INTRODUCTION:

Ramipril (RMPL), 2-[N-[(S)-1-(ethoxycarbonyl)-3-phenylpropyl)]-L-alanyl]-(1S, 3S, 5S)-2-azabicyclo [3-3-0] octane carboxylic acid, is an angiotensin-converting enzyme (ACE) inhibitor. Hydrochlorothiazide (HCTZ), 6-Chloro-3,4-dihydro-2H-1,2,4-benzothiadiazine-7-sulfonamide 1,1-dioxide is a thiazide diuretic. It increases sodium and chloride excretion in distal convoluted tubule. RMPL is frequently co-formulated with HCTZ in medicinally recommended ratios of 2.5:12.5 and 5:12.5. Literature survey reveals many spectroscopic^1-6 methods for the quantitative determination of RMPL and HCTZ alone or in combination with other anti hypertensive drugs. Few chromatographic methods^7-10for determination of RMPL and HCTZ alone or in combination with other drugs have also been reported. Only one chromatographic method¹¹ for the determination of RMPL and HCTZ has been reported. However, there is no spectroscopic available till date for the same. The aim of this work is to develop a simple, rapid, sensitive and reliable spectrophotometric method for the quality control of RMPL and HCTZ in combined dosage forms.

MATERIALS AND METHODS:

Spectrophotometric analysis was carried out on a Shimadzu 1601 double beam spectrophotometer with a fixed slit width (2nm). The system software of the instrument was used for obtaining the ratio spectra and tracing the 1st derivative of the ratio spectra. Standard RMPL and HCTZ was a gift sample from Torrent Research Centre, Ahmedabad, and M/s. Sun Pharma Advanced Research Center, Vadodara, India respectively. The drugs were used as standard without further purification. All the chemicals used in the experiments were of analytical grade. Silicagel-G (E. MERCK KGaA) and commercial pharmaceutical preparations (Hopace-H^® cardicare and Hopecard-H^® Aristo) were procured from commercial source.

Preparation of standard stock solutions of RMPL and HCTZ:

HCTZ and RMPL, 50 mg each, were accurately weighed and dissolved separately in 50 ml of methanol. Five ml of the above solution was diluted to 50 ml with 0.1 N HCl in double distilled water to produce 100 µg ml^-1 each of HCTZ and RMPL in methanolic HCl.

Preparation of standard solutions of RMPL and HCTZ for divisor spectra:

Suitable aliquots of the standard stock solution were diluted with methanolic HCl to produce standard solutions of 7 µg ml^-1 of HCTZ and 90 µg ml^-1 of RMPL for obtaining divisor spectra.

Preparation of binary mixtures of RMPL and HCTZ:

Different binary mixture solutions containing fixed amount of RMPL and varying amount of HCTZ (RMPL:HCTZ = 3:1, 3:2, 3:4, 3:8, 3:10, 3:12, 3:14 µg ml^-1) and fixed amount of HCTZ and varying amount of RMPL (RMPL:HCTZ = 1:10, 2:10, 4:10, 6:10, 8:10, 10:10 µg ml^-1) for preparation of calibration curve, were prepared by diluting different aliquots of the stock solutions with methanolic HCl.

Figure 1 (A): Ratiospectra obtained when different mixture spectra containing 1, 2, 4, 8 and 10 µg ml^-1 were divided by a standard spectra of 7 µg ml^-1 of HCTZ.

Figure 1 (B): 1^st derivative of the ratio spectra obtained when mixture spectra different mixture spectra containing 1, 2, 4, 8 and 10 µg ml^-1 were divided by a standard spectra of 7 µg ml^-1 of HCTZ.

