Int J Pharm Pharm Sci, Vol 16, Issue 1, 22-29Original Article

STABILITY INDICATING UPLC METHOD FOR ESTIMATION OF BENAZEPRIL AND HYDROCHLOROTHIAZIDE IN BULK AND COMBINED DOSAGE FORM

SHRADDHA S. BODKE1*, CHARUSHILA J. BHANGALE1, SANGITA N. BHANDARE2

1Department of Pharmaceutical Quality Assurance, PRES’s College of Pharmacy (For Women), Chincholi, Nashik, India. 2Department of Pharmaceutical Chemistry, PRES’s College of Pharmacy (For Women), Chincholi, Nashik, India
*Corresponding author: Shraddha S. Bodke; *Email: charushila.bhangale@pravara.in

Received: 10 Oct 2023, Revised and Accepted: 15 Nov 2023

ABSTRAC

Objective: The main objective was to develop stability indicating UPLC technique for simultaneous estimation of Benazepril and Hydrochlorothiazide in bulk and formulation.

Methods: 0.1% Triethylamine phosphate: Methanol (25:75v/v) was used as the mobile phase. Benazepril linearity was found to be 4-20 µg/ml and Hydrochlorothiazide linearity was found to be 5-25 g/ml. The detection wavelength was 236 nm, and the retention period of Benazepril was 3.4 min and Hydrochlorothiazide was 5.4 min with a flow rate of 1.0 ml/min. According to the ICH guidlines, the proposed method was validated and stress studies revealed that the drugs are prone to alkali and peroxide stress conditions.

Results: The calibration curve was plotted, and the regression equations for Benazepril were y = 2,01,491.67x+60,532.30 with a correlation coefficient (r2) of 0.9997 and Hydrochlorothiazide were y = 64,635.86x-74,607.10 with a correlation coefficient (r2) of 0.9994. According to the accuracy research, the percent recovery of Benazepril is 99.09-100.69 % and that of Hydrochlorothiazide is 98.27-101.88%, both of which are within the ICH recommendations. Benazepril has a limit of detection of 0.08 g/ml-0.24 g/ml and Hydrochlorothiazide has a limit of quantitation of 0.03 g/ml-0.10 g/ml. The procedure was found to be straightforward, linear, rapid, exact, repeatable, and robust. It was determined that the % RSD was within ICH norms. Stress degradation tests showed the drug's vulnerability to oxidative, thermal, photolytic, acid, basic, and neutral hydrolysis stress conditions. Under the circumstances of alkali and peroxide stress, it was discovered that the drug degraded most quickly.

Conclusion: The developed chromatographic technique under consideration was suitable for the accurate, precise, and quick simultaneous measurement of hydrochlorothiazide and benazepril in both their bulk and medicinal dose forms.

Keywords: Benazepril, Hydrochlorothiazide, UPLC method, ICH Guidelines, Stability testing

© 2024 The Authors. Published by Innovare Academic Sciences Pvt Ltd. This is an open access article under the CC BY license (https://creativecommons.org/licenses/by/4.0/)
DOI: https://dx.doi.org/10.22159/ijpps.2024v16i1.49457. Journal homepage: https://innovareacademics.in/journals/index.php/ijpps

INTRODUCTION

A prodrug called benazepril (BZP) is used to treat hypertension and heart failure, to lessen proteinuria and renal disease in nephropathies patients, and to prevent stroke, myocardial infarction, and cardiac mortality in high-risk individuals when hydrolyzed by esterases. Benazepril and benazeprilat inhibit the angiotensin-converting enzyme (ACE) in both humans and animals [1]. Angiotensin I is converted to the vasoconstrictor chemical angiotensin II by the angiotensin converting enzyme (ACE). Aldosterone is also stimulated by angiotensin II to be produced in the adrenal cortex. BZP's chemical structure is seen in fig. 1.

