Journal of Advanced Pharmacy Education & Research

2 (3) 146-176 (2012) ISSN 2249-3379

Method Development and Validation- A Review

Sudha T.*, Krishana Kanth V.1, Nukala Poorana Chandra Sainath1, Mishal1, Saloman
Raja T2, Ganesan V2

1. Department of Pharmaceutical Analysis, The Erode College of Pharmacy & Research,

Erode, India
2. Department of Pharmaceutics, The Erode College of Pharmacy & Research, Erode, India

*Corresponding author: [email protected]

ABSTRACT
Analytical method development followed by method validation is an important process in
the drug discovery. Although the drug shows good potency, lack of validated analytical
method will not allow the drug to enter into the market. This is to ensure the quality and
safety of the drug. The main objective of this review is to give an idea about the old and
novel techniques available for the analysis of drugs in their raw material and formulated
forms, check the stability of the drugs in the presence of the excipients and other stress
conditions experienced during their shelf life period. The review work puts a light on the
hyphenated techniques for the analysis and impurity profiling of drugs like LC-MS-MS, LC-
NMR-MS, GC-MS and LC –MS. This review also deals with the bioanalytical method
development for the quantitative determination of the drugs in the various biological
matrices. It also provides a means to determine the biological safety of the drugs by
dealing with the SIAMs (stability indicating assay methods).

Keyword: Validation, Stability indicating, Impurity, bioanalytical, HPLC, HPTLC.

INTRODUCTION
Quality control and quality assurance are the major areas in the pharmaceutical industry
dealing with the analysis of materials starting from the raw material, intermediate products,
APIS and finished products. Now and then new techniques are being developed all over the
world. As a result, classical methods have changed to instrumental methods and finally to
hyphenated technique. Each technique is found to be superior to the pervious technique.

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Enormous effort have been put the scientists to reach this stage of the analysis. Many
methods are developed for a product or a raw material to finally reach their quantity and
quality. Now it becomes necessary to find which method is the most suitable and is giving
the exact result. Here comes the role of validation. Method validation is the repeated
analysis of the materials by the developed method to confirm the accuracy and precision of
the results. Once validated the method could be used in routine analysis of the drugs. In
brief the objective of this review is to put a light on the various hyphenated techniques that
could be adopted for the estimation of pharmaceuticals and their validation along with the
degradation studies.

USING DIFFERENT METHODS

A. STABILITY – INDICATING METHOD

Critical issues related to development of SIAMs, such as separation of all degradation
products, establishment of mass balance, stress testing of formulations, development of
SIAMs for combination products, etc are also addressed. The stability – indicating assay is a
method that is employed for the analysis of stability samples in pharmaceutical industry.
The guidelines explicitly require conduct of forced decomposition studies under a variety of
conditions, like pH, light, oxidation, dry heat, etc and separation of drug from degradation
products.
Unfortunately, none of the ICH guideline provides an exact definition of a stability-
indicating methodology. However guidelines are provided in the United States-Food and
drug Administration (US-FDA) stability guideline of 1987 (ICH) and the draft guideline of
1998 (FDA).
The major changes brought in the new guidelines are with respect to (i) introduction of the
requirements of validation, and (ii) the requirements of analysis of degradation products
and other components, apart from the active ingredients(s). The literature was found to be
replete with publications on developments of stability-indicating assays of specific drugs.
While the current requirement is of subjecting the drug substance to variety of stress
condition and then separation of drug from all degradation products, many studies have
just shown the separation of drug from known synthetic impurities and /or potential
degradation products without subjecting it to any type of stress (Table-1). There are also
reports in which drug has been decomposed by exposing it to one (Table-2), two (Table-3)

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conditions among acidic, neutral or alkaline hydrolysis, photolysis, oxidation and thermal
stress. They are some reports where directly the formulation, instead of the drug substance,
has been subjected to stress studies for establishment, of the stability-indicating behaviour
(Table-4).

Table 1: Selected reports of stability indicating methods where no stress testing has
been done
Separations Drugs Methodology
Separations from process
Benazepril HCL HPLC
impurities
Canrenone
HPLC
Phenylbutazone
Separations from known / HPLC
Homatropine
potential degradation products UV- spectrophotometry
methylbromide
SFC
Felodipine
Benzodiazepine HPLC
Separations from known /
Piroxicam HPTLC
potential degradation products and
Fenclorac GLC
process impurities
Azathioprine CE

Table 2: Selected reports of ‘Stability-Indicating’ methods where only one stress

condition have been employed
Stress conditions Drugs Methodology
Dyclonine HLC HPLC
Acid
Lisinopril UV spectrophotometry
Allantoin HPLC
Alkali Benzapril UV
Carbachol IR
Neutral Physostigmine salicylate HPLC
Oxidation Nortriptyline HCL UV
Atenolol HPLC
Light Nifedipine HPTLC
Ranitidine HCL Spectrodensitometric TLC

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Table 3: Selected reports of ‘stability indicating’ methods where five (and additional)
stress conditions have been employed
Stress conditions Drugs Methodology
Acid, alkali, oxidation, dry heat,
Elanapril maleate HPLC
light
Acid, alkali, oxidation, dry heat,
light (separation from synthetic Sildenafil citrate HPLC
impurities also seen)

Acid, neutral, alkali, oxidation, light Nicardipine HCL HPLC

Acid, alkali, oxidation, dry heat,

Paroxetine HPLC
wet heat, light dry, light wet
Acid, alkali, oxidation, dry heat,
Cyproterone acetate HPLC
light, reduction

Acid, alkali, oxidation, dry heat,

Buspirone HCL HPLC
light, moisture, sonication

On critical evaluation the literature reports that titrimetric, spectrophotometric and

chromatographic techniques have been commonly employed in the analysis of stability
samples. In these methods, usually the objective is the analysis of the drug of interest along
in the matrix of excipients, additives, degradation products and impurities etc., and also
other drugs in case of the combination products. Their advantage is the low cost and
simplicity, though sometimes they are not sensitive.
Chromatographic methods have taken precedence over the conventional methods of
analysis. Other than separation of multiple components, the advantage of chromatographic
methods is that these possess greater accuracy and sensitivity for even small quantities of
degradation products produced.
HPLC has been very widely employed. It has gained popularity in stability studies due to
high-resolution capacity, sensitivity and specificity. Various chromatographic methods that
have been used are thin-layer chromatography (TLC), high-performance thin-layer
chromatography (HPTLC) and gas chromatography (GC), HPLC and newer technique like
capillary electrophoresis [1-2]. TLC is a simple technique that has been used in the past for
developing SIAMs.

