RP-HPLC Method Development and Validation for Simultaneous Estimation of Prazosin and Polythiazide in Bulk and Pharmaceutical Dosage Form

 

Uttam Prasad Panigrahy*, K. Naga Vishnu Kumari, T. Ram Mohan Reddy, K. Abbulu

Department of Pharmaceutical Analysis and Quality Assurance, CMR College of Pharmacy,

Kandlakoya, Medchal, Hyderabad-501401, Telangana, India

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

 

ABSTRACT:

A new isocratic RP-HPLC method was developed and validated for simultaneous estimation of Prazosin and Polythiazide in bulk and pharmaceutical dosage form with stability studies as per ICH guidelines. In this method Symmetry C18 column (150mm4.6mm, 5mm particle size), Waters Alliance e2695 HPLC system with PDA detector and the mobile phase contained a mixture of 0.01M Potassium dihydrogen orthophosphate buffer (pH adjusted to 3.48 with orthophosphoric acid) and Acetonitrile (50:50, v/v) was used. The flow rate was set to 1mL/min with the responses measured at 265nm. The retention time of Prazosin and Polythiazide was found to be 2.989min and 2.134min respectively with resolution of 5.1. Linearity was established for Prazosin is 25-150g/mL and for Polythiazide is 6.25-37.5g/mL with correlation coefficients (r2=0.999). The percentage recoveries for Prazosin are 100.34% and Polythiazide is 100.32% respectively. Prazosin and Polythiazide are more sensitive towards acidic, basic and oxidative degradation condition. The developed method was successfully applied for the quantification of Prazosin and Polythiazide in bulk and pharmaceutical dosage form.

 

KEYWORDS: Prazosin, Polythiazide, RP-HPLC, ICH.

 

 

 

INTRODUCTION:

Prazosin is a selective α-1-adrenergic receptor antagonist used to treat hypertension. It has also been used to decrease urinary obstruction and relieve symptoms associated with symptomatic benign prostatic hyperplasia1. Prazosin is chemically known as [4-(4-Amino-6, 7-dimethoxyquinazolin-2-yl) octahydroquinoxalin-1(2H)-yl] (furan-2-yl) methanone were shown in (Figure 1).

 

 

Polythiazide is a diuretic which inhibits active chloride re-absorption at the early distal tubule via the thiazide-sensitive Na-Cl co-transporter (TSC), resulting in an increase in the excretion of sodium, chloride, and water. It also inhibits sodium ion transport across the renal tubular epithelium through binding to the thiazide sensitive sodium-chloride transporter. This results in an increase in potassium excretion via the sodium-potassium exchange mechanism. The antihypertensive mechanism of polythiazide may be mediated through its action on carbonic anhydrases in the smooth muscle or through its action on the large-conductance calcium-activated potassium (KCa) channel, also found in the smooth muscle2. Polythiazide is chemically known as 6-chloro-2-methyl-1, 1-dioxo-3-(2, 2, 2-trifluoroethylsulfanylmethyl)-3, 4-dihydro-1λ6, 2, 4-benzothiadiazine-7-sulfonamide was shown in (Figure 2).

 

Prazosin and Polythiazide is a fixed dose combination drug for treatment of hypertension. Literature review reveals that very few analytical methods has been reported for the determination of Prazosin and Polythiazide individually and with other combinations which includes high performance liquid chromatography (HPLC)3-19, UV-Visible-Spectrophotometric20-23 and LC-MS24,25. The present study was intended to develop a new validated method for the simultaneous estimation of Prazosin and Polythiazide with forced degradation studies as per ICH guidelines26.

 

MATERIALS AND METHODS:

Chemicals and reagents:

Prazosin (API) and Polythiazide (API) was obtained from Synthokem Labs Private Ltd., Hyderabad, India. HPLC grade of Potassium dihydrogen orthophosphate was obtained from Rankem Ltd., India and HPLC grade of Acetonitrile was obtained from Merck Specialities Private Limited, India. HPLC grade of Water and Ortho phosphoric acid was obtained from Rankem Ltd., India. Minizide 432 capsule contains Prazosin 2mg and Polythiazide 0.5 mg were kindly supplied by Pfizer Labs, Inc.

