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

Forms, O⁄ce of Training and Communications, Division of Communications

and Management, Drug Information Branch, HFD 210, 5600 Fishers Lane,

Rockville, Maryland 20857, September, 1997.

5. US Food and Drug Administration,Center for Drug Evaluation and Research.

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.

5460-3 Shargel Ch03 R1 040304

6. US Food and Drug Administration,Center for Drug Evaluation and Research.

Reviewer Guidance: Validation of Chromatographic Methods, Division of

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

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