Different ratio spectra methods for simultaneous determination of Olmesartan Medoxomil, Hydrochlorothiazide and Amlodipine Besylate

Hamed M. EL-Fatatry¹, Mokhtar M. Mabrouk¹, Sherin F. Hammad¹, Salwa H. Elsheikh¹

1. Introduction

Olmesartan Medoxomil [OLM] [Figure 1 a] is chemically known as N-[p-[o-1H- Tetrazol-5-ylphenyl] benzyl]-N-valeryl-L- valine [1]. It is a potent and selective angiotensin AT1 receptor blocker [2], It has been approved for the treatment of © hypertension in the United States, Japan and European countries. The drug contains a Medoxomil ester moiety which is cleaved rapidly by an endogenous esterase to release the active Olmesartan [3].

Hydrochlorothiazide [HCTZ] [Figure 1 b] is chemically known as 6-chloro- 3, 4- dihydro-2H-1, 2, 4-benzothiadiazine-7- sulphonamide-1, 1-dioxide [1]. It is a diuretic of the class of benzothiadiazines widely used in antihypertensive pharmaceutical formulations, alone or in combination with other drugs, which inhibits Na Cl transport.

Amlodipine Besylate [AMLB] [Figure 1 c] is chemically known as 2-[[2- aminoethoxy] methyl]-4-[2- chlorophenyl]-1, 4-dihydro-6-methyl-3, 5- pyridine carboxylic acid 3-ethyl 5-methyl ester] [1].

[a] Structure of Olmesartan Medoxomil [OLM]

[b] Structure of Hydrochlorothiazide [HCTZ]

It is a dihydropyridine calcium channel blocker used in the treatment of hypertension and angina pectoris [4].

Recently, OLM has been marketed in combination with AMLB and HCTZ in ® tablet dosage form [TRIBENZOR tablets]. The triple combination of OLM, AMLB and HCTZ is intended for oral administration for the treatment of hypertension.

AMLB is official in British Pharmacopoeia [6], HCTZ is official in British Pharmacopoeia [6] and United States Pharmacopoeia [7].

Olmesartan, Amlodipine and Hydrochlorothiazide combination is not official in any pharmacopoeia so no official method is available for estimation of these drugs in Literature survey reveals that few methods are available for the simultaneous analysis of OLM, AMLB and HCTZ combination. There is one reported stability indicating UPLC method for the determination of OLM, HCTZ, and AMLB in pharmaceutical dosage form[8], HPLC [9- 11] and spectrophotometry [12-16].

Therefore, the aim of this work was directed to the development of simple, sensitive, selective and validated spectrophotometric methods for the simultaneous determination of OLM, 167 AMLB and HCTZ in their combined dosage form.

Theoretical back ground

The main problem of spectrophotometric multi-component analysis is the difficulty of simultaneous determination of two or more active compounds in the same mixtures without preliminary separation. Several spectrophotometric determination methods have been used for resolving mixtures of compounds with overlapping spectra, such as classical derivative spectrophotometry [17–21], Vierordt’s method[22]and its modified version [23], orthogonal function method [24], Fourier functions method [25-26] dual wavelength spectrophotometry [27-28], pH-induced differential spectrophotometry [29], and least square method [30], multi-component analysis program [31-32], principle component regression [PCR] [33], multi-wavelength linear regression analysis [MLRA] [34] and H- point standard addition method [HPSAM] [35-36].

However, sometimes the treatment of the normal absorbance data cannot cope with the level of interference especially when the spectra are strongly overlapped, thus special data treatment is required. Recently, the treatment of absorbance ratio spectra has been the basis of some analytical procedures so as to generate signals for the mixture depending only on a single analyte.