 

Instrumentation:

The analysis was performed by using a chromatographic system from Waters Alliance e2695 HPLC system with 2998 PDA detector. The HPLC system was equipped with Empower 2 software. Denver electronic balances, Ultrasonic bath sonicator (BVK enterprises, India), Digital pH meter (BVK enterprises, India) and Whatmann filter paper No. 41 (Whatmann International Ltd., England) were used in the study.

 

Chromatographic conditions:

Prazosin and Polythiazide was analysed in Symmetry C18 column (150mm4.6mm, 5mm particle size) column for the chromatographic separation. The mobile phase was composed of of 0.01M Potassium dihydrogen orthophosphate buffer (pH adjusted to 3.48 with orthophosphoric acid) and Acetonitrile (50:50, %v/v). Filtered through 0.45m nylon membrane filter under vacuum filtration and pumped at ambient temperature, at a flow rate of 1 mL/min with PDA detection wavelength at 265nm. Injection volume was 10μL. The run time was 6 min and the retention time of Prazosin and Polythiazide was found to be 2.989min and 2.134min respectively with resolution of 5.1.

 

Chromatographic Parameters:

Equipment

:

Waters Alliance e2695 HPLC system with 2998 PDA detector

Column

:

Symmetry C18 column (150mm4.6mm, 5mm particle size)

Flow rate

:

1mL/min

Wavelength

:

265nm

Injection volume

:

10 mL

Column oven

:

Ambient

Run time

:

6 Minutes

Solutions and sample preparation:

Preparation of Ammonium acetate buffer:

A 0.01M Potassium dihydrogen orthophosphate buffer was prepared by dissolving 1.36 gm of Potassium dihydrogen orthophosphate in 1000mL of HPLC grade water and pH was adjusted to 3.48 with orthophosphoric acid. The buffer was filtered through 0.45μm nylon membrane filter to remove all fine particles and gases.

 

Preparation of mobile phase:

The above prepared Potassium dihydrogen orthophosphate buffer and Acetonitrile HPLC grade were mixed in the proportion of 50:50, %v/v and was filtered through 0.45μm nylon membrane filter and degassed by sonication.

 

Preparation of diluent:

Mobile phase was used as diluent.

 

Preparation of standard stock solutions of Prazosin and Polythiazide:

Standard stock solutions of Prazosin and Polythiazide were prepared by dissolving 10mg of Prazosin and 2.5mg of Polythiazide in 10mL of diluent into a 10mL clean dry volumetric flask and the standard solutions was filtered through 0.45 μm nylon membrane filter and degassed by sonicator to get the concentration of 1000g/mL of Prazosin and 250g/mL of Polythiazide.

 

Preparation of standard solutions of Prazosin and Polythiazide for assay:

From the above standard stock solution of 1000g/mL of Prazosin and 250g/mL of Polythiazide further pipette 1mL and transferred into a 10mL volumetric flask and dilute up to the mark with diluent to get the concentration of 100g/mL of Prazosin and 25g/mL of Polythiazide.

 

Preparation of sample solutions of Prazosin and Polythiazide:

Twenty capsules were accurately weighed and capsule powder equivalent to 2mg of Prazosin and 0.5mg of Polythiazide were taken into 10mL clean dry volumetric flask, diluent was added and sonicated to dissolve it completely and volume was made up to the mark with the same diluent and filtered through 0.45 μm nylon membrane filter. Further pipette out 5mL from the above Prazosin and Polythiazide sample stock solution into a 10mL volumetric flask and diluted up to the mark with diluent to get the concentration of 100g/mL of Prazosin and 25g/mL of Polythiazide. 10mL from standard and sample solution were injected into the chromatographic system and the peak areas were measured for Prazosin and Polythiazide which was shown in (Figure 3 and 4) and the % assay was calculated by comparing the peak area of standard and sample chromatogram by using the formula given below and the assay results was shown in (Table 1).

