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Chapter

Drug Analysis
Shaza W. Shantier

Abstract

Instrumental methods are widely used for the analysis and stability studies of
compounds in bulk and pharmaceutical forms. They vary in their sensitivity, tech-
niques and reagents involved. This chapter will overview those different techniques
and the application of the analytical methods. It will also describe how to design
and develop simple, sensitive and accurate method for routine quality control of
specified compound depending on its molecular structure. Quality control and
assurance of the analytical process will be discussed. Furthermore, the chapter
will describe a number of factors affecting the chemical and physical stability of
Pharmaceutical formulations and how to develop stability-indicating methods to
qualify and quantify the drug degradation.

Keywords: instrumental, development, quality control, validation, stability

1. Introduction

Drug discovery and development process can be divided into two major stages:
drug discovery which involves isolation of the active constituent, purified, and
standardized. The second major stage, drug development, starts with a solitary
compound, which at that point progresses through different studies intended to
support its endorsement as a new drug [1]. The new drug will then be formulated as
an appropriate pharmaceutical dosage form.
Pharmaceutical product is medicine intended for human or veterinary use
in cure, alleviation, prevention or diagnosis of disease. The use of ineffective,
harmful or poor-quality drugs will cause health hazards and waste of funds. The
problem is aggravated by adverse climatic conditions and weak drug supply sys-
tem (including storage and transport). These lead to deterioration of drug quality,
loss of activity and may be formation of harmful degradation products [2]. All
this made it a must that any pharmaceutical product should be subjected to differ-
ent analytical procedures in order to ensure its efficacy and safety. Therefore, an
effective drug quality assurance and assessment system should be developed and
maintained.

2. Pharmaceutical analysis

Broadly speaking, this is the application of a process in order to identify a drug

(single or combined) in its bulk or pharmaceutical dosage form. Testing phar-
maceutical product involves chemical, physical and sometimes microbiological
analysis [3].

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Pharmaceutical Formulation Design - Recent Practices

Pharmaceutical analysis can be divided into two types of methods:

a. Qualitative methods: these methods usually are used to ascertain the presence
or identity of a component and\or impurities (predicted or expected).

b. Quantitative methods: determine how much of known drugs are present in

bulk form or in a formulation.

Since the judgment for quality of a drug depends on the method of analysis
used, the validity or control of the method used is required. The methods used in
pharmaceutical analysis should be capable of:

a. Correct identification of the drug in bulk form or as a formulated product.

b. Indicating the percentage of the stated content of a drug present in formula-

tion within acceptable stated limits.

c. Indicating the stability of the drug in the formulation and hence the shelf life
i.e. indicating the presence of a drug in its intact form and or the presence of
any impurities (whether as drug precursors, decomposition products due to
chemical or photochemical causes).

d. Application in the dissolution rate studies i.e. at what rate is the drug released
from its formulation so that can be absorbed by the body (bioavailability studies).

e. Ensuring that the identity and purity of pure drug (bulk form) meet official
standards or monograph.

f. Ensuring that the identity and purity of excipients used in formulation meet
specifications set by official standards.

g. Indicating the concentration of the specified impurities in the pure drug

substance (limit test application).

3. Drug stability

The stability of a pharmaceutical product is defined as the capability of the

product, in a specific container, to retain its efficacy, properties and characteristics
throughout its shelf-life [4]. The recommended shelf-life (expiry date) for a com-
mercial pharmaceutical product is 3–5 years. During this time, the concentration
of the drug should not be reduced more than 95% of its value when originally
prepared [5].
There are five types of stability that concern the pharmacist in the manufactur-
ing of drugs:

a. Chemical (including photochemical): the product retains its chemical integrity

and potency.

b. Physical: the conformity of the pharmaceutical product (color, appearance,

dissolution, etc.) does not change upon storage or handling.

c. Microbiological: sterilized products should remain sterile (no pyrogenicity).

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d.Therapeutic: the therapeutic effect remains unchanged within the specified

dosage regimen.

e. Toxicological: no significant increase in a predetermined toxicity effect is noted.