Examples of these methods are the ratioderivative spectrum method, derivative ratio spectra-zero-crossing method [DRSZ] [37-40], Salinas et al. proposed the spectrophotometric method termed ratioderivative spectrophotometry, for the simultaneous determination of two compounds in binary mixtures, Berzas Nevado et al. [41] developed a method for the analysis of ternary mixtures by derivative ratio spectra-zero-crossing technique. In this method, the simultaneous determination of three compounds in ternary mixtures is realized by measuring of the amplitude at the zerocrossing points in the derivative spectrum of the ratio spectra. These two methods were further studied and applied for simultaneous determination of binary or ternary mixtures [37-42].

Double divisor ratio spectra derivative method [DDRD] [39-43], Recently, Dinc et al. [44-45] has proposed a new simultaneous determination of ternary mixtures. This method is called “the double divisor ratio spectra derivative method”. This method is based on the use of the coincident spectra of the derivative of the ratio spectra obtained by using a “double divisor” [sum of two spectra] and measuring at either the maximum or minimum wavelengths. Successive derivative ratio spectra [46] and a method based on the mean centering of ratio spectra [47].

Fourier function-ratio spectrum method [48-49]. Unfortunately, the advantages of derivative spectra are at least partially offset by degradation in signal-to-noise ratio that accompanies obtaining derivatives. Recently, Wahbi et al. introduced the Fourier function-ratio spectrum method for determination of binary mixtures [50-51].

2. Materials and Methods

Apparatus

Spectrophotometric measurements were recorded using a Jasco [UV-530] –UV is a double beam spectrophotometer with two matched 1 cm Quartz cells and connected to personal computer loaded with [Jasco]- [spectra manager] software.

Pure drugs

168 Olmesartan Medoxomil [99.9%], Hydrochlorothiazide [99.9%] %], and Amlodipine Besylate [99.9%] Nutra Specialties private Limited; all were gifted from Chemipharm –Egypt.

Reagents &Materials

Acetonitrile [analytical grade], Starch, Microcrystalline cellulose, Magnesium stearate, and Silicon dioxide all were purchased from [Sigma Aldrich].

Stock Standard Solutions

Stock standard solution was prepared by dissolving OLM in acetonitrile to obtain solution having a concentration of 1.00 mg.

-1 mL . One mL of this solution was transferred into 10 mL volumetric flasks, completing to the volume with acetonitrile to obtain stock standard solution of OLM -1 having a concentration of 100.0 μg.mL . The solution was stable for one week when refrigerated at 4°C.

In the same manner prepare each HCTZ, and AMLB stock standard solution with a -1 concentration of 100.0 μg.mL .

Working Standard Solutions

Working OLM standard solutions were prepared by accurately transferring different volumes of stock standard OLM solution into 10ml volumetric flask, completing to volume with ACN to obtain solutions in the concentration range of [5- -1 50 μgmL ] In the same manner prepare HCTZ, and AMLB solutions.

Laboratory prepared mixture

Synthetic mixture simulating to tablet was prepared and an aliquot equivalent to one tablet of [40mg of OLM, 13.9mg of AMLB [equivalent to 10 mg of AML base], and 25 mg of HCTZ] with excipients [140 mg of Starch, 156.322 mg of Microcrystalline cellulose, 1.6 mg Magnesium stearate, and 3.19 mg of Silicon dioxide] were dissolved in 100 ml ACN with the aid of sonication and filtered.

5ml of the filtrate was transferred into 50- ml volumetric flask and diluted with ACN then the final solution of the laboratory prepared mixture was assayed using the proposed method.

In the same manner we prepare another solution representing dosage form with different ratio of [OLM 20mg -HCTZ 12.5mg - AMLB 6.95mg] with excipients [70 mg of Starch, 78.161 mg of microcrystalline cellulose, 0.8 mg Magnesium stearate and 1.595 mg of Silicon dioxide].

