1. food and drink
2. beverages
3. non alcoholic beverages
4. bottled water

IMPURITIES IN PHARMACEUTICALS
A
FOR ELECTIVE SUBJECT
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SUBMITTED TO THE
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PROJECT REPORT
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HEMCHANDRACHARYA NORTH GUJARAT UNIVERSITY, PATAN
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IN PARTIAL FULFILLMENT OF
THE REQUIREMENT FOR THE DEGREE OF
BACHELOR OF PHARMACY
SUBMITTED BY
Hardi K. Modi
DEPARTMENT OF PHARMACEUTICAL TECHNOLOGY
SHREE S.K. PATEL COLLEGE OF
PHARMACEUTICAL EDUCATION AND RSEARCH.
GANPAT VIDYANAGAR
KHERVA-GUJARAT
2004-2005
CERTIFICATE
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This is to certify that the project work for elective subject entitled
“Impurities in Pharmaceuticals” represents the bonafide work
of Hardi K. Modi carried out under my guidance and supervision
in the department of pharmaceutical Technology of Shree S.K.
Patel college of pharmaceutical education and
research, Ganpat Vidyanagar, during the academic year of
2004-2005. She has collected literature very sincerely and
methodically. This work is up to my satisfaction.
GUIDE
Lect. Harsha V. Patel
(M.Pharm.)
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Dept. of Pharmaceutics,
Shree S.K. Patel college of
Pharmaceutical education
and research, Ganpat
Vidyanagar Kherava,
Mehsana.
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PRINCIPAL
Prof. Dr. N.J. Patel
(M.Pharm., Ph.D)
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Shree S.K. Patel college of
Pharmaceutical education and research,
GanpatVidyanagar,
Kherava ,Mehsana.
Date:
Place:
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ACKNOWLEDGEMENT
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฀฀
This thesis on “Impurities in Pharmaceuticals.” has been prepared for
partial fulfillment of the academic requirements leading to the
Bachelor’s degree in pharmacy.
Numerous people have been Instrumental in enabling me to give a
concrete shape to my thesis constraints of time & space precludes
the mention of all of them here. However, I must mention the names
of a few people who have made a catalytic impact on the
development of this thesis.
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

 
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The chief person who help everyone in solving any problem is a
teacher. First and Foremost, I would like to acknowledge the
continuous encouragement & help extended to me by Mrs. Harsha V.
Patel for preparing this thesis. Right from the day I started working on
it till it was completed, be has been my sole guide philosopher and
friend throughout the period of my work. But for the spontaneous
support & expert guidance provided by her, this project would lot have
seen the light of day in its present form her extensive knowledge of
the subject & the way she imparted the same to me has enabled me
to develop the thesis in a cohesive manner & has kindled within me a
passion for the subject.
I also express my profound gratitude to Dr. N.J. Patel sir, our
principal who has been a constant source of inspiration to steer me
forward throughout the four years of my study.
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I take the opportunity to place on record my indebtedness to shri.
P.D.Bharadia, Shri. J. K. Patel, Shri.R.P.Patel, Shri. V.M.Patel, and all the
others faculties members who have also contributed a lot at various
stages of my academic carrier in the institute in term of valuable
knowledge inputs.
I am also thankful to the members of my family not only for their
support and encouragement in my work but also for patiently
tolerating my ling and irregular working hours during the hectic period
in which this work was under preparation. I owe a special thank to my
dad, whose help has been invaluable at various stages, and
especially during the finalization of the thesis.
Dhwanoo is such a person in my life. She raises my hopes with her
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enthusiasm. She stairs up my spirit of enterprise to superior
achievement. She is the only one person to whom I can share my
joys and sorrows without any hesitation during this study.
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I personally thank to our librarian to help me in finding and getting
more and more information regarding my thesis.
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Lastly, I wish to thank my friends. They all are very helpful to me
during my work. I also thankful my classmates with whom I spent
joyful time of four years.
Hardi K. Modi
INDEX
2. REVIEW.
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1.1 About The Impurities.
1.2 Classification Of Impurities.
1.3 Rationale For The Reporting And Control Of Impurities.
1.4 Impurities Present In Drugs.
1.5 Effect Of Impurities.
1.6 10 'Rules To Remember' For Impurities.
1.7 Specification Limits For Impurities.
1.8 Qualification Of Impurities.
1.9 New Impurities.
1.10 Impurity Decision Tree.
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1. INTRODUCTION.
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2.1 Sources Of Impurity.
2.2 Permissible Impurity In Pharmaceutical Substances.
2.3 Review.
2.4 Terminology.
2.5 Limit Tests.
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3. PURITY AND MANAGEMENT.
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3.1 Pharmaceutical Chemicals: Purity & Management.
3.2 Management.
3.3 Test For Purity.
3.4 Usp/Nf Chemical And Physical Tests.
3.5 Bioburden Testing.
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4. ANALYTICAL PROCEDURE.
4.1 Advantages Of Analytical Procedures.
4.2 Identification Tests.
4.3 Analytical Procedure.
4.3.1 Thin Layer Chromatography (Tlc)
4.3.2 High Performance Thin Layer Chromatography (Hptlc)
4.3.3 Infra-Red (Ir) Spectroscopy
4.3.4 Mass Spectroscopy(Ms).
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6. SUMMARY
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7. REFERENCES
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5.1 Validation Of Pharmaceutical Test Method.
5.2 Validation Terminology Definitions.
5.3 Validaton Requirements Of The Method.
5.4 Validation Documentation.
5.5 Validation Protocol.
5.6 Validation Experimentation.
5.7 Revalidation.
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5. VALIDATION.
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Introduction
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I. INTRODUCTION1
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The substances that are used in the pharmaceutical field ,should be almost pure so that they
can be used safely. It is rather difficult to obtain an almost pure substance. We find substances
and chemicals, with varying degree of purity. For example, substances like cane-sugar
(sucrose), dextrose, common salt and many in organic salts, Organic compounds are found
with over 99%purity while many others only contain traces of impurities. The purity of
substances depends upon several factors, such as their methods of manufacture& purification.
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This guidance provides recommendations for including information in abbreviated new drug
applications (ANDAs) and supporting drug master files (DMFs) on the identification and
qualification of impurities in drug substances produced by chemical syntheses for both
monograph and nonmonograph drug substances.
Specific guidance is provided for:
Qualifying impurities found in a drug substance used in an ANDA by a comparison with
impurities found in the related U.S. Pharmacopeia (USP) monograph, scientific literature, or
innovator material; Qualifying impurities found at higher levels in a drug substance used in an
ANDA than found in the related USP monograph, scientific literature, or innovator material;
Qualifying impurities in a drug substance used in an ANDA that are not found in the related
USP monograph, scientific literature, or innovator material.
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This
guidance
is
not
applicable
to
biological/biotechnological,
peptide,
oligonucleotide,radiopharmaceutical, fermentation and semisynthetic products derived there
from, herbal products, or crude products of animal or plant origin. The recommendations in this
guidance are effective on publication and should be followed in preparing new applications and
supplements for changes in drug substance synthesis or process.
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There should be a 0.1 percent threshold above which isolation and characterization of
individual impurities should apply to chemically synthesized drug substances including drug
substances used in generic drug products.
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II. CLASSIFICATION OF IMPURITIES (2,3,4)
(A) Organic Impurities (Process and Drug Related)
(B) Inorganic Impurities
(C) Residual Solvents
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Impurities can be classified into the following categories:





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(A) Organic impurities may arise during the manufacturing process and/or storage of the
drug substance.
They may be identified or unidentified, volatile or nonvolatile, and include:
Starting materials
By-products
Intermediates
Degradation products
Reagents, ligands, and catalysts
(B) Inorganic impurities may derive from the manufacturing process. They are normally
known and identified and include:
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

