M. Pharm. Practical Course Lab. Manual

Transcription

1 PH 5103: Pharmaceutical Quality Control and Analytical Method Validation Lab M. Pharm. Practical Course Lab. Manual Fall Semester 2015 By To Dr. Hasina Yasmin Associate Professor Department of Pharmacy State University of Bangladesh

2 INDEX SI NO. Date Name of the Experiment Page No 01 UV-Spectrophotometric Determination of Paracetamol in Pharmaceutical Dosage Forms (tablet) using single concentration of standard 02 UV-Spectrophotometric Determination of Paracetamol in Pharmaceutical Dosage Forms using Calibration Curve Validation of UV-Spectrophotometric method for determination of Paracetamol in Pharmaceutical Dosage Forms: Construction of Calibration Curve and Error Analysis Validation of UV-Spectrophotometric Method for determination of Ranitidine : LOD, LOQ, Linearity range Validation of UV-Spectrophotometric Method for determination of Ranitidine : Intraday and Inter-day precision Validation of UV-Spectrophotometric Method for determination of Ranitidine : Accuracy by recovery study Dr. Hasina Yasmin 2

3 Dr. Hasina Yasmin 3

4 Experiment 1: UV-Spectrophotometric Determination of Paracetamol in Pharmaceutical Dosage Forms using single standard concentration Objective Scan an absorbance spectrum using the spectrophotometer. Choose a suitable wavelength for photometric determination. Determine the concentration of paracetamol in dosage forms (Napa tablet). Calculate potency (%) of the provided commercial product. Principle Electrons in n or molecular orbitals are excited to higher energy levels upon absorbance of electromagnetic radiation. Molecules containing at least two conjugated double bonds (allowing mesomerism) absorb ultraviolet ( = nm) or visible ( = nm) light; this part of a molecule is called chromophore. The larger the chromophoric system, the less energy is required for excitation, the longer the respective absorbance wavelength: 1 h h c E. Molecular absorption spectroscopy is based on the measurement of transmittance T or absorbance A of solutions contained in transparent cells having a path length of b cm. Ordinarily, the concentration c of the absorbing species, the analyte, is linearly related to absorbance as represented by the following equation. LAMBERT S AND BEER S Law: A =-log T =log po/p =εbc, where p: radiation intensity at the detector p0: initial intensity of the radiation. Dr. Hasina Yasmin 4

5 ε: molar absorptivity Absorbance is directly proportional to concentration of the light absorbing species in the sample. LAMBERT S AND BEER S law is obeyed for monochromatic light. Numerous reagents react selectively with non-absorbing species to yield products that absorb strongly in the ultraviolet or visible region. For standard of a compound, Astd = εbc std...i For sample of the same compound, Asam = εbc sam II Dividing equation I by equation II gives, Astd/Asam = c std /c sam Or, c sam = (c std Asam)/ Astd The unkown concentration of a solution can be calculated by using the above formula. Apparatus & Glassware UV / Vis Spectrophotometer (Shimadzu-1700) 4 Volumetric flasks 100-mL 2 Pipette 5-mL Pipette filler Chemicals / Sample Sodium hydroxide solution: 0.1 M Standard paracetamol Napa tablets (Beximco Pharmaceuticals Ltd.) Indication: Paracetamol is used as an analgesic and antipyretic. composition: Labeled content 500 mg paracetamol / tbl. C8H9NO2 (Mr = 151.2) N-(4-Hydroxyphenyl)acetamide / 4-Hydroxyacetanilide / para-acetamidophenol / Acetaminophen Specifications of paracetamol according to B.P.: appearance: white, crystalline powder. solubility: sparingly soluble in water, freely soluble in alcohol, very slightly soluble in methylene chloride. Dr. Hasina Yasmin 5

