1. Lecture 10 ANALYTICAL METHOD DEVELOPMENT AND VALIDATION IN HPLC AND GC. Lecture 10 – Chromatography, 11-12-2012 Dr. Rasha Hanafi 1© Dr. Rasha Hanafi, GUC

2. METHOD DEVELOPMENT PLAN FOR HPLC & GC The goal of a new chromatographic method is to obtain adequate separation in reasonable time. The procedure should be RUGGED. STEPS: 1. To determine the goal of analysis (qualitative / quantitative) 2.Select a method for sample preparation (sample clean up: Solid phase extraction, liquid-liquid extraction) 3.Choice of the right detector (sensitivity range, destructive/ non- destructive/ responsiveness to analytes). 4.Choice of column (diameter, length, particle size, thickness/type of st. phase, RP is adequate to separate low m.wt. neutral or charged organic compounds) 5.Choice of injection technique (in GC: split/splitless/on column. In HPLC: Rheodyne manual injector/ autosampler). 6.Method Validation © Dr. Rasha Hanafi, GUCLecture 10 – Chromatography, 11-12-20122

3. CRITERIA FOR ADEQUATE SEPARATION 1.Capacity factor 0.5<K‘<20. If K' is too small, the 1 st peak is distorted by the solvent front. 2.If K' is too large, the run takes too long and the peak usually suffers from broadening. 3.For quantitation, min. R s = 1.5. For better ruggedness R s = 2 is better. 4.Avoid exceeding upper operating pressure of the hardware. Keeping pressure close to 150 bar in HPLC prolongs the life of pumps, valves, seals and autosampler used for automatic injection. 5.All peaks should be symmetric, with an asymmetry factor in the range of 0.9-1.5. © Dr. Rasha Hanafi, GUC3Lecture 10 – Chromatography, 11-12-2012

4. OPTIMIZATION OF 1 ORGANIC SOLVENT OF THE MOBILE PHASE IN HPLC Generally the mobile phase is composed of acetonitrile, methanol, tetrahydrofuran with water or aqueous buffer (for specific pH) which provides sufficient types of interactions with analytes to separate a vast number of compounds in RP. Advantages/ disadvantages of each of the 3 organic solvents??? 1.Acetonitrile low cut off…yet expensive 2.Methanol cheap…yet relatively high cut off 3.Tetrahydrofuran need to be handled with a higher level of work place safety and environmental control © Dr. Rasha Hanafi, GUC4Lecture 10 – Chromatography, 11-12-2012

5. GRADIENT ELUTION IS A FINE WAY TO BEGIN METHOD DEVELOPMENT IN HPLC Quickest way: run a broad gradient. Ex: from 10% to 90% B in t G = 40 min. Δt is the difference between the first and the last eluting peaks and t G the gradient time over which the solvent composition is changed. Use gradient if Δt/ t G > 0.25 Use isocratic if Δt/ t G < 0.25 (meaning that all peaks are eluting over a narrow solvent range). A good start is the point halfway through the interval Δt (i.e. if the first peak elutes at 10 min and the last peak elutes at 20 min, a reasonable isocratic solvent composition would be at 15 min in the gradient). © Dr. Rasha Hanafi, GUCLecture 10 – Chromatography, 11-12-20125

6. METHOD VALIDATION The primary regulatory concerns in analysis relate to data validity, specifically how data can be verified scientifically and documented in a legally defensible manner. Method validation is the process of ensuring that a test procedure is accurate, reproducible, and robust within the specified analyte range for the intended application. Main validation parameters: specificity, selectivity, linearity, range, robustness, LOD, LOQ. © Dr. Rasha Hanafi, GUCLecture 10 – Chromatography, 11-12-20126

7. SPECIFICITY, ACCURACY Specificity is the ability of a method to discriminate between the intended analyte(s) and other components in the sample. Specificity of the HPLC method is demonstrated by the separation of the analytes from other potential components such as impurities, degradants, or excipients. Accuracy is the closeness in agreement of the accepted true value or a reference value to the actual result obtained. Accuracy studies are usually evaluated by determining the recovery of a spiked sample of the analyte into the matrix of the sample (a placebo). © Dr. Rasha Hanafi, GUC Lecture 10 – Chromatography, 11-12-2012 7

8. LOD, LOQ Limit of detection (LOD) is the smallest amount or concentration of analyte that can be detected. There are a number of ways for the calculation of LOD. The simplest method to calculate LOD is to determine the amount (or concentration) of an analyte that yields a peak height with a signal-to-noise ratio (S/N) of 3. Limit of quantitation (LOQ) is the lowest level that an analyte can be quantitated with some degree of certainty (e.g., with a precision of ±5%).The simplest method for calculating LOQ is to determine the amount (or concentration) of an analyte that yields a peak with a signal-to-noise ratio of 10. Thus, LOQ is roughly equal to 3 times of LOD. © Dr. Rasha Hanafi, GUC Lecture 10 – Chromatography, 11-12-2012 8

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10. LINEARITY, RANGE Linearity of a method is its ability to obtain test results that are directly proportional to the sample concentration over a given range, using the relationship between detector response (peak area or height) and sample concentration (or amount). The range of an analytical method is the interval between the upper and lower analytical concentration of a sample that has been demonstrated to show acceptable levels of accuracy, precision, and linearity. © Dr. Rasha Hanafi, GUC Lecture 10 – Chromatography, 11-12-2012 10

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12. PRECISION Method 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). Intermediate precision, synonymous with the term “ruggedness,” 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. It 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 method transfer (e.g., from a research facility to quality control of a manufacturing plant). © Dr. Rasha Hanafi, GUC Lecture 10 – Chromatography, 11-12-2012 12

13. ROBUSTNESS Robustness is a measure of the performance of a method when small, deliberate changes are made to the specified method parameters. The intent of robustness validation is to identify critical parameters for the successful implementation of the method. In the case of liquid chromatography, examples of typical variations are: Influence of variations of pH in a mobile phase Influence of variations in mobile phase composition Different columns (different lots and/or suppliers) Temperature Flow rate In the case of gas-chromatography, examples of typical variations are: Different columns (different lots and/or suppliers) Temperature Flow rate © Dr. Rasha Hanafi, GUC Lecture 10 – Chromatography, 11-12-2012 13

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15. REFERENCES © Dr. Rasha Hanafi, GUCLecture 10 – Chromatography, 11-12-201215 1.“Quantitative Chemical Analysis, 7 th ed. By Harris” Chapter- section 25-3 “Method Development for Reversed-Phase separations”. 2.“Quantitative Chemical Analysis, 7 th ed. By Harris” Chapter- section 24-5 “Method development in Gas Chromatography”. 3.“Quantitative Chemical Analysis, 7 th ed. By Harris” Chapter- section 29-2 “Method Validation”.