A Prospective Validation of Hydrocholorothiazide and Bisoprolol Fumerate in its Pure and Bulk Dosage Forms by using RP- HPLC Technique

 

S. Janet Beula1*, Dr. R. Suthakaran1, Prem Kumar Bichala1, CH. Shankar1, Syed Ghouse1,

K. Suneetha2

1Vijaya College of Pharmacy, Munuganoor, RR District, India.

2 SSJ College of Pharmacy, Gandipet, RR District, India.

*Corresponding Author E-mail: Pharmjanet123@gmail.com

 

ABSTRACT:

A simple, specific and accurate RP-HPLC method was developed for the simultaneous estimation of Bisoprolol fumerate and Hydrochlorothiazide in bulk dosage forms. The chromatographic conditions were successfully developed for the separation of Bisoprolol fumerate and Hydrochlorothiazide by using ACE C18 column (4.6×150mm) 5µ, flow rate was 1.2ml/min mobile phase ratio was (70:30 v/v) methanol: Phosphate buffer pH 3 (pH was adjusted with orthophosphoric acid), detection wavelength was 240nm. The method was validated according to ICH guidelines for specificity, LOD, LOQ, Precision, Accuracy and Linearity. The method showed good reproducibility and recovery with % RSD less than 2.

 

KEYWORDS: ACE C18 column, Hydrochlorothiazide and Bisoprolol, RP HPLC.

 

 

 

INTRODUCTION:

Combination therapy or poly therapy is therapy that uses more than one medication. Typically, these terms refer to using multiple therapies to treat a single disease. Poly therapy is a related term, referring to the use of multiple medications1. Hydrochlorothiazide one of the oldest thiazide diuretics and is used in the treatment of hypertension patients either alone or with other antihypertensive drugs such as angiotensin converting enzyme inhibitors and beta blockers2-4. It is a modestly intense diuretic and applies its diuretic impact by lessening the reabsorption of electrolytes from the renal tubules along these lines expanding the discharge of sodium and chloride particles and consequently of water.

 

Bisoprolol is a cardio selective beta blocker used in the treatment of hypertension and angina pectoris. But the combination of Hydrochlorothiazide and Bisoprolol RP-HPLC method validation was still now not et al performed.5-8 This method was validated according to ICH guidelines for specificity, LOD, LOQ, Precision, Accuracy, and Linearity.9,10 The method showed good reproducibility and recovery with % RSD less than 2. Hence we had made an attempt to develop a simple accurate and precise RP-HPLC method for the simultaneous estimation of Hydrochlorothiazide and Bisoprolol in bulk and in tablet dosage form.

 

DRUG PROFILE:

Hydrochlorothiazide:

Structure:            

 

IUPAC name:

6-chloro-1, 1-dioxo-3, 4-dihydro-2H-1, 2, 4-benzothiadiazine-7-sulfonamide.

 

Category:

Antihypertensive Agents, Diuretics, Sodium Chloride Symporter Inhibitors.

 

Bisoprolol Structure:

 

IUPAC name:

[2-hydroxy-3-(4-{[2-(propan-2yloxy) ethoxy] methyl} phenoxy) propyl] (propan-2-yl) amine.

 

Category:

Antihypertensive Agents, Adrenergic beta-1 Receptor Antagonists, Sympatholytics.

 

MATERIALS AND METHODS:

Drug samples:

Pharmaceutically pure sample of Hydrochlorothiazide and Bisoprolol drug was obtained from Awamedica Company (in Erbil city, Kurdistan region of Iraq). Commercial tablet of Hydrochlorothiazide and Bisoprolol (100mg) was procured from the local drug market. Acetonitrile and water, Methanol, Ortho phosphoric acid, KH2PO4,, K2HPO4 were HPLC from merck KGaA, 64271 Darmstadt, Germany.

 

Instruments:

Analytical HPLC-auto sampler –UV detector Separation   module 2695, UV. Detector 2487 Empower-software version-2 Waters, U.V double beam spectrometer UV 3000+, Digital weighing balance(sensitivity 5mg), pH meter, Sonicator.

 
Preparation of the Reserpine and Hydrochlorothiazide standard and sample solution:
Sample solution preparation:

Accurately weigh and transfer 59.8mg of Hydrochlorothiazide and Bisoprolol Tablet powder into a 10mL clean dry volumetric flask add about 7mL of diluent and sonicate to dissolve it completely and make volume up to the mark with the same solvent. (Stock solution). Further pipette 0.6ml of Hydrochlorothiazide and Bisoprolol the above stock solution into a 10ml volumetric flask and dilute up to the mark with diluent.

 

Standard solution preparation:

Accurately weigh and transfer 12.5mg & 8mg of Hydrochlorothiazide and Bisoprolol working standard into a 10mL clean dry volumetric flask add about 7mL of diluent and sonicate to dissolve it completely and make volume up to the mark with the same solvent. (Stock solution) Further pipette 0.6ml of Hydrochlorothiazide and Bisoprolol the above stock solution into a 10ml volumetric flask and dilute up to the mark with diluent.