Reagents, ligands, and catalysts
Heavy metals
Inorganic salts
Other materials (e.g., filter aids, charcoal)
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(C) Residual solvents are solvents that are used during the manufacturing process and may
be detected after the product is in its final form. Some of the common solvents are..
benzene, chloroform, 1,4-dioxane, methylene chloride, and trichloroethylene. Residual
solvents in the active ingredient or drug product can come from many different stages in
the manufacturing process (active substance granulation, milling, or drug product
coating). The most common technique for measuring residual solvents in gas
chromatography (GC) because of the small size and volatile nature of solvent molecules.
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As Impurities are generally of known toxicity, the selection of appropriate controls is
easily accomplished.
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(1) extraneous contaminants, which should not occur in drug
substances and are more appropriately addressed as good manufacturing practice
issues;
(2) polymorphic form, a solid state property of the drug substance; and
(3) enantiomeric impurities.
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III. RATIONALE FOR THE REPORTING AND CONTROL OF IMPURITIES
A. Organic Impurities
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The actual and potential impurities most likely to arise during the synthesis, purification,
and storage of the drug substance, should be based on sound scientific appraisal of the
chemical reactions involved in the synthesis, impurities associated with raw materials that
could contribute to the impurity profile of the drug substance, and possible degradation
products.
It should include test results of materials manufactured during the development process
and batches from the proposed commercial process, as well as results of intentional
degradation studies used to identify potential impurities that arise during storage. Assessment
of the proposed commercial process may be deferred until the first batch is produced for
marketing. The impurity profile of the drug substance lots intended for marketing should be
compared with those used in development and any differences discussed.
The studies (e.g., NMR, IR, and MS) conducted to characterize the structure of actual
impurities present in the drug substance at or above an apparent level of 0.1 percent (e.g.,
calculated using the response factor of the drug substance) should be described. All recurring
impurities at or above an apparent level of 0.1 percent in batches manufactured by the
proposed commercial process should be identified. Degradation products observed in stability
studies at recommended storage conditions should be similarly identified. When identification
of an impurity is infeasible, Where attempts have been made to identify impurities below the
0.1 percent level, it is useful also to report the results of these studies.
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Identification of impurities below apparent levels of 0.1 percent is generally not
considered necessary. However, identification should be attempted for those potential
impurities those are expected to be unusually potent, producing toxic or pharmacologic effects
at a level lower than 0.1 percent. Although it is common practice to round analytical results of
between 0.05 and 0.09 percent to the nearest number (i.e., 0.1 percent), for the purpose of this
guidance, such values should not be rounded to 0.1 percent in determining whether to identify
the impurities.
B. Inorganic Impurities
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Inorganic impurities are normally detected and quantitated using pharmacopeial or other
appropriate procedures. Carryover of catalysts to the drug substance should be evaluated
during development. Acceptance criteria should be based on pharmacopeial standards or
known safety data.
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C. Residual Solvents
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IMPURITIES PRESENT IN DRUGS (FOR EXAMPLE: MORPHINE)
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The control of residues of solvents used in the manufacturing process for the drug substance
should be discussed. Any solvents that may appear in the drug substance should be quantified
using analytical procedures with an appropriate level of sensitivity. Pharmacopeial or other
appropriate procedures should be used. Acceptance criteria should be based on
pharmacopeial standards or known safety data, taking into consideration dose, duration of
treatment, and route of administration. Particular attention should be given to quantitation of
toxic solvents.
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EFFECT OF IMPURITIES
It can be seen that, almost pure substances are difficult to get and that some amount of
impurity is always present in the material. The impurities present in the substances may have
the following effects:
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1. Impurities which have a toxic effect, can be injurious when present above certain limits.
2. Impurities, even when present in traces may show a cumulative toxic effect after a
certain period.
3. Impurities are sometimes harmless, but are present in such a large proportion, that the
active strength of the substance is lowered. The therapeutic effect of drug is decreased.
4. Impurities may bring about a change in the physical & chemical properties of the
substances, thus making it medically useless.
5. Impurities may cause technical difficulties in the formulation & use of the substances.
6. Impurities may bring about an incompatibility with other substances.
7. Impurities may lower the shelf life of the substance.
8. Impurities, though harmless in nature, may bring about changes in odour, colour, taste
etc., thus making the use of the substance unethical, as well as unhygiene.
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10 'RULES TO REMEMBER' FOR IMPURITIES
Rule No.1 - Evaluate the RLD impurity profile (i.e. get a baseline).
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Rule No.2. Treat with CAUTION or REJECT a vendor profile HIGHER than the innovator
material.
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Rule No.3. LOOK at impurity profiles in the major pharmacopoeia (USP / BP / JP) and
compare with vendor's dedicated synthesis (comparing profiles is important)
Rule No.4. 'Approved vendors' may have unique impurities due to the purifying process.
LOOK for these 'specified impurities' in the actives chromatograms (i.e. "Stress the Active
material").
Rule No.5. Unknown impurities must not exceed 0.1% (if they do, go back to active vendor to
clean up material).
Rule No.6. Organic impurities are the main focus in impuritiy profiles
(Note: residual solvents have there own guideline and limits).
Rule No.7. Do get the DMF holder to state the 'specific impurities' and the potential impurities
(i.e. those impurities which do arise and those which can arise).
Rule No.8. Always stress the active in-house to see which impurities do occur.
Rule No.9. In drug development, if the active has an unknown >0.1% - and it can not be
reduced - Look for an alternative supply with a better profile.
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Rule No.10. REMEMBER an unknown impurity close to 0.1% may grow to >0.1% on stability
(ageing). There's no such concept as a safe unknown >0.1%
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ICH provides recommendations for
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(1) inclusion of information regarding specified impurities in certain new drug
applications (NDAs) (identified and unidentified impurities in new drug
substance specifications) and,
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(2) qualification of impurities (the process of acquiring and evaluating
data that establishes the biological safety of individual impurities or a given
impurity profile at the level(s) specified).
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REPORTING IMPURITY CONTENT OF BATCHES
of the new substance used for
representative of the proposed
unidentified and total impurities,
be reported with the analytical
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Analytical results should be provided for all batches
clinical, safety and stability testing, as well as for batches
commercial process. The content of individual identified and
observed in these batches of the new substance, should
procedures indicated.
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A tabulation (e.g., spreadsheet) of the data is recommended. Impurities should be
designated by code number or by an appropriate descriptor, e.g., retention time. Levels of
impurities which are present but are below the validated limit of quantitation need not be
reported. When analytical procedures change during development, reported results should be
linked with the procedure used, with appropriate validation information provided.
Representative chromatograms should be provided. Chromatograms of such
representative batches, from method validation studies showing separation and detectability of
impurities (e.g., on spiked samples), along with any other impurity tests routinely performed,
can serve as the representative impurity profiles. The applicant should ensure that complete
impurity profiles (i.e., chromatograms) of individual batches are available if requested. A
tabulation should be provided which links the specific new substance batch to each safety
study and each clinical study in which it has been used. For each batch of the new substance,
the report should include:
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• Batch Identity and Size
• Date of Manufacture
• Site of Manufacture
• Manufacturing Process
• Impurity Content, Individual and Total
• Use of Batches
• Reference to Analytical Procedure Used
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SPECIFICATION LIMITS FOR IMPURITIES
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The specifications for a new substance should include limits for impurities. Stability
studies, chemical development studies, and routine batch analyses can be used to predict
those impurities likely to occur in the commercial product. The selection of impurities to include
in the new substance specifications should be based on the impurities found in batches
manufactured by the proposed commercial process. Those impurities selected for inclusion in
the specifications for the new substance are referred to as “specified impurities”. Specified
impurities may be identified or unidentified and should be individually listed in the new
substance specifications.
A rationale for the inclusion or exclusion of impurities in the specifications should be
presented. This rationale should include a discussion of the impurity profiles observed in the
safety and clinical development batches, together with a consideration of the impurity profile of
material manufactured by the proposed commercial process.
Specific identified impurities should be included along with recurring unidentified
impurities estimated to be at or above 0.1%. For impurities known to be unusually potent or to
produce toxic or unexpected pharmacological effects, the quantitation/detection limit of the
analytical methods should be commensurate with the level at which the impurities must be
controlled. For unidentified impurities, the procedure used and assumptions made in
establishing the level of the impurity should be clearly stated.
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Unidentified impurities included in the specifications should be referred to by some
appropriate qualitative analytical descriptive label e.g., “unidentified A” unidentified with relative
retention of 0.9", etc.). Finally, a general specification limit of not more than 0.1% for any
unspecified impurity should be included.Limits should be set no higher than the level which can
be justified by safety data, and, unless safety data indicate otherwise, no lower than the level
achievable by the manufacturing process and the analytical capability.
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In other words, where there is no safety concern, impurity specifications should be
based on data generated on actual batches of the new substance allowing sufficient latitude to
deal with normal manufacturing and analytical variation and, the stability characteristics of the
new substance. Although normal manufacturing variations are expected, significant variation in
batch to batch impurity levels may indicate that the manufacturing process of the new
substance is not adequately controlled and validated.
The new substance specifications should include, where applicable, limits for:
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• Organic Impurities
• Each Specified Identified Impurity
• Each Specified Unidentified Impurity at or above 0.1%
• Any Unspecified Impurity, with a limit of not more than 0.1%
• Total Impurities
• Residual Solvents
• Inorganic Impurities
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QUALIFICATION OF IMPURITIES
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A summation of assay value and impurity levels generally may be used to obtain mass balance
for the test sample. The mass balance need not add to exactly 100% because of the analytical
error associated with each analytical procedure. The summation of impurity levels plus the
assay value may be misleading, e.g., when the assay procedure is nonspecific (e.g.,
potentiometric titrimetry) and the impurity level is relatively high.
Qualification is the process of acquiring and evaluating data which establishes the
biological safety of an individual impurity or a given impurity profile at the level(s) specified.
The applicant should provide a rationale for selecting impurity limits based on safety
considerations. The level of any impurity present in a new substance which has been
adequately tested in safety and/or clinical studies is considered qualified. Impurities which are
also significant metabolites present in animal and/or human studies do not need further
qualification.
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A level of a qualified impurity higher than that present in a new substance can also be
justified based on an analysis of the actual amount of impurity administered in previous safety
studies. If data are not available to qualify the proposed specification level of an impurity,
studies to obtain such data may be needed when the usual qualification threshold limit s given
below are exceeded:
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Higher or lower threshold limits for qualification of impurities may be appropriate for
some individual substances based on scientific rationale and level of concern, including
product class effects and clinical experience. For example, qualification may be especially
important when there is evidence that such impurities in certain substances or therapeutic
classes have previously been associated with adverse reactions in patients. In these
instances, a lower qualification threshold limit may be appropriate.
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Conversely, a higher qualification threshold limit may be appropriate for individual
substances when the level of concern for safety is less than usual based on similar
considerations (patient population, substance class effects, clinical considerations, etc.).
Technical factors (manufacturing capability and control methodology) may be considered as
part of the justification for selection of alternative threshold limits.
NEW IMPURITIES
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In some cases, decreasing the level of impurity below the threshold may be simpler
than providing safety data. Alternatively, adequate data may be available in the scientific
literature to qualify an impurity. If neither is the case, additional safety testing should be
considered. The studies desired to qualify an impurity will depend on a number of factors,
including the patient population, daily dose, route and duration of medicinal product
administration. Such studies are normally conducted on the new substance containing the
impurities to be controlled, although studies using isolated impurities are acceptable.
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During the course of a substance development program, the qualitative impurity profile
of the new substance may change, or a new impurity may appear as a result of synthetic route
changes, process optimisation, scale-up, etc. New impurities may be identified or unidentified.
Such changes call for consideration of the need for qualification of the level of the impurity
unless it is below the threshold values as noted above.
When a new impurity exceeds the threshold, the “Decision Tree for Safety Studies”
should be consulted. Safety studies should compare the new substance containing a
representative level of the new impurity with previously qualified material, although studies
using the isolated impurity are also acceptable (these studies may not always have clinical
relevance).
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IMPURITY DECISION TREE5: -
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SOURCES OF IMPURITIES (4,6,7)

Raw Material Employed in Manufacture
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The type & amount of impurity present in the chemicals or pharmaceutical substances
depends upon several factors. Some factors are discussed below:

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When substances or chemicals are manufactured, the raw materials from which these are
prepared, often contain impurities .The impurities get incorporated into the final product.
Impurities are hence found in substances.It is therefore necessary to employ pure chemicals &
substances as the raw materials for the manufacturing process.
Methods or the Process used in Manufacture

