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Chapter 1 Introduction

School of Pharmaceutical Sciences, Shobhit University 1

1. Introduction

Analytical chemistry plays an important role in nearly all aspects of pharmaceutical sciences. It

seeks ever improved means of measuring the chemical composition of natural and artificial

materials. The techniques of this science are used to identify the substances which may be

present in a material and to determine the exact amounts of the identified substances1.

1.1. Introduction to Analytical Methods

The ability to provide timely, accurate, and reliable data is central to the discovery, development,

and manufacture of pharmaceuticals. Analytical data is used to screen potential drug candidates,

aid the development of drug synthesis, support formulation studies, monitor the stability of bulk

pharmaceuticals and formulated products, and test final products for release. The quality of

analytical data is a key factor in the success of a drug development program.

Problems increase as additional persons, laboratories and equipments are used to perform the

method. When the method is used in the developer’s laboratory, a small optimization can usually

be made to develop the method work. The flexibility to change it is lost once the method is

transferred to other laboratories or used for official product testing. Where methods are

submitted to regulatory agencies, changes may require formal approval before they can be

implemented for official testing. The best way to minimize method problems is to perform

adequate validation experiments during development2.

The need of sophisticated analytical instruments and determinations using them is almost a

routine process for the modern analytical laboratories. Analytical methods are generally

classified as physical and chemical. Physical analysis includes measurement of particle size,

dimension, thickness of a solid dosage forms etc. Basically chemical analysis can be divided into

three broad categories3;

Qualitative analysis: analysis, which identifies the component(s) in a sample.

Quantitative analysis: analysis, which finds out total amount of the particular species

present in a sample.

Structural analysis: analysis which helps in finding the spatial arrangement of atoms in

a molecule and the presence or position of certain organic functional group in a given

compound.



Chemical analysis has some basic steps like choice of method, sampling, preliminary sample

treatment, separations, final measurement and assessment of results. It is with the first step, viz.

choice of method, that care should be exercised to select the proper instrument to carry out

fruitful analysis. A wrong selection at this point will lead to a meaningless analysis. Analytical

methods are broadly classified as physical, chemical and instrumental analysis.

Physical observation includes description of the compound, measurements of its

dimension (shape, size), color, odor etc.

Chemical analysis includes titrimetric analysis of the compound such as potentiometry,

iodometry, argentometry etc.

Instrumental methods of chemical analysis have become the backbone of the

experimental chemistry. The choice of an instrumental method for the determination of a

specific element or compound involves:

1. the instrument to be used, and

2. the chemical system.

The growth of instrumental analysis is related to the developments in the field of electronics

because the generation, transduction, amplification and display of a signal can be done in a

convenient manner4.

Various instrumental methods along with their major applications in pharmaceutical field are

presented in Table 1.1.

Table 1.1: Various instrumental methods of analysis4

S.N. Instrumental Method Property Measured Applications

1.

UV- Visible

Spectrophotometry

Absorption of radiation Identification of the

functional groups

Quantitation of unsaturated

compounds

2. FTIR Spectroscopy Absorption of radiation Quantitative analysis of

organic compound at high

concentration level

3.

Atomic Absorption

Spectroscopy

Absorption of radiation Quantitation of metals or

metalloids

4. Flame Photometry Emission of radiation Quantitation of alkali

metals or alkaline earth

metals



S.N. Instrumental Method Property Measured Applications

5.

X-Ray Diffraction Diffraction of radiation Identification of crystal

lattice structures

Determination of

percentage of crystallinity

in polymers

6. Nuclear Magnetic

Resonance (LC-NMR)

Nuclear spin energy level

of a molecule in an

applied magnetic field.

Identifies types of

hydrogen and carbon in

organic molecules

Analysis of trace impurities

and degradants

7.

Thermal Analysis

(DTA \ DSC)

Difference in

temperature\ heat energy Determination of melting

point, polymorphism

Drug – excipients

compatibility

8. Mass Spectrometry

(LC-MS \ GC-MS)

Mass to charge ratio

(m\e) Molecular weight

determination

Quantification of the

analyte in liquid or gas

samples

Analysis of the biological

samples

Analysis of trace impurity

and degradants

1.2. History of Chromatography

The word chromatography comes from the Greek words for color “chroma” and write

"graphein". So chromatography means 'to write with color'. Chromatography was first developed

and defined by the Russian botanist Mikhail Tswett (1872-1919) in 1903. He separated colorful

plant pigments using a column of calcium carbonate (chalk). Tswett stated that:

“Chromatography is a method in which the components of a mixture are separated on an

adsorbent column in a flowing system”.

The history of chromatography did not stop with this 'adsorption chromatography' but the

technique has been modified in many ways5. This resulted in different types of chromatography.

General classification of chromatography is presented in Table 1.2.



Table 1.2: General classification of chromatography5

General Classification Specific Method Stationary Phase Type of Equilibrium

Liquid chromatography

Liquid-liquid or partition

Liquid adsorbed on a

solid

Partition between

immiscible liquids

Liquid-solid or adsorption

Solid

Adsorption

Ion exchange

Ion exchange resin

Ion exchange

Size exclusion Liquid in interstices

of a polymeric solid

Partition/sieving

Gas chromatography

Gas –liquid

Liquid adsorbed in

solid

Partition between gas

and liquid

Gas –solid

Solid Adsorption

Supercritical –fluid

chromatography

Supercritical fluid – bonded

surface

Organic species

bonded to a solid

surface

Partition between

supercritical –fluid

and bonded surface

Chromatography is a physical method of separation in which the components to be separated are

distributed between two phases, one of which is stationary (stationary phase) while the other

(mobile phase) moves in a definite direction.

Chromatography involves a sample or sample extract dissolved in a mobile phase which may be

a gas, liquid or supercritical fluid. The mobile phase is then mode to flow through an immobile,

immiscible stationary phase, which is fixed in place in column or on solid surface. The two

phases are selected so that the components of the sample distribute themselves between the

mobile and stationary phases to varying degrees. Those components strongly retained by the

stationary phase move slowly with the flow of mobile phase. In contrast, components that are

weakly held by the stationary phase travel rapidly. As a consequence of these differences in

migration rates, sample components separate into discrete bands or zones that can be analyzed

qualitatively and quantitatively.

These techniques are useful in qualitative and quantitative analysis. Paper and thin layer

chromatography are useful for identification purposes, because of their convenience and

simplicity. Column chromatography offers wide choice of stationary phases and is useful for the

separation of various compounds. Both gas chromatography and high performance liquid



chromatography require more elaborate apparatus and usually provides high resolution that will

identify and quantitate very small amounts of materials (in micro range) 6

.

1.3. High Performance Liquid Chromatography

HPLC originated from the fact that high pressure was needed to generate the flow required for

liquid chromatography in packed columns. This is considered the most powerful approach; the

sample passes through a column or a device containing appropriate particles. These particles are

called the chromatographic packing material, stationary phase or “adsorbent”. Solvent flows

continuously through the column. At a point in time, an “injection” of the sample solution is

made into the solvent stream, which then carries the sample through the column. For improved

separation power, smaller particle sizes (<10 microns) are required. These cause greater

resistance to flow, resulting in higher pressures needed to maintain the required solvent flow.

