4  

CHAPTER II

INTRODUCTION TO HPLC METHOD DEVELOPMENT

AND VALIDATION

Pharmaceutical analysis simply means analysis of pharmaceuticals.

Webster’ dictionary defines pharmaceutical as a medical drug. It is generally

known that a pharmaceutical is a therapeutic interest. A more appropriate term for

a pharmaceutical is active pharmaceutical ingredient (API) or active ingredient to

distinguish it from a formulated product or drug product is prepared by

formulating a drug substance with inert ingredient (excipient) to prepare a drug

product that is suitable for administration to patients. It is well known in the

pharmaceutical industry that pharmaceutical analysts in research and development

(R&D) play a very comprehensive role in new drug development and follow up

activities to ensure that a new drug product meets the established standards is

stable and continue to approved by regulatory authorities ,assuring that all batches

of drug product are made to the specific standards utilization of approved

ingredients and production method becomes the responsibility of pharmaceutical

analysts in the quality control (QC) or quality assurance department .

The methods are generally developed in an analytical R&D

department and transferred to QC or other departments as needed. At times they

are transferred to other divisions.

5  

By now it should be quite apparent that pharmaceutical analysts play a

major role in assuring the identity, safety, efficacy, and quality of drug product

safety and efficacy studies required that drug substance and drug product meet two

critical requirements.

1. Established identity and purity.

2. Established bio availability/dissolution1.

SCOPE AND SIGNIFICANCE OF PHARMACEUTICAL

ANALYSIS:

Pharmaceutical companies rely upon both qualitative and quantitative

chemical analysis to ensure that the raw material used meet all the desired

specifications, and also to check the quality of the final product. The examination

of raw material is carried out to ensure that there is no unusual substance present

which might deteriorate the manufacturing process or appear as a harmful

impurity in the final product. The quantity of required ingredient in raw material is

determined by a procedure known as Assay.

The final manufactured product is subjected to quality control2

to ensure that desired components are present within a range and impurities do not

exceed certain specified limits.

Some specific use of analysis is under mentioned:

(i) Quantitative analysis of air, water, soil samples to determine the level of

pollution.

6  

(ii) Chemical analysis to assist diagnosis of illness and monitoring the

condition of patients.

(iii) In farming, nature of soil and level of fertilizer application is analyzed.

(iv) In geology, composition of the rock and soil is carried out.

Types of Analysis:

In general analysis is divided into two major parts:

(a) QUALITATIVE ANALYSIS(what substances are present in the given

sample)

(b) QUANTITATIVE ANALYSIS(to determine the quantity of each

component in the given sample)

Qualitative:

• Qualitative inorganic analysis seeks to establish the presence of a

given element or inorganic compound in a sample.

• Qualitative organic analysis seeks to establish the presence of a

given functional group or organic compound in a sample.

Quantitative:

Quantitative analysis seeks to establish the amount of a given element or

compound in a sample.

The factors which must be taken into account when selecting an appropriate

method of analysis are:

(a) The nature of the information sought

7  

(b) The size of sample available and the proportion of the constituent to be

determined

(c) The purpose for which the analytical data is required.

INTRODUCTION TO CHROMATOGRAPHY

The term chromatography 3(Greek kromatos –colour & graphos-written

means colour writing .Mikhail Twestt (1906) - invented the chromatography. The

IUPAC has defined chromatography as “a method used primarily for the

separation of component of a sample, in which the component are distributed

between two phases, one of which is stationary while the other moves .the

stationary may be a solid or liquid supported on a solid or a gel and may be packed

in a column, spread as a layer or distributed as a film. The mobile phase may be

gaseous or liquid”.

Types of Chromatographic Methods.3

Based on modes of chromatography:

Normal phase chromatography

Reverse phase chromatography

Based on principle of separation:

Partition chromatography

Adsorption chromatography

Ion exchange chromatography

Size exclusion chromatography

8  

Affinity chromatography

Chiral phase chromatography

Base on elution technique:

Isocratic separation

Gradient separation

Based on the scale of operation:

Analytical HPLC

Preparative HPLC

Partition Chromatography 

Partition chromatography was the first kind of chromatography that

chemists developed. The partition coefficient principle has been applied in paper

chromatography, thin layer chromatography, gas phase and liquid-liquid

applications. The 1952 Nobel Prize in chemistry was earned by Archer John Porter

Martin and Richard Laurence Millington Synge for their development of the

technique, which was used for their separation of amino acids. Partition

chromatography uses a retained solvent, on the surface or within the grains or

fibres of an "inert" solid supporting matrix as with paper chromatography; or takes

advantage of some coulombic and/or hydrogen donor interaction with the solid

support. Molecules equilibrate (partition) between a liquid stationary phase and

the eluent. Known as Hydrophilic Interaction Chromatography (HILIC) in HPLC,

9  

this method separates analytes based on polar differences. HILIC most often uses

a bonded polar stationary phase and a non-polar, water miscible, mobile phase.

Partition HPLC has been used historically on unbonded silica or alumina supports.

Each works effectively for separating analytes by relative polar differences,

however, HILIC has the advantage of separating acidic, basic and neutral solutes

in a single chromatogram.

The polar analytes diffuse into a stationary water layer associated with the

polar stationary phase and are thus retained. Retention strengths increase with

increased analyte polarity, and the interaction between the polar analyte and the

polar stationary phase (relative to the mobile phase) increases the elution time.

