HPLC METHOD DEVELOPMENT AND VALIDATION OF LERCANIDIPINE HCL AND ATENOLOL, CHARACTERIZATION OF ITS DEGRADANTS BY LC-MS/MS

Objective: An assay method was developed and validated for the simultaneous estimation of Lercanipine HCl and atenolol using RP-HPLC.
Methods: An effective chromatographic separation was achieved using waters symmetry C18 column of dimensions 150x4.6 mm, 3.5 μm, as a stationary phase. 0.1 percent ortho phosphoric acid and acetonitrile in 50:50 v/v was used as a mobile process with a rate of flow 1 ml/min and UV detection was carried out at 230 nm, respectively. Isocratic chromatography at ambient temperature was performed.
Results: Lercanidipine HCl and atenolol were separated by a running time of around 8 min. at 2.925 min. and 6.482 min. Respectively. By injecting the norm six times, device suitability parameters were studied and the outcomes were well under the acceptance criteria. The linearity analysis was performed at levels ranging from 10% to 150% and the R2 value was found to be 0.999.
Conclusion: Assay method validation was performed by using the marketed formulation and found to be within the limit. Degradation tests were conducted and the degradants were characterized by using LC-MS/MS.


INTRODUCTION
Lercanidipine is an anti-hypertensive [1,2] drug and belongs to the calcium channel blocker [3,4] class of dihydropyridine, which works by relaxing, and by relaxing expanding vessels of blood so that blood can flow around the body more openly it lowers blood pressure and makes it possible for the heart to function more effectively [5]. The medicattion have less adverse effects, but a comparatively high drug interaction potential. Lercanidipine is used to treat hypertension (high blood pressure). Lercanidipine, like other dihydropyridine, is contraindicated in patients with unstable angina [6,7], uncontrolled heart failure [8,9], immediately after myocardial infarction [10,11], and in patients with obstruction of outflow of left ventricular. During and in women who have become pregnant may become pregnant, it is also contraindicated because there is a lack of safety evidence as well as in patients with extreme liver and kidney disease, for the unborn.
Atenolol is a beta-blocker drug [12,13] used mainly for the treatment of elevated blood pressure and chest pain [14,15] associated with the heart. However, atenolol does not seem to increase survival in patients with elevated blood pressure [16,17]. Additional applications include migraine prevention [18] and the treatment of some irregular heartbeats [19]. It is taken by mouth or into a vein by injection. Other serious side effects include bronchospasm [20]. Tired feelings, heart failure, dizziness, anxiety and shortness of breath are common side effect. Use during breastfeeding. Is not recommended and alternative medications are used for breast feeding. It functions by blocking the hearts β1adrenergic receptors, thereby reducing the heart rate and load. Fig. 1 shows the chemical representations of Lercanidipine HCl and Atenolol. This paper proposes a novel sensitive stabilityindicating RP-HPLC procedure for the assessment of Lercanidipine HCl and Atenolol combination. The process proposed enables the rapid assessment of the Lercanidipine HCl and Atenolol in bulk drugs and formulation preparations without sample pre-treatment with high precision and specificity and with no excipient intervention.  There are some HPLC methods [21][22][23][24][25] reported in the literature, but these methods are developed only for routine analysis of Atenolol and Lercanidipine HCl in bulk and formulation studies. The developed HPLC method was utilized for the estimation of the combined drugs by in vitro method.

Chemicals
Acetonitrile, orthophosphoric acid and water all are of HPLC grade, were purchased from Merck India pvt ltd., Worli, Mumbai, India.
APIs of Lercanidipine HCl, atenolol were purchased from Spectrum Parma research solutions pvt ltd., Hyderabad.

HPLC
The chromatographic device of Waters quaternary pump alliance e-2695, PDA detector 2998 and chromatographic software Empower-2.0 were used.

Preparation of buffer
In 1 It of HPLC Water, 1 ml of orthophosphoric acid was dissolved and filter through 0.45 µ filter paper.

Preparation of mobile phase
Acetonitrile and buffer were mixed in 50:50 v/v ratio and sonicated for 5 min. After that filtered it by using 0.45μm membrane filter paper.

Diluent
Mobile phase.

Preparation of standard solution
Standard stock solution of Lercanidipine HCl and Atenolol was prepared by appropriately estimating about 10 mg and 50 mg drug 100 ml volumetric flask. Then the drug was liquified insolvent and filter through a 0.45μ filter. Standard stock solution concentrations of 100μg/ml and 500μg/ml were obtained.

Preparation of the solution for samples
Ten lercanidipine HCl and Atenolol tablets were accurately weighed and triturated to get a fine powder. A 10 mg Lercanidipine HCl and 50 mg Atenolol equivalent weight tablet powder was transferred into a 100 ml volumetric flask and dissolved in the diluent. The solution was ultra-sonicated for 10 min and made the volume with diluent. The tablet sample solution was then filtered through 0.45 micron syringe filter and utilized for preparing sample solution for the assay.

Optimization of chromatographic conditions
Various combinations of mobile phases were screened with respect to resolution, theoretical plate count, tailing and other system suitability parameters. Finally, the separation was performed with freshly prepared mobile step is composed of Acetonitrile and buffer in 50:50 v/v ratio with a 1 ml/min flow rate. Wavelength of 230 nm with injection volume 10 µl and ambient temperature was maintained during the entire process to obtain a symmetric peak of Lercanidipine HCl and Atenolol.