Fig. 1: Structure of benazepril

Fig. 1 shows the chemical structure of BZP. Chemically, BZP is 2-[(3S)-3-{[(2S)-1-ethoxy-1-oxo-4-phenylbutan-2-yl]amino}-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepin-1-yl]acetic acid [2, 3]

Hydrochlorothiazide (HTZ) is a thiazide diuretic used to treat edoema caused by a variety of diseases, as well as hypertension. The most commonly given thiazide diuretic is hydrochlorothiazide. Both high blood pressure and edoema are treated with it. Although hydrochlorothiazide is still frequently used, angiotensin-converting enzyme drugs are replacing it. There are several possible combinations of angiotensin converting enzyme inhibitors or angiotensin II receptor blockers with hydrochlorothiazide [4]. Chemical structure of HZT is shown in fig. 2.

Fig. 2: Structure of hydrochlorothiazide

Fig. 2 shows structure of HTZ. Chemically, HTZ is 6-chloro-1,1-dioxo-3,4-dihydro-2H-1lambda6,2,4-benzothiadiazine-7-sulfonamide. Hydrochlorothiazide and benazepril are prescribed to patients with high blood pressure. Hydrochlorothiazide and benazepril are prescribed to patients with high blood pressure. By lowering certain molecules that harden blood vessels, it increases blood flow. Few UV [5-9], HPTLC [10-12], and HPLC [13-25] procedures for BZP and HTZ in bulk and formulations with other medications have been described, according to a study of the literature. In order to estimate BZP and HTZ in bulk and formulations, a stability-indicating chromatographic approach has been developed. The suggested approach may be used to routinely assess BZP and HTZ in pharmaceutical dosage forms and bulk samples since it is quick, easy, accurate, and reproducible. The formulation sample recoveries were in good agreement with their label claims, and no formulation excipients interfered with the estimate. As a result, this procedure is straightforward and convenient for testing bulk pharmaceuticals in pure form and mixes on a regular basis.

MATERIALS AND METHODS

Instruments

An Agilent Technologies UPLC system, a variable wavelength programmable UV identifier, and a Rheodyne injector with a 20l fixed circle were all used throughout the chromatographic process. The opposing stage was a Cosmosil C18 (250 mm x 4.6ID, 5 micron). For Spectrophotometric assessments and gauging, the PGB 100 electronic equilibrium balance from Wenser High Precision Balance Model was employed.

Reagents and chemicals

Benazepril and Hydrochlorothiazide was procured from Pharma Tech Solutions as a gift sample. The tablet Benazepril+HCTZ 20/25 mg tablet of Sandoz PVT LTD purchased from local market. HPLC quality Methanol and water were purchased from Merck Specialities Private Limited, Mumbai.

Chromatographic conditions

Cosmosil C18 (250 mm x 4.6ID, particle size: 5 micron) was employed in the chromatographic procedure at a wavelength of 236 nm. 0.1% triethylamine buffer pH 4.8: Methanol (25:75 v/v) was used as the mobile phase for elution, and the same solvent was used to prepare the standard and sample solutions. By injecting the 20 µl and increasing the flow rate to 1.0 ml/min, the elution was evaluated.

Preparation of standard stock solutions

Accurately Benazepril and hydrochlorothiazide working standards were weighed and placed into a 100 ml clean, dry volumetric flask. A 3/4 volume of diluent was added, and the mixture was sonicated for 5 min before being made up to the final volume using diluents. Benazepril's final concentration is 40 µg/ml, whereas Hydrochlorothiazide's ultimate concentration is 50 µg/ml. These medications' working standard solutions were created by properly diluting the corresponding stock solution with mobile phase.

Selection of mobile phase

Benazepril (40 µg/ml) and Hydrochlorothiazide (50 µg/ml) standard solutions were introduced into the RP-HPLC equipment and ran in different solvent systems. Initially, different mobile phase solutions, such as phosphate buffer and methanol were explored in the isocratic mode to discover the optimal conditions.