The interaction strength depends on the functional groups in the analyte molecule

which promote partitioning but can also include coulombic (electrostatic)

interaction and hydrogen donar capability. Use of more polar solvents in the

mobile phase will decrease the retention time of the analytes, whereas more

hydrophobic solvents tend to increase retention times.

Normal Phase Chromatography

Also known as normal-phase HPLC (NP-HPLC), or adsorption

chromatography, this method separates analytes based on adsorption to a

stationary surface chemistry and by polarity. It was one of the first kinds of HPLC

that chemists developed. NP-HPLC uses a polar stationary phase and a non-polar,

non-aqueous mobile phase, and works effectively for separating analytes readily

10  

soluble in non-polar solvents. The analyte associates with and is retained by the

polar stationary phase. Adsorption strengths increase with increased analyte

polarity, and the interaction between the polar analyte and the polar stationary

phase (relative to the mobile phase) increases the elution time. The interaction

strength depends not only on the functional groups in the analyte molecule, but

also on steric factors. The effect of sterics on interaction strength allows this

method to resolve (separate) structural isomers.

The use of more polar solvents in the mobile phase

will decrease the retention time of the analytes, whereas more hydrophobic

solvents tend to increase retention times. Very polar solvents in a mixture tend to

deactivate the stationary phase by creating a stationary bound water layer on the

stationary phase surface. This behavior is somewhat peculiar to normal phase

because it is most purely an adsorptive mechanism (the interactions are with a

hard surface rather than a soft layer on a surface).

Displacement Chromatography

The chromatography matrix (the displacer) will compete effectively for

binding sites, and thus displace all molecules with lesser affinities.There are

distinct differences between displacement and elution chromatography. In elution

mode, substances typically emerge from a column in narrow, Gaussian peaks.

Wide separation of peaks, preferably to baseline, is desired in order to achieve

maximum purification. The speed at which any component of a mixture travels

11  

down the column in elution mode depends on many factors. But for two

substances to travel at different speeds, and thereby be resolved, there must be

substantial differences in some interaction between the biomolecules and the

chromatography matrix. basic principle of displacement chromatography is: A

molecule with a high affinity for Operating parameters are adjusted to maximize

the effect of this difference. mode chromatography, especially at the preparative

scale, are operational complexity, due to gradient solvent pumping, and low

through put, due to low column loadings. Displacement chromatography has

advantages over elution chromatography in that components are resolved into

consecutive zones of pure substances rather than “peaks”. Because the process

takes advantage of the nonlinearity of the isotherms, a larger column feed can be

separated on a given column with purified components recovered at significantly

higher concentrations.

Size Exclusion Chromatography

Size exclusion chromatography (SEC), also known as gel permeation

chromatography or gel filtration chromatography, separates particles on the

basis of size. It is generally a low resolution chromatography and thus it is often

reserved for the final, "polishing" step of a purification. It is also useful for

determining tertiary structure and quaternary structure of purified proteins. SEC is

used primarily for the analysis of large molecules such as proteins or polymers.

SEC works by trapping these smaller molecules in the pores of a particle. The

12  

larger molecules simply pass by the pores as they are too large to enter the pores.

Larger molecules therefore flow through the column quicker than smaller

molecules, that is, the smaller the molecule, the longer the retention time.

This technique is widely used for the molecular weight

determination of polysaccharides. SEC is the official technique (suggested by

European pharmacopeia) for the molecular weight comparison of different

commercially available low-molecular weight heparins.

Ion- Exchange Chromatography

In ion-exchange chromatography (IC), retention is based on the attraction

between solute ions and charged sites bound to the stationary phase. Ions of the

same charge are excluded. Types of ion exchangers include:

Polystyrene resins – These allow cross linkage which increases the

stability of the chain. Higher cross linkage reduces swerving, which increases the

equilibration time and ultimately improves selectivity.

Cellulose and dextran ion exchangers (gels) – These possess larger

pore sizes and low charge densities making them suitable for protein separation.

Controlled-pore glass or porous silica

In general, ion exchangers favour the binding of ions of higher charge and

smaller radius.An increase in counter ion (with respect to the functional groups in

resins) concentration reduces the retention time. A decrease in pH reduces the

13  

retention time in cation exchange while an increase in pH reduces the retention

time in anion exchange. By lowering the pH of the solvent in a cation exchange

column, for instance, more hydrogen ions are available to compete for positions on

the anionic stationary phase, thereby eluting weakly bound cations.

This form of chromatography is widely used in the

following applications: water purification, preconcentration of trace components,

ligand-exchange chromatography, ion-exchange chromatography of proteins,

high-pH anion-exchange chromatography of carbohydrates and oligosaccharides,

and others.

Bioaffinity Chromatography

This chromatographic process relies on the property of biologically active

substances to form stable, specific, and reversible complexes. The formation of

these complexes involves the participation of common molecular forces such as

the Van der Waals interaction, electrostatic interaction, dipole-dipole interaction,

hydrophobic interaction, and the hydrogen bond. An efficient, biospecific bond is

formed by a simultaneous and concerted action of several of these forces in the

complementary binding site.

Aqueous Normal-Phase Chromatography

Aqueous normal-phase chromatography (ANP) is a chromatographic

technique which encompasses the mobile phase region between reversed-phase

chromatography (RP) and organic normal phase chromatography (ONP). This

14  

technique is used to achieve unique selectivity for hydrophilic compounds,

showing normal phase elution using reverse-phase solvents. Normal – phase

chromatography uses a polar (hydrophilic) stationary phase and a non-polar

(usually with no water) mobile phase.

