Stability Indicating RP-HPLC Method Development and Validation for Simultaneous Estimation of Metoprolol Succinate and Chlorthalidone in Bulk and in Tablet Dosage Form RAMESHWAR GHOLVE*, SANJAY PEKAMWAR and TUKARAM KALYANKAR

RP-HPLC method has been developed along with stability indicating attribute for simultaneous estimation of Metoprolol Succinate and Chlorthalidone in bulk and in tablet dosage form with minimized drug extraction steps. The chromatographic analysis was performed isocratically by using Oyster ODS3, 150-4.6 mm column having particle size of 5 μm (Merck & Co.) as stationary phase maintained at ambient temperature (about 25°C) with 1.0 mL/min of flow rate and 20 mM phosphate buffer having pH 2.3 (adjusted with10% orthophosphoric acid) and Acetonitrile (650:350, v/v) as Eluent at wavelength 225 nm. Individual drug substances as well as combination drug product was subjected to acid, alkali, oxidative, photolytic, thermal and humidity degradation, the peaks due to degraded product were significantly separated out from active analytes peak. The method was validated for the specificity, linearity, detection limit, quantitation limit, precision, accuracy, robustness and solution stability as per ICH guidelines and successfully used for regular analysis.


INTRODUCTION
Survey of literature reveals that few methods are available for simultaneous estimation of MTL and CTD by UV-Spectrophotometric 5-6 and HPLC [7][8][9][10][11] techniques. The existing methods have one of the drawbacks like time consuming procedures, long run time, less sensitivity and low resolution. Among all these reported methods, two methods reported for assay of Metoprolol Succinate and Chlorthalidone in which forced degradation study was performed but with poor experimental design. In addition to this, stability of mobile phase preparation, standard and sample solution along with some robustness parameters need to be performed. Hence, an attempt has been made to perform forced degradation study with proper experimental design on individual drug substances and combination drug product to develop a validated RP-HPLC method with stability indicating attribute for the simultaneous estimation of MTL and CTD which is more simple, rapid, sensitive, accurate, precise and robust enough.
To understand the inherent stability characteristics of active component(s), stress testing needs to be carried out. 12 Impurities and/or related substances are generated from the manufacturing process and/or degradation products from improper storage or handling or as metabolites which could be active, inactive or even toxic that will significantly impact the results with respect to quality, safety, Technologies Limited, Model: WPS211) was used for collecting the Ultrapure water for the experiment. The method was developed on Oyster ODS3, 150-4.6 mm column having particle size of 5 µm (P/N: S670153, Make: Merck & Co.) column connected to a HPLC system (Make: Shimadzu, Model: SCL-10Avp) equipped with UV detector having rheodyne sample injection port with 20 μL loop. The chromatographic instrument was controlled by LC Solution software version 1.25 and same used for the chromatographic data handling.

Chromatographic conditions
The details of chromatographic conditions maintained for analysis during the experimental work are given in Table 1. sonication, allowed to attain room temperature and with diluent, made up to the mark and mixed well (Concentration of Metoprolol Succinate = 190 μg/ mL; Concentration of Chlorthalidone = 50 µg/mL).
Standard solution prepared in duplicate to confirm the suitability of standard.

Preparation of sample solution
Average weight of Metoprolol Succinate and Chlorthalidone Tablets 47.5/12.5 mg (Revelol ® -CH 50/12.5) was determined from weight of 20 tablets. Powdered these tablets by using mortor and pestle, and then transferred 376.5 mg (equivalent to MTL 47.5 mg and CTD 12.5 mg) of this fine powder into a 250 mL dry volumetric flask. Added 175 mL diluent and sonicated for 25 min with intermediate shaking. After sonication, allowed to attain the room temperature and with diluent, made up to the mark and mixed well. Finally, filtered by using Whatman filter paper (Cat No.: 1001-125, Make: GE Healthcare UK Ltd.) by discarding initial 5 mL of the filtrate and used as sample solution for assay.