 

                    AT       WS       DT       P           Avg. Wt

Assay % = x x x x X 100

                    AS        DS       WT     100     Label Claim

 

 

 

Where:

AT = Average peak area of sample preparation

AS= Average peak area of standard preparation

WS = Weight of standard taken in mg

WT=Weight of sample taken in mg

P = Percentage purity of working standard

DS= Dilution factor for standard preparation

DT=Dilution factor for sample preparation

 

 

Table 1: Assay of marketed formulation of Prazosin and Polythiazide

Drug

Minizide 432 capsule Label Claim (mg)

Amount Found (mg) (n=6)

% Label Claim % RSD (n=6)

Prazosin

2

2.006

100.28 0.9

Polythiazide

0.5

0.505

100.81 1.0

 

 

 

RESULTS AND DISCUSSION:

Method Development:

To optimize the RP-HPLC parameters, several mobile phase compositions were tried. A satisfactory separation and good peak symmetry for Prazosin and Polythiazide were obtained with a mobile phase containing a mixture of 0.01M Potassium dihydrogen orthophosphate buffer (pH adjusted to 3.48 with orthophosphoric acid) and Acetonitrile (50:50, %v/v) was delivered at a flow rate of 1mL/min to get better reproducibility and repeatability. Quantification was achieved with PDA detection at 265nm based on peak area. The retention time of Prazosin and Polythiazide was found to be 2.989min and 2.134min respectively with resolution of 5.1. Linearity was established for Prazosin and Polythiazide in the range of 25-150g/mL for Prazosin and 6.25-37.5g/mL for Polythiazide with correlation coefficients (r2=0.999) and the percentage recoveries for Prazosin are 100.34% and Polythiazide is 100.32% respectively, which indicate accuracy of the proposed method. The % RSD values of accuracy for Prazosin and Polythiazide were found to be < 2 %. The % RSD values of method precision are 0.9% and 1% for Prazosin and Polythiazide respectively and % RSD values of system precision are 1% and 0.9% for Prazosin and Polythiazide respectively. The % RSD values of intermediate precision are 0.8% and 1.4% for Prazosin and Polythiazide respectively, reveal that the proposed method is precise. LOD values for Prazosin and Polythiazide were found to be 0.491g/mL and 0.03g/mL respectively and LOQ values for Prazosin and Polythiazide were found to be 1.487g/mL and 0.1g/mL respectively. The % RSD values of robustness studies were found to be < 2% reveal that the method is robust enough. These data show that the proposed method is specific and sensitive for the determination of Prazosin and Polythiazide.

 

Method validation:

The developed method for the simultaneous estimation of Prazosin and Polythiazide was validated as per the ICH guidelines for the parameters like system suitability, specificity, linearity, accuracy, precision, ruggedness, robustness, limit of detection (LOD) and limit of quantitation (LOQ) 26.

 

System suitability test:

At first the HPLC system was optimized as per the chromatographic conditions. One blank followed by six replicates of a single calibration standard solution of 100g/mL of Prazosin and 25g/mL of Polythiazide was injected to check the system suitability. To ascertain the system suitability for the proposed method, the parameters such as retention time, theoretical plates, peak asymmetry and resolution were taken and results were presented in (Table 2).

 

Table 2: System suitability parameters for Prazosin and Polythiazide

Parameter (n=6)

Prazosin

Polythiazide

Retention Time (Mins)

2.989

2.134

Theoretical plates

8536

4920

Tailing factor

1.34

1.09

Resolution

 

5.1

 

Specificity:

The effect of excipients and other additives usually present in the combined capsule dosage form of Prazosin and Polythiazide in the determination under optimum conditions was investigated. The specificity of the RP-HPLC method was established by injecting the blank and placebo solution into the HPLC system. The representative chromatogram of blank and placebo was shown in (Figure 5 and 6).

 

Linearity and range for Prazosin and Polythiazide:

Aliquots of 0.25, 0.5, 0.75, 1, 1.25 and 1.5mL of mixed standard working solutions of Prazosin and Polythiazide was pipetted out from the standard stock solution of 1000g/mL of Prazosin and 250g/mL of Polythiazide and transferred into a series of 10mL clean dry volumetric flask and make volume up to the mark with the same diluent to get the concentration of 25, 50, 75, 100, 125 and 150g/mL of Prazosin and 6.25, 12.5, 18.75, 25, 31.25 and 37.5g/mL of Polythiazide.