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There are a number of drugs & chemicals, which are manufactured from different raw
materials by adopting different methods or processes. Some impurities get incorporated into
the materials during the manufacturing process. The type and amount of impurity present in
the drugs or chemicals varies. Furthermore, for certain drugs a multiple-step-synthesis
procedure is used, which producer intermediate compounds. The purification of the
intermediates is also essential, otherwise impurities present in the intermediates will get into
the final compound. Often, side reactions take place during the synthesis. Impurities of the side
product are also found in the substances.
CHEMICAL PROCESS AND PLANT MATERIALS EMPLOYED THE PROCESS
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In the synthesis of drugs, many chemical reactions like nitration, halogenation,
oxidation, reduction, hydrolysis etc. are involved. In these chemical process, different solvents,
chemicals etc. are used When chemical reactions are carried out in vessels or containers, the
materials of these vessels (like iron, copper, tin aluminium etc.) are reacted upon by the
solvents and chemicals and reaction products are formed. These reaction products derived
from the plant material occur as impurities in the final product. Thus impurities of iron, lead,
heavy metals, coppers etc. in substances are due to the above mentioned reason.
Storage Condition
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
The chemicals, substances when prepared, are stored in different types of
containers, depending upon the nature of materials, batch size & the quality.Various types of
materials are used for storage purpose. These may be plastic, polythene, iron vessels,
stainless steel, aluminium, copper etc. Reaction of these substances with the material of the
storage vessel takes place & the products formed, occur as impurities in the stored material.
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The reaction may take place directly or by the laeching out effect on the storage vessel.
Alkalies stored in ordinary glass containers, extract lead from it, which occurs in the final
product. Similarly strong chemicals react with iron containers, & extract iron.

Decomposition
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Some substances decompose on keeping & the decomposition is greater in the
presence of light, air or oxygen. The result of decomposition causes contamination of the final
product. Many substances loose water of crystallization when kept open, while deliquescent
substances absorb water from the atmosphere, &get liquefied. Crude vegetable drugs are
especially susceptible to decomposition. A number of organic substances get spoiled, because
of decomposition on exposure to the atmosphere e.g. amines, phenols, potent drugs etc. The
decomposition products thus appear as impurities in the substances.
PERMISSIBLE IMPURITIES IN PHARMACEUTICAL SUBSTANCES2
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Since it is not possible to avoid impurities, it is necessary to have
substances that are reasonably pure. The pharmacopoeial committee takes the following
points into consideration with respect to the problem caused by impurities in the substances.
 For impurities which are of harmful type e.g. lead, arsenic etc. a low permissible
limit is prescribed. This is based upon, how much of these can be tolerated?
Which itself is based upon, how much of the impuritiy is harmful.
 For impurities that are harmless, the aim is to fix their limits so that, their presence
does not interfer in the therapeutic usefulness of the drug. Here, again, the limits
are prescribed & fixed. This is done depending upon the nature of the impurity, the
type of the substance, use of the substance, etc.
 Another consideration is the practicablity of obtaining substances without
impurities, at reasonable costs. It may be possible to prepare substances
(through a series of steps of purification) without any impurities, but this may be
achieved at an exorbitant cost.
Considering this aspect,limits of various impurities are fixed.
 Deliberate adulteration by using materials having similar qualities also accounts
for the presence of the impurities in the substance, e.g. adulteration of sodium salt
with potassium salt, calcium salts with magnesium salts etc. Such adulteration
which brings impurities into the substances, need not exhibit less therapeutic
activity but it is reasonable to expect unadulterated material from an ethical point
of view. Pharmacopoeias guard against this type of impurity by employing tests
for identification.
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Review
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Review
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Inorganic, organic, biochemical, isomeric or polymeric components can all be
considered impurities. Microbiological species or strains are sometimes described
in similar terms of resolving into more than one component.
Terminology8:-
Foreign substances:- which are introduced by contamination or adulteration, are not
consequence of the synthesis or preparation of compendial articles and thus cannot be
anticipated when monograph tests and assays are selected. The presence of
objectionable foreign substances not revealed by monograph tests and assays
constitutes a variance from the official standards.

Toxic Impurities:- Toxic impurities have significant undesirable biological activity, even
as minor component, and require individual identification and quantitation by specific
tests. This impurities may arise out of the synthesis, preparation, or degradation of
compendial articles.

Concomitant component:- are characteristic of many bulk pharmaceutical chemicals
and are not considered to be impurities in the pharmacopoieal sense. Example of
Concomitant components are geometric and optical isomers (or racemates) and
antibiotics that are mixtures. Any component that can be considered a toxic impurity
because significant undesirable biological effect is not considered to be a Concomitant
component.

Signal impurity:- are distinct from ordinary impurities in that they require individual
identification and quantitation by specific tests. Based on validation data, individualized
tests and specification are selected. Signal impurities may include some process related
impurities or degradation products that provide key information about the process, such
as diazotizable substances in thiazides.
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Ordinary impurities:- ordinary impurities are those species in bulk pharmaceutical
chemicals that are innocuous by virtue of having no significant, undesirable biological in
the amount present. These impurities may arise out of the synthesis, preparation or
degradation of compendial articles. Selection of tests and assays allow for anticipated
amounts of impurities that are unobjectionable for the customary use article.
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Unless otherwise specify in individual monograph, estimation of the amount and number of
ordinary impurities is made by relative methods rather than by strict comparison to individual
reference standards. The value of 2.0% was selected as the general limit of the ordinary
impurities in monograph where documentation did not support adaptation of other values.
16
Related substance:- Are structurally related to a drug substance. They may be identified
or unidentified degradation products or impurities arising from manufacturing or
during storage of a material.

Process contaminants:- are identified or unidentified substances ( excluding related
substances and water), including reagents, inorganics, row-materials and
solvents. They are introduced during manufacturing.
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
17
Limit tests9
Heavy Metals Limit Test
in
In general, Limit test are quantitative or semi-quantitative test particularly put forward to
identify and control invariably small quantities of impurities that are supposed to be present in
a pharmaceutical substance.
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The Heavy Metals Limit Test is designed to determine the allowable total limit of
heavy metals contained as impurities in a sample. In this test, the “heavy metals”
mean the metallic substances that are darkened with sodium sulfide TS in its acidic solution,
and the total content of them is expressed in terms of the quantity of lead (Pb).
Here in after in the Monographs, such a specification as not more than 20 µg/g as Pb (1.0 g,
Method 1, Control solution Lead Standard Solution 2.0 ml) indicates that when determined by
weighing 1.0 g of the test substance and proceeding as directed in Method 1, using 2.0 ml of
Lead Standard Solution for the preparation of the control solution, the content of heavy metals
in the substance is not more than 20 µg/g as Pb.
Procedure: Preparation of Test Solution and Control Solution Unless Otherwise specified,
proceed as directed one of the methods below.
Method 1
Test Solution Weigh the specified amount of the sample, transfer
into a Nessler tube, dissolve in about 40 ml of water, add 2 ml of diluted acetic acid (1:20) and
water to make 50 ml. Control Solution Measure the specified amount of Lead Standard
Solution, transfer into another Nessler tube, add 2 ml of diluted acetic acid (1:20) and water to
make 50 ml.
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Method 2
Test Solution Weigh the specified amount of the sample, place into a quartz or porcelain
crucible, cover it loosely, and carbonize by gently heating. Cool, add 2 ml of nitric acid and 5
drops of sulfuric acid, heat until no white fumes are any longer evolved, and ignite at 450 550
to incinerate. Cool, add 2 ml of hydrochloric acid, evaporate to dryness on a water bath, add 3
drops of hydrochloric acid to the residue, add 10 ml of boiling water, and warm for 2 minutes.
Cool, add 1 drop of phenolphthalein TS, and add ammonia TS until the solution becomes
slightly red. Then transfer it quantitatively into a Nessler tube using water, add 2 ml of diluted
acetic acid (1:20) and water to make 50 ml. Control Solution Place 2 ml of nitric acid, 5 drops
of sulfuric acid, and 2 ml of hydrochloric acid into a crucible of the same quality as used for the
sample, heat to evaporate to dryness, and add 3 drops of hydrochloric acid to the residue.
Then, proceed as directed in the preparation for the test solution, transfer it quantitatively into
another Nessler tube, add the specified amount of Lead Standard Solution,2 mlof diluted acetic
acid (1:20) and water to make 50 ml. If the test solution is not clear, filter both the test solution
and control solution under the same conditions.
18
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Method 3
Test Solution:- Weigh the specified amount of the sample, place into a quartz or porcelain
crucible, and heat gently with care, then ignite to incinerate. Cool,add 1 ml of aqua regia, and
evaporate to dryness on a water bath. Moisten the residue with 3 drops of hydrochloric acid,
add 10 ml of boiling water, and warm for 2 minutes. Then, add 1 drop of phenolphthalein TS,
add anmonia TS until the solution becomes slightly red, and add 2 ml of diluted acetic acid
(1:20). Filter the solution if necessary, wash with 10 ml of water, take both the filtrate and
washings into a Nessler tube, and add water to make 50 ml.
Control Solution :-Take 1 ml of aqua regia into a crucible with the same quality
as for the sample, evaporate on a water bath. Proceed as directed in the preparation for the
test solution, take both the filtrate and washings into a Nessler tube, and add the specified
amount of Lead Standard Solution and water to make 50 ml.
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Method 4
Test Solution Weigh the specified amount of the sample, place into a platinum, quartz, or
porcelain crucible, add 10 ml of a solution of magnesium nitrate in ethanol(1:10), and mix.
Ignite and burn the ethanol, and carbonize by heating gradually. Cool, add 1 ml of sulfuric acid,
heat carefully, ignite at 500 600 toincinerate. Moisten with a small amount of sulfuric acid if a
carbonized matter remains and ignite to incinerate. Cool, dissolve the residue with 3 ml of
hydrochloric acid, evaporate to dryness on a water bath. Moisten the residue with 3 drops of
hydrochloric acid, add 10 ml of water, and dissolve by warming. Then, add 1 drop of
phenolphthalein TS, add ammonia TS until the solution becomes slightly red, and transfer
quantitatively into a Nessler tube using water. Add 2 ml of diluted acetic acid (1:20) and water
to make 50 ml. Control Solution Take 10 ml of a solution of magnesium nitrate in ethanol (1:10)
into a crucible of the same quality as for the sample, ignite and burn the ethanol. Cool, add 1
ml of sulfuric acid, proceed as directed in the preparation for the test solution, and take
quantitatively into another Nessler tube. Add the specified amount of Lead Standard Solution,
2 ml of diluted acetic acid (1:20), and water to make 50 ml. If the test solution is not clear, filter
both the test solution and the control solution under the same conditions. (2) Test Unless
otherwise specified, add 2 drops of sodium sulfide TS to each of the test solution and the
control solution, mix thoroughly, and allow to stand for 5 minutes. Then, observe the tubes
from above and from the side against a white background to compare the colors of both
solutions. The color of the test solution is not darker than that of the control solution.
19
Chloride Limit Test
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The Chloride Limit Test is designed to determine the allowable limit of chloride
contained in a sample. Hereinafter in the Monographs, such a specification as not more than
0.041% as Cl (0.30 g, Control solution 0.01 mol/l hydrochloric acid 0.35 ml) indicates that when
determined by weighing 0.30 g of the test substance as the sample and proceeding as directed
in the following procedure, using 0.35 ml of 0.01 mol/l hydrochloric acid in the preparation of
the control solution, the chloride content of the substance is not more than 0.041% as Cl.
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Procedure: Preparation of Test Solution and Control Solution Unless otherwise specified,
proceed as follows: When only the quantity of the sample is specified, measure the specified
quantity of the sample, transfer into a Nessler tube, and dissolve in about 30 ml of water.
Neutralize it with diluted nitric acid (1:10) if the solution is alkaline. Add 6 ml of diluted nitric
acid (1:10) and water to make 50 ml, and use this solution as the test solution. When the
preparation of a sample solution is directed, transfer the sample solution into a Nessler tube,
add 6 ml of diluted nitric acid (1:10) and water to make 50 ml, and use this solution as the test
solution. Measure the specified amount of 0.01 mol/l hydrochloric acid, and transfer into
another Nessler tube. Add 6 ml of diluted nitric acid (1:10) and water to make 50 ml. Use this
solution as the control solution. If the test solution is not clear, filter both solutions under the
same procedure.
(2) Test Unless otherwise specified, add 1 ml of silver nitrate solution (1:50) to the test solution
and to the control solution, mix thoroughly, and allow to stand
for 5 minutes, protecting from direct sunlight. Then observe both Nessler tubes from the side
and from above against a black background, and compare the turbidity. The turbidity
developed in the test solution is not thicker than that of the control solution.
20
in
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PURITY
&
MANAGEMENT
21
Pharmaceutical chemicals: Purity & Management1
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Since the second world war a rapid development of pharmaceutical chemicals, and ultimately
drugs, has made a quatum process. Medicinal chemist, pharmacologist, biochemist, analytical
chemist and medical professionals have paved the way with their single goal objective to
combat the suffering of human being. In this integrated effort the role of an analyst vis-a-vis the
chemical purity of pharmaceutical sciences and drugs made therefrom and the finally the
dosage that are usually available for direct patient’s usage, has become not only extremely
crucial but also equally important and vital. As on date product safety has to be an integral part
of all product research in pharmaceutical substances. However, the risk-benefit ratio has got to
be pegged to a bare minimum level. Therefore, it has become absolutely to lay emphasis on
product safety research and development which is very crucial in all developmental stages of a
new product.
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It is however, pertinent to mention here that pharmaceutical chemicals must maintain a very
high degree of chemical purity. It is quiet obvious that a state of absolute purity may not be
achievable but a sincere effort must be exercised to obtain the maximum freedom from foreign
substances. Bearing in mind the exorbitant operation costs to attain the ‘highest standards’ of
purity, perhaps some of this processes are not economically viable. Therefore, a compromise
has got to be made to strike a balance between the purity of a substance at reasonably viable
cost and at the same time its purity being fully acceptable for all pharmaceutical usages. In
short, a most of impurities in pharmaceutical chemicals do occur that may be partially
responsible for toxicity chemical interference and general instability.
22