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC)

High performance liquid chromatography(HPLC)3-7 is a process, which

separates mixture containing two or more components under high pressure. in this

the stationary phase is packed in a column one end of which is attached to a source

of pressurized liquid mobile phase.High performance liquid chromatography is the

fastest growing analytical technique for the analysis of drugs. Its simplicity, high

specificity and wide range of sensitivity makes it ideal for the analysis of many

drugs in both dosage forms and biological fluids.

HPLC is also known as high pressure liquid

chromatography .It is essential form of column chromatography in which the

stationary phase is consist of small particles (3-50μm) pickings contained in a

column with a small pore(2-5mm) one end of which is attached to a source of

pressurized liquid eluent (mobile phase). The three form of high performance

liquid chromatography most often used are ion-exchange, partition and adsorption.

Advantages of HPLC

i. It provides a specific, sensitive and precise method for analysis of

different

15  

complicated samples.

ii. There is speed of analysis.

iii. The analysis by HPLC is specific, accurate and precise.

iv. It offers advantage over gas chromatography in analysis of many polar

substances, metabolic products and thermo labile as well non-volatile substances.

It is presently used in pharmaceutical research and developments in the

following ways:

To purify synthetic or natural products,

To characterize metabolites,

To assay active ingredients, impurities, degradation products and

in dissolution assays

In pharmacodynamics and pharmacokinetic studies.

Fig.1: Instrumentation of HPLC

16  

HPLC includes

1) Mobile phase reservoir and solvent treatment systems

2) Pumps:

i. Displacement pumps

ii. Reciprocating pumps

iii. Pneumatic pumps

3) Precolumn

4) Sample injectors

a. Syringe injection

b. Stop flow injection

c. Solvent flowing

5) Liquid chromatographic columns

a) Analytical columns

b) Preparative columns

6) Column packing materials

a) Pellicular

b) Porous particle

7) Detectors

a) Photometric detectors

b) Fluorescence detectors

c) Refractive index detectors

d) Electrochemical detectors

17  

8) Recorders

Mobile phase reservoir and solvent treatment systems

A modern HPLC apparatus is equipped with one or more glass or stainless steel

reservoirs each of which contain 500 ml or more of solvent. The reservoirs are

often equipped with a means of removing dissolved gases usually O2andN2 that

interfere by forming bubbles in the columns and detector systems. These bubbles

cause band spreading; in addition they interfere with the performance of the

detector.

Isocratic And Gradient Elution

A separation in which the mobile phase composition remains constant throughout

the procedure is termed isocratic (meaning constant composition). The word was

coined by Csaba Horvath who was one of the pioneers of HPLC.

The mobile phase composition does not have to remain constant. A separation in

which the mobile phase composition is changed during the separation process is

described as a gradient elution.One example is a gradient starting at

10% methanol and ending at 90% methanol after 20 minutes. The two components

of the mobile phase are typically termed "A" and "B"; A is the "weak" solvent

which allows the solute to elute only slowly, while B is the "strong" solvent which

rapidly elutes the solutes from the column. In reverse-phase chromatography,

solvent A is often water or an aqueous buffer, while B is an organic solvent

miscible with water, such as acetonitrile, methanol, THF, or isopropanol.

18  

Pumps:

The pumps are used to pass mobile phase through the column at high pressure and

at controlled flow rate .in addition to this its performance directly effects the

retention time, reproducibility and detector sensitivity.

The pumps used in HPLC should have the following features:

I. The generation of pressures up to 6000psi

II. Flow rates ranging from 0.1 to 10 ml/min

III. Flow control and flow reproducibility of ±0.5%

IV. It should be composition resistant and give a pulse free out put.

Fig. 2: Structure of pump

a. Displacement pumps

It consists of a large, syringe like chamber equipped with a plunger that is

activated by a screw driven mechanism powered by a stepping motor.

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b. Reciprocating pumps:

It consists of a small chamber in which the solvent is pushed back force with the

help of a motor driven piston or pressure may be transmitted by a diaphragm

which is hydraulically pumped by a reciprocating piston.

c. Pneumatic pumps:

In this the mobile phase is contained in a collapsible container housed in a vessel

that can be pressurized by a compressed gas.

Pre column

Some HPLC instruments are equipped with a precolumn, which contains a

packing chemically identical to that in a analytical column. Particle size is a large

hence the pressure drop across the pre column is negligible with respect to the

analytical column. The precolumn is mainly used to remove the impurities from

the solvent and thus prevent contamination of the analytical column.

Sample injectors

Often the limiting factor in the precision of liquid chromatographic measurements

lies in the reproducibility with which samples can be introduced in to column

packing. It must be noted that overloading of the sample causes band broadening.

The sample injectors are of the following types

a) Syringe injection

b) Stop flow injection

c) Solvent flowing

20  

Fig.3: Loading Mode

Inject Mode

Columns

The column is one of the most important components of the HPLC chromatograph

because the separation of the sample components is achieved when those

components pass through the column. HPLC columns are made of high quality

stainless steel, polished internally to a mirror finish. Standard analytical columns

are 4-5mm internal diameter and 10-30cm in length.Normally, columns are filled

with silica gel because its particle shape, surface properties, and pore structure

help to get a good separation. Silica is wetted by nearly every potential mobile

21  

phase, is inert to most compounds and has a high surface activity which can be

modified easily with water and other agents. Silica can be used to separate a wide

variety of chemical compounds, and its chromatographic behaviours generally

predictable and reproducible.