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Table 4: Reports of ‘stability indicating’ methods on drug formulation

Stress conditions Drugs Dosage form Methodology

Admixtures
Fluconazole GC
Acid Extemporaneous
Flucytosine HPLC
solutions

Acid, alkali Gancyclovir Capsules HPLC

Sodium
Light, thermal Tablets HPLC
levothyroxine

Acid, alkali,
Pentoxifylline Suspension HPLC
oxidation

Acid, alkali,
Chlorbutanol Ointment HPLC
oxidation, thermal

Acid, alkali, Fotemustine 5% dextrose HPLC

oxidation, light Efavirenz Capsules HPLC

Acid, light,
Fentanyl Injection HPLC
oxidation, thermal

Acid, alkali, light,

Cyclosporine Oral solution HPLC
oxidation, thermal

Acid, alkali, light,

Aspirin and
thermal, 450c /75% Tablets HPLC
warfarin sodium
RH for two weeks

Aged samples (3
years at 400c and Losartan Tablets HPLC and LC-MS
75% RH

This technique overcomes the shortcomings of TLC and is reliable. Moreover many samples
can be run simultaneously using a small quantity of mobile phase thus minimising analysis
time and cost per analysis.

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Table 5: Validation criteria to be studied in pre-validation and validation steps

Criteria Pre-Validation Step Validation Step
Absolute recovery + +
Selectivity (+) (+)
Response function (+) +
Linearity + +
Accuracy (+) +
Precision (+) +
Limit of detection + +
Limit of quantification range

(+): estimation of the criteria; +: validation of the criteria

GC is stability-indicating but it is not versatile as the drug substance may be non-volatile or

thermally unstable. Further any attempt to increase the volatility of the drug and
components by increasing the temperature may lead to degradation or racemization.
Therefore, there are very few reports on GC for purpose of establishment of SIAMs. In
comparison, HPLC has been very widely employed. It has gained popularity in stability
studies due to high-resolution capacity, sensitivity and specificity. Non-volatile, thermally
unstable or polar/ionic compounds can also be analysed by this technique. Therefore, most
of the SIAMs have been established using HPLC.

A few studies have also reported the use of proton nuclear magnetic resonance (NMR)
spectroscopy for the development of SIAMs. CE is the latest entry of the techniques for the
development of SIAMs [3-6]. It has the advantage of high sensitivity, resolution and high
efficiencies with high peak dispersion.

But the information on the basic steps to be followed for the development and validation of
stability indicating methods is neither provided in the regulatory guidelines nor in
pharmacopoeias. Therefore, the practical steps involved in the development of SIAMs are
done by HPLC as it is found that 85-90% of the methods reported in literature.

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I. Critical Study of the Drug Structure to Assess the Likely Decomposition Route (S)
Much information can simply be gained from the structure by study of functional groups
and other key components. There are definite functional group categories like amides,
esters, lactams, lactones that undergo hydrolysis [7], others like thiols, thio-ethers, etc.
undergo oxidation [8] and compounds like olefins, aryl halo derivatives, aryl acetic acids
and N-oxides undergo photodecomposition.

II. Collection of information on physiochemical properties

Before method development is taken up it is generally important to know various
physiochemical parameters like pKa, log P, solubility, absorptivity and wavelength
maximum of the drug in question. Knowledge of pKa is important as most of the pH- related
changes in retention occur at pH values within ± 1.5 units of the PKa value. The ionisation
value also helps in selecting the pH of the buffer to be used in the mobile phase.

III. Stress (forced degradation) studies

The next step in the development of SIAM is the conduct of forced degradation studies to
generate degradation products of the drug. The ICH guideline Q1A suggests the following
conditions to be employed: (i) 10 0C increments above the accelerated temperatures (e.g. 50
0C, 60 0C, etc.), (ii) humidity where appropriate (e.g. 75% or greater), (iii) hydrolysis across
a wide range of pH values, (iv) oxidation and (v) photolysis. The hydrolytic degradation of a
new drug in acidic and alkaline conditions can be studied by refluxing the drug in 0.1 N
HCL/NaOH for 8h. In a similar manner, degradation under neutral conditions can be started
by refluxing the drug in water for 12 hrs. Reflux time should be increased if no degradation
is seen. If the drug is found to degrade completely, both time and temperature of study can
be decreased. To test for oxidation, it is suggested to use hydrogen peroxide in the
concentration range of 3-30%. The photolytic studies can be carried out by exposure to
light, using either a combination of cool white and fluorescent lamps, or one among the
xenon and metal halide lamps. Exposure energy should be minimum of 1.2 million lux h
fluorescent light and 200W h/m2 UV and if decomposition is not seen, the intensity should
be increased by five times. In case no decomposition still takes place the drug can be
declared as photo stable.

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The blank subjected to stress in the same manner as the drug solution, zero time sample
containing the drug which under normal conditions and drug solution subjected to stress
treatment. The comparison of results of these provides real assessment of changes.

