Chapter 1

Experimental design

AAC811S
2022
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 Content

1. The Need for Reliable Results

2. Analytical Strategy

3. Analytical Method Development and Validation

4. Considerations in Selection of an Analytical Method

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 1. The Need for Reliable Results

Measurements affect the daily lives of every citizen.

Sound, accurate and reliable measurements, be they physical, chemical or

biological, are essential to the functioning of modern society.

For these reasons, advanced nations spend up to 6% of their Gross

National Product (GNP) on measurements and measurement-related
operations.
(E. Prichard & V. Barwick, 2007)

• The results of physical and chemical measurements and analyses touch

on practically every aspect of modern life.

• For ex., we feel threatened by environmental pollution when we cannot

perceive pollutants directly with our own senses and instead picture in
our imagination water, soil, and air quality based on a system of
threshold values and analytical results.
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Analysis: determination of the composition of a material, i.e.
identification (qualitative) of its constituent parts and how much
(quantitative) of each component is present and, sometimes, in what
form (speciation).
An analysis involves several steps and operations which depend on:
•the particular problem
• the analyst’s expertise
• the apparatus or equipment available.
The analyst should be involved in every step.

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• Environmental data collection projects require systematic planning built
on broad knowledge of environmental regulations and technical
expertise of scientists.

• Planning is best carried out by a team of multidiscipline professionals,

who understand the overall project objectives and the specific
objectives of each task.

• Two major tasks of the planning phase are Data Quality Objectives
(DQOs) Development and Sampling and Analysis Plan (SAP)
Preparation.

“DQOs are qualitative and quantitative statements, developed using the

DQO process, that clarify study objectives, define the appropriate type of
data, and specify tolerable levels of potential decision errors that will be
used as the basis for establishing the quality and quantity of data needed
to support decisions.” (USEPA, 2000)

• The DQO process consists of seven consecutive steps

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7 steps of the DQO process

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Before starting work on a sample, it is vital to enquire why the work is
required, what will happen to the result(s) and to find out what
decisions will be taken based on the numerical values obtained.

Examples of social and economic impact of wrong analytical results:

• In forensic analysis, it could lead to a wrongful conviction or the guilty

going unpunished.

• In trade, it could lead to the supply of sub-standard goods and the

high cost of replacement with subsequent loss of customers.

• In environmental monitoring, mistakes could lead to hazards being

undetected or to the identification of unreal hazards.

• In the supply of drinking water, it could lead to harmful contaminants

being undetected.

• In healthcare, the incorrect medication or the incorrect content of

active ingredient in a tablet can be catastrophic for the patient. 7
The objective of analytical work is the achievement of reliable analytical
results of a defined quality.

What is quality?

 Delivering to a customer a product or service that meets the

specification agreed on with the customer, and delivering it on time.

 Satisfying customer requirements.

 Fitness for purpose.

 Getting it right the first time.

 The totality of features and characteristics of a product or service that

bear on its ability to satisfy stated or implied needs (ISO 1994).

 Degree to which a set of inherent characteristics (= distinguishing

features) fulfils requirements (= need or expectation that is stated,
generally implied, or obligatory) (ISO 2005).

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Quality characteristics of analytical processes are therefore:

• Specificity: the ability of an analytical process to register the

desired analytes in all relevant forms.

• Selectivity: the ability of an analytical process to register only the

desired analyte, while other components in or characteristics of the
sample – known as the matrix – do not influence the result.

• Sensitivity: the change in measured value per change in analyte

concentration.

• Accuracy: in the sense of both trueness (lack of systematic errors)

and precision (measurement of differences between results, as
obtained by repeated use of an established analytical process on
the same sample; imprecision is caused by random errors).

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 2. Analytical Strategy

• The process by which an analyte’s identity or the concentration level in

a sample is determined in the lab may involve many individual steps.

• These steps may vary in specifics according to the analyte, the matrix
and the chosen analytical method.

• The general organizational framework for these parts is known as the

analytical strategy (or experimental design).

There are five parts to the analytical strategy:

1) obtain the sample,
2) prepare the sample,
3) carry out the analysis method,
4) work up the data, and
5) calculate and report the results.

