Int J Pharm Pharm Sci, Vol 6, Issue 9, 318-323Original Article
DEVELOPMENT AND VALIDATION OF RP-HPLC METHOD FOR DETERMINATION OF HYDROCHLOROTHIAZIDE, OLMESARTAN MEDOXOMIL AND THEIR RELATED SUBSTANCES IN COMBINED TABLET DOSAGE FORM
URVESH M. PATEL, AVANI B. CHOKSHI, PRITESH R. DESAI
Ramanbhai Patel College of Pharmacy, Charotar University of Science and Technology, CHARUSAT Campus, Changa 388421, Ta. Petlad, Dist. Anand, Gujarat, India.
Email: urvesh.pharmacist@gmail.com
Received: 15 Jul 2014 Revised and Accepted: 21 Aug 2014
ABSTRACT
Objective: Development of RP-HPLC method for determination of Hydrochlorothiazide (HCTZ), Olmesartan medoxomil (OLM) and their related substances in combined tablet dosage form and validation of the developed method.
Methods: Gradient mobile phase system was used for estimation of drug contents and their related substances. Mobile phase A contained the mixture of Acetonitrile and 15 mM Phosphate buffer (pH adjusted to 3.4 with orthophosphoric acid) in the ratio of 20:80. Mobile phase B contained the same mixture in the ratio of 80:20. Chromatographic separation was carried out at the mobile phase flow rate of 0.8 mL/min using C18 Phenomenax inplace of Enable (250 × 4.6 mm) 5 μm column and detection was made at 254 nm.
Results: The linearity of developed method was tested in the range of 62.5-187.5 μg/mL for Hydrochlorothiazide, 100-300 μg/mL for Olmesartan medoxomil, 1-1.8 μg/mL for Hydrochlorothiazide. The % recovery was found to be 99.88-100.67 % (HCTZ), 99.14-99.91 % (OLM), 99.11-100.71% (HCTZ-IMP) and 98.13-100.83% (OLM-IMP). The assay of marketed formulation was found to be 99.78% (HCTZ) and 99.26% (OLM).
Conclusion: A simple, precise and accurate RP-HPLC method was developed for determination of Hydrochlorothiazide, Olmesartan medoxomil and their related substances.
Keywords: Hydrochlorothiazide (HCTZ), Olmesartan medoxomil (OLM), Hydrochlorothiazide Related Impurity (HCTZ-IMP), Olmesartan medoxomil Related Impurity (OLM-IMP), Reversed phase high performance liquid chromatography (RP-HPLC).
INTRODUCTION
Hydrochlorothiazide (HCTZ) is a diuretic antihypertensive drug. Chemically it is 6-chloro-3, 4-dihydro-2H-1,2,4-benzothiadiazine-7-sulphonamide 1,1-dioxide clinically used in treatment of hypertension and management of edema. It inhibits the reabsorption of sodium in the distal convoluted tubule thereby decreasing the blood pressure.
Olmesartan medoxomil (OLM) is an antihypertensive drug. Chemically it is (5-Methyl-2-oxo-1,3-dioxol-4-yl)methyl 4-(1-hydroxy-1-methylethyl)-2-propyl-1-[[2′-(1H-tetrazol-5-yl)biphenyl-4-yl]methyl]-1H-imidazole-5-carboxylate clinically used in the treatment of hypertension. Olmesartan is an angiotensin receptor blocker that selectively inhibits the binding of Angiotensin II to AT1. This results in decreased vascular resistance and blood pressure.
The literature review reveals various analytical methods for determination of Hydrochlorothiazide like UV [1,2], RP-HPLC [3,4], and HPTLC [5] and Olmesartan medoxomil UV [6,7], RP-HPLC [8,9] and HPTLC [10] individually as well as in combined dosage form with other drugs [11,12] but there was no method available for determination of these two drugs along with their related substances in combined tablet dosage form. There are various sources through which impurities (related substances & other impurities) may be generated in the drug product affecting its efficacy. It becomes necessary to have a method which can analyze such impurities in drug product.