The early 1970’s saw a tremendous leap in technology. The new “HPLC” instrument was

developed with up to 6,000psi (400 bar) of pressure, which included improved detectors and

columns. HPLC really began to take hold in the mid to late 1970s. With continued advances in

performance, the name was changed to High Performance Liquid Chromatography (HPLC)6.

High Performance Liquid Chromatography (HPLC) is now one of the most powerful tools in

analytical chemistry, with the ability to separate, identify and quantitate the compounds that are

present in any sample that can be dissolved in a liquid. HPLC can be applied to just about any

sample such as pharmaceuticals, food, cosmetics, environmental matrices, forensic samples, and

industrial chemicals7.

The techniques of HPLC can be divided into four categories depending on the nature of the

process that occur at the stationary phase as given below;

1.3.1. High Performance Adsorption Chromatography

Here the analyte is adsorbed onto the surface of polar packing. The nature of the adsorption

involves the interaction of polar molecules with a very polar solid stationary phase.

1.3.2. High Performance Partition Chromatography

It is the most widely used liquid chromatographic procedure to separate most kinds of organic

molecules. Here the components present in the analyte mixture distribute themselves between the

mobile phase and stationary phase as the mobile phase moves through the column. The

stationary phase actually consists of a thin liquid film either adsorbed or chemically bonded to

the surface of finely divided solid particles.



1.3.3. High Performance Ion-Exchange Chromatography

This method is used to separate mixture of ions and especially for protein separation. The

stationary phase consists of polymeric resin beds on which the ions get attracted or bonded

reversibly while passing through the column.

1.3.4. High Performance Size-Exclusion Chromatography

This technique is used for separating dissolved species on the basis of their size and is

particularly applicable to high molecular weight species like polymers and oligomers to

determine their relative size and molecular weight distribution8.

Different modes of separation in HPLC are presented in Table 1.3.

Table 1.3: Modes of separation in HPLC8

Mode Stationary Phase Mobile Phase Interaction Feature

Normal phase

chromatography

Silica gel Organic solvent

(n-Hexane/IPE)

Adsorption Separation of

lipid compounds

Reversed phase

chromatography

Silica-ODS Methanol/water Hydrophobic Separation of

polar compounds

Size exclusion

chromatography

i) Non- aqueous (GPC)

ii) Aqueous (GFC)

Porous polymer

Aqueous- porous

polymer

Organic solvent

Buffer solution

Gel

permeation

Gel filtration

Mol. Wt.

distribution

Protein

separation

Ion exchange

chromatography

Ion exchange gel

Buffer solution Ion exchange Separation of

ionic compounds

Affinity

chromatography

Packing with

ligands

Buffer solution Affinity Purification of

enzymes and

proteins

1.4. Instrumentation of High Performance Liquid Chromatography9-13

The liquid chromatography system consists of six basic units. The mobile phase supply system,

pump, programmer, sample valve, column, detector and finally a means of presenting and

processing the results. Schematic diagram of HPLC system is shown in Figure 1.1.



Figure 1.1: Schematic diagram of an HPLC system9

1.4.1. Mobile Phase Reservoir

The mobile phase supply system consists of number of reservoirs (200 ml to 1,000 ml in

capacity). At least two reservoirs would be necessary and are usually constructed of glass or

stainless steel and contain an exit port open to air. Stainless steel, however, is not considered

satisfactory for mobile phases buffered to a low pH and containing certain materials that can

cause corrosion. Each reservoir is usually fitted with a gas diffuser through which unwanted

gases can be bubbled.

1.4.2. Pumping System

There are a number of different types of pumps that can provide constant and optimum pressure

and flow rate required by the modern liquid chromatography. They are; reciprocating piston

pump, syringe type pump, and constant pressure pump.



Reciprocating Piston Pump

The basic principle of piston pump is to expel liquid through a one-way valve (check valve). The

pumping rate is usually adjusted by controlling the distance that piston retracts, thus limiting the

amount of liquid pushed out by each stroke.

Dual Piston Pump

A more efficient way to provide a constant and almost pulse free flow is the use of dual-headed

reciprocating pumps. Both pump chambers are driven by the same motor through a common

device; this common device allows one piston to pump while the other is refilling.

Syringe Type Pump

These are most suitable for small-bore columns because these pumps deliver only a finite volume

of mobile phase before it has to be refilled. These pumps have a volume between 250 to 500 ml.

The pump operates by a motorized lead screw that delivers mobile phase to the column at a

constant rate. The rate of solvent delivery is controlled by changing the voltage on the motor.

Constant Pressure Pump

In this type of pump system the mobile phase is driven through the column with the use of

pressure from a gas cylinder. A low-pressure gas source is needed to generate high liquid

pressures. The valve arrangement allows the rapid refill of the solvent chamber whose capacity is

about 70 ml. Constant pressure pump is responsible to deliver continuous flow rate of mobile

phase.

1.4.3. Sample Injection System

HPLC injector system divided into four types as discussed below:

Type 1 injectors utilize a completely filled sample loop to determine the injected volume. These

simple and reliable devices are six-port rotary valves. A syringe is used to push or suck an excess

of sample into a sample loop, filling it completely.

Type 2 injectors utilize a micro syringe to transfer sample into the loop. The sample size is

always smaller than the loop volume, so it is the syringe which determines the injected volume.

No sample is trapped or wasted, but the precision is not as high as type 1 injector.

Type 3 injectors utilize both complete and partial filling methods, but trap some sample. The

loop is loaded by inserting the syringe into the needle port and dispensing the contents. The

syringe is left inserted in the port until after the valve is switched. The switching action inserts

the loop into the stream without exposing the syringe to high pressure. In the injection position the



syringe is removed and some sample remains trapped in a connecting passage of the injector.

Type 4 injectors also utilize both methods, but do not trap sample. This type is similar to type 3

injector but it does not contain a connecting passage between syringe needle tip and sample loop.

It therefore not traps sample and there is no sample waste, no syringe reading error and no need

to flush between injections, except in trace analysis9.

1.4.4. Stationary Phase (Column)

In liquid chromatography stationary phase provides retention and influences the separation

mechanism of the analytes present in the sample. Characteristics of stationary phase such as

surface chemistry of column packing material, carbon chain length, end capping, base

deactivation, particle size, pore size and specific surface area play vital role in development of

efficient analytical method to control overall separation. Official monographs in pharmacopoeia

for drugs give restricted information about selection of stationary phase which draws confusion

amongst analysts to use efficient column to quantify the strength of drug.

Column is referred as heart of HPLC separation process. Stable high performance column is

essential requisite for rugged and reproducible method. For high efficiency of separation large

number of theoretical plates per unit length of the column is required. Prior to selection of

column it is necessary to understand the properties of column packing material. Silica is used

extensively for such purpose but having a draw back as it tends to dissolve above pH 8 and

cross-linking of polymeric particles takes place above this pH, for example, polystyrene or poly

methacrylates is used for separation of bases, which can withstand strongly basic mobile phase

but are somewhat less efficient. Silica particles have surface silanol groups, -SiOH which are

used for chemical bonding of stationary phases by silanization reactions with chlorosilanes.

