Introduction

Quality control

The pharmaceutical quality control laboratory serves one of the most important functions

in pharmaceutical production and control. A significant portion of the CGMP regulations

pertains to the quality control laboratory.

Quality control in pharmaceutical laboratory includes a series of analytical measurements

used to monitor the quality of the analytical data.

Quality control is useful for:

Guiding formulation development

Comparing finished products with different formulations.

Confirming the acceptability of changes to manufacturing procedures during

scale-up or post-marketing changes.

Quality control ensures that the quality of the end product is acceptable to regulatory

authorities such as FDA & it is truly required for pharmaceutical products as patients (not

the general population) use pharmaceuticals to treat their diseases or for prophylaxis to

prevent infection or disease. [5]

In the pharmaceutical quality control laboratory, the stability of the pharmaceutical

products such as solid/liquid dosage forms is studied under various conditions. Complete

analysis of the pharmaceutical products include the following tests:

Dissolution testing

Content Uniformity testing.

Impurity profiling.

In dissolution testing, the release rate of an active ingredient in a pharmaceutical product

is measured. It should be within the acceptable limits specified by the regulatory

authorities. Content uniformity testing deals with the quantification of the active

1

ingredient in a pharmaceutical product. Similarly impurity profiling, as the name

suggests, deals with the detection and the quantification of the impurities present in a raw

material or a finished product.

These tests are dealt in detail in the following chapters. These tests are performed on raw

materials & on finished products during manufacture, scaling up and after a batch is

ready to be marketed. These tests are done by using protocols such as those described, for

example, by United States phamacopoeia (USP), British Phamacopoeia (BP) or Indian

Pharmacopoeia. The pharmaceutical products for which the Q.C. protocols are available

in USP/IP/BP are called official articles and for the newly identified drug molecules

analytical methods are developed by the manufacturer, the methods are validated and

submitted to regulatory authorities.

Although all the quality control tests have different applications, all of them involve

qualitative as well as quantitative analysis of the pharmaceutical product and in many

analytical laboratories HPLC is used for such analyses & now a days ultra fast liquid

chromatographs are used to save precious time of analysis. So, before understanding the

actual quality control methods it is required to study the instrumentation, principle and

working of HPLC and UFLC and to study the analytical method development for HPLC

& UFLC.

2

High performance liquid chromatography

High-pressure liquid chromatography (HPLC), sometimes called “high-performance

liquid chromatography”, is a separation technique based on a solid stationary phase and a

liquid mobile phase.

Separations are achieved by partition, adsorption, or ion-exchange processes, depending

upon the type of stationary phase used. HPLC has distinct advantages over gas

chromatography for the analysis of organic compounds. Compounds to be analyzed are

dissolved in a suitable solvent, and most separations take place at room temperature.

Thus, most drugs, being nonvolatile or thermally unstable compounds, can be

chromatographed without decomposition or the necessity of making volatile derivatives.

Most pharmaceutical analyses are based on partition chromatography

Principle of HPLC

The basic operating principle of HPLC is to force the analyte through a column of the

stationary phase (usually a tube packed with small spherical particles with a certain

surface chemistry) by pumping a liquid (mobile phase) at high pressure through the

column. The sample to be analyzed is introduced in small volume to the stream of mobile

phase and is retarded by specific chemical or physical interactions with the stationary

phase as it traverses the length of the column. The amount of retardation depends on the

nature of the analyte, stationary phase and mobile phase composition. The time at which

a specific analyte elutes (comes out of the end of the column) is called the retention time

and is considered a reasonably unique identifying characteristic of a given analyte. The

use of pressure increases the linear velocity (speed) giving the components less time to

diffuse within the column, leading to improved resolution in the resulting chromatogram.

Common solvents used include any miscible combinations of water or various organic

liquids (the most common are methanol and acetonitrile). Water may contain buffers or

salts to assist in the separation of the analyte components, or compounds such as

Trifluoroacetic acid which acts as an ion pairing agent. [3,11,12]

3

A further refinement to HPLC has been to vary the mobile phase composition during the

analysis; this is known as “gradient elution”. A normal gradient for reversed phase

chromatography might start at 5 % methanol and progress linearly to 50 % methanol over

25 minutes, depending on how hydrophobic the analyte is. The gradient separates the

analyte mixtures as a function of the affinity of the analyte for the current mobile phase

composition relative to the stationary phase. This partitioning process is similar to that

which occurs during a liquid-liquid extraction but is continuous, not step-wise. In this

example, using a water/methanol gradient, the more hydrophobic components will elute

(come off the column) under conditions of relatively high methanol; whereas the more

hydrophilic compounds will elute under conditions of relatively low methanol. The

choice of solvents, additives and gradient depend on the nature of the stationary phase

and the analyte. Often a series of tests are performed on the analyte and a number of

generic runs may be processed in order to find the optimum HPLC method for the analyte

- the method which gives the best separation of peaks. [3]

Distribution of analytes between phases

The distribution of analytes between phases can often be described quite simply. An

analyte is in equilibrium between the two phases;

Amobile Astationary

The equilibrium constant, K, is termed the “partition coefficient”; defined as the molar

concentration of analyte in the stationary phase divided by the molar concentration of the

analyte in the mobile phase.

The time between sample injection and an analyte peak reaching a detector at the end of

the column is termed the “retention time (tR)”. Each analyte in a sample will have a

different retention time. The time taken for the mobile phase to pass through the column

is called tM.

4

A term called the “retention factor”, k', is often used to describe the migration rate of an

analyte on a column. You may also find it called the capacity factor. The retention factor

for analyte A is defined as;

k'A = ( t R – t M ) / tM

t R and tM are easily obtained from a chromatogram. When an analytes retention factor is

less than one, elution is so fast that accurate determination of the retention time is very

difficult. High retention factors (greater than 20) mean that elution takes a very long time.

Ideally, the retention factor for an analyte is between one and five.

We define a quantity called the “selectivity factor”, α, which describes the separation of

two species (A and B) on the column;

α = k 'B / k 'A

When calculating the selectivity factor, species A elutes faster than species B. The

selectivity factor is always greater than one. [2]

Band broadening and column efficiency

To obtain optimal separations, sharp, symmetrical chromatographic peaks must be

obtained. This means that band broadening must be limited. It is also beneficial to

measure the efficiency of the column. [3]

5

The Theoretical Plate Model of Chromatography

The plate model supposes that the chromatographic column contains a large number of

separate layers, called theoretical plates. Separate equilibrations of the sample between

the stationary and mobile phase occur in these "plates". The analyte moves down the

column by transfer of equilibrated mobile phase from one plate to the next.

It is important to remember that the plates do not really exist; they are a figment of the

imagination that helps us understand the processes at work in the column. They also

serve as a way of measuring column efficiency, either by stating the number of

theoretical plates in a column, N (the more plates the better), or by stating the plate

height; the Height Equivalent to a Theoretical Plate (the smaller the better).

If the length of the column is L, then the HETP is

HETP = L / N

The number of theoretical plates that a real column possesses can be found by examining

a chromatographic peak after elution;

where w1/2 is the peak width at half-height.

As can be seen from this equation, columns behave as if they have different numbers of

plates for different solutes in a mixture. [2]

6

The Rate Theory of Chromatography

A more realistic description of the processes at work inside a column takes account of the

time taken for the solute to equilibrate between the stationary and mobile phase (unlike

the plate model, which assumes that equilibration is infinitely fast). The resulting band

shape of a chromatographic peak is therefore affected by the rate of elution. It is also

affected by the different paths available to solute molecules as they travel between

particles of stationary phase. If we consider the various mechanisms, which contribute to

band broadening, we arrive at the Van Deemter equation for plate height;

HETP = A + B / u + C u

where u is the average velocity of the mobile phase. A, B, and C are factors which

contribute to band broadening.

A - Eddy diffusion

The mobile phase moves through the column, which is packed with stationary phase.

Solute molecules will take different paths through the stationary phase at random. This

will cause broadening of the solute band, because different paths are of different lengths.

B - Longitudinal diffusion

The concentration of analyte is less at the edges of the band than at the center. Analyte

diffuses out from the center to the edges. This causes band broadening. If the velocity of

the mobile phase is high then the analyte spends less time on the column, which

decreases the effects of longitudinal diffusion.

C - Resistance to mass transfer

The analyte takes a certain amount of time to equilibrate between the stationary and

mobile phase. If the velocity of the mobile phase is high, and the analyte has a strong

affinity for the stationary phase, then the analyte in the mobile phase will move ahead of

the analyte in the stationary phase. The band of analyte is broadened. The higher the

velocity of mobile phase, the worse the broadening becomes.

7

Van Deemter plots

A plot of plate height vs. average linear velocity of mobile phase.

Such plots are of considerable use in determining the optimum mobile phase flow rate.

Resolution

Although the selectivity factor, α, describes the separation of band centres, it does not

take into account peak widths. Another measure of how well species have been separated

is provided by measurement of the resolution. The resolution of two species, A and B, is

defined as

Baseline resolution is achieved when R = 1.5

It is useful to relate the resolution to the number of plates in the column, the selectivity

factor and the retention factors of the two solutes;

8

To obtain high resolution, the three terms must be maximized. An increase in N, the

number of theoretical plates, by lengthening the column leads to an increase in retention

time and increased band broadening - which may not be desirable. Instead, to increase the

number of plates, the height equivalent to a theoretical plate can be reduced by reducing

the size of the stationary phase particles.

It is often found that by controlling the capacity factor, k', separations can be greatly

improved. This can be achieved by changing the composition of the mobile phase [7,1]

The selectivity factor, α, can also be manipulated to improve separations. When α is close

to unity, optimizing k' and increasing N is not sufficient to give good separation in a

reasonable time. In these cases, k' is optimized first, and then a is increased by one of the

following procedures:

1. Changing mobile phase composition

2. Changing column temperature

3. Changing composition of stationary phase

Using special chemical effects (such as incorporating a species which complexes with

one of the solutes into the stationary phase) [4]

Types of HPLC

(A) Normal phase chromatography

Normal phase HPLC (NP-HPLC) was the first kind of HPLC chemistry used, and

separates analytes based on polarity. This method uses a polar stationary phase and a

non-polar mobile phase, and is used when the analyte of interest is fairly polar in nature.

The polar analyte associates with and is retained by the polar stationary phase.

Adsorption strengths increase with increase in 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 not only depends on the functional

groups in the analyte molecule, but also on steric factors and structural isomers are often

9

resolved from one another. Use of more polar solvents in the mobile phase will decrease

the retention time of the analytes while more hydrophobic solvents tend to increase

retention times. Particularly polar solvents in a mixture tend to deactivate the column by

occupying the stationary phase surface. This is somewhat particular 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)..

NP-HPLC had fallen out of favor in the 1970's with the development of reversed-phase

HPLC because of a lack of reproducibility of retention times as water or protic organic

solvents changed the hydration state of the silica or alumina chromatographic media.

Recently it has become useful again with the development of HILIC bonded phases

which utilize a partition mechanism which provides reproducibility.

(B) Reversed phase chromatography

Reversed phase HPLC (RP-HPLC) consists of a non-polar stationary phase and an

aqueous, moderately polar mobile phase. One common stationary phase is a silica which

has been treated with RMe2SiCl, where R is a straight chain alkyl group such as C18H37 or

C8H17. The retention time is therefore longer for molecules which are more non-polar in

nature, allowing polar molecules to elute more readily. Retention Time (RT) is increased

by the addition of polar solvent to the mobile phase and decreased by the addition of

more hydrophobic solvent. Reversed phase chromatography (RPC) is so commonly used

that it is not uncommon for it to be incorrectly referred to as "HPLC" without further

specification. The pharmaceutical industry regularly employs RPC to qualify drugs

before their release.

