ADDIS ABABA UNIVERSITY

COLLEGE OF HEALTH SCIENCES

SCHOOL OF PHARMACY

Pharmaceutical Analysis I (Phar3151) Assignment

Group V: Limit Tests

MEMBERS ID
HAYAT ABDELLA MOHAMMED UGR/0783/13
HEWAN GIRMA LEMMA UGR/6629/13
HILAL AHMED BERIHUN UGR/3823/13
HILINA ADMASS KINDIE UGR/9124/13
HIWOT NIGUSSIE BELAY UGR/8892/13
IKRAM ABDULFETA ALEMAR UGR/4018/13
KASEGN AWOKE ZEMENE UGR/3986/13
KHALID BEKRI ISSA UGR/5384/13

Submitted to: Ms. Feruza & Mr. Wondosen

Submission Date: December 4, 2022
 Limit Tests for Pharmaceutical Analysis

Table of Contents
Introduction 2
1.2 Limits on Ash Value 2
1.2.1 Ash Value (Total Ash) 3
1.2.2 Acid Insoluble Ash 3
1.2.3 Sulphated Ash 4
1.2.4 Water Soluble Ash 5
1.3 Limits on Moisture Content 6
1.3.1 Loss on Drying method 6
1.3.2 Azeotropic Distillation 7
1.3.3 Karl Fischer method 7
1.4 Limit Tests for Metals 9
1.4.1 Limit test for Lead 10
1.4.2 Limit test for Arsenic 10
1.4.3 Limit test for Iron 10
1.5 Limit Tests for Non-Metals 10
1.5.1 Limit test for Boron 11
1.5.2 Limit test for Free Halogens 11
1.5.3 Limit test for Selenium 11
1.6 Bibliography 13

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 Limit Tests for Pharmaceutical Analysis

Limit Tests

1.1 Introduction

In any drug substance exists a number of chemical entities other than the wanted compound.
These chemical entities are termed as impurities. Impurities can be harmless and impact a
drug’s therapeutic such as safety, efficacy and stability as well as be poisonous, carcinogenic,
or toxic once administered. Since eliminating impurities entirely is not an option, it raises the
need for determining their limits. For this reason, limit values are described in the
pharmacopeial monographs or by the ICH Q3 guidelines. These limits could be determined
using Limit tests, which are quantitative or semi-quantitative tests created to locate and
regulate minute amounts of impurity that are likely to be present in the substance. In order to
identify the inorganic impurities contained in a compound, a limit test is typically conducted.
Applying limit tests can allow for the examination of a wide range of impurities. To name a
few, they include nitrosamines, arsenic, and various metallic ions. These substances can enter
drugs from a variety of sources like the use of specific reagents during chemical synthesis in
the manufacturing process, contaminated water, soil uptake and microbial contamination. As
all methods have qualities which should suit their uses, Limit tests should be specific enough
to form some sort of selective reaction with the trace impurity without reacting with the drug
as well as sensitive enough to detect extremely small amounts of impurities within the intended
limits. There are various types of tests for quantitative determination of which we will cover:

• Limits tests for Ash values

• Limits tests for Moisture content
• Limit tests for Metals
• Limit tests for Non-Metals

1.2 Limits on Ash Value

Ash is the non-combustible remains of matter that was completely incinerated. On incineration,
crude drugs normally produce ash which consists of, inorganic salts of carbonates, phosphates,
silicates of sodium, potassium, calcium and magnesium. Ash values are representations of the
residue present in official herbal drugs and pharmaceutical substances. Determination of Ash
values or ashing is important so as to determine the quality and purity of the drug and to remove

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traces of organic matter which may interfere with analytical determining. Different types of
ash value are used in detection of crude drug. These values are categorized into four:

• Ash Value (Total Ash)

• Acid-Insoluble Ash
• Sulphated Ash, and
• Water-Soluble Ash

1.2.1 Ash Value (Total Ash)

Total ash method is designed to measure the total amount of material remaining after ignition.
This is used to detect adulteration of the drug with various substances such as physiological
ash which is derived from plant tissue itself and exhausted drugs and non-physiological ash
which is residue of extraneous matter like minerals and completely unrelated matter.

Procedure

1. Weigh 2 to 3 g of ground air dried drug in a tared crucible (usually of platinum or silica
dish).
2. Incinerate at a temperature not exceeding 450°C until free from carbon. The maximum
temperature should not be more than 450°C because alkali chloride that may be volatile
at higher temperature would be lost.
3. Cool and record weight.
4. Calculate the percentage of ash with reference to the air-dried drug according to the
equation.

𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑎𝑠ℎ
% Total Ash = × 100
𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑠𝑎𝑚𝑝𝑙𝑒

If a carbon-free ash could not be obtained in this way, exhaust the charred mass with hot water
then collect the residue on an ash less filter paper. Incinerate the residue and filter paper and
add the filtrate then evaporate to dryness and ignite at a temperature not exceeding 450°C.

1.2.2 Acid Insoluble Ash

Acid insoluble ash is the residue obtained after boiling with dilute hydrochloric acid and
igniting the remaining insoluble matter. This measures the amount of silica present as a sand
and siliceous earth. This method is useful as the Total ash method has various limitations.

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 Limit Tests for Pharmaceutical Analysis

Firstly, it cannot detect the amount of soil present in the drug well and the limit of excess soil
in the drug is not quite defined. Secondly, since the condition of ignition often varies across
tests consequently causing variability among results. Thus, using Acid insoluble ash proves
better to detect and limit excess soil.

Procedure

1. Place the ash in a crucible then add 15 ml distilled water and l0 ml of 3N Hydrochloric
acid.
2. Cover with watch-glass and boil for 10 minutes. After that allow it to cool.
3. Collect the insoluble matter on an ashless filter paper then wash with hot distilled water
until the filtrate is neutral and dry it.
4. Ignite at 450° to dull red color.
5. Allow to cool in a desiccator and weigh.
6. Repeat until the difference between two successive weighing is not more than l mg.
7. Calculate the percentage of acid-insoluble ash with reference to the air-dried drug

𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑎𝑐𝑖𝑑 𝑖𝑛𝑠𝑜𝑙𝑢𝑏𝑙𝑒 𝑎𝑠ℎ

% 𝐴𝑐𝑖𝑑 𝐼𝑛𝑠𝑜𝑙𝑢𝑏𝑙𝑒 𝐴𝑠ℎ = × 100
𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑠𝑎𝑚𝑝𝑙𝑒

1.2.3 Sulphated Ash

Sulphated ashes are the non-volatilized leftover material following carbonization of the sample,
sulfuric acid treatment, and constant temperature heating with this method being known as the
Sulphated Ash method. A double ignition with concentrated sulfuric acid is used to quantify
the amount of sulphated ash or how many inorganic contaminants are present in an organic
compound. Metals continue to exist as sulphides, which are typically heat-stable. The method
is somewhat precise and yields results that are somewhat more repeatable than those produced
by straightforward ignition or Total Ash method. The estimation of ‘sulphated ash’ is broadly
employed in the case of:

Unorganized medicines: such as colophony, podophyllum resin, wool alcohols, wool fat, and
hydrous wool fat.

Pharmaceuticals having inorganic contaminants: such as those of natural origin. Activated

charcoal, Frangula Bark, and spray-dried acacia

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 Limit Tests for Pharmaceutical Analysis

There are two methods to prepare sulphated ash stated by the International Pharmacopoeia
are as follows:

Method A

Accurately weigh about 1 g of the substance, or the quantity specified in the monograph, into
a suitable crucible (usually platinum) and moisten with sulfuric acid (~1760 g/l) TS. Heat
gently to remove the excess of acid and ignite at about 800 °C until all the black particles have
disappeared; again, moisten with sulfuric acid (~1760 g/l) TS and reignite. Add a small amount
of ammonium carbonate R and ignite to constant weight.

Method B

Ignite a suitable crucible (for example silica, platinum, quartz or porcelain) at 550 °C to 650
°C for 30 minutes, cool the crucible in a desiccator (silica gel or another suitable desiccant) and
weigh it accurately. Take the amount of test sample specified in the individual monograph in
the crucible and weigh the crucible accurately. Moisten the sample with a small amount
(usually 1 mL) of sulfuric acid (~1760 g/l) TS, heat gently at a temperature as low as practicable
until the sample is thoroughly charred. After cooling moisten the residue with a small amount
(usually 1 mL) of sulfuric acid (~1760 g/l) TS, heat gently until white fumes are no longer
evolved and ignite at 550 °C to 650 °C until the residue is completely incinerated. Ensure that
flames are not produced at any time during the procedure. Cool the crucible in a desiccator
(silica gel or other suitable desiccant), weigh accurately and calculate the percentage of residue.