Stability types (therapeutic, microbiological, and toxicological) are basically

dependent on the chemical and physical properties of the drug.
Knowledge of the chemical stability of a drug is of great concern for
selecting suitable storage condition against the effects of light, temperature,
humidity, etc. and for anticipation of drugs interaction with each other or with
excipients [6, 7].
A stable drug is of great concern to the pharmacist (in view of marketing, stor-
age and distribution); to the physician and patient (in view of safety and efficacy);
and to the regulator and quality control analyst (in view of quality, strength, purity
and identity).

3.1 Chemical reactions that cause drug degradation

Many drugs are derivatives of carboxylic acid or contain functional groups based
on this moiety, for example esters, amides, lactones, lactams, imides or carbamates
[5]. Accordingly, various chemical reactions can result in the degradation of the
drug. These reactions include hydrolysis, oxidation, photochemical reactions,
polymerization, isomerization, racemization and dehydration [4, 5, 8].

3.1.1 Hydrolysis (or solvolysis)

Hydrolysis forms the most common pathway by which drugs become degraded
since many drugs contain hydrolysable functional groups. It can be defined as the
process by which drug molecules interact with water to yield breakdown products
of different chemical constitution. Hydrolysis occurs more readily in liquid state
than in the solid state. It may occur in aqueous suspensions of sparingly soluble
drugs. In tablets and other solid dosage forms, there may be sufficient water to
allow hydrolysis of the drug [8].
Solvolysis is a term used for the reactions involving the decomposition of the
active drugs with their solvent present (not water).

3.1.2 Ester hydrolysis

Hydrolysis of drugs with an ester functional group (e.g. procaine, atropine, etc.)
forms one of the most common types of drug instability. It is usually a bimolecular
reaction involving acyl-oxygen cleavage. Ester hydrolysis is (H+) or (OH−) ion
catalyzed and is dependent on the specific compound and the pH of the solution.
Atropine hydrolysis is totally pH dependent and this was characterized by the slopes
of −1 and +1. In some cases, the hydrolysis of the drug can show a pH-profile with
three regions: a hydrogen ion (proton) catalyzed region, (slope = −1), an uncata-
lyzed region (solvent dependent, slope = 0) and a hydroxyl ion-catalyzed region
(slope = +1) (Figure 1) [5].

3.1.3 Amide hydrolysis

Amides are generally more stable to hydrolysis than esters. In general the rate
of hydroxyl ion-catalyzed reaction of amides is greater than the proton-catalyzed
hydrolysis [6].

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Pharmaceutical Formulation Design - Recent Practices

Figure 1.
Log rate-pH profile for the degradation of atropine at 60°C.

Penicillins and cephalosporins are amides in which the amide bond is part of
the strained four membered ß-lactam rings. Their decomposition is catalyzed by
hydrogen ion, hydroxyl ion and many buffers. Therefore, these compounds are too
unstable to be formulated as solutions. Their pH profile is generally similar to the
pH-profile shown in Figure 1.
In addition to acid-base catalyzed hydrolysis, enzyme-catalyzed hydrolysis may
take place in drugs of natural origin; for example enzymes catalyzes the hydrolysis
of cardiac glycosides in digitalis leaf [8].

3.1.4 Oxidation

Oxidation involves the removal of an electropositive atom, radical or

electron, or the addition of an electronegative atom or radical. When a reac-
tion involves molecular oxygen (O▬O), it is commonly called autoxidation
and this forms the most common pathway of oxidative decomposition of
pharmaceuticals.
Oxidative degradation by autoxidation may involve chain processes consisting
of three concurrent reactions—initiation, propagation and termination. Initiation
can be via free radicals formed from organic compounds by the action of light, heat
or transition metals such as copper and iron which are present in trace amounts in
almost every buffer [5].
Many drugs are complex molecules and can be subjected to both hydrolysis and
oxidation e.g. steroids, anti-inflammatory, polyene antibiotics (amphotericin B) etc.
Figure 2 show the oxidation of phenothiazines to the sulfoxide which involves
two single-electron transfer reactions involving a radical cation intermediate. The
sulfoxide is subsequently formed by reaction of the cation with water [5].
Oxidation in solution generally follows first or second order kinetics. Some
oxidation reactions are redox reactions that involve the loss of electrons without the
addition of oxygen e.g. oxidation of ascorbic acid, ferrous sulfate, adrenaline and
riboflavin [8].
In addition to oxidation and hydrolysis, many other degradative reactions had
been studied including addition, dehydration, polymerization, isomerization,
acylation, transesterification, etc.

