VALIDATION AND CALIBRATION OF HPLC GUIDED BY: PRESENTED BY: DR. FALGUN A. MEHTA, SWAPNIL S. DOSHI H.O.D. Pharm.

Analysis, M.Pharm(Pharm. Analysis, I-Sem) IICP 1 PowerPoint Presentation: BLOCK DIAGRAM OF HPLC 2 PowerPoint Presentation: PROCESSING CHART OF HPLC 3 PowerPoint Presentation: Validation, USP: Validation of an analytical procedure is the process by which it is established, by laboratory studies, that the performance characteristics of the procedure meet the requirements for the intended analytical applications. Validation Protocol: A written plan stating how validation will be conducted and defining acceptance criteria. For example, the protocol for a manufacturing process identifies processing equipment, critical process parameters/operating ranges, product characteristics, sampling, test data to be collected, number of validation runs, and acceptable test results. Equipment Validation: Demonstrate that equipment used in validation studies is suitable for use and is comparable to equipment used for routine analysis. 4 PowerPoint Presentation: Qualification: Action of proving and documenting that equipment or ancillary systems are properly installed, work correctly, and actually lead to the expected results. Qualification is part of validation, but the individual qualification steps alone do not constitute process validation . Parts Of The Qualification: The entire process typically consists of four parts: Design qualification (DQ), Installation qualification(IQ), Operational qualification (OQ), and Performance qualification (PQ). 5 PowerPoint Presentation: A schematic diagram illustrating the timeline and documents in the various stages in HPLC system qualification. 6 PowerPoint Presentation: Assay Impurities Identification limit quantitative + - + - Accuracy + - + - Precision + + + + Specificity - + - Detection Limit - - + - Quantitation Limit + - + - Linearity + - + - Range + + + + Robustness Validation Characteristics 7 Reference: ICH Guidelines PowerPoint Presentation: Basic Parameters for the Validation of Method: 8 PowerPoint Presentation: Accuracy: Definition: The accuracy of an analytical procedure expresses the closeness of agreement between the value that is accepted either as a conventional true value or as an accepted reference value and the value found. How To Determine?? Known amounts of Related substances and the drug substance in placebo are spiked to prepare an accuracy sample of known concentration of Related substance. According to the ICH, accuracy should be determined using a minimum of nine determinations over a minimum of three concentration levels covering the range(from 50% of the ICH reporting level to 150% of the proposed shelf life specification of the related substances) specified. e.g. Level Accuracy 125% 99.6 +/- 0.2% 75% 100 .3 +/- 0.8% 25% 99.2 +/0.7% 9 (Reference: IJPSR Determination of Voglibose by HPLC and validation of method) PowerPoint Presentation: Intrinsic Accuracy: Intrinsic accuracy indicates the bias caused by sample matrix and sample preparation. Overall Accuracy: Matrix effect Sample preparation Calculation error %relative substance (calculated) Overall accuracy = ----------------------------------------- %relative substance (theory) 10 PowerPoint Presentation: Precision : Definition : The Precision is a measure of the ability of the method to generate reproducible results. The precision of a method is evaluated for repeatability, intermediate precision, and reproducibility. Repeatability is a measure of the ability of the method to generate similar results for multiple preparations of the same homogeneous sample by one analyst using the same instrument in a short time duration (e.g., on the same day). For instance, method repeatability for pharmaceutical assays may be measured by making six sample determinations at 100% concentration, or by preparing three samples at 80, 100, and 120% concentration levels each. Intermediate precision is a measure of the variability of method results where samples are tested and compared using different analysts, different equipment, and on different days, etc. This study is a measure of the intra-laboratory variability and is a measure of the precision that can be expected within a laboratory. Reproducibility is the precision obtained when samples are prepared and compared between different testing

sites. Method reproducibility is often assessed during collaborative studies at the time of technology or method transfer. %RSD= ( s / )*100 11 PowerPoint Presentation: Limit of Detection (LOD, DL ) The LOD of an analytical procedure is the lowest amount of analyte in sample which can be detected but not necessarily quantitated as an exact value . Determination is usually based on Signal to noise ratio (~3:1) (baseline noise) or Standard deviation of response ( s ) and Slope (S) 3.3 s /S SNR = H/h Where H = height of the peak corresponding to the component h = absolute value of the largest noise fluctuation from the baseline of the chromatogram of a blank solution 12 PowerPoint Presentation: Limit of Quantitation (LOQ, QL) The LOQ is the lowest amount of analyte in a sample which can be quantitatively determined with suitable precision and accuracy The quantitation limit is used particularly for the determination of impurities and/or degradation products Determination is usually based on Signal to noise ratio (~10:1) (baseline noise) or Standard deviation of response ( s ) and Slope (S) 10 s/ S - The Q uantitation limit of a method is affected by both the detector sensitivity and the accuracy of sample preparation. 