Development and Validation of UV Spectrophotometric Method for Simultaneous Estimation of Hydrochlorothiazide and Lisinopril in Bulk Drug and Its Formulations

 

Pramod Kumar1* Babulal Saini1, Ravi Dahiya1, Jawed Akhtar2, Birendra Shrivastav1

1Department of Quality Assurance, School of Pharmaceutical Sciences, Jaipur National University, Jagatpura, Jaipur (Rajasthan) India-302025

2Department of Quality Assurance, Parul Institute of Pharmacy & Research, Baroda

 *Corresponding Author E-mail: bijarniyasadtm123@gmail.com, pharmacy_pramod@yahoo.com

 

 

ABSTRACT:

A simple, efficient, precise and accurate Vierodt’s method have been developed for the estimation of Hydrochlorothiazide and Lisinopril in pure and in fixed dose Combination. In this method UV spectrum of Hydrochlorothiazide and Lisinopril were overlained which involves the formation of Simultaneous equation at maximum wavelength 274.5 nm and 215 nm. Both the drugs obeyed Beer’s law in the concentration range 5-20 μg/ml and 1-10 μg/ml for Hydrochlorothiazide and Lisinopril, respectively. The accuracy of the method was determined by recovery studies and was found to be 100.04±0.638% and 99.13±0.404% for Hydrochlorothiazide and Lisinopril, respectively in Synthetic Mixture. The method was validated as per ICH guidelines and statically. The method showed good reproducibility and recovery with % RSD less than 2. The method was found to be simple, economic, accurate and reproducible and can be used for routine analysis of Hydrochlorothiazide and Lisinopril in pure and in fixed dose combination.

 

KEYWORDS: Simultaneous equation method, Vierodt’s method, Hydrochlorothiazide, Lisinopril, ICH guidelines, method validation

 


INTRODUCTION:

Hydrochlorothiazide (HCTZ) is diuretic and antihypertensive, which inhibits the reabsorption of sodium and calcium at the beginning of distal convoluted. It is the 3,4 dihydroderivetive of chlorothiazide. It is chemically 6-chloro-3,4-dihydro-2H-1, 2, 4- benzothiadi- azine-7-sulphonamide-1,1-dioxide.(Fig.2) The typical dose of HCTZ is 12.5 mg per day.HCT is official in IP, BP and USP. (1-3,12,18-19) Lisinopril is a lysine analog of the nonsulphydryl angiotensin-converting enzyme (ACE) inhibitor.  It is used for the treatment of hypertension, congestive heart failure and cataract. Lisinopril is chemically 1-[6-amino-2- (1-carboxy-3-phenyl-propyl) amino-hexanoyl] pyrrolidine- 2-carboxylic acid dehydrate (Fig.1) which is official in IP, BP & USP. . (1-3, 13, 16-17) Pharmaceutical analysis of such a combination is a challenge because of their extremely different physicochemical properties.

 

 

Methods proposed for analytical purposes must be as economical as possible, to enable their use in routine quality control.

 

Fig.1: Chemical Structures of Lisinopril Anhydrous   (LSP) (13)

 

 

Fig.2: Chemical Structures of Hydrochlorothiazide (HCTZ) (12)

 

 


Fig.3: Overlain Spectrum of HCTZ and LSP in 0.01M Sodium Hydroxide

 

 


Fig.3: Calibration Curve of Mixture of Lisinopril and Hydrochlorothiazide in 0.01 M Sodium Hydroxide at 212 nm and 274 nm respectively(Showing Linearity)

 

One to six milliliters of standard stock solution of Lisinopril and 0.5-5.0  ml standard stock solution of HCT were transferred into a series of six  100 ml volumetric flasks separately and made up to mark with 0.01 m sodium hydroxide. The absorbance of different concentration solutions was measured 274 nm and 212 nm against 0.01 M sodium hydroxide as blank. The calibration curve was plotted using concentration against absorbance the solutions were found to be linear with the concentration range of 1-10 μg/ml of Lisinopril and 5-20 μg/ml of HCTZ. The procedure was repeated for six times.

Both The Drug Containing 10 μg/ml; I is Lisinopril (Black); II is Hydrochlorothiazide (Blue); IV is Isobestic Point (220 nm)

Different Synthetic mixtures of the two drugs were prepared by transferring different volumes of Lisinopril and HCTZ from working standard solutions into 100 ml volumetric flasks and diluting to volume with 0.01 M sodium hydroxide. The concentrating of both Lisinopril and HCTZ were determined by means the absorbance of the prepared mixtures at 274 nm and 212 nm by using Simultaneous equation method.

