Validation of analytical methods

Performance Characteristics
Dr. B. R. Thorat
Department of Chemistry
Ismail Yusuf College, Mumbai 400060

BACKGROUND-LAB METHOD FLOW

Method
Development

Method
Validation

Method
Transfer

Approved

VALIDATION
THE PROCESS OF PROVIDING DOCUMENTED
SOMETHING DOES WHAT IT IS INTENDED TO DO.

EVIDENCE

WHY VALIDATE?

To demonstrate that the method is suitable for its intended use

Provides assurance of reliability

Part of registration application

QC
WHY ANALYTICAL
METHOD VALIDATION

Verifying system suitability

For submission to Compendia

THAT

ANALYTICAL METHOD VALIDATION PERFORMANCE

CHARACTERISTICS
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TYPES OF ANALYTICAL METHODS TO BE

VALIDATED

Identification tests.
Quantitative tests for impurities content.
Limit tests for the control of impurities.
Quantitative tests of the active in samples of the drug substance (raw material), finished
product or other selected components in the drug.

ACCURACY

Expresses the CLOSENESS of agreement BETWEEN

the value, which is accepted either as a conventional
TRUE VALUE or an accepted REFERENCE VALUE
and the VALUE FOUND i.e. individual observation or
mean of measurements
It may expressed as in terms of either absolute or

relative error.

Accuracy is often more difficult to determine because the true value is usually unknown. An
accepted or reference value must be used instead.

PRECISION
Precision (reproducibility of measurements)
The measure of the degree of agreement (degree of scatter)
among test results when the method is applied repeatedly to
multiple samplings of a homogeneous sample.
Expressed as % RSD for a statistically significant number of
samples.

The precision (variability) of an analytical procedure is

usually expressed as the standard deviation (S), variance

(S2), or coefficient of variation (= relative standard
deviation, R.S.D.) of a series of measurements.

The confidence interval should be reported for each type

of precision investigated.

Repeatability (of any process)

Repeatability expresses the precision (spread of the data, variability) under the same
operating conditions over a short interval of time. Repeatability is also termed
intra-assay precision.

Intermediate Precision and Reproducibility

Intermediate

precision

expresses

within-laboratories

variations. A, B and C: different days, different analysts,

different (manufacturing) equipment, etc.

Reproducibility

expresses

the

precision

between

laboratories A, B and C (collaborative studies, usually

applied to standardization of methodology). (Transfer of

technology).

ACCURACY & PRECISION

SPECIFICITY/SELECTIVITY
The ability of analytical method to accurately and specifically measure a signal is a
function of only the amount or concentration of analyte present in the sample. In presence of
interfering components, selectivity can be calculated as -

Where, A is analyte and I is interferent, S is the signal, k is the sensitivities and n is the
number of moles.

The selectivity of the method for the interferent relative to the analyte is defined by a
selectivity coefficient,
Selectivity may be positive or negative depending on whether the interferents effect on the
signal is opposite or negative to that of the analyte. A selectivity coefficient greater than +1 or
less than 1 indicates that the method is more selective for the interferent than for the analyte

SPECIFICITY/SELECTIVITY
The degree of interference:

Active Ingredients
Excipients
Impurities (synthetic precursors, enantiomers)
Degradation Products

Placebo Ingredients
Combination of 2 or more analytical procedures may be required to achieve
necessary level of discrimination.

Stability indicating analytical methods should always be specific.

Analysts should ascertain whether the peaks within a sample chromatogram are
pure or consist of more than one compound. Therefore should know how many

compounds are in the sample or use procedures to detect peak purity.

12

Sensitivity
The definition of sensitivity most often used is the calibration sensitivity
the change in the response signal per unit change in analyte concentration
equivalent to the proportionality constant - k
SA is the smallest increment in signal that can be measured, when the smallest difference

in the amount of analyte that can be detected is

The calibration sensitivity is thus the slope

of the calibration curve.
If the calibration curve is linear, the
sensitivity is constant and independent of
concentration.
If nonlinear, sensitivity changes with
concentration and is not a single value.