1.2.4 Water Soluble Ash

When the entire amount of ash from the experiment is cooked in water, the water-soluble part
dissolves and the water-insoluble part does not. Gravimetric analysis can be used to estimate
the insoluble ash. By deducting the weight of the insoluble matter from the weight of the ash,
the constant weight of the water-soluble ash and the residue could be calculated.

Procedure

1. Firstly, the prepared ash is boiled for 5 minutes with 25 ml distilled water.
2. Secondly, the collected insoluble material is placed on an ashless filter paper or in a
sintered glass crucible and washed with hot distilled water.
3. Then, it is ignited for 15 minutes at a temperature no higher than 450°.

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 Limit Tests for Pharmaceutical Analysis

4. Subtract the weight of the ash from the weight of the residue you just acquired. The
water-soluble ash is represented by the weight difference.
5. Now, using the air-dried medication as a reference, compute the proportion of water-
soluble ash.

1.3 Limits on Moisture Content

Moisture content or water content refers to the weight of the water contained in a certain object
or material. It is usually expressed as a percentage of weight. For instance, soil moisture
pertains to the water content of the soil. Moisture content is a critical indicator of quality, safety,
and shelf life; thus, moisture analysis serves an important quality control function. A number
of pharmaceutical substances are hygroscopic and so will absorb moisture. Too much humidity
can compromise potency and effectiveness, leading to degradation or even toxicity in some
products. The potential for danger comes when relative humidity levels reach 60 percent or
more; giving viruses, bacteria, mold, fungi and mites the opportunity to grow. Therefore,
moisture content must be limited. In order to determine moisture content, we can use the
following limit tests:

• Loss on Drying method

• Azeotropic Distillation method
• Karl Fischer method

1.3.1 Loss on Drying method

Loss on drying is the loss of weight expressed as percentage w/w resulting from water and
volatile matter of any kind that can be driven offer under specified conditions. This method is
heavily dependent on temperature and drying time. The advantages of the Loss on drying
technique include high reproducibility and reliable results, and as a result it is the standard in
many manufacturing procedures. It has the serious disadvantage of requiring a long time for
completion.

Procedure

1. The test is carried out on a well-mixed sample of the substance.

2. If the substance is in the form of large crystals, reduce the size by rapid crushing to a
powder.
3. The substance is heated until no more weight is lost, completely dry.

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 Limit Tests for Pharmaceutical Analysis

initial weight of sample−weight of sample after drying

%Loss on drying = × 100
initial weight of sample

1.3.2 Azeotropic Distillation

Azeotropic Distillation can be defined as the process of separating the components of an
azeotrope by distillation. An azeotropic mixture or an azeotrope is a mixture containing two or
more liquids that cannot be separated by simple distillation. This is because the vapors formed
from the boiling of azeotropic mixtures contain almost the same proportions of liquids as the
liquid itself. Consequently, azeotropic distillation is a specialized type of distillation which
uses specific techniques to break the azeotropes. The most common method involves the
addition of a material separation agent that has the ability to change the molecular interactions
between the components of the azeotrope. The addition of such a material separation agent
tends to alter the activity of the activity coefficient of the components of the azeotropic mixture,
thereby changing the relative volatility of the azeotropic mixture as a whole.

Azeotropic distillation is a less popular approach due to the high energy costs. Health and safety
concerns when working with benzene and cyclohexane, which are both flammable and
carcinogenic solvents.

1.3.3 Karl Fischer method

It is a combination of compounds for determining small amounts of water which can be found
in pharmaceuticals or organic substances. Karl Fischer who was a German chemist published
a method to determine trace amounts of water in a sample. The method makes uses of Karl
Fischer reagent which could be prepared using a combination of: Iodine, sulfur dioxide,
methanol and pyridine. The trace amount of water in a sample reacts with the Karl Fischer
reagent in a 2-stage process:

From stage 1(a) Iodine oxidizes

sulfur dioxide to yield sulfur
trioxide and hydrogen iodide by
consuming one molecule of

Fig (1.1): Chemical reactions showing the 2-stage process water. Each molecule of iodine
disappears against each
molecule of water present in a sample. In the presence of large amount of pyridine all the

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 Limit Tests for Pharmaceutical Analysis

substances in the reaction exists as complex a mentioned above. There are two determination
methods:

• Volumetric Titration method

• Coulometric Titration method

Volume 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: 100 ppm - 100 %.

