Chapter 5 : Limit tests

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 Introduction
• In the pharmaceutical f ield, there are large number of
drugs, chemicals and other substances which are
used in the formulations.
• All of these materials needs to be pure with less toxicity and safety
in drug therapy.

• However, it is almost impossible to get an absolute

pure material, as impurities get incorporated in to
them either during manufacturing, purif ic ation or

storage due to side reaction, degradation,

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 Introduction Cont…
• The amount of various impurities found in drug
substances will determine the ultimate safety of
the final pharmaceutical product.

• The identification, quantitation, qualification, and

control of impurities are now a critical part in
pharmaceutical field.

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 Impurity
• According to ICH various regulatory bodies, pharmaceuticals
impurities are the unwanted chemicals that remain with active
pharmaceutical ingredients (API) or drug product formulations.
• Any component of the new drug substance or new drug product which is not
the chemical entity.

• Impurity is the state of a chemical substance when it contains

undesirable foreign matter where as purity is the state of a
chemical substance when no impurity/(undesirable foreign matter)
can be detected or complete freedom from extraneous matter
• The impurities observed in drug substances may arise during
syn th esis or may be derived from sou rces su ch as startin g
materials, intermediates, reagents, solvents, catalysts, and reaction
by-products.

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 Sources of impurities
1. Raw materials used

• Impurities associated with the raw materials may be carried through the

manufacturing process to contaminate the final product

2. Method of Manufacturing

• Process or method of manufacture may introduce new impurities into the

f in al product arising due to contamination by reagents, catalysts and

solvents employed at various stages of the manufacturing process.

3. Instability of the product

• This can be as a result of chemical instability, changes in physical

properties, and reaction with container material

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 Effect of impurity
The impurity present in a substance may have the following effect:
• Toxicity effect when present above permissible limits and when
present in a cumulative

• lowering the strength or therapeutic effect of the drugs

• Bring change in the physical and chemical properties of the

substances and making it medically useless

• May cause technical dif ficulties and incompatibilities with other

substances in the formulation

• May lower shelf life of the substances and/or bring about

c hange in c o lo r, o do ur, taste, etc ., and thus making the
substance unethical and unhygienic.

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 Test for impurity/purity
Pharmacopoeias of various countries prescribe 'test for purity' for

substances which are to be used for medical purpose.

• It is a test for the determination of how much impurities is likely to

be harmful, or to bring about technical and other dif ficulties, when

the substance is used.

• As it is dif ficult to ensure freedom from every possible impurities in a

substance, pharmacopoeias aims to test for few major impurities, which

are likely to interfere in their use.

• Certain tests which are carried out on the substances includes test for

colour, odour, physicochemical constants, acidity, alkalinity, pH, anions,

cations, insoluble residue, heavy metals, acid radicals and etc.

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 Test for impurity cont…
• Tests for the presence of foreign substances and
impurities are provided to limit these substances
to the amounts that are unobjectionable

• P h armac op oe ias f ix limit s of tole ran c e for

impurities.
• Impurit ies present more t han 0.1% should be
identified and quantified by selective methods.

• Pharmacopoeias of various countries prescribes

limit test to be carried out by a particular method
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 Limit Test
• Limit tests are quantitative or semi-quantitative tests
designed to identify and control small amount of impurities,
which are likely to be present in the substance.

• They involve simple comparisons of opalescence,

turbidity or colour produced in test with that of f ix ed
standards.

• The test needs to be both specific and sensitive.

• Test for impurity can be classified in to two major limit tests;

1. General impurity limit test, and

2. Specific impurity limit test.

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 General impurity limit test
• These type of tests are employed for non-specif ic impurities
such as test for clarity of solution , color of solution,
insoluble matter, soluble matter, moisture, volatile matter, non-
volatile matter, residual solvents, residue on ignition, loss on
ignition, and ash values.
1. Clarity of solution :
• The degree of clarity or opalescence of solution is measured
by direct comparison with a reference solution.
• Solutions are clear if their clarity is the same as that of water or the
solvent used.

