, ARBs, Sartans, Determination.

INTRODUCTION:

The UHPLC-PDA (Ultra-high Performance Liquid Chromatography-Photo diode Array) technique is comparatively new chromatographic technique and has been used worldwide in the quantitative analysis of drug products. This technique has been successfully applied in every area of pharmaceutical analysis. UHPLC provides the fast, better chromatographic separation with shorter chromatographic run time. PDA is a type of UV detector, which provides three-dimensional information of peaks in their chromatogram. Hence this combined technique can be employed for degradation study, metabolite profiling and pharmacokinetic study of drug products^1-3.

Losartan, Telmisartan, Valsartan, Candesartan, Irbesartan and Olmesartan are antihypertensive drug, belongs to a group of Angiotensin II Receptor Blockers (ARBs). They modulate the Renin-Angiotensin-Aldosterone-System (RAAS) by blocking the activation of Angiotensin II AT₁Receptors resulting in Vasodilation and Vasodilation ultimately cause the fall in blood pressure. These drugs have been used worldwide for the treatment of hypertension^4,5. The literature survey revealed that several methods have reported for determination of Losartan by UV⁶, HPLC^7,8 and LC-MS^9-11. Determination of Telmisartan was reported by UV¹², HPLC^13,14 and LC-MS^15-21. Analysis of Valsartan was reported by UV²², HPLC²³ and LC-MS^24-27. Determination of Candesartan has reported by UV²⁸, HPLC²⁹ and LC-MS³⁰. Irbesartan determined by UV³¹, HPLC³² and LC-MS³³. Olmesartan is determined by UV³⁴, HPLC³⁵ and LC-MS³⁶. However in our earlier work an UHPLC/Q-TOF-MS method has been developed for structural identification and quantitative determination of these drugs in their pure samples. But the reported method requires costly instrument such as Q-TOF-MS mass spectrometer, costly solvents, and complicated operational steps which is not suitable for routine analysis³⁷. Hence in the presented work a new UHPLC-PDA method has been developed, validated and applied for routine analysis and pharmacokinetic study of all Sartans Antihypertensive drugs in human plasma.

EXPERIMENTAL:

Chemicals and Reagents:

Pure samples of all the drugs were kindly supplied by Systopic Pharmaceuticals Ltd. (New Delhi, India). HPLC grade water; acetonitrile, methanol, and ammonium acetate were purchased from Fluka analytical, Sigma-Aldrich Corporation, St. Louis, MO, USA.

UHPLC-PDA Conditions:

UHPLC was performed with a Waters Acquity UPLC system equipped with a binary solvent manager, an auto-sampler, column manager and a tunable MS detector. Chromatographic separation was performed on a Waters Acquity UPLC BEH C₁₈(100.0 × 2.1mm, 1.7µm) column. The mobile phase for UPLC analysis consisted of acetonitrile-2 mM ammonium acetate (50:50, v/v) which was filtered through 0.45 mm membrane filter and degassed by sonication. For isocratic elution, the flow rate of the mobile phase was kept at 0.25mL/min and 10 mL of sample solution was injected in each run. The total chromatographic run time was 3.0 min. Detection was carried out by PDA detector at 245 nm.

Preparation of Standard Solutions:

Each of the drugs was weighed accurately and transfer to 50 mL volumetric flasks separately. The powders were then dissolved with approximately 25mL of methanol and ultrasonicated for 5 min. The final volume was made up with methanol. The solutions were further diluted with methanol: water (50:50, v/v) to give a series of standard solutions containing required concentrations for each compound.

Preparation of Sample Solutions:

500μL of plasma sample was transferred to 10mL glass tube. To this 5mL of extraction solvent (diethyl ether: dichloromethane 70:30, v/v) was added. The sample was mixed by vortexer for 5 min. The organic layer was transferred to another glass tube. The solid residue was evaporated to dryness using evaporator at 40ºC under a stream of nitrogen. The dried extract was reconstituted in 200μL of diluent (methanol: water, 50:50, v/v). This solution was filtered through 0.45μm nylon membrane filter to remove all the particulate materials. 20μL aliquot was injected in to UPLC system.

Validation of Method:

The developed method was validated according to ICH validation guidelines³⁸.Different standard concentrations each drug in the range of 1-1000ng/mL (1, 10, 50, 100, 200, 500, and 1000ng/mL) was spiked to 100μL of blank human plasma separately in methanol: water (50:50, v/v). Similarly the low, medium and high concentration QC samples containing different concentrations of the drugs were prepared independently using the same procedure. The solutions were filtered through 0.20 μm nylon syringe filter and injected in to the UPLC/QTOF-MS system for analysis. Linearity graph was prepared by average peak area of each concentration. Intraday and interday precision and accuracy was also evaluated by analyzing the samples for three consecutive days. Specificity is the ability of the method to measure the analyte response in the presence of sample components or matrix such as excipients, potential impurities and degradation products. The samples were chromatographed to determine the extent to which mobile phase components and excipients could contribute to the interference with the analytes. This was done by comparing the chromatograms of each analyte with chromatograms obtained from blank solution. The specificity of the method was examined by analyzing blank plasma extract.

Analysis of Marketed Tablets:

Twenty tablets of each drug is weighed accurately and powdered. Powder equivalent to required amount of drugs was taken and separately transferred to a 50mL volumetric flask. The powder was dissolved with approximately 25 mL of methanol and ultrasonicated for 10 min. The final volume was made up with methanol. This solution was filtered through a 0.45 mm nylon membrane filter to remove all the excipients. The resultant filtrate was further diluted with methanol: water (50:50, v/v) to give a sample solution containing 100ng/mL of each drugs. The amounts of drugs were present in individual tablets determined by calibration equations obtained from the respective calibration plot.

