Lercanidipine, 5 O (1 [3,3 diphenylpropyl(methyl)amino]  2 methylpropan 2 yl) 3 O methyl 2,6 dimethyl 4  (3 nitrophenyl) l, 4 dihydropyridine 3,5 dicarboxylate is a dihydropyridine calcium antagonist [Figure 1]. It is used for the management of Stage I and Stage II hypertension and is also perhaps useful in relieving angina pectoris.[1,2] Lercanidipine HCl is soluble in dimethylformamide, dichloromethane, and methanol.[3] It has a pKa value of about 6.83.[4] It is extremely lipophilic, possess octanol: water partition coefficient (Log P) value of about 6.4.[1] Preclinical and clinical findings propose that lercanidipine may have protective effects on the kidneys, cardiovascular system, and target organs. Lercanidipine (10 mg/day) produces a smooth antihypertensive effect without unfavorable hemodynamic or sympathetic effects due to its vascular selectivity. Lercanidipine emerged as a flexible choice for antihypertensive treatment across a wide range of patients due to its favorable efficacy and safety profile. Lercanidipine attains maximum plasma concentration within 2–3 h after oral administration and exhibits a slow onset of action. However, it is extensively metabolized by cytochrome P450 3A4.[5] Its low water solubility (5 ?g/ml), poor permeability, extensive first pass metabolism, and food dependent absorption result in its low bioavailability of 10%.[2]

Chemical structures of lercanidipine HCl

MATERIALS AND METHODS Chemicals

Lercanidipine HCl hemihydrate obtained as gift sample from Lupin Limited, Research Park, Pune. HPLC grade methanol and acetonitrile were obtained from Finar Chemicals, Gujarat (India). All the surfactants and/or polymers were procured from Loba Chemie Ltd. Mumbai and Sigma Aldrich Bengaluru, India

Instrumentation

Instrumentation Liquid chromatographic system from Shimadzu (LC20AT) comprising of manual injector, double reciprocating plunger pump LC-20ATVp for constant flow and constant pressure delivery and Photodiodearray detector SPD-M20A connected to software LC solution for controlling the instrumentation as well as processing the data generated was used.

Method validation

The developed method was validated for the parameters such as accuracy, precision, repeatability, intermediate precision, specificity, LOD, LOQ, linearity, range, system suitability, and robustness as described below.

Linearity

The linearity of an analytical method is its capability to obtain a test result, which has a certain mathematical relationship to the concentration of analytes. A standard solution of lercanidipine HCl (500 µg/mL) was prepared by dissolving

Reagents and chemicals      

Analytically pure sample of LER was kindly supplied by Glenmark Pharmaceuticals Ltd. (Nashik, India). Acetonitrile, Potassium dihydrogen phosphate, Disodium hydrogen phosphate was of HPLC grade supplied by Merck Ltd., India. The pharmaceutical dosage form used in this study was a Lotensyl AT (Sun Pharmaceuticals Industries Ltd. Mumbai) tablets containing 50 mg 10 mg lercanidipine hydrochloride were obtained from the local drug market. Triple distilled water was generated in house.

Chromatographic condition

The isocratic mobile phase consisted of ACN/phosphate buffer (pH 3.6) in the ratio of (60:40 v/v), flowing through the column at a constant flow rate of 0.5 ml/ min. A Luna C18 column (5 µm, 150mm x 4.60mm) was used as the stationary phase. Although the LER have different ?max viz 224, 275 and 238, 358 nm respectively, but considering the chromatographic parameter, sensitivity and selectivity of method for two drugs, 235 nm was selected as the detection wavelength for UV-PDA detector.

Diluent

Mobile phase

Preparation of standard solution

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

Preparation of the solution for samples

Ten lercanidipine HCl tablets were accurately weighed and triturated to get a fine powder. A 10 mg Lercanidipine HCl equivalent weight tablet powder was transferred into a 100 ml volumetric flask and dissolved in the diluent. The solution was

RESULTS AND DISCUSSION

Chromatography The mobile phase was chosen after several trials with methanol, isopropyl alcohol, acetonitrile, water and buffer solutions in various proportions and at different pH values. A mobile phase consisting of acetonitrile/phosphate buffer (60:40, v/v, pH 3.6) was selected to achieve maximum separation and sensitivity. Flow rates between 0.5 and 1.5/min were studied. A flow rate of 0.5 ml/min gave an optimal signal to noise ratio with a reasonable separation time. Using a reversed-phase C18 column, the retention times for Lercanidipine Hydrochloride were observed to be 2.27 and 5.97 min respectively. Total time of analysis was less than 6 min. The maximum absorption of Lercanidipine Hydrochloride together as detected at 235 nm and this wavelength was chosen for the analysis (Figure 2).

System suitability

System suitability parameters such as number of theoretical plates, HETP and peak tailing are determined. The results obtained are shown in Table-1. The number of theoretical plates for LER were 7023 respectively.

Linearity

LER showed a linearity of response between 50-250 and 10-50µg/ml respectively. The linearity was represented by a linear regression equation as follows.  Y (LER)= 90997 conc. - 6978 (r2 =0.9999)

Accuracy

 Accuracy of the method was calculated by recovery studies at three levels by standard addition method Table-2. The mean percentage recoveries obtained for Lercanidipine Hydrochloride were 99.47±0.32%, respectively

Each value is the Mean ± S.D of six determinations

Mean of Nine determinations (3 replicates at 3 concentration level)

Five dilutions in three replicates were analyzed in same day for repeatability and results were found within acceptable limits (RSD ‹ 2) as shown in Table3.

Intermediate Precision

Five dilutions in three replicates were analyzed on two different days and by two analysts for day to day and analyst to analyst variation and results were found within acceptable limits (RSD ‹ 2)

. Robustness

As per ICH norms, small, but deliberate variations, by altering the pH or concentration of the mobile phasewere made to check the method’s capacity to remain unaffected. The change was made in the ratio of mobile phase, instead of acetonitrile: phosphate buffer (pH 3.6) (60:40v/v), acetonitrile: phosphate buffer (pH 3.6) (55:45 v/v), was used as a mobile phase. Results of analysis were summarized in Table- 4.

Tablet Analysis

Content of LER found in the tablets by the proposed method are shown in Table-5. The low values of R.S.D. indicate that the method is precise and accurate

Table-3 Result of precision

Mean of six determinations

Table-4 Results of robustness

* Mean of fifteen determinations (3 replicates at 5 concentration level)