reverse-phase high-performance liquid...

Drug Invention Today | Vol 10 • Issue 2 • 2018202

Reverse-phase high-performance liquid chromatographic method development and validation for the simultaneous estimation of Hydrochlorothiazide and Propranolol in bulk and Pharmaceutical dosage form in biorelevant dissolution mediaPalani Shanmugasundaram1*, S. K. Kamarapu2

INTRODUCTIONHydrochlorothiazide is a short-acting thiazide diuretic often considered the prototypical member of this class.[1,2] The physiologic effect of Hydrochlorothiazide[3,4] is by means of increased diuresis. It reduces the reabsorption of electrolytes from the renal tubules. This results in increased excretion of water and electrolytes including sodium, potassium, chloride, and magnesium. It is widely used in the treatment of several disorders including hypertension, edema, diabetes insipidus,

1Director, School of Pharmaceutical Sciences, Vels Institute of Science, Technology and Advanced Studies, Vels University, Chennai, Tamil Nadu, India, 2Department of Pharmaceutical analysis, School of Pharmaceutical Sciences, Vels Institute of Science, Technology and Advanced Studies, Vels University, Chennai, Tamil Nadu, India

*Corresponding author: Palani Shanmugasundaram Director, School of Pharmaceutical Sciences, Vels Institute of Science, Technology and Advanced Studies, Vels University, Chennai, Tamil Nadu, India. E-mail: [email protected]

Received on: 26-09-2017; Revised on: 19-10-2017; Accepted on: 24-01-2018

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Website: jprsolutions.info ISSN: 0975-7619

and hypoparathyroidism.[5-7] Propranolol is a widely used non-cardioselective beta-adrenergic antagonist. Propranolol is used in the treatment or prevention of many disorders including acute myocardial infarction, arrhythmias, angina pectoris, hypertension, hypertensive emergencies, hyperthyroidism, migraine, pheochromocytoma, menopause, and anxiety.[8-11]

Hydrochlorothiazide is chemically 6-chloro-3, 4-dihydro-2H-1, 2, 4-benzothiadiazine-7-sulphonamide 1, 1-dioxide and its molecular formula C7H8ClN3O4S2.

It is official in IP-2007[12] and BP-2009.[13] The Chemical Structure of Hydrochlorothiazide is shown in Figure 1.

Propranolol is chemically 1-(naphthalen-1-yloxy)-3-[(propan-2-yl) amino] propan-2-ol and its molecular

ABSTRACT

Objective: A simple, rapid, and precise reverse-phase high-performance liquid chromatographic (RP-HPLC) method for simultaneous analysis of Hydrochlorothiazide and Propranolol in a bulk and tablet dosage form in biorelevant dissolution medium has been developed and validated. Methods: The chromatographic separation was achieved using reverse phase C18 column; Symmetry C18 column (250 mm × 4.6 mm × 5μm). The mobile phase used was a mixture of acetonitrile:potassium dihydrogen phosphate solution (0.01 M, pH 3.0 adjusting with Orthophosphoric acid) in a ratio of 60:40 at isocratic mode and eluents were monitored at 275 nm using PDA detector. Results: By the method, Hydrochlorothiazide and Propranolol were eluted with retention times of 2.033 min and 3.280 min, respectively. The method was continued and validated accordance with ICH guidelines. Validation revealed the method is rapid, specific, accurate, precise, reliable, and reproducible. Calibration curve plots were linear over the concentration ranges 20–60 μg/mL for Hydrochlorothiazide and 20–60 μg/mL for Propranolol. Limits of detection was 0.73 and 0.95 μg/ml and limits of quantification was 2.19 and 3.01 μg/mL for Hydrochlorothiazide and Propranolol, respectively. Conclusion: The statistical analysis was proves the method is suitable for the analysis of Hydrochlorothiazide and Propranolol as a bulk and tablet dosage form in biorelevant dissolution media (Fasted State Simulated Intestinal Fluid-FaSSIF) without any interference from the excipients.