RPC operates on the principle of hydrophobic interactions, which result from repulsive

forces between a polar eluent, the relatively non-polar analyte, and the non-polar

stationary phase. The binding of the analyte to the stationary phase is proportional to the

contact surface area around the non-polar segment of the analyte molecule upon

association with the ligand in the aqueous eluent. This solvophobic effect is dominated by

10

the force of water for "cavity-reduction" around the analyte and the C18-chain versus the

complex of both. The energy released in this process is proportional to the surface tension

of the eluent (water: 73 erg/cm², methanol: 22 erg/cm²) and to the hydrophobic surface of

the analyte and the ligand respectively. The retention can be decreased by adding less-

polar solvent (MeOH, ACN) into the mobile phase to reduce the surface tension of water.

Gradient elution uses this effect by automatically changing the polarity of the mobile

phase during the course of the analysis.

Structural properties of the analyte molecule play an important role in its retention

characteristics. In general, an analyte with a larger hydrophobic surface area (C-H, C-C,

and generally non-polar atomic bonds, such as S-S and others) results in a longer

retention time because it increases the molecule's non-polar surface area, which is non-

interacting with the water structure. On the other hand, polar groups, such as -OH, -NH2,

COO- or -NH3+ reduce retention as they are well integrated into water. Very large

molecules, however, can result in an incomplete interaction between the large analyte

surface and the ligands alkyl chains can have problems entering the pores of the

stationary phase.

RT increases with hydrophobic - non-polar - surface area. Branched chain compounds

elute more rapidly than their corresponding linear isomers because the overall surface

area is decreased. Similarly organic compounds with single C-C-bonds elute later than

the ones with a C=C or C-C-triple bond, as the double or triple bond is shorter than a

single C-C-bond.

Aside from mobile phase surface tension (organizational strength in eluent structure),

other mobile phase modifiers can affect analyte retention. For example, the addition of

inorganic salts causes a moderate linear increase in the surface tension of aqueous

solutions (ca. 1.5 erg/cm² pro Mol for NaCl, 2.5 erg/cm² pro Mol for (NH4)2SO4), and

because the entropy of the analyte-solvent interface is controlled by surface tension, the

addition of salts tend to increase the retention time. This technique is used for mild

separation and recovery of proteins and protection of their biological activity in protein

analysis (hydrophobic interaction chromatography, HIC).

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Another important component is the influence of the pH since this can change the

hydrophobicity of the analyte. For this reason most methods use a buffering agent, such

as sodium phosphate, to control the pH. A volatile organic acid such as formic acid or

most commonly trifluoroacetic acid is often added to the mobile phase, if mass

spectrometry is applied to the eluent fractions. The buffers serve multiple purposes: they

control pH, neutralize the charge on any residual exposed silica on the stationary phase

and act as ion pairing agents to neutralize charge on the analyte. The effect varies

depending on use but generally improve the chromatography.

Reversed phase columns are quite difficult to damage compared with normal silica

columns, however, many reversed phase columns consist of alkyl derivatized silica

particles and should never be used with aqueous bases as these will destroy the

underlying silica particle. They can be used with aqueous acid, but the column should not

be exposed to the acid for too long, as it can corrode the metal parts of the HPLC

equipment. The metal content of HPLC columns must be kept low if the best possible

ability to separate substances is to be retained. A good test for the metal content of a

column is to inject a sample which is a mixture of 2,2'- and 4,4'- bipyridine. Because the

2,2'-bipyridine can chelate the metal, the shape of the peak for the 2,2'-bipy will be

distorted (tailed) when metal ions are present on the surface of the silica.

(C) 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

purification. It is also useful for determining the tertiary structure and quaternary

structure of purified proteins.

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.

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(D) Ion exchange chromatography.

In Ion-exchange chromatography, retention is based on the attraction between solute ions

and charged sites bound to the stationary phase. Ions of the same charge are excluded.

Some types of Ion Exchangers include: (1) Polystyrene resins- allows cross linkage

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

increases the equilibration time and ultimately improves selectivity. (2) Cellulose and

dextran ion exchangers (gels)-These possess larger pore sizes and low charge densities

making them suitable for protein separation.(3) Controlled-pore glass or porous silica.

In general, ion exchangers favor 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. An increase in pH reduces the retention time in cation

exchange while a decrease in pH reduces the retention time in anion exchange.

This form of chromatography is widely used in the following applications: In purifying

water, preconcentration of trace components, Ligand-exchange chromatography, Ion-

exchange chromatography of proteins, High-pH anion-exchange chromatography of

carbohydrates and oligosaccharides, etc.

(E) 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 sites.[1,3]

13

Types of flow

Isocratic flow and gradient elution

With regard to the mobile phase, a composition of the mobile phase that remains constant

throughout the procedure is termed isocratic.

In contrast to this is the so called "gradient elution", which is a separation where the

mobile phase changes its composition during a separation process. One example is a

gradient in 20 min starting from 10 % Methanol and ending up with 30 % Methanol.

Such a gradient can be increasing or decreasing. The benefit of gradient elution is that it

helps speed up elution by allowing components that elute more quickly to come off the

column under different conditions than components which are more readily retained by

the column. By changing the composition of the solvent, components that are to be

resolved can be selectively more or less associated with the mobile phase. As a result, at

equilibrium they spend more time in the solvent and less time in the stationary phase, and

therefore they elute faster.[10]

Other parameters

(A) Internal diameter

The internal diameter (ID) of an HPLC column is a critical aspect that determines

quantity of analyte that can be loaded onto the column and also influences sensitivity.

Larger columns are usually seen in industrial applications such as the purification of a

drug product for later use. Low ID columns have improved sensitivity and lower solvent

consumption at the expense of loading capacity.

Larger ID columns (over 10 mm) are used to purify usable amounts of material

because of their large loading capacity.

Analytical scale columns (4.6 mm) have been the most common type of columns,

though smaller columns are rapidly gaining in popularity. They are used in

14

traditional quantitative analysis of samples and often use a UV-Vis absorbance

detector.

Narrow-bore columns (1-2 mm) are used for applications when more sensitivity is

desired either with special UV-vis detectors, fluorescence detection or with other

detection methods like liquid chromatography-mass spectrometry

Capillary columns (under 0.3 mm) which are used almost exclusively with

alternative detection means such as mass spectrometry. They are usually made

from fused silica capillaries, rather than the stainless steel tubing that larger

columns employ.

(B) Particle size

Most traditional HPLC is performed with the stationary phase attached to the outside of

small spherical silica particles (very small beads). These particles come in a variety of

sizes with 5μm beads being the most common. Smaller particles generally provide more

surface area and better separations, but the pressure required for optimum linear velocity

increases by the inverse of the particle diameter squared. This means that changing to

particles that are half as big, keeping the size of the column the same, will double the

performance, but increase the required pressure by a factor of four. Larger particles are

more often used in non-HPLC applications such as solid-phase extraction.

(C) Pore size

Many stationary phases are porous to provide greater surface area. Small pores provide

greater surface area while larger pore size has better kinetics especially for larger

analytes. For example a protein which is only slightly smaller than a pore might enter the

pore but not easily leave once inside.

(D) Pump pressure

Pumps vary in pressure capacity, but their performance is measured on their ability to

yield a consistent and reproducible flow rate. Pressure may reach as high as 6000 lbf/in2

(~40 MPa, or about 400 atmospheres). Modern HPLC systems have been improved to

15

work at much higher pressures, and therefore be able to use much smaller particle sizes in

the columns (< 2 micrometres). These "Ultra High Performance Liquid Chromatography"

systems or UHPLCs can work at up to 15,000 lbf/in² (~ 100 MPa or about 1000

atmospheres).[3]

Manufacturers of HPLC chromatographs

Agilent Technologies

Beckman Coulter, Inc.

Hitachi

PerkinElmer, Inc.

Shimadzu Scientific Instruments

Thermo Electron Corporation

Varian, Inc.

Waters Corporation

Manufacturers of HPLC columns and accessories

Agilent Technologies

Beckman Coulter, Inc.

Merck KGaA

Phenomenex

Shimadzu Scientific Instruments

Sigma-Aldrich

Thermo Electron Corporation

Tosoh Corporation

Varian, Inc.

Waters Corporation

16

HPLC instrumentation:

Apparatus:

A liquid chromatograph consists of a reservoir containing the mobile phase, a pump to

force the mobile phase through the system at high pressure, an injector to introduce the

sample into the mobile phase, a chromatographic column, a detector, and a data

collection device such as a computer, integrator, or recorder. Short, small-bore columns

containing densely packed particles of stationary phase provide for the rapid exchange of

compounds between the mobile and stationary phases. In addition to receiving and

reporting detector output, computers are used to control chromatographic settings and

operations, thus providing for long periods of unattended operation. [2]

17

Pumping Systems:

HPLC pumping systems deliver metered amounts of mobile phase from the solvent

reservoirs to the column through high-pressure tubing and fittings. Modern systems

consist of one or more computer-controlled metering pumps that can be programmed to

vary the ratio of mobile phase components, as is required for gradient chromatography, or

to mix isocratic mobile phases (i.e., mobile phases having a fixed ratio of solvents).

However, the proportion of ingredients in premixed isocratic mobile phases can be more

accurately controlled than in those delivered by most pumping systems. Operating

pressures up to 5000 psi or higher, with delivery rates up to about 10 mL per minute are

typical. Pumps used for quantitative analysis should be constructed of materials inert to

corrosive mobile phase components and be capable of delivering the mobile phase at a

constant rate with minimal fluctuations over extended periods of time. [3]

18

Injectors:

After dissolution in mobile phase or other suitable solution, compounds to be

chromatographed are injected into the mobile phase, either manually by syringe or loop

injectors, or automatically by autosamplers. The latter consist of a carousel or rack to

hold sample vials with tops that have a pierceable septum or stopper and an injection

device to transfer sample from the vials to a loop from which it is loaded into the

chromatograph. Some autosamplers can be programmed to control sample volume, the

number of injections and loop rinse cycles, the interval between injections, and other

operating variables.

A syringe can be used for manual injection of samples through a septum when column

head pressures are less than 70 atmospheres (about 1000 psi). At higher pressures an

injection valve is essential. Some valve systems incorporate a calibrated loop that is filled

with test solution for transfer to the column in the mobile phase. In other systems, the test

solution is transferred to a cavity by syringe and then switched into the mobile phase. [3]

19

Columns:

For most pharmaceutical analyses, separation is achieved by partition of compounds in

the test solution between the mobile and stationary phases. Systems consisting of polar

stationary phases and nonpolar mobile phases are described as normal phase, while the

opposite arrangement, polar mobile phases and nonpolar stationary phases, are called

reverse-phase chromatography. Partition chromatography is almost always used for

hydrocarbon-soluble compounds of molecular weight less than 1000. The affinity of a

compound for the stationary phase, and thus its retention time on the column, is

controlled by making the mobile phase more or less polar. Mobile phase polarity can be

varied by the addition of a second, and sometimes a third or even a fourth, component.

Stationary phases for modern, reverse-phase liquid chromatography typically consist of

an organic phase chemically bound to silica or other materials. Particles are usually 3 to

10 µm in diameter, but sizes may range up to 50 µm or more for preparative columns.

Small particles thinly coated with organic phase provide for low mass transfer resistance

and, hence, rapid transfer of compounds between the stationary and mobile phases.