Example: Solutions for injections should be reasonably free from

particulate matter.
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 General impurity Cont…
2. Color of solution:

• A solution may be described as colorless if it has the appearance as water

or the solvent employed in the preparation of the solution being examined.

3. Insoluble matter:

• The limit test is used to control small amounts of solvent-insoluble

contamination

Example: Determination of alcohol-insoluble matter to limit inorganic salts

in Crystal violet .

4. Soluble matter: To control impurities which are soluble in a given

solvent.

Example: The control of water-soluble barium salts in Barium sulphate.

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 General impurity Cont…
5. Non-volatile matter:
• These limits are applied to substances which are readily volatile and to control impurities such
as dust in volatile substances.

6. Residue on ignition:
• Limits the amount of residue remained after the substance is ignited.
• This test applied to pharmaceutical substances which are completely volatile when
ignited like Hg and to those which undergo total decomposition thereby leaving a
residue with a definite composition
Example: like Calamine ( basic zinc carbonate) that gives rise to ZnO as the residue.
7. Loss on ignition:
• It is the loss of weight in % w/w resulting from a volatile part of any test material
that is driven off under specified conditions.
• Generally applied to relatively stable pharmaceutical substances that are likely to
contain thermo labile impurities.
Example: From of ficial compendia the ignition temperature and prescribed limits(%)
c and 17.0-34.0 respectively
for Magnesium Trisilicate is 9000

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 Limit test for specific impurity
1. Limits on ash values:

• Ashes are inorganic residue remaining after incineration

• The determination of ash values in crude drugs, organic

compounds and certain inorganic compounds provides
valuable information regarding the extent of heavy metals
and minerals impurities.
• The ash values usually represent the inorganic residue present

in official herbal drugs and pharmaceutical substances.

• These values are categorized into four heads which

includes Total Ash, Acid-Insoluble Ash, Sulphated Ash, and
water-Soluble Ash.
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 Limits on ash values Cont..
1.1. Total Ash values:
• It is an ash obtained after prolonged ignition of a substance or the
powdered vegetable drug to be examined in the crucible to constant
mass in a muffle furnace at 600 °C ± 25 °C,

• If after prolonged ignition the ash still contains black particles, take up

with hot water, f ilter through an ash less f ilter paper and ignite the residue

and the f il ter paper. Combine the f il trate with the ash, carefully evaporate

to dryness and ignite to constant mass.

• Thes e as h val ue n orm al l y d es i g n ates the p res en ce of i n org an i c

salts(Inorganic matter derived from external sources).

Example: calcium oxalate found naturally in the drug, used to

detect and check adulteration of drugs with gingers, absence of

cardamom plant
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 Limits on ash values Cont..
1.2. Acid-Insoluble Ash:

• It is the ash remain after treating the ‘total ash’ with acid(HCl).

• Designed to measure the amount of ash insoluble to diluted

hydrochloric acid

1.3. Water-Soluble Ash:

• Water-soluble ash is specifically useful in detecting such

samples which have been extracted with water.

• A typical example of an official drug is that of ‘Ginger’, the

water-soluble ash of which is found to be not more than 6.0%.

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Limits on ash values Cont..
1.4. Sulphated Ash:

• It is determined by a double ignition with sulphuric acid(

conc. H2
SO4
).

• Metals thus remain as sulphides that are stable to heat.

• The estimation of ‘sulphated ash’ is broadly employed in

the case of pharmaceutical substances contained with
inorganic impurities such as activated Charcoal ( NMT :
5 .0 0 %), G riseofulv in (N MT : 0 .1 0 %), and Asc orb ic
acid(NMT: 0.1%).

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2. Limits on moisture content
• Many of ficial drugs contain varying amount of water either in the form of

crystallization i.e. hyadrates or in absorbed form.