About half the silanol groups are chemically bonded and the rest are end capped with trimethyl

silyl groups to render them inert. The most common non-polar bonded phases (for reversed

phase chromatography) are C18 and C8.

C18 columns are the most popular (known as ODS -octadecylsilane) and highly non polar in

nature. C8 (known as octylsilane) is intermediate in hydrophobicity. Increase in polarity of

column for RP-HPLC leads to decrease in retention of non polar sample. C18 (Octaldecylsilane –

ODS) bonded ligand is particularly useful for separation of non-polar analyte. C8 bonded ligand

provides similar selectivity and shorter run times. C4 and C3 bonded ligand are also available but

these are not as stable as longer chain C18 and C8 counterparts, but provide good separations for a



variety of protein and polypeptide analytes. CN (Cyano) bonded ligand interacts with polar

functional groups, and is used in both reversed-phase and normal phase chromatography. NH2

(amino) bonded ligand interacts with polar functional groups, commonly used for the separation

of sugars and polysaccharides. Phenyl bonded ligand exhibits a more polar nature than either the

C18 or C8, the π-electron clouds providing sites of interaction for a variety of aromatic (ring)

analytes. Several other factors that contribute to appropriate column selection are as follows:

Longer columns are used for increased resolution and shorter columns have application in

shorter analysis time, and fast chromatography. Wider diameter columns are used for greater

sample loading. Narrow columns are used for more sensitive and reduced mobile phase

consumption. Columns with spherical particles are used for lower backpressure, greater stability.

Columns with irregular particles are used for high surface area and high sample capacity; smaller

particle (3-4 µm) size of packing material are used for higher separation efficiencies and larger

particle (5-10 µm) size generate less system pressure. Columns of pore size of 150Ǻ or less are

used for sample with molecular weight less than 2000 and pore size of 300Ǻ or less are used for

sample with molecular weight greater than 2000. Columns with end-capped packing are used to

eliminate unpredictable secondary interaction with the base materials, while non-end capped

stationary phases are used for selectivity differences of polar compounds by controlling

secondary interaction. High carbon loads in column are used for greater sample capacities and

higher resolution on other end less carbon load in column provides faster analysis.

There are various columns that are secondary to the separating column or stationary phase

namely: guard column, derivatising column and capillary column.

Guard Column

It is placed anterior to the separating column. This serves as a protective factor that prolongs the

life and usefulness of the separation column. It is dependable columns designed to filter or

remove particles that clog the separation column, compounds and ions that could ultimately

cause “baseline drift”, decreased resolution, decreased sensibility and false peaks, compounds

that may cause precipitation upon contact with the stationary or mobile phase and compounds

that might co-elute and cause extraneous peaks.

Derivatising Column

Pre and/or post-primary column derrivatization can be an important aspect of the sample

analysis. Reducing or altering the parent compound to a chemically related daughter molecule or



fragment elicits potentially tangible data, which may complement other results or prior analysis.

Acetylation, silylation, or concentrated acid hydrolysis are few derivatization techniques

employed for sample analysis10

.

Capillary Column

Advances in HPLC led to smaller analytical columns. Such columns are also known as micro

columns. Capillary columns have a diameter less than a millimeter and are of three types; open

tubular, partially packed, tightly packed. The columns have advantages as they allow the user to

work with nano liter sample volumes, decreased flow rate and decreased solvent volume usage

which may ultimately results in less cost per analysis11

.

1.4.5. Detector

The detector for HPLC is the component that emits a response due to the eluting sample

compound and subsequently signals a peak on the chromatogram. It is positioned immediately

posterior to the stationary phase in order to detect the compounds as they elute from the column.

There are many types of detectors that can be used with HPLC. Most common detectors include:

Refractive Index (RI), Ultra- Violet (UV), Fluorescent, Radiochemical, Electrochemical, Near

Infra-Red (Near IR), Mass Spectroscopy (MS), Nuclear Magnetic Resonance (NMR) and Light

Scattering detectors12

.

Refractive Index Detector

Refractive index detectors measure the ability of sample molecules to bend or refract light. This

property for each molecule or compound is called its refractive index. For the most RI detectors,

light proceeds through bi-modular flow-cell to photo detector. One channel of the flow-cell

directs the mobile phase passing through the column while the other directs only the mobile

phase. Detection occurs when the light is bent due to samples eluting from the column, and this

is read as a disparity between the two channels.

Ultra-Violet Detector

Ultra-violet detectors measure the ability of a sample to absorb light. Fixed wavelength detector

works at one wavelength only. Most common wavelength employed for analysis is usually 254

nm. Variable wavelength detector works at one wavelength at a time, but can detect over wide

range of wavelength.

Diode Array Detector

Diode array detector measures a spectrum in varying wavelength simultaneously.



Fluorescence Detector

Fluorescence detectors measure the ability of a compound to absorb and then re-emit light at

given wavelengths. Each compound has characteristics fluorescence. The excitation source

passes through the flow cell to a photo detector while a monochromator measures the emission

wavelengths. It has sensitivity limit of 10-9

to 10-11

gm/ml.

Radiochemical Detector

Radiochemical detection involves the use of radio labeled material, usually tritinium (3H) or

carbon (14

C). It operates by detection of fluorescence associated with β particle ionization, and is

most popular in metabolite research. Radiochemical detectors are of two types:

Homogeneous Detector: Homogeneous detector is used when addition of scintillation fluid to

column effluent causes fluorescence.

Heterogeneous Detector: Heterogeneous detector is used when lithium silicate and fluorescence

caused by beta-particle emission interacts with the detector. It has sensitivity

between 10-9

to 10-10

gm/ml.

Electrochemical Detector

Electrochemical detector measures compounds that undergo oxidation or reduction reactions.

Usually detection of components is accomplished by measuring gain or loss of electrons from

migrating samples as they pass between electrodes at given difference in electrical potential. It

has sensitivity limit between 10-12

and 10-13

gm/ml.

Mass Spectroscopy Detector

Here the sample compound or molecule is ionized, then it is passed through a mass analyzer and

the ion current is detected. There are various methods for ionization:

Electron impact ionization: An electron current or beam created under high electric potential is

used to ionize the sample migrating off the column.

Chemical ionization: A less aggressive method which utilizes ionized gas to remove electrons

from the compounds eluting from the column.

Fast atom bombardment: Xenon atoms are propelled at high speed in order to ionize the eluents

from the column. It has the detection limit of 10-8

to 10-10

gm/ml.

Nuclear Magnetic Resonance Detector

Certain nuclei with odd numbered masses, including H and 13

C, spin about an axis in a random

fashion. However, when sample is placed between poles of a strong magnet, the spins are aligned



either parallel or anti-parallel to the magnetic field, with the parallel orientation favored since it

is slightly lower in energy. The nuclei are then irradiated with electromagnetic radiation which is

absorbed and places the parallel nuclei. Each H or C produce different spectra depending on their

location and adjacent molecules, or elements in the compound, because all nuclei in molecules

are surrounded by electron clouds which change the encompassing magnetic field and thereby

alter the absorption frequency, state, consequently, they are now in “resonance” with the

radiation.