Hydrochlorothiazide and Olmesartan medoxomil both have some related substance reported in pharmacopoeias [13,14]. They may be present in combined drug product in some amount or may get generated in the amount beyond the acceptance limit under worse conditions. So, the objective of present work was determination of Hydrochlorothiazide, Olmesartan medoxomil and their related substances in combined tablet dosage form by validated RP-HPLC method as per ICH Q2(R1) [15].
Fig. 1: Structure of HCTZ
Fig. 2: Structure of OLM
Fig. 3: Chlorothiazide (HCTZ-IMP)
Fig. 4: Olmesartan acid impurity (OLM-IMP)
MATERIALS AND METHODS
Instrumentation
HPLC: Shimadzu LC-20AT system equipped with LC solution software, PDA detector, injection volume: 20μL, column: C18 Phenomenax inplace of Enable (250 × 4.6 mm) 5 μm, Milli Q water purification system.
Materials
Standard gift samples of Hydrochlorothiazide, Olmesartan medoxomil and their related impurities were provided by Zydus Cadila Healthcare Ltd. Combined tablet dosage form OLMEZEST H-20 was purchased from local market. Acetonitrile (HPLC Grade) was procured from Loba Chemie Pvt Ltd, Mumbai. Potassium dihydrogen phosphate (HPLC Grade) was used for preparation of buffer solution.
Chromatographic conditions
The chromatographic column used was C18 Phenomenax inplace of Enable (250 × 4.6 mm) 5 μm. The gradient method was employed with mobile phase A & B using 0.8 mL/min flow rate and 254 nm detection wavelength during entire gradient program. Injection volume was 20 μL.
Mobile phase A- Acetonitrile: 15 mM Phosphate buffer (pH 3.4) in the ratio of 20:80
Mobile phase B- Acetonitrile: 15 mM Phosphate buffer (pH 3.4) in the ratio of 80:20
Table 1: Gradient Program
| Time (min) | A (% v/v) | B (% v/v) |
| 0-26 | 87→13 | 13→87 |
| 26-27 | 13→87 | 87→13 |
| 27-30 | 87 | 13 |
Preparation of Solutions
Preparation of standard stock solutions
Stock solutions of 1250 μg/mL of HCTZ and 2000 μg/mL of OLM were prepared by dissolving 125 mg of HCTZ and 200 mg of OLM in 100 mL of acetonitrile in separate 100 mL volumetric flasks respectively.
HCTZ-IMP (4 mg) was accurately weighed and transferred to a 10 mL volumetric flask and then dissolved and diluted to 10 mL with acetonitrile. From above solution 1 mL was transferred to a 10 mL volumetric flask and diluted to 10 mL with acetonitrile to obtain stock solution of 40 µg/mL.
OLM-IMP (5 mg) was accurately weighed and transferred to a 10 mL volumetric flask and then dissolved and diluted to 10 mL with acetonitrile. From above solution 2 mL was transferred to a 10 mL volumetric flask and diluted to 10 mL with acetonitrile to obtain stock solution of 100 µg/mL.
Validation of developed method
The developed method was validated according to International Conference on Harmonization (ICH) Q2(R1) guideline.
System suitability
They are used to verify that resolution and reproducibility of chromatographic system are adequate for the analysis to be done. The parameters include Resolution (R), Tailing factor (T), Theoretical plates and Precision of replicate injection.
Linearity
Linearity of response was assessed by calibration curve in terms of slope, intercept and regression coefficient values. Five standard mixture solutions of HCTZ (62.5-187.5 µg/mL), OLM (100-300 µg/mL), HCTZ-IMP (1-1.8 µg/mL) and OLM-IMP (1-3 µg/mL) were analyzed for linear regression analysis.
Precision
Intraday and interday precision were performed over 3 levels of concentration at 3 different times in a day and on 3 different consecutive days respectively. Concentration levels of standard mixture of HCTZ, OLM, HCTZ-IMP and OLM-IMP used were 50%, 100% and 150% of the test concentration.
Specificity
Specificity for drug substances was assessed by taking the overlay of chromatograms of standard and sample. Specificity for impurities was assessed by taking the overlay of chromatogram of sample (i. e. drug product) and that of the mixture of impurity standards & drug substances.