Preparation of calibration curve:

The absorption spectra of 7 µg ml^-1 of HCTZ and 90 µg ml^-1were recorded in the range of 200 nm to 250 nm and stored in the memory of the instrument as divisor spectra. The absorption spectra of the binary mixture solutions of RMPL and HCTZ were recorded in the range of 200 nm to 250 nm and were stored in the memory of the instrument. The stored spectra of the binary mixtures were divided by a previously stored divisor spectrum of 7 µg ml^-1 of HCTZ to get the ratio spectra. The first derivative of the ratio spectra were traced with Δ l = 4 intervals and the amplitude at 210.6 nm was plotted against the respective concentrations of RMPL (Figure 1A and 1B). The method shows good linearity in the range of 1 to 10 µg ml^-1 for RMPL. Similarly, the absorption spectra of binary mixtures of RMPL and HCTZ were divided by a previously stored standard divisor spectra of 90 µg ml^-1of RMPL and the first derivative of the ratio spectra (Figure 2 (A) and 2 (B)) were traced with ∆l = 4 interval. The difference in amplitudes at 224.6 and 231 nm (as it showed linearity), were then plotted against the respective concentrations of HCTZ. The method shows good linearity in the range of 1 to 14 µg ml^-1for HCTZ.

Figure 2 (A): Ratiospectra obtained when different mixture spectra containing 1, 4, 6, 8 and 14 µg ml^-1 were divided by a standard spectra of 90 µg ml^-1 of RMPL.

Figure 2 (B): 1^st derivative of the ratio spectra obtained when different mixture spectra containing 1, 4, 6, 8 and 12 µg ml^-1 were divided by a standard spectra of 90 µg ml^-1 of RMPL.

Table 1 Optimized method parameters

Method parameters	Optimized Values
Scanning range	200nm to 250nm
Slit width	2nm
Scan speed	fast
∆λ for tracing 10 derivative spectra	4nm
Divisor Spectra for determination of RMPL	7 µg ml^-1 of HCTZ
Divisor Spectra for determination of HCTZ	90 µg ml^-1 of RMPL
Analytical wavelength for determination of RMPL	209 nm
Analytical wavelength for determination of HCTZ	224.7 nm and 231 nm

Table 2 Analysis of pharmaceutical formulations

Sr. No.		Label claim			Amount recovered RMPL (mg)		% Recovery RMPL	Amount recovered HCTZ (mg)		% Recovery HCTZ
		RMPL (mg)		HCTZ (mg)
Analysis of tablet ( Cardace-H^®)
1	2.5			12.5		2.392	103.93	12.35	98.78
2	2.5			12.5		2.485	105.79	12.37	98.94
3	2.5			12.5		2.672	102.06	12.43	99.40
4	2.5			12.5		2.485	100.19	12.44	99.55
Average ± SD							99.57 ± 3.276		99.17 ± 0.366
Analysis of capsule preparation (Hopecard-H^®)
1		5	12.5			5.196	103.93	12.55	100.40
2		5	12.5			5.299	105.79	12.48	99.86
3		5	12.5			5.103	102.06	12.50	100.02
4		5	12.5			5.009	100.19	12.43	99.40
Average ± SD							102.99 ± 2.413		99.92 ± 0.414

Analysis of Tablet:

A total of 20 tablets were accurately weighed and powdered in a mortar. An amount equivalent to one tablet (Containing 2.5 mg of RMPL and 12.5 mg of HCTZ) was taken and dissolved in 10 ml of methanol by magnetically stirring it for five minutes. About 10 ml of 0.1 N HCl in double distilled water was added and stirred for further 5 minutes. The mixture was transferred to two centrifuge tubes and centrifuged at 1000 rpm for 5 minutes. The supernatant was transferred to a 100 ml volumetric flask through a Whatman No 40 filter paper. The residue was washed thrice with 0.1 N HCl in double distilled water and the combined filtrate was made up to the mark with double distilled water. The sample solution thus prepared was diluted with distilled water to get the solution containing about 2.5 µg ml^-1of RMPL and 12.5 µg ml^-1of HCTZ. The above solution was analyzed for the content of RMPL and HCTZ using the method in preparation of calibration curve.