HPLC method development

Optimisation of the RP-HPLC method

The HPLC technique was developed for the simultaneous measurement of Benazepril and Hydrochlorothiazide. Different mobile phases were investigated for process optimisation, but appropriate retention periods, hypothetical plates, and high resolution were seen with 0.1% Triethylamine Buffer pH 4.8: Methanol (25:75v/v) using Cosmosil C18 (250 mm x 4.6 mm ID, Particle size: 5 m) via Isocratic method. Results are shown in table 1.

Table 1: Optimized chromatographic conditions

Mobile phase 0.1 % Triethylamine Buffer pH 4.8: Methanol (25:75v/v)
Selection of column Cosmosil C18 (250 mm x 4.6 mm ID, Particle size: 5 µm)
Injection volume 20 μl
Flow rate 1.0 ml/min
Column temperature Room temperature
Detection wavelength 236 nm
Run Time 7.5 min
Retention time Benazepril (3.4 min) and Hydrochlorothiazide (5.4 min)

Validation of RP-HPLC method

The optimised RP-HPLC technique was validated in compliance with the ICH Q2 (R) requirements [26].

Linearity

The chromatograms were obtained after each injection of a different test solution concentration. In order to create a series of Benazepril and Hydrochlorothiazide test preparations, 1–5 ml of the stock solution containing the appropriate amounts of Benazepril (40 µg/ml) and Hydrochlorothiazide (50 µg/ml) were taken and placed in five 10 ml volumetric flasks before being topped up with mobile phase. Three injections of a 20µ l volume of each concentration into the HPLC were made under optimal chromatographic conditions.

Accuracy

Samples are typically produced to cover 50%, 100% and 150% of the nominal sample preparation concentration. These samples are analysed, and the recoveries for each are computed [27].

Precision

The intraday precision research was carried out by creating a test solution of the same concentration and analysing it three times during the day. To determine interday precision, the identical process was used on two distinct days. The outcome was given as % RSD [28, 29].

Limit of quantitation (LOQ) and limit of detection (LOD)

The LOD and LOQ were computed using the slope(s) of the calibration curve and the standard deviation (SD) of the peak areas using the formulae LOD = 3.3 s/s and LOQ = 10 s/s.

Robustness

Robustness was assessed by altering the chromatographic circumstances, such as the composition of the mobile phase, the detection wavelength, the flow rate, etc. The % RSD should be given. Small adjustments were allowed under ideal circumstances, and the method's resilience was established. We tried individual variations of detecting wavelength of±2 nm and flow rate of±0.1 ml/min. Solutions of 100 % test concentration with the required modifications were injected into the system in triplicate under ideal circumstances.

Ruggedness

Ruggedness is the investigation of the influence of external factors on the approach. To assess the ruggedness of the suggested approach, factors were purposefully altered. These factors included system variation, various analysts, and atmospheric fluctuations. Three concentrations of the test solution were injected into the HPLC system at a flow rate of 1.0 ml/min by two separate analyzers after preparing it in accordance with the test method.

Assay of marketed formulation

20 tablets of Sandoz PVT LTD's commercialised Benazepril with HCTZ Weighed and crushed into a fine powder, 20/25 mg tablets were used. The average weight of the tablet sample was measured transferred to a volumetric flask measuring 100 ml, and the capacity was then filled with diluent. Sonicate while intermittently rotating for ten minutes. Using a 0.45m membrane filter, the aforementioned solution was filtered to produce a stock solution that contained 200 ug/ml of BZP and 250 ug/ml of HTZ. To be used for analysis, 0.3 ml of the solution was taken out, diluted to 10 ml, and then injected into the system.

System suitability

The system suitability properties were examined at in order to validate the approach, column performance, and system. The system was injected with a standard solution of BZP and HTZ six times, and system suitability traits were evaluated.

Force degradation studies

The BZP and HTZ drugs combinations were subjected to forced degradation experiments [30]. The principle stress conditions were 1N HCl (1 h at 60 °C), 1N NaOH (1 h at 60 °C), 3% H2O2 (24 h), dry heat (24 h), and UV radiation (24 h) [31]. The stress conditions applied are shown in table 2.

