Method Devlpoment n Validation Final Project
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Transcript of Method Devlpoment n Validation Final Project
Introduction
Development of oral pharmaceutical drug products presents many technical
and regulatory challenges. Speci¢cally, these include proper characterization
of active pharmaceutical ingredient (API), assurance of compatibility
of inactive ingredients with the active components over the shelf life of the
product,processing andmanufacturing and quality controls and compliance
with current federal regulations and draft Federal Regulations under the
CFR provisions for comments and approval process at the Food and Drug
Administration.
Current Federal Regulations mandate that any generic drug product
intended for human usemust be approved by the Agency formarketing a generic
drug product and its multi-strengths in the United States.These current
Federal Regulations provide assurances to the consumer that these generic
drug products are safe, therapeutically equivalent and e¡ective in the same
manner as the innovator or branded drug products approved previously as
New Drug Applications (NDAs) by the Food and Drug Administration.
Additionally, the quality control information presented by a generic product
manufacturer or sponsor in the Abbreviated New Drug Applications
(ANDAs) documents the evidence that the API used in the dosage form_
may it be a parenteral, oral solid dosage, topical, implant, or a specialized
delivery system form is rigorously tested to comply with the regulatory
mandates of acceptable limits of compendial or regulatory speci¢cations
mutually agreed upon by the sponsor and the O⁄ce of Generic Drugs
Division of the Food and Drug Administration. The reader is referred
to numerous Current Federal Regulations and Guidance issues on this.[1,2,3,4]
Method Development and its Importance
Development of a generic drug product begins with full analytical testing
and reproducible characterization of the API for which there is a Drug
Master File (DMF) registered with the Agency. The DMF provided by the
API manufacturer contains details of the synthetic process, assurance of
cGMPcompliance, and information on the drug substance form and purity,
along with identity of impurities listed in theAPI speci¢cations.
Analytical method development and its validation play a very vital role
in this process of API selection for generic dosage formdevelopment.
Typically, the analytical chemist utilizes numerous literature sources
such as Summary of Basis of Approval (SBA) for the innovator drug product
NDAand technical literature in numerousmedicinal chemistry and analytical
chemistry journals, as well as Internet web sites dedicated to publication
of original articles on pharmaceutical entities and pharmaceutical drug product
development. Frequently, the API supplier provides a starting point
for a review of Material Safety Data Sheet (MSDS), a current analytical
method used by theAPImanufacturer, such as an HPLC method to identify
and quantify the active drug and presence of known and unknown impurities.
This helps the method development chemist to get a head start in completion
of preliminary method development work and establish preliminary
API speci¢cations for release of the API and support the formulation
pharmacist in developing the dosage form for an ANDA ¢ling.
Once theAPImethod is developed,the analytical chemist can begin the
Method development for thedosage form.Typically,placebosofdosage forms
such as tablets or capsules are utilized to assure that the inactive ingredients
do not interfere in the process of a speci¢c method in development for the
drug. Establishment of method speci¢city, sensitivity, linearity, reproducibility,
precision, and accuracy for quanti¢cation of the drug in a dosage form
is pursued to assure that theb method can be used for evaluation of dosage form[4-8]
Method Validation and its importance
Method validation is the process of demonstrating that the analytical
method is suitable for its intended use. The validation process establishes
documented evidence that provides a high degree of assurance that the test
method will consistently provide accurate test results that evaluate a product
against its defined specifications and quality attributes.
Validation of analytical methodologies is considered as an important task,
occurring after method development and before method utilization, and is
required in support of product registration applications.[9-11]
Additional method validation and re-validation of the test method may be
needed when there are regulatory changes and when the expectation for the
method performance characteristics is higher. Sometimes, an alternative
raw material supplier is chosen and a different impurity profile is expected
due to a different synthetic manufacturing route for the API. When an old
analysis technique is replaced by new techniques, method validation will be
required again. The last possibility is that the validated procedure requires
modification due to a discovered defect and the modified method must be
re-validated.[12,13]
Method transfer and its importance
After ANDA approval, the test methods will be applied to the validation
batches and routine product testing conducted by quality control laboratories.
Hence, the test methods must be transferred to the quality control
laboratories. There could potentially be a di!erence in the geographic
location of the R&D lab and the QC lab. The experience of the instrument
operator and experience with the application of the test methods could vary
from lab to lab.Therefore, the knowledge and experience must be passed to
the new laboratories.The receiving laboratory must demonstrate its ability
to perform the test method. A method transfer SOP or protocol must
establish the requirements for satisfactory method transfer.[14,15]
The method transfer is part of the technology transfer process.The method
transfer can improve the understanding of the analytical methodology for
both the originating and receiving laboratories. The receiving laboratory
personnel performing the test method should be trained on the test method.
The receiving laboratories must be cGMP compliant.When the receiving
laboratory is a contract lab, appropriate auditing of the lab by quality
assurance personnel is necessary.When a method transfer (crossover) study
is performed, the results from both labs can serve as intermediate precision
data[16,17,18]
Design And Development And Of Separation Method
Methods for analyzing drugs in multicomponent dosage forms can be developed,
provided one has knowledge about the nature of the sample, namely, its molecular
weight, polarity, ionic character and the solubility parameter. An exact recipe for HPLC,
however, cannot be provided because method development involves considerable trial
and error procedures. The most difficult problem usually is where to start, what type of
column is worth trying with what kind of mobile phase. In general one begins with
reversed phase chromatography, when the compounds are hydrophilic in nature with
many polar groups and are water soluble. ref
The organic phase concentration required for the mobile phase can be estimated by
gradient elution method. For aqueous sample mixtures, the best way to start is with
gradient reversed phase chromatography. Gradient can be started with 5-10% organic
phase in the mobile phase and the organic phase concentration (methanol or acetonitrile)
can be increased up to 100% within 30-45min. Separation can then be optimized by
changing the initial mobile phase composition and the slope of the gradient according to
the chromatogram obtained from the preliminary run. The initial mobile phase
composition can be estimated on the basis of where the compounds of interest were
eluted, namely, at what mobile phase composition. ref
Changing the polarity of mobile phase can alter elution of drug molecules. The elution
strength of a mobile phase depends upon its polarity, the stronger the polarity, higher is
the elution. Ionic samples (acidic or basic) can be separated, if they are present in
undissociated form. Dissociation of ionic samples may be suppressed by the proper
selection of pH. ref
The pH of the mobile phase has to be selected in such a way that the compounds are not
ionized. If the retention times are too short, the decrease of the organic phase
concentration in the mobile phase can be in steps of 5%. If the retention times are too
long, an increase of the organic phase concentration is needed.[19,20,21,22,23]
Introduction to HPLC system
A schematic diagram of HPLC equipment is given in Fig.1[24]
Figure 2: block diagram of HPLC.[24]
Various components of HPLC are:
§A solvent delivery system, including pump,
§Sample injection system,
§A chromatographic column,
§A detector,
§A strip chart recorder,
§Data handling device and microprocessor control.
a) Solvent delivery system:
A mobile phase is pumped under pressure from one or several reservoir and flows
through the column at a constant rate. For normal phase separation eluting power
increases with increasing polarity of the solvent but for reversed phase separation, eluting
power decreases with increasing polarity.