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 electrolysis (i.e., for the production of iodine). Based on the quantitative
reaction of the generated iodine with water Iodine is generated electrochemically during
titration. Water in trace amounts: 1 ppm - 5 %

The end point in a Karl Fisher titration can be located in several ways.

Visual detection: Before the end point the solution is yellow. After the end point the first
excess of the reagent imparts the characteristic brown I2 color

Photometric method: The active reagent absorbs light at 525nm. Before the end point the
absorbance remains close to zero. After the end point, the absorbance increases linearly with
volume of reagent. The end point marked by the intersection of the two straight lines.

Electrometric location of the end point: It is easy to determine the end point of Karl Fischer
titration by adopting the electrometric technique employing the dead stop end stop method. A
small quantum of emf can be applied across two platinum electrodes immersed in the reaction
mixture then a current tends to flow because of free iodine ions to remove H and depolarize
the cathode.

A situation will soon arise when practically all the traces of iodine have reacted completely
thereby setting the current to almost zero or very close to zero or attain the end-point.
Electrometric location of the end point can generate iodine in situ electrochemically. It can

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 Limit Tests for Pharmaceutical Analysis

generate it as it reacts and stop

generating when endpoint is
reached. Then measure the
moles of electrons that were
required to generate it.
Therefore: the moles of titrant
required is known.
Fig (1.2): Electrometric location process

Stability of the reagent: The reagent must be free from moisture.

Advantages
Extreme accuracy: because KF titration is based upon a chemical reaction which depends
upon the presence of water.
Specificity for water: it does not detect the loss of any other volatile substances unlike other
methods such as loss on drying which are based upon the heat-induced loss of moisture and the
resulting reduction in weight of the sample. It is suitable for water determination in solids,
liquids and gases.

Limitations

KF titration depends upon a redox reaction and thus any component of the sample which is an
active redox chemical such as dimethyl sulfoxide will react with the iodine in the reagent and
generate false results. Also, Ketones and aldehydes, boric acid and metal peroxides, as well as
and strong acids, are not suitable for this titration without modification, because their reaction
with the methanol solvent produces water, resulting in a vanishing end point and falsely high-
water content, requiring the use of methanol-free reagents with such substances. Carbonates,
oxides and hydroxides also undergo side reactions and are not suitable for KF titration.

1.4 Limit Tests for Metals

Impurities are common in pharmaceutical products. Metallic impurities such as lead, arsenic
and iron present in the pharmaceutical products. These impurities have a toxic effect and bring
unpleasant reaction. The source of these metal impurities may come from the container that
contain the product, atmospheric pollution or from the raw material. In order to control these
impurities, the pharmacopeia usually fixes certain limit of tolerance. The limit test indicates

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the known amount of impurity present on the pharmaceutical product. The following are tests
for metals:

• Limit test for Lead

• Limit test for Arsenic
• Limit test for Iron

1.4.1 Limit test for Lead

Lead is a heavy metal and it is a common impurity that may come from containers, water pipe
or from the raw material. Limit test for lead is based on the reaction of lead with diphenyl
garbatiazon or dithizone in chloroform or based on the reaction of lead and sodium sulphide.
The reaction of lead and diathizon gives dithizone complex which have violent color. The
violent color is indicating the limit test for lead. if the sample produced no color or less intense
violent color, it passes the limit test but if the sample give more color intensity or more violent
color, it fails the limit test.

1.4.2 Limit test for Arsenic

Limit test for arsenic is based on the reaction of arsine gas with hydrogen ion to form yellow
stain on mercuric chloride paper in presence of reducing agents like potassium iodide. It is also
called Gutzeit test and require special apparatus. The reagents are mixed; the arsine gas is
formed from arsenic acid and hydrogen. the arsine gas again reacts with mercurial chloride and
forms yellow stain which is the standard color containing a known amount of arsenic

1.4.3 Limit test for Iron

The limit test for iron is based on the formation of a purple color by reaction the iron with
thioglycolic acid in a solution buffered with ammonium citrate. The reaction gives ferrous
glycolate which have purple color. The color produced is compared with a standard color
containing known amount of iron (0.14mg of iron).