Pharmacokinetic Study:

The method was applied for pharmacokinetic study to determine the plasma concentrations of all the drugs from a clinical trial in which 3 healthy male volunteers received individual tablets containing each of the drugs. Tablets containing single individual drugs were taken from the market and given to 3 healthy male volunteers. The clinical study was performed in three consecutive days taking low dose of drugs. Blood samples were collected before and after 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12 h post-dosing. Plasma was separated by centrifugation and stored in refrigerator until analysis.

RESULTS AND DISCUSSION:

The isocratic mobile phase containing acetonitrile-2mM ammonium acetate (50:50, v/v) at a flow rate of 0.25 mL/min provide peaks with short retention times. The detection was carried out by PDA detector at 245 nm. All the drugs were scanned between 200-400 nm. Every drug was showed their own wavelength at which highest absorption takes place. Various drugs and their wavelength at which highest absorption occurs are presented at Table 1.

Table 1: Drugs and Maximum Absorption Wavelengths

Drugs	Absorption Maxima (nm)
Losartan	205
Telmisartan	296
Valsartan	210
Candesartan	254
Irbesartan	246
Olmesartan	257

All the drugs were showed high absorption at 245 nm. This wavelength has been selected as Isobestic Point for measurement of all of these drugs. At this wavelength an overlay UV Spectra has been taken by measuring the mixed solution of all the drugs. The retention time was found to be 0.50 min for Losartan, 1.0 min for Telmisartan, 1.25 min for Valsartan, 2.2 min for Candesartan, 1.75 min for Irbesartan and 2.5 min for Olmesartan. The total chromatographic run time was 3.0 min for each compound.

Validation of the method:

The Results obtained from Linearity, LOD, and LOQ is presented in Table 2. The obtained results indicated that higher sensitivity of the method. The RSD less than 2% were obtained for all the drugs by evaluation of intraday, interday, and different analysts precision suggested that an acceptable precision and accuracy of the method. No significant interference in the blank plasma traces was seen from endogenous substances at the retention time of both drugs, suggested that method was specific. The specificity was also determined by degradation study. Degradation was observed when the drugs were treated under stress degradation conditions. But under normal condition no any degradation was observed.

Table 2: Results Obtained from Linearity, LOD, and LOQ

Parameters	Losartan	Telmisartan	Valsartan	Candesartan	Irbesartan	Olmesartan
Linear range (ng/mL)	1-1000	1-1000	1-1000	1-1000	1-1000	1-1000
Correlation coefficient	0.9997	0.9998	0.9998	0.9997	0.9997	0.9998
LOD (ng/mL)	0.1	0.05	0.05	0.1	0.1	0.1
LOQ (ng/mL)	1	0.1	0.1	1	1	1

Table 3: Results Obtained from Pharmacokinetic Studies

Drugs	Brands	Bioavailability (%)^a	t_max (hrs)^b	C_max (ng/mL)^b	AUC (ng.h/mL)^b	Half Life (T_1/2) (hrs)
Losartan	Losakind (50 mg tab, Mankind)	25-35	1.15	390	1045	1.5- 2
Telmisartan	Telma (40 mg tab, Glenmark)	42-100	2.25	750	1250	24
Valsartan	Diovan (40 mg tab, Novartis)	25	2.3	552	950	6
Candesartan	Candesar (4 mg tab, Solus)	15	2.2	595	855	9
Irbesartan	Irbest (150 mg tab, Biochem)	60-80	1.7	775	1195	11-15
Olmesartan	Olmax 40 (40 mg tab, Glenmark)	26	2.10	695	1125	13

^aBioavailability in Literature ; ^bMean of three replicates (n = 3)

Analysis of Marketed Tablets:

The validated method was applied in the determination of each these drug in commercially available tablets as mentioned in Table 3. The content in various drug tablets was found to be between 98–100% with RSD less than 2%. The low values of RSD indicated that method was suitable for routine analysis of drugs in tablets without any interference from excipients. The low analysis time of 3 min allowed fast determination of the drugs, which is an important advantage for routine quality control analysis.

Pharmacokinetic Study:

The method was applied to pharmacokinetic study of these drugs in human plasma. The tablets containing adult dose of drugs were selected for clinical studies. The results of pharmacokinetic parameters obtained from mean plasma concentration time curve after administration of single tablets of each drug are presented in Table 3. The results obtained from pharmacokinetic parameters were not significantly different from reported methods.

CONCLUSION:

The UHPLC-PDA method has been developed, validated and applied for simultaneous determination of Sartans antihypertensive drugs. The method was found to be sensitive, fast and specific for determination of all the drugs. The quantification of drugs was performed by PDA detector at single wavelength that is 245nm. The acceptable precision and accuracy was obtained of this method for analysis. The isocratic elution of mobile phase was employed throughout the analysis. The proposed method was successfully applied in routine quality control analysis and also applied for pharmacokinetic study of these drugs in human plasma. It is suggested that the developed method should be applied for dissolution studies, degradation studies, stability studies and metabolite profiling of these drugs.

ACKNOWLEDGEMENTS:

The author is grateful to Systopic Laboratories Ltd., Delhi, India, for providing pure samples of all the Sartan antihypertensive drugs. The author is also thankful to Dean and In-charge of Instrumentation Facilities, Faculty of Pharmacy, Hamdard University, New Delhi, India, for providing opportunities to work on UPLC-PDA system.

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Received on 01.01.2020 Modified on 24.01.2020

Asian J. Pharm. Ana. 2020; 10(1):07-10.

DOI: 10.5958/2231-5675.2020.00002.2