A degasser is needed to remove dissolved air and other gases from the solvent. Special
grades of solvents are available for HPLC and these have been purified carefully in order
to remove absorbing impurities and particulate matter to prevent these particles from
damaging the pumping or injection system or clogging the column.
Pumps:
The pump is one of the most important component of HPLC, since its performance
directly affects retention time, reproducibility and detector sensitivity.
Three main types of pumps are used in HPLC to propel the liquid mobile phase through
the system.
1. Displacement pump: It produces a flow that tends to independent of viscosity and back
pressure and also output is pulse free. But it possesses limited capacity (250 ml).
2. Reciprocating pump: It has small internal volume (35 to 400 µl), their high output
pressure (upto 10,000 psi) and their constant flow rates. But it produces a pulsed flow.
3. Pneumatic or constant pressure pump: They are pulse free; suffer from limited capacity
as well as a dependence of flow rate on solvent viscosity and column back pressure. They
are limited to pressure less than 2000 psi.
(b) Sample injection system:
Insertion of the sample onto the pressurized column must be as a narrow plug so that the
peak broadening attributable to this step is negligible. The injection system itself should
have no dead (void) volume.
There are three important ways of introducing the sample into injection port.
· Loop injection: In which, a fixed amount of volume is introduced by making use of
fixed volume loop injector.
· Valve injection: In which, a variable volume is introduced by making use of an injection
valve.
· On column injection: In which, a variable volume is introduced by means of a syringe
through a septum.
(c) Chromatographic column:
The column is usually made up of heavy glass or stainless steel tubing to withstand high
pressure. The columns are usually 10-30 cm long and 4-10 mm inside diameter
containing stationary phase at particle diameter of 25 µm or less.
Columns with an internal diameter of 5 mm give good results because of compromise
between efficiency, sample capacity, and the amount of packing and solvent required.
Column packing:
The packing used in modern HPLC consist of small, rigid particles having a narrow
particle size distribution. There are three main types of column packing in HPLC.
1. Porous, polymeric beds: Porous, polymeric beds based on styrene divinyl
benzene co-polymers used doe ion exchange and size exclusion
chromatography.
2. Porous layer beds: Consisting of a thin shell (1-3 µm) of silica or modified
silica on an spherical inert core (e.g. Glass). After the development of totally porous
micro particulate packings, these have not been used in HPLC.
3. Totally Porous silica particles (dia. <10 µm): These packing have widely been used for
analytical HPLC in recent years. Particles of diameter >20 µm are usually dry packed.
While particles of diameter <20 µm are slurry packed in which particles are suspended on
a suitable solvent and the slurry so obtained is driven into the column under pressure.
(d) Detectors:
The function of the detector in HPLC is to monitor the mobile phase as it merges from
the column. Detectors are usually of two types:
1. Bulk property detectors: It compares overall changes in a physical property of the
mobile phase with and without an eluting solute. e.g. refractive index, dielectric constant
or density.
2. Solute property detectors: It responds to a physical property of the solute which is not
exhibited by the pure mobile phase. e.g. UV absorbance, fluorescence or diffusion
current. Such detectors are about 1000 times more sensitive giving a detectable signal for
a few nanograms of sample.[19,20,21,22,23]
HPLC method development
Early Stage of Method Development
During the early stage of the method development process some of the more
sophisticated system suitability tests may not be practical due to the lack of experience
with the method. In this stage, usually a more "generic" approach is used. For example,
evaluation of the tailing factor to check chromatographic suitability, and replicate
injections of the system suitability solution to check injection precision may be sufficient
for an HPLC impurities assay.
In the early method development, it may be useful to perform some additional system
suitability tests to evaluate the system performances under different method conditions.
This information will help to develop an appropriate system suitability test strategy in the
future.
As The Method Matures
As more experience is acquired for this method, a more sophisticated system suitability
test may be necessary. For HPLC impurities method intended to be stability indicating, a
critical pair for resolution determination should be identified. The critical pair is defined
as the two peaks with the least resolution in the chromatographic separation. Generally, a
minimum resolution limit is defined for the critical pair to ensure that the separations of
all other impurities are acceptable. All critical factors that will significantly impact the
method performance will need to be identified. Therefore, if the resolution test results
exceed the acceptance limit, the critical factors can be adjusted to optimize the system
performance. If % organic in the mobile phase has a significant impact on the resolution
of the critical pair, organic composition in the mobile phase can be adjusted within a
predetermined range to achieve the acceptable resolution. Therefore, system suitability
strategy not only consists of the tests and limits, but also the approach used to optimize
system performance when the original test result exceeds the limit. In addition, if the
method demands high method sensitivity (e.g. to analyze very low impurity levels), a
detector sensitivity solution may be required to demonstrate suitable signal-to-noise from
the HPLC system. These system suitability tests, combined with the typical replicate
injections of the standard solution, may be used to demonstrate the system suitability for
this method.
Methods for analyzing drugs in multicomponent dosage forms can be developed,
provided one has knowledge about the nature of the sample, namely, its molecular
weight, polarity, ionic character and the solubility parameter. An exact recipe for HPLC,
however, cannot be provided because method development involves considerable trial
and error procedures. The most difficult problem usually is where to start, what type of
column is worth trying with what kind of mobile phase. In general one begins with
reversed phase chromatography, when the compounds are hydrophilic in nature with
many polar groups and are water soluble.
The organic phase concentration required for the mobile phase can be estimated by
gradient elution method. For aqueous sample mixtures, the best way to start is with
gradient reversed phase chromatography. Gradient can be started with 5-10% organic
phase in the mobile phase and the organic phase concentration (methanol or acetonitrile)
can be increased up to 100% within 30-45min. Separation can then be optimized by
changing the initial mobile phase composition and the slope of the gradient according to
the chromatogram obtained from the preliminary run. The initial mobile phase
composition can be estimated on the basis of where the compounds of interest were
eluted, namely, at what mobile phase composition.
Changing the polarity of mobile phase can alter elution of drug molecules. The elution
strength of a mobile phase depends upon its polarity, the stronger the polarity, higher is
the elution. Ionic samples (acidic or basic) can be separated, if they are present in
undissociated form. Dissociation of ionic samples may be suppressed by the proper
selection of pH.
The pH of the mobile phase has to be selected in such a way that the compounds are not
ionized. If the retention times are too short, the decrease of the organic phase
concentration in the mobile phase can be in steps of 5%. If the retention times are too
long, an increase of the organic phase concentration is needed.
In UV detection, good analytical results are obtained only when the wavelength is
selected carefully. This requires knowledge of the UV spectra of the individual
components present in the sample. If analyte standards are available, their UV spectra can
be measured prior to HPLC method development.
The molar absorbance at the detection wavelength is also an important parameter. When
peaks are not detected in the chromatograms, it is possible that the sample quantity is not
enough for the detection. An injection of volume of 20 µl from a solution of 1 mg/ml
concentration normally provides good signals for UV active compounds around 220 nm.
