1. INTRODUCTION:

One of the main challenges facing analytical chemists is the spectrophotometric determination of two or more compounds in the same sample without preliminary separation. The generation of absorbance ratio spectra has been the basis of several analytical procedures for the simultaneous spectrophotometric determination of compounds in binary and ternary mixtures. First, binary mixtures were resolved using the ratio-spectra derivative method [1] and [2]. Then, this method was modified and extended for the determination of ternary mixtures. Examples of these modified versions are the derivative ratio spectra zero-crossing method [3] and [4], the double divisor ratio spectra derivative method [5] and the successive derivative ratio spectra method [6].

Furthermore, the ratio spectra were exploited for the development of other mathematical methods for resolution of binary and ternary mixtures such as the mean centering of ratio spectra method [7] and the ratio subtraction method [8]. In most of these methods, at least two mathematical processes (e.g., division followed by derivative curve generation) are needed in order to get the measurable amplitude that is correlated to the concentration of only one compound without interference from the others in the mixture. Obviously, some methods need more sophisticated mathematical treatment to unambiguously determine the target compound in presence of interferences [8].

Candesartan cilexetil (CAND) is an angiotensin II receptor antagonist. It is used in the management of hypertension and may also be used in heart failure in patients with impaired left ventricular systolic function [9]. Hydrochlorothiazide (HCTZ) is a moderately potent diuretic. It exerts its effect by reducing the re-absorption of electrolytes from the renal tubules, thereby increasing the excretion of sodium and chloride ions, and consequently of water. HCTZ is used in the treatment of hypertension either alone or with other antihypertensive. It is also used to treat oedema associated with heart failure and with renal and hepatic disorders [9]. The simultaneous determination of CAND and HCTZ in their binary combination was addressed in several analytical reports. These reports proposed capillary electrophoresis [10], HPTLC [11], [12] and [13] and HPLC-UV detection methods [14] and [15]. An HPLC coupled with photodiode array detector method was developed for the simultaneous analysis of CAND and HCTZ in human plasma and dosage forms [16].On the other hand, spectrophotometric methods used for the assay of this antihypertensive mixture include difference spectrophotometry [13], derivative spectrophotometry [17] and [18], the ratio-spectra derivative method [17] and the Q-absorbance method [19]. In this study, a new classical least square method was described for the determination of binary mixtures of CAND and HCTZ without prior separation. The method eliminates the derivative step, and does not require searching for zero-crossing points. The developed method was validated and successfully applied for the assay of drug combinations in their commercial tablets.

2. EXPERIMENTAL:

2.1 Instrument

A Shimadzu (UV-1800) spectrophotometer (Japan) was used as instrument. The absorption spectra of the reference and test solution were recorded over the range of 200-400 nm possessing a fixed slit width, keeping the solution in 1-cm quartz cells The spectrophotometer is connected to a computer loaded with UV-probe 2.33 software and a Canon laser jet 3300 printer were used to record the absorption spectra. The regression and statistical analysis were achieved by using the Excel 2010. All weights were taken on Denver electronic balance (Germany).

2.2 Reagents and chemicals

All chemicals were of analytical reagent grade, 0.1M Hydrochloric acid ( Merck India, Mumbai) solution was prepared by using double distilled water (Mono quartz distillation unit, Borosil^®) and Millipore water(Synergy Pak^®- ICW-3000,Bellerica). Candesartan cilexetil (CAND) and Hydrochlorothiazide (HCTZ) were obtained as gift samples from Zim Laboratories Limited, Nagpur (India).

2.3 Commercial Tablet Formulation

One Commercial tablet formulation (Candesartan H^® produced by the Ranbaxy Laboratories Limited, India), consisting 16 mg of CAND and 12.5mg of HCTZ per tablet. The tablets were procured from the local chemist shop of Raipur, Chhattisgarh.

2.4 Preparation of stock solution and working solutions

Standard stock solutions of CAND and HCTZ were prepared separately dissolving 100 mg of each drug in 100 ml of 0.1M HCl. Working solutions were prepared by further dilutions of accurate volume of CAND and HCTZ stock solutions with 0.1M HCl. to reach the concentration range of 0.1mg/mL. Stock solutions and working solutions were stable for at least one week when stored refrigerated at 4⁰C.

2.5 Preparation of sample solutions.

Twenty tablets were accurately weighed and powdered in mortar. Average weight of each tablet was dissolved in 0.1M HCl in 100ml of volumetric flask with the aid of sonication (Ultra sonicator bath) for 15 min. The solution was filtered in to 100 ml volumetric flask by using Whatman No.42 filter paper. The residue was washed three times with 0.1M HCl. The solution was diluted further with 0.1M HCl. to obtain 16µg/mL of CAND and 12.5µg/mL of HCTZ.

3. PROCEDURES:

3.1 Spectrophotometric characteristics

Aliquot portion of working solution equivalent to 20µg/ml of CAND and HCTZ were transferred into two 10 ml volumetric flasks and the volume was made up with 0.1 M HCl. The absorbance spectra of solution were recorded between 200-340 nm and absorbance range from 0.00 to 1.00 at medium scanning speed (Fig. 1).