Light Scattering Detector

When a source emits a parallel beam of light, which strikes particles in solution, some light is

reflected, absorbed, transmitted, or scattered. There are two forms of LS detection that may be

used to measure the absorbance or transmittance:

Nephelometric Detector: This is based on the measurement of light scattered by a particulate

solution. This method enables the detection of the portion of light scattered at a multitude of

angles.

Turbidimetric Detector: This is based on the measurement of the reduction of light transmitted

due to particles in solution. Therefore, it quantifies the residual light transmitted.

Near Infrared Detector: It operates by scanning compounds in a spectrum from 700 to 1100 nm.

Stretching and bending vibrations of particular chemical bonds in each molecule are detected at

certain wavelengths. This is a fast growing method which offers several advantages: speed of

analysis (sometimes less than 1 second), simplicity of preparation of sample, multiple analyses

from single spectrum, and non-consumption of the sample.

1.4.6. Data System

Since the detector signal is electronic, use of modern data acquisition techniques can aid in the

signal analysis. The main goal in using electronic data systems is to increase analysis accuracy

and precision, while reducing operator attention.

1.4.7. Backpressure Regulator

As a final system enhancement, a backpressure regulator is often installed immediately after the

detector. This device prevents solvent bubble formation until the solvent is completely through

the detector. This is important because bubbles in a flow cell can interfere with the detection of

sample components.



1.5. Applications of HPLC

High performance liquid chromatography (HPLC) is a very powerful and flexible tool that forms

the analytical backbone for the entire drug development process, from discovery to the final

release of commercial product. HPLC is widely used in pharmaceutical industry, food and

beverage industry, research and development, quality control, production process control,

environmental analyses and clinical tests. The reasons of popularity of the methods are

sensitivity, ready adaptability to accurate quantitative determination and its widespread

applicability. It exhibits high resolving power, speedy separation, continuous monitoring of

column effluent, accurate quantitative measurement, repetitive and reproducible analysis using

the same column, automation of analytical procedure and data handling, evaluating reaction

conditions, in process monitoring for reaction conversion and determining final purity14

.

1.6. General Considerations for HPLC Method Development

General considerations for HPLC method development are shown in the form of flow chart in

Figure 1.2 and preferred experimental conditions for the initial HPLC run are presented in Table

1.4.



Gather information from the literature and the other sources about the

physiochemical properties of the API (active pharmaceutical ingredients)

Determine solubility profile and select λmax

Select chromatography method

(Based on solubility study, retention of the compounds etc.)

Reverse phase chromatography Normal phase chromatography

[Water soluble API (ionic/ non-ionic), [API soluble in non-polar organic solvent,

organic soluble API (polar or non-polar) etc.] sample too hydrophilic or hydrophobic]

Perform initial run for HPLC conditions

Select gradient or isocratic mode

Perform trials to select the optimum conditions for separation

(By changing mobile phase composition, buffer, pH,

column, flow rate, temperature etc)

Perform degradation experiments to challenge the method

Define system suitability parameters

Summarize methodology

Prepare development report

Validate the method

Figure 1.2: General considerations for HPLC method development



Table 1.4: Preferred experimental conditions for the initial HPLC run

1.6.1. Various Steps for Method Development15-16

Solubility Studies

Solubility studies are performed for the establishment of solubility of API (Active

Pharmaceutical Ingredient) in a number of aqueous and organic solvents like water, buffers,

sodium hydroxide, methanol, acetonitrile, chloroform, hexane, tetrahydrofuran (THF) etc. during

the method development. The API should have good solubility in the selected diluents

(preferably 1mg/ml). UV scan is performed in the range of 200-400 nm in the selected solvent to

ascertain the spectrophotometric properties of the drug. The solvent is selected as such, which

covers the range of polarities that are commonly used in the method development.

Selection of the Sample Diluents

The selection of the sample diluents should be suitable for intended use of the method. Physical

properties of commonly used solvents are presented in Table 1.5. The ideal sample diluent

should following properties-

It should dissolve the major analyte.

It should not interfere with analyte response.

It should prevent analyte interaction with container surfaces.

Separation Variables

Initial Conditions

Column

Dimensions (length, diameter)

Particle size

Stationary phase

150x4.6mm

5µm (3.5µm alternatively)

C8 or C18

Mobile Phase

Solvent A and B

% of strong solvent

Buffer (compound, pH, and concentration)

pH range

Additives (eg. Ion pair reagent, amine modifier)

Buffer-Acetonitrile

80-100%

25mM Potassium phosphate

2.0 < 3.0

Do not use initially

Flow Rate 1.5-2.0 ml/min

Temperature 35- 450C

Sample Size

Volume

Weight

< 25µl

< 100µg



Table 1.5: Physical properties of commonly used solvents15

Selection of the Wavelength

The selection of the wavelength is a critical step in the method development. To select the

wavelength, the standard solution at the required concentration in the selected solvent is prepared

and scanned on UV- spectrophotometer. Based on the UV scan results, the test solution is

injected into HPLC system equipped with the photo diode array detector and the spectrum is

collected. The wavelength, which gives the optimum response for the drug component is

selected.

Normal Phase Chromatography

The first choice for developing the HPLC method should be reverse phase chromatography.

Normal phase chromatography is preferred if:

Sample is dissolved in non-polar solvents like hexane, chloroform, dichloromethane etc.

(will cause direct injection problem if reverse phase column is used).

The sample is too strongly retained by the reverse phase chromatography and no peak

could be obtained for 50-60 minutes, even with 100% organic phase.

The sample is not retained by the reverse phase chromatography.

Reverse phase chromatography separation is unable to achieve adequate band spacing.

Sample contains positional isomers, stereoisomers, and diastereomers.

Normal phase is useful for the compound that decomposes in the aqueous phase.

Solvent

Polar

Index

Viscosity

(Poise)

Density

(g/ml)

Refractive

Index (at 25°C)

UV Cutoff

(nm)

B. P.

(°C)

Acetic acid 6.2 1.1 1.049 1.37 230 118

Acetone 5.4 0.3 0.791 1.356 330 56

Acetonitrile 6.2 0.34 0.786 1.341 210 82

Benzene 3 0.6 0.879 1.498 280 80

Methanol 6.6 0.54 0.791 1.326 210 65

Carbon

tetrachloride

1.6 0.9 1.594 1.457 265 77

Chloroform 4.4 0.53 1.483 1.443 245 61

Cyclohexane 0 0.9 0.744 1.423 210 81

THF 4.2 0.46 0.881 1.405 220 66

Ethyl Acetate 4.3 0.43 0.9 1.37 260 77

Hexane 0.06 0.3 0.659 1.372 210 69



In normal phase chromatography, the stationary phase is more polar than the mobile phase and

hence sample retention increases as the polarity of the mobile phase decreases.

Normal phase chromatography in HPLC typically encompasses adsorption chromatography on

silica and partition chromatography on cyano and amino bonded phases. Unique separations are

provided for saccharides, isomers, steroids, and class separations of lipids and poly nuclear

aromatic hydrocarbons.

For normal phase HPLC, initial trials can be taken by using 100% strong solvent like

isopropyl alcohol (IPA) with cyano column (250 x 4.5 mm, 5µ), to ascertain that all components

elute at 0.5 < K< 20. Subsequently using hexane as the weak solvent and to decrease the % of the

IPA. A gradient of 100% IPA may alternatively be used to estimate if gradient is necessary.

