ASSAY METHOD DEVELOPMENT AND VALIDATION OF CILNIDIPINE AND RAMIPRIL, CHARACTERIZATION OF ITS DEGRADANTS BY USING LC-MS/MS

Objective: The current study focused on the development, validation of Ramipril and Cilnidipine using HPLC and characterization of forced degradation products using LC-MS/MS.
Methods: Simple, accurate, and precise method has been developed for the simultaneous estimation of Cilnidipine and Ramipril in tablet type of dosage form using X-bridge phenyl column (150x4.6 mm, 3.5m) with mobile phase of buffer and Acetonitrile in the 30:70 v/v ratio was pumped through a column with a 1 ml/min flow rate. The buffer used in this process was 0.1 percent tri ethyl amine in 1 lt of water and adjust its pH-2.5 with 0.1 percent ortho phosphoric acid. At ambient temperature, chromatography was isocratically performed and run time was 8 min. 240 nm was the optimized wave length.
Results: Cilnidipine and Ramipril retention times were 2.79 min, 5.11 min. respectively. By injecting the norm six times, device parameters of suitability have been studied and the results were found to be well under the acceptance criteria. This approach offers strong linearity over a range of 2-30 µg/ml Cilnidipine 1-15 µg/ml Ramipril concentrations. The regression coefficient R2>0.999 from the calibrated curve implies that the linearity of the system was within the range.
Conclusion: The system was validated by using HPLC in terms of accuracy, linearity, method precision, accuracy, limit of detection, limit of quantification, robustness, degradation and the degradation products were characterized by using LC-MS/MS.


INTRODUCTION
Cilnidipine is a calcium channel blocker [1]. Cilnidipine is approved to treat hypertension [2]. Accompanied by L-type calcium channel blocking [3] functions, it is a calcium antagonist. Cilnidipine in addition on the L-type calcium channel, unlike other calcium antagonists, it can operate on the N-type calcium channel Cilnidipine reduces blood pressure and is used for the prevention and comorbidity of hypertension [4,5] Cilnidipine dilates both arterioles and venues due to its blocking action at the L-type and N-type calcium channels, thus reducing the pressure in the capillary bed. Cilnidipine has a mild direct dromotropic effect, effective vasodepressor and arrhythmia-inhibiting effect [6,7] and vasoselective effect.
Ramipril is a medication used to treat blood pressure, heart failure [8,9], and kidney disease with diabetes [10,11] also used to prevent cardiovascular disease [12,13]. It is fair initial care and it is swallowed by mouth. Headaches [14], dizziness [15,16], tiredness and cough [17] are typical side effects. Liver complications [18], angioedema [19,20], kidney problems, and high blood potassium [21,22]. Use in pregnancy and breastfeeding is not recommended. It is an ACE inhibitor [23] and works by decreasing renin-angiotensinaldosterone [24] system activity. Fig. 1 shows the chemical structures of Ramipril and Cilnidipine. The aim of the study is to separate the pharma ingredients Ramipril and Cilnidipine by using RP-HPLC and characterization of its degradants using LC-MS/MS.

Chemicals
Acetonitrile, tri ethylamine, orthophosphoric acid and water (HPLC Grade) were bought from Merck India Ltd, Worli, Bombay, India. All APls of Cilnidipine, ramipril were bought from Glenmark pharmaceutical private Ltd, Andheri, Mumbai, India, were procured as reference levels.

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

Chromatographic conditions
Chromatographic separation of drugs Cilnidipine and Ramipril has been carried out in isocratic mode at room temperature using waters X-bridge phenyl column (150x4.6 mm, 3.5 µm). The mixture of 0.1% TEA pH-2.5 adjusted with OPA: acetonitrile in 30:70 v/v with a flow of 1 ml/min was used as the mobile process. The injection volume was 10 μl and at 240 nm the eluents were controlled with a running time of 8 min.

Preparation of buffer
1 ml of tri ethyl amine was transferred into 1 lt of HPLC water and mixed well; adjust the pH-2.5 with OPA. Filtered and degassed [29] through 0.45 μ membrane filter paper.

Selection of mobile phase
Prepared a buffer and Acetonitrile combination (30:70v/v). The selected mobile phase has given sharp peaks with low tailing factor.

Selection of wavelength
The absorption spectra of solution of each Cilnidipine and Ramipril were scanned over a range of 200-400 nm by using spectrophotometer and the spectra were recorded. Two drugs were absorbed maximum at a wavelength of 240 nm.