Even if the compounds exhibit higher lmax, they absorb strongly at lower wavelength.
It is not always necessary to detect compounds at their maximum absorbance. It is,
however, advantageous to avoid the detection at the sloppy part of the UV spectrum for
precise quantitation. When acceptable peaks are detected on the chromatogram, the
investigation of the peak shapes can help further method development.
The addition of peak modifiers to the mobile phase can affect the separation of ionic
samples. For examples, the retention of the basic compounds can be influenced by the
addition of small amounts of triethylamine (a peak modifier) to the mobile phase.
Similarly for acidic compounds small amounts of acids such as acetic acid can be used.
This can lead to useful changes in selectivity.
When tailing or fronting is observed, it means that the mobile phase is not totally
compatible with the solutes. In most case the pH is not properly selected and hence
partial dissociation or protonation takes place. When the peak shape does not improve by
lower (1-2) or higher (8-9) pH, then ion-pair chromatography can be used. For acidic
compounds, cationic ion pair molecules at higher pH and for basic compounds, anionic
ion-pair molecules at lower pH can be used. For amphoteric solutes or a mixture of acidic
and basic compounds, ion-pair chromatography is the method of choice.
The low solubility of the sample in the mobile phase can also cause bad peak shapes. It is
always advisable to use the same solvents for the preparation of sample solution as the
mobile phase to avoid precipitation of the compounds in the column or injector.
Optimization can be started only after a reasonable chromatogram has been obtained. A
reasonable chromatogram means that more or less symmetrical peaks on the
chromatogram detect all the compounds. By sight change of the mobile phase
composition, the position of the peaks can be predicted within the range of investigated
changes. An optimized chromatogram is the one in which all the peaks are symmetrical
and are well separated in less run time.
The peak resolution can be increased by using a more efficient column (column with
higher theoretical plate number, N) which can be achieved by using a column of smaller
particle size, or a longer column. These factors, however, will increase the analysis time.
Flow rate does not influence resolution, but it has a strong effect on the analysis time.[25]
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 quantitation 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
The wide variety of equipment, columns, eluent and operational parameters involved
makes high performance liquid chromatography (HPLC) method development seem
complex. The process is influenced by the nature of the analytes and generally follows
the following steps:
step 1 - selection of the HPLC method and initial system
step 2 - selection of initial conditions
step 3 - selectivity optimization
step 4 - system optimization
Step 5 - method validation.
Depending on the overall requirements and nature of the sample and analytes, some of
these steps will not be necessary during HPLC analysis. For example, a satisfactory
separation may be found during step 2, thus steps 3 and 4 may not be required. The extent
to which method validation (step 5) is investigated will depend on the use of the end
analysis; for example, a method required for quality control will require more validation
than one developed for a one-off analysis. The following must be considered when
developing an HPLC method:
keep it simple
try the most common columns and stationary phases first
thoroughly investigate binary mobile phases before going on to ternary
think of the factors that are likely to be significant in achieving the desired
resolution.
Mobile phase composition, for example, is the most powerful way of optimizing
selectivity whereas temperature has a minor effect and would only achieve small
selectivity changes. pH will only significantly affect the retention of weak acids and
bases. A flow diagram of an HPLC system is illustrated in Figure 1.
TableI:- HPLC detector comparison
PARAMETERS REFRACTIVE
INDEX
UV/Vis FLUORESCENCE ELECTROCHEMICAL
Detection
sensitivity(g)
10-6 10-9 10-12 10-12
Linear range 104 105 103 108
Flow sensitivity yes no no yes
Temperature
sensitivity
yes no no yes
HPLC method development Step 1 –
selection of the HPLC method and initial system. When developing an HPLC method,
the first step is always to consult the literature to ascertain whether the separation has
been previously performed and if so, under what conditions - this will save time doing
unnecessary experimental work. When selecting an HPLC system, it must have a high
probability of actually being able to analyse the sample; for example, if the sample
includes polar analytes then reverse phase HPLC would offer both adequate retention and
resolution, whereas normal phase HPLC would be much less feasible. Consideration
must be given to the following:
Sample preparation. Does the sample require dissolution, filtration, extraction,
preconcentration or clean up? Is chemical derivatization required to assist detection
sensitivity or selectivity?
Types of chromatography. Reverse phase is the choice for the majority of samples, but
if acidic or basic analytes are present then reverse phase ion suppression (for weak acids
or bases) or reverse phase ion pairing (for strong acids or bases) should be used. The
stationary phase should be C18 bonded. For low/medium polarity analytes, normal phase
HPLC is a potential candidate, particularly if the separation of isomers is required.
Cyano-bonded phases are easier to work with than plain silica for normal phase
separations. For inorganic anion/cation analysis, ion exchange chromatography is best.
Size exclusion chromatography would normally be considered for analysing high
molecular weight compounds (.2000).
Table II The basic types of analytes used in HPLC
ANALYTE
CHARAC
TERISTIC
SNeutral
No significantly acidic or basic functional groups
Weak acid Has carboxylic acid function or phenolic -OH
Weak base Aromatic amine
Strong base Non-aromatic amine
Gradient HPLC. This is only a requirement for complex samples with a large number of
components (.20–30) because the maximum number of peaks that can be resolved with a
given resolution is much higher than in isocratic HPLC. This is a result of the constant
peak width that is observed in gradient HPLC (in isocratic HPLC peak width increases in
proportion to retention time). The method can also be used for samples containing
analytes with a wide range of retentivities that would, under isocratic conditions, provide
chromatograms with capacity factors outside of the normally acceptable range of 0.5–15.
Gradient HPLC will also give greater sensitivity, particularly for analytes with longer
retention times, because of the more constant peak width (for a given peak area, peak
height is inversely proportional to peak width). Reverse phase gradient HPLC is
commonly used in peptide and small protein analysis using an acetonitrile–water mobile
phase containing 1% trifluoroethanoic acid. Gradient HPLC is an excellent method for
initial sample analysis.
Column dimensions. For most samples (unless they are very complex), short columns
(10–15 cm) are recommended to reduce method development time. Such columns afford
shorter retention and equilibration times. A flow rate of 1-1.5 mL/min should be used
initially. Packing particle size should be 3 or 5 μm.
Detectors. Consideration must be given to the following:
Do the analytes have chromophores to enable UV detection?
Is more selective/sensitive detection required (Table I)?
What detection limits are necessary?
Will the sample require chemical derivatization to enhance detectability and/or
improve the chromatography?
Fluorescence or electrochemical detectors should be used for trace analysis. For
preparative HPLC, refractive index is preferred because it can handle high concentrations
without overloading the detector.
UV wavelength. For the greatest sensitivity λmax should be used, which detects all sample
components that contain chromophores. UV wavelengths below 200 nm should be
avoided because detector noise increases in this region. Higher wavelengths give greater
selectivity.
Fluorescence wavelength. The excitation wavelength locates the excitation maximum;
that is, the wavelength that gives the maximum emission intensity. The excitation is set to
the maximum value then the emission is scanned to locate the emission intensity.