Table 2: Forced degradation conditions according to ICH guidelines

Test condition Acidic degradation Alkaline degradation Oxidative degradation Thermal degradation Photolytic degradation

Benazepril

and Hydrochlorothiazide

1N HCl,

1 h at 60 °c

1N NaOH,

1 h at 60 °c

 3%H2O2, 24 h Thermal stress for 24 h Photolytic stress for 24 h

RESULTS AND DISCUSSION

Linearity

Linearity was defined as an analytical method's capacity to provide test results that are exactly proportionate to the analyte concentration within a certain range. Plotting the peak area of the HPLC chromatograph against the pertinent concentrations resulted in the calibration graph. HTZ was shown to be linear in the concentration range of 1–5 µg/ml, while BZP was found to be linear in the 5–25 µg/ml range. Fig. 3 and 4 illustrate the graphs.

Fig. 3: Calibration curve for benazepril, fig. 3 shows calibration curve of BZP and regression equation of Benazepril was found to be y = 2,01,491.67x+60,532.30 with correlation coefficient (r2) of 0.9997

Fig. 4: Calibration curve for hydrochlorothiazide, fig. 4 shows calibration curve of HZT and regression equation of Hydrochlorothiazide was found to be y = 64,635.86x-74,607.10 with correlation coefficient (r2) of 0.9994

Accuracy

The method's accuracy specifies how near the method's findings are to the real value. The results of the accuracy tests indicated that the procedure is accurate within acceptable limits. The percentage RSD for BZP and HTZ is determined, and all findings are within acceptable bounds. With a maximum RSD of 2.0%, the allowable accuracy was within the range. Table 3 displays the results.

Table 3: Recovery studies

% Mean recovery Amount found ([µg/ml]), mean±SD [n=3] % RSD
BZP HTZ BZP HTZ BZP HTZ
50% 100.19 100.19 38086.33±0.45 31825.00±1.07 0.45 1.06
100% 99.86 99.84 84553.33±0.30 79160.67±1.85 0.30 1.85
150% 99.76 99.33 210825.33±0.72 153930.00±0.43 0.72 0.43

Data are expressed as mean±SD, n=3

Precision

The repeatability of test results is ensured by intraday and interday precision. Both BZP and HTZ exhibited % RSD values less than two. Table 4 displays the results.

Robustness

Robustness was examined using various deliberate alterations in chromatographic settings, such as variation in flow Rate and wavelength. According to the robustness investigation, the RSD for the BZP and HTZ is less than 2%. Hence it is robust and complies per ICH guidelines. Table 5 displays the results.

Ruggedness

Ruggedness was studied by different analysts. From the robustness study % RSD was found to be within the limit of 2 % for the BZP and HTZ. Hence it is complying as per ICH guidelines. Table 6 presents the outcomes.

Table 4: Precision studies (Intra-day and Inter-day)

Drug Conc. [µg/ml] Intra-day amount found [µg/ml] Inter-day amount found [µg/ml]
mean±SD [n= 3] % RSD mean±SD [n= 3] % RSD
BZP 4 88025.23±6485.06 0.74 88590.33±2924.65 1.23
12 244653.33±9883.02 0.40 244513.33±9662.60 1.07
20 410825.33±13332.38 0.32 410496.33±9353.15 0.61
HTZ 4 23825.00±9737.71 1.10 23571.00±949.10 0.40
12 89910.67±4762.32 0.19 89607.67±639.50 0.07
20 153190.00±5096.90 0.12 153980.33±4994.10 0.32

Data are expressed as mean±SD, n=3

Table 5: Data for robustness study of benazepril and Hydrochlorothiazide

S. No. Parameter Condition Benazepril, [n=3] Hydrochlorothiazide [n=3]
Area Mean SD % RSD Area Mean SD %RSD
1 Variation in Flow Rate (ml/min) 0.9 2441534 2451361 9988 0.41 896145 895817 1523 0.17
2 1 2451048 897150
3 1.1 2461503 894157
1 Change in Wavelength (nm) 234 2458791 2445190 14133 0.58 897421 895259 1948 0.22
2 236 2446202 893640
3 238 2430578 894715

