Method Validation

American University of Science and Technology

Nina Esipenko, Ph.D.

 Introduction

• Validation is a process of performing a set of

experiments that reliably estimates the efficacy and
reliability of an analytical method or modification to a
previously validated method.

• Goal is to demonstrate that a method is capable of

successfully performing at a level of its intended use
and to identify the methods limitations under normal
operation condition.
Benefits of Method Validation
Method validation is a practice which can prove to be beneficial
in a number of situations such as:
• Publication of your findings in international journals of
repute or presentation in professional conferences
• Instils a degree of self-esteem and confidence in yourself as
well as in those who use your results
• Acceptance of procedures and results by accreditation
bodies and external auditors
• Arriving at critical decisions concerning the use of data with
regards to the safe use of products by consumers
• Freedom from disputes concerning non-agreement of test
results with other laboratories
Validation becomes necessary:
• Before adopting the method in routine use

• When analytical conditions are changed such as change of

technique, change in desired concentration range or change of
sample matrix

• Whenever changes are made to an existing procedure

As a regular practice, any method needs to be validated before

adoption. However, it would need to be revalidated under the
following situations:
• Any changes made in the established procedure

• Any new accessory is added to the existing system

• The system has undergone major operational problems

which have been rectified.
Analytical method validation, thinking about the maximum
relevant processes for checking the best parameters of
analytical methods, using numerous relevant overall
performance indicators such as

 Selectivity
 Specificity
 Accuracy
 Precision
 Linearity
 Range
 Limit of detection (LOD)
 Limit of quantification (LOQ)
 Ruggedness
 Robustness
 NEW ANALYTICAL METHOD

 Analytical purpose

 Available equipment

 Analyte properties
 Sample matrix

 In-house methods
 Methods in the literature
Development of Instrumental Parameters
• Developed using reference materials

• Use analytes of interest to determine performance

Development of Sample Prep Techniques

• Evaluated and optimized using reference materials

• Use analytes of interest to determine extraction,

detection, identification and quantification.
Establishing a Validation Plan

Development of a validation plan must include:

• Instrumental parameters
• Sample preparation techniques
• Document the requirements of the method
• Document the limits of the method
• Identify target concentration and acceptance criteria for:
 Reference materials
 Quality controls
 Calibrations
 Comparison of known samples
Required Parameters
(based upon scope of method)

Scope includes:
• Screening methods
• Qualitative confirmation/identification methods
• Quantitative methods

Depending upon the scope or type of testing method,

certain studies must be performed.
 Required Parameters

Screening
• Interference studies
• LOD (limit of detection)
• Dilution integrity (if applicable)
• Stability (if applicable)

Qualitative confirmation/identification
• Carryover
• Interference studies
• Ionization suppression/enhancement (LC/MS)
• LOD (limit of detection)
• Dilution integrity (if applicable)
• Stability (if applicable)
 Required Parameters
Quantitative
• Bias (accuracy)
• Calibration model
• Carryover
• Interference studies
• Ionization suppression/enhancement (LC/MS)
• LOD (limit of detection)
• LOQ (limit of quantitation)
• Precision
• Dilution integrity (if applicable)
• Stability (if applicable)
Define the problem and select the method
of analysis accordingly

Each and every analysis requires the choice of a method

that will help you achieve the required objectives. In case
an established method is available, you should consider
yourself fortunate as this will save you the effort and time
required to validate the method before adopting it.

Several options are available to you for the selection of the

method to suit your analysis requirements. Such options
include a survey of standard analytical textbooks,
research journal publications
It is essential to know the requirements of analysis. In
making your method choice, you have to take into
consideration factors such as:

• Nature of components to be detected and quantified

• Expected concentration levels
• Nature of the sample matrix (nature of interferences that can be
present that can complicate the determination of required components)
• Required degree of accuracy and precision
• Availability of required reagents and standards
• Availability of required instrumental facilities
• Availability of trained manpower
• Time requirement for analysis
• Cost of analysis
• The selected method usually represents a compromise
between accuracy required, time and equipment
available for analysis.

Method selection depend on the facilities, equipment and

instruments available; sensitivity, precision required
and cost.

• It is important to take into consideration sample size as

well as its complexity (matrix) and possible number of
components present in the sample.
In method development and validation cycle it is important to
set minimum requirements, which are essentially acceptance
specifications for the method. A complete list of criteria
should be agreed on by the developer and the end users
before the method is developed so that expectations are clear.