To change selectivity based on the strong solvent, IPA may be replaced by the methyl chloride,

MTBE (methyl-1-butyl-ether), acetonitrile or ethyl acetate, etc. However, the selection of

solvents depends on the type of detection. Some stationary phases available for the normal phase

roughly in the order of decreasing utility for the separations are Cyano > Silica > Diol > Amino,

etc. Triethylamine and acetic acid can be added to the mobile phase for the basic and acidic

compounds respectively, to prevent tailing.

Reverse Phase Chromatography

Depending upon the nature of the API (acidic/basic/neutral), an initial exploratory run using the

gradient elution can be performed by selecting the conditions. After the initial exploratory run,

the chromatogram is evaluated before proceeding with the next injection and subsequent

adjustments should be made to the mobile phase compositions, pH of the buffer, column

packing, column temperature, flow rate etc. to get the optimum conditions for separations. Each

subsequent injection is thus based on the previous conditions, so that after a number of

injections, the proper separation conditions can be found. Separations in reversed phase

chromatography depend on the reversible adsorption/desorption of solute molecules with varying

degrees of hydrophobicity to a hydrophobic stationary phase.

Selection of Isocratic or Gradient Mode

Similar conditions govern the design of both isocratic and gradient mode. A blank gradient

should be carried out initially, to ensure that there are no problems with the baseline. The initial

run begins with a linear 5 to 100% acetonitrile-buffer gradient for 60 min at a flow rate 2.0



ml/min and then estimating the best initial and final % of strong solvent. The run is repeated to

confirm the reproducibility of the separation.

To deciding whether a gradient mode or isocratic mode would be adequate, an initial gradient

run can be performed and the ratio between total gradient time and the difference in the gradient

time between the first and last components is calculated, When the calculated ratio is less than

0.25, isocratic mode is adequate, and when the ratio is more than 0.25, gradient mode would be

beneficial15

.

Reverse Phase Chromatography with Ion Pair Reagents

The use of ion pair reagent is suggested only when the adequate separation could not be obtained

with the reverse phase chromatography. The reverse phase HPLC and the reverse phase HPLC

with ion pairing are similar, except that the latter contains an ion pair reagent in the mobile phase

to improve the selectivity of the ionic samples. However, reverse phase HPLC should be utilized

first before for the ion-pair reagent unless any reference is available in the literature.

The solubility of the ion-pair reagent may be affected depending upon the organic solvent used

in the mobile phase. Methanol is generally preferred over THF and acetonitrile. In reverse phase

HPLC with ion-pair, a suitable buffer is chosen at a concentration of about 25mM, the pH and

ion-pair concentration are varied to provide the optimum selectivity to the separation.

The pH of the mobile phase is closely associated with the ion pairing, whether the ion-pair is

positively charged or negatively charged and dependent on whether the analyte is an acid or

base. For the cationic samples or bases, use the pentane-hexane or higher hydrocarbon sulfonate

ion-pair reagent and for the anionic samples or acids, tetraethyl ammonium hydroxide can be

used as ion-pair reagent16

.

1.6.2. Optimization of the Method Development Parameters17-18

Selection of Buffer

Efficient separation of the acidic or basic components is achievable by controlling the pH of the

mobile phase. The buffer should be UV transparent at or below the wavelength of the organic

solvent. Other properties such as solubility and stability of the buffer and its reactivity to analyte

and hardware components of the chromatographic systems should be taken into consideration.

The buffer capacity is determined by pH, composition of the buffer and buffer concentration.

Optimum buffering capacity occurs at a pH equal to the pKa of the buffer. In general, most

buffers provide the adequate buffering capacity for controlling mobile phase pH only within ±1



unit of their pKa. Reverse phase chromatography generally is carried out with C8 or C18 bonded-

phase silica based columns that are less stable outside the pH range 2 to 8. Therefore, the buffer

should be able to control pH between of 2.0 to 8.0.

Selection of the Buffer pH

Depending on the pKa value of the drug molecules, pH of the mobile phase can change

retentions, e.g. acids shows an increase in the retention as the pH is reduced while bases show a

decrease. It is best to adjust the mobile phase to pH values at least +1.5 pH units above or below

the pKa to ensure practically 100% unionization for separation purposes. Generally, at the low

pH (1-4), peak tailing is minimum and method ruggedness is maximum. On the other hand,

operating in the intermediate range (pH 4-8) offers an advantage in increased analyte retention

and selectivity. Different buffers for HPLC separation are presented in Table 1.6.

.

Table 1.6: Buffers used in HPLC separation

Buffer pH range UV cutoff

Trifluoracetic acid 1.5 - 2.5 210 nm

Phosphoric acid

(mono or di phosphate)

6.2 - 8.2 <200 nm

Citric acid 11.3 - 13.3 <200 nm

Formic acid/K-formate 2.8 - 4.8 210 nm

Acetic acid/K-acetate 3.8 - 5.8 210 nm

Ammonium chloride 8.2 - 10.2 200 nm

Triethylamine-

HCl/triethylamine

10.0 -12.0 <200 nm

Pyrollidine 10.3 - 12.3 200 nm

Selection of the Mobile Phase

Most separations can be achieved by choosing the optimum mobile phase compositions. Most

widely used solvents for the reverse phase chromatography are methanol and acetonitrile.

Tetrahydrofuran is also used, but to a lesser extent. Mobile phase with tetrahydrofuran is known

to be susceptible for oxidation. Experiments are conducted with the mobile phase having buffers

of different pH and different organic phases to establish the best possible separation.



If the sample is eluted with the mobile phase of 100% organic content, and there is no separation,

the solvent strength should be decreased to get the retention. Generally the increase in organic

content will shorten the run time but lead to increased band overlap.

When the separations are complex, i.e. when many components are to be separated, and when

solvent strength is decreased and still there is no resolution between two closely eluting peaks,

another organic solvent of different polarity or even a mixture of the two organic solvents may

be tried to effect the separation.

Selection of Column

HPLC column is the heart of the method and critical in performing the separation. The

parameters that should be taken into consideration while choosing the column for the HPLC

method include;

column packing material,

size and shape of the particles,

column length and diameter,

percent carbon load,

pore volume and

end capping etc.

For reverse phase chromatography, a wide variety of the columns are available like C8, C18,

Cyano group –CN and amino group like –NH2 etc. are available. It is to be remembered that no

two columns are the same and vary from manufacturer to manufacture with respect to above

mentioned parameters. The surface area of the bonded phase support is a major factor as larger

the surface area, greater will be the retention. Sample retention normally increases for the bonded

phases of greater length, C18>C8>C3>C1. To select the type of column in the method, the

experiments using different columns with different mobile phases should be conducted to get

best possible separation. Based on the experimental data, the column which gives separation of

all the possible impurities and principal peak and which is rugged for the variation in mobile

phase should be selected.