Procedures

Direct spectrophotometric method for determination of AMLB

AMLB can be determined at 356.0 nm directly from zero order spectra where OLM and HCTZ show no absorbance. A linear calibration curve relating the absorbance at 356.0 nm to the corresponding concentration of AMLB was constructed and regression equation was computed [Figure

Derivative ratio spectrum zero-crossing method [DRSZ]

The recorded absorption spectra of OLM, AMLB and their ternary mixture with HCTZ were divided by a standard spectrum of HCTZ [10μg/ml] and the first derivative of the ratio spectra was calculated with Δλ= 3 169 nm in the range 200–400 nm. In the ternary mixture, OLM and AMLB can be determined by measuring the first derivative signals at 259.0 and 358.0 nm, respectively, similarly, the stored spectra of HCTZ and AMLB and their ternary mixture were divided by a standard spectrum of OLM [10 μg/ml] and the first derivative of the result was plotted with ∆λ= 3 nm from 200 -400 nm. The concentration of HCTZ can be determined at 279.0 nm.

Double divisor ratio spectra derivative method [DDRD]

The absorption spectra of the solutions prepared at different concentrations of OLM and of the ternary mixture were recorded and divided by the sum of the absorption spectra of solutions of HCTZ and AMLB [10μg/ml each in ACN] as “double divisor” and the ratio spectra [I] were obtained. First derivatives of the ratio spectra were plotted with ∆λ= 3 nm. The amount of OLM was determined by measuring the amplitude at 266.0 nm corresponding to a minimum in the first derivative of the ratio spectra in the spectral region selected [200–400 nm].

The absorption spectra of the solutions prepared at different concentrations of HCTZ and of the ternary mixture were recorded and divided by the sum of the absorption spectra of solutions of OLM and HCTZ [10μg/ml each in ACN] as “double divisor” to get the ratio spectra [II] and their first derivatives were plotted with ∆λ= 3 nm from the ratio spectra. The content of HCTZ was determined from the first derivative of the ratio spectra between 200 and 400 nm and measuring the signals at 317.0 nm, corresponding to a minimum.

In the same way, the absorption spectra of the solutions prepared at different concentrations of AMLB and of the ternary mixture were stored and divided by the spectrum of the standard mixture solution of OLM and HCTZ [10 μg/ml each in ACN] as “double divisor” to give the ratio spectra [III] then their first derivatives were traced with ∆λ= 3 nm from the ratio spectra.

The concentration of AMLB was determined by measuring the first derivative of the ratio spectra between 200 and 400 nm and measuring the signals at 363.0 nm, corresponding to a minimum.

Successive derivative ratio spectra method

The absorption spectra of different concentrations of OLM were recorded in the range of 200–400 nm and divided by the spectrum of the standard solution of 10 μg _1 mL of HCTZ and the ratio spectra were obtained. First derivative of the ratio spectra was obtained with ∆λ=3 nm . These vectors [first derivative of the ratio spectra] were then divided by [d/dλ][AMLB/HCTZ] corresponding to the derivative of the ratio _1 of the spectra of 10 μg mL of each and then the second ratio spectra were obtained. The first derivative of these ratio spectra obtained from which OLM was determined by measuring the peak amplitude at 239.0 nm.

In the same way, the absorption spectra of the solutions prepared at different concentrations of HCTZ were recorded in the range of 200–400 nm and divided by the spectrum of the standard solution of 10 μg _1 mL of AMLB and ratio spectra were obtained. First derivative of the ratio spectra was plotted with ∆λ= 3 nm .After that these vectors [first derivative of ratio spectra] are divided by [d/ dλ][OLM/AMLB] corresponding to the derivative of the ratio _1 of the spectra of 10 μg mL of each of OLM and AMLB and then second ratio spectra were obtained. The concentration of HCTZ was determined by measuring the peak amplitude at 276.0 nm in the first derivative of ratio spectra.

By the same way, the concentrations of AMLB were determined by measuring the peak amplitude of the first derivative of the first ratio spectra at 357.0 nm using the 170 _1 spectra of 10 μg mL of each of OLM and HCTZ to obtain the second derivative of ratio spectra.