Column polarity depends on the polarity of the bound functional groups, which range

from relatively nonpolar octadecyl silane to very polar nitrile groups. Liquid, nonbound

stationary phases must be largely immiscible in the mobile phase. Even so, it is usually

necessary to presaturate the mobile phase with stationary phase to prevent stripping of the

stationary phase from the column. Polymeric stationary phases coated on the support are

more durable.

Columns used for analytical separations usually have internal diameters of 2 to 5 mm;

larger diameter columns are used for preparative chromatography. Columns may be

heated to give more efficient separations, but only rarely are they used at temperatures

above 60 because of potential stationary phase degradation or mobile phase volatility.

Unless otherwise specified in the individual monograph, columns are used at ambient

temperature Ion exchange chromatography is used to separate water-soluble, ionizable

compounds of molecular weight less than 1500. The stationary phases are usually

synthetic organic resins; cation-exchange resins contain negatively charged active sites

20

and are used to separate basic substances such as amines, while anion-exchange resins

have positively charged active sites for separation of compounds with negatively charged

groups, such as phosphate, sulfonate, or carboxylate groups. Water-soluble ionic or

ionizable compounds are attracted to the resins, and differences in affinity bring about the

chromatographic separation. The pH of the mobile phase, temperature, ion type, ionic

concentration, and organic modifiers affect the equilibrium, and these variables can be

adjusted to obtain the desired degree of separation.

In size-exclusion chromatography, columns are packed with a porous stationary phase.

Molecules of the compounds being chromatographed are filtered according to size. Those

too large to enter the pores pass unretained through the column. Smaller molecules enter

the pores and are increasingly retained as molecular size decreases. These columns are

typically used to measure aggregation and degradation of large molecules. [3]

21

Detectors:

Many HPLC methods require the use of spectrophotometric detectors. Such a detector

consists of a flow-through cell mounted at the end of the column. A beam of UV

radiation passes through the flow cell and into the detector. As compounds elute from the

column, they pass through the cell and absorb the radiation, resulting in measurable

energy level changes. [3]

Fixed, variable, and multi-wavelength detectors are widely available. Fixed

wavelength detectors operate at a single wavelength, typically 254 nm, emitted by a low-

pressure mercury lamp. Variable wavelength detectors contain a continuous source, such

as a deuterium or high-pressure xenon lamp, and a monochromator or an interference

filter to generate monochromatic radiation at a wavelength selected by the operator. The

wavelength accuracy of a variable-wavelength detector equipped with a monochromator

should be checked by the procedure recommended by its manufacturer; if the observed

wavelengths differ by more than 3 nm from the correct values, recalibration of the

instrument is indicated. Modern variable wavelength detectors can be programmed to

change wavelength while an analysis is in progress. Multi-wavelength detectors measure

absorbance at two or more wavelengths simultaneously. In diode array multi-wavelength

detectors, continuous radiation is passed through the sample cell, then resolved into its

constituent wavelengths, which are individually detected by the photodiode array. These

detectors acquire absorbance data over the entire UV-visible range, thus providing the

analyst with chromatograms at multiple, selectable wavelengths and spectra of the eluting

peaks. Diode array detectors usually have lower signal-to-noise ratios than fixed or

variable wavelength detectors, and thus are less suitable for analysis of compounds

present at low concentrations.

22

Differential refractometer detectors measure the difference between the refractive

index of the mobile phase alone and that of the mobile phase containing

chromatographed compounds as it emerges from the column. Refractive index detectors

are used to detect non-UV absorbing compounds, but they are less sensitive than UV

detectors. They are sensitive to small changes in solvent composition, flow rate, and

temperature, so that a reference column may be required to obtain a satisfactory baseline.

Fluorometric detectors are sensitive to compounds that are inherently fluorescent or that

can be converted to fluorescent derivatives either by chemical transformation of the

compound or by coupling with fluorescent reagents at specific functional groups. If

derivatization is required, it can be done prior to chromatographic separation or,

alternatively, the reagent can be introduced into the mobile phase just prior to its entering

the detector.

23

Potentiometric, voltametric, or polarographic electrochemical detectors are useful

for the quantitation of species that can be oxidized or reduced at a working electrode.

These detectors are selective, sensitive, and reliable, but require conducting mobile

phases free of dissolved oxygen and reducible metal ions. A pulseless pump must be

used, and care must be taken to ensure that the pH, ionic strength, and temperature of the

mobile phase remain constant. Working electrodes are prone to contamination by reaction

products with consequent variable responses.

Electrochemical detectors with carbon-paste electrodes may be used advantageously to

measure nanogram quantities of easily oxidized compounds, notably phenols and

catechols.

New detectors continue to be developed in attempts to overcome the deficiencies of those

being used.[10]

Data Collection Devices

Modern data stations receive and store detector output and print out chromatograms

complete with peak heights, peak areas, sample identification, and method variables.

They are also used to program the liquid chromatograph, controlling most variables and

providing for long periods of unattended operation.

Data also may be collected on simple recorders for manual measurement or on stand-

alone integrators, which range in complexity from those providing a printout of peak

areas to those providing chromatograms with peak areas and peak heights calculated and

data stored for possible subsequent reprocessing. [10]

Procedure:

The mobile phase composition significantly influences chromatographic performance

and the resolution of compounds in the mixture being chromatographed. For accurate

quantitative work, high-purity reagents and “HPLC grade” organic solvents must be used.

24

Water of suitable quality should have low conductivity and low UV absorption,

appropriate to the intended use.

Reagents used with special types of detectors (e.g., electrochemical, mass spectrometer)

may require the establishment of additional tolerances for potential interfering species.

Composition has a much greater effect than temperature on the capacity factor, k’

In partition chromatography, the partition coefficient, and hence the separation, can be

changed by addition of another component to the mobile phase. In ion-exchange

chromatography, pH and ionic strength, as well as changes in the composition of the

mobile phase, affect capacity factors. The technique of continuously changing the solvent

composition during the chromatographic run is called gradient elution or solvent

programming. It is sometimes used to chromatograph complex mixtures of components

differing greatly in their capacity factors. Detectors that are sensitive to change in solvent

composition, such as the differential refractometer, are more difficult to use with the

gradient elution technique.

The detector must have a broad linear dynamic range, and compounds to be measured

must be resolved from any interfering substances. The linear dynamic range of a

compound is the range over which the detector signal response is directly proportional to

the amount of the compound. For maximum flexibility in quantitative work, this range

should be about three orders of magnitude. HPLC systems are calibrated by plotting peak

responses in comparison with known concentrations of a reference standard, using either

an external or an internal standardization procedure.

Reliable quantitative results are obtained by external calibration if automatic injectors or

autosamplers are used. This method involves direct comparison of the peak responses

obtained by separately chromatographing the test and reference standard solutions. If

syringe injection, which is irreproducible at the high pressures involved, must be used,

better quantitative results are obtained by the internal calibration procedure where a

known amount of a noninterfering compound, the internal standard, is added to the test

25

and reference standard solutions, and the ratios of peak responses of drug and internal

standard are compared.

Because of normal variations in equipment, supplies, and techniques, a system suitability

test is required to ensure that a given operating system may be generally applicable. [10]

Trouble shooting

Start up - Preliminary checks

Problem Possible cause Solution

No peaks or

very small

peaks

Detector off Check detector

Broken

connections to

recorder

Check connections

No sample/Wrong

sample

Check sample. Be sure it is not deteriorated.

Check for bubbles in the vials

Wrong settings on

recorder or detectorCheck attenuation. Check gain

No Flow Pump off Start Pump

Flow interrupted

Check reservoirs. Check position of the inlet

tubing. Check loop for obstruction or air.

Check degasing of mobile phase. Check

compatibility of the mobile phase

components.

LeakCheck fittings. Check pump for leaks and

precipitates. Check pump seals.

Air trapped in the Disconnect column and prime pump. Flush

26

systemsystem with 100% methanol or isopropanol.

Contact servicing if necessary.

Column and Fittings Leaks

Problem Possible cause Solution

Column end

leaks

Loose fitting

White powder at

loose fitting

Tighten or replace fitting

Cut tubing and replace ferrule; disassemble

fitting, rinse and reassemble.

Leak at detector Detector-seal failure Replace detector seal or gaskets.

Leak at injection

valve

Worn or scratched

valve rotorReplace valve rotor

Leak at pump Pump seal failureReplace pump seal; check piston for

scratches and, if necessary, replace

Change in Retention time

Problem Possible cause Solution

Changing

Retention

Times

Buffer retention timesUse buffer with concentration

greater than 20 mM.

Contamination buildupFlush column occasionally with

strong solvent

Equilibration time

insufficient for gradient run

or changes in isocratic

Pass at least 10 column volumes

through the column for gradient

regeneration or after solvent

27

mobile phase changes

First few injections - active

sites

Condition column by injecting

concentrated sample

Inconsistent on-line

mobile-phase mixing

Ensure gradient system is

delivering a constant composition;

compare with manually prepared

mobile phase; partially premix

mobile phase

Selective evaporation of

mobile-phase component

Cover solvent reservoirs; use less-

vigorous helium purging; prepare

fresh mobile phase

Varying column

temperature

Thermostat or insulate column;

ensure laboratory temperature is

constant.

Decreasing

Retention

Times

Active sites on column

packing

Use mobile-phase modifier,

competing base (basic

compounds), or increase buffer

strength; use higher coverage

column packing.

Column overloaded with

sample

Decrease sample amount or use

larger-diameter column.

Increasing flow rate Check and reset pump flow rate.

Loss of bonded stationary

phase or base silica

Use mobile-phase pH between pH

2 and pH 8

Varying column

temperature

Thermostat or insulate column;

ensure laboratory temperature is

constant

Increasing Decreasing flow rate Check and reset pump flow rate;

28

Retention

Times

check for pump cavitation; check

for leaking pump seals and other

leaks in system

Changing mobile-phase

composition

Cover solvent reservoirs; ensure

that gradient system is delivering

correct composition.

Loss of bonded stationary

phase

Use mobile-phase pH between pH

2 and pH 8

Slow column

equilibration

time

Reversed phase ion pairing

- long chain ion pairing

reagents require longer

equilibration time

Use ion-pairing reagent with

shorter alkyl chain length

Baseline

Problem Possible cause Solution

Void Time

noise

Air bubbles in mobile

phase

Degas or use back pressure restrictor on

detector

Positive-negative -

difference in refractive

index of injection solvent

and mobile phase

Normal with many samples; use mobile

phase as sample solvent

Drifting

baseline

Negative direction

(gradient elution) -

absorbance of mobile-

phase A

Use non-UV absorbing mobile phase

solvents; use HPLC grade mobile phase

solvents; add UV absorbing compound

to mobile phase B.

Positive direction Use higher UV absorbance detector

29

(gradient elution) -

absorbance of mobile

phase B

wavelength; use non-UV absorbing

mobile phase solvents; use HPLC grade

mobile phase solvents; add UV

absorbing compound to mobile phase A.

Positive direction -

contamination buildup and

elution

Flush column with strong solvent; clean

up sample; use HPLC grade solvents

Wavy or undulating -

temperature changes in

room

Monitor and control changes in room

temperature; insulate column or use

column oven; cover refractive index

detector and keep it out of air currents.

Baseline

noise

Continuous - detector

lamp problem or dirty cell

Replace UV lamp( each should last 2000

h; clean and flush flow cell.

Gradient or isocratic

proportioning - lack of

solvent mixing

Use proper mixing device; check

proportioning precision by spiking one

solvent with UV absorbing compound

and monitor UV absorbance detector

output

Gradient or isocratic

proportioning -

malfunctioning

proportioning valves

Clean or replace proportioning precision

valves; partially remix solvents.

Occasional sharp spikes -

external electrical

interference

Use voltage stabilizer for LC system; use

independent electrical circuit.