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Figure 2.
Phenothiazine oxidation process.

3.1.5 Photochemical degradation

These are the reactions that take place by absorption of the visible or ultraviolet
light. The reactant molecule absorbs photons of light (energy) and get excited. The
excited molecule then produces the photodecomposition product.
In many photochemical reactions, the reactant molecule may not absorb the
radiation directly but through a mediator which absorbs the incident radiation and
subsequently transfers its energy to the reactant molecule that becomes activated.
Such type of mediator is called photosensitizer.
At times, a molecule can act as a protector for the photolabile drug by prefer-
entially absorbing the radiant energy and produce products. These compounds are
referred to as screening agents [9].

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Pharmaceutical Formulation Design - Recent Practices

3.1.5.1 Light sources for photodegradation studies

The majority of therapeutic substances are white in appearance, which means

that they may absorb in the UV region depending on their chemical structure.
Grossweiner, 1989 [10], divided the ultraviolet radiation (UV-R) into three
sub-bands:

1. UV-C: which ranges between 200 and 290 nm and is termed shortwave
or far UV. Sunlight at the earth’s surface is devoid of this band due to its
absorption by molecular oxygen and ozone in the upper atmosphere.
Artificial radiation sources such as discharge and germicidal lamps and
welding arcs form the sources of UV-C which are used for forced drug pho-
todegradation studies (stress-conditions). These also cause serious damage
to the skin and cornea [11].

2. UV-B band: this covers the region 280–320 nm. It causes sunburn, skin cancer
and other biological effects and it is responsible for the direct photoreaction of
many chemicals in natural sunlight.

3. UV-A band: this is the long wavelength region from 320 to 400 nm, also called
near-UV because it is near the visible region.

The most commonly sources for photostability studies include: day light,
window-glass filtered day light and room light [12]. All these sources can be gener-
ated artificially. The artificial light source should have an output with spectral
power distribution (SPD) as near as possible to the sunlight. This can be achieved by
the use of arc lamps and fluorescent tubes.

3.1.5.2 Drug molecules labile to photodecomposition

A number of medicinal products have been studied for their photostability.

Carbonyl, nitroaromatic and N-oxide functions, aryl halides, alkenes, polyenes and
sulfides are certain chemical functions that are expected to introduce photoreactiv-
ity [13].
Photodegradation of a drug is considered of practical significance if the com-
pound absorbs light >300 nm and the photodegradation becomes evident in a short
period.
Factors that govern photochemical reaction rate include aerobic (most reactions
proceed in presence of oxygen) and anaerobic (N2) conditions, solvents (H2O,
organic solvents), buffers, temperature, metals, intensity of radiation and spectral
distribution of light, drug concentration and volume of the sample [14].
Thus, in formulations that contain low drug concentrations, the primary
photochemical reaction follows first-order kinetics; the kinetics is more compli-
cated at higher concentrations and in the solid state because most of the light is then
absorbed near the surface of the product.
The mechanisms of photodegradation are of such complexity as to have
been fully elucidated in only a few cases. For example, the phenothiazine chlor-
promazine (CLP) is rapidly decomposed under the action of ultraviolet light, the
decomposition being accompanied by discoloration of the solutions (Figure 3).
Chlorpromazine behaves differently towards ultraviolet irradiation under anaerobic
conditions.
A polymerization process has been proposed which involves the liberation of
HCl in its initial stages [5].

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Figure 3.
The effect of ultraviolet light on chlorpromazine (CLP).

The photodegradation of ketoprofen can involve decarboxylation to form an

intermediate which then undergoes reduction, or dimerization of the ketoprofen
itself.