IV. Preliminary separation studies on stressed samples

The samples so obtained are subjected to preliminary analyses to study the number and
types of degradation products formed. For doing so, the simplest way is to start with a
reversed-phase octadecyl column. It should be preferred to use water-methanol or water-
acetonitrile as the mobile in initial stage. The solvent can be changed if the peak shape or
separation problems are seen. Initially water : organic modifier ratio can be fixed at 50 : 50
ratio or can be suitably modified so as to obtain the capacity factor of around 5-10 for the
drug. As degradation products from the drugs are generally polar in nature (of course with
exceptions) pushing the drug peak to say ~ 15 min or somewhat more in a 25 cm column,
can result in separation of even several degradation products, when formed. Normally, the
total run time should be 2.5 times more than the drug peak. The detection wavelength can
be set based on the study of spectral behaviour of degraded samples.

V. Final method development and optimization

Subsequent to preliminary chromatographic studies, the retention time and relative
retention times (RRT) of all products formed should be tabulated for each retention
condition. Special attention is then paid to those components whose RT or RRT is very close.
It was later established that the drug was almost instantly converted when brought into
contact with alkali and the product was formed quantitatively. Therefore, if PDA or LC-MS
results suggest that any of the products are different but are co-eluting then suitable
identification should be done in the chromatographic method to achieve a satisfactory
resolution. To separate close or co-eluted peaks the method is optimised by changing the
mobile phase ratio, pH, gradient, flow rate, temperature, solvent type and the column and
its type.

VI. Identification and characterization of degradation products and preparation of

standards
Before moving to the validation of a SIAM it is necessary to identify the drug degradation
products and arrange for their standards. These are required to establish

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specificity/selectivity of the method. To identify the resolved products a conventional way

is to isolate them and determine the structure through spectral (MS, NMR, IR, etc.) and
elemental analysis. The modern approach is to use hyphenated LC techniques coupled with
spectrometry. This strategy integrates in a single instrument approach, analytical HPLC, UC
detection, full scan mass spectrometry (LC-MS) and provides a fair idea on identity of
resolving components. To isolate a product the best way is to identify a reaction condition
where it is formed selectively. If the product precipitates or crystallizes on its own on
completion of the reaction it can be recovered simply. Otherwise the reaction mixture can
be lyophilized directly. If freeze drying is done directly after neutralization of the reaction
mixture the product can be recovered by extraction with dry methanol or any other dry
solvent. The recovery can also be made by selective extraction with an organic solvent after
acidification, neutralization or basification of the solution, depending upon the initial pH.
Subsequently the extraction can be evaporated to recover the product.

VII. Validation of SIAMS

There are two stages in the validation of a SIAM. First stage is early in the development
cycle when drug substance is subjected to forced degradation studies and the SIAM is
established based on the knowledge of drug degradation behaviour. The main focus of
validation at this stage is on establishment of specificity / selectivity, followed by other
parameters like accuracy, precision, linearity, range and robustness etc, the limits of
detection and quantitation are also determined for degradation products to help in
establishment of the mass balance. In the second stage, when the SIAM so developed is
extended to formulations or other matrices, the emphasis gets limited to just prove the
pertinence of the established validation parameters in the presence of excipients or other
formulation constituents. Here only selectivity, accuracy and precision are revalidated. If
the SIAM is being developed directly for a formulation, without involving the bulk drug
route, then all validation parameters are necessary to be established. However, a better
method of determining accuracy of SIAM is by spiking the drug in the mixture of degraded
solutions. The linearity for SIAMs should be established initially in the range of 0-100%, as
the drug may fall to very low concentrations during forced decomposition studies. The
range may be 50-120% in case of injections or other formulations where the drug is prone
to degradation. Validation range for degradation products during stability studies usually
should vary from 0-20%.

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B. Impurity studies – method [9-10]

Drugs play a vital role in the progress of human civilization by curing diseases. Today a
majority of the drugs used are of synthetic origin. These are produced in bulk and used for
their therapeutic effects in pharmaceutical formulations. These are biologically active
chemical substances generally formulated into convenient dosage forms such as tablets,
capsules, suspensions, ointments and injectable. These formulations deliver the drug
substance in a stable, non-toxic and acceptable form, ensuring its bioavailability and
therapeutic activity. Safety and efficacy of pharmaceutical are two fundamental issues of
importance in drug therapy. The safety of a drug is determined by its pharmacological –
toxicological profile as well as the adverse effects caused by the impurities in bulk and
dosage forms. The impurities in drugs often possess unwanted pharmacological or
toxicological effects by which any benefits from their administration may be outweighed.
The quality and safety of a drug is generally assured by monitoring and controlling the
impurities effectively. Impurity control is more important. In the beginning of 20th century,
drug products were produced and sold having no imposed control. The impurities to be
considered for new drugs are listed in regulatory documents of the food and drug
administration, International Conference on the Harmonization of the technical
requirements for registration of pharmaceuticals for human uses and United States of
Pharmacopoeia. The USP and National Formulary (NF) are the recognised standards for
potency and purity of new drugs. The ICH which took place in Yokohama, Japan in 1995 has
released new guidelines on impurities in new drug products. These guidelines have a
number of advantages, both for the industry and the regulators. The most critical aspect of
the elaboration of the guidelines was the identification and qualification. Analytical
procedures should be able to separate all the impurities from each other and the method
should be optimized to separate and quantify them in the dosage forms. Such methods are
to be validated demonstrating the accuracy, precision, specificity, limit of detection,
quantification, linearity, ranges and interferences. The ICH has harmonized the
requirements in two guidelines. The first one summarizes and defines the validation
characteristics needed for various types of test procedures, the second one extends the
previous text to include the experimental data required and some statistical interpretation.