LOD, LOQ and SNR

Peak B
LOQ

Limit of Quantitation (LOQ)

Limit of Detection (LOD)
Signal to Noise Ratio (SNR)

Baseline

Peak A
LOD

noise

LOD: The lowest concentration of an analyte in a sample that can be detected, not quantified.
LOQ: The lowest concentration of analyte in a sample that can be determined with acceptable
precision and accuracy under stated operational conditions.

Detection Limit
The detection limit, DL, is the smallest concentration that can be reported with a
certain level of confidence.
Every analytical technique has a detection limit. For methods that require a calibration
curve, the detection limit should be defined. It is determine by using following equation -

It is the analyte concentration that produces a response equal to k (called the confidence
factor ) times the standard deviation of the blank, Sb , and m is the calibration sensitivity.
The factor k is usually chosen to be 2 or 3.
A k value of 2 corresponds to a confidence
level of 92.1%, while a k value of 3
corresponds to a 98.3% confidence level.

Linear Dynamic Range

The linear dynamic range of an analytical method most often refers to the concentration
range over which the analyte can be determined using a linear calibration curve. .
upper end is the concentration
at which the analytical signal or
the slope of the calibration
curve deviates by a specified
amount

lower limit of the

dynamic
range
is
generally considered to
be the detection limit

Deviations from linearity are common at high

concentrations because of nonideal detector responses
or chemical effects.

Example: Determination of sodium in blood serum, only a small range is needed because
variations of the sodium level in humans is quite limited

Limit of quantitation
An alternative expression for the detection limit, which minimizes type 1 and type 2
errors, is the limit of identification (SA)LOI.

Where, z is relative deviation and is the standard deviation. The limit of identification
is selected such that there is an equal probability of type 1 and type 2 errors.
The American Chemical Societys Committee on Environmental Analytical Chemistry
recommends the limit of quantitation as -

RUGGEDNESS
Random variations in experimental conditions also introduce
uncertainty.
If a methods sensitivity is highly dependent on experimental
conditions, such as Different Analysts

Different Laboratories
Different Instruments
Different Reagents

Different Days
Etc.
Expressed as % RSD

ROBUSTNESS
The number of methods are subject to a variety of chemical
and physical interferences that contribute uncertainty to the
analysis.
When a method is relatively free from chemical interferences,
it can be applied to the determination of analytes in a wide
variety of sample matrices. Such methods are considered
robust.

Measure of the capacity to remain unaffected by small (deliberate) variations in

method parameters.
Indication of reliability during normal use.

Signal to noise ratio

The term noise is used to describe signal fluctuations, and each uncontrolled variable is a
noise source.
The average value of the output of an electronic device is called the signal, and the
standard deviation of the signal is a measure of the noise.
In most instruments, the average strength of the noise N is
constant and independent on the magnitude of the signal S.
The effect of the noise on the relative error of the instrument
increases as the signal being measured decreases in magnitude.
The signal-to-noise ratio is usually defined as the ratio of the
average value of the output signal to its standard deviation.

It is the reciprocal of the relative standard deviation (RDS) of

the group measurement.

Errors in analysis
Error - the result of a measurement minus a true value of the measured.

Errors in analysis

Instrumental errors: instruments such

as pipettes, burettes, and volumetric
flasks, etc.

Absolute Error: The absolute error of a

measurement is the difference between
the measured value and the true value.

Method errors: The non-ideal chemical

or physical behavior of the reagents
and reactions

Relative Error: The relative error of a

measurement is the absolute error
divided by the true value.

Personal errors result from the

carelessness, inattention, or personal
limitations

Systematic (or determinate) error causes

the mean of a data set to differ from the
accepted value.

Random or indeterminate errors:

caused by the many uncontrollable
variables that accompany every
measurement

Constant and Proportional error

Minimization of errors

Absolute Error
It is the difference between the measured value and the true value.
The sign of the absolute error tells you whether the value in question is high or low.