 

The calibration standard solutions of Prazosin and Polythiazide were injected using a 10μL Hamilton Rheodyne injector and the chromatograms were recorded at 265nm and a calibration graph was obtained by plotting peak area versus concentration of Prazosin and Polythiazide respectively.

 

Table 3: Linearity for Prazosin and Polythiazide

Linearity of Prazosin

Linearity of Polythiazide

Concentration (g/mL)

Peak Area

Concentration (g/mL)

Peak Area

25

558377

6.25

37175

50

1092727

12.5

76265

75

1637102

18.75

118682

100

2174094

25

155120

125

2663563

31.25

195593

150

3222041

37.5

227639

 

The linearity data is presented in (Figure 7 and 8) and (Table 3). Acceptance Criteria: Correlation coefficient should be not less than 0.999.

 

Accuracy studies for Prazosin and Polythiazide:

The accuracy of the method was determined by calculating recovery of Prazosin and Polythiazide by the method of standard addition. Known amount of standard solution of Prazosin and Polythiazide at 50%, 100% and 150% was added to a pre quantified sample solution and injected into the HPLC system. The mean percentage recovery of Prazosin and Polythiazide at each level was calculated and the results were presented in (Table 4 and 5). Acceptance Criteria: The % Recovery for each level should be between 98.0 to 102.0%.

 

Table 4: Accuracy for Prazosin

% Level

Amount Spiked (μg/mL)

Amount recovered (μg/mL)

% Recovery

Mean % Recovery

50%

50

49.92

99.84

100.34%

50

50.40

100.79

50

49.74

99.47

100%

100

101.59

101.59

100

101.03

101.03

100

99.58

99.58

150%

150

150.01

100.01

150

151.53

101.02

150

149.56

99.71

 

Table 5: Accuracy for Polythiazide

% Level

Amount Spiked (μg/mL)

Amount recovered

(μg/mL)

% Recovery

Mean

% Recovery

50%

12.5

12.56

100.51

100.32%

12.5

12.41

99.27

12.5

12.37

98.93

100%

25

25.35

101.40

25

25.30

101.21

25

25.31

101.23

150%

37.5

37.81

100.81

37.5

37.56

100.17

37.5

37.27

99.37

 

 

Table 6: Method precision for Prazosin and Polythiazide

S. No

Peak Area of Polythiazide

Peak Area of  Prazosin

1

154264

2170407

2

155055

2192630

3

156271

2149939

4

156341

2179888

5

158783

2201761

6

157427

2156488

Mean

156357

2175186

Std. Dev.

1619.5

20211.4

%RSD

1.0

0.9

 

Precision studies for Prazosin and Polythiazide:

Method precision (Repeatability):

From the above standard stock solution of 1000g/mL of Prazosin and 250g/mL of Polythiazide further pipette 1mL and transferred into a 10mL volumetric flask and dilute up to the mark with diluent to get the concentration of 100g/mL of Prazosin and 25g/mL of Polythiazide was injected and analysed six times and was checked whether the method is giving consistent results. The % RSD for the assay of six replicate injections was calculated as mentioned in (Table 6). Acceptance Criteria: The % RSD for the assay of six sample injections should not be more than 2%.

 

Table 7: System precision for Prazosin and Polythiazide

S. No

Peak Area of Polythiazide

 Peak Area of  Prazosin

1.

154043

2158865

2.

153626

2203077

3.

156549

2141344

4.

153705

2172404

5.

156605

2168016

6.

155116

2158118

Mean

154941

2166971

S.D

1374.7

20664.4

%RSD

0.9

1.0

 

System precision:

The system precision was carried out to ensure that the analytical system is working properly. The standard preparation concentration of 100g/mL of Prazosin and 25g/mL of Polythiazide was injected six times into the HPLC system and the %RSD for the area of six replicate injections was calculated as mentioned in (Table 7). Acceptance Criteria: The %RSD for the peak area of six standard injections should not be more than 2%.