Table 6: Data for ruggedness study of benazepril and hydrochlorothiazide

S. No. Analyst Benazepril Hydrochlorothiazide
Area Mean area* SD % RSD Area Mean area* SD % RSD
1 Analyst-Ι 2451623 2456199 7711 0.31 896457 896160 394 0.04
2451872 895712
2465102 896312
2 Analyst-ΙΙ 2441524 2446808 4629 0.19 897421 894423 3730 0.42
2450154 895602
2448745 890245

*Data are expressed as mean±SD, n=3

Specificity

Analysing reference medications and sample drugs helped to determine the specificity of the technique. Excipients and contaminants had no effect on conventional medications. Hence the method is specific as shown in fig. 5.

Fig. 5 exhibits a well-defined peak of BZP and HTZ at the determined retention times of 3.4 min for BZP and 5.4 min for HTZ.

Assay of marketed formulation

The % Assay of Benazepril+HCTZ 20/25 mg tablet of Sandoz PVT LTD was calculated. The percentage assay was discovered to be BZP (99.77%) and HTZ (98.03 %), which was in good accord with the label claim.

System suitability parameters

System appropriateness criteria were examined to validate the system, approach, and column performance. The system was given six injections of a conventional mixture of hydrochlorothiazide and benazepril, and its suitability was assessed. Table 7 presents the results of the study.

Degradation studies

The stability and specificity of the analytical approach, as well as the predicted degradation products, may all be determined with the help of stress testing the drugs material. Tests for degradation were run on solutions containing 15 µg/ml each of BZP and HTZ. Results are displayed in table 8.

Table 7: System suitability parameter

Parameter BZP HTZ
Retention time (min) 3.4 5.4
Theoretical plates 9052 8214
Asymmetry factor 0.50 0.37
Tailing factor 1.08 1.21

Fig. 5: Chromatograph of benazepril and hydrochlorothiazide

Table 8: Results of forced degradation studies

S. No. Condition Drugs Area of sample Area of standard % Drug recovered % Degradation
1 Acid stress BZP 2346264 2440154 96.15 3.85
HTZ 869641 896145 97.04 2.96
2 Alkali Stress BZP 2266574 2440154 92.89 7.11
HTZ 795440 896145 88.76 11.24
3 Oxidative Stress BZP 1782448 2440154 73.05 26.95
HTZ 584048 896145 65.17 34.83
4 Thermal Stress BZP 2388035 2440154 97.86 2.14
HTZ 870236 896145 97.11 2.89
5 Photolytic Stress BZP 2405410 2440154 98.58 1.42
HTZ 888993 896145 99.20 0.80

Fig. 6: Acid stressed chromatogram

Fig. 7: Alkali stressed chromatogram

Fig. 8: Peroxide-stressed chromatogram

Fig. 9: Thermal stressed chromatogram

Fig. 10: Photolytic stressed chromatogram

Fig. 6, 7, 8, 9 and 10 shows some additional peaks at various Rt values. The drugs were found prone to acidic, alkali and oxidation condition.

DISCUSSION

The UPLC technique for simultaneous measurement of Benazepril and Hydrochlorothiazide was developed and validated. 0.1% Triethylamine phosphate: Methanol (25:75v/v) was used as the mobile phase. Benazepril linearity was found to be 4-20 µg/ml and Hydrochlorothiazide linearity was found to be 5-25 µg/ml. The calibration curve was plotted, and the regression equations for Benazepril were y = 2,01,491.67x+60,532.30 with a correlation coefficient (r2) of 0.9997 and Hydrochlorothiazide were y = 64,635.86x-74,607.10 with a correlation coefficient (r2) of 0.9994. The detection was performed at 236 nm at a flow rate of 1.0 ml/min. Benazepril had a retention duration of 3.4 min, while Hydrochlorothiazide had a retention time of 5.4 min. In reported method [32] the benazepril hydrochloride and Hydrochlorothiazide was 9.19 min and 3.10 min, respectively. The retention time was higher and require large amount of mobile phase. The validation in accordance with ICH principles shows the method's appropriateness for the quantitative determination of the substances. Statistics were used to validate the suggested techniques' dependability and analytical performance with regard to the constraints of linearity, range, precision, accuracy, detection and quantitation.