Method validation
The proposed chromatographic method was validated as per the ICH guideline Q2(R1). 17

Specificity
For specificity, interference of the blank solution at the retention time of MTL and CTD peak were checked. Also, specificity were studied in forced degradation studies with extended run time to the twice of actual run time which ensure that no late eluting degradant peaks, as elution mode is isocratic. In this study, the forced degradation was performed by exposing both drug substances individually and sample of drug product (Revelol ® -CH 50/12.5) with known concentration to different stress conditions like acid degradation (5 N Hydrochloric acid, 3 h at room temperature), alkali degradation (5 N Sodium hydroxide, 3 h at room temperature), oxidative degradation (6% v/v Hydrogen Peroxide, 3 h at room temperature), thermal degradation (dry heat at 90 0 C for 24 h in hot air oven), photolytic (UV light for 24 h in Photostability chamber) and humidity degradation (75% relative humidity for 48 h in stability chamber). Similarly, acid, alkali and oxidative stressed blank solutions were prepared without active component to check any interference at retention time of active analytes peaks. However, samples of stress

Preparation of mobile phase
Mixed 350 mL of Acetonitrile with 650 mL of 20 mM phosphate buffer having pH 2.3 and degassed by 10 min of sonication.

Preparation of standard solution
Weighed 47.5 mg MTL and 12.5 mg of CTD standard and transferred into a 250 mL dry volumetric flask. Added 175 mL diluent and sonicated for 10 min with intermediate shaking to dissolve. After degradation were analyzed by using the proposed chromatographic method and mass balance results (%assay + %degradant) was calculated for all the stressed samples against standard solution and compared with unstressed sample.

System Suitability and System Repeatability
The system suitability parameters like tailing factor, peak area, retention time, resolution and theoretical plates count were determined from 1 st injection of standard solution. The systems repeatability parameters are determined by injecting the five replicates of first standard solution and single replicate of second standard solution in the chromatographic system and further determining the %RSD for standard 1 and %relative difference for standard 2.

Linearity
Linearity was demonstrated at five different concentration levels prepared from standard stock solution (Concentration of Metoprolol Succinate = 948.9536 µg/mL and Chlorthalidone = 248.1285 µg/mL). It was performed from 60% to 140% of the nominal working concentration in the range of 113.8744 -265.7070 µg/mL and 29.7754 -69.4760 µg/mL for MTL and CTD respectively. The linearity graph was plotted for concentration versus peak area response and determined the squared correlation coefficient (R 2 ).

Detection Limit and Quantitation Limit
The detection limit (DL) and quantitation limit (QL) of MTL and CTD were determined based on the standard deviation (residual value) of response and the slope method. As per ICH guideline, it was determined from calibration curve of MTL and CTD by using below mentioned formulae.

DL=(3.3 x σ)/S and QL= (10 x σ)/S
Where, σ = the standard deviation of the response; S = the slope of the calibration curve

Accuracy
Accuracy were assessed by triplicate analyses of sample containing placebo mixture with Metoprolol Succinate and Chlorthalidone at three concentrations 60%, 100% and 140% of the nominal working concentration. At every concentration level, a triplicate samples were prepared and each sample was injected once, and the average recovery for triplicate samples at each concentration level was calculated.

Precision
For assay determination of MTL and CTD, precision study performed by using homogeneous samples.

Method Repeatability
Method Repeatability was demonstrated by injecting six sample preparations of MTL and CTD Tablets 47.5/12.5 mg (Revelol ® -CH 50/12.5) using batch no. GYD039001AK (Make: IPCA Laboratories Ltd., Mumbai-India) as per developed method. Assay sample preparation was made on 6 replicate samples and calculated the %Assay, %RSD and 95 %confidence interval (95% CI). Also, the system suitability and the system repeatability results were determined.

Intermediate Precision
Intermediate Precision was demonstrated from six determinations of the same sample of MTL and CTD Tablets 47.5/12.5 mg (i.e. batch, storage conditions, container, etc) tested for Method Repeatability by different analyst on different day. Assay sample preparation was made on 6 replicate samples and calculated the %Assay, %RSD, 95% confidence interval (95% CI) and compares the average results obtained in the Method Repeatability and Intermediate Precision study. Also, the system suitability and the system repeatability results were determined.

