Analytical Method Development and validation of UV-Visible spectroscopy

Method Development and validation, UV-Visible spectroscopy, wavelength 257 nm, method are accurate, precise, specified, and validated

1. Analytical Method development and validation of azelnidipine by UV-Visible Spectroscopy 6/16/2021 DOPs, SIHAS, SHUATS, Prayagraj, U.P, India Presented to : Prof. (Dr). P.Malairajan (Hod, Department of Pharmaceutical Sciences, SIHAS, SHUATS, Prayagraj, U.P, India) Presented By: Name: Imdad H. Mukeri Id No: 17BPH084 Subject: Advance Instrumentation Technique Course code: BP814PW Date of Submission: 14/06/2021 1

2. Introduction Pharmaceutical analysis comprises the procedures necessary to determine the identity, strength, quality and purity of substances of therapeutic importance. Quality is important in every product or service, but it is vital in medicines as it involves life. Analytical techniques that are generally used for drug analysis are Chromatographic methods, Spectral methods, Biological and Microbiological methods 6/16/2021 DOPs, SIHAS, SHUATS, Prayagraj, U.P, India 2

3. Introduction to UV-Visible spectroscopy UV-Visible spectroscopy is type of absorption spectroscopy in which light of ultra-violet and visible region ( 200-400nm and 400-800nm ) is absorbed by the molecule which results in the excitation of the electrons from the ground state to higher energy state . Instrumentation Figure 1: Instrumentation of double Beam UV-Visible spectroscopy 6/16/2021 DOPs, SIHAS, SHUATS, Prayagraj, U.P, India 3

4. The fundamental law that governs the quantitative spectrophotometric analysis Beer -Lambert law. It states that the intensity of a beam of parallel monochromatic radiation decreases exponentially with the number of absorbing molecules i.e absorbance is proportional to the concentration and passes through a medium of homogeneous thickness. A combination of these two laws yields the Beer-Lambert law. Mathematically , Beer Lambert law is expressed as A=a b c (Where , A=absorbance or optical density, a = absorptivity or extinction coefficient, b=path length of radiation through sample (cm), c=concentration of solute in solution. Both b and a are constant so a is directly proportional to the concentration c When c is in gm/100 ml, then the constant is called A (1%, 1 cm) 6/16/2021 DOPs, SIHAS, SHUATS, Prayagraj, U.P, India 4

5. Drug profile of azelnidipine Molecular weight: 582.646 g/mol Molecular formula: C33H34N4O Melting point and pka: 122-123c and 7.89 Absorption: Orally absorbed Bioavailability: Less than 50% Half life: 16-24 hrs Plasma protein binding and Cmax: : ~90% and 3.0-13.1 ng/ml Metabolism: Metabolized by cytochrome P450 (CYP) 3A4 in the liver and has no active metabolite(5). Storage: Stored In tightly closed container in cool, dry and well maintained area. Solubility: Slightly soluble in methanol, freely soluble in acetone , soluble in ethyl acetate, sparingly soluble in water 6/16/2021 DOPs, SIHAS, SHUATS, Prayagraj, U.P, India Figure 2: Chemical structure of azelnidipine and their IUPAC name 5

6. Objective The Objective of this dissertation work is as follows Aim of the present work is to Develop some new analytical methods for the estimation of azelnidipine and drug formulations To Develop rapid, sensitive and selective method Economic and accurate method Method validation according to ICH guidelines. 6/16/2021 DOPs, SIHAS, SHUATS, Prayagraj, U.P, India 6

7. Methodology: UV-Visible spectroscopy Selection of analytical wavelength Preparation of stock solutions Calibration curve for the Azelnidipine Sample preparation for determination of azelnidipine from dosage form Validation of Spectrophotometric methods 6/16/2021 DOPs, SIHAS, SHUATS, Prayagraj, U.P, India 7

8. Selection of wavelength (λ max ) For the selection of analytical wavelength range for method 100µg/ml azelnidipine was scanned in the spectrum mode from 200nm to 400nm against distilled methanol as blank. Wavelength range was selected around wavelength maxima (257nm). 6/16/2021 DOPs, SIHAS, SHUATS, Prayagraj, U.P, India Figure 3: λ max of azelnidipine (100μg/ml) 8