KEY WORDS: Biorelevant media (Fasted State Simulated Intestinal Fluid), Hydrochlorothiazide and Propranolol, ICH Guidelines, Method validation, reverse-phase high-performance liquid chromatographic

Research Article

Palani Shanmugasundaram and S.K. Kamarapu

203Drug Invention Today | Vol 10 • Issue 2 • 2018

formula C16H21NO2. It is official in IP-2007[14] and BP-2009[15]. The Chemical Structure of Propranolol is shown in Figure 2.

In the scientific literature, the survey reveals that various analytical methods have been reported for the assay of hydrochlorothiazide and propranolol in pure form and pharmaceutical formulations, while described in Indian Pharmacopoeia thin layer liquid chromatography method for the assay of hydrochlorothiazide. Potentiometric titration method is specified in British Pharmacopoeia for the assay of propranolol. Many methods have been reported in the literature for the estimation of Hydrochlorothiazide and Propranolol individually[16,17] and in other combination.[18-26]

The present investigation was aimed at developing a fully validated reverse-phase high-performance liquid chromatographic (RP-HPLC) method according to ICH guidelines[27] for the simultaneous estimation of Hydrochlorothiazide and Propranolol in bulk and pharmaceutical combined dosage form in biorelevant dissolution medium (Fasted State Simulated Intestinal Fluid [FaSSIF]) [28-29] that is more economical, simple, precise, and accurate than the previous methods.

MATERIALS AND METHODSExperimentalMaterials and methodsPharmaceutical grade working standards Hydrochlorothiazide and Propranolol were obtained from Syncorp Pvt. Laboratories, Hyderabad, India. All chemicals and reagents were HPLC grade and were purchased from SD Fine-Chem Limited and Loba Chemie Pvt. Ltd, Mumbai, India.

InstrumentationThe analysis was performed using HPLC (Waters-717 series) with PDA detector and data handling system EMPOWER2 software, UV-Visible double beam

spectrophotometer (Labindia), analytical balance 0.1mg Sensitivity (SHIMADZU), pH meter (Labindia), ultrasonicator. The column used is Symmetry C18 column (250x2.5mm packed with 5μm size Stationary phase) with the flow rate 1.0 ml/min (isocratic).

Preparation of blank FaSSIF[28]

Accurately weighed 1.74g of Sodium hydroxide pellets, 19.77g of Sodium dihydrogen orthophosphate, and 30.93g of Sodium chloride dissolve in 5 L of purified water and adjust the pH 6.5 exactly using 1N Hydrochloric acid.

Preparation of FaSSIF[29]

Accurately weighed 3.3 g of sodium taurocholate dissolve in 500 mL blank FaSSIF solution, add 11.8 mL of a solution to 100 mg/mL lecithin in methylene chloride, and forming an emulsion. The methylene chloride was eliminated under vacuum at 40°C. Then, draw a vacuum for 15 min at 250mbar and also followed by 15 min at 100mbar. These results gave a clear, micellar solution, having no perceptible odor for methylene chloride. After that, it was cool to room temperature and adjusts the volume up to 2L with blank FaSSIF.[32]

Preparation of Standard Stock SolutionAccurately weighed 10 mg of Hydrochlorothiazide and Propranolol working standard and separately transferred into a 10 ml clean dry volumetric flasks, add about 7 mL of biorelevant media (FaSSIF) to each volumetric flask and sonicate to dissolve it completely and make volume up to the mark with the same diluents. The standard stock solution (1000 μg/mL) was further diluted separately to obtain working standard of concentration 10 μg/mL of Hydrochlorothiazide and Propranolol each.

Study of Spectra and Selection of WavelengthEach working standard solution was scanned between the range 200 and 400 nm in 1 cm cell against blank. Maximum absorbing wavelength of Hydrochlorothiazide and Propranolol was selected from spectral data and isosbestic wavelength selected from overlain spectra of ultraviolet (UV) spectrophotometer. The λmax for Hydrochlorothiazide and Propranolol and an isosbestic point 270 nm, 279 nm, and 275 nm, respectively. UV spectrum and typical standard chromatogram of hydrochlorothiazide and propranolol are shown in Figures 3-8.

