A Novel Approach: Effect of polarity Index of mobile phase on Retention Time of Antihyperlipidemic Antihypertensive and Angiotensin inhibiting Drugs in RP-HPLC Method

Dyade G. K. ¹*, Sawant R. L.², Joshi H. A.¹, Shinde A. D.¹, Bandal R. S.¹, Gadhingleskar S. V.³

¹Dept. of Postgraduate in Pharmaceutical Sciences, SVPM College of Pharmacy,

Malegaon (BK), 413115, Pune, India.

²DVVPF’S College of Pharmacy, Vilad, Ahmednagar. Savitribai Phule Pune University, Pune India.

³FDC Limited, Jogeshwari (East), Mumbai, India

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

ABSTRACT:

The present research was dedicated to know polarity index of mobile phase (instead of stating mobile phase composition) and to study its effect, on the retention time of drugs. Objective of present research was to optimize compatible mobile phase able to separate many drugs simultaneously. Common approach adopted to optimize mobile phase is by variation in mobile phase composition, during development of analytical, bio analytical liquid chromatographic techniques. Polarity index is the cumulative term for the polarity of all solvents and their proportion constituting mobile phase. Solvents acetonitrile, methanol and water were used in varying composition and Polarity index was calculated as arithmetic sum of polarity of each solvent and volume fraction of each solvent in mobile phase. Effect of polarity index of mobile phase on some antihyperlipidemic drugs like simvastatin (SIM), rosuvastatin (RV), fluvastatin (FV) and ezetimibe (EZE); angiotensin inhibiting drug like valsartan (VAL), and anti-hypertensive drugs amlodipine besylate (AMB) and lercanidipine HCl (LC) were studied in this RP-HPLC technique. The separated eluents were monitored by keeping UV detector at suitable wavelength where all drugs shows well absorption. The study proved polarity index and pH of solvent has marked effect on retention time of drugs and system suitability parameters in the chromatography. This was beneficial in optimization of the mobile phase for weakly acidic or basic drugs; and necessity of pH adjustment in many instances. Mobile phase with polarity index 7.586 (acetonitrile: water 60:40 %v/v) and 6.1386 (methanol: water 80:20 %v/v) were efficient in distinct separation of many drugs simultaneously in this study. This study proved that efficient and compatible mobile phase was capable in separation and simultaneous detection of many drugs, saves time to restore chromatographic conditions on mobile phase changeover.

KEYWORDS: Amlodipine, rosuvastatin, fluvastatin, simvastatin, lercanidipine, valsartan, ezetimibe, System suitability Tests (SST).

INTRODUCTION:

Antihyperlipidemic agents, angiotensin inhibiting agent and antihypertensive drugs were studied to know their chromatographic behavior in optimization of mobile phase, and polarity index was used to know strength of mobile phase. The chemical, pharmacological information of drugs are described below.

Amlodipine^[1] (AMB) 2-[(2-aminoethoxy) methyl]4-(2-chlorophenyl)-1,4-dihydro-6-methyl-3,5-pyridine dicarboxylic acid 3-ethyl 5 methyl ester is a second generation 1,4-dihydropyridine derivative of the prototypical molecule nifedipine, it has greater selectivity for the vascular smooth muscle than myocardial tissue, a longer half-life (3-4 Hrs.) and less negative inotropy than the prototypical nifedipine. It is used in the treatment of chronic static angina and in the management of mild to moderate essential hypertension. It is marketed as the benzene sulfonic acid salt (besylate)^[2-6].

Ezetimibe^[1] (EZE) chemically (3R, 4 S)-1-(4-fluorophenyl) -3- ((3s)-3- (4-fluorophenyl-3-hydroxypropyl)-4- (4-hydroxyphenyl)-2-azetidinone is an antihyperlipidemic agent that has usefulness in lowering cholesterol levels. It acts by decreasing cholesterol absorption in the intestine by blocking the absorption of the sterol at the brush boarder. Specifically, the ß-lactam binds to the Niemann-pick C1 like 1(NPC1L1) protein on the gastrointestinal tract that is responsible for cholesterol absorption^[2,3,6]. Although it may be used alone, it is marketed as a combination product with simvastatin.

Fluvastatin^[1-6] (FV) chemically sodium - A 3 hydroxy 3 methyl glutaryl coenzyme A (HMG-10A) reductase inhibitor is a lipid regulator drug with action on plasma lipids similar to those of simvastatin. It is used to reduce total cholesterol, LDL cholesterol, apolipoprotein B and triglycerides and to increase HDL-cholesterol.

Lercanidipine HCl^[1-2] (LC) chemically is 1,4 dihydro-2,6 dimethyl-4(3-nitrophenyl)-3-5-pyridinedicarboxylic acid-3-[2[(3,3 diphenyl propyl)methyl amino]-1,1 dimethyl ethyl] 5 methyl ester a dihydropyridine calcium channel blocker with actions similar to those of nifedipine. It is used in the treatment of hypertension.

