Validated stability-indicating RP-HPLC method for simultaneous estimation of cilnidipine and chlorthalidone in tablet dosage form

A novel, simple, speci(cid:976)ic, accurate & precise stability-indicating Gradient reverse-phase high-performance liquid chromatography (RP-HPLC) method was developed for simultaneous estimation of Cilnidipine & Chlorthalidone in tablet dosage form, validated as per ICH guideline. The separation was achieved on Inertsil ODS column (250 mm x 4.6 mm, 5 (cid:22) m) in a gradient mode. The mobile phase consisted of Methanol, 0.025 M Potassium dihydrogen phosphate Buffer pH 5.5 adjusted by 10% v/v Ortho Phosphoric Acid (50:50 v/v) (Solution A) and Acetonitrile, 0.025 M Potassium dihydrogen phosphate Buffer pH 5.5 adjusted by 10%v/v Ortho Phosphoric Acid (75:25 v/v) (Solution B) , gradient programming for 20 min at 1 ml/min rate of (cid:976)low and response was detected at 225 nm. The retention time was found to be 3.580 min and 12.606 mins for Chlorthalidone and Cilnidipine, respectively. The method is validated according to ICH guideline, which includes linearity, speci(cid:976)icity, accuracy, precision and robustness. Linearity was obtained over the concentration range of 10-60 (cid:22) g/ml for Cilnidipine and 6.25-37.5 (cid:22) g/ml for Chlorthalidone, had a regression coef(cid:976)icient (r 2 ) almost 0.9966. The % Recovery was found to be 99.63-100.59 % and 100.24-100.51 % for Cilnidipine and Chlorthalidone, respectively. The method was found to be speci(cid:976)ic enough to separate all degradation products from the active compound. Drug samples were exposed to various stress conditions like photolysis, oxidation, heat conditions, and hydrolysis (acidic and alkaline), there was no interference of any degradants and excipient in the determination of drugs so that methods can be successfully applied for routine QC analysis.


Chemicals and reagents
CIL & CHL working standards were gifted from Pure chem. Pvt Ltd., Marketed formulation of CIL and CHL (Label claim: CIL 10 mg and CHL 6.25 mg), NEXOVAS-CH6.25 tablets (Macleod Pharmaceuticals Ltd.) was purchased from the market. Acetonitrile, methanol, water all chemicals HPLC grade and HCL, Orthophosphoric acid, H2O2, triethylamine, KH 2 PO4, NaOH, are of AR Grade, were purchased from Merck.

Preparation of standard and Sample
The standard stock solutions 500 µg/ml of CIL and 125 µg/ml of CHL were prepared in methanol and later diluted to inal concentrations as 20 µg/ml CIL and 12.5 µg/ml CHL.

Preparation of sample mixture
About 20 tablets were accurately weighed and crushed. In 50 ml Vol. lask a quantity of powder equivalent to 6.25 mg of CHL or 10 mg of CIL was added. It was sonicated and iltered after the addition of MeOH; appropriately diluted to obtain 20 µg/ml CIL and 12.5 µg/ml CHL.

Forced degradation study
CHL and CIL have undergone stress conditions as tabulated in Table 2.

System Suitability
Separately injected six replicate injections of Standard preparation of CHL and CIL solution having a

Linearity
The linearity of the method was observed in the range of 10-60 µg/ml for CIL & 6.25-37.5 µg/ml for CHL.

Accuracy
The accuracy was ascertained by spiking 3 concentration levels 50, 100, and 150 % in three replicates of the targeted concentration of the analytes. The accuracy was revealed as percent analytes recovered by the proposed method.

Precision
The intra-day and inter-day precision of the method was carried out by analyzing sample solutions in three sets each of 50, 100, and 150 % of assay concentration. It is performed within a day (three distinct timings) and 3 consecutive days.

Speci icity
The speci icity of the method was ascertained by analyzing diluent, placebo, standard, and tablet formulation to examine the % interference of excipients and their impurities with analytes peak.

Robustness
The sample solutions were prepared and then analyzed with a change in the typical analytical conditions like Flow rate ± 10%, Mobile Phase ± 5%, pH ± 0.1. Observe the area and compare it with normal conditions.

Optimized chromatographic condition
The optimized separation was achieved by the chromatographic condition stated earlier, as shown in Figure 3. The System suitability parameters of the RP-HPLC method obtained for CHL and CIL is acceptable, as described in Table 3.

Forced degradation study
As the peak purity index is almost 0.999, there is no merging of peaks with suf icient degradation, as shown in Table 4 & in Figure 4.

Method validation
The method was validated for various parameters, according to ICH Q2 (R1).

Linearity
The correlation coef icients and regression equations for CHL and CIL were found to be R 2 = 0.9965, y = 138010x -154200 and R 2 = 0.9966, y=141724x + 165176 respectively. The calibration curve of CIL and CHL are shown in Figure 5 and Figure 6.

Accuracy
The percentage recovery for CIL was found within 99.63-100.59, and that for CHL was found to be in the range of 100.24-100.51. Thus, the % recovery of both drugs is acceptable, as tabulated in Table 5.

Precision
RSD for Intraday and Inter day precision was found less than 2. Thus, the method is precise. Data for precision is demonstrated in Table 5.

Speci icity
The % interference was less than 0.5 %, thus making speci icity to be acceptable.

Robustness
RSD was found to be less than 2. Thus, the method is robust.

Assay of tablet dosage form
The result for CHL and CIL tablets assay was found to be satisfactory, are tabulated in Table 6.

CONCLUSION
A speci ic, accurate, precise, and robust stabilityindicating Gradient RP-HPLC method is developed and validated for the determination of Cilnidipine and Chlorthalidone in a tablet dosage form. Degradation was carried out at oxidation, hydrolysis, photolysis, and heat conditions. The method is good enough to separate the peaks of tablet dosage form from the degradation products (generated during stress condition) and peak purity was also passed. Order of stability for Cilnidipine and Chlorthalidone is Thermal> Oxidative >Alkali >Acidic. All these induce us to assure that stability-indicating assay can be used in a routine.