UV Spectrophotometric Determination of Hydrochlorothiazide and Olmesartan Medoxomil in Pharmaceutical Formulation

A simple, specific, accurate, precise and reproducible method has been developed and validated for the simultaneous estimation of hydrochlorothiazide and olmesartan medoxomil in combined dosage form by UV spectrophotometric method. UV spectrophotometric method includes simultaneous equation method (Method I) 271.5 nm and 257.0 nm λmax of both the drugs were selected, absorbance Ratio method (Method II) 261.5 nm an isoabsorptive wavelength and 257.0 nm were selected for estimation of hydrochlorothiazide and olmesartan medoxomil respectively and Three wavelength method (Method III), two wavelengths were selected such that hydrochlorothiazide give same absorbances (263.8 and 278.4 nm) at two selected wavelength while third wavelength (316.5 nm) was such that olmesartan gives nearly zero absorbance. The two drugs follow Beer’s law over the concentration range of 5-25 μg/mL. The % recoveries of the both the drugs were found to be nearly 100 % representing the accuracy of the proposed methods. Validation of the proposed methods was carried out for its accuracy, precision, specificity and ruggedness according to ICH guidelines. The proposed methods can be successfully applied in routine work for the determination of hydrochlorothiazide and olmesartan medoximil in combined dosage form.


Experimental
SHIMADZU double beam UV visible spectrophotometer (model 1700) with 1 cm matched quartz cuvettes were used for all absorbance measurements.Shimadzu AUX 220 balance was used for weighing the samples.All the chemicals used were of AR grade.Double distilled water and Whatman filter paper (no.41) were used throughout the experimental work.
Multicomponent tablet olmetor-H (HTZ 12.5 mg and OLM 20.0 mg) manufactured by Hetero drugs ltd, Dist-Baddi, Himachal Pradesh.All chemicals and reagents used were of analytical grade.

Standard stock solution
Standard stock solutions of HTZ and OLM were prepared individually in methanol having concentration of 100 µg/mL and 160 µg/mL respectively.Aliquot portions of the stock solutions were diluted individually with distilled water to get final concentration of 10 µg/mL and 16 µg/mL for HTZ and OLM respectively.These working standard solutions were scanned in the range of 400-200 nm in 1.0 cm cell against solvent blank.

Determination of absorptivity value
The solutions of each drug in tripilicate were read against solvent blank at the selected wavelengths and A(1% 1 cm) value were calculated using below formula: Absorbance at selected wavelengths Absorptivity, A (1% 1 cm) = Concentration in g / 100 mL

Study of linearity
Stock solutions each of HTZ and OLM having concentration of 100 µg/mL were prepared.Aliquots of each solution were appropriately diluted and the final dilutions were read at the selected wavelengths individually and in their combination.The correlation coefficient was found to be less than 1.The method was first applied to standard laboratory mixture which yielded encouraging results and the proposed methods were applied to marketed formulation.

Assay
Twenty tablets were weighed and average weight was calculated.The tablets were triturated thoroughly and mixed.Tablet powder equivalent to 12.5 mg of hydrochlorothiazide (~20.0 mg of olmesartan medoximil, on the basis of label claim) was transferred to 50.0 mL volumetric flask, dissolved by sonication for 15 min.with sufficient quantity of methanol and volume was made up to mark with methanol.The content was filtered through Whatman filter paper (no.41).A 10.0 mL portion of the above filtrate was further diluted to 50.0 mL with distilled water.A 10.0 mL portion of this solution was further diluted to 50.0 mL with distilled water.
The final dilution of all sample solutions were read at the selected wavelengths using blank methanol with distilled water.The amount of each drug was estimated by proposed methods using the following formulae:

Simultaneous equation method (Method I)
where: a x1= The absorptivity of HTZ at 257.0 nm, a x2= The absorptivity of HTZ at 271.5 nm a y1= The absorptivity of OLM at 257.0 nm, a y2= The absorptivity of OLM at 271.5 nm A 1 and A 2 are the absorbances of the diluted sample at 257.0 and 271.5 nm respectively Absorbance ratio method (Method II)  1 for all proposed methods.

Method validation
Validation of the proposed methods was carried out for its accuracy, precision, specificity and ruggedness according to ICH guidelines.

