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8/4/2019 Introduction Analysis and Validation
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CHAPTER 4
INTRODUCTION TO METHOD OF ANALYSIS AND METHOD
VALIDATION
Contents
4.1 UV- VIS SPETROPHOTOMETRIC METHOD FOR ANALYSIS OF DRUG
COMPONENTS
4.2 HPTLC METHOD FOR ANALYSIS OF DRUG COMPONENTS
4.2.1 Introduction to HPTLC
4.2.2 Steps involved in HPLC
4.3 RP-HPLC METHOD FOR ANALYSIS OF DRUG COMPONENTS
4.4 VALIDATION OF ANALYTICAL METHODS
4.4.1 Linearity
4.4.2 Precision
4.4.3 Range
4.4.4 Accuracy
4.4.5 Specificity and Selectivity
4.4.6 Limit of detection (LOD) and Limit of Quantification
4.4.7 Ruggedness
4.4.8 Robustness
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Pharmaceutical products formulated with more than one drug, typically referred to as
combination products, are intended to meet previously unmet patients need by
combining the therapeutic effects of two or more drugs in one product. These
combination products can present daunting challenges to the analytical chemist
responsible for the development and validation of analytical methods.
Development and validation of analytical method (Spectrophotometric, High
performance liquid chromatography (HPLC), & High performance thin layer
chromatography (HPTLC)) is carried out for drug products containing more than one
active ingredient
Basic criteria for new method development of drug analysis:
The drug or drug combination may not be official in any pharmacopoeias.
A proper analytical procedure for the drug may not be available in the
literature due to patent regulations,
Analytical methods may not be available for the drug in the form of a
formulation due to the interference caused by the formulation excipients,
Analytical methods for the quantification of the drug in biological fluids may
not be available,
Analytical methods for a drug in combination with other drugs may not be
available,
The existing analytical procedures may require expensive reagents and
solvents. It may also involve cumbersome extraction and separation
procedures and these may not be reliable.
Introduction to UV-VIS Spectrophotometric Methods of
Analysis for Drugs in Combination
Ultraviolet-visible spectroscopy or ultraviolet – visible spectroscopy (UV-Vis)
involves the spectroscopy of photons in the UV-visible region. This means it uses
light in the visible and adjacent (near ultraviolet (UV) and near infrared (NIR)
ranges. The absorption in the visible ranges directly affect the colour of the
chemicals involved .In the region of the electromagnetic spectrum ,molecules
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undergo electronic transitions .this technique is complementary to fluorescence
spectroscopy ,in that fluorescence deals with transitions from the excited state to
the ground state ,while absorption measures transitions from the ground state to
the excited state .
The spectrophotometric assay of drugs rarely involves the measurement of
absorbance of samples containing only one absorbing component. The
pharmaceutical analyst frequently encounters the situation where the
concentration of one or more substances is required in samples known to contain
other absorbing substances, which potentially interfere in the assay. If the formula
of the samples is known, the identity and concentration of the interfering
substance are known and the extent of interference in the assay may be
determined.
A number of modifications to the simple spectrophotometric procedure are
available to the analyst, which may eliminate certain sources of interference and
permit the accurate determination of all of the absorbing components. Each
modification of the basic procedure may be applied if certain criteria are satisfied.
The basis of all the spectrophotometric techniques for multicomponent samples is
the property that at all wavelengths:
the absorbance of a solution is the sum of absorbance of the individual
components or
The measured absorbance is the difference between the total absorbance of the
solution in the sample cell and that of the solution in the reference cell.
There are various spectrophotometric methods are available which can be used
for the analysis of a combination samples. Following methods can be used
Simultaneous equation method
Derivative spectrophotometric method
Absorbance ratio method ( Q-Absorbance method)
Difference Spectrophotometry
Absorbance ratio method
Geometric correction method
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Orthogonal polynomial method
Least square approximation
Dual wavelength spectrometry
Assay as a single – component sample
Assay using absorbance corrected for interference
Simultaneous Equation Method
If a sample contains two absorbing drugs (X and Y) each of which absorbs at the lmax
of the other (as shown in figure 1. λ1 andλ2), it may be possible to determine
both drugs by the technique of simultaneous equation (Vierodt’s method) provided
that certain criteria apply.
The information’s required are:
the absorptivities of X at λ1 and λ2, ax1 and ax2 respectively
the absorptivities of Y at λ1 andλ2, ay1 and ay2 respectively
The absorbance of the diluted sample at λ1 and λ2, A1 and A2 respectively.
Let Cx and Cy be the concentration of X and Y respectively in the diluted
samples.
Two equations are constructed based upon the fact that at λ1 andλ2, the absorbance of
the mixture is the sum of the individual absorbance of X and Y.
At λ1
A1 = ax1bCx + ay1bCy ……………. (1)
At λ2
A2 = ax2bCx + ay2bCy ………. (2)
For measurements in 1 cm cells, b =1.
Rearrange equation (2)
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Cy = (A2 - ax2 Cx) / ay2
Substituting for Cy in eq. (1) and rearranging gives
Cx = (A2 ay1 - A1 ay2) / (ax2 ay1 - ax1 ay2)
Cy = (A1 ax2 - A2 ax1) / (ax2 ay1 - ax1 ay2)
Fig. 1: The overlain spectra of substance X and Y, showing the wavelength for
the assay of X and Y in admixture by the method of simultaneous equation.