Qualification of the impurities is the process of acquiring and evaluating data that
establishes biological safety of an individual impurity. Identification of impurities is done by

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variety of Chromatographic and Spectroscopic techniques, either alone or in combination

with other techniques. There are different methods for detecting and characterizing
impurities like TLC, HPLC, HPTLC and AAS etc. Conventional liquid chromatography,
particularly HPLC, has been exploited widely in the field of impurity profiling; the wide
range of detectors and stationary phases along with its sensitivity and cost-effective
separation have attributed to its varied applications. TLC is the most commonly used
separation technique for isolation of impurities due to its ease of operation and low cost
compared to HPLC. An advancement of thin layer chromatography-HPTLC is a well-known
technique for the impurity isolation. Number of articles have stated guidelines and designed
approaches for isolation and identification of process-related impurities and degradation
products, using Mass spectrometry (MS), Nuclear Magnetic Resonance (NMR), High
Performance Liquid Chromatography (HPLC), Fourier Transform Ion Cyclotron Resonance
Mass Spectrometry [11-13].Impurity profile is the description of identified and unidentified
impurities present in new drug substances.

Origin of impurities
Impurities in drugs are originated from various sources and phases of the synthetic process
and preparation of pharmaceutical dosage forms. A sharp difference between the process
related impurities and degradation products is always not possible. The majority of the
impurities are characteristic of the synthetic route of the manufacturing process. Since
there are several possibilities of synthesizing a drug, it is possible that the same product of
different sources may give rise to different impurities.

Types of impurities
The United States Pharmacopoeia (USP) classifies impurities in various sections;
1).Organic impurities (process and drug related) 2).Inorganic impurities 3).Residual
solvents.

Organic Impurity
Organic impurities may arise during the manufacturing process or storage of the drug
substance may be identified or unidentified, volatile or non-volatile, and may include; 1.
Starting materials or intermediates 2. By-products 3. Degradation products.

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100% yield is very rare; there is always a chance of having by-products. Impurities can also
be formed by degradation of the end product during manufacturing of the bulk drugs.

Inorganic Impurity [14]

Inorganic impurities may also be delivered from the manufacturing processes used for bulk
drugs. They are normally known and identified and include the following

Reagents, Ligands and Catalysts

The chances of having these impurities are rare; however in some processes, these could
create a problem, unless the manufactures take proper care during production.

Heavy metals
The main sources of heavy metals are the water used in the processes and the reactors
where acidification or acid hydrolysis takes place. These impurities of heavy metals can
easily be avoided using demineralised water and glass-lined reaction.

In process production Impurities

Impurities can originate from several sources, such as, a) Crystallization-related impurities
b)Stereochemistry-related impurities c) Synthetic intermediates and by-products d)
Formulation-related impurities e) Impurities arising during storage f) Method related
impurity g) Mutual interaction amongst ingredients h) Functional group-related typical
degradation.

(a) Crystallization-related impurities

Based on the realization that the nature of structure adopted by a given compound upon
crystallization could exert a profound effect on the solid-state properties of that system, the
pharmaceutical industry is required to take a strong interest in polymorphism and solvato-
morphism. Polymorphism is the term used to indicate crystal system where substances can
exist in different crystal packing arrangements, all of which have the same elemental
composition [15].

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(b) Stereochemistry-related impurities

It is of paramount importance to look for stereochemistry related compounds; that is, those
compounds that have similar chemical structure but different spatial orientation, these
compounds can be considered as impurities in the API’s. Chiral molecules are frequently
called enantiomers. The single enantiomeric form of chiral drug is now considered as an
improved chemical entity that may offer a better pharmacological profile and an increased
therapeutic index with a more favorable adverse reaction profile.

(c) Synthetic intermediates and by-products

Impurities in pharmaceutical compounds or a new chemical entity (NCE) can originate
during the synthetic process from raw materials, intermediates and /or by-products.
Impurity profiling of tablets by GC-MS and MDMA (3,4-Methylene dioxy methamphetamine)
samples produced impurities in the intermediates via the reductive amination route [16].

(d) Formulation-related impurities

Many impurities in a drug product can originate from excipients used to formulate a drug
marginal product, unacceptable for reliability. Solutions and suspensions are inherently
prone substance. If the source is from an excipient, variability from lot-lot may make a to
degradation due to hydrolysis or solvolysis [17]. In general, liquid dosage forms are
susceptible to both degradation and microbiological contamination. Microbiological
growth resulting from the growth of bacteria, fungi, and yeast in a humid and warm
environment may result in unsuitability of an oral liquid product for safe human
consumption.

(e) Impurities arising during storage A number of impurities can originate during
storage or shipment of drug products. It is essential to carry out stability studies to predict,
evaluate, and ensure drug product safety [18].

(f) Method related impurity

A known impurity, 1-(2, 6-dichlorophenyl) indolin-2-one is formed in the production of a
parenteral dosage form of diclofenac sodium if it is terminally sterilized by autoclave [19].
The conditions of the autoclave method (i.e., 123 + 2oC) enforce the intramolecular cyclic

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reaction of diclofenac sodium forming an indolinone derivative and sodium hydroxide. The
formation of this impurity has been found to depend on initial pH of the formulation.

(g) Mutual interaction amongst ingredients

Most vitamins are very labile and on aging they create a problem of instability in different
dosage forms especially in liquid dosage forms. Degradation of vitamins does not give toxic
impurities; however potency of active ingredients drops below Pharmacopoeial
specifications. Because of mutual interaction, the presence of nicotinamide in a formulation
containing four vitamins (nicotinamide, pyridoxine, riboflavin, and thiamine) can cause the
degradation of thiamine to a sub-standard level within a one year shelf life of vitamin B-
complex injections.

(h) Functional group-related typical degradation

Ester hydrolysis can be explained with a few drugs like aspirin, benzocaine, cefotaxime,
ethyl paraben and cefpodoxime proxetil [20-21]. Hydrolysis is the common phenomenon
for ester type of drugs, especially in liquid dosage forms like benzyl penicillin, oxazepam
and lincomycin.

Oxidative degradation of drugs like hydrocortisone, methotrexate, hydroxyl group directly

bonded to an aromatic ring (like phenol derivatives such as catecholamines and morphine),
conjugated dienes (like vitamin A and unsaturated free fatty acids), heterocyclic aromatic
rings, nitroso and nitrite derivatives, and aldehydes (especially flavorings) are all
susceptible to oxidative degradation.