According to the Accuracy research, the percent recovery of BZP is 99.09-100.69% and that of HTZ is 98.27-101.88%, both of which are within the ICH criteria. Intraday and interday precision ensure that % RSD remained within ICH norms, i.e., NMT 2 for both BZP and HTZ. Benazepril had a limit of detection of 0.08g/ml-0.24g/ml and Hydrochlorothiazide had a limit of quantitation of 0.03 g/ml-0.10 g/ml. Robustness was examined using purposeful variation, i.e., a change in flow rate and a change in wavelength that was within 2% of RSD according to ICH norms. The ruggedness testing yields results within 2 % of the variance in Analyst examined. Sandoz BZP+HTZ Tablet 20/25 mg % test revealed Benazepril (99.77 %)-Hydrochlorothiazide (98.03 %). BZP and HTZ were treated to a variety of stresses such as acid, alkali, oxidation, dry heat, and photo light. Forced degradation studies provide knowledge about possible degradation pathways and degradation products of the active ingredients and help elucidate the structure of the degradants [33, 34]. Degradation products generated from forced degradation studies are potential degradation products that may or may not be formed under relevant storage conditions. Still, they assist in developing stability-indicating methods. The proposed methods separated the drug efficiently from its breakdown products and were thus regarded as good stability-indicating procedures.

CONCLUSION

The suggested chromatographic technique for identifying BZP and HTZ from pure and dosage forms was found to be straightforward, accurate, precise, quick, and specific. The mobile phase used in method development is easy to make and affordable. The formulation had good sample recoveries. Among all proven approaches, this strategy is the most cost-effective and has the lowest run time, allowing for quick analysis. As a result, this method may be simply and conveniently used for in vitro dissolution and routine analysis of Benazepril and Hydrochlorothiazide in Pharmaceutical dosage form.

ACKNOWLEDGEMENT

For providing the necessary resources for this research, the authors are thankful to Pravara Rural Education Society’s College of Pharmacy, For Women, Nashik.

FUNDING

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors

AUTHORS CONTRIBUTIONS

SSB contributed for experimental work. SNB did the manuscript preparation. CJB contributed in hypothesis and finalization of manuscript. All authors read and approved the final manuscript.

CONFLICT OF INTERESTS

The authors declare no conflict of interest.

REFERENCES

  1. Gengo FM, Brady E. The pharmacokinetics of benazepril relative to other ACE inhibitors. Clin Cardiol. 1991 Aug;14(8)Suppl 4:IV44-51. doi: 10.1002/clc.4960141807, PMID 1893642.

  2. Gengo FM, Brady E. The pharmacokinetics of benazepril relative to other ACE inhibitors. Clin Cardiol. 1991;14(8);Suppl 4:IV44-55. doi: 10.1002/clc.4960141807, PMID 1893642.

  3. Pickkers P, Hughes AD, Russel FG, Thien T, Smits P. Thiazide-induced vasodilation in humans is mediated by potassium channel activation. Hypertension. 1998 Jun;32(6):1071-6. doi: 10.1161/01.hyp.32.6.1071, PMID 9856976.

  4. Pickkers P, Hughes AD, Russel FG, Thien T, Smits P. Thiazide-induced vasodilation in humans is mediated by potassium channel activation. Hypertension. 1998 Dec;32(6):1071-6. doi: 10.1161/01.hyp.32.6.1071, PMID 9856976.