3.2 Linearity for the spectrophotometric methods

Aliquot portions of different concentration were accurately transferred to 10mL volumetric flasks from working solution of the 0.1mg/mL; the volume was completed with 0.1M HCl of CAND and HCTZ, respectively. The absorption spectra were recorded between 200-340nm and absorbance range from 0.00 to 1.00 at medium scanning speed. The absorbance’s measured for CAND and HCTZ at 251 and 272 nm, respectively. (Fig. 1)

3.2.1 Classical Least Square methods (CLS)

The zero-order absorption spectra for CAND and HCTZ and their binary mixture in 0.1M HCl were shown in Fig.1. The absorption data matrix and concentration matrix were obtained by measurement of absorbance between the ranges of 215-225 nm in the interval with ∆λ=1 nm at 11 wavelengths in their zero-order spectra. In the techniques, calibration or regression was obtained by using the absorbance data matrix and concentration data matrix for prediction of the unknown concentrations of CAND and HCTZ in their binary mixtures and pharmaceutical formulations.

3.3 Method Validation:[20]

Accuracy was determined by recovery study. The recovery experiment was carried out by spiking the already analyzed sample of the tablets with their different known concentration of standard CAND and HCTZ. Precision for assay were determined by repeatability, inter day, intraday precision for both drugs.

3.3.1. Recovery

To evaluate the accuracy, precision and reproducibility of the method, known amount of pure drug was added to the pre-analyzed sample of tablet powder and the mixture was analyzed for the drug content using the proposed method. The percentage recovery was found to be within range. The recovery experiments indicated the absence of interference from the commonly encountered pharmaceutical additives and excipients. Result of recovery study has been shown in Table 2 and 3.

4. RESULTS AND DISCUSSIONS:

4.1 Spectrophotometric Characteristics

Fig. 1. Shown the overlain zero order spectra of CAND (λmax=251 nm), HCTZ (λmax=272 nm) and their mixture against a blank, spectra were detected in the spectral region 200-340 nm. Since the spectra of two drugs overlap in the working wavelength range, it is not possible to determine CAND and HCTZ simultaneously in their mixture by conventional spectrophotometric methods. Here, the absorption spectra of CAND and HCTZ show a strong overlap which hinders the use of conventional UV Spectrophotometry for their simultaneous determination in a binary mixture (Fig. 1). To overcome the mutual interference of each compound in the determination of the other, a simple CLS method was applied. In these methods, a standard series of solutions of CAND and HCTZ in 0.1M HCl. were prepared. In contrast, the proposed methods can resolve bands overlapping, without physical separation.

4.2. Classical Least Square methods (CLS)

The zero-order absorption spectra for CAND and HCTZ and their binary mixture in 0.1M HCl were shown in Fig.1 as could be seen, a considerable degree of spectral overlapping occurs in the region from 210-230 nm for CAND and HCTZ. The absorption data matrix and concentration matrix were obtained by measurement of absorbance between the ranges of 215-225 nm in the interval with ∆λ=1 nm at 11 selected wavelengths in their zero-order spectra. . For this reason, the range of selected wavelength (Table 1) at the critical points, which correspond to the maximum, shoulder and minimum in the spectral range 215–225 nm were selected for the construction of the individual linear regressions for CAND and HCTZ in the binary mixture. As indicated in Table 1. The highest values for the regression coefficients (r) were obtained for all regression equations. The detection limit (LOD) (signal to noise ratio 3:1) and the quantization limit (LOQ) (signal to noise ratio 10: 1) were computed using the data obtained. The slope values obtained from the linear regression analysis for each drug in the binary mixture of HCTZ and CAND were used to create the sensitivity matrices (Table 1). Synthetic mixtures containing HCTZ and CAND were prepared with various random concentration ratios. Multivarate calibrations were obtained by measuring the zero-order absorbances at 11 points from 215 nm to 225 in the 200–340 nm wavelength range. Quantitative determinations of CAND and HCTZ in the mixtures were successfully carried out by these methods. Calculate the estimations of the standard variation of the chemo metric calibrations in the case of the mixtures investigated.

Table 1. - Data for calibration graph for binary mixtures of CAND and HCTZ by using CLS method

Parameters	Drug
Parameters	CAND	HCTZ
Wavelength (nm)	215-225	215-225
Concentration range (μg/mL)	2.5-50	1-30
Intercept^a	0.011	-0.005
Slope^b	0.020	0.034
S.D.	0.121	0.354
Correlation coefficient (r)	0.9998	0.9999
LOD^c(μg/mL)	0.55	0.32
LOQ^d(μg/mL)	1.66	0.97

^aStandard deviation of the intercept. ^bStandard deviation of the slope ^cLimit of detection. ^dLimit of quantification S.D.-standard deviation

Table 2- Results of the analysis of CAND and HCTZ in the laboratory prepared mixtures.