Periodic - pump pulses Service or replace pulse damper; purge

air from pump; clean or replace check

valves.

30

Random - contamination

buildup

Flush column with strong solvent; clean

up sample; use HPLC grade solvent

Spikes - bubble in detector Degas mobile phase; use backpressure

restrictor at detector outlet.

Spikes - column

temperature higher than

boiling point of solvent

Use lower column temperature.

Pressure

Problem Possible cause Solution

Decreasing

Pressure

Insufficient flow from pump Loosen cap on mobile phase

reservoir

Leak in hydraulic lines from

pump to column

Tighten or replace fittings;

tighten rotor in injection valve

Leaking pump check valve or

seals

Replace or clean check valves;

replace pump seals.

Pump cavitation

Degas solvent; check for

obstruction in line from solvent

reservoir to pump; replace inlet-

line frit

Fluctuating

pressure

Bubble in pumpDegas solvent; purge solvent

with helium

Leaking pump check valve or

seals

Replace or clean check valves;

replace pump seals

High Back

Pressure

Column blocked with

irreversibly adsorbed sample

Improve sample cleanup; use

guard column; reverse-flush

column with strong solvent to

31

dissolve blockage

Column particle size too small

(for example 3 micrometers)

Use larger particle size (for

example 5 micrometer)

Microbial growth on column

Use at least 10% organic

modifier in mobile phase; use

fresh buffer daily; add 0.02%

sodium azide to aqueous mobile

phase; store column in at least

25% organic solvent without

buffer

Mobile phase viscosity too high Use lower viscosity solvents or

higher temperature

Plugged frit in in-line filter or

guard column Replace frit or guard column

Plugged inlet frit Replace end fitting or frit

assembly

Polymeric columns - solvent

change causes swelling of

packing

Use correct solvent with

column; change to proper

solvent compositional consult

manufacturer's solvent-

compatibility chart use a column

with a higher percentage of

cross-linking

32

Salt precipitation (especially in

reversed-phase chromatography

with high concentration of

organic solvent in mobile phase)

concentration of organic solvent

in mobile phase)

Ensure mobile phase

compatibility with buffer

concentration; decrease ionic

strength and water-organic

solvent ratio; premix mobile

phase

When injector disconnected

from column - blockage in

injector

Clean injector or replace rotor

Increasing

Pressure

Blocked flow lines

Systematically disconnect

components from detector end

to column end to find blockage;

replace or clean blocked

component

Particulate buildup at head of

column

Filter sample; use .5 micrometer

in-line filter; disconnect and

back flush column; replace inlet

frit

Water-organic solvent systems -

buffer precipitation

Ensure mobile phase

compatibility with buffer

concentration; decrease ionic

strength or water organic solvent

ratio

Peaks

Problem Possible cause Solution

Broad

peaks

Analytes eluted early

due to sample overload

Dilute sample 1:10 and re-inject

33

Detector-cell volume

too large

Use smallest possible cell volume

consistent with sensitivity needs; use

detector with no heat exchanger in

system

Injection volume too

large

Decrease solvent strength of injection

solvent to focus solute; inject smaller

volume

Large extra column

volume

Use low- or zero-dead-volume end

fittings and connectors; use smallest

possible diameter of connecting tubing

(<0.10 in. i.d.); connect tubing with

matched fittings

Mobile-phase solvent

viscosity too high

Increase column temperature; change to

lower viscosity solvent

Peak dispersion in

injector valve

Decrease injector sample loop size;

introduce air bubble in front and back of

sample in loop

Poor column efficiency

Use smaller-particle-diameter packing,

lower-viscosity mobile phase, higher

column temperature, or lower flow rate

Retention time too longUse gradient elution or stronger

isocratic mobile phase

Sampling rate of data

system too lowIncrease sampling frequency.

Slow detector time

constant

Adjust time constant to match peak

width

Some peaks broad - late

elution of analytes

Flush column with strong solvent at end

of run; end gradient at higher solvent

34

retained from previous

injectionconcentration

Ghost

peaks

ContaminationFlush column to remove contaminants

in it; use HPLC-grade solvent

Elution of analytes

retained from previous

injection

Flush column with strong solvent at end

of run; end gradient at higher solvent

concentration

Ion-pair

chromatography - upset

equilibrium

Prepare sample in mobile phase; reduce

injection volume

Oxidation of

trifluoroacetic acid in

peptide mapping

Prepare trifluoroacetic acid solutions

fresh daily; use antioxidant

Reversed-phase

chromatography -

contaminated water

Check suitability of water by running

different amounts through column and

measure peak height of interferences as

function of enrichment time; clean water

by running it through old reversed-phase

column; use HPLC-grade water.

Unknown interferences

in sample

Use sample cleanup or prefractionation

before injection.

Negative

peaks

Refractive index

detection - refractive

index of solute less than

that of mobile phase

Reverse polarity to make peak positive

UV-absorbance

detection - absorbance

of solute less than that

Use mobile phase with lower UV

absorbance; if recycling solvent, stop

recycling when recycled solvent affects

35

of mobile phase detection

36

Peaks continued

Problem Possible cause Solution

Peak

Doubling

Blocked Frit

Replace or clean frit; install 0.5-um porosity in-

line filter between pump and injector to

eliminate mobile-phase contaminants or

between injector and column to eliminate

sample contaminants

Co elution of

interfering

compound

Use sample cleanup or prefractionation; adjust

selectivity by changing mobile or stationary

phase

Co elution of

interfering

compound from

previous injection

Flush column with strong solvent at end of ran;

end gradient at higher solvent concentration

Column overloadedUse higher-capacity stationary phase; increase

column diameter; decrease sample amount

Column void or

channeling

Replace column, or, if possible, open top end

fitting and clean and fill void with glass beads

or same column packing; repack column

Injection solvent

too strong

Use weaker injection solvent or stronger mobile

phase

Sample volume too

large

Use injection volume equal to one-sixth of

column volume when sample prepared in

mobile phase for injection

Unwept injector

flow path Replace injector rotor

Peak Channeling in Replace or repack column

37

Fronting

column

Column overloaded Use higher-capacity stationary phase; increase

column diameter; decrease sample amount

Tailing

Peaks Basic solutes -

silanol interactions

Use competing base such as triethylamine; use

a stronger mobile phase; use base-deactivated

silica-based reversed-phase column; use

polymeric column

Beginning of peak

doublingSee peak doubling

Chelating solutes -

trace metals in base

silica

Use high purity silica-based column with low

trace-metal content; add EDTA or chelating

compound to mobile phase; use polymeric

column

Silica-based

column -

degradation at high

pH

Use polymeric, sterically protected, or high-

coverage reversed-phase column; install silica

gel saturator column between pump and injector

Silica-based

column -

degradation at high

temperature

Reduce temperature to less than 50 C

Silica-based

column - silanol

interactions

Decrease mobile-phase pH to suppress silanol

ionization; increase buffer concentration;

derivatize solute to change polar interactions

Unwept dead

volume

Minimize number of connections; ensure

injector rotor seal is tight; ensure all

compression fittings are correctly seated

Void formation at Replace column, or, if possible, open top end

38

head of column

fitting and clean and fill in void with glass

beads or same column packing; rotate injection

valve quickly; use injection valve with pressure

bypass; avoid pressure shock

Spikes

Bubbles in mobile

phase

Degas mobile phase; use back-pressure

restrictor at detector outlet; ensure that all

fittings are tight

Column stored

without caps

Store column tightly capped; flush reversed-

phase columns with degassed methanol

39

Ultra Fast Liquid Chromatography (UFLC)

High Performance Liquid Chromatography, HPLC, is a proven technique that has been

used in laboratories worldwide over the past 30-plus years. One of the primary derives

for the growth of technique has been evolution of packaging materials used to effect the

separation. The underlying principles of this evolution are governed by Van Deemter

equation, which is the empirical formula that describes the relationship between linear

velocity (flow rate) and plate height (HETP or efficiency) since particle size is one of the

variable, a Van Deemter curve can be used to investigate column performance.

According to Van Deemter equation, as the particle size decreases to less than 2.5

micrometer, not only is there a significant gain in efficiency, but also the efficiency does

not diminish at increased flow rates or linear velocity. By using smaller particles, speed

and peak capacity (number of peaks resolved per unit time in gradient separations) can be

extended to new limits. This is the underlying principle of Ultra Fast Liquid

Chromatography.

Going back to the concept of resolution,

To obtain high resolution, number of theoretical plates must be maximized. An increase

in N, the number of theoretical plates, by lengthening the column leads to an increase in

retention time and increased band broadening - which may not be desirable. Instead, to

increase the number of plates, the height equivalent to a theoretical plate can be reduced

by reducing the size of the stationary phase particles. This is the underlying principle of

UFLC.

40

Differences between HPLC and UFLC

Item/

Parameter

Way to UFLC Conventional

5 μm

4.6 mm

i.d.×150 mm

Column vol.:

2.5 mL

UFLC

2.2 μm

3.0 mm i.d.×75 mm

Column vol: 0.5 mL

1 Tube ID Proportional to column

section area ratio

0.3 mm 0.1 mm

[0.3×(3.0/4.6)2 ]

2 Mixer

volume

Proportional to column

volume ratio (sample loop

volume should be

included)

1.5 mL

(including

loop)

0.3 mL (including

loop)

[1.5×(0.5/2.5)]

3 Flow rate Opt. flow rate for each

column

1.0 mL/min 1.2 mL/min

4 Gradient

time

program

Proportional to (column

volume ratio) / (flow rate

ratio)

A/B=70/30 at

0 min

A/B=30/70 at

30 min

A/B=70/30 at 0 min

A/B=30/70 at 5 min

[30×(0.5/2.5)/(1.2/1)]

5 Column

temp

No change 40℃ 40℃

6 Response (col. vol. ratio) / (flow

rate ratio)

*Sampling rate is also

changed

500 ms » 100 ms

[500×(0.5/2.5)/

(1.2/1)]

7 Injection

volume

Proportional to column

cross section area ratio

10 mL 4 mL

[10×(3.0/4.6)2 ]

41

1. Column internal diameter

Internal Diameter

(mm ID)

Optimal Flow Rate

(ml/min)Application

2.0 0.4-0.5 For fast semi micro analysis with LC-MS

3.0 0.9-1.2 For fast analysis in general purpose HPLC

4.6 2.0-2.5 For fast analysis with large sample load

2. Column length

Length Application

30mm For ultra high-speed analysis

50mm For general purpose fast analysis

75mmTo shorten analysis time from that obtained with 5µm particle diameter, 150mm

column.

100mm For high-speed, high-separation analysis

42

43

Method development for HPLC

The 3 critical components for an HPLC method are:

Sample preparation

HPLC analysis

Standardization (calculations).

During the preliminary method development stage, all individual components should be

investigated before the final method optimization. This gives the scientist a chance to

critically evaluate the method performance in each component and streamline the final

method optimization. Sample preparation for chromatography is as important as the

chromatographic conditions.

Developing an HPLC method involves, understanding the chemistry of analytes and the

drug products. The intended use of the method should be known to the scientist. During

time management for HPLC method development around 10% of the time is given for

understanding the chemistry of the analyte and the drug product. [6]

The next step is to develop a preliminary HPLC conditions to achieve minimally

acceptable separations. These methods are used during entire method development

procedure. Around 20% of the total time is given for the development of preliminary

method.

The solubility of the analyte is then studied with different solvents to develop a suitable

sample preparation scheme for the drug product. 10% of the total time is given for the

sample preparation. The method is then standardized by varying the chromatographic

conditions such as mobile phase composition, temperature of the column etc.10% of the

total time is given to the standardization step.