Selection of the Column Temperature

Generally it is preferable to optimize the chromatographic conditions with the ambient column

temperature. However, if the peak symmetry is not achieved with any combination of the column

and mobile phase at the ambient condition, then the column temperature above ambient can be



adopted. If the column temperature is increased, it generally results in the reduction in the peak

symmetry and peak retention time. Change in temperature may be a more effective tool for the

separation in the ionization of the sample and this changes pH and pKa values.

Filter Compatibility

If the filter paper is used before injecting the sample, it should be checked for the adsorption of

the drug(s). For this, the standard solutions and the sample solutions are prepared and filtered

using two and three different types of the filter papers. The filtered standard solution results are

compared to the unfiltered standard solution and further to the centrifuged/unfiltered sample

solutions. For the filters to be acceptable for the use, the results of the filtered portions should be

within +2.0% of the unfiltered standard solution and the centrifuged/unfiltered sample solution.

Selection of Test Concentration and Injection Volume

The selection of test concentration depends upon the response of the API at the selected

wavelength. The test concentration should be finalized only after it is proved that the API is

completely extractable at the selected test concentration.

Generally, the injection volume of 10 µl to 20 µl is recommended but injection volume can be

increased upto 50 μl-100 µl. Before selecting high injection volume, it is to be ensured that with

the selected higher injection volume, column is not overloaded, resolution and peak symmetry

are not compromised.

1.7. HPLC Method Validation19-23

Quality of analytical data is key factor for success of drug development programme. In the

absence of a proven measurement system, there is no way of judging whether or not the process

has done what it purported to do. If method performance is not verified, all data become

doubtful. Therefore, validation of an analytical method is essential for establishing the

confidence that the method is “fit for the purpose”.

Validation of an analytical method is the process by which it is established, through laboratory

studies, that the performance characteristics of the method meet the requirements for the

intended analytical applications. The objective of validation of an analytical procedure is to

demonstrate that it is suitable for its intended purpose. (ICH Q2)

There are two important reasons for validating analytical procedure in pharmaceutical industries.

Validation of analytical method is an integral part of the quality control system.



Regulatory bodies of different countries require validation of both compendial and non-

compendial procedures.

As a part of this initiative, the International Conference of Harmonization (ICH)

provides a tripartite guideline on Validation of Analytical Procedures: Methodology (Q2) for the

registration of pharmaceuticals in USA, Japan, and European Union. USP also provides a general

information chapter (1225) on Validation of Compendial Procedures. Analytical procedures in

USP/NF are legally recognized under section 501(b) of the Federal Food, Drug and Cosmetic

Act as the regulatory analytical procedures for the compendial items and are not required to

validate accuracy and reliability of these methods, but merely verify their suitability under actual

conditions of use.

Types of Analytical Procedures to be Validated

According to ICH Harmonized Tripartite Guidelines, the discussion of the validation of

analytical procedures is directed to the four most common types of analytical procedures;

identification tests,

quantitative tests for impurities content,

limit tests for the control of impurities and

quantitative tests of the active moiety in the samples of drug substance or drug product or

other selected component(s) in the drug product.



1.7.1. Validation Parameters

Different validation parameters required to be performed by the applicant are presented in Table

1.7.

Table 1.7: Different validation parameters

USP (1225)

ICH

CDER*

Accuracy Accuracy Accuracy

Precision Precision Precision

Repeatability Repeatability Repeatability

- Intermediate Precision Intermediate Precision

Reproducibility Reproducibility Reproducibility

Specificity Specificity Specificity

Limit of Detection Limit of Detection Limit of Detection

Limit of Quantitation Limit of Quantitation Limit of Quantitation

Linearity Linearity Linearity

Range Range Range

Ruggedness - -

Robustness Robustness Robustness

- - Stability of Solution

*CDER- Center for Drug Evaluation and Research

Selection of validation parameter is dependent on the type of method and its intended use. Before

initiation of the validation, validation protocol should be prepared, which include;

list of reagents, solvents (batch no., manufacturer etc.),

instructions for preparation of standard, sample etc.,

equipments to be used,

instrumental parameters and chromatographic conditions,

validation parameters required / methodology,

system suitability requirements,

working standard, sample and placebo details and

calculations.



1.7.1.1. Different Validation Parameters

Specificity

Specificity is the ability of the method to accurately measure the analyte response in the presence

of all potential components. The response of the analyte in the test mixtures containing the

analyte and all potential sample components (placebo formulation, synthesis intermediates,

excipients, degradation products and process impurities) is compared with the response of a

solution containing only the analyte.

Linearity

A linear relationship should be evaluated across the range of the analytical procedures. It may

either be demonstrated directly on the drug substance (by dilution of a standard stock solution)

by/or separate weighing of synthetic mixture of the drug product components, using the proposed

procedure. Linearity should be evaluated by visual inspection of a plot of signals as a function of

analytes concentration or content. If there is a linear relationship, test results should be evaluated

by appropriate statistical methods. Under most circumstances, correlation coefficient (r2) ≥

0.999. Recommended validation ranges for linearity are presented in Table 1.8.

Table 1.8: Recommended validation ranges for linearity determinations

Purpose of Analysis Typical Range of

Interest (%)

Recommended

Validation Range (%)

Release specification assay 95-105 80-120

Assay for stability 90-105 80-120

Content uniformity 72-125 60-140

Assay of preservative in stability study 50-110 40-150

Assay of degradation product 0-110 0-20

Range

The range of an analytical method is the interval between the upper and lower levels of the

analytes for which the analytical method has adequate precision, accuracy and linearity. For

major component assay, concentration of standards should measure at or near the expected target

measurement level. A good strategy is to perform studies at 50, 75, 100, 125 and 150% of targets

levels.



Precision

The precision of an analytical method describes the closeness of individual measures of an

analytes. Precision may be considered at three levels: repeatability, intermediate precision and

reproducibility.

Repeatability: It expresses the precision under the same operating conditions over a short interval

of time (within-assay variability, with-in day variability).

Intermediate precision: It was previously known as part of ruggedness. The attributes evaluate

the reliability of the method in a different environment other than that used during development

of the method. The objective is to ensure that the method would provide the same results when

similar samples are analyzed.

Reproducibility: Reproducibility expresses the precision between laboratories.

Accuracy

The accuracy of an analytical method describes the closeness of mean test results obtained by the

method to the true value (concentration) of the analyte. The deviation of the mean from the true

value serves as the measure of accuracy. Accuracy is often expressed as percentage recovery of

known, added amount of the analyte by the assay. Several methods are available for determining

accuracy of drug substance and drug product as given below.

For drug substance;

Application of an analytical procedure to an analyte of known purity (e.g. reference

material).

Comparison of the results of proposed analytical procedure with those of a second well-

characterized procedure, the accuracy of which is stated and/or defined.

Accuracy may be inferred once precision, linearity and specificity have been established.

For drug product:

Application of the analytical procedure to the drug product to which known quantities of

the drug substance have been added.

In case where it is impossible to obtain samples of all drug product components, it may

be acceptable either to add known quantities of the analytes to the drug product or to

compare the results obtained from a second, well characterized procedure, the accuracy

of which is stated and/or defined.



Accuracy may be inferred once precision, linearity and specificity have been established.