BROAD BASED HIGHEST ATTAINABLE STANDARD
NAME OF SUBSTANCE
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
Aspirin
Atropine Sulphate
Bendrofluaride
Betamethasone
Busulphan
Caffeine
Calcium Levulinate
Carbamazepine
Chloramphenicol
Dexamethazone
Ethacrynic acid
Ferrous sulphate
Griseofulvin
Nitrofurazone
Salbutamol sulphate
Thyroxine sodium
STANDARD OF PURITY
(%)
99.5-100.5
98.5-101.5
98.0-102.0
96.0-104.0
98.0-100.5
98.5-101.0
97.5-100.5
97.5-103.0
98.0-102.0
96.0-104.0
97.0-102.0
98.0-105.0
97.0-102.0
97.0-103.0
98.0-101.0
97.0-103.0

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S. NO.
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The standards for pharmaceutical chemicals and their respective dosage forms, as laid down
in various official compendia fulfill broadly the following three cardinal objectives, namely:
(a) Broad based highest attainable standard,
(b) Biological response versus chemical purity, and
(c) Official standards versus manufacturing standards
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BIOLOGICAL RESPONSE VERSUS CHEMICAL PURITY
Though chemical purity is the topmost priority, yet the biological response of
pharmaceutical substance holds an equal importance. A wild variation of active ingredients
ranging between 90% in one sample and 110% (± 10 % limit) in another sample could in
variably be observed. Therefore, it has become absolutely essential to lay down definite
standards so as to ensure that
 Different laboratories may produced reasonably reproducible products.
 Difference in active ingredients in various lots may be minimized.
 Retention of acceptable level of potency.
 Freedom of toxicity during storage before use.
23
OFFICIAL STANDARDS VERSUS MANUFACTURING STANDARDS
During the process of manufacture and unavoidable criterion is the loss of active
ingredients. Therefore all official standards for pharmaceutical chemicals and dosage
forms should accommodate such losses caused due to loss in manufacture,
unavoidable decomposition and storage under normal conditions for a stipulated
period. The official standards, in general, legislate and control the presents of toxic
impurities by prescribe ‘limit tests’ and also by more sophisticated analytical
techniques using thin layer chromatography (TLC), high performance thin layer
chromatography (HP-TLC), gas-liquid chromatography (GLC) and high performance
liquid chromatography (HPLC).
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
DESCRIPTION OF THE DRUG OR THE FINISHED PRODUCT :It may essentially include the following details namely-:o Brand name of the product
o Name of the active ingredients
o Strength of the active ingredient in dosage form
o Lot or batch no
o Date of manufacture
o Date of expiry
o Storage condition (if any)
o Separate dosage for adults and children.
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 MANAGEMENT:Various official compendia of different countries categorically specify descriptive as
well as informative details with regards to the pharmaceutical substances and
formulated dosage forms produced there from. Hence all pharmaceutical chemicals and
finished products must rigidly conform to the laid-out standard in a particular country
and are subjected to various checks at different levels either by government/state
owned drug testing laboratories or by government/state approved drug testing
laboratories.
Official compendia for pharmaceutical substances usually include the following
parameters namely :
 Description of the drug or the finished product
 Identification test
 Physical constants
 Assay of principal active ingredients
 Limit tests
 Storage condition
24
Test For Purity3

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Pharmacopoeias of various countries prescribe ‘test for purity’, for substances which are to be
used for medical purposes. The so called ‘test for purity’< are a matter of fact tests for
detecting impurities in the substances & pharmacopoeias fix the limits of tolerance for these
impurities. The governing factor for these tests, is to determine how much impurity is likely to
be harmful, or to bring about technical & other difficulties, when the substance is used.
Pharmacopoeias do not aim at ensuring freedom every possible impurity in a substance, but to
test for few major impurities, which are likely to interfere in their use. Certain tests which are
carried out on the substances are:
Colour, odour and taste
Along with other tests for purity, description of taste, odour, colour etc., are given in the
pharmacopoeias. Though they have limited value they are useful in the determining whether
the substance is reasonably pure, hygiene etc.

Physico-chemical constants
Acidity, Alkalinity,& pH
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Solubility of the substance in various solvents, determimation of melting & boiling points for
organic substances, optical rotation for optically active substances & refrective index for
liquids, are some values which tell us about the purity of substance. Determinationof the acid
value, iodine value, saponification value, acetyl value, ester value etc.,for vagetable oils are
general constants & variation in their value, signifies the presence of impurities. The extent
of the variation in these values, usually depends upon the nature & extent of impurities present
in the substances. However, a very low concentration of impurities, may fail to alter these
constants, & thus remain undetected, unless tested specifically, by special tests.
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Substances that are prepared from chemical reactions involving acids & alkalies often
considerable amounts of the acid or alkali, as an impurity. Thus, the tests for Acidity &
Alkalinity are of a great help to estimate the impurity. Furthermore, solutions of certain
sudstances have a definite pHat a given concentration. The presence of an impurity will bring
about a change in the pH & thus can be detected.
25

Anions & Cations

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A large number of synthetic drugs both inorganic & organic is prepared using strong
acids like hydrochloric, sulphuric, nitric etc. The presence of chloride & sulphate ions are thus
common impurities. Test for these ions(anions) is thus generally carried out. Simmilarly tests
for sodium, ammonium(cations) are often carried out to detect impurities in the inorganic
compounds.tests for heavy metals, like lead, iron, copper, mercury are carried out as are very
common impurities in substances.
Insoluble residues
Pure substances give a clear solution in a given solvent. When insoluble
impurities are present in a substance then the solution appears cloudy, or shows opalescence.
The measurment of turbidity or opalescence helps to determine the amount of insoluble
impurity present in the substance. If the insoluble residue is high then this can be determined
by filtering & weighing the insoluble residue.

