By : TALAL K.

HASSAN ,
MSc. food chemistry. Senior adviser in standardization and quality control tkh953@hotmail.com

Q : WHAT IS A METHOD?
A : method is a procedure for the analysis of a specific analyte (e.g. determination of Mn in water).

Q : WHAT IS METHOD VALIDATION AND HOW DOES IT APPLY TO US (AS ANALYTICAL CHEMISTS).
A :Method Validation is a way of testing a particular analytical method to see if it is suitable for its intended purpose. The goal of Method Validation is to prove that the results obtained are true and to show that the assay will perform properly under the conditions in which it is intended to be used. Method validation is an important part of analytical chemistry as well as many other fields (software design, engineering, environmental toxicology, and the pharmaceutical industry to name a few).

WHY SHOULD WE VALIDATE METHODS?

In order to answer that question, we need to look at how and why methods are developed in the first place.
1. Identify parameter that needs to be known (is it a qualitative or quantitative application?) 2. Choose type of technology to be used (e.g. HPCL, GC or Mass spectrometry) 3. Develop the method 4. Pre-validate the method (how does it perform in its intended area of use) 5. Adjust or modify method if necessary (based on results of pre-validation) 6. Develop a validation plan (determine what criteria will be required) 7. Validate 8. Report findings (Conclusion: Is this method suitable for its intended purpose?).

ACCURACY
Closeness of agreement between the value obtained by the method and the true value. (this is how teacher calculated your results marks in the lab, if you had 100% Accuracy, you got 10/10 on your Data mark).

compare your results using one technique to results obtained with a second, validated procedure prepare a placebo matrix without any analyte in it and spike it with varying concentrations of the standard and analyse

use the procedure on a pure and characterized reference standard (more later) and calculate the recovery

PRECISION
Expresses the closeness of agreement between a series of measurements obtained from multiple sampling. Precision is often expressed as the standard deviation or Relative standard deviation of replicate measurements (more later on RSD). Note that a method can be precise, but not accurate (so all your measurements may be close together, but the result is wrong).

INTERMEDIATE PRECISION: Evaluate

the variance when the same sample is analyzed by using two or more different instruments, or between different lab technician or between different days. RSD >3

Reproducibility: Similar to intermediate

precision ,only the sample will tested by two or more different laboratories and the result will be evaluated

ACCURACY AND PRECISION

These two words became source of confusion for many people .Because a method is precise dose not mean it is accurate .its is easy to evaluate precision, just make several measurement s of the same thing and compare them. Evaluating Accuracy is more difficult ,especially if you dont know the ( true ) value of analyte. The below diagram illustrate four possible scenarios

SPECIFICITY
The ability to measure the analyte in the presence of components which we expect to be present in the sample matrix. So if you are determining the concentration of Iron and Chromium in water by UV/Vis spectroscopy, if there are small amounts of Fluoride and Chloride in the sample, will that affect my measurement?

THE PROCEDURE IS MEASURING ONLY THE COMPOUND INTERST, AND THE QUNTITATION IS NOT AFFECTED BY THE MATRIX COMPONENT

DETECTION LIMIT

QUANTITATION LIMIT

LINEARITY

The range of concentration of analyte for which the procedure provides test result that are in direct correlation to the amount of analyte in the sample (remember when you studied Beers law ,that absorbance wasnt linear at higher concentration of analyte

RANGE

The same as linearity, except the result must also be accurate and precise( over the concentration tested)

LINEARITY/RANGE
The assay might not be linear over a complete range of concentrations so it is important to define linearity and what range of values for which the procedure is linear. Usually ,linearity and range are evaluated at the same time. The Range is just the concentration for which the procedure is linear, accurate and precise(so you can image that several preparations of standard would need to be prepared at each concentration and then evaluate the plotted)

ROBUSTNESS ( RUGGEDNESS) Measure of an method s capacity to remain unaffected by small but deliberate variations in method parameters . (So all of the methods we used in our lab are some what robust, because the people using them are untrained well and the equipment is not always so reliable

RSD : ( RELATIVE STANDARD DEVIATION),WHAT IS IT

The RSD is just a way of normalizing standard deviation data so that it is easier to compare the variance in two different sets of numbers that may not be of the same magnitude. For example, in experiment (Determination of Nitrite and Nitrate by Anion Exchange Chromatography) you were asked to determine which measurement was better, peak height or peak area. Data from four injections of the unknown may have looked like this

BIAS
difference between the expectation of the test results and an accepted reference value
NOTE Bias is the total systematic error as contrasted to random error. There may be one or more systematic error components contributing to the bias. A larger systematic difference from the accepted reference value is reflected by a larger bias value.
[ISO 3534-1]

Bias (don't let precision fool you!)

So, if you measure something several times and all values are close, they may still all be wrong if there is a "Bias" Bias is a systematic (built in) error which makes all measurements wrong by a certain amount.

Examples of Bias

The scales read "1 kg" when there is nothing on them You always measure your height wearing shoes with thick soles.

STANDARD UNCERTAINTY

uncertainty of the result of a measurement expressed as a standard deviation

TRUENESS

trueness closeness of agreement between the average value obtained from a large set of test results and an accepted reference value
NOTE The measure of trueness is normally expressed in terms of bias. The reference to trueness as accuracy of the mean is not generally recommended.

[ISO 3534-1]

ONGOING DEMONSTRATION OF METHOD CONTROL

+ 3 std dev + 2 std dev + 1 std dev Average - 1 std dev

Measurement

- 2 std dev
- 3 std dev

Date/Time

UNCERTAINTY
measurement

parameter, associated with the result of a measurement, that characterizes the dispersion of the values that could reasonably be attributed to the measurand

NOTE 1 The parameter may be, for example, a standard deviation (or a given multiple of it), or the half-width of an interval having a stated level of confidence. NOTE 2 Uncertainty of measurement comprises, in general, many components. Some of these components may be evaluated from the statistical distribution of the results of a series of measurements and can be characterized by experimental standard deviations. Other components, which also can be characterized by standard deviations, are evaluated from assumed probability distributions based on experience or other information. NOTE 3 It is understood that the result of the measurement is the best estimate of the value of the measured, and that all components of uncertainty, including those arising from systematic effects such as components associated with corrections and reference standards, contribute to the dispersion.

C.2 DETERMINATION OF MEAT CONTENT

C.2.1 Introduction Meat products are regulated to ensure that the meat content is accurately declared. Meat content is determined as a combination of nitrogen content (converted to total protein), and fat content. The present example accordingly shows the principle of combining different contributions to uncertainty, each of which itself arises chiefly from reproducibility estimates, as described at Clause 12. C.2.2 Basic equations

C.2 DETERMINATION OF MEAT CONTENT

C.2.1 Introduction
Meat products are regulated to ensure that the meat content is accurately declared. Meat content is determined as a combination of nitrogen content (converted to total protein), and fat content. The present example accordingly shows the principle of combining different contributions to uncertainty, each of which itself arises chiefly from reproducibility estimates, as described at Clause 12.

C.2.2 Basic equations

C.2.3 EXPERIMENTAL STEPS IN MEAT-CONTENT DETERMINATION

C.2.4 UNCERTAINTY COMPONENTS

Thank You! Lets Get Started!!