 

Intermediate precision/ruggedness:

The intermediate precision (also known as Ruggedness) of the method was evaluated by performing precision on different laboratories by different analysts and different days. The sample preparation concentration of 100g/mL of Prazosin and 25g/mL of Polythiazide was injected six times into the HPLC system and the %RSD for the assay of six replicate injections was calculated as mentioned in (Table 8). Acceptance Criteria: The % RSD for the assay of six sample injections should not be more than 2%.

 

Table 8: Intermediate precision for Prazosin and Polythiazide

S. No

Peak Area of Polythiazide

 Peak Area of  Prazosin

1.

144700

1992746

2.

147333

2026970

3.

145375

2011318

4.

149619

1994322

5.

144200

2031773

6.

145937

2000397

Mean

146194

2009588

S.D

1998.6

16724.1

%RSD

1.4

0.8

 

Limit of Detection (LOD) and Limit of Quantification (LOQ):

Limit of Detection (LOD) and Limit of Quantification (LOQ) were calculated as 3.3SD/S and 10SD/S respectively as per ICH guidelines, Where SD is the standard deviation of the response (Y-intercept) and S is the slope of the calibration curve. The LOD is the smallest concentration of the analyte that gives a measurable response (signal to noise ratio of 3). The LOD of Prazosin and Polythiazide was calculated and shown in (Table 9). The LOQ is the smallest concentration of the analyte which gives response that can be accurately quantified (signal to noise ratio of 10). The LOQ of Prazosin and Polythiazide was calculated and shown in (Table 9).

 

Table 9: LOD and LOQ for Prazosin and Polythiazide

Drug

LOD(μg/mL)

LOQ(μg/mL)

Polythiazide

0.03

0.1

Prazosin

0.491

1.487

 

Robustness:

As part of the Robustness, deliberate change in the flow rate, mobile phase proportion and temperature of 10% was made to evaluate the impact on the method. The results reveal that the method is robust. The results are summarized in (Table 10).

 

Table 10: Robustness for Prazosin and Polythiazide

S.

No

Condition

% RSD of Polythiazide

%RSD of Prazosin

1.

Flow rate (-) 0.9mL/min

1.2

0.8

2.

Flow rate (+) 1.1mL/min

0.9

1.3

3.

Mobile phase (-) 55B:45A

0.8

0.6

4.

Mobile phase (+) 45B:55A

0.7

0.8

5.

Temperature (-) 25C

0.7

0.8

6.

Temperature (+) 35C

1.1

0.4

 

Stability of solution:

The results of the solution stability experiments confirm that the sample solutions and mobile phase used during the assays were stable upto 24hours at room temperature was calculated and shown in (Table 11).

 

 

Table 11: Solution stability for Prazosin and Polythiazide

 

 

Solution stability for Polythiazide

S. No.

Time in hours

Concentration(μg/mL)

Retention time (min)

Peak Area

USP Plate Count

Asymmetry

1.

0

25

2.197

224740

2233

0.9

2.

24

25

2.124

144700

4550

1.24

Solution stability for Prazosin

S. No.

Time in hours

Concentration (μg/mL)

Retention time (min)

Peak Area

USP Plate Count

Asymmetry

1.

0

100

2.988

1775994

8732

1.3

2.

24

100

2.991

1992746

8268

1.35

 

 

 

Forced degradation studies:

Acid Degradation Studies:

To 1mL of stock solution of Prazosin and Polythiazide, 1mL of 2N Hydrochloric acid was added and refluxed for 30mins at 600C. The resultant solution was diluted to obtain 100g/mL of Prazosin and 25g/mL of Polythiazide solution and 10L solutions were injected into the HPLC system and the chromatogram were recorded to assess the stability of sample was shown in (Figure 9).

 

Alkali Degradation Studies:

To 1mL of stock solution of Prazosin and Polythiazide, 1 mL of 2N sodium hydroxide was added and refluxed for 30mins at 600C.The resultant solution was diluted to obtain 100g/mL of Prazosin and 25g/mL of Polythiazide solution and 10L solutions were injected into the HPLC system and the chromatogram were recorded to assess the stability of sample was shown in (Figure 10).