1.5 Limit Tests for Non-Metals

In medicinal chemicals, non-metallic impurities including boron, free halogens (I2, Br2, and
Cl2), and selenium. These impurities are introduced from various ways and since they
frequently cause adverse responses, cutaneous symptoms, and are poisonous to healthy tissues,

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their limits need to be monitored. The following tests are employed to monitor their
concentration.:

• Limit test for Boron

• Limit test for free Halogens
• Limit test for Selenium

1.5.1 Limit test for Boron

Boron can be found in Salbutamol Sulphate due to the use of sodium borohydride (NaBH4) in
the manufacturing process. The estimation is based on the conversion of boron to borate, after
which the organic matter is destroyed by ignition with anhydrous sodium carbonate. Finally,
the amount of boron using a colorimetric assay.

1.5.2 Limit test for Free Halogens

Free halogens are present in a wide array of compounds as impurities. A few typical examples
of pharmaceutical chemicals in which free halogens like Iodine, Bromine, Fluorine and
Chlorine are present as non-metallic impurities are given below:

A. Clioquinol: Free Iodine

Materials Required: Clioquinol 1.0 g; potassium iodide 1.0 g; H2SO4 (1 M) 1.0 ml;
chloroform 2.0 ml; sodium thiosulphate (0.005 M) 0.1 ml.
Procedure: Shake 1.0 g with a solution of 1 g potassium iodide in 20 ml DW for 30 seconds,
allow to stand for 5 minutes and filter. To 10 ml of the filtrate add 1 ml 1 M H2 SO4 and 2 ml
chloroform and shake.
Prescribed Limits: Any color in the chloroform layer is discharged on the addition of 0.1 ml
of 0.005 M sodium thiosulphate.
B. Diethylpropion Hydrochloride: Free Bromine
Test: Place 0.05 ml of a 10% w/v solution on starch-iodide paper.
Prescribed Limit: No color is produced.

1.5.3 Limit test for Selenium

Selenium is very toxic and its contamination is usually controlled by an absorptiometric method
after destruction of the organic compound with fuming nitric acid. The latter converts selenium
(Se) as selenous acid (H2 SeO3), which on subsequent treatment with 3,3′-diaminobenzidine
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 Limit Tests for Pharmaceutical Analysis

under controlled experimental parameters, results into the formation of a highly colored
compound known as 3,4-diaminophenylpiazselenol. The latter is consequently extracted with
toluene after making the aqueous solution alkaline, and the color compared with a standard
prepared likewise from a known amount of selenium

Materials Required: Selenium sulphide: 10.0 g; formic acid (2.5 M): 2.0 ml; 3,3′-
diaminobenzedine tetrahydrochloride solution (0.5% w/v in DW): 2.0 ml; ammonia (5 M): 20
ml; selenium standard solution (1 ppm Se) (Dilute 2.5 ml of a 0.00654% w/v solution of
selenous acid to 100 ml with DW): 5.0 ml.

Procedure

1. To 10 g of selenium sulphide add 100 ml DW, mix well, allow to stand for 1 hour with
frequent shaking and filter.
2. To 10 ml of the filtrate, add 2 ml of 2.5 M formic acid, dilute to 50 ml with DW, adjust
the pH to 2.0 to 3.0 with 2.5M formic acid,
3. Add 2.0 ml of a 3,3′-diaminobenzidine tetrahydrochloride in DW then allow to stand
for 45 minutes and adjust the pH to 6.0 to 7.0 with 5M ammonia.
4. Shake the solution for 1 minute with 10 ml of toluene and allow to separate. Measure
the absorbance at 420 nm.

Prescribed Limit: The measured absorbance at 420 nm is not greater than that of a solution
prepared by treating 5 ml of selenium standard solution (1 ppm Se) in the same manner (5 ppm,
calculated as Se)

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 Limit Tests for Pharmaceutical Analysis

1.6 Bibliography
Davani, B. (2017). Pharmaceutical Analysis for Small Molecules. Chennai, India: John Wiley
& Sons, Inc.

Handley, J. M. (2003). Pharmaceutical Analysis. Pondicherry, India: © Blackwell Publishing

Ltd.

Kar, A. (2005). Pharmaceutical Drug Analysis. Addis Ababa: New Age International (P)
Ltd., Publishers.

Mohd. Mumtaz Alam, M. A. (2011). Practical Pharmaceuticcal Analytical Chemistry. New

Delhi: Reed Elsevier India Private Ltd.

PEDERSEN-BJERGAARD, S. (2019). Introduction to Pharmaceutical Analytical

Chemistry. University of Oslo, Norway: © 2019 John Wiley & Sons Ltd.

The International Pharmacopoeia Ninth Edition. (2019).

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