INTRODUCTION:

Angiotensin II receptor antagonists (AIIRA's) are orally effective potent antihypertensive drugs that selectively block AT₁ receptors in many tissues leading to marked decrease in angiotensin II synthesis which is potent vasoconstrictor and then lowering the blood pressure¹. Among these drugs; Losartan potassium (Los-K) and Irbesartan (Irb) are extensively prescribed for treatment of hypertension. Fig.1. illustrates the chemical structure of the studied drugs. The investigated drugs have been approved by FDA since the middle of 1990s and are prescribed as the first line treatment of hypertension, left ventricular hypertrophy, nephropathy in type 2 diabetic patients and heart failure². Both drugs are official in the United States Pharmacopeia 2007 while only Losartan potassium is official in the British Pharmacopeia 2010.

Losartan Potassium

Irbesartan

Fig. 1. : Chemical structure of the used drugs

Several analytical methods have been reported for the analysis of the investigated AIIRA's. Different chromatographic methods were extensively used for determination of the studied drugs including high performance liquid chromatography (HPLC)^3-7, capillary electrophoresis^8-10, also voltammetric methods^{11, 12} and spectroflourimetric methods^{13, 14} were reported for analysis of the cited drugs. Spectrophotometric analysis still very important tool for the analysis of drugs due to being inexpensive, rapid, easy and accurate method in comparison to the other expensive and sophisticated methods as well as, it is available in the most of analytical laboratories. The literatures revealed different spectrophotometric methods for the analysis of the studied sartans depending on their UV absorbance or derivative spectrophotometry^15-17. Few colorimetric methods were previously published for the assay of Los-K^18,
19 and Irb²⁰. Most of these previously reported spectrophotometric methods suffered from bad sensitivity, instability and complexity. So, the development of simple spectrophotometric method with high sensitivity and good stability for the assay of the investigated AIIRA's will lead to easy, rapid and reliable method for the analysis of these drugs. The aim of the present work is to develop three, simple, rapid, sensitive, accurate, cheap and non-extractive spectrophotometric methods for the determination of the cited drugs in pure forms as well as in their pharmaceutical dosage forms. These methods were based on the reaction of the investigated drugs with either Bromocresol purple (BCP) or Cresol red (CR) to give highly coloured non-extractive ion pair complex species measured at the visible region (415, 435 nm).

EXPERIMENTAL:

Apparatus

UV- 1601 PC, UV-Visible Spectrophotometer (Shimadzu, Tokyo, Japan), sartorius handy balance-H51 (GmbH Gottingen, Germany), ultrasonic cleaner (Cole- Parmer, Chicago, U.S.A.) and MLW type thermostatically controlled water bath (Memmert GmbH, Schwabach, Germany).

Materials and reagents

All solvents and reagents used were of analytical grade. Methyl alcohol and chloroform (A.D.W.I.C., Egypt). Losartan potassium (Los-K) (Amriya Pharmaceutical Industries, Alexandria, Egypt) and Irbesartan (Irb) (Memphis Pharm. Co, Cairo, Egypt) were obtained as gifts and were used as supplied.

Bromocresol purple (BCP) (Fluka Chemie, AG, Switzerland), Cresol red (CR) (Merck, Darmstadt, Germany) were used without further purification; 3 mg/ml of BCP and 2.5 mg/ml of CR were prepared in chloroform.

Pharmaceutical formulations containing the studied drugs were purchased from local market. Losarmepha^® tablets (Sigma Pharm.Co., Cairo, Egypt) is labelled to contain 50 mg Los-K, Hysartan^® tablets (Amriya Pharm. Ind. Co., Alexanderia, Egypt) is labelled to contain 50 mg Los-K and 12.5 mg HCTZ, Aprovel^®tablets (Sanofi Winthrop, France)is labelled to contain 300 mg Irb and Co- Aprovel^® tablets (Sanofi Winthrop, France) is labelled to contain 300 mg Irb and 12.5 mg HCTZ.

Preparation of standard solutions

Stock solutions containing 5 mg/ml of either Los-K or Irb were prepared in chloroform. The working standard solutions of each drug were prepared by further dilution of suitable volumes of the stock solution with chloroform to obtain concentration ranges of 20-180 μg/ml, 20-300 μg/ml for Los-K and Irb respectively.