Analysis of Capsule:

A total of 20 Capsule was uncapped and the content of the capsule was accurately weighed. An amount equivalent to one capsule (Containing 5 mg of RMPL and 12.5 mg of HCTZ) was taken and the method for analysis of tablet was followed.

The results of tablet and capsule analysis are shown in Table-2.

RESULTS AND DISCUSSION:

Ratio spectra derivative spectrophotometry and simultaneous equation methods for simultaneous determination of RMPL and HCTZ from their binary mixture were successfully developed.

Optimization of method parameters:

The method parameters like divisor spectra and wave length interval (∆λ) of tracing the 1^st derivative of ratio spectra were optimized for the reliable determination of subject components (Table-1). Some divisor concentrations were tested for selecting the standard solution as divisor at an appropriate concentration, and it was observed that the standard solution of 7 µg ml^-1 of HCTZ and 90 µg ml^-1of RMPL were suitable for determination of RMPL and HCTZ respectively in their binary mixture by ratio spectra derivative spectrophotometric method. The influence of ∆λ in smoothing function for the first derivative spectra of ratio spectra were tested and found very appropriate to use ∆λ = 4 for tracing the first derivatives of the ratio spectra as linearity and sensitivity is concerned.

Method validation:

1. Linearity and range:

The ratio spectra derivative spectrophotometry method showed good linearity for RMPL in the range of 2 to 10 µg ml^-1of RMPL with co-relation co-efficient, intercept and slope 0.9998, 0.0011 and 0.0115 respectively and for HCTZ the linearity range was found to be 1 to 14 µg ml^-1with correlation coefficient, intercept and slope 0.9997, 0.0044 and 0.0517 respectively.

2. Precision:

Inter day and intraday precision for ratio spectra derivative spectroscopy was measured in terms of % RSD. The experiment (preparation of calibration curve) was repeated five times in a day for intra-day and on five different days for inter-day precision. The method was found precise on intraday and inter day basis as the average %RSD value for the determination of RMPL was found to be 2.050 % and 3.650 % and that of HCTZ was 2.380 % and 2.153 % respectively.

3. Accuracy:

Accuracy of the method was determined by performing recovery study from previously analyzed synthetic mixture by standard addition method at three levels. Recovery studies were performed for RMPL and HCTZ by % standard addition method. The method showed % recovery ± SD in the range of 99.688 ± 0.50 % to 102.803 ± 0.90 % for RMPL and between 98.46 ± 1.01 % to 102.88 ± 1.02% for HCTZ. The results indicate that the developed ratio spectra derivative spectrophotometric method is accurate enough for routine use.

4. Limit of detection (LOD) and Limit of quantification (LOQ):

Calibration curve was repeated for 5 times and the standard deviation (SD) of the intercepts was calculated. The values of LOD and LOQ were found to be 0.463 μg ml^-1 and 0.117 μg ml^-1for RMPL and 1.542 and 0.392 for HCTZ.

5 Applicability of the method:

Applicability of the proposed method was tested by analyzing the commercially available tablet formulations (Hopecard-H^®) and capsule formulation (Hopecard-H^®). The results shown in Table-2 indicates that the average recovery, between 96 to 106 %, was found to be satisfactory.

CONCLUSION:

The proposed ratiospectra derivative spectrophotometic method for determination of RMPL and HCTZ obeys Beer’s law in the range of 2 to 10 µg / ml of RMPL and 1 to 14 µg / ml of HCTZ with correlation-coefficient greater than 0.999. Recovery more than 98 % with low SD suggests the accuracy of the method. Low values of % RSD for intraday, inter day and robustness suggests the method is precise enough for routine analysis of both the drugs.

REFERENCE:

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Received on 12.05.2010 Modified on 20.05.2010

Asian J. Research Chem. 3(4): Oct. - Dec. 2010; Page 888-891

R. Sahu1, Patel V. B.2* and Bapna M.2

INTRODUCTION:

R. Sahu¹, Patel V. B.²*and Bapna M.²