Photolytic cleavage includes example of pharmaceutical products that are exposed to light
while being manufactured as solid or solution, packaged, or when being stored in pharmacy
shops or hospitals for use by consumers. In ciprofloxacin eye drop preparation (0.3%),
sunlight induces photo cleavage reaction producing ethylenediamine analog of
ciprofloxacin. As seen earlier, impurities in drug products can come from the drug or from
excipients or can be brought into the system through an in process step by contact with the
packaging material. For most drugs, the reactive species consist of, Water- that can
hydrolyze some drugs or affect the dosage form performance, Small electrophiles- like
aldehydes and carboxylic acid derivatives, Peroxides- that can oxidize some drugs, Metals-

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which can catalyze oxidation of drugs and the degradation pathway and leachable or
extractable- can come from glass, rubber stoppers, and plastic packaging materials. Metal
oxides such as NaO2, SiO2, CaO, MgO, are the major components leached/extracted from
glass [22]. Generally most synthetic materials contain leachable oligomers/monomers,
vulcanizing agents, accelerators, plasticizers, and antioxidants [23].

Residual solvents
Residual solvents are organic volatile chemicals used during the manufacturing process or
generated during the production. Some solvents that are known to cause toxicity should be
avoided in the production of bulk drugs. Depending on the possible risk to human health,
residual solvents are divided into three classes [24]. Especially solvents in Class I like
benzene (2 ppm limit), carbon tetrachloride (4 ppm limit), 1,2-dichloroethane (5 ppm), 1,1-
dichloroethane (8 ppm) and 1,1,1-trichloroethane (1500 ppm) should be avoided. In Class
II, solvents like N, N-dimethylformamide (880 ppm), acetonitrile (410 ppm) etc should be
limited. Class III solvents, like acetic acid, ethanol and acetone have permitted daily
exposure of 50 mg or less per day as per the ICH guidelines and are classified as solvents
with low toxic potential. Using this method, the main contaminants of each organic solvent
can be quantified. Moreover, the developed method allows the simultaneous determination
of ethanol, isopropanol, chloroform, benzene, acetone, dichloromethane, methanol and
toluene with propionitrile as the internal standard.

ICH limits for Impurities

According to ICH guidelines on impurities in new drug products, identification of impurities
below 0.1% level is not considered to be necessary, unless potential impurities are expected
to be unusually potent or toxic. According to ICH, the maximum daily dose qualification
threshold to be considered is as follows; < 2g/day 0.1 % or 1 mg per day intake (whichever
is lower) >2g/day 0.05%.

Analytical method Development

New drug development requires meaningful and reliable analytical data to be produced at
various stages of the development [25-26].
a) Sample set selection for analytical method development

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b) Screening of Chromatographic conditions and Phases, typically using the linear-solvent-

strength model of gradient elution.
c) Optimization of the method to fine-tune parameters related to ruggedness and
robustness

The impurities can be identified predominantly by following methods are: 1.Reference

standard method 2.Spectroscopic method 3.Separation method 4.Isolation method and
Characterization method

Reference standard method

Reference standard serves as the basis of evaluation of both process and product
performance and is the benchmark for assessment of drug safety for patient consumption.
These standards are needed, not only for the active ingredients in dosage forms but also for
impurities, degradation products, starting materials, process intermediates, and excipients.

Spectroscopic methods
The UV, IR, MS, NMR and Raman spectroscopic methods are routinely being used for
characterizing impurities.

Separation methods
The Capillary electrophoresis (CE), Chiral Separations, Gas Chromatography (GC),
Supercritical Fluid Chromatography (SFC), TLC, HPTLC and HPLC are regularly being used
for separation of impurities and degradation products.

Isolation methods
It is often necessary to isolate impurities. But if the instrumental methods are used isolation
of impurities is avoided as it directly characterizes the impurities. Methods that can be used
for isolation of impurities are Solid-phase extraction method, Liquid-liquid extraction
method, Accelerated solvent extraction method, Supercritical fluid extraction, Column
chromatography, Flash chromatography, TLC, GC, HPLC, HPTLC, Capillary electrophoresis
(CE) and Supercritical fluid chromatography (SFC). High performance chromatography and
the chromatographic reactor approach, with solution phase hydrolysis kinetics can be used

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for an Aprepitant prodrug and fosaprepitant dimeglumine. In loratidine the impurity was
found to be ofloratidine and other examples include celecixib and amikacin [27-30].

Characterization methods
Highly sophisticated instrumentation, such as MS attached to a GC or HPLC, are inevitable
tools in the identification of minor components (drugs, impurities, degradation products,
metabolites) in various matrices. For characterization of impurities, different techniques
are used; which are as follows;

NMR Spectroscopy
The ability of NMR to provide information regarding the specific bonding structure and
stereochemistry of molecules of pharmaceutical interest has made it a powerful analytical
instrument for structural elucidation. The ability of NMR-based diffusion coefficient
determination to distinguish between monomeric and dimeric substances was validated
using a standard mixture of authentic materials containing both monomers and dimers31.
Unfortunately, NMR has traditionally been used as a less sensitive method compared to
other analytical techniques. Conventional sample requirements for NMR are on the order of
10 mg as compared with MS which requires less than 1 mg [31].