4. Analytical methods

4.1 Method development

Prior the development of any method for the analysis of certain compound
or formulation, there are many factors must be considered before developing
the method and applying it to the intended use. The first step include collecting
information about the analyte itself (the analyte structure and its physicochemical
properties). The mode of detection should be selected (e.g. UV detection). Sample
preparation which may include centrifugation, sonication and filtration. The type
of the diluent also plays an integral role in the analysis as it should be transparent
and does not interfere in the analysis. The stability of the prepared solution, the
mobile phase; stationary phase and mode of elution in case of chromatographic
elution. All these factors and much more should be considered, optimized and the
developed method is then validated and applied for the analysis.

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Pharmaceutical Formulation Design - Recent Practices

4.2 Spectrophotometric methods

4.2.1 UV/VIS spectrophotometry

Absorption spectrophotometry is the measurement of an interaction between

electromagnetic radiation and the molecules, or atoms, of a chemical substance
[15]. Techniques frequently employed in pharmaceutical analysis include UV,
visible, IR and atomic absorption. Spectrophotometric measurement in the visible
region was referred to as colorimetry.
The procedure of UV-unmistakable spectrophotometry includes the estima-
tion of the measure of bright (190–380 nm) or noticeable (380–800 nm) radiation
consumed by a substance in arrangement. Retention of light in both the UV and
unmistakable area of the electromagnetic range happens when the vitality of the
light matches the vitality required to instigate an electronic change and it is related
with vibration and rotational progress in the atom. There are two systems of utilizing
spectroscopic estimations in medication examination, the total and the similar strat-
egies for measure, and the one utilized relies upon which side of the Atlantic Ocean
you complete the investigation. In the UK and Europe the Beer-Lambert condition
will in general be utilized in what is known as the outright technique for examine. In
this strategy the absorbance is estimated tentatively and the Beer-Lambert condition
is comprehended for c, the medication fixation. Hence, the British Pharmacopeia
and European Pharmacopeia quote A1% 1 cm qualities in medication monographs.
In the US Pharmacopeia, the near strategy for test is liked. In this sort of examine a
standard arrangement of the medication to be investigated is readied, the absorbance
of the example and the standard are estimated under indistinguishable conditions,
and the centralization of the example is determined from the relationship:

_ Astd
Atest = _ (1)
[test] [std]

Where [test] is the centralization of the example and [std] is the convergence
of the readied standard. The relative strategy for test has the bit of leeway that
it very well may be utilized regardless of whether the medication experiences a
substance response during the measure (for example development of a shaded
subsidiary to permit estimation in the obvious district of the range), yet experi-
ences the hindrance that a credible example of the medication being referred to
must be accessible for examination. When doing medication examines by spec-
troscopy it is frequently important to set up a scope of groupings of a standard
example of the analyte and measure the absorbance of every arrangement. At the
point when these information are plotted, a straight line of positive incline ought
to be acquired that goes through the inception. Developing diagrams of this sort
not just confirms that the Beer-Lambert law applies to the test at the wavelength of
estimation yet additionally enables the chart to be utilized for alignment purposes.
An answer of obscure fixation is set up in the very same manner as the benchmarks
and its absorbance is estimated at a similar wavelength as the principles. This
absorbance is then perused off the alignment chart and the fixation is determined.
Standard arrangements arranged independently from the example along these
lines are known as outer models. An increasingly thorough system includes the uti-
lization of inside models. An inside standard is an exacerbate that is comparative
in compound structure and physical properties to the example being investigated.
The inner standard ought to be added to the example being referred to before
extraction or measure initiates and is then present in the example framework all
through the consequent test. In the measure of complex examples, some example
pre-treatment is normally required and the recuperation of the example from the