Preparation of Calibration CurveCalibration standards at five levels were prepared by appropriately mixed, and further diluted standard stock solutions in the concentration range from 20 to 60 μg/mL for Hydrochlorothiazide and 20–60 μg/mL for Propranolol. Samples in triple injections were

Figure 1: Chemical Structure of Hydrochlorothiazide

Figure 2: Chemical Structure of Propranolol



made for each prepared concentration. Peak areas were plotted against the corresponding concentration to obtain the linearity graphs. Chromatograms of each solution were recorded.

Preparation of Mixed Working Standard SolutionThe above standard stock solution was containing 1000 μg/mL of each Hydrochlorothiazide and for

Propranolol in separate volumetric flasks. Then transferred the 0.1 ml of Hydrochlorothiazide and 0.1 ml of Propranolol of prepared standard stock solution into a clean 10 ml volumetric flask and made up to the mark with diluents. To get the mixed standard solution concentrations were 10 μg/mL for Hydrochlorothiazide and for Propranolol, respectively. Chromatogram of mixed standard of Hydrochlorothiazide and Propranolol is shown in Figure 9.

Preparation of Test SolutionTwenty tablets were taken, and the I.P. method was followed to determine the average weight. Finally, the weighed tablets are powdered and triturated well using mortar and pestle. A quantity of powder which is equivalent to the 100 mg of drugs was transferred to a clean and dry 100 ml of volumetric flask and add 70 ml of biorelevent media (FaSSIF), and the resulted solution was sonicated for 15 min using ultrasonicator, then the final volume was makeup to the mark with the same diluent. The final solution was filtered through a selected membrane filter (0.45 µm). From this above stock solution (1 ml) was transferred to five different 10 ml volumetric flasks and volume were made up to

Figure 3: Ultraviolet spectrum for Propranolol

Figure 4: Ultraviolet spectrum for Hydrochlorothiazide

Figure 5: Overlay spectra for Isosbestic point



10 ml with same solvent system biorelevent media (FaSSIF). The prepared solutions were injected in six

replicates into the HPLC system, and the observations were recorded.

Figure 6: Chromatogram for blank Preparation

Figure 7: Chromatogram for standard Hydrochlorothiazide

Figure 8: Chromatogram for standard Propranolol

Figure 9: Chromatogram mixed standard of Hydrochlorothiazide and Propranolol



Optimization of HPLC MethodThe selected and optimized mobile phase was acetonitrile:potassium dihydrogen phosphate buffer (0.02M, pH 3.0) (60:40v/v) and conditions optimized were flow rate (1.0 ml/min) and wavelength (275 nm, PDA-detector), run time was 8 min, and injection volume was 20 µl.

Method ValidationThe developed method for the simultaneous estimation of Hydrochlorothiazide and Propranolol was validated according to the guidelines ICH[17,18] for the method validation parameters such as system suitability, specificity, accuracy, precision linearity, ruggedness and robustness, detection limit (LOD), and quantification limit (LOQ).

Linearity and RangeAliquots of 0.2, 0.3, 0.4, 0.5, and 0.6 ml of standard working solution of Hydrochlorothiazide and Propranolol were pipetted out from the standard stock solution of 1000 µg/ml of Hydrochlorothiazide and Propranolol and transferred into a series of 10 ml clean dry volumetric flask and make volume up to the mark with biorelevent media to get the concentration of 20, 30, 40, 50, and 60 µg/ml of Hydrochlorothiazide and Propranolol.

PrecisionThe precision of each method was ascertained separately from the peak areas obtained by actual determination of five replicates of a fixed amount of drugs Hydrochlorothiazide and Propranolol.

Limit of Detection and QuantificationLOD and LOQ are measured as 3.3 × SD/S and 10 × SD/S, respectively, as per the Guidelines of ICH, Where SD is the standard deviation response (Y-intercept), and S is the slope of calibration curve. The LOD is the least amount of concentration of the analyte that gives a measurable response (signal to noise ratio of 3). The LOQ is the concentration of the analyte which gives a response that can be precisely quantified (signal to noise ratio of 10). Signal to noise ratio between 10:1 has been considered for this method.