Recommended data: Accuracy should be assessed using a minimum of 9 determinations over a

minimum of 3 concentration levels covering the specified range (e.g. 3 concentrations/ 3

replicates each of the total analytical procedure). Accuracy should be reported as percent

recovery by the assay of known added amount of analytes in the sample or as the difference

between the mean and the accepted true value together with the confidence intervals.

Limit of Detection (LOD) and Limit of Quantitation (LOQ)

Limit of detection is the lowest amount of the analyte in a sample that can be detected but not

necessarily quantitated. The results obtained at the limit of detection are not necessarily accurate

and precise. Limit of quantitation is the lowest amount of analyte in the sample that can be

quantified with suitable accuracy and precision. LOD and LOQ can be estimated by different

methods.

Based on evaluation: Visual evaluation may be used for non- instrumental methods but may

also be used with instrumental methods. The detection limit is determined by the analysis of

samples with known concentration of analyte and by establishing the minimum level at which

the analyte can be readily detected.

Based on signal to noise ratio (S/N): Determination of signal to noise ratio is performed by

comparing measured signal from sample with known concentrations of analyte with those of

blank sample and establishing the minimum concentration at which the analyte can be reliably

detected.

S/N for LOD is 3:1

S/N for LOQ is 10:1

Based on standard deviation of the response and slope: Results of LOD and LOQ are

calculated by following formulae:

Where, S = Slope of calibration curve

σ = Standard deviation of the response

LOD = 3.3 (σ / S)

LOQ = 10 (σ / S)



Robustness

The robustness of an analytical procedure is measure of its capacity to remain unaffected by

small, but deliberate variation in method parameters and provides an indication of its reliability

during normal usage. It has been suggested that the robustness of a method can be checked by

determining the specificity on at least three different batches of a column from the same supplier.

The evaluation of the robustness should be considered during the development phase

and depends on the type of procedure under study. It shows the reliability of an analysis with

respect to deliberate variation in analytical conditions.

1.7.1.2. Statistical Validation24-25

Mean

It is a measure of center of distribution if the data are symmetrically distributed below and above

the average. It is denoted by Xm:

Where, ∑Xi = Sum of all observations

N = Number of observations

Standard Deviation

It is a measure of the spread of data about the mean. It is denoted by S.

Where, ∑X 2 = Sum of each value squared

(∑X) 2 = Square of sum of all values

(∑X) 2 / N = Correction term


Xm= ∑Xi / N

S = ∑X 2

– (∑X) 2 /N

N-1



Standard Deviation of Mean

It is a measure of variability of mean. It is denoted by Sxm.

Where, Sxm= Standard deviation of mean

S = Standard deviation


Linear Regression

A common application of linear regression in analytical chemistry is to determine best linear

equation for calibration data to generate a calibration curve. The concentration of analyte in a

sample can then be determined by comparing a measurement of the unknown to the calibration

curve.

For the linear equation: y = mx + b

Where, y= Estimated response / dependent variable

m = Slope of the regression line

b = Intercept (y value when x=0)

Correlation

Correlation is a measure of the relation between two or more variables. Correlation coefficients

can range from -1.00 to +1.00. The value of -1.00 represents a perfect negative correlation while

a value of +1.00 represents a perfect positive correlation. A value of 0.00 represents a lack of

correlation24

. The most widely used type of correlation coefficient is Pearson (r), also called

linear or product-moment correlation.

Coefficient of Variation

It is a measure of relative variability.

CV = S / Xm

CV of 0.1 or 10 % means that standard deviation is one tenth of the mean.

Sxm = S / (N) ½



1.8. System Suitability26-27

System suitability tests are integral part of gas and liquid chromatography methods. They are

used to verify that the resolution and reproducibility of the system are adequate for the analysis

to be done. The test is based on the concept that the equipment, electronics, analytical operations

and sample to be analyzed constitute an integral system that can be analyzed.

No sample analysis is acceptable unless the requirements for system suitability have been met.

System suitability tests are run every time a method is used either before or during the analysis.

The results of each and every test are compared with defined acceptance criteria and, if they

pass, the method is deemed satisfactory.

1.8.1. Retention Factor

Rt (retention time), is the time between the injection point and the maximum detector response

for correspondent compound. A more useful measurement is the capacity factor (k') which is

calculated from the retention time of a peak and the dead time of the column as shown below.

Calculation of retention factor is shown in Figure 1.3.

Where, tR = Retention time of analyte

t0 = Dead time of the column.

Figure 1.3: Calculation of capacity factor

1.8.2. Efficiency

Efficiency is measure of degree of peak dispersion in particular column. Calculation of

efficiency is shown in Figure 1.4.

N= 16(tR/w)2

k' = tR-t0 / t0



Where, tR = Retention time of the peak

. W = Peak width

Figure 1.4: Calculation of theoretical plate number

1.8.3. Selectivity

Selectivity is defined as ability of chromatographic system to discriminate two different analytes.

α= k’2/k’1

1.8.4. Resolution

The resolution (Rs) of a column provides a quantitative measure of its ability to separate two

analytes. Column resolution is expressed as;

Rs = 2[(tR) B - (tR) A]/WA +WB

Where, (tR)B, and (tR)A = retention time of compound B and A, respectively

WA, and WB = width at the base of the peak in time units of peak A and B,

respectively

1.8.5. Tailing Factor

The tailing factor is measured at 5% of the peak height and largely used in the pharmaceutical

industry. Calculation of tailing factor is shown in Figure 1.5.

Figure 1.5: Calculation of tailing factor



1.8.6. Asymmetry Factor

The asymmetry factor is measured at 10% of the peak height. In most cases, the asymmetry

factor and tailing factor will be roughly the same (although rarely exactly equal). Calculation of

asymmetry factor is shown in Figure 1.6.

Figure 1.6: Calculation of asymmetry factor

1.9. Stability Indicating Methods Development28-30

Stability indicating methods are capable of discriminating between major active pharmaceutical

ingredients (API) and their degradation product(s) formed under defined storage conditions

during the stability evaluation period. The discriminating nature of method makes the method

‘stability indicating’ as well as ‘stability specific’. Stability indicating methods (SIMs) are the

quantitative analytical methods based on characteristic structural, chemical or biological

properties of each API of a drug product that are aimed to distinguish the active ingredients from

their degradation products so that the active ingredients can be accurately measured. Stability

evaluation is stability detection system that identifies potential degradation products and includes

analytical method for their quantitation.

1.9.1. Objective of Stability Studies

Stability studies are performed to establish the storage conditions and shelf life for the API and

products. In recently adopted stability guidelines, the Committee for Proprietary Medicinal

Products (CPMP) indicates that the objective of stability testing is to provide evidence on how

the quality of an API varies with time under the influence of variety of environmental factors

such as temperature, humidity and light. The stability of API does not mean “fixed” or “not

likely change” but it means “controlled, documented and acceptable change”. Forced



degradation conditions, stress agent concentration and time of stress are to be established in such

a way that they effect degradation, preferably 10-20% of the parent constituent. Stability testing

is, performed for the welfare of the patient, to protect the reputation of producer, as a

requirement of regulatory agencies to provide data that may be of value in the formulation of

other products.