  5. Durmus O, Erdal D. Determination of benazepril HCl and hydrochlorothiazide in pharmaceutical preparations using UV-visible spectrophotometry and genetic multivariate calibration methods. J Food Drug Anal 2005 Jan;13(4):301-11.

  6. Hapse SA, Wagh V, Kadaskar PT, Dokhe MD, Shirsath AS. Spectrophotometric estimation and validation of hydrochlorothiazide in tablet dosage forms by using different solvents. Pharm Chem. 2012 Jan;4(1):110-4.

  7. Parmar V, Chhalotiya U, Shah D, Bhatt K, Baldania S. Quantification of benazepril hydrochloride and hydrochlorothiazide in tablet dosage form by simultaneous equation spectrophotometric method. J Appl Chem. 2013;2013:1-5. doi: 10.1155/2013/316137.

  8. Jain N, Jain R, Thakur N, Gupta BP, Banweer J. Jains. Novel spectrophotometric quantitative estimation of hydrochlorothiazide in bulk drug and their dosage forms by using hydrotropic agent. Int J Appl Pharm 2010 Mar;2(3):11-4.

  9. Panderi IE. Simultaneous determination of benazepril hydrochloride and hydrochlorothiazide in tablets by second-order derivative spectrophotometry. J Pharm Biomed Anal. 1999 Aug;21(2):257-65. doi: 10.1016/s0731-7085(99)00134-x, PMID 10703980.

  10. Meyyanathan SN, Suresh B. HPTLC Method for the simultaneous determination of amlodipine and benazepril in their formulations. J Chromatogr Sci. 2005;43(2):73-5. doi: 10.1093/chromsci/43.2.73, PMID 15826364.

  11. El-Gindy A, Ashour A, Abdel Fattah L, Shabana MM. Application of LC and HPTLC-densitometry for the simultaneous determination of benazepril hydrochloride and hydrochlorothiazide. J Pharm Biomed Anal. 2001;25(2):171-79. doi: 10.1016/s0731-7085(00)00480-5, PMID 11275425.

  12. Naguib IA, Abdelaleem EA, Zaazaa HE, Draz ME. Simultaneous determination of hydrochlorothiazide and benazepril hydrochloride or amiloride hydrochloride in presence of hydrochlorothiazide impurities: chlorothiazide and salamide by HPTLC method. J Chromatogr Sci. 2015;53(1):183-8. doi: 10.1093/chromsci/bmu016, PMID 24771053.

  13. Mohammed NMS, Abdo HR, Hassan HM. Method development and validation of simultaneous determination of hydrochlorothiazide and losartan in tablet dosage form by RP-HPLC. Int J Pharm Sci Res 2019 Jan;10(1):227-31. doi: 10.13040/IJPSR.0975-8232.

  14. Ramakrishna NV, Vishwottam KN, Manoj S, Koteshwara M, Wishu S, Varma DP. Sensitive liquid chromatography-tandem mass spectrometry method for quantification of hydrochlorothiazide in human plasma. Biomed Chromatogr. 2005;19(10):751-60. doi: 10.1002/bmc.510, PMID 15856489.

  15. Shravan GS, Hemalatha R. Method development and validation for the simultaneous determination of benazepril and hydrochlorothiazide in bulk form and marketed pharmaceutical combined dosage form by using reverse phase-HPLC. J Pharmacreations. 2020 Apr;7(4):278-301.

  16. Rao TN, Apparao K, Krishnarao N, Vijayalakshmi AA. A new simultaneous HPLC analytical method for quantification of benazepril hydrochloride and its related impurities in bulk drug product. Asian J Res Pharm Sci. 2017 Sep;7(3):135-40. doi: 10.5958/2231-5659.2017.00022.4.

  17. Yadav SS, Rao JR. RP-HPLC method for simultaneous estimation of losartan, hydrochlorothiazide and amlodipine in tablet dosage form. Asian J Pharm Clin Res. 2014;7(1):137-40.

  18. Hermann TW, Chavan SP, Kostenbauder HB. Determination of hydrochlorothiazide in plasma by high-performance liquid chromatography (HPLC) open access. J Pharm Res. 2018;2(1):1-4.

