1. INTRODUCTION:

Telmisartan (TELM) is an angiotensin II receptor (type AT1) antagonist used in the management of hypertension. TELM prevents the constriction (narrowing) of blood vessels (veins and arteries). It is a non-peptide molecule and chemically described as 4'-[(1,7'-Dimethyl-2'-propyl- 1H,3'H -2,5'-bibenzimidazol-3'-yl) methyl]-2-biphenyl carboxylic acid [1]. Telmisartan is not official in IP, BP, and USP. Telmisartan was approved by the FDA in November 2000 [2].

Amlodipine besylate is chemically known as 3-ethyl-5-methyl-2-(2-aminoethoxymethyl)-4-(2-chlorophenyl)-1,4-dihydro-6- methylpyridine-3,5-dicarboxylate benzene sulphonate. It is a dihydropyridine calcium channel blocker used in the treatment of hypertension and angina pectoris [3]. AMLO is official in the British Pharmacopoeia (BP) which describes HPLC for its assay in the bulk powder [4]. Hydrochlorothiazide (HCTZ) is chemically known as 6-chloro-3,4-dihydro-2H-1,2,4-benzothiadiazine-7-sulphonamide- 1,1-dioxide.It is a diuretic of the class of benzothiadiazines widely used in antihypertensive pharmaceutical formulations, alone or in combination with other drugs, which inhibits NaCl transport in distal convoluted tubule and decreases blood pressure [5] HCTZ is official in I.P., B.P and U.S.P. monograph. [6-8]

Literature review reveals that some analytical methods have been reported in combination with other drugs and in single dosage forms for Telmisartan hydrochloride , amlodipine besilate and hydrochlorothiazide such as spectrophotometric methods [9-11], HPTLC method [12] chromatographic methods [13-23], stability indicating analytical methods [24,25] and methods for determination of AML and TEL in human plasma [26-28]. These methods employed intensive instrumentation (e.g. HPLC) or suffered from low robustness such as spectrophotometric because calibration procedures depend on measuring absorbances at one wavelength (univariate calibration method). So any shift in wavelength scale will lead to false results. Inclusion of many spectral wavelengths instead of using a single wavelength greatly improves the precision and predictive ability of the multivariate calibration methods. The scientific novelty of the present work is that the methods used are simple, rapid, selective, less expensive and less time consuming compared with other published HPLC [18] methods. Furthermore, these methods have high precision and accuracy as compared with the reported spectrophotometric methods because calibration procedures depend on whole spectra. So, the aim of this work was to develop simple, sensitive and validated chemometric assisted spectrophotometric methods for the simultaneous determination of TELM, AMLO and HCTZ in tablet dosage form laboratory prepared mixtures and in pharmaceutical formulation.

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). Telmisartan hydrochloride (TELM), Amlodipine besylate (AMLO) and Hydrochlorothiazide (HCTZ) were obtained as gift samples from Zim Laboratories Limited, Nagpur (India).

2.3 Commercial Tablet Formulation

One Commercial tablet formulation (TELMA-AM H^® produced by the Glenmark Laboratories Limited, India), consisting 40 mg of TELM, 5 mg of AMLO 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 TELM, AMLO 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 TELM, AMLO and HCTZ stock solutions with 0.1M HCl. to reach the concentration range of 0.05mg/mL. Stock solutions and working solutions were stable for at least one weeks 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 40µg/mL of TELM, 5µg/mL of AMLO and 12.5µg/mL of HCTZ.

3. PROCEDURES:

3.1 Spectrophotometric characteristics

Aliquot portion of working solution equivalent to 5µg/ml of TELM, AMLO 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 220-320 nm and absorbance range from 0.00 to 1.00 at medium scanning speed (Fig. 2).

3.2 Linearity for the spectrophotometric methods

Aliquot portions of different concentration were accurately transferred to 10mL volumetric flasks from working solution of the 0.05mg/mL; the volume was completed with 0.1M HCl of TELM, AMLO and HCTZ, respectively. The absorption spectra were recorded between 220-320nm and absorbance range from 0.00 to 1.00 at medium scanning speed. The absorbance’s measured for TELM, AMLO and HCTZ at 226.8, 260 and 271.4 nm, respectively. (Fig. 2)

3.3 Classical Least Square methods (CLS)

The zero-order absorption spectra for TELM and HCTZ and their binary mixture in 0.1M HCl were shown in Fig.2. The absorption data matrix and concentration matrix were obtained by measurement of absorbance between the ranges of 210-230 nm in the interval with ∆λ=2 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 TELM, AMLO and HCTZ in their ternary mixtures and pharmaceutical formulations. Here 15 samples were used to build the multivariate calibration models (training set). The concentrations chosen for each compound in 15 samples were based on the calibration range of each of the two drugs, the ratio of TELM: AMLO: HCTZ in the TELMA^® AM H tablets (8:1:2.5 respectively). (Table 1) The absorption spectra of the 15 samples were scanned from 200 - 400 nm against 0.1MHCl. as a blank (Fig. 1) and transferred to Microsoft Excel for subsequent calculations. The noisy region from 210-230 nm and the zero absorbance of TELM and HCTZ after 340 nm accounted for the rejection of these parts from the spectra.