 

Oxidative degradation Studies:

To 1mL of stock solution of Prazosin and Polythiazide, 1 mL of 20% Hydrogen peroxide (H2O2) was added and the solution was kept for 30mins at 60C. For HPLC study, the resultant solution was diluted to obtain 100g/mL of Prazosin and 25g/mL of Polythiazide solution and 10L solutions were injected into the HPLC system and the chromatogram were recorded to assess the stability of sample was shown in (Figure 11).

Thermal Degradation Studies:

The standard drug solution was placed in oven at 1050C for 6hrs to study dry heat degradation. For HPLC study, the resultant solution was diluted to 100g/mL of Prazosin and 25g/mL of Polythiazide solution and 10L solutions were injected into the HPLC system and the chromatogram were recorded to assess the stability of sample was shown in (Figure 12).

 

Photolytic degradation studies:

The photochemical stability of the drug was also studied by exposing the drug solution to UV light by keeping the beaker in UV Chamber for 7days or 200 Watt hours/m2 in photo stability chamber. For HPLC study, the resultant solution was diluted to obtain 100g/mL of Prazosin and 25g/mL of Polythiazide solution and 10L solutions were injected into the HPLC system and the chromatogram were recorded to assess the stability of sample was shown in (Figure 13).

 

Water Degradation Studies:

Stress testing under neutral conditions was studied by refluxing the drug in water for 6hrs at a temperature of 60C. For HPLC study, the resultant solution was diluted to 100g/mL of Prazosin and 25g/mL of Polythiazide solution and 10L solutions were injected into the HPLC system and the chromatogram were recorded to assess the stability of sample was shown in (Figure 14).

 

 

Table 12: Forced degradation data of Prazosin and Polythiazide in different degradation conditions

Type of degradation

Polythiazide

Prazosin

Peak Area

%Recovered

% Degraded

Peak Area

%Recovered

% Degraded

Acid

143068

92.24

7.76

2065482

95.22

4.78

Base

148136

95.51

4.49

2103021

96.95

3.05

Oxidative

150073

96.76

3.24

2115736

97.54

2.46

Thermal

150813

97.24

2.76

2117903

97.64

2.36

Photolytic

152101

98.07

1.93

2139560

98.64

1.36

Water

154402

99.55

0.45

2152613

99.24

0.76

 

 

CONCLUSION:

RP-HPLC method for the simultaneous estimation of Prazosin and Polythiazide in their combine dosage form was established and validated as per the ICH guidelines. Linearity was achieved for Prazosin and Polythiazide in the range of 25-150g/mL for Prazosin and 6.25-37.5g/mL for Polythiazide with correlation coefficients (r2=0.999). The percentage recoveries of Prazosin and Polythiazide were achieved in the range of 98-102% which was within the acceptance criteria. The %RSD was NMT 2% which proved the precision of the developed method. The developed method is simple, sensitive, rapid, linear, precise, rugged, accurate, specific, and robust. The forced degradation studies were performed by using HCl, NaOH, H2O2, thermal, UV radiation and water. Prazosin and Polythiazide are more sensitive towards acidic, basic and oxidative degradation condition which was shown in (Table 12). No interference from any components of pharmaceutical dosage form or degradation products was observed and the method has been successfully used to perform long term and accelerated stability studies of Prazosin and Polythiazide formulations. Hence it can be used for the routine analysis of Prazosin and Polythiazide in their bulk and combine dosage form.

 

ACKNOWLEDGEMENT:

The authors are thankful to CMR College of Pharmacy, Kandlakoya, Medchal, Hyderabad, Telangana, India for providing the chemicals and instruments and Synthokem Labs Private Ltd., Hyderabad, India for providing the drug samples for research.

 

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Received on 05.08.2019         Modified on 28.09.2019

Accepted on 19.11.2019         RJPT All right reserved

Research J. Pharm. and Tech. 2020; 13(4):1779-1787.

DOI: 10.5958/0974-360X.2020.00321.2