Method optimization
The current study was designed to develop a simple, reliable and rapid analytical RP-HPLC system which can be used to evaluate assay method of current estimation of Lercanidipine and atenolol pharmaceutical and bulk dosages forms. In order to have good results for the assay, the chromatographic conditions were optimized. Different combinations of Lercanidipine and atenolol have been tried to optimize the mobile process. The final working mobile phase was acetonitrile: 0.1 percent OPA at 50:50v/v. Based on its polarity, the mobile phase was selected for each drug. In order to achieve adequate sensitivity for the two smaller proportions of APIs (Lercanidipine and atenolol), the detection was carried out at several wavelengths. Finally, as a detection wavelength, the 230 nm wavelength at which the two drugs showed strong absorbance was chosen. The rate of flow was 1.0 ml/min, which is important as it affects the parameters of peak symmetry. The retention time for Lercanidipine and atenolol was 2.925 min and 6.482 min.
Respectively. The suggested approach is checked in compliance with the ICH guidelines [31] and found to be within the limit.

System suitability
In six replicates, system suitability was achieved by injecting a regular solution containing 10 µg/ml Lercanidipine and 50 µg/ml of Atenolol. The findings suggest that the criteria of system suitability were within the boundaries.  At the retention time of Lercanidipine HCl and atenolol, no intervention [38] from the blank occurred. The process is also unique.

Linearity
Linearity was determined by plotting a curve between peak areas to its respective concentration. From this calibration curve [39,40], it was noticed that the curve was linear over the 1-15 µg/ml Lercanidipine and 5-75 µg/ml atenolol concentration range. The calibration curve regression equations were Y= 131823.93x +25289.92 (R 2 =0.999) for Lercanidipine and Y=78721.38x +26974.78 (R 2 = 0.999) for atenolol.

Precision
The precision of this approach was evaluated in terms of inter and intraday variations. The intraday studies were calculated by six repeated tests of the Lercanidipine and atenolol sample solution under the same experimental conditions on the same day. In the same Laboratory, the intermediate precision of this approach was carried out by examining the analysis with various analyst and different instruments [41][42][43]. As the percent RSD values were found to be<2 percent, the method is highly accurate. At each added concentration, good recovery of the drug was achieved; suggesting that the procedure was successful. Below table represents the outcomes given.

Robustness
By varying flow rate and mobile phase composition, the robustness of the chromatographic process was calculated. It was found that RSD was within the appropriate range.

Forced degradation
The forced degradation study [47][48][49] was carried out according to ICH guidelines, include acid, base, peroxide, reduction, thermal and hydrolysis degradation. From the chromatograms, it is evident that selected drugs were stable under the applied stress conditions though the degradation peaks [50][51][52] were obtained. The formed degradants were characterized by using LC-MS/MS.

Acid degradation
Acid degradation of Lercanidipine and Atenolol were studied in 1N HCl. 12.6% of Lercanidipine and 14.7% of Atenolol degradation was observed in HPLC. Three degradation peaks were formed.

Alkali degradation
Alkali degradation of Lercanidipine and Atenolol were studied in 1N NaOH. 13.4% of Lercanidipine and 12.1% of Atenolol degradation was observed in HPLC. Three degradation peaks were formed.

Peroxide degradation
Peroxide degradation of Lercanidipine and Atenolol were studied in 30% hydrogen peroxide. 11.8% of Lercanidipine and 13.6% of Atenolol degradation was observed in HPLC. Two degradation peaks were formed.

Reduction degradation
Reduction degradation of Lercanidipine and Atenolol were studied in 30% sodium bisulphate solution. 14.5% of Lercanidipine and 12.3% of Atenolol degradation was observed in HPLC. Two degradation peaks were formed.

Thermal degradation
Sample was exposed to 105 °C for 6 h, 13.3% of Lercanidipine and 14.4% of Atenolol degradation was observed. No degradation products were formed in thermal degradation.

Hydrolysis degradation
Hydrolysis degradation of Lercanidipine and Atenolol was observed in HPLC grade water. 11.2% of Lercanidipine and 15.9% of Atenolol degradation was observed. No degradation peaks were observed in hydrolysis degradation.

MS/MS degradation product
The fragmentation mechanism of degradation product 1of m/z-315 observed under acidic, alkali degradation conditions is shown in fig.  6. Abundant substance ions are seen on the spectrum at m/z-239 (C6H5 LOSS), m/z-148 (C4H8Cl loss), m/z-78 (C4H9N loss). The proposed structures were confirmed by the accurate mass measurements and MS/MS studies.

MS/MS degradation product
The fragmentation mechanism of degradation product 4 of m/z-243 observed under acidic, alkaline degradation conditions is shown in fig. 9.

CONCLUSION
In this study a fast novel, economical, sensitive and easily available method of HPLC has been produced for the simultaneous determination of Lercanidipine and atenolol in bulk and a type of pharmaceutical dosage form. The advantage of this process was no HPLC methods were reported. This method consists of shorter run time, low price, accessibility, sensitivity, reliability and reproducibility. These properties are important when a large number of samples are to be analyzed. The validation of all the parameters like linearity, accuracy, specificity, robustness was done and found to be within the acceptance criteria. The RSD values were found to be less than 2.0 percent for all the parameters, which indicates the validity of the process and the results obtained by this process are seen to be in good agreement. So, the proposed method could be easily used for the routine analysis and pharmaceutical formulations of Lercanidipine and atenolol in quality control laboratories without any preliminary separation.

ACKNOWLEDGEMENT
The author is thankful to the management of P B Siddhartha College of Arts and Science for their encouragement to complete this research work and Shree Icon Pharmaceutical Laboratories, Vijayawada for providing laboratory equipment.

AUTHORS CONTRIBUTIONS
All the authors have contributed equally.

CONFLICTS OF INTERESTS
Declared none