Mass spectroscopy
It has an increasingly significant impact on the pharmaceutical development process over
the past several decades. Advances in the design and efficiency of the interfaces that directly
connect separation techniques with Mass Spectrometers have afforded new opportunities
for monitoring, characterizing and quantification of drug-related substances in active
pharmaceutical ingredients and pharmaceutical formulations. If single method fails to
provide the necessary selectivity, orthogonal coupling of chromatographic techniques such
as HPLC-TLC and HPLC-CE, or coupling of chromatographic separations with information
rich spectroscopic methods such as HPLC-MS or HPLC-NMR may need to be contemplated
but hopefully only as a development tool rather than a tool for routine QC use.
1. HPLC-DAD-MS
2. HPLC-DAD-NMR-MS
3. GC-MS
4. LC-MS

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An example of reverse-phase LC-MS analysis in gradient elution with two distinct soft
ionization techniques is the Atmospheric pressure ionization with electrospray source (API-
ESI). A common goal for investigation of both process and product degradation-related
impurities is to determine which of the many potential impurities are, in fact, produced in
the manufacturing process and which occur under a given set of storage conditions.

Applications
Numerous applications have been sought in the areas of drug designing and in monitoring
quality, stability and safety of pharmaceutical compounds, whether produced synthetically
extracted from natural products or produced by recombinant methods. The applications
include alkaloids, amines, amino acids, analgesics, antibacterial, anticonvulsants,
antidepressant, tranquilizers, antineoplastic agents, local anesthetics, macromolecules,
steroids and miscellaneous.

C. Bio-Analytical method
Selective and sensitive analytical methods for the quantitative evaluation of drugs and their
metabolites are crucial for the successful conduct of preclinical and/or biopharmaceuticals
and clinical pharmacology studies. Bio analytical method validation includes all of the
procedures that demonstrate that a particular method used for quantitative measurement
of analytes in a given biological matrix, such as blood, plasma, serum, or urine is reliable and
reproducible for the intended use.

The first bioanalytical method workshop was conducted in 1990. It dedicated to

investigating and harmonizing procedures varied in method validation. The 1st workshop
clearly identified 2 distinct phases of bioanalytical method validation [32].

1. Analytical method development (pre study validation) where the appropriate bio
analytical method with its various parameters is developed and the assay is defined; and 2)
application of the bio analytical to actual analysis of samples from bioavailability, bio
equivalence and pharmacokinetic studies. One of the most important outcomes of the first
workshop was that it defined the acceptance criteria for a run. The first workshop was well
received within the global pharmacokinetic community. They needed to validate bio
analytical methods and the development and acceptance of general standards for their

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conduct, brought about significant improvement in the quality of bio analytical methods and
in the submission of pharmacokinetic, bioavailability and bio equivalence studies to .the
workshop report was not an official document of the FDA. Therefore the agent decided to
develop and publish draft guidance in 1999.

The IInd workshop, cosponsored by AAPS and the FDA, was held in Jan 2000, one year after
the publication of the draft guidance by the agency. The workshop focused on discussing the
advances in analytical technology that had occurred over the part dread and reconfirmed
and updated the principles of bio analytical method validation. The second workshop
discussed the advances in hyphenated mass spectrometry and ligand binding assays and it
also discussed different categories of validation. There are partial validation, cross
validation and full validation. The workshop reemphasized that it is not necessary to have
100% recovery where using an extraction procedure. The following principles of
bioanalytical method validation provide steps for the development of a new method or for
establishing an existing method. The parameters essential to ensure the acceptability of the
performance of a bio analytical method are accuracy, precision, selectivity, sensitivity,
reproducibility and stability. The guidance provides information for determining the
parameters. The guidance also establishes requirements for a standard curve. The matrix
based standard curve should consists of a maximum of five standard points, excluding
blanks, using single or replicate samples, and should cover the entire range of expected
concentration. All these parameters used to be defined during the full validation of a bio
analytical method. The (LLOQ) should serve as the lowest concentration on the standard
curve and should not be confused with the limit of detection (LOD). There are two distinct
phases of bio analytical method validation. (a).the bioanalytical method development phase
in which the assay is defined and validated. (b). the application to actual analysis of samples
from pharmacokinetic, bioavailability, bioequivalence and drug interaction studies.

As bioanalytical tools and techniques have continued to evolve, significant scientific and
regulatory experience has been gained since the last workshop in 2000 and issuance of the
guidance in 2001.The evolution and expansion of bioanalytical tools require a critical
review of the scope, applicability, and success of the presently employed bioanalytical
guiding principles. The third work shop in this series was held in May 2000 in Arlington
USA. The purpose of this 3rd AAPS/FDA Bioanalytical Workshop was to identify, review and

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evaluate the existing practices, white papers, and articles clarify the FDA Guidance. The
workshop addressed quantitative bioanalytical methods validation and their use in sample
analysis, focusing on both chromatographic and ligand-binding assays.

The purpose of the 3rd AAPS/FDA Bioanalytical Workshop was to [33]

1. Review the scope and applicability and bioanalytical principles and procedures for the
quantitative analysis of samples from bioequivalence, pharmacokinetic and
comparability studies in both human and non human subjects.
2. Review current practice for scientific excellence and regulating compliance, suggesting
classification and improvements where needed.
3. Review and evaluate validation implementation requirements for chromatographic
ligand bound. Quantitative bio analytical assays, covering all types (sizes) of molecules.
4. Review recent advances in technology, automation regulatory and scientific
requirements and data achieving on the performance and exporting of quantitative bio
analytical work and discuss current based approaches for the conduct of quantitative
bio analytical work regardless of the size of the molecule analysed.

In these guidelines are used to various essential development and validation characteristics
for bio analytical methodology have been discussed with a view to improve the standard
and acceptance in this area of research. Characterization of the stability of analytes in
biological samples collected during clinical studies together with that of critical assay
reagents including analyte stock solutions is recognized as an important component of bio
analytical assay validation. The information in this guideline generally applies to bio
analytical procedures such as [34-39] Gas chromatography, High- pressure liquid
chromatography, Combined GC and LC mass spectrometric, LC-MS, LC-MS-MS and GC-MS
These procedures are generally performed for the quantitative determination of drugs
and/or metabolites in biological matrices such as blood, serum, plasma and urine. This
guideline also applies to other bio analytical methods, such as immunological and
microbiological procedures and to other biological matrices, such as tissue and skin
samples. This guideline provides general recommendations for bio analytical method
validation. It is essential to employ well characterized and fully validated bio analytical
methods to yield reliable results that can be satisfactorily interpreted. It is also important to
emphasize that each bio analytical technique has its own characteristics which will vary

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from analyte to analyte in these instances, specific validation criteria may need to be
developed for each analyte. When sample analysis for a given study is conducted at more
than one site, it is necessary to validate the bio analytical method at each site and provide
appropriate validation information for different sites to establish inter laboratory
reliability. For quantitative bioanalytical method validation procedure and requirements,
there was a relatively good agreement between chromatographic assays and ligand-binding
assays. It was realized that the quantitative and qualitative aspects of bioanalytical method
validation should be reviewed and applied appropriately.