9. Method development by UV-Visible spectroscopy It is the process of formulating the material, condition, and protocol for measuring the analyte To develop a suitable UV-Visible method for determination of azelnidipine with the compositions of methanol was used as diluents Finally good % RSD was found with diluents methanol. On scanning the standard solution against diluents in entire UV-Visible range of 200-800nm, good response was found at 257nm for 100µg/ml solution. There is no interference from diluents methanol through the entire UV-Visible range 200-800nm. 6/16/2021 DOPs, SIHAS, SHUATS, Prayagraj, U.P, India 9

10. Method life cycle 6/16/2021 DOPs, SIHAS, SHUATS, Prayagraj, U.P, India Figure 4 : method development life cycle 10

11. Steps involved in method development Analyte standard characterization   Method requirements   Literature search and prior methodology   Choosing a method   Instrumental setup and initial studies Optimization Documentation of analytical figures of merit   Evaluation of method development with actual samples Determination of percent RSD of actual sample and demonstration of quantitative sample analysis. 6/16/2021 DOPs, SIHAS, SHUATS, Prayagraj, U.P, India 11

12. Analytical method validation Validation of analytical procedures is the process of determining the suitability of a given methodology for providing useful analytical data . Validation is the formal and systematic proof that a method compiles with the requirements for testing a product when observing a defined procedures. Purpose of Method Validation Identification of Sources and Quantitation of Potential errors Determination if Method is Acceptable for Intended Use Establish Proof that a Method Can be Used for Decision Making Satisfy FDA Requirements 6/16/2021 DOPs, SIHAS, SHUATS, Prayagraj, U.P, India 12

13. Various validation parameters Figure 5: Eight Steps of Analytical Method Validation 6/16/2021 DOPs, SIHAS, SHUATS, Prayagraj, U.P, India 13

14. 1. Linearity Ability of an assay to elicit a direct and proportional response to changes in analyte concentration. By Visual Inspection of plot of signals vs. analyte concentration By Appropriate statistical methods Linear Regression (y = mx + c) Correlation Coefficient, y-intercept (b), slope (m ) Acceptance criteria: Linear regression r2 > 0.95 Requires a minimum of 5 concentration levels S.N Concentration (μg/ml) Absorbance 1 2 0.187 2 4 0.326 3 6 0.424 4 8 0.534 5 10 0.722 6 12 0.951 7 14 1.019 6/16/2021 DOPs, SIHAS, SHUATS, Prayagraj, U.P, India Figure 6: Calibration Curve of Azelnidipine at 257 nm 14 Table 1 : Calibration Curve of linearity

15. 2.Range The specified range is normally derived from linearity studies and depends on the intended application of the procedure e.g.. 2-14µg/ml. It is established by confirming that the analytical procedure provides an acceptable degree of linearity, accuracy and precision when applied to samples containing amounts of analyte within or at the extremes of the specified range of the analytical procedure. For Drug Substance & Drug product Assay 80 to 120% of test Concentration i.e 2-14µg/ml. 6/16/2021 DOPs, SIHAS, SHUATS, Prayagraj, U.P, India 15

16. 3.Accuracy Closeness of the test results obtained by the method to the true value. Should be established across specified range of analytical procedure. Should be assessed using a minimum of 3 concentration levels, each in triplicate (total of 9 determinations) Should be reported as %RSD of known amount added Acceptance criteria : % RSD ≤ 2% S.N SPIKE % TOTAL CONCENTRATION ABSORBANCE MEAN STD DEVIATION % RSD 1 80% 27µg/ml 27µg/ml 27µg/ml 1.001 1.010 1.023 1.011 0.011 1.09% 2 100% 30µg/ml 30µg/ml 30µg/ml 1.023 1.041 1.047 1.037 0.0124 1.20% 4 120% 33µg/ml 33µg/ml 33µg/ml 1.073 1.081 1.091 1.081 0.0090 0.83% 6/16/2021 DOPs, SIHAS, SHUATS, Prayagraj, U.P, India 16 Table 2: accuracy data

17. 4.Precision The closeness of agreement (degree of scatter) between a series of measurements obtained from multiple samplings of the same homogeneous sample . Should be investigated using homogeneous, authentic samples. Precision Considered at 3 Levels Repeatability Intraday Precision Interday precision 6/16/2021 DOPs, SIHAS, SHUATS, Prayagraj, U.P, India 17