13 PowerPoint Presentation: Noise LOD Signal to Noise = 3:1 LOQ Signal to Noise = 10:1 LOD, LOQ and Signal to Noise Ratio (SNR) PowerPoint Presentation: Range: ICH Definition: The range of an analytical procedure is the interval between the upper and lower concentrations of analytes in the for which it has been demonstrated that the analytical procedure has a suitable level of precision, accuracy, and linearity. Range For Different Tests: Assay 80 to 120% of test concentration Content uniformity 70 to 130% of test concentration Dissolution Q-20% to 120% Impurities Reporting level 120% of specification limit (with respect to test concentration of API) Assay & Impurities Reporting level to 120% of assay specification 15 PowerPoint Presentation: Linearity: Definition : Linearity of an analytical procedure is its ability (within a given range) to obtain test results which are directly proportional to the concentration of analytes in the sample. If there is a linear relationship test results should be evaluated by appropriate statistical methods like, Correlation coefficient Y-intercept Slope of regression line Plot of the Data Method To Determine Linearity Direct Weigh Method Dilution Method 16 PowerPoint Presentation: Linearity Ranges and Acceptance Criteria for Various Pharmaceutical Methods 17 Reference: Morden HPLC Michel Dong PowerPoint Presentation: SYSTEM SUITABILITY TESTING (SST) System suitability testing (SST) is used to verify resolution, column efficiency, and repeatability of the analysis system to ensure its adequacy for performing the intended application on a daily basis. Which Parameters?? Number of theoretical plates (efficiency) Capacity factor, Separation (relative retention) Resolution, Tailing factor Relative Standard Deviation (Precision) 18 PowerPoint Presentation: Plate number or number of theoretical plates (n) n=L/H, where L is Length of Column H is HETP or height of one theoretical plate Capacity factor (capacity ratio) k k= t r -t m / t m where t r is retention time t m is dead time Separation Factor (relative retention) =k1/k2 where k1 is capacity factor of compound a and k2 is capacity factor of compound b Tailing factor ,T T=W/ 2 f where W is width at 5% at peak height f, distance between max and leading edge of the peak 19 PowerPoint Presentation: 20 Reference: Morden HPLC Michel Dong PowerPoint Presentation: Robustness : Definition : Robustness is reliability of an analytical procedure with respect to deliberate variations in method parameters. If measurements are susceptible to variations in analytical conditions the analytical conditions should be suitably controlled or a precautionary statement should be included in the procedure. PARAMETERS TO BE EVALUATED FOR ROBUSTNESS Column consistency Three columns packed by bonded phases from three different silica lots Mobile Phase pH (0.10.2 units) Buffer concentration (510mM) Percentage organic modifier (12% MP) Sample Injection volume or sample concentration Solvent strength for the final solution Column temperature (5C) Detector wavelength (3nm) 21

PowerPoint Presentation: Selectivity and Specificity : Selectivity is the ability to measure accurately and specifically the analyte in the presence of components that may be expected to be present in the sample matrix. Specificity for an assay ensures that the signal measured comes from the substance of interest, and that there is no interference from excipient s and/or degradation products and/or impurities. 22 PowerPoint Presentation: Calibration: Definition (ICH): The demonstration that a particular instrument or device produces results within specified limits by comparison with those produced by a reference or traceable standard over an appropriate range of measurements . Various Calibration parameters for HPLC are: Flow rate accuracy (Pump Module) Injector accuracy Carryover System Precision Wavelength accuracy Detector linearity Injector linearity Gradient Performance Check Column oven temperature accuracy 23 PowerPoint Presentation: Flow Rate Accuracy: 1. Prime all the solvent lines with water. 2. Set the flow rate to 0.500 ml/m. 3.Wait for about 15 m to stabilize the system and ensure that the pressure is stable. 4.Insert the outlet tubing into a 10 ml volumetric flask and start the stop watch simultaneously. 5. Stop the stopwatch when the lower meniscus reaches the 10 ml mark on the flask. 6. Record the elapsed time. 7. Similarly check the flow for 1.0 ml/m and 2.0 ml/m. Acceptance criteria: The time taken to collect the water should be with in 2.0% of the actual value. 24 Reference: JBSR (Journal) PowerPoint Presentation: Pressure Test : The performance of the HPLC pump depends on the proper functioning of the check valves and the proper connection of the tubing. For pump systems that output the pressure reading in the pump head over time, a simple pressure test can be a useful qualitative test to check the condition of the check valves and to determine whether or not there are any leaks in the system. The first step of the pressure test is to plug the outlet of the pump using a dead-nut and by setting the automatic pump shutdown pressure to 6,000 psi. The pumphead pressure signal output is connected to a recorder. Pressurize the pump by pumping methanol at 1 mL/min. The pressure inside the pump head increases quickly as the outlet of the pump is blocked. As the pressure increases to about 3,000 psi, the flow rate is reduced to 0.1 mL/min. The pressure will gradually rise to the shutdown pressure if the check valves are able to hold the mobile phase in the pump chamber as would be normally expected . If the check valve is not functioning properly, the pressure will fluctuate at about 3,000 psi instead of reaching the shutdown pressure. 