3. Results and discussion

DRSZ Method

In this method, the absorption spectra of the ternary mixture solutions of OLM, HCTZ and AMLB in ACN were divided by the spectrum of the standard solution of 10 μg/ml HCTZ in the same solvent and the ratio spectra of OLM–AMLB were obtained. Figure 3 shows the first derivative of the ratio spectra plotted with intervals of ∆λ= 3 nm. The concentrations of OLM and AMLB in the ternary mixture were determined by measuring the signals at 259.0 nm for OLM [the wavelength which corresponds to the zero-crossing point of AMLB] and 358.0 nm for AMLB [the wavelength which corresponds to the zero-crossing point of

In a similar manner, the absorption spectra of the ternary mixture solutions of OLM, HCTZ and AMLB in ACN were divided by the spectrum of the standard solution of 10μg/ml OLM in the same solvent and the ratio spectra of HCTZ–AMLB were obtained by following the same procedure as above. Figure 5 shows the first derivative of the ratio spectra plotted with intervals of ∆λ=3nm from the ratio spectra. The content of HCTZ in the ternary mixture was determined by measuring the signal at 279.0 nm for HCTZ [wavelength corresponding to zero-crossing point for AMLB].

Method optimization

The influence of the wavelength interval ,∆λ, for obtaining the first derivative of the ratio spectra was tested and a value of ∆ λ = 3nm was considered suitable for OLM, HCTZ and 171 AMLB determinations maximum recovery was obtained using the selected ∆λ.

The effect of divisor concentration on the calibration graphs was studied. It affects only the slope of the standard curves while the maxima and minima remain at the same wavelengths. The standard solutions of 10μg/ml of HCTZ for determining OLM and AMLB and of 10μg/ml of OLM for the determination of HCTZ in their ternary mixtures and in the ratios present in their

DDRD method

The absorption spectra of the solutions of OLM in ACN were recorded in the range 200–400 nm and divided by the double divisor [10μg/ml HCTZ and 10μg/ml AMLB] to obtain their corresponding ratio spectra Figure 6a. First derivatives of the ratio spectra thus obtained were calculated with interval of ∆λ=3nm as shown in Figure 6b. The concentration of OLM was determined by measuring the amplitude at 266.0 nm

the Ratio spectra.

The absorption spectra of the solutions prepared at different concentrations of HCTZ in ACN were recorded between 200 and 400 nm and divided by the spectrum of the standard mixture solution of 10μg/ml OLM and 10 μg/ml AMLB The ratio spectra were thus obtained Figure 7a and their first derivatives were traced with ∆λ= 3 nm. The concentration of HCTZ was determined by measuring the amplitude at 317.0 nm corresponding to a minimum point Figure 7b.

Similarly, the absorption spectra of the solutions prepared at different concentrations of AMLB in ACN in the range 172 200-400 nm were stored and divided by the double divisor [10μg/ml OLM and 10μg/ml HCTZ]. The ratio spectra thus obtained Figure 8a were differentiated with interval of ∆λ=3 nm to the corresponding first derivatives Figure 8b. The concentration of AMLB was determined by measuring the amplitude at 363.0 nm corresponding to a

Ratio spectra.

[a]

Ratio spectra.

These coinciding points of the derivative of the ratio spectra were selected as working wavelengths, as mentioned above, for the determinations of the subject compounds in the ternary mixture.

Method optimization

The main instrumental parameter conditions were optimized for reliable determination of the active compounds. For selecting the standard binary mixture as double divisor at appropriate concentrations, a very important factor in practice, some double divisor concentrations were tried. The sum of the spectra of 10μg/ml HCTZ and 10μg/ml AMLB as a ‘double divisor’ for determining OLM; 10μg/ml OLM and 10μg/ml HCTZ as a ‘double divisor’ for determining HCTZ and of 10 μg/ml OLM and 10 μg/ml HCTZ as a ‘double divisor’ for determining AMLB were found suitable for the determination of the three drugs in the ratios present in their pharmaceutical preparations.

Furthermore, the influence of ∆λ for the first derivative of the ratio spectra was studied. It was found appropriate to use the values of ∆λ= 3 nm that gives optimum recovery for the determination of OLM, HCTZ and AMLB, respectively.