Criteria for obtaining maximum precision have been suggested by Glenn3. According
to him absorbance ratio place limits on the relative concentrations of the components
of the mixture.
(A2 /A1) / (ax2 /ax1) and (ay2 /ay1)/ (A2/A1)
The criteria are that the ratios should lie outside the range 0.1- 2.0 for the precise
determination of Y and X respectively. These criteria are satisfied only when the
λmax of the two components are reasonably dissimilar. An additional criterion is that
the two components do not interact chemically, thereby negating the initial
assumption that the total absorbance is the sum of the individual absorbance. The
additive of the absorbance should always be confirmed in the development of a new
application of this technique.
Simultaneous equation method using Matrices and Cramer's Rule can be explained as
follows:
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Consider a binary mixture of component X and Y for which the absorption spectra of
individual components and mixture are shown in figure 1.
-1 is the λmax of component X
-2 is the λmax of component Y
the total absorbance of a solution at a given wavelength is equal to the sum of the
absorbance of the individual components at the wavelength. Thus the absorbance of
mixture at the wavelength 1 and 2 may be expressed as follows:
At λ1
A1 = ax1bCx + ay1bCy ………………… (1)
At λ2
A2 = ax2bCx + ay2bCy ………………. (2)
Such equation can be solved using matrices.
From equation (1) and (2),
A1 = k x1 Cx + k y1Cy …………. (3)
A2 = k x2 Cx + k y2Cy ………………. (4)
Where k = a x b
Let A, be a column matrix with 'i' elements [i, is the number of wavelength at which
measurements are done; here two wavelength 1 and 2 are taken in to consideration, so
i=2]. Let C, be a column matrix with 'j' elements [j, is the number of components, in
this case X and Y are present, so j = 2]. Let k, be a matrix with i x j values so that it
has number of rows equals to number of wavelength and number of columns equal to
number of components ( in this case it has two rows and two columns). Hence we
have
A = k x C …………. (5)
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Since the number of wavelength equal to number of components, the equation (5) has
a unique solution.
C = k -1
x A …………. (6)
However, it will be faster to solve the equation (3) and (4) by means of Cramer’s rule.
And unknown concentration Cj of component j is found by replacing' j column of
matrix A. The determinant of the new matrix is divided by determinant of 'k' matrix.
Cx = (A1 k y2 - A2 k y1) / (k x1 k y2 - k x2 k y1) …………. (7)
Cy = (k x1 A2 - k x2 A1) / ( k x1 k y2 - k x2 k y1) ………….. (8)
Therefore
Cx = (A1 ay2 - A2 ay1) / ( ax1 ay2 - ax2 ay1) ……………(9)
Cy = (ax1 A2 - ax2 A1) / ( ax1 ay2 - ax2 ay1) …………..(10)
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4.2. HPTLC METHOD FOR ANALYSIS OF DRUG
COMPONENTS[15, 16]
4.2.1 Principles of thin-layer chromatography:
Thin-layer chromatography (TLC), also known as planar chromatography (PC), is one
of the oldest methods in analytical chemistry still in use.
In TLC, the different components of the sample are separated by their interaction with
the stationary phase (bonded to the glass, aluminium, or plastic support) and the liquid
mobile phase that moves along the stationary phase.
TLC is a fast, simple, and low-cost method suitable for any laboratory. A particularadvantage is that it allows the analysis of many samples simultaneously. In contrast to
liquid chromatography (LC), TLC offers separation without or at least with minimal sample preparation. Also, the plates are disposable, and there is no memory effect,
such as may occur in LC. TLC is also an off-line method: sample application,
separation, and detection take place in different processes. Because of its off-line
character, TLC allows the use of a number of detection methods and appropriate
derivatization reagents in sequence, which improves the reliability of the detection
Table 18. Difference between HPTLC and TLC:-
Parameters HPTLC TLC
Layer of Sorbent 100µm 250µm
EfficiencyHigh due to smaller particle
size generatedLess
Separations 3 - 5 cm 10 - 15 cm
Analysis Time
Shorter migration distance
and the analysis time is
greatly reduced
Slower
Solid support
Wide choice of stationary
phases like silica gel for
normal phase and C8 , C18
for reversed phase modes
Silica gel ,
Alumina &
Kiesulguhr
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4.2.2. Features of HPTLC:
Simultaneous processing of sample and standard - better analytical precision and
accuracy less need for Internal Standard
Several analysts work simultaneously
Lower analysis time and less cost per analysis
Low maintenance cost
Simple sample preparation - handle samples of divergent nature
No prior treatment for solvents like filtration and degassing
Low mobile phase consumption per sample
No interference from previous analysis - fresh stationary and mobile phases for
each analysis - no contamination
Visual detection possible - open system
Non UV absorbing compounds detected by post-chromatographic derivatization
4.2.3. Steps involved in HPTLC:
1. Selection of chromatographic layer
2. Sample and standard preparation
3. Layer pre-washing
4. Layer pre-conditioning
5. Application of sample and standard
6. Chromatographic development
7. Detection of spots
8. Scanning
Development
chamber
New type that require less
amount of mobile phaseMore amount
Sample spotting Auto sampler Manual spotting
Scanning
Use of UV/ Visible/ Fluorescence scanner scans
the entire chromatogram
qualitatively and
quantitatively densitometer
Not possible
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9. Documentation of chromatic plate
Selection of chromatographic layer
· Precoated plates with different support materials and different Sorbents are available
· 80% of analysis is done on silica gel GF. Activation of pre-coated plates
Plates exposed to high humidity or kept on hand for long time requires activation.