In HPLC, generally two types of columns are used; normal phase columns

and reversed phase columns. Using normal phase chromatography, particularly

polar and moderately non-polar substances can make excellent separation using

normal phase columns and polar eluents. While reversed phase chromatography,

particularly polar substances can make excellent separation using reversed phase

columns and non-polar eluents.

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

stationary phase. They are guard, Derivatizing, Capillary, and preparatory

columns.

22  

Fig.4: Structure of Column

Guard columns are placed anterior to the separating column. This serves as a

protective factor that prolongs the life and usefulness of the separation column.

They are dependable columns designed to filter or remove:

1. Particles that clog the separation column

2. Compounds and ions could ultimately cause “baseline drift”, decreased

resolution, decreased sensitivity, and create false peaks .Compounds that may

cause precipitation upon contact with the stationary or mobile phase and

3. Compound that might co-elute and cause extraneous peaks and interfere

with detection and / or quantification.

These columns must be changed on a regular basis in order to optimize their

protection function. Size of the packing varies with the type of protection needed.

23  

Derivatizing columns – Pre or post – primary column derivatization can be an

important aspect to 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. In few cases, the

derivatization step to cause data to become questionable, which is one reason why

HPLC was advantageous over gas chromatography. Because GC requires volatile,

thermally stabile, or nonpolar analytes , derivatization was usually required for

those samples, which did not contain these properties, Acetylation, Silylation, or

concentrated acid hydrolysis are a few derivatization techniques.

Capillary columns – Advances in HPLC led to smaller analytical columns. Also

known as microcolumns, capillary columns have a diameter much less than a

millimeter and there are three types: open – tubular, partially packed, and tightly

packed. They allow the user to work with nanonliter sample volumes, decreased

flow rate, and decreased solvent volume usage which may lead to cost

effectiveness.

Microbore and small – bore Columns are also analytical and small volumes

assays. A typical diameter for a small – bore column is 1-2mm. Like capillary

columns, instruments must usually be modified to accommodate these smaller

capacity columns (i.e decreased flow rate).

Fast columns – One of the primary reasons for using these columns is to obtain

improve sample through put (amount of compound per unit time). Fast columns

24  

are designed to decreases time of the chromatographic analysis without forsaking

significant deviations in results. These columns have the same internal diameter

but much shorter length than most other columns, and they are packed with

smaller particles that are typically 3 mcg/ml in diameter. Advantages include

increased sensitivity, deceased analysis time, decreased mobile phase usage, and

increased reproducibility.

Preparatory Columns - These columns are utilized when the objective its prepare

bulk (milligrams) of sample for laboratory preparatory applications. A facilitate

large volume injection into the HPLC system. Accessories important to mention

are the back-pressure regulator and the fraction collector. The back-pressure

regulator is placed immediately posterior to HPLC detector. The fraction collector

is an automated device that collects uniform increment of the HPLC output.

Microbe columns of 1-2mm internal diameter and 10-25 cm in length have

certain advantages of lower detection limits and lower consumption of solvent, the

latter being important if expensive HPLC grade solvents are used.

25  

Table 1.COLUMN DIMENSIONS

Detectors:

The most widely used detectors for liquid chromatography are based upon

absorption of ultraviolet or visible radiation. Photometers and columns are

available from commercial sources. The former often makes use of the 254 nm to

280 nm lines from a mercury source because many organic functional groups

absorb in this region. Deuterium or tungsten filament sources with interference

filters also provide a simple means of detecting absorbing species. Some modern

filters, which can be rapidly switched in to place. Spectrophotometer detectors are

considerably more versatile than photometry and are also widely used in high

performance instruments. Often these are diode-array instruments that can display

an entire spectrum as an analyte exits the column. Another detector, which has

Type

Internal

Diameter

(cm)

Length

(cm)

Particle

Size

(µm)

Analytical 0.3 - 0.46 3-28 3-10

Semimicro 0.1 – 0.21 10 – 25 3 – 18

Semipreparative 0.8 – 1.0 10 – 25 5 – 10

Preparative 2.0 – 5.0 10 – 25 10 – 20

26  

found considerable application, is based up on the changes in the refractive index

of the solvent that is caused by analyte molecules. In contrast to most of the other

detector is its some what limited sensitivity. Several electrochemical detectors

have also been introduced that are based on potentiometric, conductometric and

voltametric measurements.

Recorders

The signals from a detector are recorded as deviations from a base line. Two pen

recorder are used with instruments having two detectors. The peak position along

the curve relative to the starting point denotes the particular component .with

proper calibration, the height or area of the peak is a measure of amount of

component in a sample.

Chromatographic Parameters3,4:

Retention time (tr):

This is the time of emergence of the peak maximum of the component after

injection. This is the sum of the times the component spends in the mobile phase

(tM) and in the stationary phase.

Adjusted retention time:

It is the time the component spends in the stationary phase and is given by t1

r=tR-tM

The value of tM is obtained by measuring the time to elute an un retained

substance, e.g. air or Methane.

27  

It is the ratio of the time the component spends in the stationary phase to the time

in the mobile phase.

K= tR-tM/tM

Retention volume (VR):

This is the volume of carrier gas required to elute one

half of the compound from the column by the peak maximum and is given by:

VR=tR x f

Adjusted retention volume (VR):

This allows for the gas hold up volume of the column which is due to the

interstitial volume of the column and the volume of the injector and detector

systems .It is given by:

V'R=t'R x f

Relative retention volume:

Retention volumes for compounds are expressed relative to the retention volume

of a standard compound on the same column under the same conditions of a

standard compound examined. Therefore, this ratio is given by:

VN(Sample)/VN(Standard)=t’R(Sample)/t’R(Standard)

Relative retention volumes can there fore be represented by ratios of the distances

on the recorder chart and are the same as relative retention times.