Simvastatin^[1-3,5,6] (SIM)chemically 2-2-dimethyl butanoic acid, 1,2,3,7,8,8a-hexahydro,3,7-dimethyl-8-[2(tetrahydro-4hydroxy-6-oxo-2pyran-2-yl)ethyl]-1-naphthalenyl ester is an analog of lovastatin. In liver undergo extensive metabolism to several open ring hydroxyl acids including the active ß-hydroxy acids. They are also highly bound to plasma proteins.

Rosuvastatin^[1-4,6](RV) chemically 7-[4-(4-fluorophenyl)-6-(1-methylethyl)-2-(methyl-methylsulfonyl-amino)-pyrimidin-5-yl] 3, 5-dihydroxy-hept-6-enoic acid is one of the more recently introduced statins. Statins are HMG-CoA reductase inhibitors. The conversion of 3-hydroxy 3 methyl glutaryl (HMG)-COA to mevalonic acid is especially important, because it is a primary control site for cholesterol bio synthesis.

Valsartan^[1-6](VAL)chemically is N-(1-oxophenyl)-N-[[2¹-(1H-tetrazol-5-yl)(1-1¹-biphenyl) -4-yl]methyl]-L-valine possesses the acidic tetrazole system, which most likely plays, a role, similar to that of the acidic groups of angiotensin II in binding to the angiotensin II receptor, In addition the biphenyl system that serves to separate the tetrazole from the aliphatic nitrogen is still present, In addition there is a carboxylic acid side chain in the valine moiety that also serves to bind to the angiotensin II receptor. Chemical structure of these drug molecules is shown in (Figure No 1)

In literature survey of selected drugs for this study it was revealed that, analytical methods have been reported for the estimation of amlodipine besylate alone or in combination with other drugs these includes chemometric method^[7], RP-HPLC method^[8,9]. Similarly analytical methods have been reported for the estimation of ezetimibe includes RP-HPLC method^[10], for the estimation of lercanidipine UV spectroscopic method^[11], for the estimation of rosuvastatin RP-HPLC method^[12,13], for the estimation of simvastatin Dropping mercury electrode method^[14], and for the estimation of valsartan RP-HPLC method^[15,16], spectroscopic method^[17] have been reported. Many research articles have been published which elaborates novelty in HPLC method, rejuvenation of HPLC knowledge and new approach in development of new analytical method, bio analytical method. These articles were highlighted as^[18]discusses a general system suitability tests that monitors instrument performance throughout run,^[19] reviews HPLC is a generally accepted method for the assay of drug substance,^[20]reviews the objectives of analytical methods and the objectives of validation methods,^[21] reviews parameter study of analytical method,^[22]study to investigate the impact of mobile phase influences as important factor of selectivity during in method development,^[23] discusses improving method capability of a drug substance HPLC assay,^[24]discusses stability of new ACE inhibitors in different pH, and^[25]highlights analyse complex samples using serially coupled columns.

In chromatography optimization of mobile phase is crucial and worthy procedure for separation and subsequent measurement for complex mixtures. The solubility of substance in suitable solvent, partition coefficient of the substance in solvent mixture, and polarity of solvent has measurable impact on retention time of analyte. The solubility of a drug is due in large measure to the polarity of solvent, that is, to its dipole moment. Polar solvents dissolve ionic solvents and other polar substances. The solubility of a substance also depends on structural features such as the ratio of the polar to the nonpolar groups of the molecule. It becomes evident that the solubility of weak electrolytes is strongly influenced by the pH of the solution^[26]shown in below equation.

Where

pH_p= is the pH below which the drug separates from solution as the undissociated acid.

S₀ – molar solubility of undissociated acid

S – Initial conc. of drug

pH at which free base shows solubility in following equation.

pH_p- pH above which the drug begins to precipitate from solution as the free base.

S_o– molar solubility of free base in water

S – conc. of drug initially added as the salt.

Polarity index of mixed mobile phase is the arithmetic average of the solvent polarity index weighting factor adjusted according to the volume fraction of each solvent as is given by equation.

Where P_i is the polarity index weighting factor of solvent i and Q_i is the volume fraction of solvent i.