Activation of pre-coated plates is done by placing them in an oven at 110-120ºc for
30’ prior to spotting. Aluminum sheets should be kept in between two glass plates and
placing in oven at 110-120ºc for 15 minutes.
Application of sample and standard
· Usual concentration range is 0.1-1µg / µl
· Above this causes poor separation
· automatic applicators are available wherein nitrogen gas sprays sample and standard
from syringe on TLC plates as bands
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· Band wise application provides better separation and shows high response to
densitometer
Selection of mobile phase
- Trial and error
- One’s own experience and Literature
- When the mobile phase is polar, polar compounds would be eluted first because of
lower affinity with stationary phase
- Non-Polar compounds retained because of higher affinity with the stationary phase
for chromatographic development twin trough chambers are used where only 10 -15ml of mobile phase is required
- Components of mobile phase should be mixed thoroughly and then introduced into
the twin trough chamber
Pre- conditioning (Chamber saturation)
· Unsaturated chamber may lead to high Rf values
· Saturation of chamber is done by lining with filter paper for 30 minutes prior to
development.
. This allows uniform distribution of solvent vapors in the chamber so less solvent is
required for the sample to travel.
Chromatographic development and drying
After development, plate is removed from the chamber and mobile phase is removed
from the plate. Drying can be done either at room temperature or at alleviated
temperatures if solvents like water or acids are used.
Detection(Quantification)
1) Densitometry
Densiometry is a mean of measuring the concentration of the chromatographic zones
on the developed HPTLC layer with damaging the separated substance.
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There are three possible scanning modes ,single beam,single wavelength and double
beam combined in to a single beam .the single beam fortmat is most popular ,as the
beam of electromagnetic radiation hits the electromatographic layer, some passes into
and through the layer whilst the remainder is reflected back from the surface
reflectance occurs due to the opaqueness of the layer .this reflected radiation is
measured by the photomultiplier unit or photoelectric cell in the instrument .
The spectro densiometric scanner scan separate tracks and wavelength produces vast
amount data .these data includes peak heights and areas ,and position of zones (start,
middle and end ) for every resolved component on every chromatographic track on
the HPTLC plate .A baseline adjustment is applied so that all peaks can be accurately
integrated ready for possible quantification .
2) Video imaging and densiometry
The developed chromatogram is illuminated from above with visible ,254 nm (UV) or
366nm (UV) light ,depending on the radiation required to visualize the analytes
.Illumination from below the plate can often improve the brightness of the image with
the plate suitably lit, an image acquisition device,usually CCD (charged coupled
device) camera with zoom attachment is positioned vertically above .the CCD cameratransmits a digital signal to a computer and video printer.
4.3 RP-HPLC METHOD FOR ANALYSIS OF DRUG COMPONENTS
High Performance Liquid Chromatography is unquestionably the most widely used
analytical separation technique. HPLC has been rapidly developed with the
introduction of new pumping methods, more reliable columns and a variety of
detectors has made. HPLC is one of the commonly used analytical techniques.
HPLC can also be automated which involve automated sampling, separation,
detection, recording and calculation and printing of results. Due to its high
selectivity, specificity and sensitivity achieved by HPLC methods are mainly used
for the analysis of most drugs.
HPLC is one of the most versatile instruments used in the field of pharmaceutical
analysis. It provides the following features.
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High resolving power
Speedy separation
Continuous monitoring of the column effluent
Accurate quantitative measurement
Repetitive and reproducible analysis using the same column
Automation of the analytical procedure and data handling
In HPLC, the analyst has a wide choice of Chromatographic separation methodologies
from normal to reverse phase and a whole range of mobile phases using isocratic (or)
gradient elution techniques. Various detectors are also available for HPLC like
electrochemical detectors, refractive index detectors, fluorescence detectors,
radiochemical detectors, mass-sensitive detectors, UV detectors.
4.3.1 Characteristics of HPLC method
Efficient, highly selective, widely applicable.
Only small sample required.
May be non-destructive of sample.
Readily adapted to quantitative analysis.
High resolving power.
Speed of separation.
4.3.2 STRATEGY FOR METHOD DEVELOPMENT IN HPLC:
Everyday many chromatographers face the need to develop a high-performance liquid
chromatography (HPLC) separation.37
Method development and optimization in
liquid chromatography is still an attractive field of research for theoreticians
(researchers) and attracts also a lot of interest from practical analysts. Complex
mixtures or samples required systematic method development involving accurate
modelling of the retention behaviour of the analyte. Among all, the liquid
chromatographic methods, the reversed phase systems based on modified silica offers
the highest probability of successful results. However, a large number of (system)
variables (parameters) affect the selectivity and the resolution.