Ash, Water insoluble ash
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Determination of ash in crude vegetable drugs, organic compounds & some
inorgantc compounds, gives a good indication about the extent of impurities of heavy metals or
minerals in nature. This determination is thereforecommonly employed for anature of
substances. In certain cases, water-insoluble ash is also determined to find water-soluble
heavy metals or minerals type of impurity.
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It is thus clear that depending upon the type of material or substance, Pharmacopoeias
prescribe tests for purity of particular nature; e.g. salicylic acid in acetyl salicylic acid, phena
tidine in phenacetin, acraldehyde in glycerine, Paminophenol in paracetamol etc. In general, it
could be said that, impyrities of chloride , sulphate, iron, heavy metals, lead & arsenic, are
common in drugs & chemicals, Pharmacopoeias of various countries, therefore prescribe limit
for these to be carried out by a particular method
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USP/NF CHEMICAL AND PHYSICAL TESTS4
Northview performs USP/NF chemical and physical tests on a wide variety of raw materials
and finished products. Some of the more commonly performed tests are listed below. We
perform many more tests that are not listed. ACS, BP, EP and JP compendial procedures are
also available. All testing is performed in strict accordance with procedure details.
26
3949
3101
Acid Value
Alcohol Assay – Gas Chromatography
– 1 sample
3113
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3117
3114
3115
3116
3918
3915
3960
3121
3107
3917
3959
3916
3911
3964
3919
3920
3325
3934
3966
4114
4116
4115
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3930
3906
3110
3111
3112
Alginates Assay
Arsenic Limit Test – Method I
Arsenic Limit Test – Method II
Container Tests
Conductivity
Congealing Temperature
Disintegration Test – uncoated or plain coated tablets
Disintegration Test – other tablets and capsules
Dissolution – Stage I, Spectroscopy, 6 kettles
– initial assay, minimum
– additional assays
Dissolution – Stage I, HPLC, 6 kettles
– initial assay, minimum
– additional assays
Distilling Range
Metals – Method I
Heavy Metals – Method II
Heavy Metals – Method III
Hydroxyl Value
Iodine Value
Iron
Lead Limit Test – with sample digestion
Lead Limit Test – without sample digestion
Loss on Drying
Loss on Ignition
Melting Point or Range – Class I or Ia
Melting Point or Range – Class II
Methoxy or Ethoxy Determination
Nitrogen – Kjeldahl – Method I
Nitrogen – Kjeldahl – Method II
Nitrogen Gas Testing
Optical Rotation
Ordinary Impurities
Organic Volatile Impurities – Method I
Organic Volatile Impurities – Method IV
Organic Volatile Impurities – Method V
Particulate Matter in Injections – Light Obscuration Method
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3958
3102
3103
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– additional samples, concurrently submitted
3128
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3921
3922
3976
3924
3925
3928
3129
3933
3980
3941
3942
3970
3971
3972
3131
3143
in
– liquids (per sample or sample composite)
– powders (per sample or sample composite)
sample composition for particulate analysis
Particulate Matter in Injections – Microscopic Method
– 1 sample
– 2+ samples
Particulate Matter in Water
Peroxide Value
Ph
Readily Carbonizable Substances
Refractive Index
Residue on Ignition
Saponification Value
Selenium Limit Test
Specific Gravity
Total Organic Carbon (TOC)
Viscosity – Brookfield
Viscosity – Capillary
Water – Method I (Karl Fischer)
Water – Method II (Azeotropic – Toluene Distillation)
Water – Method III (Gravimetric)
USP Purified Water Monograph (conductivity and TOC)
USP Water for Injection Monograph (conductivity and TOC)
3125
3148
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 GENERAL ANALYTICAL TECHNIQUES
o Chromatography
o Spectrophotometry And Spectroscopy
 I.
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METHODS FOR EVALUATING APPEARANCE AND PHYSICAL PROPERTIES
o Boiling point and distillation range
o Colour (platinum-cobalt hazen scale)
o Melting range
o Ph determination
o Refractive index
o Solidificaton point
o Specific gravity
o Specific rotation
 II.
28
METHODS FOR DETERMINING INORGANIC COMPONENTS
o Acid-insoluble matter
o
o
o
o
o
o
o
o
o
METHODS FOR DETERMINING ORGANIC COMPONENTS
o Aromatic Hydrocarbons Determination (Based On Astm D 2267-67.)
o Carbon Dioxide Determination By Decarboxylation
o Chlorinated Organic Compounds Limit Test
o 1,4-Dioxane Limit Test
o Ethoxyl And Methoxyl Group Determination
o Gum Constituents Identification
o Maleic Acid Limit Test
o Oxalate Limit Test
o Readily Carbonizable Substances
o Reducing Substances (As Glucose)
o Residual Solvent
o Residual Solvent Limit Test
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 III.
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o
o
o
in
o
o
o
o
Ash
Chlorides limit test
Fluoride limit test(food chemicals codex, 3rd edition, national academy press
(1981).)
Loss on drying
Loss on ignition
Metallic impurities
Instrumental methods (tentative)
 Measurement of antimony, barium, cadmium, chromium, copper, lead and
zinc by atomic absorption
 Measurement of arsenic and antimony by atomic absorption hydride
technique
 Determination of mercury by atomic absorption cold vapour technique
Arsenic limit test
 Method i (gutzeit procedure)
 Method ii (colorimetric procedure)
Chromium limit test
Heavy metals limit test
Iron limit test
Lead limit test
Mercury limit test
Nickel limit test
selenium limit test
nitrogen determination (kjeldahl method)( iso r-937-1969 may be used as an
alternate method.)
non-volatile residue
sulfates limit test
water determination (karl fischer titrimetric method)
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o
o
 IV.
29
METHODS FOR FOOD COLOURS
Chloride As Sodium Chloride Determination
Chloroform Insoluble Matter
Colouring Matters
 Total Content By Spectrophotometry
 Total Content By Titration With Titanous Chloride
 Method Of Assay Of Certain Food Colours (Tentative “Comments Are
Invited On The Use Of This Method As An Alternative”)
 Subsidiary Colouring Matter Content
 Determination By Paper Chromatography
 Identification And Rapid Limit Test By Thin-Layer Chromatography
(Tentative*)
Ether Extractable Matter
 Method I
 Method Ii
Hydrochloric Acid Insoluble Matters In Lakes
Leuco Base In Sulphonated Triarylmethane Colours
Organic Compounds Other Than Colouring Matters
 Determination By Liquid Chromatography
 Determination By Column Chromatography
Sulfate As Sodium Sulfate
Unsulphonated Primary Aromatic Amines
Water Content (Loss On Drying)
Water Insoluble Matter
Water Soluble Chlorides And Sulfates In Aluminum Lakes
o
o
o
o
o
o
o
o
o
METHODS FOR ENZYME PREPARATIONS
o General Specifications And Considerations For Enzyme Preparations Used In
Food Processing
o Alpha-Amylase Activity, Bacterial
 Bacterial Alpha-Amylase Enzymes Used In Desizing (Atcc Test Method
103-19701, Atcc Tech. Manual, 264 (1970))
o Alpha-Amylase Activity, Fungal
o Alpha-Amylase Activity, Malt (Amer. Soc. Brewing Chemistry, 6th Ed., 169
(1958))
o Antibiotic Activity
o Catalase Activity
o Cellulase Activity
o Ethylenimine Limit Test
o Glucoamylase Activity (Amyloglucosidase Activity)
o Beta-Glucanase Activity
1
o Glucose Isomerase Activity
o Glucose Oxidase Activity
o Glutaraldehyde Limit Test
o Glutaraldehyde Determination In High Fructose Corn Syrup
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 V.
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o
o
o
30
METHODS FOR FATS AND RELATED SUBSTANCES
o Acid Value
o Congealing Range
o Free Fatty Acids (Based On Aocs “Aocs: American Oil Chemists' Society.”
Method Ca 5a-40)
o Hydroxyl Value
o Identification Tests For Functional Groups
 Fatty Acids Upon Hydrolysis (A)
 Acetic Acid (B)
 Succinic Acid (C)
 Fumaric Acid (D)
 Tartaric Acid (E)
 Citric Acid (F)
 Lactic Acid (G)
 Glycerol (H)
 Polyols
o Iodine Value (Wijs Method)
o 1-Monoglyceride And Free Glycerol Contents
o Oxyethylene Group Determination
o Polyglycerol Determination In Polyglycerol Esters
o Propylene Glycol Dimer And Trimer Determination
o Saponification
o Saponification Value
o Sorbitan Ester Content
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 VI.
Hemicellulase Activity
Milk Clotting Activity
Protease Activity, Viscometer
Proteolytic Activity, Bacterial (Pc)
Proteolytic Activity, Fungal (Hut)
Proteolytic Activity, Fungal (Sap)
Proteolytic Activity, Plant
Pullulanase Activity
in
o
o
o
o
o
o
o
o
METHODS FOR FLAVOURING SUBSTANCES
o Acetal Determination
o Acid Value
o Aldehyde Determination
o Aldehyde And Ketone Determination
o Chlorinated Compounds Limit Test
o Ester Determination
o Solubility In Ethanol
o Total Alcohols Determination
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 VIII.
31
METHODS FOR PHOSPHATES
o Cyclic Phosphate Determination
IDENTIFICATION TESTS
 Acetate
 Aluminum
 Ammonium
 Benzoate
 Bicarbonate
 Bisulfite
 Bromate
 Bromide
 Calcium
 Carbonate
 Chloride
 Citrate
 Copper
 Ferrocyanide
 Iodide
 Iron
 Lactate
 Magnesium
 Manganese
 Nitrate
 Nitrite
 Peroxide
 Phosphate
 Potassium
 Sodium
 Sulfate
 Sulfite
 Tartrate
 Thiosulfate
 Zinc
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 X.
MICROBIOLOGICAL METHODS
o Total (Aerobic) Plate Count
o Coliforms And E-Coli
o Salmonella
o Enumeration Of Yeasts And Moulds
o Media And Reagents
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 IX.
Phosphate Determination As P205
Water-Insoluble Matter
 XI.
32
in
o
o
STANDARD BUFFER SOLUTIONS
o BUFFER TEST SOLUTIONS
 Buffer TS (pH 2)
 Buffer TS (pH 5)
STANDARD SOLUTIONS
 Ammonium Standard Solution
 Barium Standard Solution
 Barium Chloride Standard Solution
 Chromium Standard Solution
 Condensed Formaldehyde Standard Solution
 Dithizone Standard Solution
 Formaldehyde Standard Solution
 Iron Standard Solution
 Lead Standard Solution
 Lead Standard Solution for Dithizone test
 Magnesium Standard Solution
 Mercury Standard Solution
 Methanol Standard Solution
 Nitrate Standard Solution
 Phosphate Standard Solution
 Potassium Phosphate, Monobasic, Standard Solution
 Selenium Standard Solution
 Thiamine Hydrochloride Standard Solution
 Zinc Standard Solution
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 XII.
STANDARD BUFFER SOLUTION
 Reagent Solutions
 Composition of Standard Buffer Solutions
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 Buffer TS (pH 5.45)
 Buffer TS (pH 6.5)
 Buffer acetate TS (pH 5.0)
 Barbital buffer solution (pH 7.6)
 Citric acid buffer solution
 Formic acid buffer solution (pH 2.5)
 Phosphate buffer solution (pH 7.0)
 Phosphate buffer solution (pH 7.5)
Bioburden Testing4
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Products or components used in the pharmaceutical or medical field require control of
microbial levels during processing and handling. Bioburden or microbial limit testing on these
products prove that the requirements are met.
Microbial limit testing of raw material as well as finished pharmaceutical products can
help to determine whether the product complies with the requirements of the BP, Ph. Eur. or
33
USP. Bioburden testing of components can show the use of adequate control measures during
the preparation and handling.
in
The satisfactory criteria for the microbial quality of pharmaceutical preparations are
specified in the relevant Pharmacopoeia. Guidelines are also given for the carrying out of
testing. All our methods are UKAS accredited and based on the Ph. Eur. The Medical Devices
Agency advises on the microbiological safety aspects of medical components.
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Pharmaceutical product types range from powder to oil and include creams, oral syrups
and capsules, in fact anything that is included in of the Ph. Eur. Components range from small
medical implants to devices used for the delivery of pharmaceutical products and may be
composed of metal or plastic.
34
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Analytical Procedure
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Analytical Procedure10
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Concepts about purity change with time and are inseparable from developments in analytical
chemistry. If a material previously considered being pure can be resolved into more than one
component, that material can be redefined into new terms of purity and impurity.
Monographs on bulk pharmaceuticals chemicals usually cite one of three types of purity
tests:
1. Chromatographic purity test coupled with a non-specific assay;
2. A chromatographic purity indicating method that serves as assay; or
35
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3. A specific tests and a limit for known impurity, an approach that usually require the
reference standards for impurity the modern separation methods clearly place the
dominant role in scientific research today because this method simultaneously separate
and measure components and fulfill the analytical ideal of making measurements only
on purified specimens. Nevertheless, the more classical method based on tritrimetry,
colourimetry, spectrophotometry, single or multiple partitions or changes in physical
constants lose none of there previous validities.
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The analytical procedures are validated and suitable for the detection and quantitation of
impurities. Organic impurity levels can be measured by a variety of techniques, including those
that compare an analytical response for an impurity to that of an appropriate reference
standard or to the response of the drug substance itself. Reference standards used in the
analytical procedures for control of impurities should be evaluated and characterized according
to their intended uses.
It is considered acceptable to use the drug substance to estimate the levels of impurities
when the response factors of the drug substance and impurities are close. In cases where the
response factors are not close, this practice may still be acceptable, provided a correction
factor is applied or the impurities are, in fact, being overestimated. Analytical procedures used
to estimate identified or unidentified impurities are often based on analytical assumptions (e.g.,
equivalent detector response).
Advantages of analytical procedures:
Automated, fast and sensitive detection of impurities
Improved detection certainty of key impurities
Higher analytical productivity
Improved product at lower cost
Identification Tests1:-
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