Accuracy
Recovery studies were carried out at four different levels by adding the pure drug to previously analysed tablet powder sample.From the amount of drug total drug found, percentage recovery was calculated by proposed three methods and results are shown in Table 2a and 2b

Inter-day precision
It was done by analysing the solutions by same analyst on alternate days till 5 th day.The % RSD is shown in Table 3. Results indicate that the solution is stable up to 1day, thereafter degradation may have taken place leading lower percent label claim.

Intra day precision
It was done by analysing the solutions by same analyst within a day.The % RSD is shown in Table 3. Results indicate that the solution is stable up to 4 h and thereafter, degradation may have taken place in the solution.

Linearity and range
Accurately weighed quantities of tablet powder equivalent to 80, 90, 100, 110 and 120% of label claim of HTZ were taken in a series of 50 mL volumetric flasks and dilutions were made as under sample solution.The graphs of % label claim vs. absorbance were plotted for Method I and II were plotted, were found to be linear.

Ruggedness
It was done by analysing the samples solutions by three different analysts.The % RSD by proposed methods is shown in Table 3 and was found to be within limits.

Stability
Sample solution: Accurately weighed quantities of tablet content equivalent to about 12.5 mg HCTZ, were transferred to 50.0 mL volumetric flask.These entire samples were stored for 24 h under following different conditions.At 50 0 C after addition of 1.0 mL of 1N HCl (Acid) At 50 0 C after addition of 1.0 mL of 1N NaOH (Alkali) At 50 0 C after addition of 1.0 mL of 6% H 2 O 2 (Oxide) At 60 0 C (Heat) After the specified period, the results were analysed by proposed methods and results are shown in Table 4.

Results and Discussion
The study of spectrum recorded (Figure 1) showed that the HTZ exhibited two λ max at 271.6 and 316.5 nm while OLM showed at 257.0 nm.Simultaneous Equation was constructed using 271.5 nm and 257.0 nm λ max of both the drugs, Absorbance Ratio method utilises 261.5 nm an isoabsorptive wavelength and 257.0 nm for estimation of hydrochlorothiazide and Olmesartan medoxomil respectively and Three-wavelength method, two wavelengths were selected such that hydrochlorthiazide give same absorbance (263.8 and 278.4 nm) at two selected wavelength while third wavelength (316.5 nm) was such that olmesartan gives nearly zero absorbance.The difference in absorbance of olmesartan at the former two selected wavelength was used for estimation of OLM while at latter wavelength HCTZ was estimated.The two drugs follow Beer's law over the concentration range of 5-25 µg/mL.The values of relative standard deviation were found satisfactory and the recovery studies were close to 100%.The stability studies showed that the drugs are susceptible to stress condition that indicates, the drugs are not intrinsically stable towards such conditions.

Conclusion
The proposed methods were found to be accurate, simple, rapid and reproducible.Thus all the methods can be applied in the routine analysis of the hydrochlorothiazide and Olmesartan medoxomil in formulations.
Absorbance ratio of sample mixture at 257.0/261.5 nm Q x = Ratio of absorptivity of OLM at 257.0/261.5 nm Q y = Ratio of absorptivity of HTZ at 257.0/261.5 nm a x = Absorptivity of HTZ at 257.0 , a y = Absorptivity of OLM at 261.5 A = Absorbance of sample mixture at isobestic wavelength (261.5 nm) Three wavelength method (Method III) of unknown, Cs = Concentration of standard As = (A1-A2), were A1 = Absorbance of standard OLM at 263.8 nm A2 = Absorbance of standard OLM at 278.4 nm Au = (A1-A2), were A1 = Absorbance of sample OLM at 263.8 nm A2 = Absorbance of sample OLM at 278.4 nm of unknown, Cs = Concentration of standard As = Abs.Of standard at 316.5 nm, Au = Abs. of unknown at 316.5 nm The results are shown in Table

Table 1 .
Results of estimation in marketed formulation.
Method I-Simultaneous equation method, Method-II-Absorbance ratio method, Method III-Three wavelength method.

Table 2a .
. Results of recovery studies.

Table 2b .
Results of recovery studies.

Table 3 .
Results of ruggedness studies.

Table 4 .
Results of stability studies.
Method I-Simultaneous equation method, Method-II-Absorbance ratio method, Method III-Three wavelength method.