6 Procedure Serial dilutions of standard solution preparation 1- Weigh accurately about 10 mg of paracetamol standard into a 100 ml volumetric flask. 2- Add about 50ml of 0.1N sodium hydroxide and shake for 15 minutes to mix. 3- Make the volume upto the mark with water and mix well. 4- Pipet 4 ml of aliquot into a 100 ml volumetric flask and add make up the volume with water and mix well. Test solution preparation Tablet 1- Weigh and powder 10 tablets. 2- Take a quantity of the powder equivalent to 10 mg of paracetamol into a 100 ml volumetric flask. 3- Add 50 ml 0.1N sodium hydroxide, dilute with water and sonicate for 5 min. 4- Make up the volume with water. 5- Mix well and filter through filter paper. 6- Transfer 4 ml of the filtrate to a 100 ml volumetric flask and add make up the volume with water. Syrup 1- Pipette accurately about 0.42 ml paracetamol syrup equivalent to 10 mg of paracetamol into a 100 ml volumetric flask. 2- Follow the same procedure used for tablet preparation from step 3. No need to sonicate. Blank preparation: Use water as blank. Taking the absorbance: Take the absorbance of standard and sample solutions at 257 nm using water as the blank. Dr. Hasina Yasmin 6

7 Experiment 2: UV-Spectrophotometric method for determination of paracetamol using a calibration curve Objective Scan an absorbance spectrum using the spectrophotometer. Choose a suitable wavelength for photometric determination. Construct a calibration curve for standard paracetamol solution. Principle Electrons in n or molecular orbitals are excited to higher energy levels upon absorbance of electromagnetic radiation. Molecules containing at least two conjugated double bonds (allowing mesomerism) absorb ultraviolet ( = nm) or visible ( = nm) light; this part of a molecule is called chromophore. The larger the chromophoric system, the less energy is required for excitation, the longer the respective absorbance wavelength: 1 h h c E. Molecular absorption spectroscopy is based on the measurement of transmittance T or absorbance A of solutions contained in transparent cells having a path length of b cm. Ordinarily, the concentration c of the absorbing species, the analyte, is linearly related to absorbance as represented by the following equation. LAMBERT S AND BEER S Law: A =-log T =log po/p =εbc, where Dr. Hasina Yasmin 7

8 p: radiation intensity at the detector p0: initial intensity of the radiation. ε: molar absorptivity Absorbance is directly proportional to concentration of the light absorbing species in the sample. LAMBERT S AND BEER S law is a relation between absorbance and concentration which is a straight line passing by the origin at a constant path length, b, and at certain wavelength,. LAMBERT S AND BEER S law is obeyed for monochromatic light. Numerous reagents react selectively with non-absorbing species to yield products that absorb strongly in the ultraviolet or visible region. Figure 1: Calibration curve Path length (distance that light travels through the solution) is determined by the cuvette that the sample is placed in spectrophotometers. Usually the cuvettes with a path length of 1 cm. Apparatus & Glassware UV / Vis Spectrophotometer (Shimadzu-1700) 8 Volumetric flasks 100-mL Pipette 10-mL Pipette 5-mL Pipette filler Procedure Standard paracetamol solution: Take 10.0 mg paracetamol standard in a 100 ml volumetric flask. Add 50 ml of 0.1 M sodium hydroxide, add 25 ml of water, shake for 15 minutes and make the volume with water to produce ml. Dr. Hasina Yasmin 8

9 Serial dilutions of standard solution preparation 1. Into a 100-mL volumetric flask, pipette 16.0 ml of the standard paracetamol stock solution, and make the volume to ml with water. The concentration of the solution is 16.0 ug/ml. For serial dilution, take 50 ml of the above solution (16 ug/ml) in a 100 ml volumetric flask and volume up to 100 ml with water. The concentration of the solution is 8.0 ug/ml. Similarly, prepare the 4.0 ug/ml solution from 8.0 ug/ml solution and 2.0 ug/ml solution from 4.0 ug/ml solution. 2. Repeat the total process to prepare another two sets of the solutions of 2.0, 4.0, 8.0 and 16.0 ug/ml. 3. Use water as the reagent blank. 4. Record the absorbance readings of the each standard solution for 3 times at 257 nm using the reagent blank. 5. Construct a calibration curve. Test solution preparation 1- Weigh and powder 5 tablets. 2- Take a quantity of the powder equivalent to 10 mg of paracetamol into a 100 ml volumetric flask. 3- Add 50 ml 0.1N sodium hydroxide, dilute with water and sonicate for 5 min. 4- Make up the volume with water. 5- Mix well and filter through filter paper. 6- Transfer 10 ml of the filtrate to a 100 ml volumetric flask and add make up the volume with water. 7- Record the absorbance readings of the each standard solution for 3 times at 257 nm using the reagent blank. 8- Calculate the potency using calibration curve. Dr. Hasina Yasmin 9