Finally the robustness of the method is checked by repeated injections of the analyte.

Relative standard deviation for the analyte peak is tried to be kept minimum. Method

performance under different conditions – different instruments, different samples is

studied. 20% of the time is given to this step. [8]

The developed method is then validated according to ICH guidelines. It is a time

consuming step and takes about 30% of the required time. There’s no specific end to the

44

method development procedure. According to the objective of the method being

developed there are some points to be taken care of:

For a related substance method, determining the “significant and relevant” related

substances is very critical. With limited experience with the drug product, a good way to

determine the significant related substances is to look at the degradation products

observed during stress testing. Significant degradation products observed during stress

testing should be investigated in the method development..

Based on the current ICH guidelines on specifications, the related substances method for

active pharmaceutical ingredients (API) should focus on both the API degradation

products and synthetic impurities, while the same method for drug products should focus

only on the degradation products. In general practice, unless there are any special

toxicology concerns, related substances below the limit of quantitation (LOQ) should not

be reported and therefore should not be investigated.

In this stage, relevant related substances should be separated into 2 groups:

Significant related substances: Linearity, accuracy and response factors should be

established for the significant related substances during the method validation. To

limit the workload during method development, usually 3 or less significant

related substances should be selected in a method

Other related substances: These are potential degradation products that are not

significant in amount. The developed HPLC conditions only need to provide good

resolution for these related substances to show that they do not exist in significant

levels.[7,10]

Resolution (Rs)

A stability indicating method must resolve all significant degradation products from each

other. Typically the minimum requirement for baseline resolution is 1.5. This limit is

valid only for 2 Gaussian-shape peaks of equal size. In actual method development, Rs =

2.0 should be used as a minimum to account for day-to-day variability, non-ideal peak

shapes and differences in peak sizes. [3]

45

Limit of Quantitation (LOQ)

The desired method LOQ is related to the ICH reporting limits. If the corresponding ICH

reporting limit is 0.1%, the method LOQ should be 0.05% or less to ensure the results are

accurate up to one decimal place. However, it is of little value to develop a method with

an LOQ much below this level in standard practice because when the method is too

sensitive, method precision and accuracy are compromised. [3]

Precision, Accuracy

Expectations for precision and accuracy should be determined on a case-by-case basis.

For a typical related substance method, the RSD of 6 replicates should be less than 10%.

Accuracy should be within 70 % to 130% of theory at the LOQ level. [3]

Analysis time

A run time of about 5-10 minutes per injection is sufficient in most routine related

substance analyses. Unless the method is intended to support a high-volume assay,

shortening the run time further is not recommended as it may compromise the method

performance in other aspects (e.g., specificity, precision and accuracy.) [8]

Adaptability for Automation

For methods that are likely to be used in a high sample volume application, it is very

important for the Method to be “automatable”. The manual sample preparation procedure

should be easy to perform. This will ensure the sample preparation can be automated in

common sample preparation workstations. [3]

Understand the Chemistry

Similar to any other research project, a comprehensive literature search of the chemical

and physical properties of the analytes (and other structurally related compounds) is

essential to ensure the success of the project.

Chemical Properties

Most sample preparations involve the use of organic-aqueous and acid-base extraction

46

techniques. Therefore it is very helpful to understand the solubility and pKa of the

analytes. Solubility in different organic or aqueous solvents determines the best

composition of the sample solvent. pKa determines the pH in which the analyte will exist

as a neutral or ionic species. This information will facilitate an efficient sample extraction

scheme and determine the optimum pH in mobile phase to achieve good separations. [7]

Potential Degradation Products

Subjecting the API or drug product to common stress conditions provides insight into the

stability of the analytes under different conditions. The common stress conditions include

acidic pH, basic pH, neutral pH, different temperature and humidity conditions,

oxidation, reduction and photo-degradation. These studies help to determine the

significant related substances to be used in method development, and to determine the

sample solvent that gives the best sample solution stability.

In addition, the structures of the analytes will indicate the potential active sites for

degradation. Knowledge from basic organic chemistry will help to predict the reactivity

of the functional groups. For example, some excipients are known to contain trace level

of peroxide impurities. If the analyte is susceptible to oxidation, these peroxide impurities

could possibly produce significant degradation products. [2]

Sample Matrix

Physical (e.g., solubility) and chemical (e.g., UV activity, stability, pH effect) properties

of the sample matrix will help to design an appropriate sample preparation scheme. For

example, Hydroxypropyl Methylcellulose (HPMC) is known to absorb water to form a

very viscous solution; therefore it is essential to use mostly organic solvents in sample

preparation. [2]

Preliminary HPLC Conditions

In order to develop preliminary HPLC conditions in a timely fashion, scientists should

use artificial mixtures of active pharmaceutical ingredients and related substances at

relatively high concentrations (e.g., 1-2% of related substance relative to API) to develop

the preliminary HPLC conditions. The concentration ratio between API and the related

47

substances should be maintained to ensure the chromatography represents that of a real

sample. Alternatively, a highly stressed sample (e.g., 5% degradation) can also be used at

this stage. With the known composition and high levels of degradation products in the

sample, one can evaluate the chromatography to determine whether there are adequate

separations for all analytes. The high concentrations of related substances are used to

ensure all peaks will be detected.

Computer assisted method development can be very helpful in developing the

preliminary HPLC conditions quickly. Since the objective at this stage is to quickly

develop HPLC conditions for subsequent method development experiments, scientists

should focus on the separation of the significant related substances (section 3.1.1) instead

of trying to achieve good resolution for all related substances. These significant related

substances should be baseline resolved from each other with Rs > 2.0. After the

preliminary method development the HPLC conditions can be further fine-tuned at a later

stage to achieve the required specificity for the other related substances. [2]

Aged HPLC Column

An aged HPLC column should be used to develop the initial HPLC conditions. Usually it

is more difficult to achieve the required resolution with an aged column (e.g., column

with about 200 injections). This will reflect the worst-case scenario likely to be

encountered in actual method uses, and help the long-term method robustness.

In general, develop all methods with HPLC columns from the same vendor. The

preferred brand of HPLC column should be selected primarily based on the long term

stability and lot to lot reproducibility. [2]

Sample Preparation

Selection of Sample Solvent

This stage focuses on the selection of the sample solvent (for extraction) and the proper

sample preparation procedures. Investigate the effect of sample solvents of different %

organic, pH, extraction volume and extraction procedure on accuracy, precision,

sensitivity (LOQ) and the changes in the chromatography (e.g., peak shape, resolution).

Whenever possible use the mobile phase in the sample preparation. This will ensure that

48

there will not be any compatibility issues between the sample solution and the HPLC

conditions. [6,8]

Accuracy:

To investigate the accuracy in sample preparation (i.e., extraction efficiency), prepare a

spiked solution by adding known amounts of related substances into a sample matrix.

Compare responses of the spike solutions and the neat standard solutions to assess the

recovery from the sample preparation. In this stage, since only one particular step is being

investigated (i.e., sample preparation), close to theoretical recovery should be observed at

this point (e.g., 90-110%). [6,8]

Precision:

Use the stressed sample to represent the worst-case scenario and perform replicate

sample preparations from the same sample composite. Investigate the consistency of the

related substance profile (i.e., any missing peaks?) and the repeatability results from these

preparations. Another objective is to determine the sample concentration that gives an

acceptable LOQ (Signal to Noise ratio, S/N) in low-level spike concentrations. The

sample concentration should be low enough to maintain linearity and precision, but high

enough to achieve the desired LOQ. For example, if the ICH reporting limit for this drug

product is 0.1%, the LOQ of the method should be less than 0.05% (i.e., desired LOQ, in

%). By using spike sample solutions of very diluted concentrations for the significant

related substances, estimate the concentrations that give a S/N of about 10 for the

significant related substances. This estimated concentration is the approximate LOQ

concentration (i.e., estimated LOQ concentration, in g/mL). The following equation can

be used to estimate the target sample concentration for the method

Target sample concentration =estimated LOQ concentration (g/mL) x 1/desired LOQ

(%) x 100% [6,8]

Standardization

Area % method

If the response of the active pharmaceutical ingredient is linear from LOQ to the nominal

sample concentration, use the % area approach where the related substance is reported as

49

% area. This is the most straightforward approach, and doesn’t require the preparation of

standard solutions. It also has the highest precision since preparation to preparation

variation will not affect the results. However, in order to ensure the concentration is

linear within this range, the sample concentration is usually limited and this will reduce

the method sensitivity (i.e., increase LOQ) In general, use this approach as long as the

desired LOQ can be achieved. [10]

External Standard method

Use the external standard method if the response of the active pharmaceutical ingredient

is not linear throughout the whole range, or the desired LOQ cannot be achieved by the

area % method. The concentration of standard solution should be high enough to ensure

the standard solution can be prepared accurately and precisely on a routine basis, it

should be low enough to approximate the concentration of related substance in the

sample solution. In general, the standard concentration should correspond to about 5 % of

related substances. [10]

Wavelength Selection and Relative Response Factor

Generate the linearity plot of API and related substances at different wavelengths. At this

point, Photodiode Array Detector can be used to investigate the linearity of the active

pharmaceutical ingredient and related substances in the proposed concentration range. By

comparing the linearity slopes of the active pharmaceutical ingredient and the related

substances, one can estimate the relative response factors of the related substances at

different wavelengths. Disregard of whether Area % or External Standard approach is

used, if the relative response factors of some significant related substances are far from

unity, a response factor correction must be applied.

The optimum wavelength of detection is the wavelength that gives the highest sensitivity

for the significant related substances and minimizes the difference in response factors

between those of the active pharmaceutical ingredient and the related substances.

After the optimum wavelength is determined, use a highly stressed sample (e.g., 5%

degradation) to verify that the selected wavelength will give the highest % related

substance results. [6]

Overall accuracy

50

A final check of the method performance is to determine the overall accuracy of the

method. Unlike the accuracy from sample preparation (section 6.1.1), which simply

compares the response of the analyte with and without spiking with matrix, the overall

accuracy compares the % related substances calculated from an accuracy solution with

that of the theoretical value. The accuracy solutions are the solutions spiked with known

concentrations of related substances and matrix. Since the extraction efficiency, choice of

wavelength and the bias in standardization influence the calculated related substance

result, this is the best way to investigate the accuracy of the method. Overall accuracy

reflects the true accuracy of the method [6]

Method Optimization

Robustness

After the individual components of the method are optimized, perform the final

optimization of the method to improve the accuracy, precision and LOQ. Use an

experimental design approach to determine the experimental factors that have significant

impact on the method. This is very important in determining what factors need to be

investigated in the robustness testing during the method validation. To streamline the

method optimization process, use Plackett Burmann Design (or similar approach) to

simultaneously determine the main effects of many experimental factors.

Some of the typical experimental factors that need to be investigated are:

HPLC conditions: % organic, pH, flow rate, temperature, wavelength, and column age.

Sample preparation: % organic, pH, shaking/sonication, sample size, and sample age.

Calculation/standardization: integration, wavelength, standard concentration, response

factor correction.

Typical responses that need to be investigated are:

Results: precision (%RSD), % related substance of significant related substances, total

related substances. [8]

51

Method validation

Validation can be defined as “ establishing documented evidence which provides a high

degree of assurance that specific process will consistently produce a product meeting its

predetermined specification and quality attributes.” Method validation comes into play

after method development and begins with installation and qualification of instruments.

Since HPLC methods are used for different purposes, the method validation may also be

different. For e.g. several publications outline guides to validate pharmaceutical methods

such as USP, ICH ,FDA guidelines

The first step in development and validation of HPLC method should be to set clear

understandable minimum requirements that are acceptable to the chromatographer and

the end user.