Selection of the initial system could, therefore, be based on assessment of the nature of
sample and analytes together with literature data, experience, expert system software and
empirical approaches.
Step 2 - selection of initial conditions.
This step determines the optimum conditions to adequately retain all analytes; that is,
ensures no analyte has a capacity factor of less than 0.5 (poor retention could result in
peak overlapping) and no analyte has a capacity factor greater than 10–15 (excessive
retention leads to long analysis time and broad peaks with poor detectability). Selection
of the following is then required.
Mobile phase solvent strength. The solvent strength is a measure of its ability to pull
analytes from the column. It is generally controlled by the concentration of the solvent
with the highest strength; for example, in reverse phase HPLC with aqueous mobile
phases, the strong solvent would be the organic modifier; in normal phase HPLC, it
would be the most polar one. The aim is to find the correct concentration of the strong
solvent. With many samples, there will be a range of solvent strengths that can be used
within the aforementioned capacity limits. Other factors (such as pH and the presence of
ion pairing reagents) may also affect the overall retention of analytes.
Gradient HPLC. With samples containing a large number of analytes (.20–30) or with a
wide range of analyte retentivities, gradient elution will be necessary to avoid excessive
retention.
Determination of initial conditions. The recommended method involves performing
two gradient runs differing only in the run time. A binary system based on either
acetonitrile/water (or aqueous buffer) or methanol/water (or aqueous buffer) should be
used.
TABIE III:-HPLC optimization parameters
ANALYTES HPLC
METHOD
OPTIMIZE
neutral Reverse
phase
Solvent strength,solvent type
Weak bases
and/or weak
bases
Ion
suppression
ph,solvent strength,solvent type
Strong acid
and/or strong
bases
Ion pairing Ion pairing reagent concentration,ph solvent
strength,solvent type
Inorganic
anions/cations
Ion
exchange
Eluting ion concentration
Step 3 - selectivity optimization.
The aim of this step is to achieve adequate selectivity (peak spacing). The mobile phase
and stationary phase compositions need to be taken into account. To minimize the
number of trial chromatograms involved, only the parameters that are likely to have a
significant effect on selectivity in the optimization must be examined. To select these, the
nature of the analytes must be considered. For this, it is useful to categorize analytes into
a few basic types (Table II).
Once the analyte types are identified, the relevant optimization parameters may be
selected (Table III). Note that the optimization of mobile phase parameters is always
considered first as this is much easier and convenient than stationary phase optimization.
Selectivity optimization in gradient HPLC. Initially, gradient conditions should be
optimized using a binary system based on either acetonitrile/water (or aqueous buffer) or
methanol/water (or aqueous buffer). If there is a serious lack of selectivity, a different
organic modifier should be considered.
Step 4 - system parameter optimization. This is used to find the desired balance
between resolution and analysis time after satisfactory selectivity has been achieved. The
parameters involved include column dimensions, column-packing particle size and flow
rate. These parameters may be changed without affecting capacity factors or selectivity.
Step 5 - method validation.
Proper validation of analytical methods is important for pharmaceutical analysis when
ensurance of the continuing efficacy and safety of each batch manufactured relies solely
on the determination of quality. The ability to control this quality is dependent upon the
ability of the analytical methods, as applied under well-defined conditions and at an
established level of sensitivity, to give a reliable demonstration of all deviation from
target criteria.
Analytical method validation is now required by regulatory authorities for marketing
authorizations and guidelines have been published. It is important to isolate analytical
method validation from the selection and development of the method. Method selection is
the first step in establishing an analytical method and consideration must be given to what
is to be measured, and with what accuracy and precision.
Method development and validation can be simultaneous, but they are two different
processes, both downstream of method selection. Analytical methods used in quality
control should ensure an acceptable degree of confidence that results of the analyses of
raw materials, excipients, intermediates, bulk products or finished products are viable.
Before a test procedure is validated, the criteria to be used must be determined.
Analytical methods should be used within good manufacturing practice (GMP) and good
laboratory practice (GLP) environments, and must be developed using the protocols set
out in the International Conference on Harmonization (ICH) guidelines (Q2A and
Q2B).1,2 The US Food and Drug Administration (FDA)3,4 and US Pharmacopoeia (USP)5
both refer to ICH guidelines. The most widely applied validation characteristics are
accuracy, precision (repeatability and intermediate precision), specificity, detection limit,
quantitation limit, linearity, range, robustness and stability of analytical solutions.
Method validation must have a written and approved protocol prior to use.6
HPLC instrumentation The HPLC systems used for the validation studies consisted of
Series 200 UV/Visible Detector, Series 200 LC Pump, Series 200 Autosampler and Series
200 Peltier LC Column Oven (all Perkin Elmer, Boston, Massachusetts, USA). The data
were acquired via Total Chrom Workstation (Version 6.2.0) data acquisition software
(Perkin Elmer), using Nelson Series 600 LINK interfaces (Perkin Elmer).
All chromatographic experiments were performed in the isocratic mode. The mobile
phase was a methanol/water solution (75:25 v/v). The flow rate was 1.5 mL/min and the
oven temperature was 40 ºC. The injection volume was 20 μL and the detection
wavelength was set at 254 nm. The chromatographic separation was on a 25034.6 mm
ID, 10 μm C18 μ-Bondapak column (Waters, Milford, Massachusetts, USA). [26-35]
System Suitability Tests For Chromatographic Methods
System suitability is the checking of a system to ensure system performance before or
during the analysis of unknowns. Parameters such as plate count, tailing factors,
resolution and reproducibility (%RSD retention time and area for six repetitions) are
determined and compared against the specifications set for the method. These parameters
are measured during the analysis of a system suitability "sample" that is a mixture of
main components and expected by-products. Lists of the terms to be measured and their
recommended limits obtained from the analysis of the system suitability sample are given
below.
Definition
The purpose of the system suitability test is to ensure that the complete testing system
(including instrument, reagents, columns, analysts) is suitable for the intended
application. The USP Chromatography General Chapter states:
"System suitability tests are an integral part of gas and liquid chromatographic methods.
They are used to verify that the resolution and reproducibility of the chromatographic
system are adequate for the analysis to be done. 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."
Evolution of System Suitability
Similar to the analytical method development, the system suitability test strategy should
be revised as the analysts develop more experience with the assay. In general,
consistency of system performance (e.g., replicate injections of the standard) and
chromatographic suitability (e.g. tailing factor, column efficiency and resolution of the
critical pair) are the main components of system suitability.
Long Term System Suitability Strategy
During the final stage of method development, there is a need to define the long-term
strategy for system suitability requirements, and the practicalities for all laboratories
using this method. If the system suitability test involves the use of any reference sample
(i.e. isolated and characterized impurity), the laboratory needs to have enough supply of
this reference sample to complete the system suitability test. However, maintaining the
supply of this reference sample in the long term is usually not an easy task. If the
reference sample is a degradation product of the drug substance, it is desirable to generate
the reference sample in-situ by artificially degrading the drug substance in order to
streamline the method. Therefore, extensive investigations must be done to evaluate the
best approach to generate the reference sample, and to identify the critical factors needed
to ensure that the degradation process is reproducible.