10 Experiment 3: Method Validation: UV-Spectrophotometric method for determination of paracetamol : Error analysis Objective Construct a calibration curve for standard paracetamol solution. Calculate standard deviation and show in the curve. Principle Same as experiment 2. Standard deviation calculation In statistics, the standard deviation (SD, also represented by the Greek letter sigma, σ for the population standard deviation or s for the sample standard deviation) is a measure that is used to quantify the amount of variation or dispersion of a set of data values. A standard deviation close to 0 indicates that the data points tend to be very close to the mean of the set, while a high standard deviation indicates that the data points are spread out over a wider range of values. Error bars are a graphical representation of the variability of data and are used on graphs to indicate the error, or uncertainty in a reported measurement. They give a general idea of how accurate a measurement is, or conversely, how far from the reported value the true (error free) value might be. Error bars often represent one standard deviation of uncertainty, one standard error, or a certain confidence interval (e.g., a 95% interval). Apparatus & Glassware UV / Vis Spectrophotometer (Shimadzu-1700) 8 Volumetric flasks 100-mL Pipette 10-mL Pipette 5-mL Pipette filler Chemicals / Sample Same as experiment 2. Dr. Hasina Yasmin 10

11 Procedure Serial dilutions of standard solution preparation 1. Into a 100-mL volumetric flask, pipette 16.0 ml of the standard paracetamol stock solution, and make the volume to ml with water. The concentration of the solution is 16.0 ug/ml. For serial dilution, take 50 ml of the above solution (16 ug/ml) in a 100 ml volumetric flask and volume up to 100 ml with water. The concentration of the solution is 8.0 ug/ml. Similarly, prepare the 4.0 ug/ml solution from 8.0 ug/ml solution and 2.0 ug/ml solution from 4.0 ug/ml solution. 2. Repeat the total process to prepare another two sets of the solutions of 2.0, 4.0, 8.0 and 16.0 ug/ml. 3. Use water as the reagent blank. 4. Record the absorbance readings of the each standard solution for 3 times at 257 nm using the reagent blank. 5. Construct a calibration curve. 6. Calculate the standard deviation using Microsoft Excel. 7. Insert the error bars in the curve showing the standard deviation. Dr. Hasina Yasmin 11

12 Experiment 4: Method validation: UV-Spectrophotometric Determination of Ranitidine: LOD, LOQ & Linearity range Objective Scan an absorbance spectrum using the spectrophotometer. Choose a suitable wavelength for photometric determination. Determine the concentration of ranitidine in different dosage forms (tablet). Determine the range of linearity, LOD, LOQ, Principle Analytical Method Validation Validation is defined by the International Organization for Standardization (ISO) as verification, where the specified requirements are adequate for an intended use, where the term verification is defined as provision of objective evidence that a given item fulfills specified requirements. The method can be validated for use as a screening (qualitative), semi-quantitative (e.g ppm) or quantitative method. The various validation parameters include accuracy, precision (repeatability, interday and intraday precision, and reproducibility), specificity, detection limit, quantitation limit, linearity, range, robustness, system suitability testing etc. The validation of an analytic method demonstrates the scientific soundness of the measurement or characterization. It is required to varying extents throughout the regulatory submission process. The validation practice demonstrates that an analytic method measures the correct substance, in the correct amount, and in the appropriate range for the intended samples. It allows the analyst to understand the behavior of the method and to establish the performance limits of the method. Validation Parameters 1. Linearity range The linearity of an analytical procedure is its ability (within a given range) to obtain test results which are directly proportional to the concentration (amount) of analyte in the sample. A linear relationship should be evaluated across the range of the analytical procedure. Dr. Hasina Yasmin 12

