Dyade et al. QbD and Chemometric method for estimation of Metoprolol and Cilnidipine Online First Indian J Pharm Drug Studies | 1 Original Article QBD Approach and Chemo metric Method Development for the estimation of Metoprolol succinate and Cilnidipine in combined solid dosage form G. K. Dyade1, R. B. Jadhav2, Sanket Repal3, Shital Uparwad3, Prashant Suryawanshi3, Aashish Taware3, Sonali Khapne3, Shweta Kokare3, Anuse Dnyaneshwar3 From, 1Asso. Professor, 2Principal, 3M. Pharma Students, Dept of Post Graduate Studies in Pharmaceutical Quality Assurance, SVPM’S College of Pharmacy, Malegaon (BKII)-413115 Baramati Dist Pune, Maharashtra, India. ABSTRACT Background: Development of several pharmaceutical processes including analytical methods by applying Quality by design assists in ensuring the robustness of the method. USFDA and other regulatory agencies have recommended implementation of Quality by design (QbD) a systematic process for pharmaceutical development along with its significance. Objective: Chemo metric assisted UV-VIS spectrophotometric analytical method based on QbD was developed for the estimation of metoprolol succinate (MET) and cilnidipine (CIL) from the combined dosage forms. Method: Nature of spectra focused applicability of absorbance correction and amplitude modulation methods for estimation of both drugs from the formulations; and 50 % alcohol was being the common solvent. For both this method 221 nm and 242 nm was the wavelength for measurement of absorbance of metoprolol and cilnidipine respectively. Effect of input variables on spectrum characteristics were studied for selection of critical parameters and developed method was validated as per ICH Q 2 R1 regulatory guidelines. Result and conclusion: Linearity of the drugs was ascertained over the conc range 1-36 μg/ml (microgram/ml) for MET and 1-16 μg/ml for CIL. The percentage purity of assay in method II was found 103.773 % for MET and 96.825 % for CIL; and the accuracy study data of method I were varied 2.04859 for MET and 1.26321 for CIL. Precision study was shown acceptable data as % RSD in method I data varied 0.58894 for MET and 1.15116 for CIL. The developed method is rigid, robust and efficient for the estimation of MET and CIL from the composition of dosage form. Keywords: Metoprolol succinate, Cilnidipine, QbD, ICH, Absorbance correction method, Amplitude modulation method ilnidipine (CIL) chemically 1, 4-Dihydro-2,6- dimethyl-4-(3-nitrophenyl)-3,5-pyridine dicarboxylic acid 2-methyloxyethyl (2E)-3-phenyl-2-propenyl ester [1] is a dihydropyridine calcium channel blocker given orally in the management of hypertension [2] Reported analytical methods for estimation of cilnidipine were includes alone by UV spectrophotometric method [3], UV spectrometric methods with metoprolol [4, 5] alone UV-HPLC method [6] , Stability indicating HPLC [7] QbD based HPLC [8] chromatographic methods [9-12] and have been found in the literature. Metoprolol succinate (MET) is a beta blocker; used in the treatment of hypertension, angina and to reduce myocardial infarction [2]. Chemically it is (RS)-1-(Isopropy Access this article online Received – 20th May 2024 Initial Review – 13th June 2024 Accepted – 15th July 2024 Quick Response Code lamino)-3-[p-(2- Methoxyethyl) phenoxy]-2-propan-2-ol succinate [1]. Literature survey revealed that various analytical methods have been reported for estimation of MET such as alone UV spectrophotometric method [13-16], with other drugs UV spectrophotometric method [17-22], bio analytical method [23], MET alone by RP-HPLC [24], with other drug by RP- HPLC [25-30], stability indicating HPLC [31, 32], designed and eco-friendly TLC densitometry [33] and HPTLC [34] in combination with other drug. Cilnidipine and Metoprolol succinate are official in Indian Pharmacopoeia [35], whereas Metoprolol is official in BP [36]. Chemical structures of both drugs are shown in (Fig 1). Quality by design concept is applied for the development of pharmaceutical processes to assure a predefined product quality. __________________________________________________ Correspondence to: G. K. Dyade, Dept of Post Graduate studies in Pharmaceutical Quality Assurance, SVPM’S College of