RobustnessRobustness is intentional smallest changes in the flow rate. Temperature (±2°C), flow rate (±0.1 ml/min), and wavelength (±2 nm) were made to assess the influence on the developed method.

System SuitabilityThe system suitability parameters with respect of tailing factor, theoretical plates, repeatability, and resolution between Propranolol and Hydrochlorothiazide peaks were defined.

Forced degradation studyThe API (Hydrochlorothiazide and Propranolol) was subjected to keep in some stress conditions in various ways to observe the rate and extent of degradation that is likely to occur in the course of storage and/or after administration to the body. The different types of forced degradation pathways/studies studied here are acid hydrolysis, basic hydrolysis, thermal degradation, and oxidative degradation. Degradation studies were done to find the loss of drug by acid, alkali, thermal, photo, and oxidation was carried out.

Acid DegradationThe mixture sample was taken in a 10 ml volumetric flask and treated with 5 ml of 0.1 N hydrochloric acids and kept for 24 h. The solution was neutralized with 5 ml of 0.1 N NaOH, made the volume up to the mark with biorelevant media. From that, 0.1 ml was taken into a 10 ml volumetric flask and makeup to the mark with biorelevent media, then injected into the HPLC system against a blank of HCl.

Alkali DegradationThe mixture sample was taken in a 10 ml volumetric flask and treated with 5 ml of 0.1 N NaOH and kept for 24 h. The solution was neutralized with 5 ml of 0.1 N HCl, made the volume up to the mark with biorelevant media. From that, 0.1 ml was taken into a 10 ml volumetric flask and makeup to the mark with biorelevent media, and then injected into the HPLC system against a blank of NaOH.

Oxidative DegradationThe mixture sample was taken in a 10 ml volumetric flask and treated with 5 ml of 3% H2O2 and kept for 24 h. After 24 h made the volume up to the mark with biorelevant media. From that, 0.1 ml was taken into a 10 ml volumetric flask and make up to the mark with biorelevent media, and then injected into the HPLC system against a blank of 3% H2O2.

Photolytic DegradationThe samples were taken in a Petri dish and kept under UV light for 24 h at 254 nm. After 24 h 10 mg of the sample taken into a 10 ml volumetric flask then made the volume up to the mark with biorelevant media. From that, 0.1 ml was taken into a 10 ml volumetric flask and makeup to the mark with biorelevent media, and then injected into the HPLC system.

Thermal DegradationSamples were heated at 50°C for 24 h, then finally injected into the HPLC system against a blank of mobile phase.

AccuracyThe accuracy of the method was determined by calculating recovery of Hydrochlorothiazide and



Propranolol by the method of standard addition. Known amount of standard solution of Hydrochlorothiazide and Propranolol at 50%, 100%, and 150% was added to a pre-quantified sample solution and injected into the HPLC system.

Analysis of Marketed FormulationThe marketed formulation was assayed by above description. The peak areas were monitored at 275 nm and determination of sample concentrations were using by multilevel calibration developed on the same HPLC system under the same conditions using linear regression.

RESULTS AND DISCUSSIONOptimized MethodTo develop a precise, linear, specific, and suitable stability indicating RP-HPLC method for analysis of Hydrochlorothiazide and Propranolol, different chromatographic conditions were applied. The mobile phase used was a mixture of acetonitrile:potassium dihydrogen phosphate buffer (0.01M, pH 3.0) (60:40v/v) and conditions optimized were flow rate (1.0 ml/min) and wavelength (275 nm, PDA-detector), run time was 8 min, and injection volume was 10 μl. The proposed chromatographic conditions were found appropriate for the quantitative determination of the drugs and also suitable for determination in biorelevant media. The results are shown in Table 1.

