A Guide to Validation in HPLC
Based on the work of G. M. Hearn PERKIN ELMER
Sometimes you may wonder who was the first to make an experimental procedure for a specific analysis. The route from an idea to an actual standard operating procedure (SOP) is not easy. In the early days it was a long period oftrial and error to establish a document where common users could benefit from. Nowadays the use of computer programmes like Drylab or HIPAC makes it easier to optimize the intended procedure to a practical analysis for other laboratories and schools. This document will try to explain some of the basic jargon and glossary concerning HPLC. At the end you may understand the long route to a methodthat has been validated as have many of the experiments and analytical procedures you will use within StandardBase.
"Validation of an analytical method is the process by which it is established by laboratory studies, that the performance characteristics of the method meet the requirements for the intended analytical application - "(6) Validation is required for any newor amended method to ensure that it is capable of giving reproducible and reliable results, when used by different operators employing the same equipment in the same or different laboratories. The type of validation programme required depends entirely on the particular method and its proposed applications.
Typical analytical parameters used in assay validation include: 1. 2. 3. 4. 5. 6. 7. 8. 9.Precision Accuracy Linearity Range Ruggedness Limit of detection Limit of quantitation Selectivity Specificity
R.A. van Iterson Drenthe College Emmen Holland for www.standardbase.com 2.1 Precision "The precision of an analytical method is the degree of agreement among individual test results obtained when the method is applied to multiple sampling of a homogenous sample -"(6) Precision is ameasure of the reproducibility of the whole analytical method (including sampling, sample preparation and analysis) under normal operating circumstances. Precision is determined by using the method to assay a sample for a sufficient number of times to obtain statistically valid results (ie between 6 - 1 0). The precision is then expressed as the relative standard deviation std dev x 100% %RSD =────────── mean 2.2 Accuracy "Accuracy is a measure of the closeness of test results obtained by a method to the true value. " (6) Accuracy indicates the deviation between the mean value found and the true value. lt is determined by applying the method to samples to which known amounts of analyte have been added. These should be analysed against standard and blank solutions to ensure that no interferenceexists. The accuracy is then calculated from the test results as a percentage of the analyte recovered by the assay. Accuracy and precision are not the same, as the diagram below indicates. A method can have good precision and yet not be accurate.
Errors in measurement can be divided into two general categories: systematic errors and
R.A. van Iterson Drenthe College Emmen Holland forwww.standardbase.com random errors. Systematic errors result from sources that can be traced to the methodology, the instrument or the operator, and affect both the accuracy and the precision of the measurement. Random errors only affect the precision, and are difficult to eliminate, because they are the result of random fluctuations in the measured signal, due to noise and other factors. Whilst systematicerrors are proportional to the sum of individual contributions, random errors are proportional to the root of the sum of the squares of the individual contributions. Thus, the imprecision of the entire procedure is often dominated by the random errors of the most imprecise step. 2.3 Linearity This is the method's ability to obtain results which are either directly, or after mathematical...