If the measurement result is low, the sign is negative;

if the measurement result is high, the sign is positive.

The absolute error E in the measurement of a quantity x is given by the equation.

Where, xt and xi are the true and measured value respectively.

The term absolute has a different meaning in this text than it does in mathematics. An
absolute value in mathematics means the magnitude of a number ignoring its sign.
As used in this text, the absolute error is the difference between an experimental result
and an accepted value including its sign.

Relative Error
The relative error of a measurement is the absolute error divided by the true value.
Relative error may be expressed in percent, parts per thousand, or parts per million,
depending on the magnitude of the result.
The relative error is often a more useful quantity than the absolute error. The percent
relative error is given by the expression

E indicate error of measurement. Chemical analyses are affected by at least two types of
errors - determinate and indeterminate errors.

Systematic (or determinate) error

Systematic (or determinate) error causes the mean of a data set to differ from the
accepted value. It affect the accuracy of results.
In general, a systematic error in a series of replicate measurements causes all the results to
be too high or too low.
Example: The loss of a volatile analyte while heating a sample.

Systematic errors have a definite value, an assignable cause,

and are of the same magnitude for replicate measurements
made in the same way.
They lead to bias in measurement results. Bias measures the
systematic error associated with an analysis. It has a negative
sign if it causes the results to be low and a positive sign
otherwise.

Instrumental errors
Sources of
Systematic
Errors

Method errors
Personal errors

Instrumental errors
Errors occurs due to instruments or apparatus used for the accurate measurement such as
pipettes, burettes, and volumetric flasks may hold or deliver volumes slightly different
from those indicated by their graduations.
These differences arise from using glassware at a temperature that differs significantly
from the calibration temperature,
from distortions in container walls due to heating while drying,
from errors in the original calibration,
from contaminants on the inner surfaces of the containers.
Electronic instruments are also subject to systematic errors.
Errors may emerge as the voltage of a battery-operated power supply decreases with use.
Errors can also occur if instruments are not calibrated frequently or if they are calibrated
incorrectly.

Method errors
The non-ideal chemical or physical behavior of the reagents and reactions on which an
analysis is based often introduce systematic method errors.
Such sources of non-ideality include the slowness of some reactions,
the incompleteness of others,
the instability of some species,
the lack of specificity of most reagents,
the possible occurrence of side reactions that interfere with the measurement process.

Example
The analytical method used involves the decomposition of the organic samples in hot
concentrated sulfuric acid, which converts the nitrogen in the samples to ammonium
sulfate. Often a catalyst, such as mercuric oxide or a selenium or copper salt, is added to
speed the decomposition.

Personal Errors
Many measurements require personal judgments.
estimating the position of a pointer between two scale divisions,
the color of a solution at the end point in a titration,
the level of a liquid with respect to a graduation in a pipette or burette.

Random or indeterminate errors

It can never be totally eliminated and are often the major source of uncertainty in a
determination.
They are caused by the many uncontrollable variables that accompany every measurement.
Usually, most contributors to random error cannot be positively identified. Even if we
can identify random error sources, it is often impossible to measure them because most are
so small that they cannot be detected individually.
The accumulated effect of the individual uncertainties, however, causes replicate results to
fluctuate randomly around the mean of the set

Constant error: a type of Systematic errors

The magnitude of a constant error stays essentially the same as the size of the quantity
measured is varied. In general, Constant errors are independent of the size of the sample
being analyzed.
The effect of a constant error becomes more serious as the size of the quantity measured
decreases.
e.g. The effect of solubility losses on the results of a gravimetric analysis, the excess of
reagent needed to bring about a color change during a titration (this volume, usually small,
remains the same regardless of the total volume of reagent required for the titration).
One way of reducing the effect of constant error is to increase the sample size until the error is
acceptable.