Preparation of sample solutions

Ten tablets of each formulation containing Los-K or Irb alone or combined with HCTZ were accurately weighed and finely powdered. A quantity equivalent to 250 mg of Los-K or Irb was transferred into separate 50 ml volumetric flasks, dissolved in about 25 ml chloroform, swirled, sonicated for about 10 min and the flasks were completed to the mark with chloroform, shacked well and then filtered. The first portion of the filtrate was discarded. Further dilutions with chloroform were prepared in order to obtain the working sample solutions with concentrations that lie in the linear range of each studied drug as mentioned under preparation of standard solutions.

General assay procedures

Into a series of 10 ml volumetric flasks, accurately measured 1.0 ml of the working standard or sample solutions of either Los-K or Irb (in chloroform) was transferred, then 1.0 ml of BCP (3 mg/ml) or CR (2.5 mg/ml) was added, mixed well and then completed to the volume with chloroform. The absorbance was measured at 415 nm or 435 nm against reagent blank treated similarly for BCP or CR respectively.

Determination of molar ratio

The Job’s method of continuous variation²¹was employed. Master equimolar solutions of each drug and reagents were prepared. The concentrations of these solutions were 2.17x10^-4 M of Los-K or 2.33x10^-4 M of Irb was prepared in chloroform for both methods. Series of 10 ml portions of the master solutions of each drug with the respective reagent were made up comprising different complementary proportions (0:10, 1:9,…….., 9:1, 10:0) in 10 ml volumetric flasks. The absorbance of the resulting ion pair complexes was measured at the selected λ_max for each method against reagent blank treated similarly.

RESULTS AND DISCUSSION:

Absorption spectra

The presence of basic cationic nitrogen atoms in all the investigated AIIRA’s allow them to interact with acidic anionic dyes such as BCP or CR to form highly coloured products through formation of non-extractive ion pair complexes. Los-K and Irb reacted with BCP to form highly coloured products exhibited maximum absorbance at 415 nm for both drugs, Fig. 2 represents the absorption spectra of the reaction between Irb and BCP as a representative example. CR reacted with the studied drugs producing highly coloured products with maximum absorbance at 435 nm for both drugs.

Optimization of the reaction variables

Acidic dyes were reported for the quantitative analysis of several pharmaceutical compounds with different chemical structures having a basic nitrogen atom. Most of the reported methods are based on using these dyes as analytical reagent in buffered aqueous solution containing the basic drug. The resulting coloured ion pair complex is extracted with organic solvent and then determined spectrophotometrically^22-24. The ion pair extraction technique has some difficulties arising from incomplete extraction and emulsions formation between the two immiscible layers, furthermore; it is time consuming. Few methods have been reported for the analysis of pharmaceutical compounds through ion pair formation without extraction²⁵. In the present study; preliminary investigations revealed that the reactions between the studied drugs and the used acidic dyes occurred spontaneously without using buffer (with different pH values) or extraction with organic solvent. All reactions variables such as reactions time, concentration of reagents, effect of diluting solvents and temperature were studied to achieve complete reactions formation with maximum sensitivity and highest stability.

Concentration of the reagent

The effect of concentration of each reagent on the intensity of the developed colour was tested using constant drug concentration and different concentrations of each studied dye, (0.4-4 mg/ml) of BCP and (0.5-3.5 mg/ml) of CR were used. The results showed that, the absorbance of the formed ion pair complexes of each drug and its corresponding reagent was increased by increasing the reagent concentration. By increasing the reagent concentration over 2.5 mg/ml of BCP or 2.0 mg/ml of CR the absorbance of the formed complexes remained constant. For more accurate measurements, the best chosen concentration of BCP was 3 mg/ml which gave maximum colour development of the ion pair complexes with either Los-K or Irb, 2.5 mg/ml of CR for determination of either Los-K or Irb. Fig.3. represents the effect of each reagent concentration on the developed colour.

Reaction time and temperature

Optimum reaction time was determined by following the colour development for all the ion pair complexes of the studied drugs at room temperature and in thermostatically controlled water bath at different temperatures (15˚C – 60˚C). It was observed that at higher temperature the absorption was decreased rapidly for all the coloured complexes which mean that the formed complexes may be dissociated by elevation the temperature. Room Temperature (25°C ±5°C) was chosen as the optimum reaction temperature for all methods. At room temperature, the effect of time before dilution on the reaction products was studied for about 30 minutes. The colour occurred spontaneously and remained stable for more than one hour.