Height equivalent to a theoretical plate (HETP):

The column is considered as being made up of a large number of parallel layers or

‘theoretical plates’, and when the mobile phase passes down the column the

28  

components of a mixture on the column distribute themselves between the

stationary and mobile phases in accordance with their partition that equilibrium is

established with in each plate. The equilibrium however is dynamic and the

components move down the column at a definite rate depending on the rate of

movement of the mobile phase.

A column may be considered as being made up of a large number of theoretical

plates where distribution of sample between liquid and gas phase occurs.

The number of theoretical plates (n) in a column is given by the relationship.

n=16(tR/W)2=5.54(tR/W1/2)2

W= peak width, i.e the segment of the peak base formed by projecting the straight

sides of the peak to the base line.

W1/2=peak width at half height

Resolution:

Chromatographers measure the quality of separation by resolution of adjacent

bands. T1 and t2retention times of the first and second adjacent bands.

RS=2(t2-t1)/W1-W2

W1 and W2 are the base line band width.

Column Efficiency (N):

Two related terms are widely used as quantitative measures of the efficiency of the

chromatographic columns.

I. Plate height

II. Number of theoretical plates

29  

The two are related by the equation

N =L/H

Selectivity:

It measures relative retention of two components selectivity is the function of

chromatographic surface (column), melting point and temperature.

α =K’2/K’1=V2-V0/V1-V0

Method:

For achieving of stable base line Equilibration of the column with the prescribed

mobile phase and flow rate and room temperature or at the temperature specified

in the monograph and preparation of sample solution and to be examined and the

reference solution is require d the solutions must be free from solid particles.

Optimization of the method:

During optimization of the method5 the initial set of conditions have evolved from

the first stages of development are improved or maximized in terms of resolution

and peak shape plate counts asymmetry capacity elution time detection limits limit

of quantification and overall ability to quantify the specific analyte of interest.

The various parameters is that include to be optimized during method

development

Modes of separation

Selection of stationary phase

Selection of mobile phase

Selection of the detector

30  

Selection modes of separation:

Reverse phase mode the mobile phase is comparatively more polar than the

stationary phase for the separation of polar or moderately polar compounds the

most preferred mode is reverse phase The nature of the analyte is the primary

factor in the selection of modes of separation. The second factor is based on nature

of the matrix.

Selection of stationary phase /column:

Selection of the column is the first and the most important step in method

development. The appropriate choice of separation column includes three different

approaches

1. Selection of separation system

2. The particle size and the nature of the column packing

3. The physical parameters of the column i.e the length and the diameter

The important parameters which should be selected the chromatographic column

Length and diameter of the column

Packing material

Shape of the particles

Size of the particles

% of the carbon loading

Pore volume

Surface area

End capping

31  

The column is selected depending on the nature of the solute and the information

about the Analyte.Reverse phase mode of chromatography facilitates a wide range

of columns like dimethyl silane(C2) butylsilane(C4) octylsilane

(C8),octadecacylsilane(C18),base deactivated silane , BDS

phenyl,cyanopropyl(CN) nitro amino etc c18 was for this study since it is most

retentive one. The sample manipulation becomes easier with this type of column.

Due to higher theoretical plates the higher columns provide

better separation surface area available for coating increases as the particle size

decreases for the better efficacy reproducibility and reliability size of 5 μm the

column which we have selected should be of 5m and internal diameter of 4.6 mm.

Peak shape is equally important in method development columns

that provide symmetrical peaks are always preferred while peaks with poor

asymmetry result in

• Accurate plate number and resolution measurement

• Imprecise quantitation

• Degraded and undetected minor bands in the peak tail

• Poor retention reproducibility

A useful and practical measurement of peak shape is peak asymmetry factor and

peak tailing factor peak asymmetry is measured at 10% of full peak height and

peak tailing factor 5% reproducibility of retention times and capacity factor is

important for developing a rugged and repeatable method.

32  

Selection of mobile phase:

The primary objective in selection and optimization of mobile phase is to achieve

optimum separation of all the individual impurities and degradants from each

other and from analyte peak.

In liquid chromatography the solute retention is governed by the

solute distribution factor, which reflects the different interactions of the solute-

stationary phase, solute-Mobile phase and the mobile phase –stationary phase for

the given stationary phase the retention of the given solute depends directly up on

the mobile phase, the nature and the composition of which has to be judiciously

selected in order to get appropriate and required solute retention.

The mobile phase has to be adopted in terms of elution strength

(solute retention) and the solvent selectivity (solute separation) solvent polarity is

the key word in chromatographic separations since a polar mobile phase will give

rise to low solute retention in normal phase and high solute retention in reverse

phase LC.

The selectivity will be particularly altered if the buffer pH is close to

the pKa of the Analytes: the solvent strength is a measure of its to pull an analyte

from the column it is generally controlled by the concentration of the solvent with

the highest strength.

The following parameters which shall be taken in to consideration while selecting

and optimizing the mobile phase.

33  

• Buffer

• pH of the buffer

• Mobile phase composition

Selection of the detector:

The detector was chosen depend up on the some characteristic property of the

Analyte like UV absorbance, Fluorescence Conductance, Oxidation, Reduction

etc.