 

Accurately weighed portion of Lisinopril (10 mg) and Hydrochlorothiazide  (10 mg) was transferred to a separate 100 ml volumetric flask and dissolved and diluted to the mark with 0.01 M Sodium Hydroxide to obtain standard solution having concentrations of  Lisinopril (100 µg/ml)  and  Hydrochlorothiazide (100 µg/ml). A quantity of powder equivalent to 20 mg Lisinopril and 12.5 mg Hydrochlorothiazide (transferred to 100 ml volumetric flask. 0.01 M Sodium Hydroxide (50 ml) was added to it and sonicated for 20 min. The volume was adjusted up to the mark with 0.01 M Sodium Hydroxide after filtration of sonicated solution. The absorbance was measured at their selected wavelengths and the concentrations of two drugs were estimated by using Simultaneous Equation method as follows.

 


 

Table 1: LINEAR REGRESSION DATA FOR CALIBRATION CURVES

Parameter

Hydrochlorothiazide

Lisinopril

273.4 nm

212nm

273.4nm

212nm

Beer’s Law Limit (µg/ml)

5-20

2-80

1-45

1-10

Molar Absorptivity (Lmol-1 cm-1)

17.398

4.98

6.678

40.106

Sandell’s Sensitivityµg/cm2 / .001 A.U.

17.126

81.434

44.598

10.104

Correlation Coefficient (r)

0.999

0.999

0.996

0.999

Regression Equation (Y=mx+c)

y= 0.022x - 0.000

y = 0.012x - 0.000

y = 0.022x - 0.000

y = 0.099x - 0.002

 


Simultaneous Determination (Vierodt’s Method):-Sometimes we come across a situation where in the analyte contains two species which have overlapping spectra. In order to determine these species we need to find two wavelengths where molar absorptivities of two species is different. In such a case, measurements are made on the solution of the analyte at two different wavelengths. This gives a set of simultaneous equations which could be solved for the concentrations of the individual constituents. For best results it is desirable to select two such wavelengths where the ratio of molar absorptivities is largest the absorbance’s of the mixed standard solutions were measured at the selected wavelengths. A set of two simultaneous equations were used for obtaining the concentrations of X and Y are as follows;

 

Where, A1 and A2 are absorbance of mixture at X nm and Y nm respectively, ax1 and ax2 are absorptivities of X  at λ1and λ2 respectively and ay1 and ay2 are absorptivities of Y  at λ1 and λ2 respectively. Cx and Cy are concentrations of X and Y respectively. The concentration of NF and TZ in mixed standard and tablets formulation can be obtained by solving equation (i) and (ii).(20)

 

The accuracy of the proposed method was confirmed by recovery studies. To the pre analyzed formulation, a known amount of raw material was added and it can be analyzed by the proposed method.

 

To an accurately weighed quantity of the Synthetic Mixture powder equivalent to 20 mg of Lisinopril 8, 10, 12 and 5, 6.25 and 7.50 mg of HCTZ raw materials were added into a series of 100 ml volumetric flasks. The procedure was repeated as per the analysis of formulation. The amount of each drug recovered was calculated. The procedure was repeated for three times for each concentration. The accuracy of the method was determined by recovery studies and was found to be 100.04±0.638% and 99.13±0.404% for Hydrochlorothiazide and Lisinopril, respectively in Synthetic Mixture is shown in Table 2 and 3.

 

In quantitative estimation of two components of Lisinopril and HCT by Simultaneous method absorbance were measured at the selected wavelength maximum of drugs. From the overlain spectra of Lisinopril and HCTZ absorbance were measured at selected wavelengths i.e., 220 nm (Isobestic point) and at 274 nm, the max of HCT. The absorptivities coefficients of each drug at both wavelengths were determined. Both LSP and HCTZ Obeyed Beers law in the concentration range of 1-10 µg/ml and 5-20 µg/ml respectively. The optical characteristics such as correlation coefficient, slope, intercept, Molar absorptivities and Sandell’s sensitivity were calculated and are shown Table 1. The Correlation Coefficient for LSP found to be 0.999 and 0.996 at 212 nm and 274nm and for HCTZ it was found to be 0.999 and .0999 at 274 nm and 212 nm. This indicates that the drugs were linear with the selected concentration range.