• Moisture plays remarkable negative role in pharmaceutical product,

particularly for solid dosage forms.

• Both physical and chemical stability of some drugs are affected by

moisture.

• Moisture absorbed on the surface of solid drugs increases the rate of

decomposition, causes agglomeration and dissolution of drugs.

• Presence of moisture possesses a critical challenge on drug

stability.

• Because moisture accelerates the hydrolysis of drug, facilitates

reaction with other excipients thereby affecting stability and shelf life of
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the final product, it is important to specify limits of moisture content.
 Limits on moisture content
• There are various methods of moisture

content determination. The most common

of these are:

1. Loss on drying method,

2. Karl- Fischer titration method

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Limits on moisture cont…
1. Loss on drying(LOD):
• It is the loss of weight in % w/w resulting from water & volatile matters
that are lost under specified conditions.
 Loss in weight when the pharmaceutical chemical is subjected to drying
under specified conditions
• The amount of heat to which the substance is submitted varies
considerably according to the nature of the substance
• The temperature must be sufficiently high to produce the required result
within a reasonable time
• But not so high as to cause decomposition
• Ref lects the net weight of a pharmaceutical substance being dried
at a specif ie d temperature either at an atmospheric or under reduced
pressure.
Advantages: easy to use, relatively rapid, many samples can be
analyzed simultaneously.
Disadvantages: destructive and time consuming.
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Loss on drying cont…
• loss on drying is an unspecif ic analytical technique removing not only

water but all other volatile impurities like alcohol etc. from a sample.

• In cases there are additional traces of other volatile impurities

present, like alcohol; LOD may be higher than water content.

• LOD or total moisture content of Pharmaceutical products can

include both bound (e.g. water of hydration) & free water.

• In other cases, LOD may be lower than water content, as bound

crystal water may not be removed by heating(% LOD = % Water

content - % water molecule in the API)

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 Karl Fischer Method
• K arl Fische r de scribe d a spe cif ic t it rim e t ric m e t hod for wat e r

determination

• Where water content is critical or where it is important to distinguish

between moisture & other volatile matter, water may be determined

titrimetric ally with Karl Fischer reagent

• Karl Fischer titration is the method of choice for water determination

in most kinds of samples

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Karl Fischer Method
• It is a method to determine water content in sample materials,

ut ilizing t he fact t hat wat e r re act s wit h iodine and sulfur dioxide

quantitatively in the presence of a lower alcohol such as methanol, and an

organic base such as pyridine.

• The regent is called Karl Fischer reagent(composed of iodine,

sulphur dioxide, pyridine and methanol).

• Water present in the analyte reacts with the KF reagent in three a three-

stage process as:

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Karl Fischer Cont…
• In f irst step, the oxidation of sulphur dioxide takes place by iodine
to yield sulphur trioxide and hydrogen iodide thereby consuming
one mole of water.(i.e. one molecule of iodine disappears
against each molecule of water present in the given sample)

• In this method, iodine is previously dissolved in a reagent for water

determination, and water content is determined by measuring the
amount of iodine consumed as a result of reaction with water.

• The optimal pH range for the Karl Fischer reaction is from5 to 8,

• Water content is determined by the Karl Fischer titration method

and it consists of only water i.e moisture content.
• The results does not contain other volatile matter except the water.