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HPLC method development follows a series of steps which are summarized as below
Information on a sample, define separation goals
Need for special HPLC procedure, sample pretreatment, etc.?
Choose detector and detector settings
Choose LC method; preliminary run; estimate best separation conditions
Optimize separation conditions
Check for problems or requirement for special procedure
Recover purified material Quantitative calibration Qualitative method
Validate method for release to routine laboratory
Fig.2: Steps in HPLC method development
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Table 19. Preferred Experimental Conditions for the Initial HPLC Separation
Separation Variable Preferred initial choice
Column
Dimensions(Length × ID) 15 × 0.46 cm
Particle Size 5µm
Stationary Phase C8 or C18
Mobile Phase
Solvents A and B Buffer-Acetonitrile
% B 80-100%
Buffer
(compound, pH, concentration)
10 - 25mM Phosphate Buffer
2.0 < pH
< 3.0
Additives(e.g., amine modifiers,
ion-pair reagents)Do not use initially
Flow-rate 1.5-2.0 ml/min
Temperature 35 – 45°C
Sample Size
Volume
< 25 µl
Weight
< 100 µg
4.3.3. Getting Started on Method Development
“Best column, best mobile phase, best detection wavelength, efforts in separation
can make a world of difference while developing HPLC method for routine
analysis. Determining the ideal combination of these factors assures faster
delivery of desired results – a validated method of separation. ”
a) The Best Mobile Phase
In reverse-phase chromatography, the mobile phase is more polar than the stationary
phase. Mobile phase in these systems is usually mixtures of two or more individual
solvents with or without additives or organic solvent modifiers. The usual approach
is to choose what appears to be the most appropriate column, and then to design a
mobile phase that will optimize the retention and selectivity of the system.
Separations in these systems are considered to be due to different degrees of
hydrophobicity of the solutes. The polarity of organic modifier and its proportion
control the rate of elution of the components in the mobile phase. The rate of elution
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is increased by reducing the polarity. The simple alteration of composition of the
mobile phase or of the flow rate allows the rate of the elution of the solutes to be
adjusted to an optimum value and permits the separation of a wide range of the
chemical types. First isocratic run followed by gradient run is preferred.
Since the mobile phase governs solute-stationary phase interaction, its choice is
critical.
Practical considerations dictate that it should not degrade the equipment or the
column packing. For this reason, strong acids, bases and halide solutions should
be avoided.
Chemical purity of solvents is an important factor. Since large volumes of solvent
are pumped through the column, trace impurities can easily concentrate in
column and eventually be detrimental to the results. Spectro or HPLC grade
solvents are recommended.
Volatility should be considered if sample recovery is required.
Viscosity should be less than 0.5 centipoises, otherwise higher pump pressures
are required and mass transfer between solvent and stationary phase will be
reduced.
LC/MS-only volatile buffers.
b) The Best Detector
The next consideration should be the choice of detector. There is little use in
running a separation if detector one uses cannot “see” all the components of
interest, or conversely, if it “sees” too much. UV-visible detectors are the most
popular as they can detect a broad range of compounds and have a fair degree of
selectivity for some analytes. Unfortunately UV-visible detectors are not
universal detectors so it is worthwhile to look at the chemical structure of the
analyte to see if it has suitable chromophores, such as aromatic rings, for UV-
visible detection.
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Table-20. Detector options
Detector AnalytesSolvent
RequirementsComments
UV-visibleAny with
chromophores
UV-grade non-UV absorbing
solvents
Has a degree of
selectivity and is useful
for many HPLC
applications
FluorescenceFluorescent
compounds
UV-grade non-
UV absorbing
solvents
Highly selective and
sensitive. Often used to
analyze derivatized
compounds
Refractive Index
(RI)
Compounds with
a different RI to
the mobile phase
Cannot run
mobile phase
gradients
Virtually a universal
detector but has limited
sensitivity
Electrochemical
Readily oxidized
or reduced
compounds,
especially
biological
samples
Mobile phase
must be
conducting
Very selective and
sensitive
Evaporative Light
Scattering (ELSD)
Virtually all
compounds
Must use volatile
solvents and
volatile buffers
A universal detector
which is highly
sensitive. Not selective
Mass
Spectrometer
(MS)
Broad range of
compounds
Must use volatile
solvents and
volatile buffers
Highly sensitive and is a
powerful 2nd
dimensional analytical
tool. Many modes
available. Needs trained
operators
c) The Best Column LengthMany chromatographers make the mistake of simply using what is available. Often this
is a 250 × 4.6mm C18 column. These columns are able to resolve a wide variety of
compounds (due to their selectivity and high plate counts) and are common to most
laboratories. While many reverse phase separations can be carried out on such column,
its high resolving capabilities are often unnecessary, as illustrated in
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Figure 2. Method development can be streamlined by starting with shorter columns; 150,
100 or even 50mm long. This is simply because they have proportionally shorter run
times.