MATERIALS AND METHODS:

Instrumentation:

This study was performed with a Shimadzu (Japan) prominence chromatograph equipped with an LC - 20 AT solvent delivery system, a universal loop injector (Rheodyne 7725), of injection capacity of 20µl, and an SPD - 20 A UV – Visible detector set at wavelength. The equipment was controlled by a PC work station with clarity software. Compounds were separated on a Phenomenex Luna C₁₈ column (250mm × 4.6mm i.d., 5-μm particle size) under reversed phase partition conditions. The mobile phase was selected Acetonitrile: Water, Methanol: Water in varying composition and pH adjusted with acetic acid. The mobile phase was pumped at a flow rate of 0.8ml/min during run time. Before chromatography both the mobile phase and sample solutions were degassed by the use of a sonicator (Lab man scientific Instruments Chennai) and filtered through a 0.22μm filter (Pall corporation, Mumbai). Chromatography was performed in an ambient temperature maintained at 40±1°C. For selecting the working wavelength of detection, the UV spectrum of SMV, RV, FV, EZE, VAL and AMB was taken using a Shimadzu–1700 Double beam UV-Visible spectrophotometer (Shimadzu, Kyoto, Japan). For weighing of drug Afcoset balance (The Bombay Burmah Trading Corpo Ltd) with accuracy ±0.1mg Model No. ER 200A was used throughout study.

Reagents and Chemicals:

Pharmaceutically pure samples of RV from Ajanta pharma Aurangabad India, AMB from Cure pharma Pune India, VAL from FDC Mumbai India and EZE, SIM, FV and LC from Swapnaroop Lab Aurangabad India were procured. HPLC grade acetonitrile, methanol, water and acetic acid were obtained from Qualigens India Pvt Limited Mumbai and Merck India Ltd.

Table No.1 Characteristics of solvents

Sr No	Name of solvent	Formula	Boiling point	Dielectric constant	Density g/ml	Dipole moment	Polarity index
1	Acetonitrile MCN	CH₃C=N	82	37.5	0.786	3.92	5.8
2	Methanol	CH₃OH	65	33	0.791	1.70	5.1
3	Acetic acid	CH₃COOH	118	6.2	1.049	1.74	6.2
4	water	H₂O	100	80	1.000	1.85	10.2

Preparation of Stock Solution of pure drug:

Five mobile phases were prepared constituting composition of acetonitrile: water and ranges from 60:40 to 85: 15 and 3 mobile phases were prepared constituting composition of methanol: water and ranges from 75:25 to 85:15, and used throughout this study.

Each drug 5mg was accurately weighed separately and transferred into separate 10ml volumetric flasks. Each drug was dissolved in ‘A’ mobile phase acetonitrile: water 60:40 (pH adjusted to 4.6±0.1 with acetic acid) and the volume were made up to the 10ml with mobile phase. From this standard stock solution, the aliquots of solution were further diluted with mobile phase to obtain standard solution in working conc. range. The standard solutions were filtered through syringe filter (pore size 0.22u). Before analysis both the mobile phase and solutions were degassed by the use of sonicator for 10 minutes and injected 20μl standard solutions each time with Hamilton syringe. The flow rate was kept at 0.8 ml/min and the run time was 12 min.

Retention time and system suitability parameters were measured for every injected solution.

The above procedure i.e. weighing of drug, dilution with the mobile phase, preparation of solutions from aliquots, and injecting of solution were treated for other mobile phases B, C, D, E, F, G and H.

Preparation of Mixed solution:

Capability of mobile phase to resolve many analyte was tested by preparing mixed standard solution simulated to marketed pharmaceutical dosage form. The mixed standard solutions were filtered through syringe filter (pore size 0.22u). The solutions were degassed by the use of sonicator for 10 minutes and injected 20 μl mixed solutions each time with Hamilton syringe. Retention time was measured to specify drug in the mixture.

Calculation of polarity index of each mobile phase:

Calculated polarity index of each mobile phase above equation was applied and obtained data were tabulated under result and discussion section. For calculation the reported values of polarity index of solvent are accounted here and resourced by^[27] shown in Table-1.

RESULTS AND DISCUSSION:

The effectiveness of a chromatographic column in separating two solutes depends in part upon the relative rates at which the two species are eluted. These rates are determined by the magnitude of the equilibrium constants for the reactions by which the solutes distribute themselves between the mobile and stationary phases. The time analyte takes after sample injection for the analyte peak to reach the detector is called the retention time and is given by the symbol t_R.

The polarity index of each mobile was known (Table No 2) from the equation and it was found that as proportional amount of water in mobile phase decreases, the polarity index also decreases as water bears highest polarity index. Here the polarity index was read instead of composition of mobile phase, and retention time of drug was shown in chart type graph in individual mobile phase.