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Karl Fischer Cont…
• However, loss on drying (LOD) is determined by heating the
sample bellow its melting point in an oven and it includes all
volatile matter including water content and solvents.
• There are two Karl Fischer titration for determination :
• Volumetric titration and coulometric titration methods
1. Volumetric titration method:
• water content is determined by measuring the amount of
iodine consumed as a result of reaction with water in a sample
• from the burette volume of the iodine containing KF solution and water as a
major component

• End point can be determined either by visual indication (yellow-to

brown) or photometrically
• Used to determine from 2%- 100% water content
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Karl Fischer Cont…
2. Coulometric titration method:
• First, iodine is produced by electrolysis of the
reagent containing iodide ion, and then, the
water content in a sample is determined by
measuring the quantity of electricity which is
required for the electrolys is (i.e., for the
production of iodine).
• Based on the quantitative reaction of the
generated iodine with water.
• Iodide(HI) is generated electrochemically during
titration.
• As all the water consumed and no more iodide
formation the flow of electric current will be zero.
• This method used to determine water in trace
amounts: 1 ppm - 2 %.
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 KFR cont…

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Limit tests for heavy metals
• T h e l i m i t t es t for h ea v y m et a l s i s d es i g n ed t o
determine the content of metallic impurities that are
colored by hydrogen sulphide or sodium sulphide
under the condition of the test.

• The metallic impurities may be iron, copper, lead,

nickel, cobalt, bismuth, antimony etc.
• The limit for heavy metals is indicated in the individual
monograph in term of ppm of lead i.e. the parts of lead
per million parts of the substance being examined.
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 Limit Tests for Lead(Pb):
• The of ficial test is based on the conversion of traces of lead salts
present in the pharmaceutical substances to lead sulphide.

• By the addition of sodium sulphide in an alkaline medium. The

reaction may be expressed as follows :

• The brown colour, caused due to colloidal lead sulphide in the test
solution is compared with that of a standard.

• The color produced in test solution is not more intense than that of
the standard.

Example: Limit for Pb from the of fic ial compendium( Nicotinic

Acid, NMT20), (Pentobarbitone Sodium, NMT30), and (Piperazine
Hydrate,NMT20).

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 Limit Test for Arsenic(As):
• The test is based on the fact that all arsenic present is duly
converted into arsine gas (AsH3) by reduction with zinc &
hydrochloric acid.

• Reaction of the gas, arsine, with mercuric chloride

(HgCl2) paper produces a yellow stain.

• The intensity of which is directly proportional to the

quantity of arsenic present.

• The color of test solution is compared with that produced

from a known amount of arsenic.
Example: Limit for As from the of ficial compendium of Barium Sulphate
(NMT 1), Benzoic Acid(NMT2), and Calcium Lactate(NMT 2)
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 Limit Test for Iron(Fe):
• The limit tes t for iron is bas ed on the
formation of pale pink to deep reddish purple
colour by reaction of iron with thioglycollic
acid in the presence of citric acid in a solution
made alkaline with ammonia solution.

• W hi c h i s sub se q ue nt l y c o m p a re d w i t h t he
standard colour.
• Any colour produced is not more intense than the
standard colour
• The colour is due to the formation of the co-
ordination compound, ferrous thioglycolate
Fe(HSCH COO)2 2.

• Thioglycollic acid converts the entire Fe into Fe

3+ 2+

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 Limit tests for some non-metals acidic
•
radical
Non-metallic impurities, such as free halogens (I , Br and Cl) and
sulphate in pharmaceutical substances usually contribute untoward
reactions, skin manifestations and are found to be toxic to healthy
tissues.

• A few typical examples are described below:

1. Limit Test for Chlorides:

• The limit test for chlorides is based on its precipitation with silver
nitrate in the presence of dilute HNOand comparing the opalescence
produced due to the formation of AgCl with a standard opalescence
achieved with a known quantity of Cl ions.

• If the opalescence produced in the test is less intense than that of

standard opalescence, the sample passes the limit test for chloride
and vice versa.
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• The equation may be expressed as:
 Limit Test for Sulphates:
• This test is designed to control sulphate impurity
primarily in inorganic substance.

• Based upon the simple reaction between barium

chloride and soluble sulphate in the presence of
acetic acid to give insoluble barium sulphate.

• Thus, the opalescence caused by the sample is

compared immediately with a standard turbidity
produced with a known amount of the sulphate ion
(SO4
). 2 -

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 Thank You

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