Fig.3: Effect of Column length
d) The Best Stationary Phase
Selecting an appropriate stationary phase can also help to improve the efficiency
of method development. For example, a C8 phase (reversed phase) can provide a
further time saving over a C18, as it does not retain analytes as strongly as the
C18 phase. For normal phase applications, cyano (nitrile) phases are most
versatile.
e) The Best Internal Diameter
By selecting a shorter column with an appropriate phase, run times can be
minimized so that an elution order and an optimum mobile phase can be
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quickly determined. It can also be advantageous to consider the column
internal diameter. Many laboratories use 4.6mm ID columns as a standard, but
it is worth considering the use 4.6mm ID columns as an alternative. These
require only 75% of the solvent flow that a 4.6mm column uses. This
translates to a 25% solvent saving over the life of the column and can be even
more significant if a routine method is developed for such a column.
f) Gradient Programming
The fastest and easiest way to develop a method is to use a mobile phase
gradient. Always start with a weak solvent strength and move to a higher
solvent strength. To begin, use a very fast gradient (e.g.10 minutes) and then
modify the starting and finishing mobile phases to achieve a suitable separation.
Of course the choice of solvents and buffers may need to be modified during
method development. (Different HPLC instruments will give different results
for the same gradient, so if a method is to be validated for use by several
different laboratories, isocratic methods are recommended). Optimizing the
mobile phase for an analysis will help to improve the separation. A number of
factors depend upon the solvents chosen.
g) Retention
Analytes may be too strongly retained (producing long run times). If this occurs,
the solvent strength should be increased. In reverse phase analysis this means a
higher % of organic solvent in the mobile phase.
h) Poor Separation
Analytes often co-elute with each other or impurities. To overcome this, the
analysis should be run at both higher and lower solvent strengths so the bestseparation conditions may be determined. Varying solvents may help - try
methanol instead of acetonitrile for reversed phase analysis. Using buffers and
modifying the pH (within the column’s recommended pH range) can also assist
the separation. When the optimum conditions have been achieved, improving
the resolution is often just a case of changing to a longer column and/or one
with a smaller particle size to increase the column efficiency. (For reversed
phase analysis, having started with a 100mm C8 column there is also the option
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of trying C18 columns to get better resolution. The important point is having
used a short column for this stage of the development a lot of time was saved).
i) Peak Shape
This is often a problem, especially for basic compounds analyzed by reversed
phase HPLC. To minimize any potential problems always use a high purity
silica phase such as Wakosil II. These modern phases are very highly
deactivated so secondary interactions with the support are minimal. Buffers can
be used effectively to give sharp peaks. If peak shape remains a problem, use an
organic modifier such as triethylamine, although this should not be necessary
with modern phases like Wakosil. One point often forgotten is the effect of
temperature changes on a separation. To maximize the reproducibility of a
method, it is best to use a column heater to control the temperature of the
separation. A temperature of 35 – 40°C is recommended.
j) Buffer selection
In reverse phase HPLC, the retention of analytes is related to their
hydrophobicity. The more hydrophobic the analyte, the longer it is retained.
When an analyte is ionized, it becomes less hydrophobic and, therefore, it
retention decreases. When separating mixtures containing acid and/or bases by
reversed phase HPLC, it is necessary to control the pH of mobile phase using
appropriate buffer in order to achieve reproducible results.
When separating acids and bases a buffered mobile phase is recommended to
maintain consistent retention and selectivity. A buffered mobile phase, by
definition, resists changes in pH so that the analytes and silica will be
consistently ionized, resulting in reproducible chromatography. If the sample isneutral, buffers or additives are generally not required in the mobile phase.
Acids or bases usually require the addition of a buffer to the mobile phase. For
basic or cationic samples, “less acidic” reverse-phase columns are
recommended and amine additives for the mobile phase may be beneficial.
Optimum buffering capacity occurs at a pH equal to the pKa of the buffer.
Beyond that, buffering capacity will be inadequate.
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Buffers play an additional role in the reproducibility of a separation. The buffer
salts reduce peak tailing for basic compounds by effectively masking silanols.
They also reduce potential ion-exchange interactions with unprotonated silanols
(Figure 3). To be most effective, a buffer concentration range of 10 - 50 mM is
recommended for most basic compounds.
Fig.-4: Peak Tailing Interaction
Table21. Commonly used Buffers for reversed phase HPLC
Buffer PKa
(25°C)Maximum
Buffer RangeUV Cut-off (nm)
TFA 0.3 - 210
Phosphate,pK
1 H2PO4 2.1 1.1-3.1 < 200
Phosphate,pK
2 HPO42-
7.2 6.2-8.2 < 200
Phosphate,pK
3 PO43-
12.3 11.3-13.3 < 200
Citrate, pK
1
C3H5O(COOH)2(COO)1-
3.1 2.1-4.1 230
Citrate, pK
2
C3H5O(COOH)1(COO)2-
4.7 3.7-5.7 230
Citrate, pK
3
C3H
5O(COO)
3-
6.4 4.4-6.4 230
Carbonate, pK
1
HCO31-
6.1 5.1-7.1 < 200
Carbonate, pK
2
CO32-
10.3 9.3-11.3 > 200
Formate 3.8 2.8-4.8 210
Acetate 4.8 3.8-5.8 210
Ammonia 9.3 8.3-10.3 200
Borate 9.2 8.2-10.2 N/A
TEA 10.8 9.8-11.8 < 200
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k) Selection of pH
The pH range most often used for reversed-phase HPLC is 1 - 8 and can be
divided into low pH (1 - 4) and intermediate pH (4 - 8) ranges. Each range has a
number of advantages. Low pH has the advantage of creating an environment inwhich peak tailing is minimized and method ruggedness is, maximized. For this
reason, operating at low pH is recommended.