Accuracy (n=3)
Accuracy was determined in terms of percentage recovery. Accuracy was determined by spiking 3 different known concentrations of standard to sample solution to obtain three levels of concentrations (80%, 100% and 120%).
Limit of Detection (LOD) and Limit of Quantitation (LOQ)
LOD and LOQ were determined using following equations.
LOD = 3.3 σ/S
LOQ = 10 σ/S
Where, σ = Standard deviation of response
S = Slope of calibration curve
Robustness
The developed HPLC method was tested for robustness using factorial design (23-1) with four experiments. Three factors selected were Flow rate, Mobile phase ratio in line A and pH of buffer solution. The selection of factors was based on observation during method development. Each factor was studied at two levels. The levels of factors studied were selected according to error ranges which would be typically encountered in an analytical laboratory.
Table 2: Selected factors and levels
| S. No. | Factor | Low level (-1) | High level (+1) |
| 1. | Mobile phase ratio in line A | 15:85 | 25:75 |
| 2. | pH of buffer solution | 3.2 | 3.6 |
| 3. | Flow rate | 0.7 | 0.9 |
Table 3: Factorial design 23-1
| S. No. | Mobile phase in line A | pH of buffer solution | Flow rate |
| 1. | 15:85 | 3.2 | 0.7 |
| 2. | 25:75 | 3.2 | 0.9 |
| 3. | 15:85 | 3.6 | 0.9 |
| 4. | 25:75 | 3.6 | 0.7 |
Analysis of marketed formulation
Twenty tablets were powdered and the powder quantity equivalent to 200 mg of OLM and 125 mg of HCTZ was accurately weighed and transferred to a 100 mL volumetric flask. 50 mL of acetonitrile was added to dissolve the drug content and solution was filtered. The filtrate solution was diluted to 100 mL using acetonitrile. From above solution, 1 mL was transferred to a 10 mL volumetric flask and diluted to 10 mL with mixture of acetonitrile and water (50:50) to obtain final solution of 200 µg/mL (OLM) and 125 µg/mL (HCTZ). The amount of both the drugs was calculated from regression equation of calibration curve and percentage assay was calculated.
Forced degradation of marketed formulation
Sample solution of marketed formulation was prepared containing 2500 µg/mL OLM and 1562.5 µg/mL HCTZ. This solution was used as stock solution for degradation.
Acidic degradation
10 mL of above stock solution was transferred to a 100 mL volumetric flask and 10 mL of 0.1N HCl was added to it. Solution was kept for 60 min at room temperature and then it was neutralized. The final volume was made up to 100 mL using mixture of acetonitrile and water (50:50).
Alkaline degradation
10 mL of stock solution was transferred to a 100 mL volumetric flask and 10 mL of 0.1N NaOH was added to it. Solution was kept for 30 min at room temperature and then it was neutralized. The final volume was made up to 100 mL using mixture of acetonitrile and water (50:50).
Oxidative degradation
10 mL of stock solution was transferred to a 100 mL volumetric flask and 10 mL of 5% H2O2 was added to it. Solution was kept for 60 min at room temperature. The final volume was made up to 100 mL using mixture of acetonitrile and water (50:50).
The degraded samples were subjected to HPLC analysis using developed method and the percentage impurity of interest was calculated using following equation.
% Impurity = (Area of that impurity peak / Total Area of all peaks) * 100
RESULTS AND DISCUSSION
Optimization of chromatographic condition
The retention time was found to be 6.194 min (HCTZ), 8.389 min (HCTZ-IMP), 15.226 min (OLM) and 19.309 min (OLM-IMP).
Fig. 5: Chromatogram of Mixture of HCTZ, HCTZ-IMP, OLM & OLM-IMP (125, 1.4, 200 & 2 μg/mL in ACN:Water-50:50 respectively)
System Suitability Test
Results of theoretical plate, tailing factor, resolution and precision of injection repeatability have been shown below (Table 4).