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extraction procedure may not be 100%. On the off chance that an inner standard
is utilized, misfortunes in test will be reflected by comparative misfortunes in the
standard and the proportion of test to standard ought to stay consistent. Inner
measures are especially utilized in chromatographic examination (particularly gas
chromatography and elite fluid chromatography), where fluctuations in instru-
mental parameters (for example flow rate of versatile stage) influence precision. In
certain spectroscopic examinations a comparable way to deal with the utilization
of inner benchmarks is utilized. This is the strategy of standard augmentations
and includes expansion of expanding volumes of a standard arrangement of the
analyte to a fixed volume of the example and development of an alignment dia-
gram. The diagram in a standard expansion examine is of positive incline however
converges they-pivot at a positive estimation of absorbance. The measure of medi-
cation in the example is found by extrapolation of the alignment chart back to the
point where the line crosses the x-pivot (for example at the point when y 0 in the
condition of the line). The strategy for standard increments is generally utilized in
nuclear spectroscopy (for example assurance of Ca2+ particles in serum by nuclear
emanation spectrophotometry) and, since a few aliquots of test are examined
to create the alignment chart, should expand the exactness and accuracy of the
measure. The chief advantage of colorimetric and spectrophotometric methods is
that they provide a simple means for determining minute quantities of substances
[16, 17]. Although spectral interference (degradation products, excipients, etc.)
can often occur, the selectivity and sensitivity of these methods can be improved
by employing an instrumental technique such as derivative spectrophotometry.

4.2.2 Derivative spectrophotometry

In derivative spectrophotometry the absorbance (A) of a sample is differentiated

with respect to wavelength (λ) to generate the first, second or higher order deriva-
tive [18].
In the context of derivative spectrophotometry, the normal absorption spectrum
is referred to as the fundamental, zero order or 0D spectrum.

A = f(λ) dA/dλ = f(λ) d2A/λ2 = f ∆(λ), etc.

Zero order first order second derivative

The first derivative (1D) spectrum is a plot of the rate of change of absorbance
with wavelength against wavelength, i.e. a plot of the slope of the fundamental
spectrum against wavelength. The second derivative (2D) spectrum is a plot of the
curvature of the 0D spectrum against wavelength.
The first order derivative spectrum of an absorption band is characterized by a
maximum, a minimum and a crossover point at λmax of the absorption band. This
bipolar function is characteristic of all odd-order derivatives.
The second derivative spectrum is characterized by two satellite maxima and an
inverted band of which the minimum corresponds to the λmax of the fundamental
band.
A derivative spectrum is therefore gives better resolution of overlapping bands
than the corresponding fundamental spectrum and may permit the accurate deter-
mination of the λmax of the individual bands. Secondly, it discriminates in favor of
substances of narrow spectral band width against those with broad bandwidth. And
consequently, substances with narrow spectral bandwidth display larger derivative
amplitude than those with broad bandwidth [15].
These advantages of enhanced resolution and band width discrimination found
in derivative spectrophotometry permit the selective determination of certain

9
Pharmaceutical Formulation Design - Recent Practices

absorbing substances in samples in which non-specific interference may limit the

application of simple spectrophotometric methods. Ephedrine hydrochloride in
ephedrine hydrochloride elixir is assayed by second derivative spectrophotometry,
which eliminates the broad band absorption of the excipient.
Derivative spectrophotometry has found significant application in clinical,
forensic and biomedical analysis. It has been widely applied in the analysis of
different pharmaceutical dosage forms. It solves the problem of analysis associated
with drug combination, stability studies of drug and degradation products, drug
impurities and interference of excipient in drugs [19, 20]. It also solves the problem
of analysis of drugs in biological fluids.

4.2.3 Difference spectrophotometry

Both selectivity and accuracy of spectrophotometric analysis of samples, which

contain absorbing interferons, may be greatly improved by the technique of dif-
ference spectrophotometry. In difference spectrophotometry assays the measured
value is the difference in absorbance (∆A) between two equimolar solutions of the
analyte, in different chemical forms which exhibit different spectral characteristics.
It is sometimes referred to as differential spectrophotometry.
Certain criteria are required for applying difference spectrophotometry for the
analysis of a substance in the presence of other absorbing substances:

1. Reproducible changes are induced in the spectrum of the analyte by the

addition of one or more reagents.

2. The absorbance of the interfering substances is not altered by the addition of

such reagents.

The simplest and most commonly used techniques for altering the spectral prop-
erties of the analyte is the adjustment of the pH of the solution by means of aqueous
solution of acids, alkali or buffers [21]. The measured value (∆A) in a quantitative
difference spectrophotometric assay can be proportional to the concentration of the
analyte and so it obeys Beer’s law. A modified equation may be derived.