18. A. Repeatability Express the precision under the same operating conditions over a short interval of time . Also referred to as Intra-assay precision Should be assessed using minimum of 9 determinations(3 concentrations/ 3 replicates) Acceptance criteria : % RSD ≤ 2% S.N Concentration (µg/ml) Absorbance Mean Std. deviation % RSD 1 15µg/ml 0.771 0.805 0.067 8.37% 2 15µg/ml 0.784 3 15µg/ml 0.816 4 15µg/ml 0.804 5 15µg/ml 0.730 6 15µg/ml 0.929 6/16/2021 DOPs, SIHAS, SHUATS, Prayagraj, U.P, India Table 3 : Repeatability data 18

19. B. Intraday precision D etermined by repeating the above methods at different time intervals (morning, afternoon and evening) on the same day (Intraday precision) and on three consecutive days (interday precision). The intraday variation for the estimation of Azelnidipine was carried out at three different concentration levels of 5, 15and 25 µg/ml as shown in table . Acceptance criteria : % RSD ≤ 2% C . Interday precision D etermined by repeating the above methods on three consecutive days (interday precision). The interday variation for the estimation of Azelnidipine was carried out at three different concentration levels of 5, 15and 25 µg/ml as shown in table. Acceptance criteria : % RSD ≤ 2% 6/16/2021 DOPs, SIHAS, SHUATS, Prayagraj, U.P, India 19

20. S.N Concentration ( µg/ml ) Absorbance Mean Std deviation % RSD 1 2 3 1 5 µg/ml 0.436 0.441 0.432 0.436 0.0045 1.03% 2 15 µg/ml 1.267 1.311 1.289 1.289 0.022 1.71% 3 25 µg/ml 1.344 1.389 1.357 1.363 0.023 1.70% 6/16/2021 DOPs, SIHAS, SHUATS, Prayagraj, U.P, India S.N Concentration (µg/ml ) Absorbance Mean Std deviation % RSD 1 2 3 1 5 µg/ml 0.436 0.452 0.439 0.442 0.0085 1.92% 2 15 µg/ml 1.267 1.319 1.309 1.298 0.027 2.13% 3 25 µg/ml 1.344 1.382 1.446 1.390 0.0515 3.71% Table 5: Interday Precision data Table 4: Intraday Precision data 20

21. 5. Detection Limit (LOD)/ Quantitation Limit (LOQ) Detection Limit (LOD ) Lowest amount of analyte in a sample that can be detected but not necessarily quantitated. Acceptance criteria: Less than 2 Based on Standard Deviation of the Response and the Slope. From the formula, we calculate LOD = (3.3*SD)/slope. Where , SD= the standard deviation of y-intercept of 5 calibration curves. Slope= the mean slope of the 5 calibration curves. Quantitation Limit (LOQ) Lowest amount of analyte in a sample that can be quantified with suitable accuracy and precision . Estimated by Signal to Noise Ratio of 10:1 Based on Standard Deviation of the Response and the Slope The LOQ may be calculated as LOQ = 10 × (σ/S). Where , σ = Standard deviation of the Y- intercepts of the five calibration curves . S = Mean slope of the five calibration curves. 6/16/2021 DOPs, SIHAS, SHUATS, Prayagraj, U.P, India 21

22. 6.Robustness It is a measure of its capacity to remain unaffected by small, but deliberate variations in method parameters and provides an indication of its reliability during normal usage. It is carryout by doing deliberate variation in method parameter is done (i.e. change in wavelength, analysis by person to person and changing room temperature). Absorbance of any one concentration 15 µg/ml is measured at three different wavelengths i.e. 256,257,258 nm and calculate % RSD. Acceptance criteria : % RSD ≤ 2% 6/16/2021 DOPs, SIHAS, SHUATS, Prayagraj, U.P, India 22

23. S.N Parameter Absorbance Mean Std. Deviation % RSD 1 Wavelength (256,257,258 nm) 256 257 258 0.494 0.4963 0.0068 1.37% 0.491 0.504 2 Analysis by person to person 1 st 2 nd 3 rd 0.476 0.484 0.0065 1.34% 0.481 0.491 3 By changing T emperature 38c 43c 40c 0.436 0.442 0.0052 1.92% 0.452 0.439 6/16/2021 DOPs, SIHAS, SHUATS, Prayagraj, U.P, India Table 6: Measure Robustness (15µg/ml) of the method 23