Effect of diluting solvent

Trials have been made to increase the intensity of the developed colour of the reactions products between Los-K or Irb with either BCP or CR by diluting the reaction mixture with different organic solvents such as chloroform, carbon tetrachloride, 1,4 dioxan, methylene chloride, 1,2 dichloroethane and methanol. As illustrated in Table 1, it was found that chloroform was the best diluting solvent for both BCP and CR methods. The developed colours of the ion pair complexes for both drugs using both reagents were stable for more than one hour.

Molar ratios determination

Job's method of continuous variation²¹ of equimolar solutions of each studied drug and its corresponding reagent (BCP or CR) was used for determination of the molar ratios of the reactions. The results showed that, the maximum values for all formed complexes are at a mole fraction of 0.5 which indicated that; the ratios are 1:1 (drug: reagent).

Table 1: Effect of diluting solvent on the absorption intensities of the reaction products of Los-K and Irb with BCP or CR

	Absorbance^*
	BCP method (λ_max 415 nm)		CRmethod (λ_max 435nm)
Solvent	Los-K (10 μg/ml)	Irb (10 μg/ml)	Los-K (10 μg/ml)	Irb (15 μg/ml)
Chloroform	0.541	0.480	0.441	0.356
Carbone tetrachloride	0.462	0.397	0.432	0.347
1,4 dioxane	----	----	----	----
Methylen chloride	0.390	0.350	0.350	0.280
1,2 dichloroethane	----	----	----	----
Methanol	----	----	----	----

^*Average of three determinations.

Table 2: Quantitative parameters for the analysis of the studied drugs by the proposed methods

Parameter	BCP method		CR method
Parameter	Los-k	Irb	Los-k	Irb
Linear range (µg/ml)	2-16	2-20	3-18	3-30
Intercept (a) ± SD^a	-0.0218 ±0.0071	-0.0054 ±0.0105	0.0359 ±0.0078	0.0251 ±0.0058
Slope (b) ±SD^a	0.0545 ±0.0005	0.0479 ±0.0008	0.0435 ±0.0003	0.0224 ±0.0002
Correlation coefficient (r)	0.9997	0.9998	0.9999	0.9971
ε×10⁴ (L/mol/cm)	2.49	2.02	2.15	0.99
LOD (µg/ml)^b	0.43	0.72	0.59	0.85
LOQ (µg/ml) ^c	1.30	2.19	1.79	2.59

^aAverage of five determinations, ^bLOD is the limit of detection and ^c LOQ is the limit of quantitation.

Calibration curves

Under the specified optimum reaction conditions, the calibration curves for the reaction products of Los-K or Irb with the used reagents were constructed by plotting absorbance versus concentrations for each studied drug. The data were analyzed using least square method. A linear relationship was found between absorbance and the concentration of each drug in the range of 2-16 µg/ml, 2-20 µg/ml for Los-K and Irb respectively using BCP; however when CR was used, the linearity range were 3-18 µg/ml for Los-K and 3-30 µg/ml for Irb. The correlation coefficients in all methods were ranged from 0.9971-0.9999. All the quantitative parameters are shown in Table 2 indicating good linearity of all the proposed methods.

Validation of the proposed methods

The proposed methods were validated according to ICH guidelines on the validation of analytical methods²⁶ and complied with USP 2007 validation guidelines.

Sensitivity

The limit of detection (LOD) and limit of quantitation (LOQ) for all the proposed methods were calculated using the formula:

LOD or LOQ = kS D_a /b; Where: k= 3.3 for LOD and 10 for LOQ, SD_a is the standard deviation of the intercept, b is the slope. The values of LOD and LOQ which are listed in Table 2 indicate good sensitivity of the proposed methods.

Precision and accuracy

In order to determine the precision of the proposed methods, six determinations of each drug were performed using the corresponding proposed method according to the USP recommendations. The results showed in Table 3 revealed that, the relative standard deviations didn't exceed 2% indicating good reproducibility of the proposed methods and ability of those methods for application in quality control laboratories. Moreover, the good percentage of recoveries of each standard drug confirms excellent accuracy.

Table 3: Precision and accuracy of the proposed methods for analysis of the studied drugs

Method	Drug	Drug Concentration (μg/ml)	Mean (%)±SD ^a	% RSD ^b
BCP	Los-K	10	98.56±0.48	0.43
BCP	Irb	10	100.00±0.92	0.21
CR	Los-K	10	99.28±0.05	1.20
CR	Irb	15	98.59±0.64	1.19

^a SD is the standard deviation for the mean of six determinations.