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 Summary of the
analytical process

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 3. Analytical Method Development and Validation
METHOD DEVELOPMENT

The process of establishing an analytical procedure for the reliable

qualitative and/or quantitative assessment of a target entity (analyte).

Reasons for developing new methods of analysis:

.There may not be a suitable method for a particular analyte in the specific sample
matrix.

.Existing methods may be too error-, artifact-, and/or contamination-prone, or they may
be unreliable (have poor accuracy or precision).

.Existing methods may be too expensive, time consuming, or energy intensive, or they
may not be easily automated.

.Existing methods may not provide adequate sensitivity or analyte selectivity in

samples of interest.

.Newer instrumentation and techniques may have evolved that provide opportunities
for improved methods, including improved analyte identification or detection limits,
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greater accuracy or precision, or better return on investment.
 New or improved methods of analysis include:

.Qualitative identification of the specific analyte(s) of interest, providing some

structural information to confirm " general behavior" (e.g., retention time, color
change, pH).

.Quantitative determination, at trace levels when necessary, that is accurate,

precise, and reproducible in any laboratory setting when performed according
to established procedures.

.Ease of use, ability to be automated, high sample throughput (maximum rate at

which samples can be processed), and rapid sample turnaround time.

.Decreased cost per analysis from using simple quality assurance and quality
control procedures.

.Sample preparation that minimizes time, effort, materials, and volume of sample
consumed.

.Direct output of qualitative or quantitative data to laboratory computers in a format

usable for evaluation, interpretation, printing out, and transmission to other
locations via a network.

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 OPTIMISATION General criteria:

.Analytical Method resolution is adequate.

.For most samples, limits of detection are lower by at least one order of magnitude than
needed.

.Calibration plots are linear over several orders of magnitude, beginning with limits of
quantitation.

.Sample throughput is increased, with minimal instrument equilibration (before run and
from run to run.).

.Sample preparation before analysis is minimized.

.Interference is minimized and identified, and approaches are established to circumvent

such problems.

.Data is acquired by computer and can be manipulated, translated, interpreted, printed, or

stored in various forms (computer software allows for rapid acquisition, storage, and
manipulation of data).

.Reproducibility of analytical figures of merit is demonstrated, with acceptable accuracy

and precision.
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.Cost per analysis is minimized.
Method validation: process used to confirm that the analytical procedure
employed for a specific test is suitable for its intended use.
 Results from method validation can be used to judge the quality, reliability
and consistency of analytical results; it is an integral part of any good
analytical practice.
The process consists of at least 4 distinct steps:
1. Software validation
2. Hardware (instrumentation) validation/quantification
3. Method validation
4. System suitability
Each step is critical to the overall success of the process.

Hardware Method
Validation

VALIDATION

Software System
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Suitability
 ANALYTICAL FIGURES OF MERIT
STEPS OF METHOD VALIDATION, ANALYTICAL PERFORMANCE PARAMETERS

• ACCURACY Measure of exactness of analytical methods

The closeness of agreement between the measured value and a “true value”

• PRECISION Measure of the degree of repeatability (presented as SD or %RSD)

• SPECIFICITY The ability to measure the analyte accurately and specifically in the
presence of other components (matrix)

• LIMIT OF DETECTION (LOD) The lowest concentration that can be detected

• LIMIT OF QUANTIFICATION (LOQ) The lowest concentration that can be determined with
good precision and accuracy

• LINEARITY The results are directly proportional to concentration

• RANGE Interval between the lowest and highest levels of concentration

• RUGGEDNESS Reproducibility under variety of conditions

• ROBUSTNESS Capacity of insusceptibility of a method to small variations in method

parameters
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 VALIDATION
APPROACHES

USP:
United States
Pharmacopeia

ICH:
International
Council for
Harmonisation

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 ASSAY VALIDATION

USP Data Elements Required for Assay Validation

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ICH Validation Characteristics versus
Type of Analytical Procedure

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 METHOD VALIDATION APPROACHES
Validation of optimized method to demonstrate its suitability for the given
analyte under real analytical conditions.