Complete list of criteria should be made and evaluated before the method is validated.

The Statistics generated from validation studies should be similar and predictive of the

range of values gathered from real sample analysis. Validtion is of three types:

Full validation

Partial validation

Cross validation

Full validation

Full validation is important while developing and implementing an analyitical method for

the first time. It is important for a new drug entity. A full validation of the revised assay

is important if metabolites are added to an existing assay for quantification.

Partial Validation:

Partial validations are modifications of already validated analytical methods. Typical

analytical method changes that fall into this category include,

Change in analytical methodology (e.g. change in detection system).

Change in sample processing procedures.

Change in relevant concentration range.

Change in instruments or software.

52

Cross Validation.

Cross validation is a comparison of validation parameters when two or more analytical

methods are used to generate data within the same study or across different studies.

An example of cross validation would be a situation where an original validated

analytical method serves as the reference and the revised analytical method is the

comparator. The comparisons should be done both ways. [7]

PARAMETERS FOR VALIDATION OF HPLC METHODS

Selectivity

Specificity

Linearity

Accuracy

Precision

Detection limit [LOD]

Limit of quantification

Range

Ruggedness

Robustness

Selectivity:

Selectivity is the ability of an analytical method to differentiate and quantify the analyte

in the presence of other components in the sample. Each blank sample should be tested

for interference and selectivity should be ensured at the Lower Limit Of Quantification

(LLOQ).

Before any sample is introduced into a chromatographic system, the appropriate

resolution criteria must be outlined and satisfied. Generally the ability to resolve

individual components is generally a limiting factor for number of analytes that can be

measured using a single procedure. If appropriate resolution cannot be achieved, the

unresolved components at their maximum expected concentration should be validated to

demonstrate that these components would not affect the final result.

53

Specificity:

Specificity is a measure of the capability of the analytical method to be perfectly selective

for an analyte or group of similar analystes. .

Linearity:

Validation requires linearity to be established to verify that the analyte response is

linearly proportional to the concentration range of interest. A linearity study is generally

performed by preparing analyte solutions at various concentration levels and these

solutions should be prepared and analyzed at least three times.

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. Accuracy is

determined by replicate analysis of samples containing known amounts of the analyte.

A minimum of 3 concentrations in the range of expected concentrations is recommended.

The mean value should be within 15% of the actual value except at LLOQ, where it

should not deviate by more than 20%. The deviation of the mean from the true value

serves as the measure of accuracy.

Precision:

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

an analyte when the procedure is applied repeatedly to multiple aliquots of a single

homogenous volume of the sample

Precision should be measured using a minimum of 5 determinations per concentration

& minimum of 3 concentrations in the range of expected concentration is recommended.

The precision determined at each concentration level should not exceed 15% of the co

efficient of variation (CV) expect for the LLOQ, where it should not exceed 20% of the

CV.

According to ICH guidelines the measured standard deviation is subdivided into three

categories: repeatability, intermediate precision and reproducibility.

54

Repeatability of a method is obtained if the analysis is carried out in one laboratory by

one operator using the same equipment over a relatively short period of time.

Intermediate precision is measured in one laboratory but over several days and/or using

different analysts.

Reproducibility is defined as the variability of the measurement process in different

laboratories with different instruments.

Limit of detection:

It is the lowest conc. of the analyte that can be detected, but not necessarily quantifies, in

chromatography the detection limit is the injected amount that results in a peak with a

height at least twice as high as baseline noise. It is determined experimentally.

Limit of quantitation:

The limit of quantitation is the injected amount, which results in a reproducible

measurement of peak areas (equivalent amounts). Peak heights are typically required to

be about 10 to 20 times higher than the baseline noise.

Range of the method:

The working range of the method generally gives an optimum concentration range for

quantitative analyses. In practice, the linear range is generally determined by analysis or

samples of varying concentrations of the analyte of interest and plotting concentration

versus detector response.

Ruggedness:

Ruggedness is a measure of reproducibility of the results under normal, expected

operational conditions from laboratory to laboratory and from analyst to analyst i.e. the

chromatographer must be certain that the new method holds up under other conditions for

which the method has been validated.

Robustness:

55

It is the ability of the method to allow the analyte to remain unaffected by small

changes in the parameters such as ionic strength of the sample, detector temperature,

temperature of the sample and injection volume.

For the determination of a method’s robustness pH, flow rate, column temperature,

injection volume, detection wavelength or mobile phase composition is varied within a

realistic range and the quantitative influence of the parameter is within the specified

tolerance, the parameter is said to be within the method’s robustness range. [8]

56

Quality control parameters analyzed using HPLC

Dissolution

Dissolution testing is used to measure the release rate of an active component from a

solid dosage form under controlled conditions. This technique is used to assess the

performance of tablets, capsules and other solids.

Dissolution testing is useful for:

Guiding formulation development

Assessing the quality of a sample by determining whether the release of active

ingredient from the formulation is within acceptable limits (often used for release

and stability testing)

Comparing finished products with different formulations

Confirming the acceptability of changes to manufacturing procedures during

scale-up or post-marketing changes [9]

Dissolution testing involves dissolution of a solid dosage form in an appropriate

solvent and the conc. Of the active ingredient is measured at regular intervals of

time using HPLC. Two different types of apparatus are used for dissolution

testing. These are as described below:

Apparatus 1— The assembly consists of the following:

A covered vessel made of glass or other inert, transparent material; a motor; a metallic

drive shaft; and a cylindrical basket. The vessel is partially immersed in a suitable water

bath of any convenient size or placed in a heating jacket. The water bath or heating jacket

permits holding the temperature inside the vessel at 37 ± 0.5 during the test and keeping

the bath fluid in constant, smooth motion. No part of the assembly, including the

environment in which the assembly is placed, contributes significant motion, agitation, or

vibration beyond that due to the smoothly rotating stirring element. Apparatus that

permits observation of the specimen and stirring element during the test is preferable. The

vessel is cylindrical, with a hemispherical bottom and with one of the following

dimensions and capacities: for a nominal capacity of 1 L, the height is 160 mm to 210

57

mm and its inside diameter is 98 mm to 106 mm; for a nominal capacity of 2 L, the

height is 280 mm to 300 mm and its inside diameter is 98 mm to 106 mm; and for a

nominal capacity of 4 L, the height is 280 mm to300 mm and its inside diameter is 145

mm to 155 mm. Its sides are flanged at the top. A fitted cover may be used to retard

evaporation. The shaft is positioned so that its axis is not more than 2 mm at any point

from the vertical axis of the vessel and rotates smoothly and without significant wobble.

A speed-regulating device is used that allows the shaft rotation speed to be selected and

maintained at the rate specified in the individual monograph, within ±4%. Shaft and

basket components of the stirring element are fabricated of stainless steel. The entire

assembly is as shown in Figure 1

Fig. 1. Basket Stirring Element

58

Unless otherwise specified in the individual monograph, use 40-mesh cloth. A basket

having a gold coating 0.0001 inch (2.5 µm) thick may be used. The dosage unit is placed

in a dry basket at the beginning of each test. The distance between the inside bottom of

the vessel and the basket is maintained at 25 ± 2 mm during the test. [10]

Apparatus 2— the assembly from Apparatus 1 is used, except that a paddle formed from

a blade and a shaft is used as the stirring element. The shaft is positioned so that its axis is

not more than 2 mm at any point from the vertical axis of the vessel and rotates smoothly

without significant wobble. The vertical centerline of the blade passes through the axis of

the shaft so that the bottom of the blade is flush with the bottom of the shaft. The paddle

conforms to the specifications shown in Figure 2.

Fig. 2. Paddle Stirring element

59

The distance of 25 ± 2 mm between the blade and the inside bottom of the vessel is

maintained during the test. The metallic or suitably inert, rigid blade and shaft comprise a

single entity. A suitable two-part detachable design may be used provided the assembly

remains firmly engaged during the test. The paddle blade and shaft may be coated with a

suitable inert coating. The dosage unit is allowed to sink to the bottom of the vessel

before rotation of the blade is started. A small, loose piece of nonreactive material such

as not more than a few turns of wire helix may be attached to dosage units that would

otherwise float. Other validated sinker devices may be used. [10]

Dissolution Testing Apparatus

60

Impurity profiling

Impurities In Official articles

Concepts about purity change with time are inseparable from developments in analytical

chemistry. If a material previously considered to be pure can be resolved into more than

one component, that material can be redefined into new terms of purity and impurity.

Inorganic, organic, biochemical, isomeric, or polymeric components can all be

considered impurities. Microbiological species or strains are sometimes described in

similar terms of resolving into more than one component.

Monographs on bulk pharmaceutical chemicals usually cite one of three types of purity

tests: (1) a chromatographic purity test coupled with a nonspecific assay; (2) a

chromatographic purity-indicating method that serves as the assay; or (3) a specific test

and limit for a known impurity, an approach that usually requires a reference standard for

that impurity. Modern separation methods clearly play a dominant role in scientific

research today because these methods simultaneously separate and measure components

and fulfill the analytical ideal of making measurements only on purified specimens.. The

purity profile of a specimen that is constructed from the results of experiments using a

number of analytical methods is the ultimate goal.

Definitions

Foreign Substances

Foreign substances, which are introduced by contamination or adulteration, are not

consequences of the synthesis or preparation of compendial articles and thus cannot be

anticipated when monograph tests and assays are selected. Examples of foreign

substances include ephedrine in Ipecac or a pesticide in an oral liquid analgesic.

Residual Solvents

Residual solvents are defined as organic volatile chemicals that are used or produced in

the manufacture of drug substances or in the preparation of drug products. The solvents

are not completely removed by practical manufacturing techniques. Appropriate selection

61

of the solvent for the synthesis of a drug substance may enhance the yield or determine

characteristics such as crystal form, purity, and solubility and, as such, may be a critical

parameter in the synthetic process. Because there is no therapeutic benefit from residual

solvents, they should be removed to the extent possible to meet product specifications,

good manufacturing practices, or other quality-based requirements. Drug products should

contain no higher levels of residual solvents than can be supported by safety data.

Toxic Impurities

Toxic impurities have significant undesirable biological activity, even as minor

components, and require individual identification and quantitation by specific tests. These

impurities may arise out of the synthesis, preparation, or degradation of compendial

articles. Based on validation data, individualized tests and specifications are selected.

These feature comparison to a Reference Standard of the impurity, if available. It is

incumbent on the manufacturer to provide data that would support the classification of

such impurities as toxic impurities.

Concomitant Components

Concomitant components are characteristic of many bulk pharmaceutical chemicals and

are not considered to be impurities in the Pharmacopeial sense. Examples of concomitant

components are geometric and optical isomers (or racemates) and antibiotics that are

mixtures. Any component that can be considered a toxic impurity because of significant

undesirable biological effect is not considered to be a concomitant component.

Signal Impurities

Signal impurities are distinct from ordinary impurities in that they require individual

identification and quantitation by specific tests. Based on validation data, individualized

tests and specifications are selected. These feature a comparison to a reference standard

of the impurity, if available.

Signal impurities may include some process-related impurities or degradation products

that provide key information about the process, such as diazotizable substances in

thiazides. It is incumbent on the manufacturer to provide data that would support the

classification of such impurities as signal impurities rather than ordinary impurities.

62

Ordinary Impurities

Ordinary impurities are those species in bulk pharmaceutical chemicals that are

innocuous by virtue of having no significant, undesirable biological activity in the

amounts present. These impurities may arise out of the synthesis, preparation, or

degradation of compendial articles. Tests for related substances or chromatographic

purity might also control the presence of ordinary impurities.