How to Set Limits
Numerous approaches can be used to set limits for system suitability tests. This depends
on the experience with the method, material available and personal preference. During
method development, it may be useful to perform some system suitability tests with no
acceptance limit. Firstly, it is premature to set any limit during the very early stage of
method development. Secondly, since experimental conditions will be varied
intentionally during method development, collecting system suitability data in these
experiments will help the analyst to evaluate the impact of results generated under
different method conditions. This information will be used to set appropriate system
suitability limits in the future.
Default Values from Regulatory Guidelines
There are numerous guidelines which detail the expected limits for typical
chromatographic methods. In the current FDA guidelines on "Validation of
Chromatographic Methods" , the following acceptance limits are proposed as initial
criteria:
These suggested limits may be used as a reference to set up the initial system suitability
criteria in the early method development process.
Simulated Conditions
Ideally the analyst should observe the results from a "deteriorating" system and determine
the situations under which the results are no longer acceptable. One way to simulate the
deterioration of the system is to use an old or artificially degraded column in the analysis.
Typically, a column can be degraded artificially by numerous injections or heating at
extreme pH conditions. These old columns will provide the information about the
changes
System Suitability Parameters and Recommendations
Parameter Recommendation
Capacity Factor (k’) The peak should be well-resolved from other peaks and
the void volume, generally k’>2.0
Repeatability RSD </= 1% for N >/= 5 is desirable.
Relative retention Not essential as long as the resolution is stated.
Resolution (Rs) Rs of > 2 between the peak of interest and the closest
eluting potential interferent (impurity, excipient, degradation product, internal standard,
etc.
Tailing Factor (T) T of </= 2
Theoretical Plates (N) In general should be > 2000
If the results are adversely affected by the changes in column performance (e.g.
unacceptable precision of results due to overlapping peaks), the system suitability results
from these experiments will help to determine the limits for system suitability criteria.
This approach facilitates the investigation of the worst case scenario, which reflects
minimum performance standard used to ensure that the chromatography is not adversely
affected.
The parameters that are affected by the changes in chromatographic conditions are:
§ Resolution (Rs),
§ Capacity factor (k’),
§ Selectivity (a),
§ Column efficiency (N) and
§ Peak asymmetry factor (As).
1. Resolution (Rs): Resolution is the parameter describing the separation power of the
complete chromatographic system relative to the particular components of the mixture.
The resolution, Rs, of two neighboring peaks is defined as the ratio of the distance
between two peak maxima. It is the difference between the retention times of two solutes
divided by their average peak width. For baseline separation, the ideal value of Rs is 1.5.
It is calculated by using the formula,
Fig. 5: Resolution between two peaks.
where, Rt1 and Rt2 are the retention times of components 1 and 2 and
W1 and W2 are peak width of components 1 and 2.
There are three fundamental parameters that influence the resolution of a
chromatographic separation:
·capacity factor (k')
·selectivity (α)
·column efficiency (N)
These parameters provide you with different means to achieve better resolution, as well
as defining different problem sources
2. Capacity Factor (k’): Capacity factor is the ratio of the reduced retention volume to
the dead volume. Capacity factor, k’, is defined as the ratio of the number of molecules of
solute in the stationary phase to the number of molecules of the same in the mobile phase.
Capacity factor is a measure of how well the sample molecule is retained by a column
during an isocratic separation. The ideal value of k’ ranges from 2-10. Capacity factor
can be determined by using the formula,
Fig. 6: Retention Factor
Where, tR = retention volume at the apex of the peak (solute) and
t0 = void volume of the system.
Capacity Factor (k') changes are typically due to:
· Variations in mobile phase composition
· Changes in column surface chemistry (due to aging)
· Changes in operating temperature.
In most chromatography modes, capacity factor (k') changes by 10 percent for a
temperature change of 5 C.
Adjusting Capacity Factor (k')
Good isocratic methods usually have a capacity factor (k') in the range of 2 to 10
(typically between 2 and 5). Lower values may give inadequate resolution. Higher
values are usually associated with excessively brood peaks and unacceptably long run
times.
If the analytes fall outside their specified windows run the initial column test protocol to
compare the results obtained with a new column.
Capacity Factor (k') values are sensitive to:
· solvent strength
· composition
· purity
· temperature
· column chemistry
· sample
If the shift in Capacity Factor (k') value is observed with both analytes and the column
test solution, the problem is most likely due to change in the column, temperature or
mobile phase composition. This is particularly true if the shift occurred gradually over a
series of runs. If, however the test mixture runs as expected, the problem is most likely
sample-related.
3. Selectivity(a): The selectivity (or separation factor), a, is a measure of relative
retention of two components in a mixture. Selectivity is the ratio of the capacity factors of
both peaks, and the ratio of its adjusted retention times. Selectivity represents the
separation power of particular adsorbent to the mixture of these particular components.
This parameter is independent of the column efficiency; it only depends on the nature of
the components, eluent type, and eluent composition, and adsorbent surface chemistry. In
general, if the selectivity of two components is equal to 1, then there is no way to
separate them by improving the column efficiency.
The ideal value of a is 2. It can be calculated by using formula,
a= V2 – V1 / V1 – V0 = k1’/ k2’
Where, V0 = the void volume of the column,
V1 and V2 =the retention volumes of the second and the first peak respectively.
Fig. 7: Selectivity
Adjusting selectivity (α)
When troubleshooting changes in Selectivity (α), the approach is similar to the approach
used to troubleshoot changes in Capacity Factor (k').
When Selectivity (α) is affected, the corrective action depends on whether the problem is
mobile phase or column related.
Be sure to compare results obtained with the test solution to those observed when the
column was new. Use these results to distinguish column changes from problems with
mobile phase or other operating parameters.
Selectivity (α) values are sensitive to:
·changes in mobile phase composition (pH ionic strength)
·purity
·temperature
4. Column Efficiency/ Band broadening: Efficiency, N, of a column is measured by the
number of theoretical plates per meter. It is a measure of band spreading of a peak.
Similar the band spread, higher is the number of theoretical plates, indicating good
column and system performance. Columns with N ranging from 5,000 to 100,000
plates/meter are ideal for a good system. Efficiency is calculated by using the formula,
Fig. 8: Number of Theoretical Plates
Where, tR is the retention time and
W is the peak width.
A decline in measured efficiency may be due to:
·age and history of the column
· extra column band broadening (such as due to malfunctioning injector or improper
tubing ID)
· inappropriate detector settings (for example, time constant)
· change in flow rate and solvent viscosity.
You can recognize problems in your separation due to a loss of column efficiency when
the width and/or shape of all peaks are affected.
If the measured efficiency has degraded, either the column has degraded, or system
bandbroadening has increased. At this point, check system bandspreading against
established benchmarks.
Methods of measuring column efficiency (N)
Methods used for the measurement and calculation of column include (in order to
sensitivity to abnormal peak shape):
·
Asymmetry-based (Most sensitive to tailing or fronting)
· 5 sigma
· 4 sigma
· Tangent
· 3 sigma
· ½ height
· 2 sigma (infection) (Least sensitive to tailing or fronting)
Choose the method that best suits your operating requirements. It is critical that the same
method always be used and executed reproducibly.