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The true identification of a drug of ways, namely :Determination of physical constants, chromatographic tests and finally the chemical tests.
The physical constants essentially include:



36
The melting point,
The boiling point,
Refractive index,
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Weight per milliliter,
Specific optical rotation,
Light absorption,
Viscosity,
Specific surface area,
Swelling power,
Infra-red absorption,
Sulfated Ash,
Loss on Drying,
Clarity and colour of solution,
Heavy metals.
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










The chromatographic tests include specific spot-tests by Thin-layer chromatography (TLC) of
pure drug or its presence in a multi-component system.
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However, the most specific and reliable are the chemical tests which may be categorized
separately under tests for inorganic substances and organic substances.
Analytical Procedure:-
Generally impurities are identified by:
Thin layer chromatography (TLC)

High performance thin layer chromatography (HPTLC)

Infra-red (IR) spectroscopy

Mass spectroscopy(MS).
37
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
Thin layer chromatography (TLC)(11,12): The adsorbent used in TLC is a thin, uniform layer (normally 0.24mm thick) of a dry, finely
powdered material applied to an appropriate support, such as a glass plate or an aluminum
sheet or a plastic foil. Subsequently, the mobile phase is permitted to move across the surface
of the plate (usually by capillary action) and the chromatographic phenomenon may soley
depend upon adsorption, partition, or a combination of both, depending on adsorbent, its
treatment, and the nature of solvents employed. During the chromatographic separation
procedure the TLC plate is placed in a chromatographic chamber, mostly made up of glass to
enable clear observation of the movement of the mobile phase of the plate that is presaturated with solvent vapour. The inert solid supports invariably employed are, namely :
alumina, silica gel, kieselghur and cellulose, to these may be added appropriate substances,
for instance : calcium sulphate (gypsum) so as to provide adequate adhesion to the solid
support, example : silica gel G. The prepaid layer may be impregnated with suitable materials
to achieve specific purpose, namely :
1. Buffering materials
2. Silver nitrate
3. Ionexchange materials
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APPLICATION OF TLC IN PHRMACEUTICAL ANALYSIS: To Identify the presence of undesirable specific organic compounds present
as impurities in a number of pharmaceuticals substances, namely: morphine
in apomorphine hydrochloride; hydrazide in carbidopa; 3-aminopropranol in
dexapanthenol; etc.
 Related substances present in official drugs, namely :- related substances
present in a wide number of potent pharmaceuticals substances e.g.
aminophylline, baclofen, chloramphenicol, carbamazepine.
 Foreign alkaloids present in alkaloidal drugs, for instance: atropine sulphate,
38