Preparation of diluent
The mobile phase was used as diluent.

Standard solution preparation
Weighed 20 mg Cilnidipine, 10 mg of Ramipril and transferred into 100 ml volumetric flask. 70 ml diluents was added and sonicated to dissolve for 15 min and made up to the mark with diluents. 5 ml of the above solution was further diluted to 50 ml with diluents.

Suitability
To check the performance of the system, system suitability parameters were calculated. The parameters include the count of USP plates, USP tailing [35] and % RSD can be calculated and found to be within the limit.

Specificity
Specificity is the ability to assess unequivocally in the presence of an analyte, other impurities, and elements such as, expedients which may be assumed to be available in the sample and standard solution. It was checked by examining chromatograms of blank standard and the standard spiked [36] with Cilnidipine and Ramipril.

Accuracy
Accuracy is the closeness of the test result to the true value. The therapy was assessed by the study at three different concentration levels. A minimum of three injections have been given at each stage, the percentage recovery and the corresponding standard deviation, and the RSD have been determined.

Precision
The degree of agreement among individual tests results is the precision of an analytical technique. In terms of repeatability, intraday and intra-day variations, the precision of the present method was evaluated. Analysis of the standards at different time intervals of the same day and on different days was carried out.

Linearity
The Linearity of an analytical method is its ability to obtain result is directly proportional to the analytes concentration. For the evaluation of the linearity range, seven series of standard solution were selected. Using peak area versus concentration of the standard solution, the calibration curve was plotted and the regression equations were measured using the method with the least squares.

LOD and LOQ
LOD is the lowest analyte quantity in the sample that can be detected, while LOQ is the lowest analyte quantity in the sample that can be determined with appropriate accuracy and precision. Based on the calibration curves, LOD and LOQ were separately determined. The LOD and LOQ were calculated according to ICH guidelines as 3.3σ/s and 10σ/s, respectively, where σ/s indicate the ratio of signal to noise.

Robustness
Analytical procedures robustness is a measure of its ability to remain unaffected by small but deliberate variations in parameters of the method and provides an indication of its reliability during normal use. The robustness study was carried out by injecting the standard solution into the HPLC system and modifying the flow rate (±0.2 ml/min), organic phase (±10 percent) of chromatographic conditions. The separation factor and retention time and maximum asymmetry have been measured.

Degradation
Stress degradation should be no interference between the peaks obtained for the forced degradation chromatogram. According to ICH guidelines [37], stress degradation studies were conducted. The peaks of degradation should be well separated and the resolution between the peaks should be at least 1.0 and the peak purity of the principal peaks [38] should pass.

Method development and optimization
Initially, the RP-HPLC system was optimized using an X-Bridge phenyl (150x4.6 mm, 3.5 µm) column. To satisfy the system suitability parameters, a mixture of acetonitrile and TEA (70:30v/v) as the mobile phase (Flow rate 1.0 ml/min) was found to be more appropriate. Table 1 summarizes the optimized chromatographic conditions.

System suitability
To get stable baseline, the HPLC system was stabilized for 60 min. six replicate injections mixture containing 20 µg/ml of Cilnidipine and 10 µg/ml of Ramipril were performed and assessed to check the suitability of the system. From six replication injections, the system suitability parameters were evaluated. The research concludes that the system suitability and outcomes of the HPLC system being used are summarized below in table 2 [39].

Specificity
There was no interference [40] from the blank during the retention time of Cilnidipine and Ramipril. The process is, therefore, specific.

Accuracy
The accuracy of the method was performed by calculating the recovery experiments at three levels (50 percent, 100 percent 150 percent). APls were prepared at concentrations of 10, 20, and 30 µg/ml of Cilnidipine and 5, 10, 15 µg/ml of Ramipril, respectively. For each spike level, the test solution was injected three times and an assay was performed as per the test method. The recovery results were close to 100 percent and the RSD values were less than 2 percent. Recovery values showed that, within the desired range, the method was accurate. The results were summarized below in table 4 [41].

Precision
In terms of intra-day and inter-day differences, the accuracy of this method was assessed. Six repeated analyses of the standard solution of Cilnidipine and Ramipril on the same day under the same experimental conditions were used to determine intraday studies. In the same laboratory, the intermediate precision of the method was carried out by studying the analysis with different analysts with different instruments. As the each concentration level, good recovery values were obtained, indicating that the procedure was accurate.