4. RESULTS AND DISCUSSION:

TELMA^®AM H tablets are combined dosage form containing the angiotensin II receptor blocker TELM, the calcium channel blocker AMLO and the diuretic HCTZ. It has been used in the treatment of hypertension. The ratio of TELM: AMLO: HCTZ in TELMA^®AM H tablets are 8:1:2.5 respectively. This study was designed to develop simple, robust and accurate Classical least square method for the simultaneous determination of TELM, AML and HCTZ in TELMA^®AM H tablets. Because of the practical simplicity, and wide availability of spectrophotometry in quality control laboratories, it was attempted in this study. Multivariate calibration method (CLS) is very useful in spectral analysis because the simultaneous inclusion of many spectral wavelengths instead of using a single wavelength greatly improves the precision and predictive ability of this methods.

4.1 CLS model

The zero-order absorption spectra for TELM, AMLO and HCTZ and their ternary 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 TELM, AMLO and HCTZ. The absorption data matrix and concentration matrix were obtained by measurement of absorbance between the ranges of 210-230 nm in the interval with ∆λ=2 nm at 11 selected wavelengths in their zero-order spectra. In the techniques, calibration or regression equation was obtained by using the absorbance data matrix and concentration data matrix for prediction of the unknown concentrations of TELM, AMLO and HCTZ in their ternary mixtures and pharmaceutical formulations.

After optimization of parameters and calibration step, CLS method was applied successfully for analysis of TELM, AMLO and HCTZ in laboratory prepared mixtures. The recoveries [29] mean recoveries, standard deviation values are summarized in Tables 1.The proposed method was then applied for the simultaneous determination of the three analytes in TELMA^®AM H tablets (Table 2). This fact was further assessed by the statistical comparison of t and F values of the proposed models and the reference HPLC method [18] (Table 3) showing that there is a no significant difference between CLS and the reference one regarding both accuracy and precision.

Fig. 1. Zero order overlain Absorption spectra for TELM, AMLO and HCTZ against 0.1M HCl. as a blank (10 μg /mL each).

Table 1. Recovery results of laboratory prepared mixture for TELM, AMLO and HCTZ by CLS methods

Mixture No.	Added (µg			Found(µg			Recovery (%)
Mixture No.	TELM	AMLO	HCTZ	TELM	AMLO	HCTZ	TELM	AMLO	HCTZ
1	40	5	12.5	39.96	4.95	12.5	99.9	98.9	99.97
2	40	3	10	40	2.94	9.99	100	98.07	99.89
3	40	4	11.25	39.99	3.98	11.23	99.98	99.47	99.79
4	40	6	13.75	39.91	5.99	13.76	99.78	99.99	100.1
5	40	7	10	39.90	6.99	9.98	99.76	99.87	99.87
6	25	3	15	24.67	2.97	14.89	98.67	99.25	99.25
7	15	4	12.5	14.98	4.01	12.53	99.89	100.2	100.2
8	17.5	3	11.25	17.59	2.99	11.25	100.5	99.98	99.98
9	17.5	4	13.75	17.32	3.98	13.67	98.98	99.40	99.4
10	15	7	11.25	14.99	7.01	11.35	99.99	100.1	100.9
11	17.5	6	15	17.5	5.99	14.98	100	99.89	99.89
12	15	7	11.25	14.96	7.13	11.34	99.76	101.9	100.8
13	17.5	5	10	17.38	5.01	10.01	99.34	100.01	100.01
14	17.5	6	15	17.49	6	15.02	99.98	100.0	99.98
15	15	2	12.5	14.99	2.01	12.49	99.99	100.1	100.1
MEAN (%)							99.76	99.80	100.01
S.D.							0.435	0.787	0.409
RSD							0.436	.0788	0.409

Table 2. Determination of TELM, AMLO and HCTZ in marketed formulation by CLS methods

Drug in (µg/mL)			Found(µg/mL)			CLS (% Recovery)
TELM	AMLO	HCTZ	TELM	AMLO	HCTZ	TELM	AMLO	HCTZ
40	5	12.5	39.99	4.94	12.5	99.98	98.9	99.97
40	5	12.5	39.91	4.99	12.53	99.25	99.89	99.8
40	5	12.5	39.90	5.01	12.51	99.89	100.01	98.9
Mean (%)						99.70	99.6	99.55
SD						0.324	0.497	0.469
RSD						0.325	0.499	0.471

Table 3.Statistical comparisons of the results obtained by CLS and reference HPLC method for the analysis of the tablet formulation

Parameter	CLS method			Reported method^*
% Recovery	TELM	AMLO	HCTZ	TELM	AMLO	HCTZ
	99.98	98.9	99.97	99.38	100.48	99.33
	99.25	99.89	99.8	98.68	98.91	98.38
	99.89	100.01	98.9	99.50	99.95	99.28
Mean	99.70	99.6	99.55	99.18	99.78	98.99
SD	0.324	0.497	0.469	0.44	0.79	0.53
No. of sample	3	3	3	3	3	3
t- value	1.61	1.13	0.57	-	-	-
f-ratio	2.28	0.09	0.47	-	-	-

For the degrees of freedom (1,4) the critical value of t and f are 6.31 and 7.71 , respectively at (p=0.05 level)

* reported method is HPLC published in literature [18]