The value of 2.0% was selected as the general limit on ordinary impurities in monographs

where documentation did not support adoption of other values.

Related Substances

Related substances are structurally related to a drug substance. These substances may be

identified or unidentified degradation products or impurities arising from a manufacturing

process or during storage of a material.

Process Contaminants

Process contaminants are identified or unidentified substances (excluding related

substances and water), including reagents, inorganics (e.g., heavy metals, chloride, or

sulfate), raw materials, and solvents. These substances may be introduced during

manufacturing or handling procedures. [11]

63

Materials and methods

Objective 1:

Complete analysis of Enalapril Maleate as per USP

Materials required:

Dissolution testing:

Dissolution testing apparatus: apparatus 2

Dissolution Medium: phosphate buffer pH 6.8

Glass wares- Measuring cylinder – 1000mL, test tubes, pipettes (not required if

the dissolution tester has an auto sampler), 100mL volumetric flask, agilent 1200

series sample vials with septa

Sonicator

Requirements for chromatography:

Standard preparation: 10 mg of USP Enalapril Maleate RS was transferred to a

100 mL volumetric flask & diluted using pH 6.8 phosphate buffer. The final

concentration thus became 0.1 mg of USP Enalpril Maleate per mL.

Test preparation: filtered portion of the dissolved tablet was used as the test

preparation.

Buffer solution: 1.38 gms of monobasic sodium phosphate was dissolved in about

800 mL water,pH was adjusted to 2.2 & the solution was diluted to 1000mL with

water.

Mobile phase: a filtered and degassed mixture of buffer solution and acetonitrile-

75:25 was made and used as the mobile phase.

Chromatographic system:

Instrument: Agilent 1200 series HPLC

Column: 4.6mm x 25 cm x 5 packing L7

Temperature: 50

Flow rate: 2mL/minute

64

Content uniformity testing:

Glass wares- test tubes, 100mL volumetric flasks 2, agilent 1200 series sample

vials with septa

Sonicator

Requirements for chromatography:

Buffer solution: 1.38 gms of monobasic sodium phosphate was dissolved in about

800 mL water,pH was adjusted to 2.2 & the solution was diluted to 1000mL with

water.

Standard preparation: 10 mg of USP Enalapril Maleate RS was transferred to a

100 mL volumetric flask & diluted buffer solution. The final concentration thus

became 0.1 mg of USP Enalpril Maleate per mL.

Test preparation: one tablet of 10 mg enalapril maleate was transferred to a 100

mL volumetric flask & volume was adjusted with buffer solution.

Mobile phase: a filtered and degassed mixture of buffer solution and acetonitrile-

75:25 was made and used as the mobile phase.

Chromatographic system:

Instrument: Agilent 1200 series HPLC

Column: 4.6mm x 25 cm x 5 packing L7

Temperature: 50

Flow rate: 2mL/minute

Assay:

Glass wares- test tubes, 100 mL beaker, 100mL volumetric flasks 2, 25 mL

volumetric flask, agilent 1200 series sample vials with septa

Sonicator

Requirements for chromatography:

Buffer solution: 1.38 gms of monobasic sodium phosphate was dissolved in about

800 mL water, pH was adjusted to 2.2 & the solution was diluted to 1000mL with

water.

65

Enalaprilat standard solution: 40 mg of USP Enalarilat RS was taken in a 100 mL

volumetric flask and the volume was adjusted using distilled water. The conc of

the solution thus became 0.4 mg of USP Enalaprilat RS per mL..

Enalprilapril diketopiperazine solution: 20 mg of USP enalapril Maleate RS was

placed in a100 mL beaker to form a mound on the bottom of the beaker. The

beaker was then placed on a hot plate at 70-80c to melt the solid. When melting

was observed, the beaker was immediately removed from the hot plate and

allowed to cool. To the cooled residue, 50 mL of acetonitrile was added and the

solution was sonicated to dissolve the residue. The solution typically contained, in

each mL, between 0.2 mg & 0.4 mg of enalapril diketopiperazine.

Standard preparation: 20 mg of USP Enalapril Maleate RS was transferred to 100

mL volumetric flask.0.5 mL of Enalaprilat standard solution was added to the

flask & volume was adjusted with buffer solution. The solution had a known

conc. of about 0.2 mg of USP Enalapril Maleate RS per mL and 0.002 mg of USP

Enalaprilat RS per mL.

Assay preparation: 10 tablets of enalapril maleate were transferred to 500 mL

volumetric flask and the vol. was adjusted with buffer solution.

System suitability solution: 0.5 mL of enalapril diketopiperazine solution was

transferred to a 25 mL volumetric flask and diluted with standard preparation to

volume.

Mobile phase: a filtered and degassed mixture of buffer solution and acetonitrile-

75:25 was made and used as the mobile phase.

Chromatographic system:

Instrument: Agilent 1200 series HPLC

Column: 4.6mm x 25 cm x 5 packing L7

Temperature: 50

Flow rate: 2mL/minute

Related substances:

Glass wares- test tubes, 100 mL beaker, 100mL volumetric flasks 3, agilent 1200

series sample vials with septa

66

Sonicator

Requirements for chromatography:

Buffer solution: 1.38 gms of monobasic sodium phosphate was dissolved in about

800 mL water, pH was adjusted to 2.2 & the solution was diluted to 1000mL with

water.

Enalaprilat standard solution: 40 mg of USP Enalarilat RS was taken in a 100 mL

volumetric flask and the volume was adjusted using distilled water. The conc of

the solution thus became 0.4 mg of USP Enalaprilat RS per mL..

Enalprilapril diketopiperazine solution: 20 mg of USP enalapril Maleate RS was

placed in a100 mL beaker to form a mound on the bottom of the beaker. The

beaker was then placed on a hot plate at 70-80c to melt the solid. When melting

was observed, the beaker was immediately removed from the hot plate and

allowed to cool. To the cooled residue, 50 mL of acetonitrile was added and the

solution was sonicated to dissolve the residue.the solution typically contained, in

each mL, between 0.2 mg & 0.4 mg of enalapril diketopiperazine.

Standard preparation: 20 mg of USP Enalapril Maleate RS was transferred to 100

mL volumetric flask.0.5 mL of Enalaprilat standard solution was added to the

flask & volume was adjusted with buffer solution. The solution had known conc.

of about 0.2 mg of USP Enalapril Maleate RS per mL and 0.002 mg of USP

Enalaprilat RS per mL.

Related compounds standard solution: 1 mL of standard preparation was

transferred to 100 mL volumetric flask and diluted with buffer solution to volume.

Test preparation: assay preparation was used as the test sample.

Mobile phase: a filtered and degassed mixture of buffer solution and acetonitrile-

75:25 was made and used as the mobile phase.

Chromatographic system:

Instrument: Agilent 1200 series HPLC

Column: 4.6mm x 25 cm x 5 packing L7

Temperature: 50

Flow rate: 2mL/minute

67

Objective 2:

To study separation pattern of Parabens mixture (methyl, ethyl, butyl

parabens) using conventional HPLC and to transfer the method to UFLC

and study the separation pattern.

Materials required:

Glass wares: 100mL volumetric flask.

Sonicator:

Requirements for conventional liquid chromatography:

Sample preparation: 5mg each of methyl, ethyl and butyl Parabens is dissolved in

100 mL water.

Mobile phase: methanol: water (60:40)

Chromatographic system:

Instrument: Agilent 1200 series HPLC

Column: Shimpack XR-ODS (C18) (4.66mm. x 150mm x 2.2 µm)

Temperature: 30℃

Flow rate: 1.00 mL/min

Requirements for UFLC

Sample preparation: 5mg each of methyl, ethyl and butyl Parabens is dissolved in

100 mL water.

Mobile phase: methanol: water (60:40)

Chromatographic system:

Instrument: Shimadzu prominence UFLC

Column: Shimpack XR-ODS, 3mm x 50mm x 1µm

Temperature: 30℃

Flow rate: 1.00 mL/min

68

Methods:

Objective 1:

Complete analysis of Enalapril Maleate as per USP

Dissolution testing:

1000 mL of dissolution med. was placed in the vessel of apparatus 2. The dissolution

med. was equilibrated to 37 ± 0.5. One tablet was placed in the apparatus taking care to

exclude air bubbles from the surface of the tablet and the apparatus was operated at 50

rpm for 30 minutes. After 30 minutes a specimen from a zone midway between the

surface of the dissolution medium and the top of the vessel was withdrawn and used as a

test sample after filtration. The conc of enalapril maleate was then determined using

HPLC.

About 50µl of the test preparation and the standard preparation were separately injected

in the chromatograph. The column temperature was maintained at 50℃ and the flow rate

was about 2mL/min. The responses for major peaks were then recorded. The quantity of

enalapril maleate in each tablet was calculated by the formula;

(TC/D)(rU / rS),

in which T is the labeled quantity, in mg, of enalapril maleate in the Tablet; C is

the concentration, in mg per mL, of USP Enalapril Maleate RS in the Standard

preparation; D is the concentration, in mg per mL, of enalapril maleate in the Test

preparation, based upon the labeled quantity per Tablet and the extent of dilution; and rU

and rS are the enalapril peak responses obtained from the Test preparation and the

Standard preparation, respectively.

Content uniformity testing:

About 50µl of the test preparation and the standard preparation were separately

injected in the chromatograph. The column temperature was maintained at 50℃ and the

flow rate was about 2mL/min .The responses for major peaks were then recorded. The

quantity of enalapril maleate in each tablet was calculated by the formula;

(TC/D)(rU / rS),

69

in which T is the labeled quantity, in mg, of enalapril maleate in the Tablet; C is

the concentration, in mg per mL, of USP Enalapril Maleate RS in the Standard

preparation; D is the concentration, in mg per mL, of enalapril maleate in the Test

preparation, based upon the labeled quantity per Tablet and the extent of dilution; and rU

and rS are the enalapril peak responses obtained from the Test preparation and the

Standard preparation, respectively.

Assay:

About 50µl of the assay preparation and the standard preparation were separately injected

in the chromatograph. The column temperature was maintained at 50℃ and the flow rate

was about 2mL/min .The responses for major peaks were then recorded. The quantity of

enalapril maleate in each tablet was calculated by the formula;

(CV/N)(rU / rS),

in which C is the concentration, in mg per mL, of USP Enalapril Maleate RS in the

Standard preparation; V is the nominal capacity, in mL, of the volumetric flask

containing the Assay preparation; N is the number of Tablets taken for the Assay

preparation; and rU and rS are the enalapril peak responses obtained from the Assay

preparation and the Standard preparation, respectively.

Related substances:

About 50µl of the test preparation, the standard preparation, the related compounds

standard solution and the buffer solution were separately injected in the chromatograph.

The column temperature was maintained at 50℃ and the flow rate was about 2mL/min.