Figure above illustrates the use of the different peak widths of a Gaussian peak for the
calculation of column efficiency (N).
When measuring Column Efficiency, use test conditions identical to those used in the
established benchmark performance (such as test sample, flow rate, mobile phase
composition and so on). Measure the column efficiency against the established
performance.
5. Peak asymmetry factor (Tf): Peak asymmetry factor, Tf, can be used as a criterion of
column performance. The peak half width, b, of a peak at 10% of the peak height, divided
by the corresponding front half width, a, gives the asymmetry factor.
Fig. 9A: Asymmetric Factor
Fig. 9B: Asymmetric Factor.
For a well packed column, an asymmetry factor of 0.9 to 1.1 should be achievable.[25]
Validation of Analytical Method
Method validation is the process to confirm that the analytical procedure employed for a
specific test is suitable for its intended use.
Methods need to be
Validated or revalidated as follows:
Before their introduction into routine use
Whenever the conditions change for which the method has been validated
(e.g., instrument with different characteristics)
Whenever the method is changed, and the change is outside the original
Scope of the method
When quality control indicates an established method is changing with
Time
In order to demonstrate the equivalence between two methods (e.g., a new
method and a standard)
Method validation has received considerable attention in the literature and from
Industrial committees and regulatory agencies. The international standard ISO/
IEC [1] requires validation of nonstandard methods, laboratory designed/developed
methods, standard methods used outside their intended scope, and amplifications
and modifications of standard methods to confirm that the methods are
suitable for their intended use. The Guidance on the Interpretation of the EN
45000 Series of Standards and ISO/IEC Guide 25 includes a chapter on the
validation of methods [2] with a list of nine validation parameters. The International
Conference on Harmonization (ICH) of Technical Requirements for the
Registration of Pharmaceuticals for Human Use [3] has developed a consensus
text on the validation of analytical procedures. The document includes definitions
for eight validation characteristics. An extension with more detailed methodology
is in preparation and nearly completed . The U.S. Environmental
Protection Agency (U.S. EPA) prepared a guidance for methods development
and validation for the Resource Conservation and Recovery Act (RCRA).
The American Association of Official Analytical Chemists (AOAC), the U.S.
Environmental Protection Agency (EPA), and other scientific organizations provide
methods that are validated through multilaboratory studies.
Validation is an act of proving that any procedure, process, equipment, material, activity
or system performs as expected under given set of conditions and also give the required
accuracy, precision, sensitivity, ruggedness, etc.
When extended to an analytical procedure, depending upon the application, it means that
a method works reproducibly, when carried out by same or different persons, in same or
different laboratories, using different reagents, different equipments, etc.
The various validation parameters are:
§accuracy,
§precision(repeatability and reproducibility),
§linearity and range,
§limit of detection(LOD)/ limit of quantitation(LOQ),
§selectivity/ specificity,
§robustness/ ruggedness and
§Stability and system suitability studies.
Steps in Method Validation
1. Develop a validation protocol or operating procedure for the validation.
2. Define the application, purpose, and scope of the method.
3. Define the performance parameters and acceptance criteria.
4. Define validation experiments.
5. Verify relevant peformance characteristics of equipment.
6. Qualify materials (e.g., standards and reagents).
7. Perform prevalidation experiments.
8. Adjust method parameters or/and acceptance criteria if necessary.
9. Perform full internal (and external) validation experiments.
10. Develop SOPs for executing the method in the routine.
11. Define criteria for revalidation.
12. Define type and frequency of system suitability tests and/or analytical quality control
(AQC) checks for the routine.
13. Document validation experiments and results in the validation report.
Copyright © 2003 Marcel Dekker, Inc.
Advantages of Analytical method Validation:-
§The biggest advantage of method validation is that it builds a degree of confidence, not
only for the developer but also to the user.
§Although the validation exercise may appear costly and time consuming, it results
inexpensive, eliminates frustrating repetitions and leads to better time management in the
end.
§Minor changes in the conditions such as reagent supplier or grade, analytical setup are
unavoidable due to obvious reasons but the method validation absorbs the shock of such
conditions and pays for more than invested on the process.
Analytical method validation: The Regulatory Perspective
In the US, there was no mention of the word validation in the cGMP’s of 1971, and
precision and accuracy were stated as laboratory controls. It was only in the cGMP
guideline of March 1979 that the need for validation was implied. It was done in two
sections: (1) Section 211.165, where the word ‘validation’ was used and (2) section
211.194, in which the proof of suitability, accuracy and reliability was made compulsory
for regulatory submissions. Another guidance on validation of chromatographic methods
was released by CDRE on 1st Nov. 1994.
The WHO published a guidelines under the title, ‘Validation of analytical procedures
used in the examination of pharmaceutical materials’. It appeared in the 32nd report of the
WHO expert committee on ‘specifications for pharmaceutical preparations’ which was
published in 1992.
The international Conference on Harmonization (ICH), which has been on the forefront
of developing the harmonized tripartite guidelines for adoption in the US, Japan and EC ,
has issued two guidelines under the titles-‘ Text on validation of Analytical
procedures(Q2A) and validation of Analytical procedure Methodology (Q2B)’.
Among the pharmacopoeias, USP XXII 1225 (1995) carries a section which describes
requirements of validation of compendial methods. The British Pharmacopoeia includes
the definition of method validation in 15 latest editions, but the term is completely
missing form the Indian Pharmacopoeia. (1996).
Key parameters of the Analytical method validation:-
It is important for one to understand the parameters or characteristics involved in the
validation process. The various Performance parameters, which are addressed in a
validation exercise, are grouped as follows.
(1) Accuracy: -
The accuracy of an analytical method may be defined as the closeness of the test results
obtained by the method to the true value. It is the measure of the exactness of the
analytical method developed. Accuracy may often express as percent recovery by the
assay of a known amount of analyte added.
Accuracy may be determined by applying the method to samples or mixtures of
excipients to which known amount of analyte have been added both above and below the
normal levels expected in the samples. Accuracy is then calculated from the test results as
the percentage of the analyte recovered by the assay. Dosage form assays commonly
provide accuracy within 3-5% of the true value.
The ICH documents recommend that accuracy should be assessed using a minimum of
nine determinations over a minimum of three concentration levels, covering the specified
range (i.e. three concentrations and three replicated of each concentration).
(2) Precision: -
The precision of an analytical method is the degree of agreement among individual test
results when the method is applied repeatedly to multiple samplings of homogenous
samples. This is usually expressed as the standard deviation or the relative standard
deviation (coefficient of variation). Precision is a measure of the degree of reproducibility
or of the repeatability of the analytical method under normal operating circumstances.
Repeatability involves analysis of replicates by the analyst using the same equipment and
method and conducting the precision study over short period of time while
reproducibility involves precision study at
§ Different Occasions,
§ Different Laboratories,
§ Different Batch of Reagent,
§ Different Analysts,
§ Different Equipments.