Robustness
The method robustness was demonstrated by doing conscious changes in method parameters. Filter compatibility was demonstrated for MTL and CTD Tablets 47.5/12.5 mg (Revelol ® -CH 50/12.5) using three sample preparations. Each sample solution was divided into three parts. First part was filtered by using Whatmann filter (Cat no. 1001-125, Make: GE Healthcare UK Ltd.) by discarding initial 5 mL of the filtrate as per method. The second part was filtered by using 0.45 µm PVDF Syringe filter (Cat no. SYVF0602MNXX104, Make: Advanced Microdevices Pvt. Ltd.) by discarding initial 5 mL of the filtrate and third part was filtered by using 0.45 µm Nylon Syringe filter (Cat no. SYNN0602MNXX104, Make: Advanced Microdevices Pvt. Ltd.) by discarding initial 5 mL of the filtrate and used as the sample solution, the %assay and %relative difference were calculated.
The extraction efficiency of method was established by doing conscious changes into sonication time for sample preparation from 20 min to 30 minutes. Change in sonication time for sample preparation was checked with three replicate sample preparations of MTL and CTD Tablets 47.5/12.5 mg (Revelol ® -CH 50/12.5) for each changed condition, the %assay and %relative difference were calculated.
As part of robustness study, deliberate change in chromatographic parameters with respect to change in flow rate (± 0.1 mL/min) from 0.9 mL/min to 1.1 mL/min; change in the organic composition of mobile phase (± 10%) from 650:315, v/v to 650:385, v/v; change in pH (± 0.1) of mobile phase buffer from pH 2.2 to pH 2.4; change in the quantity of Potassium Dihydrogen Phosphate for mobile phase buffer (± 10%) from 2.448 g/1000 mL to 2.992 g/1000 mL and each changed condition impact on the method was assessed. The system suitability and system repeatability results were checked for each changed condition.

Solution Stability
The stability of standard solutions carried out on two preparations and evaluated after day 1 and day 2, storage at room temperature and in refrigerator (2-8°C). The stored standard solution results were compared with freshly prepared standard solution; the %relative difference was calculated.
The stability of sample solution carried out on triplicate sample solutions and evaluated after day 1 and day 2, storage at room temperature and in refrigerator (2-8°C) as per test method. Results of stored sample solution were compared with initial sample solutions and the %relative difference between the %assays was calculated.
Stability of mobile phase preparation was evaluated at room temperature after day 1 and day 2. The results for change in appearance, system suitability and system repeatability were checked during evaluation of mobile phase stability.

Range
Linearity as well as accuracy for Metoprolol Succinate and Chlorthalidone was checked from 60 to 140% of the nominal working standard solution. The method range is checked, based on suitable linearity, accuracy and precision results.

Method Development and Chromatographic Conditions Optimization
The consideration of specificity, linearity, accuracy, precision, robustness and solution stability parameters for development and validation of stability indicating method for MTL and CTD in bulk and in tablet dosage form. The Eluent was selected and optimized after use of a number of changed compositions followed by optimization of detection wavelength and flow rate. Method optimization performed by using the columns like Oyster ODS3, 150 -4.6 mm column having particle size of 5 µm (P/N: S670153, Make: Merck & Co.) and ODS Hypersil, 250 -4.0 mm column having particle size of 5 µm (P/N: 30105-254030, Make: Thermo Scientific). The organic modifiers like acetonitrile and methanol were used with 20 mM Phosphate buffer at a different pH level as 2.3, 2.5 and 3.0 to obtain best peak with optimum resolution.
Finally, the Eluent comprised in the ratio of 650:350, v/v of 20 mM phosphate buffer pH 2.3 ± 0.05 (adjusted with 10% orthophosphoric acid) and Acetonitrile was selected for simultaneous estimation of MTL and CTD because it retain both the peak efficiently in short time with satisfactory resolution, tailing factor (symmetry factor) and plate count (number of theoretical plates). Assay was carried out with 1.0 mL/min flow rate with ambient column temperature (about 25°C) and response recorded by UV detector at 225 nm. All quantitative assay calculations of MTL and CTD were done based on peak area response.

Method Validation Specificity
Specificity was established by demonstrating that, there is no blank interference with Metoprolol and Chlorthalidone peak (Figure 2).

Fig. 2. Chromatograms of (a) Blank; (b) Standard; (c) Sample
The forced degradation study was performed and no interference was observed from the peaks The forced degradation study results are summarized in Table 2. The chromatograms of stressed samples (thermal stressed) were showed degradation product peak significantly distinguishable from the drugs peak, indicating that the method is specific (Figure 3).

System Suitability and System Repeatability
The reproducibility attribute of any chromatographic system has measured through system suitability and system repeatability parameters (Table 3).

Linearity
The method was found to be linear for MTL and CTD from 60% to 140% of the nominal working concentration in the range of 113.8744 -265.7070 µg/mL and 29.7754 -69.4760 µg/mL with 0.999 as squared correlation coefficient (R 2 ) in both cases. Results of linearity showed an excellent linear relationship in the studied concentration range for MTL and CTD, indicating the fitness of method for analysis. Linearity study results are summarized in Table 4 and plot is shown in Figure 4.

Detection Limit and Quantitation Limit
The detection limit was 3.0169 µg/mL for MTL and 0.9908 µg/mL for CTD respectively, indicating that even small quantities of the MTL and CTD can be detected.
The quantitation limit was 9.1420 µg/mL for MTL and 3.0024 µg/mL CTD respectively, indicating that even small quantities of the MTL and CTD can be determined.