 

To study the mutual interference, if any, in the simultaneous estimation of Lisinopril and HCTZ, synthetic mixtures containing various proportions of Lisinopril and HCTZ, synthetic mixtures containing various proportions of Lisinopril and HCTZ were prepared and the contents were estimated by the proposed method. The percentage recovery varied from 98.67% to 99.39 % for Lisinopril and 99.49% to 100.74% for HCTZ indicating that no mutual interference in different ratios for both the drugs is shown in Table 3.

 

 

Table 2: Summary of validation and SST (System Suitability Test) Parameters

Parameter

Hydrochlorothiazide

Lisinopril

LOD(µg/ml)

0.0527

0.2215

LOQ(µg/ml)

0.1597

0.6712

Recovery (%)

(Synthetic Mxture)

100.04±0.638

99.13±0.404

Precision (%RSD)

Interday (n=3)

0.29%

0.12

Intraday (n=3)

0.1

0.292

Specificity

No interferences

No interferences

 

 

Table 3: Recovery data for the proposed method (n=3)

Drug

Amount present in formulation

(µg/ml)

Amount added

(µg/ml)

Amount found

(µg/ml)

% Recovery

(n=3)

LSP

10

10

10

8

10

12

11.352

18.52

20.32

98.67

99.39

99.35

 

HCTZ

6.25

6.25

6.25

5

6.25

7.5

9.5

10.75

11.82

99.82

100.74

99.49

Abbreviations; LSP-Lisinopril; HCTZ-Hydrochlorothiazide

 

Table 4: Results of statistical Analysis for Dosage form (Synthetic Mixture)

Parameters

Hydrochlorothiazide

Lisinopril

% Conc.± S.D.

99.97±0.235

99.57±0.307

% Coefficient of Variance

0.00235

0.00308

Standard error Mean

0.963

0.1258

 

The amount of Lisinopril and HCTZ in combined Synthetic Mixture was determined and it was found to 99.57±0.307 The amount of Lisinopril and HCTZ in combined Synthetic Mixture was determined and it was found to and 99.97±0.235 for Lisinopril and HCT respectively are shown in table 4. To study the precision of the method the analysis of formulation was carried out for six times. The % RSD values for Inter-day was found to be 0.12 for Lisinopril and 0.29 for HCTZ is shown in Table 2. The % RSD values for Intra-day was found to be 0.292 for Lisinopril and 0.1 for HCTZ is shown in Table 2. The low % RSD values indicated that the amount found was in good agreement with the label claim. Hence the precision of the method was confirmed. Further. The precision was confirmed by intermediate precision. The analysis of formulation was carried out for three times in the same day and on three successive days. The % RSD values for inter day and intraday analysis of formulation was found to be less than 2%. The ruggedness was confirmed by different analysts and different instruments. The % RSD values for different analysts and different instruments were found to be less than 2%. Hence, the intermediate precision and ruggedness were confirmed.

 

The accuracy of method was confirmed by recovery studies. To the pre analyze formulation a known quantity of raw material was added in different concentrations. The amount of drug recovered was calculated and the percentage recovery was found to be in the range of 98.67-99.39% for Lisinopril. 99.49-100.74% for HCTZ is shown in Table 3. The procedure was repeated for three times for each concentration and the % RSD values were calculated. The low % RSD values ensure that the excipients used in formulation are not interfering in the analysis of LSP and HCTZ. LOD was found 0.2215 for LSP and 0.0527 for HCTZ, LOQ was found 0.6712 for LSP and 0.1597 for HCTZ, are shown Table 2. Specificity were calculated by adding excipients in API Solution by checking the interferences is shown Table 2.

 

CONCLUSION:

The proposed method is simple, accurate, precise and selective for the simultaneous estimation of LSP and HCTZ in pure and in fixed dose combination. The method is economical, rapid and do not require any sophisticated instruments contrast to chromatographic method. Hence it can be effectively applied for the routine analysis of Lisinopril and HCTZ in pure and in fixed does combination.

 

ACKNOWLEDGEMENTS:

The authors wish to thank Dr. Birendra Shrivastav, Ravi Dahiya and Dr. Jawed Akhtar for their kind help and providing all necessary facilities. The authors also thankful to Torrent Pharmaceutical, Ahmadabad for providing the souvenir sample of Lisinopril and Hydrochlorothiazide.