At a mobile phase pH greater than 7, dissolution of silica can severely shorten
the lifetime of columns packed with silica-based stationary phases.
The pKa value (acid dissociation [ionization] constant) for a compound is the
pH at which equal concentrations of the acidic and basic forms of the molecule
are present in aqueous solutions. Analytes may sometimes appear as broad or
tailing peaks when the mobile phase pH is at, or near, their pKa values. A more
rugged mobile phase pH will be at least 1 pH unit different from the analyte
pKa. This shifts the equilibrium so that 99% of the sample will be in one form.
The result is consistent chromatography.
Dramatic changes in the retention and selectivity (peak spacing) of basic and
acidic compounds can occur when the pH of the mobile phase is changed. This
is often a result of different interactions between the column and the analytes
when the ionization of these compounds changes. It is important to evaluate
these changes when a method is developed in order to select the mobile phase
pH that provides the most reproducible results.
4.4. ANALYTICAL MEHOD VALIDATION TERMINOLOGY:
[6-9]
“Doing thorough method validation can be tedious, but the consequences of
not doing it right are wasted time, money, and resources.”
4.4.1 Definition:
Validation is a process of establishing documented evidence, which provides a
high degree of assurance that a specific activity will consistently produce a
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desired result or product meeting its predetermined specifications and quality
characteristics.
Method validation is the process of demonstrating that analytical procedures are
suitable for their intended use and that they support the identity, quality, purity,
and potency of the drug substances and drug products. Simply, method validation
is the process of proving that an analytical method is acceptable for its intended
purpose. A successful Validation guarantees that both the technical and
regulatory objectives of the analytical methods have been fulfilled. The transfer
of a method is best accomplished by a systematic method validation process. The
real goal of validation process is to challenge the method and determine limits of
allowed variability for the conditions needed to run the method.
4.4.2 Objective of validation
The objective of validation of analytical procedure is to demonstrate that it is
suitable for its intended purpose. Validation is documented evidence, which
provide a high degree of assurance for specific method. Any developed method
may be influenced by variables like different elapsed assay times, different days,
reagents lots, instruments, equipments, environmental conditions like
temperature, etc so it is expected that after the method has been developed and
before it is communicated or transferred from one lab to the other, it is properly
validated and the result of validity tests reported.
Two steps are required to evaluate an analytical method.
1) First determine the classification of the method.
2) The second step is to consider the characteristics of the analyticalmethod
For analytical method validation of pharmaceuticals, guidelines from the
International Conference on Harmonization (ICH), United States Food and Drug
Administration (US FDA), American Association of Official Analytical Chemists
(AOAC)United States Pharmacopoeia (USP), and International Union of Pure
and Applied Chemists (IUPAC) provide a framework for performing such
validations in a more efficient and productive manner50.
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The primary objective of validation is to form a basis for written procedure for
production and process control which are designed to assure that the drug
products have the identity, strength, quality and purity they purport or are
represented to possess quality, safety and efficacy must be designed to build into
the product. Each step of the manufacturing process must be controlled to
maximize the probability that the finished products meet all quality and design
specification.
4.4.3 Data Elements Required for Assay Validation
Both the USP and ICH recognize that is it not always necessary to evaluate every
analytical performance parameter. The type of method and its intended use
dictates which parameters needed to be investigated, as illustrated in Table 4
Table-22. ICH Validation Guideline
Type of analytical
procedure
Characteristics
IDENTIFICATION
TESTING
FOR
IMPURITIES
Quantitative
Limit
ASSAY
-dissolution
(measurement only)
-content/potency
Accuracy
Precision
Repeatability
Interm.Precision
Reproducibility
Specificity (3)
Detection Limit
Quantitation Limit
Linearity
Range
-
-
-
-
+
-
-
-
-
+ -
+ -
+(1) -
- (2) -
+ +
- +
+ -
+ -
+ -
+
+
+(1)
- (2)
+(4)
-
-
+
+
- Signifies that this characteristic is not normally evaluated.
+ Signifies that this characteristic is normally evaluated.
1. Intermediate precision is not needed in some case, when
reproducibility is checked.
2. May be needed in some cases.
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3. Lack of specificity of one analytical procedure could be
compensated by other supporting analytical procedure(s).
4. May not be needed in some cases.
The different parameters of analytical method development are discussed below
as per ICH guideline:-
1) Specificity:
Specificity is the ability to assess unequivocally the analyte in the presence of
components which may be expected to be present. Typically these might include
impurities, degradants, matrix, etc.
Method:
When the impurities are available: Spiking of pure substance (drug
substance or drug product) with appropriate levels of impurities/excipients
and demonstrate the result is unaffected.
When the impurities are not available: Comparing the test results of sample
containing impurities or degradation product to second well-characterized
procedure. These comparisons should include sample under relevant stress
condition.
In chromatographic method: Peak purity test to be done by diode array and
mass spectrometry.
Expression/calculation:
Proof of discrimination of analyte in the presence of impurities. e.g. for
chromatography chromatogram should be submitted.