Accuracy
The mean %recovery results obtained from triplicate samples at each level were found to be 100.09, 98.76 and 99.41% for Metoprolol Succinate and 98.79, 99.25 and 99.16% for Chlorthalidone at 60%, 100% and 140% of nominal working concentration respectively, indicating that the method is accurate and showing that no interference from the excipients in the estimation (Table 5).

Precision
Method repeatability and intermediate precision results were shown that, the less than 2.0 %RSD values for MTL and CTD, signifying the method is precise and reproducible (Table 6).

Robustness
Results obtained from sample filtered by using 0.45 µm Nylon Syringe filter was meet acceptance criteria for %relative difference while 0.45 µm PVDF Syringe filter does not meet acceptance criteria for %relative difference (should be not more than 3.0%) with results obtained from sample filtered through Whatmann filter paper (as per method). Results of 0.45 µm Nylon Syringe filter are acceptable whereas results 0.45 µm PVDF Syringe filter are not acceptable. Hence, in addition to Whatmann filter paper, only 0.45 µm Nylon Syringe filter are useful for assay samples (Table 7).  The extraction efficiency results (Table 8) of MTL and CTD Tablets 47.5/12.5 mg (Revelol ® -CH 50/12.5) were not affected by changing the sample sonication time from 20 min to 30 min as the results were found within the acceptable range for %relative difference (should be not more than 3.0%).  The results of system suitability, system repeatability and change in retention time were checked for each changed method parameter during robustness. Each chromatographic method parameter fulfills the acceptance criteria even after making the deliberate changes into it, indicating its robustness (Table 9).

Solution stability
The results of standard solution stability (Table 10 and Table 11) were meets the acceptance criteria (%relative difference between initial and studied timepoint is NMT 2.0%) revealed that standard solutions were stable for 2 days at bench top (room temperature) and in refrigerator (2-8°C).
The sample solutions were stable for 2 days at bench top (room temperature) and in refrigerator (2-8°C) as the %relative difference between the %assay results obtained (Table 12) from stored sample solutions and initial sample solutions meets the acceptance criteria (the %relative difference between initial and studied timepoint is NMT 3.0%).
The mobile phase appearance was found to be clear and free of visible particles during estimation of mobile phase stability. Also, the system suitability and system repeatability results (Table 13) complies the acceptance criteria, hence the mobile phase was stable for 2 days at bench top (room temperature).

Range
The method range is from 60 to 140% of the nominal working standard solution has been derived for Metoprolol Succinate and Chlorthalidone, based on acceptable linearity, accuracy and precision study results.

Comparison with reported methods for simultaneous estimation of MTL and CTD
After, doing in depth study in present research work in comparison with previously reported work, it was found that the present research work is having various advantages like less time consuming process, short run time and improved resolution. The system suitability and system repeatability results established with two standard solution preparations. Robustness for change in sonication time and filter compatibility study provides additional benefit. The solution stability was reported for mobile phase preparations, standards and samples as well. The forced degradation study also performed on drug substance and drug product with suitable experimental design. Also, it is more economic as require less time of analysis with simple solution preparations. The results obtained from the validation suggest that this method was more simple, rapid, specific, precise, accurate, linear and robust enough as compare to earlier reported methods.

CONCLUSION
Stability indicating RP-HPLC method for simultaneous determination of MTL and CTD in bulk and in tablet dosage form was developed and validated as per guidelines of ICH. Experimental results confirmed that the chromatographic method is linear in the range of studied concentration as well as precise, specific, accurate and robust. The forced degradation study results reveals that all the peaks due to degradant were significantly resolved from the active components peaks, demonstrating the method is stability indicating and specific. The results of %recovery for dosage forms demonstrate that, no interference from the excipients in the determination of active analytes. The values of %RSD were < 2.0 for method repeatability and intermediate precision signifying the high level of method precision. The detection limits and quantitation limits values found to be extremely low, which provides an additional benefit. The robustness results were also demonstrates the working dimension of the method with changes made in different parameters. The solution stability results showed that standard, samples as well as mobile phase solution are stable for 2 days.
Also, analysis of drugs is rapid and cost-effective as method has simple isocratic elution with easy extraction as well as sample preparation process. The proposed method can be used for routine analysis and stability studies of MTL and CTD in the quality control of finished product as well as in bulk manufacturing also. However, the separation and structural characterization of degradation products were not carried out.