13 It may be demonstrated directly on the drug substance (by dilution of a standard stock solution) and/or separate weighings of synthetic mixtures of the drug product components, using the proposed procedure. The latter aspect can be studied during investigation of the range. Linearity should be evaluated by visual inspection of a plot of signals as a function of analyte concentration or content. If there is a linear relationship, test results should be evaluated by appropriate statistical methods, for example, by calculation of a regression line by the method of least squares. In some cases, to obtain linearity between assays and sample concentrations, the test data may need to be subjected to a mathematical transformation prior to the regression analysis. Data from the regression line itself may be helpful to provide mathematical estimates of the degree of linearity. The correlation coefficient, y-intercept, slope of the regression line and residual sum of squares should be submitted. A plot of the data should be included. In addition, an analysis of the deviation of the actual data points from the regression line may also be helpful for evaluating linearity. For the establishment of linearity, a minimum of 5 concentrations is recommended. Other approaches should be justified. 2. Limit of Detection (LOD) LOD of an individual analytical procedure is the lowest amount of analyte in a sample which can be detected but not necessarily quantitated as an exact value. Several approaches for determining the detection limit are possible, depending on whether the procedure is a non-instrumental or instrumental. Approaches other than those listed below may be acceptable. Based on Signal-to-Noise This approach can only be applied to analytical procedures which exhibit baseline noise. Determination of the signal-to-noise ratio is performed by comparing measured signals from samples with known low concentrations of analyte with those of blank samples and establishing the minimum concentration at which the analyte can be reliably detected. A signal-to-noise ratio between 3 or 2:1 is generally considered acceptable for estimating the detection limit. Based on the Standard Deviation of the Response and the Slope The limit of detection (LOD) may be expressed as: LOD Where, σ = the standard deviation of the response and S = the slope of the calibration curve. Dr. Hasina Yasmin 13

14 The slope S may be estimated from the calibration curve of the analyte. The estimate of σ may be carried out in a variety of ways, for example: Based on the Standard Deviation of the Blank - Measurement of the magnitude of analytical background response is performed by analyzing an appropriate number of blank samples and calculating the standard deviation of these responses. Based on the Calibration Curve - A specific calibration curve should be studied using samples containing an analyte in the range of DL. The residual standard deviation of a regression line or the standard deviation of y-intercepts of regression lines may be used as the standard deviation. 3. Limit of Quantitation (LOQ) LOQ of an individual analytical procedure is the lowest amount of analyte in a sample which can be quantitatively determined with suitable precision and accuracy. The quantitation limit is a parameter of quantitative assays for low levels of compounds in sample matrices, and is used particularly for the determination of impurities and/or degradation products.several approaches for determining the quantitation limit are possible, depending on whether the procedure is a non-instrumental or instrumental. Approaches other than those listed below may be acceptable. Based on Signal-to-Noise Approach This approach can only be applied to analytical procedures that exhibit baseline noise. Determination of the signal-to-noise ratio is performed by comparing measured signals from samples with known low concentrations of analyte with those of blank samples and by establishing the minimum concentration at which the analyte can be reliably quantified. A typical signal-to-noise ratio is 10:1. Based on the Standard Deviation of the Response and the Slope The limit of Quantitation (LOQ) may be expressed as: LOQ Where, σ = the standard deviation of the response and S = the slope of the calibration curve. The slope S may be estimated from the calibration curve of the analyte. The estimate of σ may be carried out in a variety of ways as shown for LOD. Dr. Hasina Yasmin 14