Pharmacy, Malegaon (BKII)-413115 Baramati Dist Pune, Maharashtra, India. Email: pharmacyresearchsvpmcop@gmail.com C pharmacyresearchsvpmcop@gmail.com Dyade et al. QbD and Chemometric method for estimation of Metoprolol and Cilnidipine Online First Indian J Pharm Drug Studies | 2 Fig 1: Chemical structure of Drug molecule QBD concepts are mentioned in ICH guidelines Q 8(R2) (Pharmaceutical development), Q9 (Quality risk management), and Q10 (Pharmaceutical quality system) [37- 39] shown in Fig 2. ICH guidelines Q8 (R2) [40] defines QBD as a “a systematic approach to development that begins with predefined objectives and emphasizes product and process understanding and process control, based on sound science and quality risk management”. QBD approach in analytical method summarizes a complete understanding of how the analytical technique attributes and operating conditions affect the analytical performance. Factors to study in analytical quality by design (AQbD) approach may include the type of analytical technique chosen, reagents used and instrument parameters. QbD was applied to build rigid robust method through risk assessment at early stage and defining the design space at the later stage. Fig 2: Analytical QbD approach There are similar advantages of applying QbD principles to analytical methods as to manufacturing processes and product [41]. A QbD approach can be beneficial in the development of suitable, robust, low cost and eco-friendly (eco-friendly solvent, chemicals) method which is applicable at any stage of the lifecycle of the product. Also some regulatory guidelines have mentioned flexibility of changing analytical method without revalidation if the AQbD approach has been implemented during analytical method development. The first stage of AQbD approach is to fix an analytical target profile (ATP) for the method. ATP defines the goal of the analytical method development process and it is the sign of method performance [42, 43]. For analytical method validation ICH Q2 (R1) has given various method performance characteristics for an analytical method. Thus a QbD based UV spectrophotometric was developed, QbD approach was implemented with the study of the effect of method input variables on spectral shape, intensity of absorbance, and absorbance maxima λmax and critical parameters were selected for the proposed method and method was validated as per ICH guidelines Q2 (R1). MATERIALS AND METHODS Instrumentation: Analysis was performed with a Shimadzu Double beam UV-Visible spectrophotometer 1900i and 1700 (Shimadzu, Kyoto, Japan) with spectral bandwidth of 2 nm and wavelength accuracy of ± 1 nm with 10 mm matched Quartz cells was used. Electronic balance Afcoset balance (The Bombay Burmah Trading corpo Ltd) with accuracy ±0.1 mg Model No. ER 200A was utilised for weighing and for degassing the solution Digital Ultrasonic cleaner 1.8 Ltr (Labman scientific Instruments Chennai) was used. Reagents and Chemicals: Pharmaceutically pure sample of MET was procured from Macleods Pharmaceuticals Ltd. Mumbai and CIL from Swapnroop drugs and pharmaceuticals, Aurangabad, Maharashtra, India procured as a gift samples and the commercial formulation Cilacar-M manufactured by J B Chemicals and Pharmaceuticals containing cilnidipine 10 and metoprolol 50 mg was procured from the local market. AQbD approach application in method development: AQbD approach was applied to study the influence of input variable parameters on spectrophotometric analytical method performance shown in (Fig 3). Solvent selection: CIL is very soluble in N, N-Dimethyl acetamide, freely soluble in acetone, soluble in methanol and practically insoluble in water, whereas MET is freely soluble in water, ethanol, methanol and ethyl acetate;. Although the solubility of the procured drugs were studied in alcohol 90%, 0.1 N HCl and 0.1 N NaOH separately; and found that CIL is insoluble in both these solvents however MET is soluble in 0.1 N NaOH. Alcohol 90% was selected as common solvent as both drugs have solubility. Each solution with known conc of analyte was scanned in UV range of 400 nm to 200 nm. It Dyade et al. QbD and Chemometric method for estimation of Metoprolol and Cilnidipine Online