INTRODUCTION:

Lisinopril, (S)-1-[N2-(1-Carboxy-3-phenylpropyl) - L-lysyl]-L-proline dihydrate, is an angiotensin converting enzyme inhibitor that is used in the treatment of hypertension and heart failure.^[1] It acts on the renin– angiotensin aldosterone system. It inhibits the conversion of the inactive angiotensin I to the highly potent vasoconstrictor, angiotensin II, and and also reduce the degradation of bradykinin.^[2] Amlodipine (AMLO), chemically, 2-[(2- aminoethoxy) methyl]-4-(2-chlorophenyl)-1, 4-dihydro- 6-methyl-3, 5- pyridinedicarboxylic acid 3-ethyl, 5-methyl ester, is an anti-hypertensive and an antianginal agent in the form of the besylate salt, Amlodipine besylate. The official methods for the determination of lisinopril are potentiometric acid–base titration^[3] and HPLC.^[4]

Various spectrophotometric methods have been reported for the determination of lisinopril in pharmaceutical tablets using different reagents.^[5] First, second derivative spectrophotometric ^[5,6] and spectrofluorometric methods^[5] were applied. The chromatographic techniques of analyses, HPLC,^[6]micellar electro kinetic chromatography^[7] and gas liquid chromatography^[8] have been employed. Capillary electrophoresis was applied to the determination of lisinopril in pharmaceutical tablets.^[9] The literature review reveals that few mehods for determination of lisinopril in biological fluids. These include HPLC,^[10] fluoroimmunoassay,^[11] radioimmunoassay,^[12] Titrimetric assay^[16] and fluoroenzymatic assay.^[13] Many spectrophotometric methods have been applied for the simultaneous determination of lisinopril and hydrochlorothiazide in binary mixture.^[14]Vierordt method, first derivative spectrophotometry, ratio spectra first derivative spectrophotometry.^[15]

Various analytical methods have been reported for the assay of Amlodipine besylate^[17] in pure form as well as in pharmaceutical formulations. They include HPLC,^[18-23]RP-HPLC,^[24-27] HPTLC,^[28-31] GC,^[32] GC-MS,^[33,40]LC-MS^[34] and fluorimetry,^[35] Derivative spectroscopy^[36,37] simultaneous multicomponent mode of analysis and difference spectrophotometry.^[38-39]

To the best of our knowledge, no study has been described for the simultaneous determination of this combination in pharmaceutical formulation by Stability study. Lisinopril is frequently co- formulated with Amlodipine. Analysis of such mixture is challenging, because Lisinopril (the minor component) is poorly absorbing light in the UV region while, Amlodipine (the major component) is strongly absorbing light in UV region. Hence the aim of the present investigation was to develop and validate stability indicating RP-HPLC method for Lisinopril and Amlodipine for its Pharmaceutical Preparation. Therefore it is desirable to develop simple and reproducible analytical methods.

EXPERIMENTAL:

Materials and Reagents:

Lisinopril (LSN) reference standard and amlodipine besylate (AML) reference standard were received as gift sample from Cipla Pharmaceutica Ltd, India. The pharmaceutical preparations of combination of lisinopril and amlodipine that is AMLOPRESS-L (Cipla) contains 5 mg of Lisinopril and 5 mg of amlodipine equivalent to amlodipine besylate was obtained from local market. Acetonitrile of HPLC grade was purchased (RANKEM ,India). Sodium Dihydrogen phosphate of analytical grade (Merck, india). All the solutions were protected for light and were analyzed on the day of preparations.

Instrument Condition:

A Shimadzu HPLC instrument (LC_2010 CHT) [software LC Solution, equipped with prominence diode array detector (SPD-M20A), Auto-sampler].The chromatographic separation was performed using a Hypersil BDS C18 (150 mm ´4.6 mm, 5 µm particle size), mobile phase consisting Buffer(0.05 M Sodium dihydrogen phosphate):Acetonitrile (50: 50, v/v) with apparent pH adjusted to 3.5 adjusted with Ortho-phosphoric acid, filtered through 0.45µm nylon filter and degassed in ultrasonic bath prior to use and UV detection at 258 nm using photo diode array detector. For analysis of forced degradation samples, the photodiode array detector was used in scan mode with a scan range of 220–400 nm and desired peak coverage of 100%. Peak homogeneity was expressed in terms of peak purity values, and was obtained directly from the spectral analysis report obtained using the above-mentioned software.