Table 4: Data of System Suitability Test (n=6)
| Parameter | HCTZ | OLM | HCTZ-IMP | OLM-IMP |
| Retention Time (min) ±SD | 6.194±0.021 | 15.16±0.115 | 8.380±0.014 | 19.279±0.053 |
| Tailing Factor ±SD | 1.343±0.030 | 1.481±0.065 | 0.929±0.043 | 1.346±0.014 |
| Theoretical Plate ±SD | 2363.281 ± 214.089 | 29127.175 ± 1128.663 | 1797.433 ± 91.445 | 51571.147 ± 1031.876 |
| Resolution | - | 11.908 | 3.373 | 11.717 |
| %RSD (Injection repeatability) | 0.95 | 1.72 | 0.64 | 0.54 |
Linearity
The data of linearity of HCTZ, OLM, HCTZ-IMP and OLM-IMP has been shown below (Table 5). The results are within the acceptance criteria.
Precision
The % RSD for Intraday and Interday precision were found to be less than 2 for HCTZ, OLM, HCTZ-IMP and OLM-IMP which indicates that the developed method is precise.
The results of precision study have been shown below (Intraday: Table 7 & Table 8) (Interday: Table 9 & Table 10).
Fig. 6: Overlay Chromatogram for Linearity of HCTZ, OLM, HCTZ-IMP, OLM-IMP.
Specificity
Overlay of chromatograms for specificity has been shown below (fig. 7 and fig. 8).
Fig. 7: Overlay Chromatogram of Standard & Sample for Specificity of HCTZ & OLM.
Accuracy
Accuracy study was performed at three levels for HCTZ, OLM, HCTZ-IMP and OLM-IMP (Table 11 and 12). The values of % recovery were found in the acceptance limit of 98-102 % with low % RSD, which justifies that, the method is accurate and free from the interference of excipients used in formulation and is applicable for analysis of marketed formulation.
Table 5: Linear regression data of HCTZ, OLM, HCTZ-IMP, OLM-IMP
| Parameter | HCTZ | OLM | HCTZ-IMP | OLM-IMP |
| Regression equation | y = 30,571.4944x + 291,450.4 | y = 47,953.4380x - 57,777.4 | y = 188,952.50x - 73,059.9 | y = 35,586.80x + 104,769.8 |
| Regression coefficient mean ±SD | 0.997±0.00037 | 0.998±0.00032 | 0.999±0.00030 | 0.997±0.00089 |
| Mean of intercept ±SD | 291,450.4 ± 12269.537 | 57,777.4 ± 3303.837 | 73,059.9 ± 2436.1 | 104,769.8 ± 1141.972 |
| 95% Confidence interval for intercept | 277565.705 to 305335.095 | 54038.647 to 61516.153 | 70303.113 to 75816.687 | 103477.499 to 106062.101 |
| Slope ±SD | 30,571.4944 ± 196.99 | 47,953.4380 ± 489.83 | 188,952.50 ± 1563.52 | 35,586.80 ± 692.49 |
| 95% Confidence interval for Slope | 30185.3944 to 30957.5944 | 46993.372 to 48913.504 | 185888.001 to 192016.999 | 34803.151 to 36370.449 |
Fig. 8: Overlay Chromatogram of Standard spiked with impurities and Sample for Specificity of HCTZ-IMP & OLM-IMP
Table 6: LOD and LOQ data
| HCTZ | OLM | HCTZ-IMP | OLM-IMP | |
| LOD (μg/mL) | 1.32 | 5.08 | 0.04 | 0.10 |
| LOQ (μg/mL) | 4.01 | 15.39 | 0.13 | 0.32 |
Robustness
The effect of 3 factors on the response was analyzed using design expert software and p-value was obtained for each factor. The p-value for factors Mobile phase ratio in line A and pH of buffer solution were found to be greater than 0.05 which indicates that they are non significant factors within the study range of factors for robustness. The p-value of factor Flow rate was found to be less than 0.05 for HCTZ and OLM which indicates that flow rate is a significant factor affecting the response of HCTZ and OLM.