Proportional error: a type of Systematic errors

Proportional errors increase or decrease according to the size of the sample taken for
analysis. A common cause of proportional errors is the presence of interfering
contaminants in the sample.
e.g. Iodometric Estimation of copper is the reaction of copper(II) ion with potassium iodide
to give iodine. The quantity of iodine is then measured and is proportional to the amount of
copper.
Iron(III), if present, also liberates iodine from potassium iodide. The size of this error is
fixed by the fraction of iron contamination, which is independent of the size of sample
taken. If the sample size is doubled, for example, the amount of iodine liberated by both the
copper and the iron contaminant is also doubled. Thus, the magnitude of the reported
percentage of copper is independent of sample size.

Minimization of errors
There are steps that can be taken to ensure accuracy in analytical procedures.
Most of these depend on minimizing or correcting errors that might occur in the
measurement step.
The overall accuracy and precision of an analysis might not be limited by the
measurement step and might instead be limited by factors such as sampling, sample
preparation, and calibration.
Dilution and Matrix
Matching

Internal Standard
Methods

Standard Addition Methods

Minimization
of
errors

Saturation, Matrix
Modification, and Masking

Separations

Separations
Sample cleanup by separation methods is an important way to minimize errors from
possible interferences in the sample matrix.
Techniques such as filtration, precipitation, dialysis, solvent extraction, volatilization, ion
exchange, and chromatography are all very useful in ridding the sample of potential
interfering constituents.

Most separation methods are, however, time consuming and may increase the chances
that some of the analyte will be lost or that the sample can be contaminated.
In many cases, though, separations are the only way to eliminate an interfering species.

Saturation, Matrix Modification, and Masking

The saturation method involves adding the interfering species to all the samples, standards,
and blanks so that the interference effect becomes independent of the original
concentration of the interfering species in the sample.
For example, a buffer might be added to keep the pH within limits regardless of the sample
pH. Sometimes, a masking agent is added that reacts selectively with the interfering species
to form a complex that does not interfere.
In both these methods, care must be taken that the added reagents do not contain significant
quantities of the analyte or other interfering species.

Dilution and Matrix Matching

The dilution method can sometimes be used if the interfering species produces no
significant effect below a certain concentration level. So, the interference effect is
minimized simply by diluting the sample.
The matrix-matching method attempts to duplicate the sample matrix by adding the
major matrix constituents to the standard and blank solutions.
e.g. Analysis of seawater samples for a trace metal, the standards can be prepared in a
synthetic seawater containing Na+, K+, Cl-, Ca2+, Mg2+, and other components. The
concentrations of these species are well known and fairly constant in seawater.
In some cases, the analyte can be removed from the original sample matrix, and the
remaining components used to prepare standards and blanks.

Internal Standard Methods

In the internal standard method, a known amount of a reference species is added to all the
samples, standards, and blanks. The response signal is then not the analyte signal itself but
the ratio of the analyte signal to the reference species signal. A calibration curve is prepared
where the y-axis is the ratio of responses and the x-axis is the analyte concentration in the
standards as usual. The internal standard method can compensate for certain types of errors
if these influence both the analyte and the reference species to the same proportional
extent.
e.g. if temperature influences both the analyte and reference species to the same extent,
taking the ratio can compensate for variations in temperature.

Standard Addition Methods

The standard addition method is used when it is difficult or impossible to duplicate the sample
matrix.
In the single-point standard addition method, two portions of the sample are taken. One
portion is measured as usual, but a known amount of standard analyte solution is added to the
second portion. The responses for the two portions are then used to calculate the unknown
concentration assuming a linear relationship between response and analyte concentration.
In the multiple additions method, additions of known amounts of standard analyte solution
are made to several portions of the sample, and a multiple additions calibration curve is
obtained.
The multiple additions method verifies to some extent that the linear relationship between
response and analyte concentration holds.

Mean
The mean, also called the arithmetic mean or the average, is obtained by dividing the sum
of replicate measurements by the number of measurements in the set:

Where, xi represents the individual values of x of ith component making up the set of N
replicate measurements.

median
The median is the middle result when replicate data are arranged in increasing or
decreasing order.
There are equal numbers of results that are larger and smaller than the median.
For an odd number of results, the median can be found by arranging the results in order
and locating the middle result.
For an even number, the average value of the middle pair is used.
In ideal cases, the mean and median are identical.