High Performance Thin Layer Chromatography (HPTLC)13
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
codeine
Foreign steroids present in steroidal drugs for example: betamethasone
valerate.
Ninhydrin positive substances in official amino acids e.g. glutamic acid,
leucine.
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HPLC is a versatile separation technique and is official in most of the pharmacopoeias
for determining content uniformity, purity profile, assay values and dissolution rates in unlimited
number of monographs. It is precisely for these reasons that almost every laboratory today is
equipped with HPLC system. However, it cannot be denied that more than often, the systems
are working beyond their capacities and mostly dedicated. Who would like to change a wellrunning stabilized column and prepare fresh solutions only because few assorted samples
even through urgent are required to be analyzed. Analyst usually has the tendency to wait until
large number of similar samples are accumulated, even risking the product development work
because of non-availability of analytical results.
The HPTLC, the answer in such situation. It can simultaneously handle several samples
even of divergent nature and composition supporting several analysts at a given time. HPTLC
is the most simple separation technique today available to the analyst. It can be considered a
time machine that can speed your work and allows you to do many things at a time usually not
possible with other analytical techniques.
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Various steps involved in TLC/HPTLC/planar chromatography
 Selection of TLC/HPTLC plates and sorbent
 Sample preparation including any clean up and pre-chromatographic derivatization
 Application of sample
 Development (separation)
 Detection including post-chromatography derivatization
 Quantitation
 Documentation
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TLC/HPTLC is often found more troublesome than GLC/HPLC as quantitative TLC is an offline technique, hence automation is difficult and because of its open character, is highly
influenced by environment factors. It is, therefore, essential that each step which may require
specific approach must be carefully validated to determine potential source of error.
Sample and standard preparation
39
Selection of chromatography layer
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Layer pre-washing
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Layer pre-conditioning
Application of sample and standard
Chromatographic development
Detection of spots
Scanning and documentation of chromatoplate
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Fig- Schematic procedure for HPTLC
40
Story of TLC/HPTLC
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 Applications of HPTLC: -
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1. Identification:o HPTLC is used for identification of compounds.
o Relative method of identification uses standard compound along with
Unknown or test solution.
o Test is identified by comparison with the spots of standard
o Absolute method used ratio front(Rf) or RX value for identification RF or Rx values are
constants for compound.
o Eg. Amiloride HCL IP’96 is identified using HPTLC using Relative method of
identification spot in developed using U.V.light.
41
o Eg. Cyproheptadene HCL IP’96 is identified using HPTLC using Relative method of
identification.
o Eg. Ibuprofen HCL IP’96 is identified by HPTLC
o Eg. Doxycyline HCL IP’96 is identified by HPTLC
Infra-red (IR) spectroscopy14
u.
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2. Limit tests of Impurities:o TLC is widely used to find out type and conc. of impurities present in a drug.
o We can detect number of impurities by a single analysis using HPTLC & so
Pharmacopeia prescribes HPTLC to detect impurities.
o Previously Pharmacopoeia used to give name of impurities now it gives or states ‘Limits
of related impurities to be measured by HPTLC’
o Eg: Amodiaquine HCL – impurities of related substances is determined by HPTLC.
o Alprazolam is analysed for amount of related impurities by HPTLC.
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Principle:- The infra-red spectrum provides the largest number of characteristic properties of a
compound. It also serves as a powerful analytical tool for the extensive and intensive study of
molecular structure. The underlyling principle Infra-red (IR) spectroscopy is based upon the
molecular vibrations which is further compose of the stretching and the bending vibrations of
the molecule.
The vibrations of molecules are of two types, namely:
a) stretching
b) bending
42
Stretching:- Vibration causes stretching where by the distance between the two atoms
increases or decreases, but the atoms remain in the same bond axis.
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Bending:- Vibration causes bending where by the position of the atom charges relative to the
original bond axis.
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 APPLICATION OF INFRA-RED (IR) SPECTROSCOPY IN PHRMACEUTICAL
ANALYSIS:* A host of pharmaceuticals substances can be identified and critically examined with
the help of IR spectroscopy. Hence the latest version of British Pharmacopeia (BP) and United
states Pharmacopia (USP) contain the complete IR spectrum of such pure pharmaceuticals
substances that are essentially included in the respective official compendium. This authentic
IR spectra are profusely used in many well-equipped Quality Assurance Laboratory in
checking the purity of commercial available drugs before employing them in various
formulations.
Examples of compounds for which the analytical assays are done by IR spectroscopy
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Ampicillin
sodium
Amylobarbitone
Betamethasone
Carbenicillin
Chloroquine
43
Colchcine
Mebendazole
Rifampicin
Dexamethasone
Erythromycin
Ethambutol
hydrochloride
Ethirylestradiol
Methotrexate
Nalidixic acid
Niclosamide
Spironolactone
Sulphamethizole
Thiabendazole
Nitrofurantoin
Ethiosuimide
Nitrofurazone
Primidone
Clofibrate
Fludrocortisone
Acetate
Fluphenazine
Clofazimime
Clonidine
hydrocholide
Ibuprofen
Lincomycin
hydrochloride
Progunil
hydrochloride
Pyrazinamide
Pyrimethamine
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phosphate
Chloroquine
sulphate
Cemetidine
 APPLICATION OF INFRA-RED (IR) SPECTROSCOPY IN ANALYSIS OF
PHRMACEUTICAL DOSAGE FORMS: Determination of Aspirin, Phenacetin, and caffeine in tablets
 Determination of Codeine phosphate in tablets
 Determination of Meprobamate in tablets
 APPLICATION OF INFRA-RED (IR) SPECTROSCOPY IN ANALYTICAL CHEMISTRY: Determination of Cis-trans isomers ratio in clomiphene citrate
 Distinguish and characterize the pri-,sec-,and tert-amine salts from one another
 Infra-red (IR) spectroscopy in the study of complex formations
(a) Ninhydrin forms blue complex with amino acid
(b) 1:10 – phenanthrolin reacts with Fe2+ ion quantitatively to give rise to deep red
complex.
 IR spectroscopy in the identification of functional groups
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Mass Spectroscopy (MS)15: MS studies the arrangement of mass in different compounds. Molecules subjected to
bombardment by high energy electrons. High energy electrons have energy of about 70 eV. 1
eV = 23 Kcal / mole. Strength of normal covalent bond is 100Kcal /mole . and so 4-5 eV energy
Better
Obtain most
is sufficient to break normal covalent
bond molecule is converted
into molecular ion by
sample
Appropriate sample
breaking a ionization.
Run LC-MS
Reproduce chromatography
Obtain mol mass and fragmentation
Confident
Answer
change
conditions
no
Yes
Compare with parent
Drug substance
44
Simple
difference
Consult with
Colleagues
Identify additional
no
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Yes
no
no
Propose
Synthesis
Synthesize
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Yes
no
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Yes
Unknown Degradant/impurity
Fig – Mass spectrometry
process
flow chart for identification
Problem
Identified
Of Impurities11
in
no
Discuss w/
Project Team
Contact Degradation/MS/NMR
Groups Access Timeliness for
Completion
<0.1%
Impurity
Level ?
>0.1%
HPLC/UV
LC/MS
PRI/Deg.STD
RRT Match ?
45
Develop LC-MS
MS Compatible
MS Data ?
Confirm
Structure
No
No
Confirm Structure
(MS and RRT)
Yes
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No
Standards?
Yes
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No
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Discuss possible structures
with project team. Determine
if information is suitable or if
isolation is required.
No
Degradant/
Impurity?
Evaluate
Process Stage
Scale –Degardant
up
Degradation
Drug Substance or
Drug Product?
Yes
Isolate
Degradant/Impurity
For NMR Studies
Synthesize
Mother Liquor
Sample?
Reasonable
Synthesis?
No
Must be most
Efficient route
Obtain Bulk
Fig – Impurities /Degradant isolation and identification process flow chart. PRI, processrelated impurity; STD, standard; RRT, relative retention time; MW, molecular weight. 9
46
47
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Validation
Validation of Pharmaceutical Test Method16
gn
Analytical method validation is a matter of establishing documented evidence that
provides a high degree of assurance that the specified method will consistently provide
accurate test results that evaluate a product against its defined specification and quality
attributes. The requirements of validation have been clearly documented by regulatory
authorities the approach to validation is varied and open to interpretation. The following
approach will focus on the International conference on Harmonization (ICH) guidelines.
48
in
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Return to
Method Development
49
Completed Method Development
Documented evidence that the developed Method
1) Proven to be robust
2) Adequate system suitability parameters
3) Successfully completedMethod
pre-validation experiments
Transfer
4) Satisfied “Customer labs”
that method meets objectives
To
Control
Laborator
Categorize Validation Requirements
Generalized Validation Flowchart.
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Fig- Generalized
Flow by
chart
Analysis need to ensure the accuracy and reliability of theValidation
data generated
their test
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methods as show in Above figure, there are required and fundamental controls that ensure the
overall quality of the analytical test data.
50
Properly
developed
and robust
methods
Data from
accurate and
reliable analytical
methodology
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Validated
methodologySystem
suitability
Certified
reference
standards
in
Properly
executed
method
transfers
Qualified and
trained
laboratory
analysts
Qualified and
calibrated
Laboratory
Instruments
Accurate
reporting and
recording of
data
VALIDATION TERMINOLOGY DEFINITIONS :
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II]
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Unified elements that ensure reliability of data from analytical methodology
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It is important to define the terms used in regulatory guidelines when discussing method
validation.

51
ACCURACY: The accuracy of an analytical procedure expresses the closeness of
agreement between the value which is accepted either as a conventional true value or
an accepted reference value and the value found.
PRECISION: The precision of an analytical procedure expresses the closeness of
agreement between a series of measurements from multiple sampling of the same
homogeneous sample under prescribed conditions. Precisions may be considered at 3
levels: Repeatability, intermediate precision, and reproducibility.

REPEATABILITY: Repeatability expresses the precision under the same operating
conditions over a short interval of time.

INTERMEDIATE PRECISION: Intermediate precision expresses within laboratories
variations: Different days, different analysts, different equipment, etc.,

REPRODUCIBILITY:

SPECIFICITY:
Specificity is the ability to assess unequivocally the analyte in the
presence of components, which may be expected to be present.

DETECTION LIMIT: The detection limit of a individual analytical procedure is the lowest
amount of analyte in a sample which can be detected but not necessarily quantities as
an exact value.

QUANTITATION LIMIT:
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
Reproducibility expresses the precision between laboratories.
The quantitation limit of an individual analytical procedure is
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the lowest amount of analyte in a sample, which can be quantitatively determined with
suitable precision and accuracy.
LINEARITY: The linearity of an analytical procedure is its ability (within a given range)
to obtain test results, which are directly proportional to the concentration (amount) of the
analyte in the sample.
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

52
RANGE:
The range of an analytical procedure is the interval between the up per
and lower concentration (amounts) of the analyte in the sample (including these
concentrations) for which it has been shown that the analytical procedure has a suitable
level of precision, accuracy, and linearity.
ROBUSTNESS:
The robustness of an analytical procedure is the measure of its
capacity to remain unaffected by small, but deliberate various in method parameters
and provides an indication of its reliability during normal usage.
RUGGEDNESS:
The degree of reproducibility of test results obtained by the analysis
of the same sample under a variety of normal test conditions such as different it is a
measurer of reproducibility of test results under normal expected operational conditions
from laboratory to laboratory and from analyst to analyst.

SENSITIVITY:
The sensitivity of an analytical method is equal to the slope of the
calibration line in a linear system.
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
For general HPLC assay / purity methods the following validation parameters will typically
gn
be evaluated:
53

Specificity

Linearity

Accuracy

Range

Precision

Limit of detection / quantitation.