First Indian J Pharm Drug Studies | 3 was found that suitable solvent is alcohol 90% with respect to stable, robust and precise in producing result. Fig 3: Diagram showing the relationship between input variable parameters and the spectrophotometric method performance characteristics Preparation of stock solutions and standard solutions: 10 mg each of drug CIL and MET were separately and accurately weighed; and transferred into separate 25 ml volumetric flask. Dissolved into alcohol 90% solvent and volume was made to 25 ml with solvent. Subsequent standard solution of drug MET with conc 16μg/ml and CIL with conc 8μg/ml were prepared by diluting aliquot of stock solution to 10 ml with 50% alcohol into 10 ml capacity volumetric flask. Selection of method, wavelength and conc range: From UV spectra it was found that CIL has measurable absorbance at 242 nm (Fig 4) and less interference was observed by MET; similarly MET has maximum absorbance at 221 nm (Fig 4) and measurable interference having constant absorptivity by CIL was accounted. Chemo metric method i.e. absorbance correction method was applied and which was reasonable remedy to overcome interference at each other’s absorbance, and other method was amplitude modulation method. From the nature of spectra to study linearity, working conc range 1 to 16 μg/ml for CIL and 1 to 36μg/ml for MET was selected. Also combined drug solution was prepared simulated to marketed formulation. Selected critical parameters based upon above discussion, observations are listed in and by using these; method was validated as per ICH guidelines and by analysing marketed preparations. Experimental Method for estimation: From the overlain spectra absorbance correction method was applicable for estimation of both the analytes from the combined dosage form. Method-I: Absorbance correction method for estimation of MET and CIL: MET was shown maximum absorbance i.e. λmax at 221 nm where measurable interference with constant absorptivity by CIL found and CIL has maximum absorbance i.e. λmax at 242 nm where negligible interference observed by MET. At 242 nm the λmax of CIL, MET was shown consistency in the absorptivity; hence two wavelengths 221 and 242 nm were considered as 1 and 2 respectively for the said method to estimate MET and CIL. The equation A= abc was applied for x (MET) and y (CIL) determination. Conc of working standard solutions of MET and CIL containing 28 mcg/ml and 8 mcg/ml conc respectively were separately prepared and used for the method. Where CX = Conc of MPS in sample solution A1 and A2 = absorbance of sample solution at 1 and 2 wavelength ay1 and ay2 = absorptivity of CIL at 1 and 2 wavelength of standard solution ax1 and ax2 = absorptivity of MET at 1 and 2 wavelength of standard solution Cy = Conc of CIL in sample solution As = Absorbance of Sample solution at 2 wavelength Method-II: Amplitude Modulation method: This method comprises the conversion of zero order/normal spectra to its first, second or higher derivative spectrum. The amplitude is directly proportional to the conc of solution provided Beer’s law is obeyed by spectrum. In derivative method zero crossing wave length for both drugs is found such that at the zero crossing of one drug the other drug should show substantial absorbance. Here standard solutions 16 µg/ml each of metoprolol and cilnidipine were prepared in 10 ml volumetric flask and scanned from 400 to 200 nm wavelength range against ethanol 50% as blank. Absorption spectra of both drugs were recorded and the found λmax of MET and CIL were stated in earlier discussed method. From overlain spectra CIL has substantial interference due to absorption at wavelength 221 nm and MET shows negligible absorption at 242 nm, hence it was decided to apply derivative method. Dyade et al. QbD and Chemometric method for estimation of Metoprolol and Cilnidipine Online First Indian J Pharm Drug Studies | 4 Zero order spectra of both drug was modulated and converted to first order derivative spectra. From overlain spectra, it clearly shows that no interference or zero absorbance of MET was found at 263 nm hence it was selected λmax of CIL and 229.5 nm was zero crossing of CIL, so it was selected as λmax