15 Apparatus & Glassware UV / Vis Spectrophotometer (Shimadzu-1700) 8 Volumetric flasks 100-mL 2 Pipette 5-mL Pipette filler Chemicals / Sample Standard ranitidine Procedure Linearity range 1- Weigh accurately about 50 mg of ranitidine standard into a 50 ml volumetric flask. 2- Add about 40 ml of water and shake thoroughly for 15 minutes to mix. 3- Make the volume upto the mark with water and mix well. Concentration of the solution is 1 mg/ml. 4- Prepare two different solutions of 128 and 96 ug/ml (1 mg/ml) by taking 12.8 ml and 9.6 ml from the stock solution in two different 100 ml volumetric flasks. Volume the flasks up to the mark with water and mix well. 5- Prepare two different sets of concentrations of 100 ml each from the above two solutions as follows by serial dilution: 1, 2, 4, 8, 16, 32, 64 ug/ml from 128 ug/ml and 1.5, 3, 6, 12, 24, 48 ug/ml from 96 ug/ml solution. 6- Measure the absorbance of the solutions at 314 nm and draw a scatter diagram by plotting absorbance Vs concentration (1, 1.5, 2, 3, 4, 6, 8, 12, 16, 24, 32, 48, 64, 96, and 128 ug/ml). It will be a hyperbolic curve and give non-linear relation between concentrations and absorbance. 7- Select the concentration range from the above range and draw a calibration curve that will give a linear relation with R 2 value approximates This is the linearity range. LOD and LOQ 1- The slope of the calibration curve will be the s 2- Take absorbance of the reagent blank (H2O) for six different times (n = 6). 3- Calculate the standard deviation of the six absorbance values and it will be the σ. 4- Calculate LOD and LOQ using the formula. Dr. Hasina Yasmin 15

16 Experiment 5: Method validation: UV-Spectrophotometric Determination of Ranitidine: Intraday and Inter day precision Objective Scan an absorbance spectrum using the spectrophotometer. Choose a suitable wavelength for photometric determination. Determine the concentration of ranitidine in different dosage forms (tablet). Determine Intraday and inter day precision. Principle Analytical Method Validation Validation is defined by the International Organization for Standardization (ISO) as verification, where the specified requirements are adequate for an intended use, where the term verification is defined as provision of objective evidence that a given item fulfills specified requirements. The method can be validated for use as a screening (qualitative), semi-quantitative (e.g ppm) or quantitative method. The various validation parameters include accuracy, precision (repeatability, interday and intraday precision, and reproducibility), specificity, detection limit, quantitation limit, linearity, range, robustness, system suitability testing etc. The validation of an analytic method demonstrates the scientific soundness of the measurement or characterization. It is required to varying extents throughout the regulatory submission process. The validation practice demonstrates that an analytic method measures the correct substance, in the correct amount, and in the appropriate range for the intended samples. It allows the analyst to understand the behavior of the method and to establish the performance limits of the method. Validation Parameters Precision The precision of an analytical procedure expresses the closeness of agreement (degree of scatter) between a series of measurements obtained from multiple sampling of the same homogeneous sample under the prescribed conditions. Precision may be considered at three levels: repeatability, intermediate precision Dr. Hasina Yasmin 16

17 and reproducibility. Precision should be investigated using homogeneous, authentic samples. However, if it is not possible to obtain a homogeneous sample it may be investigated using artificially prepared samples or a sample solution. The precision of an analytical procedure is usually expressed as the variance, standard deviation or coefficient of variation of a series of measurements. i) Reproducibility The measure of agreement between results obtained with the same method on identical test or reference material under different conditions (execution by different persons, in different laboratories, with different equipment and at different times). The measure of reproducibility R is the standard deviation of these results SR, and for a not too small number of data (n 8) R is defined by (with 95% confidence): R = 2.8 SR (where 2.8 = 2 and is derived from the normal or gaussian distribution; ISO 5725). Thus, reproducibility is a measure of the spread of results when a sample is analyzed by different laboratories. This parameter can obviously not be verified in daily practice. For that purpose the next two parameters are used (repeatability and within-laboratory reproducibility). ii) Repeatability Repeatability expresses the precision under the same operating conditions over a short interval of time. Repeatability is also termed intra-assay precision. Repeatability should be assessed using: a minimum of 9 determinations covering the specified range for the procedure (e.g. 3 concentrations / 3 replicates each); or a minimum of 6 determinations at 100% of the test concentration. iii) Intermediate Precision Intermediate precision expresses within-laboratories variations: different days, different analysts, different equipment, etc. The extent to which intermediate precision should be established depends on the circumstances under which the procedure is intended to be used. The applicant should establish the effects of random events on the precision of the analytical procedure. Typical variations to be studied include days, analysts, equipment, etc. It is not considered necessary to study these effects individually. The use of an experimental design (matrix) is encouraged. ** Precision is usually determined for the sample analysis. Dr. Hasina Yasmin 17