 Zero-blind method: Single analyst uses Fast, cheap, good 1st approximation,
the method with sample @ known levels of … but high risk of bias
analyte concentration to demonstrate
recovery, accuracy and precision.

 Single-blind method. 2 analysts: one Risk of bias during data comparison

prepares samples @ varying levels
unknown to the second who analyses them

 Double-blind method. 3 analysts: 1st Most objective approach. No risk of

prepares,2nd analyses &3rd compares both bias from analyst #3
sets of data received separately.

 Analysis of Standard Reference Risk of bias when SRMs values are

Material (SRM) known by the analyst.

 Interlaboratory collaborative study Most widely accepted… but costly, time

consuming and hard to coordinate

 Comparison with a currently accepted Uses results of the accepted method to

method verify new method results.
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 METHOD DEVELOPMENT, OPTIMIZATION, AND VALIDATION

1. CHARACTERIZATION OF STANDARD ANALYTE

2. REQUIREMENTS FOR THE ANALYTICAL METHOD

3. LITERATURE SEARCH AND PRIOR METHODOLOGY

4. CHOOSING A METHOD

5. INSTRUMENTATION AND PILOT STUDIES

6. OPTIMIZATION

7. DETERMINATION AND OPTIMIZATION OF ANALYTICAL FIGURES OF MERIT

8. EVALUATION OF DEVELOPED METHOD WITH ACTUAL SAMPLES: DERIVATION OF FIGURES OF

MERIT

9. VALIDATION OF FIGURES OF MERIT

10. QUANTITATIVE SAMPLE ANALYSIS AND DETERMINATION OF PERCENT RECOVERY

11. METHOD VALIDATION

12. PREPARATION OF METHOD MANUAL – PROTOCOLS AND PROCEDURES

13. INTERLABORATORY COLLABORATIVE STUDIES – TRANSFER OF METHOD TECHNOLOGY

14. COMPARISON OF INTERLABORATORY COLLABORATIVE STUDIES

15. SUMMARY REPORT ON VALIDATION RESULTS 22

16. SUMMARY REPORT OF METHOD, VALIDATION PROCEDURES AND RESULTS
4. Considerations in Selection of an Analytical Method
Accuracy
Precision
Selectivity
Limit of Detection
Interferences
Availability of Qualified Personnel
Number of Analyses or Determinations
Time per Analysis
Equipment Availability
Matrix
Cost of Analysis
Safety

ANALYTICAL TECHNIQUES

Gravimetric Analysis
Volumetric Analysis
Separations
Spectroscopy
Electrochemistry
Hyphenated Techniques 23
 SEPARATION
SPECTROSCOPY ELECTROCHEMISTRY
Chromatography
Atomic Potentiometry Thin Layer Chromatography
Atomic Absorption (AA) I. pH (TLC)
Atomic Emission (AE) 2. Ion Selective Electrodes Liquid Chromatography
I. Flame Polarography (HPLC)
2. Plasma (ICP ) Anodic Stripping Ion Chromatography (IC)
3. Arcs & Sparks Voltammetry Gas Chromatography (GC)
Atomic Fluorescence Conductometric Methods Electrophoresis
X-ray Fluorescence Extraction
Neutron Activation Liquid -Liquid
SPE
Molecular SLM
Infrared Physical
Raman Distillation
Mass Recrystallization
Nuclear Magnetic Headspace
Resonance
Ultraviolet- Visible
Absorption HYPHENATED METHODS
Fluorescence
Separation Detection
Gas Chromatography Mass Spectrometry (GC-MS)
Gas Chromatography Fourier Transform Infrared
Spectroscopy (GC-FTIR)
Liquid Chromatography Electrochemistry 24
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Summary & Conclusions
• Nowadays it is not only important to have state-of-the art analytical
equipment but it also is critical to produce quality analysis.

• The need for reliable data underlines the importance of analytical

method development and validation.

• Validation is a constant, evolving process that starts before an

instrument installation and continues long after method development
and transfer.

• A well-defined and documented validation process provides regulatory

agencies with evidence that the system and method is suitable for its
intended use.

• Method development, optimization and validation – more efficient and

productive use of laboratory resources.
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