Bio analytical method development and validation

The process by which a specific bio analytical method is developed, validated and used in
routine sample analysis can be divided into 1.Reference standard 2.Bio analytical method
development and establishment of assay procedure 3.Application of validated bio analytical
method to routine drug analysis and acceptance criteria for the analytical run and/or batch.

Reference standard
Analysis of drugs and their metabolites in a biological matrix is carried out using samples
spiked with calibration standards and using quality control samples.
Three types of reference standards are usually used. They are:

1. Certified reference standards

2. Commercially supplied reference standard applied from a reputable commercial source
3. Other materials of documented purity custom-synthesized by an analytical laboratory.

Bio analytical method development

Typical method development and establishment for a bio analytical method include
determination of selectivity, sensitivity, accuracy, precision, stability of analyte in spiked
samples.

A) Selectivity
It is the analytical method to differentiate and quantify the analyte in the presence of other
components in the sample. For selectivity, analysis of blank samples of the appropriate

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biological matrix should be obtained from at least six sources. Each blank sample should be
tested for interference and selectivity, should be ensured at the lower limit of quantification.

B) Accuracy, precision, and recovery[40]

Accuracy
Accuracy is determined by replicate analysis of samples containing known amounts of the
analyte. Accuracy should be measured by minimum of five determinations per
concentration. The mean value should be within 15% of the actual value except at LLOQ,
where it should not deviate by more than 20%. The deviation of the mean from the true
value serves as a measure of accuracy.

Precision
It should be measured using a minimum of five determinations per concentration. The
precision determined at each concentration level should not exceed 15% of the coefficient
of variation except for LLOQ where it should not exceed 20% of the CV.

Recovery
The recovery of analyte in the assay is the detector response obtained from an amount of
the analyte added to and extracted from the biological matrix compared to the detector
response obtained from the true concentration of the pure authentic standard. Recovery of
the analyte need not to be 100%, but the extent of recovery of an analyte and of the internal
standard should be precise and reproducible.

Calibration/ standard curve

A calibration curve is the relationship between instrument response and known
concentrations of the analyte. A calibration curve should be generated for each analyte in
the sample. A calibration curve should be prepared in the same biological matrix as the
samples in the intended study by spiking the matrix with known concentrations of the
analyte.
Concentrations of the standard should be chosen on the basis of concentration range
expected in a particular study. A calibration curve should consist of i) A blank sample ii) A
zero sample iii) Six to eight non-zero samples covering the expected range, including LLOQ.

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Lower limit of quantification (LLOQ)

The lowest standard on the calibration curve should be accepted as the limit of
quantification if the following conditions are met 1).The analyte response at the LLOQ
should be at least 5 times the response compared to blank response.11).Analyte peak
should be identifiable, discrete and reproducible with a precision of 20% and accuracy of
80-120%

Calibration curve/standard curve-concentration-response.

The simplest model that adequately describes the concentration-response relationship
should be used. The following conditions should be met in developing a calibration curve
1). 20% deviation of the LLOQ from normal concentration. 2).15% deviation of the standard
other than LLOQ from normal concentration.

Stability in a biological fluid

The stability of an analyte in a particular matrix and container system is relevant only to
that matrix and container system and should not be extrapolated to other matrices and
container systems. Stability procedures should evaluate the stability of the analytes during
sample collection and handling, after long term (frozen at the intended storage
temperature) and short term (bench top, room temperature) storage, after going through
freeze and thaw cycles and the analytical process. All stability determinations should use
asset of samples prepared from a freshly made stock solution of the analyte in the
appropriate –analyte free, interference free biological matrix. Stock solutions of the analyte
for stability evaluation should be prepared in an appropriate solvent at known
concentrations.

Freeze and Thaw stability

Analyte stability should be determined after three freeze and thaw cycles. At least three
aliquots at each of the low and high concentrations should be stored at the intended storage
temperature for 24hrs and thawed unassisted at room temperature. When completely
thawed the samples should be re-frozen for 12-24hrs under the same conditions. The
freeze-thaw cycle should be repeated two more times then analysed on the third cycle. If an
analyte is unstable at the intended storage temperature, the stability sample should be
frozen at -700c during the three freeze and thaw cycles.

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Short-Term Temperature stability

Three aliquots of each of the low and high concentrations should be thawed at room
temperature and kept at this temperature from 4-24 hrs (based on the expected duration
that samples will be maintained at room temperature in the intended study) and analysed.

Long-Term Stability
The storage time in a long-term stability evaluation should exceed the time between the
date of first sample collection and the date of last sample analysis. Long-term stability
should be determined by storing at least three aliquots of each of the low and high
concentrations under same conditions as the study samples. The volume of samples should
be sufficient for analysis on three separate occasions. The concentrations of all the stability
samples should be compared to the mean of back-calculated values for the standards at the
appropriate concentrations from the first day of long term stability testing.

Stock solution stability

The stability of stock solutions of drug and the internal standard should be evaluated at
room temperature for at least 6 hrs. If the stock solutions are refrigerated or frozen for the
relevant period, the ability should be documented. After completion of the desired storage
time, the stability should be tested by comparing the instrument response with that of
freshly prepared solutions.