^bRSD is the relative standard deviation.

Selectivity

The selectivity of the proposed methods for determination of the studied drugs in the presence of frequently encountered excipients such as starch, glucose, lactose, magnesium steareate and gum-acacia was studied as well as the co-formulated HCTZ. The good percentage of recoveries revealed that; there is no inferences could be observed from the commonly added excipients or the co-formulated HCTZ. So, the proposed methods were considered as selective methods. This selectivity may be attributed to the basic nitrogen present in the cited drugs, rather than HCTZ, which doesn't have sufficient basicity to achieve ion pair formation²⁷.

Table 4: Determination of the studied drugs in pharmaceutical dosage forms using the proposed and the reported methods

Method	Drug	Dosage form (Tablets)	Recovery (%) ±SD^a		t-value^b	F-value^b
Method	Drug	Dosage form (Tablets)	Proposed method	Reported method	t-value^b	F-value^b
BCP	Los-K	Losarmepha^®	98.23±0.85	98.84±0.38	2.16	5.00
	Los-K	Hysartan^®	98.49±0.49	98.84±0.44	0.44	1.24
	Irb	Aprovel^®	101.43±0.54	100.13±0.51	0.02	1.12
	Irb	CoAprovel^®	98.81±0.37	98.74±0.21	0.17	3.10
CR	Los-K	Losarmepha^®	98.42±0.79	98.84±0.38	0.16	4.32
	Los-K	Hysartan^®	100.72±0.41	98.84±0.44	0.02	0.87
	Irb	Aprovel^®	101.87±0.80	100.13±0.51	0.04	2.46
	Irb	CoAprovel^®	101.62±0.94	98.74±0.70	0.18	1.80

^a Average of five determinations.

^b The tabulated values of t- and F- at 95% confidence level are 2.78 and 6.39 respectively.

Robustness and ruggedness

Robustness was examined by evaluating the influence of small variation in the experimental parameters for each method; such as the concentration of reagents and the reaction time on the analytical performance of the method. In these experiments, one experimental parameter was changed while the other parameters were kept unchanged. The percentage of recoveries was calculated each time. The small variation in any of the variables didn’t significantly affect the results.

Ruggedness of the proposed methods was assessed by applying the procedures using two different instruments in two different laboratories at different elapsed time. Results obtained from lab-to-lab and day-to-day variation was found to be highly reproducible.

Applications

The suggested methods were successfully applied for the analysis of the studied drugs in their pharmaceutical preparation and the results obtained were validated by comparison with those of the reported methods[18, 20]. No significant difference was found between the results obtained by the proposed methods or reported methods by applying t-test and F-test at 95% confidence level as showed in Table 4. The obtained results indicate good accuracy and precision for the determination of the investigated drugs in their pharmaceutical dosage forms using the presented methods.

Suggested mechanism of the reaction

Some of the investigated AIIRA’s have only one positively charged site that is capable for electrostatic attraction with the negatively charged anion of the used reagents to give coloured ion pair complexes. These sites are the univalent positively charged species of the basic nitrogen atoms present in the imidazole ring of both Los-K and Irb.

Los-K and Irb contain an imidazole ring with two nitrogen atoms. One of the nitrogen atoms of the imidazole is involving in the aromaticity of the ring (pyrole nitrogen) and doesn’t have any basic properties while the other atom (pyrimidine type) is responsible for the basic characters of the imidazole ring and consequently the basicity of both Los-K and Irb²⁸. This basic nitrogen allowed the electrostatic attraction between the electronegative sulphoxide group of either BCP or CR.

CONCLUSION:

The methods depend on formation of non extractive ion pair between AIIRA's drugs and anionic dyes. The proposed methods were advantageous over other reported methods with respect to high sensitivity, simplicity, low cost. The results demonstrated that the useful use of BCP or CR for the colorimetric analysis of the studied antihypertensive drugs. The proposed methods could be performed in any analytical laboratory due to availability of the instruments and low cost of the methods. It can be applied in quality control laboratories for the analysis of the investigated drugs.

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Received on 11.12.2012 Accepted on 24.01.2013

Asian J. Pharm. Ana. 3(1): Jan.-Mar. 2013; Page 03-08