24. 7.Assay of Azelnidipine Tablet In assay of the azelnidipine tablet formulation. At first Measure the absorbance of standard concentration three times (1%) at 1000µg (10 µg/ml) was found to be mean value 325 and sample concentration at 10µg (0.01 µg/ml) was found to be mean value 0.319 at 257 nm. Calculate the % assay of the sample of the drug. % assay = sample abs × Std. Conc. / Std. Abs × test Conc. × 100% 6/16/2021 DOPs, SIHAS, SHUATS, Prayagraj, U.P, India S.N Std.Conc Abs (1000 µg) Sample Conc.(10 µg) % Assay 1 325 0.319 98.87% Table 7: Assay of azelnidipine tablet 24

25. 8.Result and Discussion S.N PARAMETER NORMAL RANGE RESULT 1 Linearity (R 2 ) 0.999 0.9826 Slope (m) - 0.072 Y-intercept - 0.017 2 Accuracy % RSD ≤ 2% 2.42 % 3 Precision Repeatability % RSD ≤ 2% 8.37 % 1.03-1.48 % 1.92-5.58 % Intraday Interday 4 Limit of detection (LOD) Less than 2 0.77 5 Limit of Quantification (LOQ) Less than 2 2.36 6 Robustness % RSD ≤ 2% 1.37-1.92% 7 Assay of azelnidipine % Assay ±99.98% 98.87% 6/16/2021 DOPs, SIHAS, SHUATS, Prayagraj, U.P, India Table 8: Summary of Validation Parameter 25

26. The method was tested and validated for various parameters according to ICH guidelines. In validation of UV-Visible Spectroscopy, it can be concluded that spectroscopic method was found to be simple and rapid method. Correlation coefficient of Linearity and The value of % RSD for intra-day were within normal range and the accuracy was found to be nearby normal range but in the interday precision was found to be more than 2. This value was out of normal range. The value of % assay was found to be more than 98.87% for this method. Shows that the method is almost accurate and free from the in interference used in formulation. 6/16/2021 DOPs, SIHAS, SHUATS, Prayagraj, U.P, India 26

27. The limit of detection and limit of quantification of APL were found to be 0.77 and 2.36. The excipients in the commercial tablet preparation did not interfere with the % assay . The developed UV-Visible Spectrophotometric method was found to be least accurate, sensitive, precise, so this method was almost accurate to apply in pharmaceutical tablet formulation for quantitative estimation of Azelnidipine. The developed UV method was subjected to stability indicating studies for Azelnidipine. It was found that the interfering peak from solvent did not interfere with estimation of drug and the developed method was found to be specific for estimation of Azelnidipine. 6/16/2021 DOPs, SIHAS, SHUATS, Prayagraj, U.P, India 27

28. 9. Conclusion The proposed method development of UV-Visible SPECTROSCOPY was found to be least rapid, precise, accurate and sensitive in comparison to other . Many samples cannot be suitably analyzed by this method. Hence developed method cannot be used for routine analysis of AZP in various formulations . It was concluded that developed method is simple, almost accurate, precise and reliable. 6/16/2021 DOPs, SIHAS, SHUATS, Prayagraj, U.P, India 28

29. 10.References 1. Guy RC. International Conference on Harmonisation . Encycl Toxicol Third Ed. 2014;2:1070–1072. 2. Thakare L, Ahmad S, Shastry VM. Development and Validation of Uv -Visible Spectrophotometric Method for Estimation of Cilnidipine and Telmisartan in Bulk and Dosage Form. Indo Am J Pharm Res. 2017;7(04):8552–8559. 3. Jenisha Modi , Shivangi K. Patel, Namrata Parikh, Shreya R. Shah* PKP and UMU. World Journal of Pharmaceutical Research. Infection. 2014;5(2):831-847. 4. Rane AS, Mahajan SK. Validation and Forced Stability-Indicating Hptlc Method for Determination of Azelnidipine. Rane al World J Pharm Res World J Pharm Res SJIF Impact Factor 6 [Internet]. 2016;5(9):1053–1062. Available from: www.wjpr.net 6/16/2021 DOPs, SIHAS, SHUATS, Prayagraj, U.P, India 29

30. Thank you! Any question? 6/16/2021 DOPs, SIHAS, SHUATS, Prayagraj, U.P, India 30