For example, is it critical that method precision (RSD) be 2%?

Does the method need to be accurate to within 2% of the target
concentration? Is it acceptable to have only one supplier of the
HPLC column used in the analysis? During the actual studies
and in the final validation report, these criteria will allow clear
judgment about the acceptability of the analytical method.
 Obtain Representative Samples

• To produce meaningful information, an analysis must be

performed on a sample that has the same composition as the bulk
(population) of material from which it was taken.

• Sampling is the process of collecting a small mass of a material

whose composition accurately represents the bulk (population) of
the materials being analyzed.

 When the bulk is large and heterogeneous (parts can be

distinguished) great effort is required to get a representative
sample.

 When the bulk is homogeneous, the sample population is

reduced in size to an amount that can be conveniently
handled in the laboratory.
 Prepare Laboratory Samples

Depending on the sample, sometime sample processing is required.

Keep the INTEGRITY of the sample !
• A solid sample is ground to decrease particle size, mixed to
ensure homegeneity, and stored before analysis. Precaution:
Control the water adsorption content.
• For liquid samples. Precaution: avoid volume changes and cross-
contamination.
• For gas dissolved in a liquid. Precaution: Isolate the container and
avoid contamination.

The reliability of the final results will be often limited by this step.
 Define Replicate Samples
Laboratory replicate sample (replicate) is a apportion of the
representative sample used for laboratory quality control purposes
to demonstrate reproducibility. It is prepared and analyzed in the
identical manner as the representative sample. The results from
replicate analyses are used to evaluate analytical precision.

• Quantitative measurements on replicates are usually

averaged, and various statistical tests are performed on the
results to establish reliability.

Your confidence on results reported increases with the replication

of analysis. Statistically speaking the larger the number of replicates
the closer will be the experimental mean to the true mean. Generally,
minimum of 3 replicate samples are required.
 Significant Figures
(sf)

Significant figures are the digits of value which carry

meaning towards the resolution of the measurement.

The number of significant figures in a result is simply the

number of figures that are known with some degree of reliability

Significant figures can affect how useful a reported number is.

The same number can be reported in multiple ways, with some
ways being more specific and reliable.
• Any digit that is not zero is significant
1.234 kg 4 significant figures
• Exact numbers have an infinite number of s.f.
• Zeros between nonzero digits (captive zeros) are significant
606 m 3 significant figures
• Zeros to the left of the first nonzero digit (leading zeros) are not
significant
0.08 L 1 significant figure
• If a number is greater than 1, then all zeros to the right of the
decimal point are significant
2.0 mg 2 significant figures
• If a number is less than 1, then only the zeros that are at the end
and in the middle of the number are significant
0.00420 g 3 significant figures
•Trailing zeros are counted only if it is a decimal point
100 (1 s.f.)
 Determine the number of significant figures in the
following measurements:
(a) 478 cm
(b) 6.01 g

(c) 0.825 m
(d) 0.043 kg
(e) 1.310 × 1022 atoms
(f) 7000 mL

(g) 0.00000003 cm
(h) 2900.0 g

(i) 60,104 tons

 Significant Figures (sf)
Addition or Subtraction
The number of digits after decimal point in the result is set
by the original number that has the smallest number of
digits after decimal point.

89.332
+ 1.1 one significant figure after decimal point
90.432 round off to 90.4

3.70 two significant figures after decimal point

-2.9133
0.7867 round off to 0.79
 Significant Figures (sf)
Multiplication or Division
The number of significant figures in the result is set by
the original number that has the smallest number of
significant figures.

4.51 x 3.6666 = 16.536366 = 16.5

3 s.f. round to
3 s.f.

6.8 ÷ 112.04 = 0.0606926 = 0.061

2 s.f. round to
2 s.f.
Carry out the following arithmetic operations to the correct
number of significant figures:
11,254.1 g + 0.1983 g

66.59 L − 3.113 L

8.16 m × 5.1355

0.0154 kg  88.3 mL
Carry out the following operations and express each answer in
the correct units with the correct number of significant figures:
5.6792 m + 0.6 m + 4.33 m

3.90 g – 2.9133 g

4.51 cm × 3.6666 cm

(3 × 104 g + 6.827 g) / (0.043 mL – 0.021 mL)

(3.26 × 105 dm) + (7.74 × 107 dm)

(7.8 m – 0.34 m) / (1.15 s + 0.82 s)

7.310 km ÷ 5.70 km