Standard and sample preparation:

The standard stock solutions 250 µg/ml of AML and LSN were prepared by dissolving working standard in small proportion of mobilephase and later diluted to desired volume with mobile phase. The standard calibration solution of AML and LSN having concentration in the range of 12.5-37.5 µg/ml were prepared by diluting stock solution with mobile phase.

Ten tablets were weighed, their mean weight determined, and crushed in mortar. An amount of powdered mass equivalent to 25 mg of AML and LSN content was transferred into a 100ml volumetric flask containing 10 ml of methanol, mechanically shaken for 10 min, ultrasonicated for 5 min, and then diluted to volume with mobile phase (sample stock solution). 5 ml aliquot diluted to 50 ml with mobile phase (sample solution). A small portion of sample solution was filtered through 0.45µm nylon filter and used for injection on HPLC.

Procedure for forced degradation study:

Forced degradation of each drug substances and the drug product was carried out under acid/base hydrolytic, thermal and oxidative stress conditions.

For Acid hydrolysis, accurately weighed quantity of 25 mg AML and 25 mg LSN were transferred into 100 ml volumetric flask, dissolve with 30 ml of mobile phase and dilute up to mark with 0.1 N HCl (250 µg/ml). It was kept for 30 minutes at room temperature. From this solution 5 ml was taken and transferred into 50 ml volumetric flask and diluted up to mark with mobile phase (25µg/ml). Aliquots (20 μL) of the stressed samples were injected into the HPLC system as described under chromatographic conditions, and the chromatograms were recorded.

For base hydrolysis, accurately weighed quantity of 25 mg AML and 25 mg LSN were transferred into 100 ml volumetric flask, dissolve with 30 ml of mobile phase and dilute up to mark with 0.1 N NaOH (250 µg/ml). It was kept for 30 minutes at room temperature. From this solution 5 ml was taken and transferred into 50 ml volumetric flask and diluted up to mark with mobile phase (25µg/ml). Aliquots (20 μL) of the stressed samples were injected into the HPLC system as described under chromatographic conditions, and the chromatograms were recorded.

For thermal degradation, thin layer of AML (25 mg) and LSN (25 mg) were distributed over a glass plate to evaluate the effect of heat. The plate was kept in an oven at 105 ^◦C for 4 hours. After storage AML and LSN were transferred in a 50 mL volumetric flask and dilutions were made as per sample preparation to obtain the degraded AML and LSN solution (25 µg/mL). Aliquots (20 μL) of the stressed samples were injected into the HPLC system as described under chromatographic conditions, and the chromatograms were recorded.

For oxidative hydrolysis, Accurately weighed AML (25 mg) and LSN (25mg) were transferred to 100 mL volumetric flasks and add 20 ml of 3% H₂O₂for oxidattive hydrolysis, solution were kept for 1 Hour at room temperature and make up volume with methanol (250 µg/mL). From these, dilutions were made as per sample preparation to obtain the degraded AML solution (25 µg/mL). Aliquots (20 μL) of the stressed samples were injected into the HPLC system as described under chromatographic conditions, and the chromatograms were recorded.

For test Preparation, twenty tablets were crushed and weight equivalent to 25 mg of AML and LSN are taken in 100 ml volumetric flask. For each degradation test procedure was followed as per standard procedure. Aliquots (20 μL) of the stressed samples were injected into the HPLC system as described under chromatographic conditions, and the chromatograms were recorded.

RESULT AND DISCUSSION:

This method describes a reversed phase HPLC procedure employing a Shimadzu HPLC instrument (LC_2010 CHT) [software LC Solution, equipped with prominence diode array detector (SPD-M20A), Auto-sampler]. The chromatographic separation was performed using a Hypersil BDS C18 (150 mm ´4.6 mm, 5 µm particle size), mobile phase consisting Buffer(0.05 M Sodium dihydrogen Phosphate):Acetonitrile (50: 50, v/v) with apparent pH adjusted to 3.5 adjusted with Ortho-phosphoric acid, filtered through 0.45µm nylon filter and degassed in ultrasonic bath prior to use and UV detection at 258 nm using photo diode array detector.