Solution stability (recommended)
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(repeatability intermediate reproducibility)
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Start
54
1)
2)
3)
4)
5)
6)
1)
2)
3)
4)
Approval Protocol
Scope
Responsibilities
Define reagents and working solutions
Procedures
Acceptances criteria
QA & Management Approval
Define Method Specificity
Degrade Samples
Collect “active peak”
Re-inject on non correlated system
On-Line analysis using LC-MS or Diode
Array UV
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VALIDATON REQUIREMENTS OF THE METHOD:
gn
A]
NON COMPENDIAL METHODS
In developing HPLC methodology these validation requirements stipulated that stability
indicating impurity methods be designed and validated to:
[1]
55
Simultaneously separate identify and quantity degradates/ impurities from the “active”
drug substance.
[2]
Be free from interference from the excipient materials.
On the other hand content uniformity and dissolution methods may not be required to be
Stability indicating because separation of the active compound and impurities may be
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through separate validated methods.
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determined not to be critical to these tests, particularly impurities are determined
B]
COMPENDIAL ANALYTICAL PROCEDURES:
It is important for all compendia analytical methods that each individual
laboratory performs a scaled back validation of the method of verification of the
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methods suitability in its laboratory.
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VALIDATION DOCUMENTATION:
The validation documentation typically consists of a protocol, test data and a final
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report. This protocol may have data tables to enter the test results, requiring only a
short executive summary to summaries the results and a reference or attachment of raw
data.
Validation protocol:
56
Method principle / objective.
2]
List of responsibilities [laboratories involved and their role in the validation]
3]
Method categorization according the ICH or USP.
4]
List of reagents [including test lots] and standards required.
5]
Test procedures to evaluate each validation parameter and proposed acceptance
criteria.
Plan or procedure when acceptance criteria are not met.
7]
Requirements for the final report.
APPENDIXES
:
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6]
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1]
Method development report
2]
Method procedure.
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1]
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VALIDATION EXPERIMENTATION:
Depending on the requirements of the validation there can be a preferred order to
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efficiently perform the validation experiments.
1]
DETERMINATION OF METHOD SPECIFICITY
Specificity is one of the most important characteristics of a “stability indicating” method a
and should be determined as one of the first validation items. A specific method can accurately
57
measure the analyte of interest even in the presence of potential sample components (place of
ingredients, impurities, degradation products etc) when criteria for specificity are not met, this
often indicates that the method is not sufficiently developed furthermore it is likely that criteria
for accuracy precision and linearity may also not be fulfilled. A major objective of determining
in
specificity is to ensure “ Peak Purity” of the main compound to be determined, in other words,
confirm that no related compound or product ingredient correlates and intervals with the
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measurement of the assayed. Compound stressed stability samples are often specified in
validation protocols to evaluate purity, in addition the ICH outlines two approaches to further
evaluating method specificity for when impurities are and are not available.
A]
WHEN IMPURITIES ARE AVAILABLE:
Knowledge of degrading and synthetic impurities can be derived from the historical
information that has accumulated for the drug substance/product. Ideally a library of impurity
and degradation compound reference standards is synthesized and characterized and
sufficient quantities are made available. These compounds can be spiked in to sample matrix
(Placebo) to determine if the matrix interferes with the quantitation of the compound (s) of
interest.
WHEN IMPUIRITIES ARE NOT AVAILABLE:
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[B]
When impurities are not available to check method specificity on e federal guideline
defines several conditions under which various drug substances/product types should be
in
stressed to support the suitability of the method. Depending on the matrix and packaging these
include extremes of acid and heat and high oxygen exposure and light exposure in the case of
drug substances, heat (50 C) light (600fc) acid (0.1 N HCL) and oxidant (3% H2O2) are often
u.
used for drug products heat light humidity (85%) are used as stress conditions. Analytic peaks
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are evaluated for peak purity upon sufficient stress to effect 10 -15% degradation.
[2]
EVALUATION OF PEAK PURITY:
The peak purity in degraded or spiked samples should be determined by using specific
detection techniques, such as diode array UV or HPLC – MS. A less direct, but perhaps more
58
persuasive approach is to isolate the peak of interest and re-inject on a chromatographic
system that based on different “ Non Correlated “ separation mechanism. Capillary
electrophoresis (CE) has also been used as a non-correlated analytical technique to evaluate
[3]
in
peaks isolated from reversed – phase methodology.
DEMONSTRATION OF LINEARITY AND RANGE: DETERMINATION OF RELATIVE
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RESPONSE FACTOR.
Linearity is the ability to obtain results that are directly or indirectly proportional to the
concentrations of a substance in a sample within a given range.
[a]
Linearity of the active component: -
The linearity can be demonstrated by analyzing five or more; concentrations the
active compound in the presence of matrix: For example, 50 %, 75 %, 100 %, 125 %
and 150 % of the normal concentration for a stability – indicating method.
[b]
Linearity of the related compounds: -
The Linearity should be demonstrated by analyzing five concentrations in the
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presence of the matrix: at LOQ (Limit of quantitation) at the specification level, at an
upper level above specification, and at two
intermediate concentrations
DETERMINATION OF DETECTION AND QUANTITAION LIMITS: -
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[4]
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(e.g. 0.1 %, 0.25 %, 0.55 %, 0.75 % and 1.0 %)
LOD = 3.3 X Std. Error
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Slope
59
LOQ = 10 X Std. Error
Slope
In Pharmaceutical analysis of the active drug substance, the target value for the LOQ is
typically set at 0.05 %.
[5]
DEMONSTRATION OF THE ACCURACY OF THE METHOD: The accuracy is usually examined by determination of the trueness of a rest
of test results and true result or an accepted reference value.
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result, which is the closeness agreement between the average value of a large number
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The accuracy of a method can be determined by performing recovery
experiments implementing standard addition calibration procedures testing reference
materials etc., it is also possible to compare the test results of a new method with those
of an existing fully validated reference method through loss validation experiments.
Accuracy in often determined by recovery studies in which the analyses are
DETERMINATION OF METHOD PRECISION:
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[6]
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spikes in to a solution containing the matrix.
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The precision of a method may be considered at 3 levels: -

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Injection Repeatability: If indicates the performance of the HPLC instrument using the
chromatographic condition on one particular day and in one lab.

Analysis Repeatability: If expresses the precision under the same operating conditions
over a short interval of time.
Intermediate Precision: If expresses the effects of random events on the precision of
the analytical procedure within the same lab. The procedure requires repeating the
analysis of one technician by a qualified second technician on a different instrument
using different material on a different day.
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
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Example Acceptance Criteria for an HPLC Assay/purity
Linearity
Correlated coefficient
Y intercept (relative to
the active or related
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Para
meter
Limit (active
ingredient)
Limit (related
compounds)
>0.99
>0.98
2%
3.0%
Linear
15.0%
10.0%
Linear
10.0%
5.0%
Linear
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>0.99
96.0-104.0%
98.0-102.0%
50.0-150.0%
70.0-130.0%
80.0-120.0%
90.0-110.0%
50.0-150.0%
70.0-130.0%
80.0-120.0%
90.0-110.0%
RSD
1.0%
2.0%
3.0%
RSD
25.0%
15.0%
10.0%
5.0%
RSD
25.0%
15.0%
10.0%
5.0%
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compound)
RSD response ratios
Visual
Linearity over the whole range
Correlated coefficient
Y intercept (relative to
the active)
RSD response ratios
Visual
Accuracy
Accuracy ingredient
Recovery of each
over the whole range
Mean recovery per
Concentration
Related compounds
Mean recovery
0.05%x<0.1%
0.1%x<0.5%
0.5%x<1.0%
1.0%
Precision
Active ingredient
Injection repeatability
Analysis repeatability
Intermediate precision
Related compounds (analysis
Repeatability)
0.05%x<0.1%
0.1%x<0.5%
0.5%x<1.0%
1.0%
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REVALIDATION:
According to the validation life cycle test methods may require additional validation or
revalidation when regulatory agencies issue new requirements or when changes are made to
the methodology. Method changes and additional validation activities may be required when
there are.
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[2]
Product changes
[3]
Method modifications
[4]
Analyst changes
[5]
Outdated technology
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Instrument changes
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[1]
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Summary
Summary: -
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Inorganic, organic, biochemical, isomeric or polymeric components can all be considered
impurities. Microbiological species or strains are sometimes described in similar terms of
resolving into more than one component.
There should be a 0.1 percent threshold above which isolation and characterization of
individual impurities should apply to chemically synthesized drug substances including drug
substances used in generic drug products.
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CLASSIFICATION OF IMPURITIES
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(1) Organic Impurities (Process and Drug Related)
(2) Inorganic Impurities
(3) Residual Solvents
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Impurities can be classified into the following categories:
SPECIFICATION LIMITS FOR IMPURITIES
A summation of assay value and impurity levels generally may be used to obtain mass balance
for the test sample. The mass balance need not add to exactly 100% because of the analytical
error associated with each analytical procedure. The summation of impurity levels plus the
assay value may be misleading, e.g., when the assay procedure is nonspecific (e.g.,
potentiometric titrimetry) and the impurity level is relatively high.
SOURCES OF IMPURITIES
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1. Raw Material Employed in Manufacture
2. Methods or the Process used in Manufacture
3. Chemical Process And Plant Materials Employed The
Process
Limit tests
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limit test are quantitative or semi-quantitative test particularly put forward to identify and
control invariably small quantities of impurities that are supposed to be present in a
pharmaceutical substance.
1. Heavy Metals Limit Test
2. Chloride Limit Test
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Test For Purity
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It is however, pertinent to mention here that pharmaceutical chemicals must maintain a very
high degree of chemical purity. It is quiet obvious that a state of absolute purity may not be
achievable but a sincere effort must be exercised to obtain the maximum freedom from foreign
substances. Bearing in mind the exorbitant operation costs to attain the ‘highest standards’ of
purity,
In short, a most of impurities in pharmaceutical chemicals do occur that may be partially
responsible for toxicity chemical interference and general instability.
Pharmacopoeias of various countries prescribe ‘test for purity’, for substances which are to be
used for medical purposes.
1. Colour, odour and taste
2. Physico-chemical constants
3. Acidity, Alkalinity,& pH
4. Anions & Cations
5. Ash, Water insoluble ash
6. Bioburden Testing
Analytical Procedure: -
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Generally impurities are identified by:
Thin layer chromatography (TLC)

High performance thin layer chromatography (HPTLC)

Infra-red (IR) spectroscopy

Nuclear magnetic resonance (NMR) spectroscopy
Validation: Due to the current accuracy and precision in analytical instrumentation regents and
u.
capabilities of modern data processing systems even poor methods may validate to be
acceptable validation does not necessarily certify a method as good Robust or suitable for a
control environment these must be established within the method during development. It is
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however a necessary and important step in both providing and documenting the capabilities of
the method. The ensure that the data are both accurate and reliable qualified and trained
laboratory analysts must performs methods on qualified equipments using suitable standards.
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References
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References
1. Ashutosh Kar, Pharmaceutical drug analysis, Minerva Press, 2001, 5-8.
2. Ahuja S., Scypinski S., Handbook of Modern pharmaceutical analysis, VOL-3,
Academic Press, 2001, 1-9.
3. Kasture A.V., Wadodkar S.G., Pharmaceutical chemistry-I, First edn.,
Nirali Prakashan, 1993, 9-22.
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4. Google.com
5. Ahuja S., Scypinski S., Handbook of Modern pharmaceutical analysis, VOL-3,
Academic Press, 2001, 119.
6. Ahuja S., Scypinski S., Handbook of Modern pharmaceutical analysis, VOL-3,
Academic Press, 2001, 336-338.
7. Mahandik K.R., Kuchekar B.S., Consice inorganic Pharmaceutical
Chemistry Second edn., Nirali Prakashan, 1995, 157-182.
8. USP24 NF19, United states Pharmacopeia-the national formulary-Asian
Edn., 2000, <466>, <467>, 1876-1888.
9. Beckette A.H.,Stenklake, Practical Pharmaceutical Chemistry Vol-1 Third edn.
10. Mendham J., Denney R., Barnes J., Thomas M., Vogel’s textbook of
quantitative chemical Analysis, 6th edn., person education Publisher,
India, 2003, 251-281.
11. Joel S., HPLC-Practical and Industrial Applications, CRS Press, 1997,
130-142.
12. Shethi P.D., HPLC-Quantitative Analysis of Pharmaceutical formulations,
first edn., CBS Publishers and distributors, 2001, 3-7.
13. Sethi P., HPTLC-Quantitative Analysis of Pharmaceutical formulations,
CBS Publishers and distributors, 1996, 3-4.
14. Ashutosh Kar, Pharmaceutical drug analysis, Minerva Press, 2001, 397-422.
15. Ahuja S., Scypinski S., Handbook of Modern pharmaceutical analysis, VOL-3,
Academic Press, 2001, 129.
16. Ahuja S., Scypinski S., Handbook of Modern pharmaceutical analysis, VOL-3,
Academic Press, 2001, 415-442.
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