 

Procedure:

10mL of the blank, standard and sample were injected into the chromatographic system and areas for the Hydrochlorothiazide and Bisoprolol the peaks were used for calculating the % assay by using the formula.

 

Optimized chromatographic conditions:

By performing the trails the chromatographic conditions were optimized as follows

Column

:

“ACE C18 (4.6×150 mm) 5.0µm”

Mobile phase

:

 “Methnol: Pb (70: 30 % v/v)”

Wavelength

:

240 nm

Flow rate

:

1.2 ml/min

Injection volume

:

10µl

Column Temperature

:

 Ambient

Rt

:

2.449 & 3.191 mins

 

Chromatographic trial for simultaneous estimation of Hydrochlorothiazide and Bisoprolol by RP- HPLC

 

Fig.No.1 Optimized chromatographic conditions

RESULTS AND DISCUSSIONS:

Method Development:

The detection wavelength was hand-picked by”. “The ensuing resolution was scanned in U.V vary from 200-400nm”. “The overlay and Hydrochlorothiazide and Bisoprolol showed at 240 nm”.

      

 

Fig.No.2 The scan spectrums

 

 

Fig.No.3. Chromatogram showing blank preparation (mobile phase)

 

 

Fig.No.4. Chromatogram showing standard injection

 

 

Fig.No.5. Chromatogram showing sample injection.

 

The specificity check was performed for Hydrochlorothiazide and Bisoprolol. It had been found that there was no interference of impurities in retention time of analytical peak.

 

Assay calculation for Hydrochlorothiazide and Bisoprolol:

The assay study was performed for the Hydrochlorothiazide and Bisoprolol The chromatograms area unit shown in Fig. No.6 and results area unit tabulated in Table. No.1

 

 

Fig.No.6. Chromatogram showing assay of sample injection-1, 2

 

The specificity check was performed for Hydrochlorothiazide and Bisoprolol. It had been found that there was no interference of impurities in retention time of analytical peak.

 

Table.No.1. Showing assay results:

S. No

“Name of compound”

“Amount taken”

“%purity”

1

“Hydrochlorothiazide”

754.7

99.24

2

“Bisoprolol”

735.6

101.04

 

 

 

 

Linearity:

The one-dimensionality study was performed for the concentration of 50 ppm to 250 ppm for Hydrochlorothiazide” and 10ppm to 50ppm for Bisoprolol”. Every level was injected into activity system. The world of every level was used for calculation of correlation. The results area unit tabulated in Table. No.2-3. Standardization graph area unit shown in Fig.No.7- 8.

Table No.2 Linearity Results for Hydrochlorothiazide

S. No

Linearity Level

Concentration

Area

1

I

50 ppm

471543

2

II

100 ppm

656277

3

III

150 ppm

794999

4

IV

200 ppm

946124

5

V

250 ppm

1002139

Correlation Coefficient

0.999

 

 

 

 

Fig.No.7. Showing calibration graph for Hydrochlorothiazide

 

 

Fig.No.7. Showing calibration graph for Bisoprolol

 

 

Table No.3. Linearity Results for Bisoprolol

S. No

Linearity Level

Concentration

Area

1

I

10 ppm

220102

2

II

20 ppm

258503

3

III

30 ppm

299655

4

IV

40ppm

344622

5

V

50 ppm

389792

 

Correlation Coefficient

0.999

 

 

The linearity range of 50.µg-250µg and 10µg-50µg of Hydrochlorothiazide and Bisoprolol and the correlation coefficient was found to be 0.999 and 0.999. (NLT 0.999)”.

 

Accuracy:

The accuracy study was performed for five hundredth 100% and a hundred and fifty you take care of Hydrochlorothiazide and Bisoprolol.

 

Table.No.4. Showing accuracy results for Hydrochlorothiazide: 

%Concentration

(at specification level)

Average

area

Amount added

(mg)

Amount found

(mg)

%

Recovery

Mean recovery

50%

656659

5

4.96

99.91%

99.56%

100%

1304258

10

   9.98

99.18%

150%

1854608

15

15.02

99.60%

 

Table.No.5. Showing accuracy results for Bisoprolol:

%Concentration

(at specification level)

Average

area

Amount added

(mg)

Amount found

(mg)

% Recovery

Mean recovery

50%

65312

0.5

0.99

99.53%

99.47%

100%

124509

1.0

1.05

99.38%

150%

178517

1.5

1.495

99.52%

 

 

For teenagers recovery of Hydrochlorothiazide and Bisoprolol. The be 99.56% and 99.47% severally (NLT ninety eight and NMT 102%).

 

Precision:

Repeatability:

The standard resolution was injected for 5 times and measured the world for all 5 injections in HPLC”. The % RSD is among the desired limits”. The % RSD and results area unit tabulated in Table.6-7.

 

Intermediate precision:

The intermediate exactitude study was performed for 5 injections of Hydrochlorothiazide and Bisoprolol”. The result area unit tabulated in table 7-8.  