Figure 1: Chromatogram of Standard Amlodipine Besylate and Lisinopril (acid stressed).

Figure 2: Chromatogram of Test Sample (acid stressed).

Figure 3: Chromatogram of Standard Amlodipine Besylate and Lisinopril (alkali stressed).

Figure 4: Chromatogram of Test Sample (alkali stressed).

Figure 5: Chromatogram of Standard Amlodipine Besylate and Lisinopril (oxidative stressed).

Figure 6: Chromatogram of Test Sample (oxidative stressed).

Figure 7: Chromatogram of Standard Amlodipine Besylate and Lisinopril (Thermal stressed).

Figure 8: Chromatogram of Test Sample (Thermal stressed).

METHOD VALIDATION:

The present method was validated for specificity, Accuracy and intermediate precision, LOD and LOQ. The nominal concentrations of standard and test solutions for AML and LSN were 25 µg/ml. The standard solutions for linearity were prepared five times and inter-run precision for slope of regressed line was found to be 0.99% R.S.D. for AML and LSN. The correlation coefficients were found to be more than 0.998 for AML and LSN respectively (Table 2). Accuracy and precision of the method was determined by performing the recovery experiment. This experiment was performed at three levels, in which sample stock solutions were spiked with standard drug solution containing 80%, 100% and 120% of labeled amount of both the drug in tablet. Three replicate samples of each concentration level were prepared and the %recovery at each level (n = 3), and mean %recovery (n=9) were determined (Table 3). The mean recovery in the range of 100.06-100.13 % for AML and 99.61-100.31 % for LSN. Based on the peak purity results, obtained from the analysis of forced degraded samples using the described method, it can be concluded that the method is specific for estimation of AML and LSN in presence of degradants (table-1). The method has linear response in stated range and is accurate and precise (table-4 and 5). The described method can be used as stability indicating method for assay of AML and LSN in pharmaceutical dosage form.

Table 2: Method validation data for AML and LSN

Regression Analysis	Statistics Value AML	LSN
Linearity range (µg/ml)	12.5-37.5	12.5-37.5
Regression Equation	y = 64.94x + 0.356	y = 91.47x - 12.10
Correlation Co-efficient (r²)	0.999	0.999
Slope	64.94	91.47
Intercept	0.356	12.10

Table 3: Results of accuracy for AML

	AML
%Spiked	80 %	100%	120%
Amount spiked	20	25	30
Total Conc.	45	50	55
Amount recovered	45.03	50.07	55.01
% Recovery	100.06	100.13	100.01
Mean ± S.D	100.07±0.06
% RSD	0.059

Table 4: Results of accuracy for LSN

	LSN
%Spiked	80 %	100%	120%
Amount spiked	20	25	30
Total Conc.	45	50	55
Amount recovered	44.82	50.16	55.03
% Recovery	99.61	100.31	100.05
Mean ± S.D	99.99±0.35
% RSD	0.35

Table 5: Results of LOD and LOQ

PARAMETER	AML(µg/ml)	LSN(µg/ml)
LOD(µg/ml)	0.324 µg/ml	0.160 µg/ml
LOQ(µg/ml)	0.984 µg/ml	0.484 µg/ml

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Received on 01.05.2013 Modified on 22.05.2013

Research J. Pharm. and Tech 6(7): July 2013; Page 784-789

Optimized Degradation condition	Matrix	%Degradation		Peak Purity
Optimized Degradation condition	Matrix	AML	LSN
2 ml of 0.1 N HCl for 30 minutes	API	13.4	14.5	999	998
2 ml of 0.1 N HCl for 30 minutes	Tablet	17.0	12.2	999	997
2 ml of 0.1 N NaOH for 30 minutes	API	11.2	14.9	999	998
2 ml of 0.1 N NaOH for 30 minutes	Tablet	13.3	16.9	999	999
3% H₂O₂Fresh for 1 hour	API	12.4	16.8	999	999
3% H₂O₂Fresh for 1 hour	Tablet	12.3	18.0	997	998
105 ^◦C for 4 hours	API	8.3	1.9	999	999
105 ^◦C for 4 hours	Tablet	6.7	0.3	998	999