Validation ResultsLinearity and RangeThe calibration standard solutions of Hydrochlorothiazide and Propranolol were injected into the HPLC system, and the chromatograms were recorded at 275 nm, and a calibration graph was obtained by plotting peak area versus concentration of Hydrochlorothiazide and Propranolol. Linearity range was found to be 20–

60 µg/ml for Hydrochlorothiazide and 20–60 µg/ml for Propranolol. The correlation coefficients were found to be 0.9988 and 0.9996; the slopes were found to be 17234 and 78021 and intercept was found to be 977181 and 927466 for Hydrochlorothiazide and Propranolol, respectively. The results are shown in Tables 2 and 3. The graphs of area versus concentration recorded and overlay chromatogram for both the drugs are shown in Figures 10-13.

Precision: The percentage relative standard deviations were calculated for Hydrochlorothiazide and Propranolol as presented in Table 4.

LOD and LOQ: The LOD was found to be 0.73 mg/ml and 0.95 mg/ml and LOQ was found to be 2.19 mg/ml and 3.01 mg/ml for Hydrochlorothiazide and Propranolol, respectively, which shows that the sensitivity of the proposed method is high.

Robustness: The obtained results make known that the method is robust. The gained results are summarized in Tables 5 and 6.

System suitability: To establish the system suitability for the proposed method and the parameters such as retention time, peak asymmetry, and theoretical

Table 1: Optimized chromatographic conditions

Mobile phase ratio Phosphate Buffer: Acetonitrile 40: 60

Column Phenomenex Gemini C18 (4.6×250mm) 5 µ

Column temperature 37°CWavelength PDA-DETECTOR, 275 nmFlow rate 1 ml/minInjection volume 10 µlRun time 8 min

Table 2: Linearity data for Hydrochlorothiazide

Linearity of Hydrochlorothiazide

Concentration (µg/ml) Peak area20 132174330 148581640 167356950 185049960 2001110

Table 3: Linearity data for Propranolol

Linearity of Propranolol

Concentration (µg/ml) Peak area20 251807330 326214940 401824550 478566160 5657356

Table 4: Repeatability data for Hydrochlorothiazide and Propranolol

Concentration (μg/ml)

Hydrochlorothiazide Propranolol

10 1818827 534624710 1795700 529754210 1799909 532731610 1807064 535744410 1816744 5376521Average 1807649 5341014SD 10132.81 30147.66%RSD 0.560552 0.564456RSD: Relative standard deviation, SD: Standard deviation

Table 5: Result of method robustness test for Hydrochlorothiazide

Change in parameter % RSDFlow (1.1 ml/min) 0.98Flow (0.9 ml/min) 0.92Temperature (27°C) 0.94Temperature (23°C) 0.97Wavelength of detection (277 nm) 0.59Wavelength of detection (273 nm) 0.57RSD: Relative standard deviation



the degradation (stress) studies indicated that the specificity of the developed method. The Propranolol was stable in thermal and photolytic (degradation) stress conditions, and the Hydrochlorothiazide was comparatively stable in oxidation degradation. The obtained results of forced degradation studies are given in Tables 8 and 9.

Accuracy: The mean recoveries were found to be 98.5%, 99.679%, and 100.145% for Hydrochlorothiazide and 99.346%, 100.2%, and 10.209% for Propranolol. The limit for mean percentage recovery is 98–102%, and as both the values are within the limit; hence, it can be said that the proposed method was accurate. The results are shown in Tables 10 and 11.

Analysis of marketed formulation: The amount of drugs in Ciplar H tablet was found to be 24.96 (99.84 ± 0.26%) mg/tab for Hydrochlorothiazide and 39.86 (99.65 ± 0.19%) mg/tab for Propranolol. The results are shown in Table 12. Chromatogram for marketed formulation was shown in Figure 11.