Peak purity test helps in demonstrating that the peak is not attributable to
more than one component.
For assay two results should be compared and for impurity tests two
profiles should be compared.
Acceptance criteria:
a) Interference from blank, placebo and impurities:
There should not be any interference from blank, placebo and impurities
peak with the main peaks.
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Peak purity factor for the main peaks in standard preparation, unspiked
sample preparation and spiked sample preparation with known
impurities should be equal to or more than 995.
Assay difference of spiked and unspiked samples should not be morethan 2.0% absolute.
b) Interference from degradation products by stress study:
Degradation impurities in all degraded API preparations and sample
preparations should be separated from the main peak.
Peak purity factor for the main peaks in all unstressed and degraded API
and sample preparations should be equal to or more than 995.
2) Linearity:
The linearity of an analytical procedure is its ability (within given range) to
obtain test results, which are directly proportional to the concentration
(amount) of analyte in the sample.
Method:
Drug (different dilution) and/or separately weighed synthetic mixture.
Measurement of response and plot response vs. concentration of analyte and
demonstration of linearity by
Visual inspection of plot
Appropriate statistical methods
Recommendation:
Minimum of 5 concentrations are recommended
Expression/calculation:
Correlation coefficient, y-intercept, slope of regression line, residual sum of
squares.
Acceptance criteria:
The correlation co-efficient (r) value should not be less than 0.995 over the
working range.
3) Range:
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The range of analytical procedure is the interval between the upper and
lower concentration (amounts) of analyte in the sample (including these
concentrations) for which it has been demonstrated that the analytical
procedure has a suitable level of precision, accuracy and linearity.
Method:
Drug (different dilution) and/or separately weighed synthetic mixture.
Measurement of response and plot response vs. concentration of analyte and
demonstration of linearity by
Visual inspection of plot
Appropriate statistical methods
Recommendation:
Assay of drug/finished product: 80 – 120% of test concentration.
For content uniformity: 70 – 130% of test concentration.
For dissolution testing: ± 20% over specified range.
For impurity: from reporting level to 120% of specification.
Expression/calculation:
Correlation coefficient, y-intercept, slope of regression line, residual sum of
squares.
Acceptance criteria:
Not specified
4) Accuracy:
The accuracy of analytical procedure 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. This is sometimes termed
trueness.
Method:
Application of procedure to analyze synthetic mixture of known purity.
Comparison of result with already established procedure.
Accuracy may be inferred once precision, linearity and specificity have
been established.
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Recommendation:
Minimum of nine determinations
Low concentration of range × 3 replicates
Medium concentration of range × 3 replicates
High concentration of range × 3 replicates
Expression/calculation:
Percent recovery by the assay of known added amount of analyte
Mean – Accepted true value with confidence interval
The % recovery was calculated using the formula,
100
)(covRe%
b
abaery
Where,
a – Amount of drug present in sample
b – Amount of standard added to the sample
Acceptance criteria:
Individual and mean % recovery at each level should be 98.0% to 102.0%.
5) Precision:
The precision of an analytical procedure expresses the closeness of agreement
(degree of scatter) between the series of measurements obtained from multiple
sampling of the same homogeneous sample under the prescribed conditions.
Method:
Determination of % relative standard deviation (RSD) of response of
multiple aliquots.
Recommendation:
a) Repeatability (Same operating condition over short interval of time):
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Minimum of nine determinations
Low concentration of range × 3 replicates
Medium concentration of range × 3 replicates
High concentration of range × 3 replicates
(Or)
At target concentration × 6 determinations
Acceptance Criteria:
RSD for assay of six determinations should not be more than 2.0%.
b) Intermediate precision (within laboratory variation):
Different Days
Different Analysts
Different Equipment etc.
Expression/calculation:
Standard deviation, % RSD and confidence interval
Acceptance criteria:
RSD for assay of six determinations should not be more than 2.0%.
Difference between the mean assay value obtained in the intermediate
precision study and method precision study should not be more than 2.0%
absolute.
6) Detection Limit:
The detection limit of an individual analytical procedure is the lowest
amount of analyte in a sample, which can be detected but not necessarily
quantitated under stated experimental conditions.
Method:
1. By visual evaluation
2. Based on S/N ratio
Applicable to procedure, which exhibit baseline noise.
Actual lowest concentration of analyte detected in compared with
blank response
3. Based on S.D. of response and slope
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LOD = 3.3 σ/s
s = Slope of calibration curve
σ = S.D. of response; can be obtained by
Standard deviation of blank response
Residual standard deviation of the regression line
Standard deviation of the y-intercept of the regression line
Sy/x i.e. standard error of estimate
Expression/calculation:
If based on visual examination or S/N ratio – relevant chromatogram is to
be presented.
If by calculation/extrapolation – estimate is validated by analysis of suitable
no. of samples known to be near or prepared at detection limit.
Acceptance criteria:
S/N ratio > 3 or 2:1; not specified in other cases.
7) Quantitation Limit:
The quantitation limit of an individual analytical procedure is defined as the
lowest amount of analyte in a sample, which can be quantitatively determined
with suitable precision and accuracy.