Determination of Repeatability:- Repeatability can be defined as the precision of the
procedure when repeated by same analyst under the same operating conditions (same
reagents, equipments, settings and laboratory) over a short interval of time.
It is normally expected that at least six replicates be carried out and a table showing each
individual result provided from which the mean, standard deviation and co-efficient of
variation should be calculated for set of n value. The RSD values are important for
showing degree of variation expected when the analytical procedure is repeated several
time in a standard situation. (RSD below 1% for built drugs, RSD below 2% for assays in
finished product).
The ICH documents recommend that repeatability should be assessed using a minimum
of nine determinations covering the specified range for the procedure (i.e. three
concentrations and three replicates of each concentration or using a minimum of six
determinations at 100% of the test concentration).
Determination of reproducibility:- Reproducibility means the precision of the procedure
when it is carried out under different conditions-usually in different laboratories-on
separate, putatively identical samples taken from the same homogenous batch of material.
Comparisions of results obtained by different analysts, by the use of different
equipments, or by carrying out the analysis at different times can also provide valuable
information.
(3) Linearity and range:-
The linearity of an analytical method is its ability to elicit test results that are directly (or
by a well defined mathematical transformation) proportional to the analyte concentration
in samples within a given range. Linearity usually expressed in terms of the variance
around the slope of regression line calculated according to an established mathematical
relationship from test results obtained by the analysis of samples with varying
concentrations of analyte.
The linear range of detectability that obeys Beer’s law is dependent on the compound
analyzed and the detector used. The working sample concentration and samples tested for
accuracy should be in the linear range. The claim that the method is linear is to be
justified with additional mention of zero intercept by processing data by linear least
square regression. Data is processed by linear least square regression declaring the
regression co-efficient and b of the linear equation y= ax + b together with the correlation
coefficient of determination r. For the method to be linear the r value should be close to1.
The range of an analytical method is the interval between the upper and lower levels of
the analyte (including these levels) that have been demonstrated to be determined with
precision, accuracy and linearity using the method as written.
(4) Limit of Detection and limit of Quantitation:-
Limit of detection:- The limit of detection is the parameter of limit tests. It is the lowest
level of analyte that can be detected, but not necessarily determined in a quantitative
fashion, using a specific method under the required experimental conditions. The limit
test thus merely substantiates that the analyte concentration is above or below a certain
level.
The determination of the limit of detection of instrumental procedures is carried out by
determining the signal-to-noise ratio by comparing test results from the samples with
known concentration of analyte with those of blank samples and establishing the
minimum level at which the analyte can be reliably detected. A signal-to-noise ratio of
2:1 or 3:1 is generally accepted.
The signal-to-noise ratio is determined by dividing the base peak by the standard
deviation of all data points below a set threshold. Limit of detection is calculated by
taking the concentration of the peak of interest divided by three times the signal-to-noise
ratio.
For spectroscopic techniques or other methods that rely upon a calibration curve for
quantitative measurements, the IUPAC approach employs the standard deviation of the
intercept (Sa) which may be related to LOD and the slope of the calibration curve, b, by
LOD = 3 Sa / b
Limit of quantitation:- Limit of quantitation is a parameter of quantitative assays for low
levels of compounds in sample matrices such as impurities in bulk drugs and degradation
products in finished pharmaceuticals. The limit of quantitation is the lowest concentration
of analyte in a sample that may be determined with acceptable accuracy and precision
when the required procedure is applied.
It is measured by analyzing samples containing known quantities of the analyte and
determining the lowest level at which acceptable degrees of accuracy and precision are
attainable. Where the final assessment is based on an instrumental reading, the magnitude
of background response by analyzing a number of blank samples and calculating the
standard deviation of this response. The standard deviation multiplied by a factor (usually
10) provides an estimate of the limit of quantitation. In many cases, the limit of
quantitation is approximately twice the limit of detection.
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(5) Selectivity and Specificity:-
The selectivity of an analytical method is its ability to measure accurately and
specifically the analyte of interest in the presence of components that may be expected to
be present in the sample matrix.
If an analytical procedure is able to separate and resolve the various components of a
mixture and detect the analyte qualitatively the method is called selective. On the other
hand, if the method determines or measures quantitatively the component of interest in
the sample matrix without separation, it is said to be specific.
Hence one basic difference in the selectivity and specificity is that, while the former is
restricted to qualitative detection of the components of a sample, the latter means
quantitative measurement of one or more analyte.
Selectivity may be expressed in terms of the bias of the assay results obtained when the
procedure is applied to the analyte in the presence of expected levels of other
components, compared the results obtained when the procedure is applied to the analyte
in the presence of expected levels of other components, compared to the results obtained
on the same analyte without added substances. When the other components are all known
and available, selectivity may be determined by comparing the test results obtained on the
analyte with and without the addition of the potentially interfering materials. When such
components are either unidentified or unavailable, a measure of selectivity can often be
obtained by determining the recovery of a standard addition of pure analyte to a material
containing a constant level of the other components.
(6) Robustness and Ruggedness:-
Robustness:- The robustness of an analytical method is a measure of its capacity to
remain unaffected by small but deliberate variation in method parameters and provides an
indication of its reliability during normal usage. The determination of robustness requires
that methods characteristic are assessed when one or more operating parameter varied.
Ruggedness:- The ruggedness of an analytical method is the degree of reproducibility of
test results obtained by the analysis of the same samples under a variety of normal test
conditions such as different laboratories, different analysts, using operational and
environmental conditions that may differ but are still within the specified parameters of
the assay. The testing of ruggedness is normally suggested when the method is to be used
in more than one laboratory. Ruggedness is normally expressed as the lack of the
influence on the test results of operational and environmental variables of the analytical
method.
For the determination of ruggedness, the degree of reproducibility of test result is
determined as function of the assay variable. This reproducibility may be compared to the
precision of the assay under normal condition to obtain a measure of the ruggedness of
the analytical method.
(7) Stability and System suitability tests:-
Stability of the sample, standard and reagents is required for a reasonable time to
generate reproducible and reliable results. For example, 24 hour stability is desired for
solutions and reagents that need to be prepared for each analysis.
System suitability test provide the added assurance that on a specific occasion the method
is giving, accurate and precise results. System suitability test are run every time a method
is used either before or during analysis. The results of each system suitability test are
compared with defined acceptance criteria and if they pass, the method is deemed
satisfactory on that occasion. The nature of the test and the acceptance criteria will be
based upon data generated during method development optimization and validation
experiments.[36,37,38]
Data Elements Required For Assay Validation
There are various analytical methods used for the examination of pharmaceutical
materials. Not all the characteristics referred above will need to be considered in all
cases. Analytical methods may be broadly classified as Per WHO as follows:
Class A: Tests designed to establish identity, whether of bulk drug substances or of a
particular ingredient in a finished dosage form.
Class B: Methods designed to detect and quantitative impurities in a bulk drug substance
or finished dosage form.
Class C: Methods used to determine quantitatively the concentration of a bulk drug
substance or of a major ingredient in a finished dosage form.
Class D: Methods used to assess the characteristic of finished dosage forms, such
as dissolution profiles and content uniformity.