Characteristics that are to be fulfilled by a detector to be used in HPLC

determination are

Higher sensitivity, Facilitating trace analysis

Negligible base line noise to facilitate lower detection

Large linear dynamic range

Low dead volume

Non destructive to sample

Inexpensive to purchase and operate

Pharmaceutical ingredients do not all absorb UV light equally. So the selection of

detection wave length is important .An understanding of the UV light absorptive

properties of the organic impurities and the active pharmaceutical ingredient is

very helpful.

Fur the greatest Sensitivity λmax should be used .UV wave lengths

below 200nm should be avoided because detector noise increases in this region.

Higher wavelengths give greater selectivity.

34  

HPLC METHOD DEVELOPMENT:

Systematic approach to HPLC method development5 should be based on the

knowledge of the chromatographic process. In most cases, a considerable amount

of experimentation may be needed. A good method development strategy should

require only as many experimental runs as are necessary to achieve desired final

result.

Fig. 5: Flow chart for Method development

1. Information on sample defines separation

Goals

2. Need for special HPLC procedure sample

Pre-treatment etc.

3. Choose of detector.

4. Choose LC method; preliminary runs; estimate

Best separation conditions.

5. Optimize separation conditions.

35  

6. Requirements for separation procedures

7a. Recovery of purified material 7b.Quantitative method &

7c. Qualitative method

8. Validated method for laboratories, released to routine

36  

Table 2. Choice of operating conditions to obtain the adequate

resolution of the mixture

Separation Variable Preferred Initial Choice

Column

Dimensions (length, ID) 15 X 0.46 cm

Particle size 5 μma

Stationary phase C8 or C18

Mobile phase

Solvents A and B Buffer- acetonitrile

% B 80-100%b

Buffer (compound, PH, concentration)

25mM potassium phosphate, 2.0<pH<3.0c

Additives (e.g., amine modifiers, ion- pair reagents)

Do not use initially

Flow rate 1.5-2.0 mL/min

Temperature 35-45oC

Sample Size

Volumed < 25μL

Weight <100 μg

a: 3.5 μm particles are an alternative, using a 7.5 cm column. b : For an initial isocratic run; an initial gradient run is preferred c No buffer required for neutral samples; for pH <2.5, pH-stable columns are recommended. d Smaller values required for smaller-volume columns (e.g., 7.5 x 0.46-cm, 3.5-μm column).

37  

HPLC METHOD VALIDATION

Definition:

Method validation8-10 is defined as a process of providing that an analytical

method is acceptable for its intended use. Method validation provides the method

development extremely specific, linear, precise, accurate and sensitive.

OBJECTIVE OF THE VALIDATION

The primary objective of validation is to from a basis for written procedure

for production and process control which are designed to assure that the

drug products have the identity, quality, and purity they purport are

represented to possess.

Assurance of Quantity

Government Regulation

CONCEPT OF VALIDATION

The basic principle of quality assurance has as goal the production of

articles that are fit for their intended use.The principle may be stated as quality,

safety and effectiveness must be designed and built into the product and quality

cannot be inspected or tested to the finished product.Each step of the

manufacturing process must be controlled to maximize the probability that the

finished product meets all quality and design specification.

38  

IMPORTANCE OF VALIDATION

As the quality of product cannot always be assured by routine quality

control because of testing of statically insignificant number of sample, the

validation thus should provide adequacy and reliability of a system or

product to meet the pre – determined criteria or attributes to provide high

degree of confidence that the same level of quality is consistently built into

each of finished product from batch to batch and to take action in case of

non compliance.

Retrospective Validation is useful for trend comparison of results

complains to cGMP to cGLP.

Fig.6: Types of Validation

VALIDATION 

Analytical Method validation  

Instrumental Validation  

Process  Validation  

Prospective Validation 

Retrospective Validation  

Revalidation 

39  

ANALYTICAL METHOD VALIDATION

Method validation is the process for establishing that performance

characteristics of the analytical method are suitable for the intended application.

Chromatographic methods need to be validation before first routine use. To obtain

the most accurate results, all of the variables of the method should be considered,

including sampling procedure, sample preparation, chromatographic separation,

detection and data evaluation, using the same matrix as that of the intended

sample. The validity of an analytical method can only be verified by laboratory

studies. All validation experiments used to make claims or conclusions about

validity of the method should be documented in report.

Types of analytical procedures to be validated

Identification test for impurities

Quantitative test for impurities

Limit test control of impurities

Quantitative test for the active moiety in samples of drug substance or drug

product, or other selected components (s) in the drug product.

Dissolution testing.

BENEFITS OF VALIDATION

Regulatory compliance

Minimize rejection and reworking

Minimize utility cost

40  

Minimize complaints

Reduce testing requirements

More rapid and reliable start – up new equipment

Easier scale – up from development

Easier maintenance of equipment

More rapid automation

The different parameters of analytical method development are discussed below:

System Suitability:

System suitability tests are an integral part of chromatographic methods.

These tests are used to verify that the resolution and reproducibility of the system

are adequate for the analysis to be performed. System suitability tests are based on

the concept that the equipment, electronics, analytical operations, and samples

constitute an integral system that can be evaluated as a whole. The purpose of the

system suitability test is to ensure that the complete testing system (including

instrument, reagents, columns, analysts) is suitable for the intended application.

Similar to the analytical method development, the system suitability test

strategy should be revised as the analysts develop more experience with the

assay. In general, consistency of system performance (e.g., replicate injections of

the standard) and chromatographic suitability (e.g. tailing factor, column

efficiency and resolution of the critical pair) are the main components of system

suitability.