Sr. No.	CAND		HCTZ		CLS (Recovery (%))
Sr. No.	Added (µg/ml)	Found (µg/ml	Added (µg/ml)	Found (µg/ml)	CAND	HCTZ
1.	4	4.02	6	5.85	100.6	97.5
2.	6	5.79	4	4.02	99.6	100.5
3.	8	8.01	10	10.01	100.1	100.1
4.	10	9.95	8	8.02	99.5	100.2
5.	12	11.85	10	9.99	98.75	99.93
6.	10	10	12	11.98	100	99.8
7.	15	15	12	12.0	100	100
8.	12	11.97	15	15.03	99.75	100.2
9.	16	15.97	12.5	12.49	99.8	99.93
10.	15	14.96	10	9.97	99.75	99.7
11.	12.5	12.49	16	15.99	99.9	99.93
Mean					99.79	99.80
S.D.					0.45	0.79
R.S.D.					0.45	0.79

Table 3.

Determination of CAND and HCTZ in tablet using the proposed method

Parameter	Recovery (%) ±S.D.
Parameter	CAND	HCTZ
Tablet	99.6±0.1	99.83±0.057
RSD(%)	0.1004	0.0578

S.D.: standard deviation, RSD: relative standard deviation

Table 4. Result of application of the standard addition techniques to the simultaneous determination of CAND and HCTZ in tablet by proposed methods

Claimed amount taken (µg/ ml)		Standard added (µg/ ml)		Recovery of added standard (%)a ± S. D.
CAND	HCTZ	CAND	HCTZ	CAND	HCTZ
16	12.5	8	6.126	100.1±0.654	100.03±0.253
16	12.5	16	12.5	99.98±0.126	99.89±0.981
16	12.5	24	18.75	99.9±0.234	101.01±0.251
Mean				99.99	100.34
Mean standard deviation				0.338	0.495

Table 5.- Analysis of variance (ANOVA) result of precision for the simultaneous determination of CAND and HCTZ of developed method

Parameter	CLS
Parameter	CAND	HCTZ
Between day variance	5.68	6.33
Within day variance	3.55	4.57
F ratio	1.60	1.38
Mean value	3.99	2.05
Between day RSD%	1.62	2.12
Within day RSD%	1.21	1.57

Between-day and within-day degrees of freedom 2 and 27, respectively

The critical F-ratio value for 2 and 27 degrees of freedom at 95% confidence level is 4.21

Table 2 and Table 3 summarizes the results obtained for the suggested binary mixtures and tablet formulation. Recoveries were within 99.7%, with standard deviations ranging from 0.5–0.8% of CAND and HCTZ, respectively. The results for each drug were obtained with as average of six replicates and repeated three times.

4.3 Accuracy

Accuracy of the method was confirmed by recovery study from marketed formulation at three level of standard addition from 80 % to 120 % of label claim. The results are shown in Table 4. Recovery greater than 98 % with low SD (standard deviation) justifies the accuracy of the method

4.4 Precision

Precision was estimated by the determination of the repeatability of the method. Binary mixtures containing three different concentrations were prepared with 0.1M HCL. The experiment was repeated three times in a day (intra-day precision) and repeated on three different days (inter-day precision. The precision was determined by means of a one-way ANOVA including 10 replicates carried out on three successive days using two chemometric methods for synthetic mixtures. Snedecor F values below the tabulated levels were obtained in all cases (F = 4.15, n₁ = 2, n₂ = 27, Table 5 ) so there were no significant differences between the result obtained in the determination of each drug in the presence of the other on different days. ). The average % RSD (relative standard deviation) values of the results were calculated which were found to be less than 2%, which confirms that the method is precise.

Regarding these results and the recovery studies done for the pure compounds added to the commercial tablets which indicate the absence of the interferences due to the presence of the excipients and additives, we can conclude that the proposed methods are sufficiently accurate and precise in order to be applied to the pharmaceutical dosage forms.

5. CONCLUSION:

The contents of several laboratory prepared mixtures and commercial tablets were simultaneously determined using UV-spectrophotometric measurements together with CLS method. The good recoveries obtained in all cases as well as the reliable agreement with the reported procedures proved that, the proposed procedures could be applied efficiently for determination of studied drugs simultaneously in their binary mixtures as well as in the commercial dosage forms with satisfactory precision. Hence, the proposed procedures are rapid and sufficiently precise and suitable for quality control laboratories, where the economy and time are important factors.

6. ACKNOWLEDGEMENT:

The authors thank to Zim Laboratories Limited, Nagpur (India), for providing the gift samples of drugs for conducting the study. The authors also thank the Director, University Institute of Pharmacy, Pt. Ravishankar Shukla university, Raipur for providing the necessary facilities.

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Received on 28.05.2015 Accepted on 22.06.2015

Asian J. Pharm. Res. 5(2): April-June 2015; Page 90-95

DOI: 10.5958/2231-5691.2015.00013.1