of MET. Both drugs were obeying Beer’s law in first order derivative mode at the respective wavelength. Fig 4: UV spectra of Cilnidipine and Metoprolol succinate in ethanol Validation of the Method: Selected critical parameters should meet the performance characteristics of the analytical method so as to attain analytical target profile of the method. An ICH guideline Q2 R1 was applied to study methods performance with critical parameters in order to implement AQbD approach. The method was validated as per ICH guidelines. System suitability: System suitability is studied to demonstrate the suitability of the developed procedure under consideration for the analytical method. Six replicates of working standard solutions with conc16 μg/ml and 8 μg/ml each of MET and CIL respectively were prepared separately and absorbance was recorded, SD and % RSD of the response was calculated. Linearity: The linearity of an analytical method is its ability to obtain response i.e. absorbance which is directly proportional to the conc of analyte. Series of working standard solutions were prepared in conc. range of 1-36μg/ml for MET and 1-16 μg/ml for CIL and scanned in 400 to 200 nm range in spectrum mode of the spectrophotometer, absorbance of the standard solutions were recorded at their respective wavelength; i.e. 221 for MET and 242 nm for CIL in spectrum order. Microsoft office excel software tool was used to obtain the standard regression curve and its analysis as slope, intercept, and correlation coefficient. Assay of formulation: Assay was carried out by proposed methods and assay was validated by statistical parameters. Estimation of formulations by absorbance correction and amplitude modulation method: Tablet powder equivalent to 12.5 mg MET and 2.5 mg CIL was weighed and transferred into 25 ml volumetric flask. Dissolved into 90% alcohol, mixed well for 10 mins and volume was made to 25 ml with the solvent. Solution was filtered through what man filter paper and aliquots of solution were further diluted with the 50% alcohol to obtain tablet sample solution. Solution was scanned in the range of 400 to 200 nm to obtain absorbance of tablet solution at 221 nm and 242 nm in spectrum order. Also spectrum converted to first order derivative and absorbance was recorded at 229.5 and 263 nm. Obtained absorbance were utilised to estimate unknown conc of formulation; and results were statistically validated to obtain % of nominal conc, standard deviation and % of RSD. Accuracy and Precision: The accuracy of an analytical method expresses the closeness of an agreement between test result and true result. Accuracy study was performed by recovery study i.e. standard addition method; diluted standard solutions of MET and CIL were prepared and standard solutions added in 100 % proportionate to the tablet solution. Three replicates at this level was prepared, absorbance measured and % of conc, SD and RSD were calculated. The precision study was carried out by performing assay of tablet six times; also the reproducibility in result was studied by inter day and intraday precision. Limit of Detection (LOD) and Limit of Quantitation (LOQ): The LOD and LOQ of MET and CIL by the proposed method were determined using calibration graph method and calculated as 3.3σ/s and 10 σ/s for LOD and LOQ respectively; σ is the standard deviation of calibration curve and s is the slope of regression line. Robustness and Ruggedness: It is measure of capacity of analytical procedure to remain unaffected by small but deliberate variations in method parameter. RESULTS Method development comprises numerous steps, and of which solvent selection, selection of method for measurement are significant one. Uses of aqueous solvents, eco-friendly solvents like hydrotropic have got remarkable weightage due to low cost, readily available and environmentally sound. Drugs underlying analysis must have appreciable solubility in the selected solvent. Chemical structure of the drug and physico-chemical properties available in the literature guides about use of appropriate solvent in the method. From UV spectra two wavelengths were selected as 221nm Dyade et al. QbD and Chemometric method for estimation of Metoprolol