41  

During the early stage of the method development process some of the

more sophisticated system suitability tests may not be practical due to the lack of

experience with the method. In this stage, usually a more "generic" approach is

used. For example, evaluation of the tailing factor to check chromatographic

suitability, and replicate injections of the system suitability solution to check

injection precision may be sufficient for an HPLC impurities assay. As the method

matures more experience is acquired for this method, a more sophisticated system

suitability test may be necessary.

System suitability is the checking of a system to ensure system

performance before or during the analysis of unknowns. Parameters such as plate

count, tailing factors, resolution and reproducibility (%RSD retention time and

area for six repetitions) are determined and compared against the specifications set

for the method. These parameters are measured during the analysis of a system

suitability "sample" that is a mixture of main components and expected by-

products.

The following table lists the terms to be measured and their recommended

limits obtained from the analysis of the system suitability sample as per current

FDA guidelines on "Validation of Chromatographic Methods".

42  

Table 3.System Suitability Parameters and Recommendations

Parameter Recommendation

Capacity Factor (k’) The peak should be well-resolved from other peaks and the void volume, generally k’>2.0

Repeatability RSD < 1% for N > 5 is desirable.

Relative retention Not essential as long as the resolution is stated.

Resolution (Rs) Rs of > 2 between the peak of interest and the closest eluting potential interferent (impurity, excipient, degradation product, internal standard, etc.

TailingFactor(T) T of < 2

Theoretical Plates (N)

N > 2000

Accuracy:

Accuracy is the measure of exactness of an analytical method, or the

closeness of agreement between the value which is accepted either as a

conventional true value or an accepted reference value and the value found. It is

measured as the percent of analyte recovered by assay, by spiking samples in a

blind study. For the assay of the drug substance, accuracy measurements are

obtained by comparison of the results with the analysis of standard reference

material or by comparison to a second, well – characterized method. For the assay

of the drug product, accuracy is evaluated by analyzing synthetic mixtures spiked

with known quantities of components. For the quantitation of impurities, accuracy

43  

is determined by analyzing samples (drug substance or drug product) spiked with

known amounts of impurities are not available, see specificity.)

To document accuracy the ICH guideline on methodology recommends

collecting data from a minimum of nine determinations over a minimum of three

concentration levels covering the specified range (for example, three

concentrations, three replicates each).The data should be reported as the percent

recovery of the known, added amount, or as the difference between the mean and

true value with confidence intervals.

Precision:

Precision is the measure of the degree of repeatability of an analytical

method under normal operation and is normally expressed as the percent relative

standard deviation for a statistically significant number of samples. According to

the ICH precision should be performed at three different levels: repeatability,

intermediate precision and reproducibility. Repeatability is the results of the

method operating over a short time interval under the same conditions (inter-assay

precision). It should be determined from a minimum of nine determinations

covering the specified range of the procedure (for example, three levels, three

repetitions each) or from a minimum of six determinations at 100 % of the test or

target concentration. Intermediate precision is the results from within lab variation

due to random events such as different days, analysts, equipment, etc. In

determining intermediate precision, experimental design should be employed so

that the effects (if any) of the individual variables can be monitored.

44  

Documenting precision:

Reproducibility refers to the results of collaborative studies between

laboratories. Documentation in support of precision studies should include the

standard deviation relative standard deviation, coefficient of variation, and the

confidence interval.

Specificity:

Specificity is the ability to measure accurately and specifically the analyte

of interest in the presence of the other components that may be expected to b

present in the sample matrix. It is a measure of the degree of interference from

such things as other active ingredients, excipients, impurities and degradation

products, ensuring that a peak response is due to a single component only, i.e. that

no co- elution exist.

Specificity is measured and documented in a separation by the resolution,

plate count (efficiency), and tailing factor. Specificity can also be evaluated with

modern photodiode array detectors that compare spectra collected across a peak

mathematically as an indication of peak homogeneity ICH also use the term

specificity, and divide it in to two separate categories: identification and

assay/impurity tests.

For identification purposes, specificity is demonstrated by the ability to

discriminate between compounds of closely related structures, or by comparison to

known reference materials. For assay and impurity tests, specificity is

demonstrated by the resolution of the two closest eluting compounds. These

45  

compounds are usually the major component or active ingredient and an impurity.

If impurities are available, it must be demonstrated that the assay is unaffected by

the presence of spiked materials (impurities and /or excipients). If the impurities

are not available, the test results are compared to a second well- characterized

procedure. For impurity tests, the impurity profiles are compared head- to-head.

In case of the assay, demonstration of specificity requires that the

procedure is unaffected by the presence of impurities or excipients. In practice,

this can be done by spiking the drug substances or product with appropriate levels

of impurities or excipients and demonstrating that the assay is unaffected by the

presence of these extraneous materials. If the degradation product impurity

standards are unavailable, specificity may be demonstrated by comparing the test

results of samples containing impurities or degradation products to a second well-

characterized procedure. These comparisons should include samples stored under

relevant stress conditions (e.g. light, heat humidity, acid/base hydrolysis,

oxidation).

Limit of Detection:

The limit of detection (LOD) is defined as the lowest concentration of an

analyte in a sample that can be detected, not quantitated. It is a limit test that

specifies whether are not an analyte is above or below a certain value. It is

expressed as concentration at a specified signal-to-noise ratio, usually two-or

46  

three-to-one. The ICH has recognized the signal- to- noise ratio convention, but

also lists two other options to determine LOD: Visual non- instrumental methods

and a means of calculating the LOD. Visual non- instrumental methods may

include LOD’S determined by techniques such as thin layer

chromatography(TLC) or titration .LOD’s may also be calculated based on the

standard deviation of the response (SD) and the slope of the calibration curve(S) at

levels approximating the LOD according to the formula :

LOD=3.3(SD/S)

The standard deviation of the response can be determined based on standard

deviation of the blank, on the residual standard deviation of the regression line, or

the standard deviation of y- intercepts of regression lines.

