UV Spectrophotometric Determination of Hydrochlorothiazide...

ISSN: 0973-4945; CODEN ECJHAO

E-Journal of Chemistry

http://www.e-journals.net 2010, 7(4), 1156-1161

UV Spectrophotometric Determination of

Hydrochlorothiazide and Olmesartan Medoxomil in

Pharmaceutical Formulation

A.T. HEMKE*, M.V. BHURE, K.S. CHOUHAN,

K.R. GUPTA and S.G. WADODKAR

Department of Pharmaceutical Chemistry,

S.K.B. College of Pharmacy New Kamptee, Nagpur, M.S. India.

[email protected]

Received 6 November 2009; Accepted 2 January 2010

Abstract: 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.

Keywords: Hydrochlorothiazide, Olmesartan medoxomil, UV spectroscopy, Simultaneous Equation

method, Absorbance Ratio method.

Introduction

Hydrochlorothiazide (HTZ) is a drug used in the treatment of hypertension. It blocks the

reabsorption of Na+ in the distal convoluted tubules by inhibiting the luminal membrane

1157 A.T. HEMKE et al.

bound Na+/Cl

- co-transport system. HTZ

1, chemically

it is 6-chloro-3,4-dihydro-2H-1,2,4-

benzothiadiazine-7-sulphonamide1, 1 dioxide. Olmesartan medoxomil (OLM) is used in the

treatment of hypertension. It is angiotensin II receptor antagonists. OLM2, chemically it is

2,3-dihydroxy- 2-butenyl -4- (1-hydroxy-1-methylethyl)- 2-propyl-1- [p-(o-1H-tetrazol-5-yl-

phenyl) benzyl] imidazole-5-corboxylate, cyclic 2,3-corbonate. Literature survey revealed

that the methods reported earlier were only for the analysis of single drug like UV-

spectrophotometry3 for HTZ and LC

4, UV

5 and Capillary electrophoresis

6 for determination

of OLM. In combination methods are reported for HTZ with telmisartan7 irbesartan

8,

valsartan9 and lisinopril

10 while OLM with ramipril

11 for their determinations in

pharmaceutical formulation. HPTLC12

method is reported for the HTZ with OLM

combination. The paper presents two simple, accurate, reproducible, rapid and economical

methods for simultaneous analysis of the two components in tablet formulation.

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

UV-Spectrophotometric Determination of Hydrochlorothiazide 1158

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)

C HTZ = A2ay1 – A1ay2 / ax2ay1 – ax1ay2

C OLM = A1ax2 – A2ax1 / ax2ay1 – ax1ay2

where:

ax1= The absorptivity of HTZ at 257.0 nm, ax2= The absorptivity of HTZ at 271.5 nm

ay1= The absorptivity of OLM at 257.0 nm, ay2= The absorptivity of OLM at 271.5 nm

A1 and A2 are the absorbances of the diluted sample at 257.0 and 271.5 nm respectively

Table 1. Results of estimation in marketed formulation.

Label Claim % Label Claim

Method I Method II Method III

HTZ OLM HTZ OLM HTZ OLM HTZ OLM

101.2 99.8 101.7 99.8 100.5 98.1

99.8 102.0 100.0 101.9 100.9 98.6

100.9 100.0 101.6 100.2 99.0 98.2

101.2 100.4 101.2 100.1 101.0 99.0

12.5 20.0

101.6 101.2 102.1 100.1 101.8 98.7

Mean 100.94 100.68 101.32 100.66 100.64 98.52

S.D. (±) 0.61 0.82 0.70 0.82 1.03 0.37

% R.S.D

0.60 0.81 0.69 0.81 1.02 0.38

Method I- Simultaneous equation method, Method-II- Absorbance ratio method, Method III- Three

wavelength method.

Absorbance ratio method (Method II)

vxv

vmOLNE

xvx

vmHTZ

a

Ax

QQ

QQC

a

Ax

QQ

QQC

−

−=

−

−=

Where:

Q m = 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

ax = Absorptivity of HTZ at 257.0, ay = Absorptivity of OLM at 261.5

A = Absorbance of sample mixture at isobestic wavelength (261.5 nm)

Three wavelength method (Method III)

( ) xCsAs

AuCuCOLME =,

Cu = Concentration 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

( ) xCsAs

AuCuCHCTZ =,

Cu = Concentration 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 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.

Table 2a. Results of recovery studies.

Amount of pure drug recovered, mg % Recovery Amount of pure

drug added, mg Method I Method II Method I Method II

HTZ OLM HTZ OLM HTZ OLM HTZ OLM HTZ OLM

2.6 3.9 2.62 3.91 2.68 3.89 100.7 100.2 103.0 99.7

5.0 7.4 5.1 7.32 5.10 7.30 102.0 98.9 102.0 98.7

7.3 11.6 7.39 11.51 7.39 11.43 101.3 99.2 101.3 98.5

10.0 15.7 9.95 15.59 9.95 15.40 99.5 99.3 100.0 98.1

Mean 100.87 99.4 101.57 98.75

± S.D 0.92 0.48 1.09 0.59

% R.S.D (CV) 0.91 0.49 1.07 0.56

Table 2b. Results of recovery studies.

Amount of pure drug

recovered, mg % Recovery Amount of pure

drug added, mg Method III Method III

HTZ OLM HTZ OLM HTZ OLM

2.4 3.7 2.43 3.69 101.2 100.0

4.8 7.2 4.82 7.10 100.4 98.6

7.2 11.1 7.27 10.89 100.9 98.1

9.0 14.0 8.98 13.89 99.8 99.2

Mean 101.57 98.75

± S.D 1.09 0.59

% R.S.D (CV) 1.07 0.56


wavelength method.

Precision

Inter-day precision

It was done by analysing the solutions by same analyst on alternate days till 5th

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.

UV-Spectrophotometric Determination of Hydrochlorothiazide 1160

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.

Table 3. Results of ruggedness studies.

Mean percent of label claim ±%R.S.D

Method I Method II Method III Parameter


Precision

Intra-day

(n=3) 97.63±0.80 97.10±1.02 96.00±0.88 97.13±0.83 97.10±1.99 96.43±5.36

Inter-day

(n=3) 87.66±10.41 92.63±7.42 81.53±17.38 95.36±5.15 87.36±14.31 101.40±1.78

Analyst-

Analyst 101.40±0.12 99.33±0.66 101.0 ±0.81 99.23 ±0.88 100.16±0.76 100.86±2.70


wavelength method.

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 0C after addition of 1.0 mL of 1N HCl (Acid)

At 50 0C after addition of 1.0 mL of 1N NaOH (Alkali)

At 50 0C after addition of 1.0 mL of 6% H2O2 (Oxide)

At 60 0C (Heat)

After the specified period, the results were analysed by proposed methods and results

are shown in Table 4.

Table 4. Results of stability studies.

Percent of un-degraded drug

Method I Method II Method III Sample


1N HCl 75.5 69.19 63.0 73.3 75.3 78.0

1N NaOH ---- ---- ---- ---- ---- ----

6% H2O2 69.7 102.4 80.3 98.7 78.1 91.3

60 0C 97.3 95.9 97.3 95.9 100.3 94.3


wavelength method.


Results and Discussion

The study of spectrum recorded (Figure 1) showed that the HTZ exhibited two λmax 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 λmax 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.

Figure 1. Overlain spectrum of hydrochlorothiazide and olmesartan medoxomil.

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.

Acknowledgments

The authors are thankful to Alembic Pharmaceutical Ltd. for providing standard drug samples and also to S.K.B.College of Pharmacy, Kamptee for providing the facilities to carry out the work.

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