Table 7: Intraday Precision Data of HCTZ & OLM (n=3)
| HCTZ | OLM | ||||
| Conc. (μg/mL) | Mean Area ± SD | %RSD | Conc. (μg/mL) | Mean Area ± SD | %RSD |
| 62.5 | 2098427 ± 4553.48 | 0.22 | 100 | 4585538 ± 1659.25 | 0.04 |
| 125 | 4144189 ± 9575.98 | 0.23 | 200 | 9648941 ± 83119.86 | 0.86 |
| 187.5 | 5967782 ± 24816.77 | 0.42 | 300 | 14137073 ± 65355.89 | 0.46 |
Table 8: Intraday Precision Data of HCTZ-IMP & OLM-IMP (n=3)
| HCTZ-IMP | OLM-IMP | ||||
| Conc. (μg/mL) | Mean Area ± SD | %RSD | Conc. (μg/mL) | Mean Area ± SD | %RSD |
| 1 | 114895 ± 1537.68 | 1.34 | 1 | 138950 ± 425.61 | 0.31 |
| 1.4 | 192754 ± 614.51 | 0.32 | 2 | 175622 ± 88.15 | 0.05 |
| 1.8 | 265152 ± 170.28 | 0.06 | 3 | 211829 ± 1407.87 | 0.66 |
Table 9: Interday Precision Data of HCTZ & OLM (n=3)
| HCTZ | OLM | ||||
| Conc. (μg/mL) | Mean Area ± SD | %RSD | Conc. (μg/mL) | Mean Area ± SD | %RSD |
| 62.5 | 1930766 ± 10293.61 | 0.53 | 100 | 4543694 ± 56438.02 | 1.24 |
| 125 | 4090065 ± 39388.14 | 0.96 | 200 | 10149030 ± 119970.5 | 1.18 |
| 187.5 | 6222916 ± 654.82 | 0.01 | 300 | 15401259 ± 39407.02 | 0.26 |
Table 10: Interday Precision Data of HCTZ-IMP & OLM-IMP (n=3)
| HCTZ-IMP | OLM-IMP | ||||
| Conc. (μg/mL) | Mean Area ± SD | %RSD | Conc. (μg/mL) | Mean Area ± SD | %RSD |
| 1 | 113193 ± 2015.28 | 1.78 | 1 | 121755 ± 1495.97 | 1.22 |
| 1.4 | 215568 ± 1808.22 | 0.84 | 2 | 180156 ± 1315.37 | 0.73 |
| 1.8 | 281837 ± 1262.29 | 0.46 | 3 | 220241 ± 2198.62 | 0.99 |
Analysis of marketed formulation
Marketed formulation OLMEZEST H-20 containing 20 mg of OLM and 12.5 mg of HCTZ was used for assay purpose. Tablet powder was dissolved, filtered and diluted to make the final concentration of 200 μg/mL (OLM) and 125 μg/mL (HCTZ). Along with the peaks of HCTZ and OLM, a small peak at 8.386 min was obtained which is a peak of HCTZ Related substance (i. e. HCTZ-IMP) present in marketed formulation.
Forced degradation of marketed formulation:
The tablet sample was subjected to acidic, basic and oxidative degradation to generate the impurities in sample. The impurities of interest were quantified using regression equation obtained from calibration curves of impurity standards. In all three conditions for degradation of marketed formulation, HCTZ-IMP was generated in more amounts and OLM-IMP was generated in very less amount because the chromatogram of marketed formulation without degradation shows that the HCTZ Related Impurity was already present within the acceptance limit and after degradation its amount got increased.