and Cilnidipine Online First Indian J Pharm Drug Studies | 5 (λmax of MET) and 242 nm (λmax of CIL) shown in Fig 5 for calculation of both drugs in combined solution shown. Fig 5: Overlaid spectra of MET and CIL System Suitability: The absorbances of six replicates of standard solutions of tabulated respective conc are reported in Table No 1. The SD and % RSD was found for MET and CIL and meets the system suitability requirements indicates method was suitable for analysis. Table No 1: System suitability study of MET and CIL Sr No Conc in μg /ml Absorbance of MET* Conc in μg /ml Absorbance of CIL* 1 16μg/ml 0.5157 8 μg /ml 0.6519 2 SD RSD % 0.004676 0.9060 SD RSD 0.004809 0.73774 *Mean of six determinations Linearity: The calibration curve of both drugs was found to be linear in the conc range of 1-36 μg/ml for MET and 1-16 μg/ml for CIL in normal spectrum as shown in Fig 6 and in first order derivative mode Fig 7. The regression equation of line and its parameters slope, r2 value and intercept are tabulated in Table No 2, which proved the linear relationship between conc and obtained response. Assay: The assay was carried out by the proposed method. The overlaid spectra obtained in amplitude modulation method are shown in Fig 8. The assay of formulation was carried out by proposed method and calculated % of nominal conc and RSD was found within acceptable limits are summarized in Table No 3. The results indicated applicability of the method for estimation of formulation. Fig 6: Normal spectrum kind Calibration curve of MET and CIL Fig 7: Calibration curve of MET and CIL in First order derivative Accuracy and Precision: The results of accuracy are summarised in Table No 4, the obtained results were within acceptable limit; and methods accuracy was justified by calculating % drug content. The precision study was carried out by performing assay of solutions; further the reproducibility in result was studied by interday and intraday precision. The values obtained SD and % RSD was shown methods precision and are summarised in Table No 4. Limit of Detection (LOD) and Limit of Quantitation (LOQ) and Robustness and Ruggedness: The LOD and LOQ of CIL and MET were found in acceptable limits by the proposed method. Robustness was studied and capacity of analytical procedure to measure analyte was remain unaffected by small but deliberate variations in method Dyade et al. QbD and Chemometric method for estimation of Metoprolol and Cilnidipine Online First Indian J Pharm Drug Studies | 6 parameter. The analytical method was found rugged during development; similarity the result was produced by performing the analysis by different analyst. Table No 2: Parameters of regression equation obtained in Microsoft excel Parameters MET CIL MET CIL Linearity study in Normal Linearity in derivative Detection wavelength 221 242 229.5 263 Beer’s law limit (μg/ml) 1–36 μg/ml 1–16 μg/ml 1–36 μg/ml 1–16 μg/ml Correlation coefficient (r2) 0.9984 0.9994 0.9991 0.9972 Regression equation (y = mx + c) Y = 0.0317X + 0.0097 Y = 0.0797X + 0.0145 Y=- 0.0006X- 0.0001 Y=- 0.0015X- 0.0003 Table No 3: Results of assay of formulation by proposed method Formula tion Dr ug Label Claim (mg/Ta blet; n=6) Amou nt found/ mg Drug Cont ent % Std Deviat ion % RSD Method- I ME T 50 47.084 94.16 7 4.1614 6 4.419 2 CI L 10 9.681 96.78 7 1.7606 3 1.809 67 Method- II ME T 50 51.886 103.7 73 1.8865 1 1.817 91 CI L 10 9.682 96.82 5 2.7493 4 2.839 49 Table No 4: Results of accuracy and precision Sr. No. Parame ter Level of study Dru g Na me % Mean S.D. % RSD Meth od - I Precisi on Intrada y precisi on ME T 94.877 % 0.588 94 0.620 74 CIL 97.744 % 1.151 16 1.177 12 Accura cy 100% ME T 96.308 % 2.048 59 2.127 12 CIL 94.845 % 1.263 21 1.331 88 Meth od - II Precisi on Intrada y precisi on ME T 102.06 6% 1.665 33 1.622 08 CIL 99.476 % 2.260 24 2.272 15 Accura cy 100% ME T 97.646 % 1.176 75 1.205 11 CIL 96.667 % 1.159 71 1.194 52 Fig 8: Overlaid spectra obtained in amplitude modulation method Dyade et al. 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Funding: None; Conflicts of Interest: None Stated Keywords: Metoprolol succinate, Cilnidipine, QbD, ICH, Absorbance correction method, Amplitude modulation method