 

REFERENCES:

1.     Indian Pharmacopoeia, the Controller of Publication Government of India, Ministry of Health and Family Welfare. New Delhi. Vol. 2, 2007. p. 688-690, 576-578.

2.       British Pharmacopoeia 2009 .Department of Health and Social Services for Northern Ireland. London: 1993. p.3509-12, 2982-2985

3.       United State Pharmacopoeia 30 and National Formulary 25. Vol. 2. Asian ed. Rockville MD: United State Pharmacopoeial Convention Inc: 2007. p. 2482-83,2288

4.       D Ivanovic et al. Validation of an Analytical Procedure for Simultaneous Determination of Hydrochlorothiazide, Lisinopril, and Their Impurities. Acta Chromatographica. 18; 2007: 143-156.

5.       Hapse SA et al. Spectrophotometric estimation and validation of hydrochlorothiazide in tablet dosage forms by using different solvents. International Journal of Pharmaceutical Sciences and Drug Research. 2(3); 2010: 182-187.

6.       Divyakant Patel et al. RP-HPLC Method for Development and Validation of Lisinopril Tablets. Pharma Science Monitor An International Journal Of Pharmaceutical Sciences.  2(3)Suppl-1; 2011: S17-23.

7.       CM Jamakhandi et al. Spectrophotometric Determination of Lisinopril Dosage Form by Condensation Reaction. International Journal of Pharmaceutical Sciences and Drug Research. 3(2); 2011: 182-187.

8.       Nafisur Rahman, Manisha Singh and Md Nasrul Hoda. Optimized and Validated Spectrophotometric Methods for the Determination of Lisinopril in Pharmaceutical Formulations Using Ninhydrin and Ascorbic Acid. Journal Of The Brazilian Chemical Society. 16(5); 2005: 1001-1009.

9.       Kanakapura Basavaiah et al.. Spectrophotometric Determination of Lisinopril in Pharmaceuticals Using Ninhydrin- a Modified Approach. Journal of Food and Drug Analysis. 17(2); 2009: 93-99.

10.    Asad Raza, Tariq Mahmood Ansaria and Atta-ur-Rehman. Spectrophotometric Determination of Lisinopril in Pure and Pharmaceutical Formulations. Journal of the Chinese Chemical Society. 52; 2005: 1055-1059.

11.    A. Mohammad, S Sharma and SA Bhawani. Identification and Quantification of Lisinopril from Pure, Formulated and Urine samples by Micellar Thin Layer Chromatography. International Journal of PharmTech Research. 1(2); 2009: 264-272.

12.    Klaus Flory. Analytical Profile of Drug Substance Vol. 10 p.405-441.

13.    Klaus Flory Analytical Profile of Drug Substance Vol. 21 p.223-227.

14.    ICH guidelines Q2A; Text on Validation of analytical procedures: Methodology International Conference on Harmonization. Geneva. March 1994. p. 1-5

15.    ICH guidelines Q2B; Text on Validation of analytical procedures: Methodology International Conference on Harmonization. Geneva. March 1996. p. 1-8.

16.    Available from: http://en.wikipedia.org/wiki/Lisinopril [Last accessed on 2012 Aug 07].

17.    Available from: http://www.drugs.com/lisinopril.html Lisinopril [Last accessed on 2012 Aug 07].

18.    Available from: http://en.wikipedia.org/wiki/Hydrochlorothiazide Lisinopril [Last accessed on 2012 Aug 07].

19.    Available from: http://www.drugs.com/hydrochlorothiazide.html Lisinopril [Last accessed on 2012 Aug 07].

20.    Beckett AH and Stenlake JB. Practical Pharmaceutical Chemistry, New Delhi. 2007; 4th ed Part 2: pp. 157-167.

21.    Devika GS, M Sudhakar and J Venkateshwara Rao. Isocratic RP-HPLC Method for Simultaneous Estimation of Spirinolactone and Hydrochlorthiazide in Oral Solid Dosage Form. Research Journal of Pharmacy and Technology. 5(8); 2012: 1050-1053.

 

 

 

 

Received on 10.01.2013       Modified on 13.01.2013

Accepted on 20.01.2013      © RJPT All right reserved

Research J. Pharm. and Tech. 6(2): Feb. 2013; Page 212-215