Table 11: Accuracy Data of HCTZ & OLM
| Sample (μg/mL) | Std added (μg/mL) | Total
(μg/mL) |
Mean Area ± SD | %
RSD |
Total found (μg/mL) | %
Reco-very |
|
| HCTZ | 75 | 60 | 135 | 4446142±54748.6 | 1.23 | 135.90 | 100.67 |
| 75 | 75 | 150 | 4871521±57179.3 | 1.17 | 149.82 | 99.88 | |
| 75 | 90 | 165 | 5354073±43326.3 | 0.81 | 165.60 | 100.36 | |
| OLM | 120 | 96 | 216 | 10211399±139896.2 | 1.37 | 214.15 | 99.14 |
| 120 | 120 | 240 | 11370489±89519.63 | 0.79 | 238.32 | 99.30 | |
| 120 | 144 | 264 | 12591083±10306.66 | 0.08 | 263.77 | 99.91 |
Table 12: Accuracy Data of HCTZ-IMP & OLM-IMP
| (HCTZ + OLM) (μg/mL) | Impurity Std spiked
(μg/mL) |
Mean Area±SD | % RSD | Spiked Std found
(μg/mL) |
% Recovery | |
| HCTZ IMP | 125 + 200 | 1.12 | 137886±822.50 | 0.59 | 1.11 | 99.11 |
| 125 + 200 | 1.4 | 192577±622.73 | 0.32 | 1.41 | 100.71 | |
| 125 + 200 | 1.68 | 243042±1200.65 | 0.49 | 1.67 | 99.40 | |
| OLM IMP | 125 +200 | 1.6 | 160769±1087.36 | 0.67 | 1.57 | 98.13 |
| 125 + 200 | 2 | 175133±434.17 | 0.25 | 1.97 | 98.50 | |
| 125 + 200 | 2.4 | 191004±763.04 | 0.39 | 2.42 | 100.83 |
Table 13: p-values of factors for robustness
| p-value of factors | |||
| Mobile Phase Ratio in line A | pH of buffer solution | Flow rate | |
| HCTZ | 0.9166 | 0.8075 | 0.0223 |
| OLM | 0.9700 | 0.8175 | 0.0173 |
| HCTZ-IMP | 0.7276 | 0.2360 | 0.4151 |
| OLM-IMP | 0.6794 | 0.7586 | 0.0841 |
p-value < 0.05: Factor has significant effect on response, p-value > 0.05: Factor has no significant effect on response
Fig. 9: Chromatogram of marketed formulation
Fig. 10: Acidic degradation of tablet sample
Fig. 11: Alkaline degradation of tablet sample
Fig. 12: Oxidative degradation of tablet sample
Table 14: Data of Marketed formulation Analysis
| Mean Area±SD | % RSD | % Assay | |
| HCTZ (125 μg/mL) | 4104467±5848.43 | 0.14 | 99.78 % |
| OLM (200 μg/mL) | 9461639±88939.41 | 0.94 | 99.26 % |
| HCTZ-IMP | 139486±727.99 | 0.52 | 1.01 % |
Table 15: Acidic degradation data
| Impurity | Retention Time (min) | Peak Area | Conc. found (μg/mL) | % Impurity |
| HCTZ-IMP | 8.278 | 850913 | 4.89 | 5.79 % |
| OLM-IMP | 18.795 | 110533 | 0.16 | 0.75 % |
Table 16: Alkaline degradation data
| Impurity | Retention Time (min) | Peak Area | Conc. found (μg/mL) | % Impurity |
| HCTZ-IMP | 8.148 | 664499 | 3.90 | 4.10 % |
| OLM-IMP | 19.466 | 119840 | 0.42 | 0.74 % |
Table 17: Oxidative degradation data
| Impurity | Retention Time (min) | Peak Area | Conc. found (μg/mL) | %Impurity |
| HCTZ-IMP | 8.537 | 396041 | 2.48 | 4.94 % |
| OLM-IMP | 19.550 | 117986 | 0.37 | 1.47 % |
CONCLUSION
The developed method was validated as per ICH Q2(R1) guideline and was found to be within the prescribed limit. It concludes that the developed method is simple, accurate, sensitive and precise and suitable for routine quality control analysis of Hydrochlorothiazide, Olmesartan medoxomil and their related substances in combined tablet dosage form.
CONFLICT OF INTERESTS
Declared None
ACKNOWLEDGEMENT
The authors are very thankful to Dr. R. H PARIKH; Principal, Ramanbhai Patel College of Pharmacy, Changa, for providing the research facilities. We are also thankful to Zydus Cadila Healthcare Ltd for providing gift samples of Hydrochlorothiazide and Olmesartan medoxomil.
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