∆A = ∆abc (2)

Where ∆a is the difference absorptivity of the substance at the wavelength of

measurement.
The accuracy and selectivity of the method was found to be increased by con-
version of normal zero-order or differential UV spectra into higher order [21, 22].
Therefore, the application of difference spectrophotometry is expected to have the
totality of advantages of both derivative spectrophotometry (first, second, etc.)
combined with delta spectrophotometry [23].
On the other hand, the stability-indicating property, coupled with the selectivity
and simplicity of application, of the derivative spectrophotometry (first, second,
etc.) and ∆D1 make these methods more preferable to use for drug analysis than the
costly HPLC methods, especially in developing countries.

4.2.4 Colorimetric method

Colorimetric methods, although are generally dependent on functional group

in the drug molecule (NH2, OH, SH), are sometimes utilized as stability-indicating

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methods. This can be achieved by selectively transforming a drug, its degradation

product or its impurity into a derivative so that the spectrum of the derivative is
shifted to the visible region.
There are several parameters, which require careful and critical consideration
in colorimetry. Firstly, the color reagent should be selective for the drug molecule
itself, discriminating degradation products which might be present. Secondly, the
effect of any parameters which can affect the development and stability of the color
should be established. Moreover, the time required to establish the chromophore
should be carefully monitored and assessed.

4.3 Chromatographic methods (HPLC and TLC)

Chromatography is essentially a group of techniques for the separation

of the compounds of mixtures by their continuous distribution between two
phases. One of the two phases is the fixed (stationary) phase, which can be
solid or a liquid supported on a solid. The other phase is a moving (mobile)
phase which can be gas or a liquid that flows continuously around the station-
ary phase.
According to the nature of the mobile phase, chromatography is subdivided into
liquid chromatography (LC) where the mobile phase is a liquid, and gas chromatog-
raphy (GC) where the mobile phase is a gas [15].

4.3.1 Liquid chromatography

There are four classifications for liquid chromatography:

a. Adsorption chromatography: liquid solid chromatography (LSC).

b. Partition chromatography: liquid-liquid chromatography (LLC).

c. Ion exchange chromatography: an ionic liquid mobile phase and a solid poly-
meric stationary phase containing replaceable ions.

d.Size-exclusion chromatography.

The adsorption and partition chromatography (most widely used in pharmaceu-

tical analysis) are subdivided to:

a. Column chromatography and thin-layer chromatography (TLC): solid station-

ary phase and liquid mobile phase.

b. Column, paper and thin-layer chromatography: liquid stationary phase and

liquid mobile phase.

High performance liquid chromatography (HPLC) belongs to the category of

column chromatography and it covers four classes of chromatography: adsorption
(LSC), partition (LLC), ion exchange and size-exclusion.

4.3.2 Thin-layer chromatography (TLC)

Thin-layer chromatography has developed into a very sophisticated technique

for identification of compounds and for determination of the presence of trace

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Pharmaceutical Formulation Design - Recent Practices

impurities. Separation in TLC occurs by either adsorption or partition. For adsorp-

tion, the stationary phase consists of a thin layer of sorbent (e.g. silica) which is
activated by heating at 105°C to evaporate water and the mobile phase is devoid of
water (usually a mixture of organic solvents).
The term retention time used in TLC is referred to as Rf value which is the
distance traveled by the compound from the origin (where the compound is spot-
ted on the plate) divided by the distance traveled by the solvent. Although TLC
is widely used for qualitative analysis, it does not in general provide quantitative
information of high precision and accuracy. Changes in the practice of TLC have
resulted in improved performance of separation and quantitative measurement.
These developments are referred to as high-performance thin-layer chromatogra-
phy (HPTLC) [24].
TLC is widely used in pharmaceutical analysis for:

a. Identification of the components of a mixture by comparing their Rf values

with those of reference standard.

b. Detection of any impurities (synthetic route, stability during manufacturing

process or storage).

c. Separation of a mixture of compounds and recovery by elution technique.

d.Following synthetic reactions for their completion.

e. Forensic application in drug poisoning or addiction.