  19. Kavathia A, Misra M. Development and validation of RP-HPLC and UV-spectrophotometric methods for rapid simultaneous estimation of amlodipine and benazepril in pure and fixed-dose combination. Arab J Chem. 2017 Feb;10(2):S3021-8. doi: 10.1016/j.arabjc.2013.11.043.

  20. Mahesh M, Kumanan R, Jayaveera KN. Isocratic RP-HPLC, UV method development and validation for the simultaneous estimation of hydrochlorothiazide and ramipril in tablet dosage form and bulk durg. Int J Curr Pharm Res 2011 Jan;3(2):119–23.

  21. Sarat M, krishna PM, Rambabu C. Development and validation of RP-HPLC method for simultaneous estimation of amlodipine besyate and benazepril HCl in tablet. Int J Curr Pharm Res. 2012;4(3):80-4.

  22. Neelima MS, Gandhi BM, Raju VB, Sumanth KS, Mounika KS, Lakshmi PJN. Development and validation of stability indicating reverse-phase high-performance liquid chromatography method for simultaneous estimation of atenolol, hydrochlorothiazide and losartan in bulk and pharmaceutical dosage form. Asian J Pharm Clin Res. 2016 Aug;9(2):118-24.

  23. Hassib ST, El-Sherif ZA, El-Bagary RI, Youssef NF. Reversed-phase high-performance liquid chromatographic and thin layer chromatographic methods for the simultaneous determination of benazepril hydrochloride and hydrochlorothiazide in cibadrex tablets. Anal Lett. 2000 Jun;33(15):3225-37. doi: 10.1080/00032719.2000.10399497.

  24. Vonaparti A, Kazanis M, Panderi I. Development and validation of a liquid chromatographic/electrospray ionization mass spectrometric method for the determination of benazepril, benazeprilat and hydrochlorothiazide in human plasma. J Mass Spectrom. 2006;41(5):593-605. doi: 10.1002/jms.1014, PMID 16541390.

  25. ICH. Q2A validation of analytical procedures: consensus guidelines; ICH harmonized tripartite guidelines; 1994.

  26. ICH. Q2B validation of analytical procedures: methodology, consensus guidelines ICH harmonized tripartite guidelines; 1996.

  27. ICH. Q2 (R1) validation of analytical procedure: test and methodology, international Conference on Harmonization, Geneva; 2005.

  28. Rashmin M. An introduction to analytical method development for pharmaceutical formulations. pharma info. Net. 2008 Jun;6(4):1-11.

  29. Prathap B, Rao GHS, Devdass G. Review on stability indicating HPLC method development. Int J Innov Pharm Res. 2012;3(3):229-37.

  30. Sutar SV, Yeligar VC, Patil SS. A review: stability indicating forced degradation studies. Res J Pharm Technol 2019 Feb;12(2). doi: 10.5958/0974-360X.2019.00152.5.

  31. Venkataraman S, Manasa M. Forced degradation studies: regulatory guidance, characterization of drugs, and their degradation products. Drug Invent Today 2018 May;10(2):137.

  32. Sunil LB KK. Stability indicating LC method for the estimation of benazepril HCl and hydrochlorthiazide in pharmaceutical dosage form. J Chromat Separation Techniq 2014;5(2):1-7. doi: 10.4172/2157-7064.1000216.

  33. Alsante KM, Ando A, Brown R, Ensing J, Hatajik TD, Kong W. The role of degradant profiling in active pharmaceutical ingredients and drug products. Adv Drug Deliv Rev. 2007;59(1):29-37. doi: 10.1016/j.addr.2006.10.006, PMID 17187892.

  34. Blessy M, Patel RD, Prajapati PN, Agrawal YK. Development of forced degradation and stability indicating studies of drugs-a review. J Pharm Anal. 2014;4(3):159-65. doi: 10.1016/j.jpha.2013.09.003, PMID 29403878.