18 Procedure Intraday precision Serial dilutions of standard solution preparation 1- Weigh accurately about 10 mg of ranitidine standard into a 100 ml volumetric flask. 2- Add about 40 ml of water and shake thoroughly for 15 minutes to mix. 3- Make the volume up to the mark with water and mix well. 4- Pipette 2, 4, 8, & 16 ml of aliquot into a 100 ml volumetric flasks and make up the volume with water and mix well. Concentrations of the solutions will be 2, 4, 8, & 16 ug/ml. 5- Repeat the total process to prepare another two sets of the solutions of 2.0, 4.0, 8.0 and 16.0 ug/ml. 6- Take the absorbance of all the above solutions at 314 nm using water as the blank. 7- Construct a calibration curve. 8- Calculate the standard deviation using Microsoft Excel. 9- Insert the error bars in the curve showing the standard deviation. Test solution preparation 1- Calculate the average weight of the tablets and grind them into fine powder. 2- Weigh accurately 10 mg ranitidine equivalent powder into a 100 ml volumetric flask. 3- Add 40 ml water, and sonicate for 5 min. 4- Make up the volume with water. Mix well and filter through filter paper. 5- Transfer 10 ml of the filtrate to a 100 ml volumetric flask and make up the volume with water. Prepare 5 different solutions of the same concentration (10 ug/ml). 6- Take the absorbance of sample solutions at 314 nm using water as the blank. 7- Calculate the concentration of the solutions using the equation of calibration curve. 8- Determine the standard deviation of the sample concentrations. Inter day precision 1- Repeat the above process on another day and determine the standard deviation of the sample concentrations. 2- Determine the standard deviation between the average sample concentrations of Day 1 and Day 2. Dr. Hasina Yasmin 18

19 Experiment 6: Method validation: UV-Spectrophotometric Determination of Ranitidine: Accuracy Objective Scan an absorbance spectrum using the spectrophotometer. Choose a suitable wavelength for photometric determination. Determine the concentration of ranitidine in different dosage forms (tablet). Determine the accuracy by recovery study. Principle A dart board is a good way to illustrate precision and accuracy. Accuracy refers to the extent that all measurements agree with the true value of what is being measured. Accuracy refers to how close a value is to the true value. The difference from the true method is called the bias. Precision refers to the magnitude of random errors and the reproducibility of measurements. In other words, if you run a test many times on the same sample, precision will be a measure of how close all the test results are to each other. Figure 1 shows results that are both precise (close together) and accurate (close to the true value.) Figure 2 illustrates a series of results that are again very precise, but this time they are all clustered away from the bull s eye, so they are not accurate (e.g. the results for a new method are consistently clustered away from the reference or comparison method results). In Figure 3, you can see that the results are scattered around the bull s eye, but only one of them is on the mark. This illustrates that a test can t really be very accurate, unless it is precise. Dr. Hasina Yasmin 19

20 Figure 2 Figure 1 Figure 3 Procedure Test solution preparation 1- Calculate the average weight of the tablets and grind them into fine powder. 2- Weigh accurately 10 mg ranitidine equivalent powder into a 100 ml volumetric flask. 3- Add 40 ml water, and sonicate for 5 min. Make up the volume with water. 4- Mix well and filter through filter paper. 5- Transfer 4 ml of the filtrate to each of 4 different 100 ml volumetric flasks. Standard solution preparation 1- Weigh accurately about 10 mg of ranitidine standard into a 100 ml volumetric flask. 2- Add about 40 ml of water and shake thoroughly for 15 minutes to mix. 3- Make the volume up to the mark with water and mix well. 4- Pipette 0, 2, 4 & 6 ml of aliquot into the above four 100 ml volumetric flasks containing the sample solutions. 5- Make up the volume with water and mix well. 6- Final concentration of the solutions will be 4, 6, 8, and 10 ug/ml. 7- Take the absorbance of solutions at 314 nm using water as the blank. 8- Calculate the percent of standard recovered. Dr. Hasina Yasmin 20