4.3.3 High performance liquid chromatography (HPLC)

HPLC is the most commonly used technique for the quantification of drugs in
formulations. The principal advantages of HPLC compared to column chromatogra-
phy are improved resolution of the separated substances, faster separation time and
the increased accuracy, precision and sensitivity.
HPLC is based on the same separation modes of column chromatography i.e.
adsorption and partition. Unmodified silica (silanol group) is the most widely used
in adsorption HPLC. Partition HPLC is divided into two categories, normal-phase
and reverse-phase, based on the relative polarities of the stationary and mobile
phases.
In order to develop HPLC method and to select the appropriate column type, the
analyst needs to know:

a. Suitable solvent for the drug.

b. Molecular structure.

c. Nature of analysis: whether for quantification analysis or stability-indicating

method.

The following diagram (Figure 4) gives a general guide to the selection of a

chromatographic method for separation of compounds of molecular weight ˂ 2000;
for samples of higher molecular weight the method of choice would be size-exclu-
sion [25, 26].

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Figure 4.
General guide for selection of chromatographic method.

5. Quality assurance and quality control

In the pharmaceutical Industry, quality management is defined as the aspect of

management function that determines and implements the quality policy.
The major elements of quality management are:

a. A quality system describing the organizational structure, procedures, pro-

cesses and resources.

b. A systematic action or actions necessary to ensure adequate confidence that a

product (or service) will satisfy given requirement for quality.

The concepts of quality assurance, GMP and quality control are interrelated
aspects of quality management. They are inter-related and have fundamental
importance to the production and control of pharmaceutical products.
In fact quality assurance covers quality control in exactly the same manner as
it covers other functions such as manufacturing and ware-housing. It approves
methods and standards and sees to it that good laboratory practices are operative.
Each manufacturing unit must have a quality control department independent
from the production and other departments and under the control of a qualified
and experienced personnel and has one or several quality control laboratories at his
or her disposal.
Quality control is integral part to all modern industrial processes and the
pharmaceutical industry is not an exception. Testing a pharmaceutical product
involves physical, chemical and sometimes microbiological analysis. It is a critical
function of any business offering a product or service to consumers. In the field of
pharmaceutical chemistry, quality control is vital to the successful development,
manufacturing, and use of drugs meant to save lives. It determines the quality
and stability of drug products via pharmaceutical analysis; it includes areas such
as method validation, handling raw materials and finished products, documenta-
tions, inspections that impact the development of pharmaceutical products that are
governed by specified rules.
Pharmaceutical products are developed and produced according to GMP
requirements and other associated codes e.g. good laboratory practices (GLP), and
good clinical laboratory practices (GcLP), … etc.

13
Pharmaceutical Formulation Design - Recent Practices

Production and control operations are clearly specified in a written form i.e.
standard operating procedures (SOP’s) and GMP requirements are adopted.
Control procedures on starting materials, intermediate products and finished
products and other in process controls should be carried out according to written
and validated procedures.
The finished products should be correctly processed, checked, packed according
to defined procedures.
Finished pharmaceutical products should not be sold or supplied unless they are
released by an authorized person.

6. Method validation and statistical interpretation of the analytical

method

The function of the analyst is to obtain a result as near to the true value
as possible by the correct application of the analytical procedure employed.
Quantitative analysis is not simply a case of taking sample, carrying out a single
determination and then claim that the value obtained is irrefutable. It also
requires knowledge of the chemistry involvements and the possibilities of the
interferences from other ions, elements and compounds as well as of the statisti-
cal distribution of values [27].
Different errors may occur during the analysis process which should be well
noticed and overcome. There are three types of errors:

a. Gross errors: easily recognized as it leads to definite unreliable results: could be due
to contaminated reagents, defective instruments, accidental loss of crucial sample
etc. It is defined as a serious error so that there is no way to correct the experiment.

b. Random error (in determinate error): the average of the results are very close
to the true value, so there is no evidence of bias i.e. some results could be high
and some results could be low. Arises from sources that cannot be corrected i.e.
the degree of sensitivity of the balance: fourth decimal, fifth decimal. Types of
burettes or pipettes (A, B, C types) etc.

c. Systematic (determinant errors): this causes all the results to be in error in the
same sense (constant error). May be due to (1) faults in analytical procedure or
(2) the equipment used. Observed result could be too low or too high i.e. inac-
curacy should be constant (all answers are 10% too high or too low). Example:
True value for three samples were 25, 20 and 30% assay result was 35, 30 and
40% respectively i.e. 10% too high. It makes the assay precise but inaccurate
sometimes the inaccuracy may be proportional to the true answer, giving rise
to proportional error such as 10% of the answer i.e. for the above example for
25% result is 27.5%, 22% for 20% and 33% for30%.