If LOD is determined based on visual evaluation or based on signal to noise ratio,

the presentation of the relevant chromatograms is considered acceptable for

justification.

In cases where an estimated value for the detection limit is obtained by

calculation or extrapolation, this estimate may subsequently be validated by the

independent analysis of a suitable number of samples known to be near or prepared

at the detection limit.

Limit of quantitation:

The limit of quantitation (LOQ) is defined as the lowest concentration of an

analyte in a sample that can be determined with acceptable precision and accuracy

47  

under the stated operational conditions of the method. Like LOD, LOQ is

expressed as a concentration with the precision and accuracy of the measurement

also reported. Sometimes a signal- to-noise ratio of ten-to- one is used to

determine LOQ. That is as the LOQ concentration level decreases the precision

increases .If better precision is required, a higher concentration must report for

LOQ. This compromise is dictated by the analytical method and its intended use.

The ICH has recognized the ten-to-one-signal-to-noise ratio as typical, and also,

like LOD, lists the same two additional options that can be used to determine

LOQ, visual non-instrumental methods and a means of calculating the LOQ. The

method is again based on the standard deviation of the response (SD) and the slope

of the calibration curve (S) according to the formula:

LOQ=10(SD/S)

Again, the standard deviation of the response can be determined based on the

standard deviation of the blank, on the residual standard deviation of the

regression line, or the standard deviation of y intercepts of regression lines.

Linearity and Range:

Linearity is the ability of the method to elicit test results that are directly

proportional to analyte concentration within a given range .Linearity is generally

reported as the variance of the slope of the regression line. Range is the interval

between the upper and lower levels of analyte (inclusive) that have been

demonstrated to be determined with precision, accuracy and linearity using the

method as written. The range is normally expressed in the same units as the test

48  

results obtained by the method. The ICH guidelines specify a minimum of five

concentration levels, along with certain minimum specified ranges.

For assay, the minimum specified range is from 80-120 % of the target

concentration. For an impurity test, the minimum range is from the reporting level

of each impurity, to 120 % of the specification (for toxic or more pote nt

impurities, the range should be commensurate with the controlled level).

For content uniformity testing, the minimum range is from 70-130 % of the

test or target concentration, and for dissolution testing ±20 % over the specified

range of the test. That is, in the case of an extended release product dissolution

test, with a Q- factor of 20 % dissolved after six hours, and 0 % dissolved after 24

hrs, the range would be 0-100 %.

Ruggedness:

Ruggedness, according to the USP, is the degree of reproducibility of the

results obtained under a variety of conditions, expressed as % RSD. These

conditions include different laboratories, analysts, instruments, reagents, days, etc.

In the guide line on definitions and terminology, the ICH did not address

ruggedness specifically. This apparent omission is really a matter of semantics,

however, as ICH chose instead to cover the topic of ruggedness as precision, as

discussed previously.

Robustness:

Robustness is the capacity of a method to remain unaffected by small

deliberate variations in method parameters. The robustness of a method is

49  

evaluated by varying method parameters such as mobile phase ratio, pH, ionic

strength, temperature, flow rate etc and determining the effect (if any) on the

results of the method. As documented in the ICH guidelines, robustness should be

considered early in the development of the method. In addition, if the results of a

method or other measurements are susceptible to variations in method parameters,

these parameters should be adequately controlled and a precautionary statement

included in the method documentation.

50  

REFERENCES

1. Satinder, A., Stephen, S., Hand Book of Modern Pharmaceutical Analysis.,

Published by

Academic Press, London., (2001),(3), 1-2.

2. Yang H, Feng Y and Luan Y., Simultaneous Determination of Simvastatin and

Ezetimibe in

Tablets by HPLC.,J Chromatogr B., 2003, 785, 369.

3. Srivastava, VK., and Srivastava, KK., Introduction to Chromatography Theory

and Practice,

14th Edition., S.Chand and Company limited, New Delhi., (1991), 66-67.

4. Sethi PD., Quantitative Analysis of Pharmaceutical Formulations., 1st ed., CBS

Publishers

and Distributors, New Delhi. (2001), 3-5.

5. Snyder, LR., Joseph Kirkland, J., Joseph Glajch, L., Practical HPLC Method

Development.,

2nd ed., John Wiley and Sons, INC, Canada., (1997), 2-11.

6. Melani L, Mills R and Hassman D., Efficacy and safety of ezetimibe co-

administered with

pravastatin in patients with primary hypercholesterolemia: a prospective,

randomized, Double-

blind trial., Eur Heart J., 2003, 24, 717-728.

51  

7. Curlucci G, Mazzeo P, Biordi L and Bologna M., Simultaneous determination

of simvastatin

and its hydroxy acid form in human plasma by high performance liquid

chromatography with

UVdetection., J. Pharm. Biomed. Anal., 1992, 10 (9),693-697.

8. International Conference on Harmonization, Draft Guideline on Validation of

Analytical

Procedures: Definitions and Terminology, Federal Register, 60 (1995)

11260.,1996(1-8).

9. Center for Drug Evaluation and Research, Food and Drug

Administration,Reviewer Guidance,

Validation of Chromatographic Methods. 1994.

10. Guideline for Submitting Samples and Analytical Data for Methods

Validation. Food and

Drug Administration, 1987.