LOD and LOQ
Using the calibration curve process, LOD and LOQ were calculated separately. By injecting a progressively lower concentration of standard solution using the established RP-HPLC process, the LOD and LOQ of the compounds were calculated. The LOD values of Cilnidipine and Ramipril were 0.6µg/ml, 0.3µg/ml and s/n values were 6, 3 respectively. Cilnidipine and Ramipril LOQ values were 2.0 µg/ml, 1.0 µg/ml s/n values were 26, 24 respectively.

Robustness
As per ICH norms, small but intentional differences were made in the parameters of the method, for instance, like the change in the rate of flow (±0.2 ml/min) and organic phase (±10%) to check the method capacity to remain unaffected [42].

Forced degradation
The proposed analytical method can be used for release and stability studies [43] for successful evaluations for and can be considered as a stability-indicating method. The survey of forced degradation [44] was conducted according to ICH guidelines includes acid, base, peroxide, reduction, thermal and hydrolysis degradation [45][46][47]. From the chromatograms, it is evident that selected drugs were stable under the applied stress conditions though the degradation peaks were obtained. The degradation samples were characterized by using LCMS.

Acid degradation
Acid degradation of Cilnidipine and Ramipril was studied in 1N HCl. 15.1% of Cilnidipine and 15.6% of Ramipril degradation was observed in HPLC, two degradation peaks of D1 and D4 were formed.

Alkali degradation
Alkali degradation of Cilnidipine and Ramipril was studied in 1N NaOH. 14.9% of Cilnidipine and 14.4% of Ramipril degradation were observed in HPLC, two degradation peaks of D1 and D4 were formed.

Peroxide degradation
Peroxide degradation of Cilnidipine and Ramipril was studied in 30% hydrogen peroxide. 13.2% of Cilnidipine and 11.3% of Ramipril degradation was observed in HPLC, two degradation peaks of D2 and D5 were formed.

Reduction degradation
Reduction degradation of Cilnidipine and Ramipril were studied in 30% sodium bisulphate solution. 12.8% of Cilnidipine and 11.7% of Ramipril degradation was observed in HPLC, two degradation peaks of D3 and D6 were formed.

Thermal degradation
In thermal degradation sample was exposed to 105 °C for 6 h, 1.4% Cilnidipine and 2.3% Ramipril degradation was observed in HPLC.
No degradation products were formed.

Hydrolysis degradation
Hydrolysis degradation of Cilnidipine and ramipril was observed in HPLC water. 1.2% Cilnidipine and 0.8% Ramipril degradation was observed. No degradation products were formed.

MS/MS degradation peak
The proposed fragmentation mechanism of degradation product 5 of m/z-564 observed under the condition of peroxide degradation is shown in fig. 10. Abundant productions are seen on the spectrum at m/z-373 (loss of C7H9O7 from m/z-564), m/z-296 (loss of C6H5 from m/z-373), m/z-209 (loss of C4H7O2 from m/z-296), m/z-137 (loss of C3H6NO from m/z-209), m/z-328 (loss of C13H18NO3 from m/z-564). The proposed structures were confirmed by the MS/MS tests in combination with accurate mass measurements. Fig. 11: Proposed fragmentation mechanism of DP6 (m/z-398)

MS/MS degradation peak
The proposed fragmentation mechanism for degradation product 6 of m/z-398 observed under the reduction degradation condition is shown in fig. 11. The spectrum shows abundant product ions at m/z-293 (loss of C8H9 from m/z-398), m/z-208 (loss of C4H7O2 from m/z-293). The proposed structures were verified in combination with accurate mass measurements by the MS/MS experiments.

CONCLUSION
In this a novel, simple, fast, economical, sensitive and easily available method of HPLC was developed for the simultaneous determination of Cilnidipine and Ramipril in the form of tablet dosage. The main advantages of this method were there is no HPLC methods were reported. In this method, shorter run time, low price, accessibility, sensitivity, reliability and reproducibility these properties were important when a large number of samples are to be analyzed. The validation of all the parameters like linearity, accuracy, specificity, robustness, stability was done and found to be within the acceptable limit. For all the parameters, RSD were found to be less than 2 percent which the validity of the process is suggested and the results obtained by this process are in good agreement. So the proposed procedure was easily applied for routine analysis and pharmaceutical formulations of Cilnidipine and Ramipril in quality control laboratories without any preliminary separation.

ACKNOWLEDGEMENT
The author was thankful to the management of P B Siddhartha college of Arts and Science for their encouragement and Shree Icon Pharmaceutical Laboratories for providing laboratory equipment.