Table No 2. Polarity index of used mobile phase

Sr No.	Alphabetical code of Mobile Phase, composition ratio and pH	Polarity index of mobile phase
1	A [Acetonitrile: water (60:40) pH 4.9]	7.586
2	B [Acetonitrile: water (70:30) pH 4.7]	7.1386
3	C [Acetonitrile: water (75:25) pH 4.6]	6.9015
4	D [Acetonitrile: water (80:20) pH 4.4]	6.6986
5	E [Acetonitrile: water (85:15) pH 4.5]	6.4786
6	F [Methanol : Water (75:25) pH 4.5]	6.375
7	G [Methanol: water (80: 20) pH 4.5]	6.1386
8	H [Methanol : Water (85:15) pH 4.6]	5.865

From obtained chromatograph ezetimibe, valsartan and simvastatin were shown more retention time ( Figure No 2) in mobile phase A with polarity index 7.586, and then marginal decrease in retention time was observed in B mobile phase, afterwards gradual decrease in retention time (Figure No 3) was observed in mobile phase C.

Figure No 2 Chromatograph of Ezetimibe and Valsartan in Mobile Phase A

Table No 3a. At glance study Retention time of drug in mobile phase

Mobile Phase	Polarity index	Retention time of drugs
Mobile Phase	Polarity index	EZE	RV	VAL	AMB	SIM
A	7.586	7.754	5.760	8.985	2.453	>24
B	7.1386	6.670	5.313	6.032	3.327	18.917
C	6.9015	4.701	3.963	5.017	2.453	14.568
D	6.6986	4.321	3.751	4.252	2.958	12.68
E	6.4786	3.957	3.630	3.852	2.527	8.102

Table No 3b. At glance study Retention time of drug in mobile phase

Mobile Phase	Polarity index	Retention time of drugs
Mobile Phase	Polarity index	EZE	RV	VAL	FV	LC
F	6.375	7.317	5.108	8.928	12.947	3.248
G	6.1386	6.602	5.005	6.932	11.100	2.842
H	5.865	5.328	4.43	6.217	9.658	2.453

Table No 4a. System suitability parameters of drugs in selective mobile phase

Mobile phase composition	Name of Drug	Retention time*	*Theoretical plates**	Resolution	Tailing factor	Asymmetry factor*
Acetonitrile: water (60:40) pH 4.9	EZE	7.754	19055	3.472	1.067	1.103
	RV	5.760	14323	4.252	1.119	1.200
	VAL	8.985	18218	18.298	1.078	1.125
	AMB	2.453	5928	2.060	1.250	1.429
Acetonitrile: water (75:25) pH 4.6	EZE	4.701	11445	2.360	1.426	1.456
	RV	3.963	7375	2.623	1.477	1.572
	VAL	5.017	13598	11.538	1.132	1.200
	AMB	2.524	6066	-	1.000	1.045
	SIM	14.568	16656	34.899	0.963	0.971
Acetonitrile: water (85:15) pH 4.5	EZE	3.957	9956	4.003	1.413	1.425
	RV	3.630	11483	-	1.157	1.273
	VAL	3.852	11478	6.311	1.220	1.140
	AMB	2.52	2894	-	1.317	1.479
	SIM	8.10	17097	26.870	1.295	1.487

Table No 4b. System suitability parameters of drugs in selective mobile phase

Mobile phase composition	Name of Drug	Retention time*	*Theoretical plates**	Resolution	Tailing factor	Asymmetry factor*
Methanol: Water (75:25)	EZE	7.317	10528	7.714	1.172	1.320
	RV	5.108	5998	6.933	0.827	0.757
	VAL	8.928	9658	3.419	1.330	1.356
	FV	12.947	12222	15.00	1.209	1.303
	LC	3.248	5034	2.684	1.221	1.326
Methanol: Water (80:20)	EZE	6.602	13900	14.038	1.103	1.153
	RV	5.005	8779	3.452	1.167	1.273
	VAL	6.932	9959	7.356	1.105	1.172
	FV	11.100	14563	4.225	1.047	1.068
	LC	2.842	5634	-	1.313	1.457

CONCLUSIONS:

Polarity index of mobile phase is rather simple term and explains the strength of mobile phase. Here it was concluded that as polarity of mobile phase increases, there is increase in retention time and vice versa. Mobile phase A and G were shown good resolution and acceptable system suitability parameters. The polarity index against retention time chart would be novel approach if accepted during optimisation of mobile phase. The efficient unique mobile phase A and G were able to separate and simultaneously detect five drugs; such approach would be beneficial in keeping consistency of chromatographic conditions.

CONFLICT OF INTEREST:

The authors declared that they have no conflicts of interest.

ACKNOWLEDGEMENTS:

We the authors express gratitude to Dr. P.Y. Pawar, Principal, Dr VVPF’S College of Pharmacy Vilad, Ahmednagar, India for providing the necessary facilities to endeavor the research work. Also expresses thankfulness to FDC Pharma Mumbai India, Ajanta Pharma India, Swapnaroop Lab Ahmadabad India for providing pure drugs.

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Received on 31.10.2019 Modified on 25.12.2019

Research J. Pharm. and Tech. 2020; 13(7): 3065-3071.

DOI: 10.5958/0974-360X.2020.00544.2