173

Successive derivative ratio spectra method

The absorption spectra of different concentrations of OLM were recorded in the range of 200–400 nm and divided by the spectrum of the standard solution of 10 μg _1 mL of HCTZ and the ratio spectra were obtained. First derivative of the ratio spectra was obtained with ∆λ =3 nm. These vectors [first derivative of the ratio spectra] were then divided by [d/dλ][AMLB/HCTZ] corresponding to the derivative of the ratio _1 of the spectra of 10 μg mL of each and then the second ratio spectra were obtained. The first derivative of these ratio spectra was obtained from which OLM was determined by measuring the peak amplitude at 239.0

μg/ml EACH] μg/ml]} in acetonitrile, and [b] the first derivative of their second ratio spectra obtained with Δλ = 3 nm.

In the same way, the absorption spectra of the solutions prepared at different concentrations of HCTZ were recorded in the range of 200–400 nm and divided by the spectrum of the standard solution of 10 μg -1 .mL of AMLB and ratio spectra were obtained. First derivative of the ratio spectra was plotted with ∆λ= 3 nm. After that these vectors [first derivative of ratio spectra] are divided by [d/dλ] [OLM/AMLB] corresponding to the derivative of the ratio -1 of the spectra of 10 μg mL each of OLM and AMLB and then second ratio spectra were obtained. The concentration of HCTZ was determined by measuring the peak amplitude at 276.0 nm in the first

μg/ml EACH] μg/ml]} in acetonitrile, and [b] the first derivative of their second ratio spectra obtained with Δλ = 3 nm.

174 By the same way, the concentrations of AMLB were determined by measuring the peak amplitude of the first derivative of the first ratio spectra at 357.0 nm using the -1 spectra of 10 μg mL of each of OLM and HCTZ to obtain the first derivative of ratio

μg/ml EACH] μg/ml]} in acetonitrile, and [b] the first ratio derivative of their second ratio spectra obtained with Δλ = 3 nm.

Validation of Method

As per ICH guideline the method is validated and following parameters were evaluated.

Linearity

Under the above described experimental conditions, the graphs obtained by plotting the first derivative absorbance ratio[DRSZ method], first derivative double divisor absorbance ratio [DDRD method] and first derivative of successive ratio spectra versus concentration for OLM, HCTZ and AMLB show linear relationships in the range of [5- -1 50 μg ml ] for the three drugs. Using the method of least squares, regression equations, correlation coefficients, limits of detection and quantitation were calculated for each of the three drugs and the results are summarized in Table 1. The high values of the correlation coefficients with negligible intercepts indicate the good linearity of the calibration graphs.

Precision

Separate determinations at different concentration levels were carried out for each drug to test reproducibility of measured analytical signals. The percentage relative standard deviations [R.S.D. %] were found to be less than 2% as shown in tables [2,3,&4].

Accuracy

In order to assess the accuracy of the three previously discussed methods, several synthetic mixtures, covering the linearity ranges, with different ratios of OLM, HCTZ and AMLB were analyzed. The percentage recovery for each mixture is calculated as shown in [Table 5]. It is obvious that the three methods could be applied to resolve the three drugs in their ternary mixtures with good accuracy.

The mixture solutions of OLM, HCTZ and AMLB combination in the concentration ratios simulating tablets ratios, 40:25:13.9 and 20:12.5:6.95 [OLM: HCTZ: AMLB], were prepared in six replicates.

Each mixture solution was resolved independently for its OLM, HCTZ and AMLB, by measuring its analytical signals, each at selected wavelengths. The proposed methods successfully determine the three drugs in the dosage form without previous separation or extraction procedures as shown in [Table 5].

175

Sa =standard error of the intercept, Sb=standard error of the slope, S y/x=residual standard deviation of regression line

LOD=limit of detection, LOQ=limit of quantitation

The proposed methods are simple, sensitive and easy to be applied for simultaneous determination of OLM, HCTZ, and AMLB, in their ternary mixture with good precision.

These methods don’t need tedious extraction procedures or sophisticated instrumentation such as HPLC.

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