25 PowerPoint Presentation: 26 Reference: Analytical Method Validation and Instrument Performance Verification-Herman Lam and Y.C. Lec PowerPoint Presentation: Injector Accuracy: 1. Connect the pump and detector inlet with union. 2. Prepare mobile phase consisting of a mixture of water and Methanol (70:30) 3. Set a flow rate of 0.5 ml/m and a run time of 1 m. 4. Set the column temperature at 25 2C. 5.Fill a standard HPLC vial to 2/3rd with water. Seal the vial properly with a cap. 6. Weigh the vial and record the weight as W1 grams. 7. Place the vial in the chromatographic system and perform 6 injections of 50l volume from this vial. 8. Weigh the vial again and note the weigh after the injections as W2 grams. 9. Calculate the mean volume injected per injection as follows: Mean injected volume (l) = (W1 W2) 100/6 Acceptance criteria: The mean injected volume should be 50.01.0 l. 27 Reference : JBSR (Journal) Analytical Method Validation and Instrument Performance Verification -Herman Lam and Y.C. Lec PowerPoint Presentation: Carryover : Small amounts of analyte may get carried over from the previous injection and contaminate the next sample to be injected. The carryover will affect the accurate quantitation of the subsequent sample. The problem is more serious when a dilute sample is injected after a concentrated sample. In order to avoid cross contamination from the previous sample injection, all the parts in the injector that come into contact with the sample (the injection loop, the injection needle and the needle seat) have to be cleaned effectively after the injection. The effectiveness of the cleaning can be evaluated by injecting a blank after a sample that contains a high concentration of analyte. The response of the analyte found in the blank sample expressed as a percentage of the response of the concentrated sample can be used to determine the level of carryover. 28 PowerPoint Presentation: Detector Module: UV-Visible Detector Module: Wavelength Accuracy Wavelength accuracy is defined as the deviation of the wavelength reading at an absorption or emission band from the known wavelength of the band. The deuterium line at 656 nm and the absorption bands at 418, 453 and 536 nm in a Holmium oxide filter are often used . (B) PDA Detector Accuracy: Select 3D mode and set the wavelength range as 200-400nm. Inject 20 l Holonium oxide of standard preparation once into the chromatographic system. Extract and record the chromatograms at wavelengths of 202 to 208nm with an interval of 1nm and at 269 to 275 nm with an interval of

1nm. Note down the height or absorbance. Acceptance criteria: The maximum absorbance should be at 2052nm and 2722nm. 29 Reference : JBSR (Journal) PowerPoint Presentation: 30 You can Calibrate the PDA Detector for 5 Parameters: 1-Baseline Noise and PDA Drift. 2- Your Lamp Intensity. 3- You have to check for the Wavelength Accuracy. You can inject Pyrene with Methanol as eluent at a flow of 1 ml/min. The characteristically maximum of pyrene is determined at 333 nm and compared to a theoretical value. 4- Lastly- You must check your PDA's Linearity you can inject 5 caffeine solutions at different concentrations. Concentrations and peak heights can be represented in a graph. The regression coefficient of the resulting line and the deviations from it indicate indicates the linearity- Usually you R square value should be above 99.90% . Ref: Scientist solution.com Chemicalforum.com PowerPoint Presentation: Automated Detector Calibration From column To waste Filter with a known Absorbance(s) Flow cell 31 Automated Detector Calibration: Automated Detector Calibration From column To waste Filter with a known Absorbance(s) Flow cell 32 Holmium Oxide: Holmium Oxide 33 PowerPoint Presentation: Noise and Drift : Drift: upward &/or downward movement of base line. Noise : any unwanted signal recorded by the detector. Electronic, pump and photometric noise, poor lamp intensity, dirty flow cell, and thermal instability contribute to the overall noise and drift in the detector. Excessive noise can reduce the sensitivity of the detector and hence affect the quantitation of low-level analytes. Detector drift may affect the baseline determination and peak integration. Overcomes for avoiding Noise and Drift: The detector should be warmed up prior to the test, and any temperature fluctuations should be avoided during the test. For a dynamic testing condition, methanol is passed through the flow cell at 1 mL/min. A backpressure of about 500 psi is maintained to prevent bubble formation. 34 PowerPoint Presentation: Column Heating Module : The efficiency of a HPLC column varies with column temperature. capacity factor k decreases with temperature, and hence the retention of the analysis decreases with temperature . Retention drops by 1 to 3% for each increase of 1C. How To Check?? The temperature accuracy of the column heater is evaluated by placing a calibrated thermometer in the column compartment to measure the actual compartment temperature. The thermometer readings are compared to the preset temperature at 40 C and 60C. Reference: Chemistpub.org 35 PowerPoint Presentation: 36 Reference: Morden HPLC Michel Dong PowerPoint Presentation: 37 References: Morden HPLC by Michel Dong, Chapter 9. Analytical Method Validation and Instrument Performance Verification by Herman Lam and Y.C. Lec, Chapter 3 and Chapter 11 . Net surf. Scientist solution.com Chemicalforum.com Chemistpub.org 4. Pharmaceutical Journals, IJPSR JBSR PowerPoint Presentation: 38 PowerPoint Presentation: 39