TableIV: Characteristic that should be considered for different types of analytical
procedure
Class A Class B Class C Class D
Quantitative
tests
Limit tests
Accuracy X X X
Precision X X X
Robustness X X X X X
Linearity and
range
X X X
Selectivity X X X X X
Limit of
Detection
X X
Limit of
Quantitation
X
Where, X indicates the tests to be performed.
As per USP:
Category I: Analytical methods for quantitation of major components of bulk drug
substances or active ingredients including preservatives in finished pharmaceutical
products.
Category II: Analytical methods for determination of impurities in bulk drugs or for
determination of degradation compounds in finished pharmaceutical products.
Category III: Analytical methods for determination of performance characteristics (e.g.
dissolution, drug release).
Category IV: Identification tests.
Table V: Data Elements Required for Assay Validation:
Analytical
Performance
Characteristics
Assay
Category I
Assay Category II Assay
Category III
Assay
Category IVQuantitative
tests
Limit
tests
Accuracy X X May be May be
Precision X X X
Specificity X X X May be X
Limit of Detection X May be
Limit of Quantitation X May be
Linearity X X May be
Range X X May be May be
Where, X indicates the tests to be performed.
Method Validation Results
Making use of the method validation results is yet another approach. During the
robustness testing of method validation, critical method parameters such as mobile phase
composition, column temperature are varied to mimic the day-to-day variability.
Therefore, the system suitability results from these robustness experiments should reflect
the expected range for the system suitability results. As a result, the limits for system
suitability tests can be determined from these experiments. This is a very effective
approach since the required system suitability results can be generated during method
validation, and no special study is required. However, these results only reflect the
expected performance of the system, but not necessarily the minimum "performance
standard" for acceptable results. For example, the minimum resolution of the critical pair
from method validation may be 3.5; however, a resolution of 2.0 may still be acceptable
as long as they are baseline resolved, and all other chromatographic parameters remain
acceptable.[39,40]
Conclusion:
The efficient development and validation of analytical methods are critical elements in
the development of pharmaceuticals. Success in these areas can be attributed to several
important factors, which in turn will contribute to regulatory compliance. Experience is
one of these factors both the experience level of the individual scientists and the
collective experience level of the development and validation department.
Development of accurate and reliable analytical methods is an important
element of pharmaceutical development. Good analytical methods support
correct decisions being made from data for formulation development and
stability studies. All analytical methods must be validated before they are
used to generate data which will support a regulatory decision.
Analytical development can proceed efficiently if a thorough literature
search is made of the available information on the API and drug product,
including related compounds. A good source of information is the portion
of the DMF that the API manufacturer is willing to share with its customers.
It is a good idea to work closely with the lab personnel from the API manufacturer
in developing methods for the API and in identifying unknown
impurities in the API.
Analytical development and validation must follow a timeline keyed to
the other activities in developing a drug product. Analytical methods
will usually be needed to support other plant activities such as cleaning
validation or packaging development. The analytical method should be
evaluated for robustness and reliability prior to committing the time and
effort to validate a method.
Avalidated method can still be updated for special situations encountered
during the method application. Such update may or may not involve
an addendum or supplement to the method validation. This is usually part
of the life cycle of the test method application.
The validation report is necessary for documenting the capability
of the test method. All data that support the validation must be clearly
identi¢ed and audited. These data will be scrutinized by the regulatory
agency granting a drug product approval in a pre-approval inspection.[41,42,43]
Reference:
1. US Food and Drug Administration, Title 21 CFR 314.94, O⁄ce of Federal
Register,National Archives and Records Administration, 2003.
2. US Food and Drug Administration,Center for Drug Evaluation and Research.
Guidance for Industry: Dissolution testing of Immediate Release Solid Oral
Dosage Forms,O⁄ce of Training andCommunications,Division of Communications
and Management, Drug Information Branch, HFD 210, 5600 Fishers
Lane, Rockville, Maryland 20857, August, 1997.
3. US Food and Drug Administration, Center for Drug Evaluation and Research.
Guidance for Industry: Extended Release Oral Dosage Forms: Development,
Evaluation and Application of InVitro^InVivo Correlations,O⁄ce ofTraining
and Communications, Division of Communications and Management, Drug
Information Branch, HFD 210, 5600 Fishers Lane, Rockville, Maryland
20857, September, 1997.
4. US Food and Drug Administration,Center for Drug Evaluation and Research.
Guidance for Industry: SUPAC-MR: Modi¢ed Release Solid Oral Dosage
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and Management, Drug Information Branch, HFD 210, 5600 Fishers Lane,
Rockville, Maryland 20857, September, 1997.
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Guidance for Industry: Analytical Procedures and Methods Validation,
Chemistry, Manufacturing and Controls Documentation, O⁄ce of Training
and Communications, Division of Communications and Management, Drug
Information Branch, HFD 210, 5600 Fishers Lane, Rockville, Maryland
20857, August, 2000.
50 Gao et al.
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Communications and Management, Drug Information Branch, HFD 210,
5600 Fishers Lane, Rockville, Maryland 20857,November, 1994.
7. US Food and Drug Administration,Center for Drug Evaluation and Research.
Draft Guidance: Stability Testing of Drug Substances and Drug Products,
Division of Communications and Management, Drug Information Branch,
HFD 210, 5600 Fishers Lane, Rockville, Maryland 20857, June, 1998.
8. The International Conference on Harmonization. The Common Technical
Document (M4). (Internet).
9. The International Conference on Harmonization of Technical Requirements
for Registration of Pharmaceuticals for Human Use. Q3B (R), Impurities in
New Drug Products,October 1999.
10. The International Conference on Harmonization of Technical Requirements
for Registration of Pharmaceuticals for Human Use. Q3C, Impurities in New
Drug Products, July 1997.
11. The International Conference on Harmonization of Technical Requirements
for Registration of Pharmaceuticals for Human Use. Q3A (R) Impurities in
NewDrug Substances,October1999.
12. Miller JM, Crowther JB, eds. Analytical Chemistry in a GMP Environment.
NewYork: Wiley, 2000.
13. USP 26=NF 21, h724iDrug Release and h711iDissolution.
14. Snyder LR, Kirkland JJ, Glajch JL. Practical HPLC Method Development.
2nd ed. NewYork:Wiley, 1997.
15. Jenke DR. J Liq Chromatogr RelatTech 1996; 19(5):719^736.
16. Jenke DR. J Liq Chromatogr RelatTech Mar 1996; 19(5):737^757.
17. Jenke DR. Instrum Sci Technol 1998-01; 26(1):19^35.
18. Krull I, Swartz M.LC-GC North America 2000-06; 18(6):620, 622^625.
19. http://hplc.chem.shu.edu/NEW/HPLC_Book
20. Szepei Gabor., HPLC in pharmaceutical Analysis, Volume I, (1990), 101-173.
21. Jeffery G.H., Bassett J., Vogels textbook of Quantitative Chemical Analysis, 5th
edition, (1991), 217-235.
22. Willard Hobart. H., Merritt L.L., Dean John.A., Instrumental Methods of Analysis, 7 th
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