1.9.2. Stability Protocols31

Although the stability protocols contain a schedule for testing samples stored at one or more

controlled storage conditions as depicted in Tables 1.9 and 1.10, the protocol specification can

differ significantly from one product to another. Stability samples are stored in chamber in which

temperature and humidity conditions are carefully controlled. Stress conditions are applied to

drug product to approximate long –term stability in short period.

Table 1.9: Storage conditions

Test Conditions Minimum Period

Long term

25˚C ± 2˚C

60% ± 5%

12 months

Intermediate 30˚C ± 2˚C

60% ± 5%

6 months

Accelerated 40˚C ± 2˚C

75% ± 5%

6 months



Table 1.10: Proposed storage guidelines

Storage Condition Tolerance Testing Zone Guidelines

25˚C / 60% RH ± 2˚C / ± 5% RH Long term (II) ICH, WHO

30˚C / 60% RH ± 2˚C / ± 5% RH Intermediate ICH, WHO

40˚C / 75% RH ± 2˚C / ± 5% RH Accelerated ICH, WHO

30˚C / 35% RH ± 2˚C / ± 5% RH Long term (III) WHO

30˚C / 70% RH ± 2˚C / ± 5% RH Long term (IV) WHO

2 - 8˚C Not specified Refrigeration ICH, WHO

-5 to -20˚C Not specified Freezer WHO

Below -18˚C Not specified Deep freezer WHO

Light 1.2 millions lux

Hours/ 200 watt

Hours (UV)

Stress ICH

1.9.3. Degradation Pathways and their Role in SIM Development

Forced degradation studies typically involve the exposure of representative sample of drug

substance and drug product to relevant stress conditions of light, heat, humidity, acid/ base

hydrolysis and oxidation. These experiments play an important role in drug development

process. The results of forced degradation studies can facilitate SIM development, drug

formulation design, selection of storage conditions and packaging. It can also provide a better

understanding of drug molecule chemistry and solutions for stability related problems.

According to ICH guideline stress testing is likely to be carried on a single batch of material and

to include effect of temperature in 10˚C increments above the accelerated temperature test

condition (e.g. 50˚C, 60˚C etc.), humidity where appropriate (75% RH or greater), oxidation and

photolysis on the drug substance plus its susceptibility to hydrolysis across a wide range of pH

values when in solution and suspension. Both FDA and ICH recommended forced degradation or

stress testing of drug substance and drug product. Acid and base hydrolysis, temperature,

photolysis and oxidation are recommended for these studies. However, neither ICH nor FDA



guidelines specify how to perform these forced degradation studies. Experimental conditions and

design of these studies have been left to discretion of pharmaceutical companies. A generic

protocol for these studies is presented in Table 1.11 and 1.12.

Table 1.11: Recommended stress conditions for drug substances

Stress Type Conditions Time

Acid hydrolysis

1mg mL¯1 in 0.1M (upto1.0M) HCl,

RT or higher

1-7 days

Base hydrolysis 1mg mL¯1 in 0.1M (upto1.0M) NaOH,

RT or higher

1-7 days

Thermal hydrolysis Aqueous solution ; 70˚C 1-7 days

Oxidative solution O2 +initiator(AIBN)* in

acetonitrile/H2O 80/20; 40˚C

1-7 days

Oxidative solution 0.3% (upto 3%) H2O2; RT*; protected

from light

Few hours to 7 days

Thermal 70˚C Upto 2 weeks

Thermal humidity 70˚C/ 75% RH Upto 2 weeks

*AIBN- 2, 2-azobisisobutyronitrile

*RT- room temperature (25˚C)

Table 1.12: Recommended stress conditions for drug product

Stress Conditions Time

Thermal 70˚C Upto 3 weeks

Thermal / humidity 70˚C/ 75% RH Upto 3 weeks

Photo-degradation Fluorescent and UV light >2 × ICH



In stress testing minimum four samples should be generated for every stress condition

viz.

Blank solution stored under normal conditions.

Blank solution subjected to stress in the same manner as the drug solution.

Zero time sample containing the drug which is stored under normal conditions.

Drug solution subjected to stress treatment.

The comparison of the results of these provides real assessment of the changes. Furthermore, it is

advised to withdraw samples at different time periods for each reaction condition. By doing so,

one can get a clear idea on the number of products formed, their relative strengths and whether

they are stable or unstable, resulting in further degradation products. This information is

essential in establishment of SIMs. Potential degradation pathways include hydrolysis, oxidation,

photo degradation etc.

Hydrolysis

Hydrolysis is one of the most common reactions seen in the API having ester and amide

functional groups within their structure. Hydrolytic degradation is performed in hydrochloric

acid and sodium hydroxide in solution. If the compound is poorly water soluble, organic co-

solvents may be used in combination with acid or base. Stress is first initiated at room

temperature, if no degradation occurs, an elevated temperature is applied (50-70˚C). The

hydrolytic degradation of new drug in acidic and alkaline conditions can be studied by refluxing

the drug in the 0.1 M HCl / NaOH for 8 hours. If reasonable degradation occurs, testing can be

stopped, if no degradation is seen under these conditions, the drug should be refluxed in a

stronger acid / alkali and for longer duration.

Oxidation

Oxidation is a well established degradation pathway for APIs. Oxygen which is required in most

oxidation reactions and is abundant in environment to which pharmaceuticals are exposed,

during either processing or long term storage. Oxidation reactions may be performed under

several conditions. Hydrogen peroxide concentration may be adjusted as necessary to obtain 5-

20% degradation. The disadvantage of using hydrogen peroxide is that it is non- selective and

relatively non predictable in its results. Stress with hydrogen peroxide often leads to secondary

degradation of the primary degradants making result interpretation more difficult.



Photo Degradation

Photo degradation has been reported for large number of drug substances and mechanisms for

these reactions are generally complex. The photolytic studies should be carried out by exposure

to light, using a combination of cool white and UV fluorescent lamps.

The desired target extent of degradation is approximately 5-20%. This is achieved by varying the

stress conditions. Over stressing may destroy the compound or may lead to further degradation

of relevant primary degradants. Under such circumstances stressing may fail to generate an

important degradation product. The degradation studies should be terminated after the maximum

recommended time/ stress conditions, even if sufficient degradation has not been achieved.

1.9.4. Need of Stress Testing in SIMs

When large number of degradation products is formed during forced decomposition of drug even

in one stress condition, it may be truly difficult or impossible to develop a selective stability-

indicating assay method if degradation products formed under all conditions are simultaneously

taken into consideration. Moreover, it has been mentioned that some degradation products

formed during forced decomposition are never developed in the stability samples. So a tagged

question is ‘should only major degradation products are targeted while developing a SIAM,

instead of all degradation products formed under the ICH suggested test conditions?’

Interestingly, the dilemma is well answered in the ICH guideline itself where the clarification is

provided in the statement ‘However, it may not be necessary to examine specifically for certain

degradation products if it has been demonstrated that they are not formed under accelerated or

long term storage conditions.’ Therefore, it emerges that a SIAM separating all types of possible

degradation products should normally be developed through stress testing under different ICH

suggested conditions. In case, it is not possible to develop a ‘selective SIAM’ due to the complex

nature of degradation, one can target for a method that takes into account degradation products

formed only under accelerated and long-term storage conditions.



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