Post-preparative stability
The stability of processed samples, including the resident time in the auto-sampler, should
be determined. The stability of the drug and the internal standard should be assessed over
the anticipated run time for the batch size in validation samples by determining
concentrations on the basis of original calibration standards. Although the traditional
approach of comparing analytical results for stored samples with those for freshly prepared
samples has been referred to in this guidance, other statistical approaches based on
confidence limits for evaluation of analyte stability in a biological matrix can be used. SOPs
should clearly describe the statistical method and rules used. Additional validation may
include investigation of samples from dosed subjects.

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Bioanalytical method Validation [41]

Pre-validation study
Before going for the actual validation process so called pre-validation is done. In pre-
validation study large numbers of replicate spiked matrix samples are analyzed in single
batch before a method validation is started. The utility of this pre-validation approach is
illustrated using actual laboratory data. The process of interpreting the results and drawing
conclusions about assay viability is demonstrated. The resulting conclusions provide
sufficient background information to indicate if an assay is ready to enter the validation
process. Following validation criteria is studied in pre-validation and validation steps.
(Table-5)

Full validation
1. Full validation is important when developing and implementing a bio analytical method
for the first time.
2. Full validation is important for a new drug entity.
3. A full validation of the revised assay is important if metabolites are added to an existing
assay for quantification.

Partial validation
Partial validations are modifications of already validated bio analytical methods. Partial
validation can range from as little as one intra-assay accuracy and precision determination
to a nearly full validation. Typical bio analytical method changes that fall into this category
include, but are not limited to: 1.Bio analytical method transfers between laboratories or
analysts 2.Change in anticoagulant in harvesting biological fluid.3.Change in analytical
methodology (e.g., change in detection systems).4.Change in matrix within species (e.g.,
human plasma to human urine).5.Change in sampling processing procedures.6. Change in
species within matrix (e.g., rat plasma to mouse plasma) 7. Change in relevant concentration
range 8. Changes in instruments and/ or software platforms 9. Limited sample volume (e.g.,
pediatric study) 10. Selectivity demonstration of an analyte in the presence of specific
metabolites.

Cross-validation

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Cross-validation is a comparison of validation parameters when two or more bio analytical

methods are used to generate data within the same study or across different studies. An
example of cross validation would be a situation where an original validated bio analytical
method serves as the reference and the revised bio analytical method is the comparator.
The comparisons should be done both ways. When sample analyses within in a single study
are conducted at more than one site or more than one laboratory, cross validation with
spiked matrix standards and subject samples should be conducted at each site or laboratory
to establish inter laboratory reliability. Cross validation should also be considered when
data generated using different analytical techniques (e.g., LC-MS-MS vs. ELISA) in different
studies are included in a regulatory submission.

Documentation
The validation of an analytical method should be established and verified by laboratory
studies, and documentation of successful completion of such studies should be provided in
the assay validation report. General and specific SOPs and good record keeping are an
essential part of a validated analytical method. The data generated for bio analytical method
establishment and the QCs should be documented and available for data audit and
inspection. Documentation for submission to the agency should include:

1. Summary information
2. Method development and establishment
3. Bio analytical reports of the application of methods to routine sample analysis
4. Other information applicable to method development and establishment and/ or to
routine sample analysis.

Documentation for method establishment

Documentation for method development and establishment should include:
1. An operational description of the analytical method 2.Evidence of purity and identity of
drug standards, metabolite standards, and internal standards used in validation
experiments 3.A description of stability studies and supporting data 4.A description of
experiments conducted to determine accuracy, precision, recovery, selectivity, limit of
quantification, calibration curve (equations and weighing functions used, if any) and

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relevant data obtained from these studies 5.Documentation of intra- and inter-assay
precision and accuracy.

Application of validated method to routine drug analysis

1. Assays of all samples of an analyte in a biological matrix should be completed within the
time period for which stability data are available. In general, biological samples are
analysed with a single determination without duplicate or replicate analysis if the assay
method has acceptable variability as defined by validation data. The following
recommendations should be noted in applying a bio-analytical method to routine drug
analysis
2. Response Function: Typically, the same curve fitting, weighing, and goodness, of fit
determined during restudy validation should be used for the standard curve within the
study. Response function is determined by appropriate statistical tests based on actual
standard points during each run in the validation. Changes in the response function
relationship between pre-study validation and routine run validation indicate potential
problems.
3. The QC samples should be used to accept or reject the run. These QC samples are matrix
spiked with analyte.
4. System Suitability
5. Based on the analyte and technique, a specific SOP (or sample) should be identified to
ensure optimum operation of the system used.

CONCLUSION
Analytical methodology provides to an analyst the required data for a given analytical
problem, sensitivity, accuracy, range of analysis, precision i.e. the minimum requirements
which essentially are the specifications of the method for the intended purpose to be able to
analyse the desired analyte in different matrices with surety and certainty. Analytical
methods need to be validated before their introduction into routine use; whenever the
conditions change for which the method has been validated (e.g., an instrument with
different characteristics or samples with a different matrix); and whenever the method is
changed, the change is outside the original scope of the method. The stability indicating
assays have been developed for a large number of drugs but most of them fail to meet
current regulatory requirements of separation and analysis of individual degradation

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products. So the discussion provided would be general and of wide use. Nowadays, it is a
mandatory requirement in various pharmacopoeias to know the impurities present in API’s.

Isolation and characterization of impurities are required for acquiring and evaluating data
that establishes biological safety which reveals the need and scope of impurity profiling of
drugs in pharmaceutical research. The aim of this article is to provide a simple way to use
approaches with a correct scientific background to improve the quality of the bioanalytical
method development and validation. Applications of bio analytical method in routine drug
analysis are also taken into consideration in this article. Method development involves a
series of simple steps. All the conditions are optimized as required for the purpose of the
separation and the method is validated using ICH guidelines. The validated method and data
can then be documented.

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