Method:
1. By visual evaluation
2. Based on S/N ratio
Applicable to procedure, which exhibit baseline noise.
Actual lowest concentration of analyte detected in compared with
blank response
3. Based on S.D. of response and slope
LOQ = 10 σ/s
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s = Slope of calibration curve
σ = S.D. of response; can be obtained by
Standard deviation of blank response
Residual standard deviation of the regression line
Standard deviation of the y-intercept of the regression line
Sy/x i.e. standard error of estimate
Recommendation:
Limit should be validated by analysis of suitable no. of samples known to
be near or prepared at quantitation limit.Expression/calculation:
Limits of quantitation and method used for determining should be
presented.
Expresses as analyte concentration.
Acceptance criteria:
S/N ratio > 10:1; not specified in other cases.
8) Robustness:
The robustness of an analytical procedure is a measure of its capacity to
remain unaffected by small, but deliberate variations in method parameters
and provides an indication of its reliability during normal usage.
Method:
It should show the reliability of an analysis with respect to deliberate variations inmethod parameters.
In case of liquid chromatography, examples of typical variations are
Influence of variations of pH in a mobile phase,
Influence of variations in mobile phase composition,
Different columns (different lots and/or suppliers),
Temperature,
Flow rate.
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Recommendation:
Robustness should be considered early in the development of a method.
If the results of a method or other measurements are susceptible to
variations in method parameters, these parameters should be adequatelycontrolled and a precautionary statement included in the method
documentation.
Expression/calculation:
Effect of these changed parameters on system suitability parameters.
Acceptance criteria:
System suitability criteria should meet as per test procedure.
The difference between assay value of sample analyzed as per test
procedure and analyzed by applying proposed changes should not be more
than 2.0% absolute.
9) Ruggedness:
The ruggedness of an analytical method is the degree of reproducibility of test
results obtained by analysis of the same samples under a variety of conditions.
Method:
Analysis of aliquots of homogenous lots in different laboratories by
different analysts under different operational and environmental
conditions.
Expression/calculation:
% RSD
Note: In the guideline on definitions and terminology, the ICH did not address
ruggedness specifically. This apparent omission is really a matter of semantics,
however, as ICH chose instead to cover the topic of ruggedness as part of
precision, as discussed previously.
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10) Solution stability:
Prepare standard and sample as per test procedure and determine initial assay
value. Store the standard and sample preparation up to 48 hours at room
temperature. Determine the assay of sample preparation after 24 hours and 48
hours storage against freshly prepared standard and determine % response of
standard preparation after 24 hours and 48 hours storage against initial standard
response. The assay value of sample and % response of standard calculated after
24 hours and 48 hours storage should be compared with the initial value and
recorded.
If the stability of solution fails to the acceptance criteria at 24 hour interval at
room temperature, repeat the experiment and injecting after standing for 2, 4, 8,
12, and 18 hours at room temperature.
If the stability of solution is found to be less than 24 hours at room temperature,
then establish the solution stability at 5°C±3°C as per the above procedure.
Calculation: Calculate results as follows:
Standard preparation stability: Calculate the % response of the Standard
preparation after specified period using the formula;
% Response =TA
SA× 100
Where,
TA = the peak area of standard preparation after standing for specified period,
SA = the initial peak area of standard preparation subjected for solution stability,
Sample preparation stability: Calculate the % Assay of the Sample preparation
after specified period as per the test procedure against freshly prepared standard.
Calculate the difference of the result obtained after each interval against initial
result.
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Acceptance criteria:
The difference in the response of standard preparation should not be more
than ± 2.0% from the initial value at any time interval.
The absolute difference in the assay value of sample should not be more
than ± 2.0% from the initial value at each time point.
11) System Suitability Testing:
The system has to be tested for its suitability for the intended purpose. System
suitability testing is an integral part of many analytical procedures. The tests are
based on the concept that the equipment, electronics, analytical operations and
samples to be analyzed constitute an integral system that can be evaluated as
such.
Numerous approaches may be used to set the limits for system suitability tests.
This depends on experience with the method, material available and personal
preference. Parameters such as plate count, tailing factors, resolution and
reproducibility (% RSD retention time and area for six repetitions) are determinedand compared against the specifications set for the method.
Table- 23. System Suitability Parameters and their recommended limits
Parameter Recommendation
Capacity Factor (K’) The peak should be well-resolved from other peaks and
the void volume, generally K’ > 2
Repeatability RSD ≤ 1% ; N ≥ 5 is desirable
Relative Retention Not essential as the resolution is stated.
Resolution(Rs)
Rs of > 2 between the peak of interest and the closest
eluting potential interferent (impurity, excipients,
degradation product, internal standard, etc.)
Tailing Factor(T) T ≤ 2
Theoretical Plates(N) In general should be > 2000.
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Table -24. Characteristics to be validated in HPLC
Characteristics Acceptance Criteria
Accuracy/trueness Recovery 98-102% (individual)
Precision RSD < 2%
Repeatability RSD < 2%
Intermediate Precision RSD < 2%
Specificity / Selectivity No interference
Detection Limit S/N > 2 or 3
Quantitation Limit S/N > 10
Linearity Correlation coefficient r2
> 0.999
Range 80 – 120 %
Stability > 24 h or >12 h