 

 

 

Table.No.6. Showing% RSD results for Hydrochlorothiazide Peak Name: Hydroclorothaizide

 

Peak Name

RT

Area (µV*Sec)

Height (µV)

1

Hctz

2.343

1302729

248455

2

Hctz

2.344

1302729

248699

3

Hctz

2.344

1302947

249526

4

Hctz

2.345

1303977

246695

5

Hctz

2.345

1303236

250012

Mean

 

 

1304529.8

 

Std. Dev.

 

 

2361.1

 

% RSD

 

 

0.2

 

 

Table.No.7. Showing %RSD results for Bisoprolol

Peak Name: Bisoprolol

 

Peak Name

RT

Area (µV*Sec)

Height (µV)

1

Bisoprolol

3.285

124263

19458

2

Bisoprolol

3.287

124487

19634

3

Bisoprolol

3.287

124175

19600

4

Bisoprolol

3.288

124894

19327

5

Bisoprolol

3.288

124495

19540

Mean

 

 

124462.7

 

Std. Dev.

 

 

278.6

 

% RSD

 

 

0.2

 

 

 

 

Table.No.7. Showing results for intermediate precision of Hydrochlorothiazide

Peak Name: Hydroclorothaizide

 

Peak Name

RT

Area (µV*Sec)

Height (µV)

1

Hctz

2.342

1305937

247870

2

Hctz

2.343

1306476

246764

3

Hctz

2.344

1304520

245696

4

Hctz

2.344

1300148

247140

5

Hctz

2.345

1308271

247280

Mean

 

 

1305070.2

 

Std. Dev.

 

 

3061.8

 

% RSD

 

 

0.2

 

 

 

 

Table.No.8. Showing results for intermediate precision of Bisoprolol

Peak Name: Bisoprolol

 

Peak Name

RT

Area (µV*Sec)

Height (µV)

1

Bisoprolol

3.278

122962

19165

2

Bisoprolol

3.281

122487

19115

3

Bisoprolol

3.281

122632

19073

4

Bisoprolol

3.281

122626

19003

5

Bisoprolol

3.283

122702

19123

Mean

 

 

122681.8

 

Std. Dev.

 

 

174.8

 

% RSD

 

 

0.1

 

 

The intermediate precision was performed for % RSD of Hydrochlorothiazide and Bisoprolol was found to be 0.2 and 0.1. From the system precisions studies it is observed that all parameters like %RSD of retention time and peak areas are within limits.

 

Detection limit:

LOD’s will be calculated supported the quality deviation of the response (SD) and also the slope of the standardization curve (S) at levels approximating the LOD consistent with the formula. The quality deviation of the response will be determined supported the quality deviation of y-intercepts of regression lines.

 

Formula

LOD = 3.3 X σ/S

Where

σ - Standard deviation (SD)

S – Slope

 

 

Fig.No.8. Showing Results of LOD

 

The LOD was performed for Hydrochlorothiazide and Bisoprolol was found to be 3.17and 0.0172 respectively.

 
Quantitation limit:

LOQ’s may be calculated supported the quality deviation of the response (SD) and also the slope of the activity curve (S) in line with the formula. Again, the quality deviation of the response may be determined supported the quality deviation of y-intercepts of regression lines.

 

Formula

LOQ = 10 X σ/S

Whereσ - Standard deviation

S - Slope

 

 

Fig.No.9. Showing Results of LOQ

The LOQ was performed for Hydrochlorothiazide and Bisoprolol was found to be 5.80 and 0.212 severally.

 

SUMMARY AND CONCLUSION:

The developed method was successfully applied for simultaneous estimation of Hydrochlorothiazide and Bisoprolol in compound tablet formulation. The proposed method was found to be simple, accurate and precise. There is no analytical method available to determine the same combination of drugs. Therefore, this method may be useful for routine analysis of Hydrochlorothiazide and Bisoprolol in API drugs and pharmaceutical dosage forms.

 

ACKNOWLEDGEMENT:

I express my sincere thanks to Dr. Suthakaran and Management of Vijaya College of Pharmacy for encouragement throughout my research work. And also thankful to Awamedica Company providing the Reserpine and Hydrochlorothiazide pure drugs.

 

REFERENCE:

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7.      Arjun G, Sathis KD, Bindu MB, Naga MM, Ramalingam R, Nath AR. Asimple HPLC Method for Quantitation of Bisoprolol Fumarate in Tablet Dosage Form. Indian Drugs, 2009; 46:39-42. 7.

8.      Yadav SS, Rao JR. Simultaneous HPTLC analysis of Bisoprolol Fumarate and Hydrochlorothiazide in Pharmaceutical Dosage Form. Int J Pharm Sci, 2013; 5:286-90.

9.      International Conference on Harmonization. Draft Guideline on Validation of Analytical Procedures: Definitions and Terminology, Federal Register. 60, 1995, 11260.

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Received on 09.01.2019       Modified on 22.01.2020

Accepted on 18.02.2020      ©Asian Pharma Press All Right Reserved

Asian J. Pharm. Ana. 2020; 10(1):01-06.

DOI: 10.5958/2231-5675.2020.00001.0