CONCLUSIONThe result shows the developed method is yet another suitable method for assay and stability studies which can help in the analysis of Hydrochlorothiazide and

Figure 10: Chromatogram for marketed formulation of Hydrochlorothiazide and Propranolol

Figure 11: Calibration curve for Propranolol

Table 6: Result of method robustness test for Propranolol

Change in parameter % RSDFlow (1.1 ml/min) 0.97Flow (0.9 ml/min) 0.96Temperature (27°C) 0.85Temperature (23°C) 0.46Wavelength of detection (257 nm) 0.92Wavelength of detection (253 nm) 0.71RSD: Relative standard deviation

Table 7: System suitability test parameters for Hydrochlorothiazide and Propranolol

Parameter Hydrochlorothiazide PropranololRetention Time (Min)

2.033 3.28

Resolution (Rs>2)

3.16 3.25

Asymmetry (T £ 2)

0.19 0.28

Theoretical plates

3896 6987

Tailing factor 1.56 1.95

plates, and tailing factor were taken and obtained results were presented in Table 7.

Forced degradation study: The obtained results of



the developed method is specific for the estimation of Hydrochlorothiazide and Propranolol in the presence of degradation products. Further, the proposed RP-HPLC method has excellent sensitivity, precision, and reproducibility.

Figure 13: Overlay chromatogram for linearity

propranolol in biorelevent media and formulations. Based on peak purity results, obtained from the analysis of force degradation samples using the described method, it can be concluded that the absence of coeluting peak along with the main peak of Hydrochlorothiazide and Propranolol indicated that

Figure 12: Calibration curve for HTZ

Table 8: Results of forced degradation studies of Hydrochlorothiazide API

Stress condition Time (h) Assay of active substance (Hydro)

Assay of degraded products (Hydro)

Mass Balance (%)

Acid Hydrolysis (0.1N HCl) 24 80.223 19.777 100Basic Hydrolysis (0.IN NaOH) 24 82.411 17.589 100Thermal degradation (50°C) 24 83.084 16.916 100UV (254 nm) 24 79.223 20.777 1003% Hydrogen peroxide 24 87.766 12.234 100UV: Ultraviolet

Table 9: Results of forced degradation studies of Propranolol API

Stress condition Time (h) Assay of active substance (Prop)

Assay of degraded products (Prop)

Mass balance (%)

Acid Hydrolysis (0.1N HCl) 24 80.303 19.697 100Basic Hydrolysis (0.IN NaOH) 24 76.847 23.153 100Thermal degradation (50°C) 24 98.997 1.003 100UV (254 nm) 24 99.007 0.993 1003% Hydrogen peroxide 24 77.877 22.123 100



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Table 10: Recovery study data of Hydrochlorothiazide

Sample name (%) Amount added (µg/ml) Amount found (µg/ml) % Recovery Statistical analysisS1:50 50 49.149 98.298 Mean-98.5S2:50 50 49.329 98.658 S.D-0.183989S3:50 50 49.272 98.544 %RSD-0.186791S4:100 100 99.367 99.367 Mean-99.679S5:100 100 100.175 100.175 S.D-0.43429S6:100 100 99.495 99.495 %RSD=0.435689S7:150 150 150.21 100.14 Mean-100.1453S8:150 150 150.294 100.196 S.D-0.048222S9 :150 150 150.15 100.1 %RSD-0.048152RSD: Relative standard deviation, SD: Standard deviation

Table 11: Recovery study data of Propranolol

Sample name (%) Amount added (µg/ml) Amount found (µg/ml) % Recovery Statistical analysisS1:50 50 49.548 99.096 Mean-99.34667S2:50 50 49.836 99.672 S.D-0.29517S3:50 50 49.636 99.272 %RSD-0.297111S4:100 100 99.433 99.433 Mean-100.2S5:100 100 100.715 100.715 S.D-0.677133S6:100 100 100.452 100.452 %RSD=0.675781S7:150 150 150.371 100.247 Mean-100.2093S8:150 150 150.434 100.289 S.D-0.103761S9 :150 150 150.139 100.092 %RSD-0.103544RSD: Relative standard deviation, SD: Standard deviation

Table 12: Assay of marketed formulation of Hydrochlorothiazide and Propranolol

Drug (Ciplar H) Label claim (mg) Amount found (mg) % Label Claim±% RSD (n = 3)Hydrochlorothiazide and Propranolol 25/40 24.96/39.86 99.84 ± 0.26/99.65 ± 0.19RSD: Relative standard deviation



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Source of support: Nil; Conflict of interest: None Declared

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