Mode
The "Mode" for a data set is the element that occurs the most often.
It is not uncommon for a data set to have more than one mode. This happens when two or
more elements occur with equal frequency in the data set.
A data set with two modes is called bimodal.
A data set with three modes is called trimodal.

Measure of dispersion
Population: The population is the collection of all measurements of interest and must be
carefully defined by the experimenter. In some cases, the population is finite and real, while
in others, the population is hypothetical or conceptual in nature.
Sampling: The population is finite, we usually would not have the time or resources to test
all indiduals. Hence, we select a sample for analysis according to statistical sampling
principles. We then infer the characteristics of the population from those of the sample.
Example: 1. The determination of calcium in a community water supply to determine water
hardness. 2. Determining glucose in the blood of a patient, we could hypothetically make an
extremely large number of measurements if we used the entire blood supply.

Correlation coefficient
Correlation coefficients (denoted r) are statistics that quantify the relation between X and
Y in unit-free terms.
When all points of a scatter plot fall directly
on a line with an upward incline, r = +1;
When all points fall directly on a downward
incline, r = -1.
To calculate a correlation
coefficient, you normally need three different
sums of squares (SS). The sum of squares for
variable X, the sum of square for variable Y,
and the sum of the cross-product of XY

Sample Mean

The sample mean

population of data.

is the arithmetic average of a limited sample drawn from a

Where, N represents the number of measurements in the sample set.

The population mean

The population mean is the true mean for the population.

Where, N represents the total number of measurements in the population.

standard deviation

The difference between sample mean and population goes decreases as sample size goes on
in increasing and it negligible when N is 20 30. In most of cases, we do not know the
population mean which is then decided from sample mean.
The (xi - ) is the deviation (d) of data value xi from the mean of population and (xi
sample mean) is the deviation of data value xi from the sample mean. The average of the
deviation like mean is defined by the following equation -

The population standard deviation , is a measure of the precision of the population, is given
by the equation Where, N is the number of data points
making up the population

The standard deviation for the data set yielding

the broader but lower curve B is twice that for
the measurements yielding curve A. The
breadth of these curves is a measure of the
precision of the two sets of data. Thus, the
precision of the data set leading to curve A is
twice as good as that of the data set
represented by curve B.

Figure-B shows another type of normal error curve in which

the x-axis is now a new variable z, which is defined as -

Where, z is the relative deviation of a data point from

the mean, that is, the deviation relative to the standard
deviation.
(x - ) = Z

The sample standard deviation s is given by the equation

Where, the quantity (xi sample mean) represents the deviation di of value xi from mean, (N - 1) is
called the number of degrees of freedom.

When (N 1) is used instead of N, s is said to be an unbiased estimator of the population

standard deviation . If this substitution is not used, the calculated s will be less on average
than the true standard deviation i.e. that is, s will have a negative bias.

Relative standard deviation

Relative standard deviation by dividing the standard deviation by the mean value of the
data set. The relative standard deviation, RSD, is sometimes given the symbol sr -

The result is often expressed in parts per thousand (ppt) or in percent by multiplying this ratio
by 1000 ppt or by 100%.

The relative standard deviation multiplied by 100% is called the coefficient of variation
(CV).

Relative standard deviations often give a clearer picture of data quality than do absolute
standard deviations.
Example: Suppose that a copper determination has a standard deviation of 2 mg. If the
sample has a mean value of 50 mg of copper, the CV for this sample is 4%. For a sample
containing only 10 mg, the CV is 20%.

Sample variance (s2)

It is an estimate of the population variance 2. It is equal to the square of the standard
deviation.
Note that the standard deviation has the same units as the data, while the variance has the
units of the data squared.

Spread or Range (w)

The spread, or range, w, is another term that is sometimes used to describe the precision of a
set of replicate results. It is the difference between the largest value in the set and the
smallest.