Validation of methods for the quantitative analysis of drugs involves determin-

ing as a minimum, their selectivity, and limit of detection, limit of quantification,
linearity, working range, accuracy and precision [28].

6.1 Accuracy

Accuracy is a measure of how closely the result of an experiment agrees with

the expected result. The difference between the obtained result and the expected

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result is usually divided by the expected result and reported as a percent relative
error [29].

6.2 Precision

Precision is a measure of how close a set of results are to each other [30]. It
is often measured under repeatable (same analyst, same day, same instruments
and same materials) and reproducible conditions. Precision always accompanies
accuracy, but a high degree of precision does not imply accuracy.

6.3 Linearity

For any developed analytical method, standard curve is constructed to verify the
linear relationship between the concentration and a characteristic parameter for a
component such as peak area, peak height or peak ratio in chromatographic analysis
or UV-absorption in spectrophotometry.
Most analytical methods are based on processes where the method produces
a response that is linear and which increases or decreases linearly with analyte
concentration. In other words, it is the ability of the method to elicit test results that
directly proportional to the concentration of analyte within a given range.
Statistical application is important in evaluating calibration graphs in instru-
mental analysis. The equation of a straight line takes the form:

y = a + bx (3)

Where a is the intercept of the straight line with the y axis and b is the slope of
the line.
The statistical measure of the goodness of the fit of the line through the data
is the correlation coefficient “r”. It falls in the range −1 ≤ r ≥+1. Negative r-values
indicate negative slope and vice-versa. It is important to note that calculated
r-values can be sometimes misleading and a calibration curve must be physically
plotted to ensure the shape of the plot. From the calculated regression line data,
the concentration of the analyte can be estimated by interpolation. Each value of
y is subjected to random error and likely an error in the slope and intercept values
can occur. This can be resolved by calculating standard deviations of the slope (Sb)
and intercept (Sa). Sb and Sa are obtained from a calculated statistic value Sy/x [29].
The values of Sb and Sa are used to calculate the confidence limits for the slope and
intercept using a t-value at a desired confidence level, normally 95% level. These
limits are important to indicate if there is a significant difference between these
values and certain true values, which reflects the effect of random or systemic
errors.

6.4 Limit of detection

The limit of detection is the lowest content of analyte that can be distinguished
from background noise and measured with reasonable statistical certainty. It can be
calculated by the reduced formula:

3SB / b (4)

Where SB = Sy/x (calculated from the regression analysis data), b is slope [29].

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Pharmaceutical Formulation Design - Recent Practices

6.5 Limit of quantification

The lower limit of quantification is the amount equal to or greater than the
lowest concentration point on the calibration curve that can be measured with an
acceptable level of accuracy and precision [29].
It can be calculated by the equation:

10SB / b (5)

Where SB = Sy/x (calculated from the regression analysis data), b is slope.

6.6 Method comparison

The comparison of two methods should be carried out using a suitable statisti-
cal procedure to test if there are significant differences between them. The t-test
provides a simple check on accuracy and the F-test on precision. These tests require
the knowledge of what is known as the number of degrees of freedom.

7. Conclusion

Pharmaceutical products must be analyzed regularly to ensure their safety and

effectiveness. This chapter described the quality assurance and quality control of
materials and finished products. The requirements to develop a suitable method and
its validation. Also different analytical methods and their application in the field of
pharmaceutical analysis was also discussed.

Conflict of interest

The author declares that there is no conflict of interest.

Author details

Shaza W. Shantier
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of
Khartoum, Sudan

*Address all correspondence to: [email protected]

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms
of the Creative Commons Attribution License (http://creativecommons.org/licenses/
by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,
provided the original work is properly cited.

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