The responses for all the peaks greater than 0.1 % of the enalapril peak that were not

observed in the buffer solution were measured. The percentage of anhydrous enalaprilat

present in the portion of tablets was calculated by the formula;

(492.52/348.39)(CV/N)(rU / rS)(100/L),

in which 492.52 and 348.39 are the molecular weights of enalapril maleate and

anhydrous enalaprilat, respectively; C is the concentration, in mg per mL, of USP

Enalaprilat RS in the Standard preparation; V is the nominal capacity, in mL, of the

volumetric flask containing the Test preparation; N is the number of Tablets taken for the

70

Test preparation; rU and rS are the enalaprilat peak responses obtained from the Test

preparation and the Standard preparation, respectively; and L is the labeled amount of

enalapril maleate in theTablet.

the percentage of enalapril diketopiperazine (as enalapril maleate) present in the portion

of Tablets was calculated from the formula,

(492.52/358.44)(C¢V/N)(rU /1.25 rS)(100/L),

in which 492.52 and 358.44 are the molecular weights of enalapril maleate and enalapril

diketopiperazine, respectively; C¢is the concentration, in mg per mL, of USP Enalapril

Maleate RS in the Related compounds standard solution; V is the nominal capacity, in

mL, of the volumetric flask containing the Test preparation; N is the number of Tablets

taken for the Test preparation; rU is the enalapril diketopiperazine peak response

obtained from the Test preparation; 1.25 is the response for enalapril diketopiperazine

relative to that for enalapril maleate; rS is the enalapril peak response obtained from the

Related compounds standard solution; and L is the labeled amount, in mg, of enalapril

maleate in the Tablet.

the percentage of any other related compound was calculated by the formula:

(C¢V/N)(rR / rS)(100/L),

in which rR is the sum of the responses of any related compound, other than

those from maleic acid, enalapril, enalaprilat, and enalapril diketopiperazine obtained

from the Test preparation; rs is the enalapril peak response obtained from the Related

compounds standard solution; and C¢, V, N, and L are as defined above: the sum of all

related compounds including those from enalaprilat and enalapril diketopiperazine is not

greater than 5.0%.

71

Objective 2:

To study separation pattern of Parabens mixture (methyl, ethyl, butyl

Parabens) using conventional HPLC and to transfer the method to UFLC

and study the separation pattern.

Procedure:

For conventional HPLC:

About 15µl of the sample preparation was injected in the chromatograph. The column

temperature was maintained at 30℃ and the flow rate is about 1mL/min. The responses

for major peaks were then recorded.

For UFLC:

About 8µl of the sample preparation was injected in the chromatograph. The column

temperature was maintained at 30℃ and the flow rate is about 1mL/min. The responses

for major peaks were then recorded.

The changes in the separation pattern of the same sample i.e. parabens mixture were

studied by comparing the chromatograms of HPLC and UFLC.

72

Results and calculations

Objective 1:

Complete analysis of Enalapril Maleate tablets 10 mg using HPLC.

Dissolution testing:Chromatogram for the standard preparation:Data file: C:\EZchrom Elite\Data\August2007\Enalapril Maleate tablets 10mg\10807_001.datMethod: C:\EZchrom Elite\Data\August2007\Enalapril Maleate tablets 10mg\RS.metAcquired: 1/08/07 12:10:01PMName: standardVial no: 1Injection vol: 50µLUser: TGK

Results:

Retention Time

Name Area Area %

Theoretical plates

Asymmetry

1 1.710 11740 0.194 522 0.92 2.132 184 0.003 10877 1.03 8.790 ENALAPRIL MALEATE 6038812 99.80 870 1.3

73

Chromatogram for the test preparation:Data file: C:\EZchrom Elite\Data\August2007\Enalapril Maleate tablets 10mg\10807_003.datMethod: C:\EZchrom Elite\Data\August2007\Enalapril Maleate tablets 10mg\RS.metAcquired: 1/08/07 12:10:01PMName: test 2Vial no: 3Injection vol: 50µLUser: TGK

Results:

Retention Time

Name Area Area % Theoretical plates

Asymmetry

1 1.474 2299296 79.151 2789 0.82 1.769 11458 0.394 16345 1.13 2.158 198 0.006 2675 1.04 8.689 ENALAPRIL MALEATE 593963 20.447 1018 1.2

74

Calculations:

The amount of enalapril maleate in mg released in the dissolution medium is given by the

formula:

(TC/D)(rU / rS),

in which T is the labeled quantity, in mg, of enalapril maleate in the Tablet; C is

the concentration, in mg per mL, of USP Enalapril Maleate RS in the Standard

preparation; D is the concentration, in mg per mL, of enalapril maleate in the Test

preparation, based upon the labeled quantity per Tablet and the extent of dilution; and rU

and rS are the enalapril peak responses obtained from the Test preparation and the

Standard preparation, respectively.

Substituting the values from the chromatogram, we get,

Amt of enalapril maleate released = 10 X 0.1/0.01 X 593963/6038812

= 9.835 mg

According to USP not less than 80% of the labeled amount of the tablet should be

dissolved in 30 minutes and from the calculations it can be seen that 9.85 mg of the 10

mg i.e. 98.5% of the labeled quantity is released in the dissolution medium after 30

minutes. Thus the batch passes the dissolution test.

75

Content uniformity testing:Since the buffer solution and the mobile phase for dissolution testing and content uniformity testing is same, no separate injection of the standard preparation is required.The chromatogram for the test preparation is given below:

Data file: C:\EZchrom Elite\Data\August2007\Enalapril Maleate tablets 10mg\10807_002.datMethod: C:\EZchrom Elite\Data\August2007\Enalapril Maleate tablets 10mg\RS.metAcquired: 1/08/07 12:36:07PMName: test 1Vial no: 2Injection vol: 50µLUser: TGK

Results:

Retention Time

Name Area Area % Theoretical plates

Asymmetry

1 1.467 22992679

79.167 2569 0.8

2 1.780 11578 0.039 16329 1.03 2.111 175 0.0006 2696 1.24 8.780 ENALAPRIL MALEATE 6038808 20.793 1016 1.2

76

Calculations:

Amount of Enalapril maleate, in mg, per tablet is given by the formula:

(TC/D)(rU / rS),

in which T is the labeled quantity, in mg, of enalapril maleate in the Tablet; C is

the concentration, in mg per mL, of USP Enalapril Maleate RS in the Standard

preparation; D is the concentration, in mg per mL, of enalapril maleate in the Test

preparation, based upon the labeled quantity per Tablet and the extent of dilution; and rU

and rS are the enalapril peak responses obtained from the Test preparation and the

Standard preparation, respectively.

Substituting the values from the chromatogram, we get,

Amt of enalapril maleate per tablet = 10 X 0.1/0.1 X 6038798/6038812

= 9.999 mg

According to USP the tablets should contain not less than 90% and more than 110% of

the labeled amount of enalapril maleate.

Since the calculations indicate that the tablets contain 9.999 mg i.e. 99.99% of the labeled

amount of enalapril maleate, the batch passes the content uniformity testing.

77

Assay:

Chromatogram for the standard preparation:

Data file: C:\EZchrom Elite\Data\August2007\Enalapril Maleate tablets 10mg\10807_005.datMethod: C:\EZchrom Elite\Data\August2007\Enalapril Maleate tablets 10mg\RS.metAcquired: 1/08/07 1:47:03PMName: assay stdVial no: 5Injection vol: 50µLUser: TGK

Results:

Retention Time

Name Area Area %

Theoretical plates

Asymmetry

1 1.689 24148 0.396 15896 1.22 2.213 8107 0.133 3698 1.33 2.487 ENALAPRILAT 6733 0.110 2698 1.94 3.341 1134 0.019 7569 1.25 4.146 234 0.003 9658 16 4.710 271 0.004 16598 1.47 5.418 709 0.012 7698 1.08 8.451 ENALAPRIL MALEATE 6050899 99.321 968 1.2

78

Chromatogram for assay preparation:

Data file: C:\EZchrom Elite\Data\August2007\Enalapril Maleate tablets 10mg\10807_007.datMethod: C:\EZchrom Elite\Data\August2007\Enalapril Maleate tablets 10mg\RS.metAcquired: 1/08/07 03:10:01PMName: test 1Vial no: 7Injection vol: 50µLUser: TGK

Results:

Retention Time

Name Area Area %

Theoretical plates

Asymmetry

1 1.467 21482506

77.920 2516 0.7

2 1.710 22388 0.081 16329 1.13 2.041 7406 0.027 2923 1.34 2.595 ENALAPRILAT 9526 0.035 2013 2.15 3.333 1059 0.004 4250 2.26 4.446 245 0.001 14494 0.87 4.602 262 0.001 13766 1.48 5.384 726 0.003 7786 1.49 8.353 ENALAPRIL MALEATE 6038798 21.905 866 1.3

79

10 12.867 DIKETOPIPERAZINE 5035 0.018 6230 1.2

Calculations:

The quantity of enalapril maleate in each tablet is calculated by the formula;

(CV/N)(rU / rS),

in which C is the concentration, in mg per mL, of USP Enalapril Maleate RS in the

Standard preparation; V is the nominal capacity, in mL, of the volumetric flask

containing the Assay preparation; N is the number of Tablets taken for the Assay

preparation; and rU and rS are the enalapril peak responses obtained from the Assay

preparation and the Standard preparation, respectively.

Substituting the values from the chromatogram we get,

Amt of enalapril maleate per tablet = 0.2 X (500/10) X (6038798/6050899)

= 9.980mg

According to USP the tablets should contain not less than 90% and more than 110% of

the labeled amount of enalapril maleate.

Since the calculations indicate that the tablets contain 9.980 mg i.e. 99.80 % of the

labeled amount of enalapril maleate, the batch passes the content uniformity testing.

80

81

Related substances:Chromatogram for the blank solution:Data file: C:\EZchrom Elite\Data\August2007\Enalapril Maleate tablets 10mg\10807_004.datMethod: C:\EZchrom Elite\Data\August2007\Enalapril Maleate tablets 10mg\RS.metAcquired: 1/08/07 01:10:01PMName: blankVial no: 4Injection vol: 50µLUser: TGK

Results:

Retention Time

Name Area Area %

Theoretical plates

Asymmetry

1 1.695 22741 68.584 17588 1.12 2.012 7698 23.216 3658 1.33 3.128 1235 3.725 7856 2.24 4.487 198 0.597 10256 0.85 4.692 305 0.919 15256 1.46 5.394 850 2.563 7896 1.47 23.693 131 0.395 0 1.7

82

Since the buffer solution and the mobile phase for assay and related substances testing is same, no separate injection of the standard preparation is required.The chromatogram for the related compounds standard preparation is given below:

Data file: C:\EZchrom Elite\Data\August2007\Enalapril Maleate tablets 10mg\10807_006.datMethod: C:\EZchrom Elite\Data\August2007\Enalapril Maleate tablets 10mg\RS.metAcquired: 1/08/07 2:17:03PMName: referenceVial no: 6Injection vol: 50µLUser: TGK

Results:

Retention Time

Name Area Area %

Theoretical plates

Asymmetry

1 1.670 21270 27.381 15785 1.02 2.211 8813 11.345 3647 1.23 2.510 ENALAPRILAT 689 0.886 2698 1.64 3.180 1041 1.340 7125 1.05 4.210 310 0.399 9741 1.16 4.711 280 0.360 15963 1.27 5.532 678 0.873 7752 1.18 8.472 ENALAPRIL MALEATE 44602 57.415 903 1.0

Analyst:

83

Chromatogram for the test preparation:

Data file: C:\EZchrom Elite\Data\August2007\Enalapril Maleate tablets 10mg\10807_007.datMethod: C:\EZchrom Elite\Data\August2007\Enalapril Maleate tablets 10mg\RS.metAcquired: 1/08/07 03:10:01PMName: test 1Vial no: 7Injection vol: 50µLUser: TGK

Results:

Retention Time

Name Area Area %

Theoretical plates

Asymmetry

1 1.467 21482506

77.920 2516 0.7

2 1.710 22388 0.081 16329 1.13 2.041 7406 0.027 2923 1.34 2.595 ENALAPRILAT 9526 0.035 2013 2.15 3.333 1059 0.004 4250 2.26 4.446 245 0.001 14494 0.87 4.602 262 0.001 13766 1.48 5.384 726 0.003 7786 1.49 8.353 ENALAPRIL MALEATE 6038798 21.905 866 1.310 12.867 DIKETOPIPERAZINE 5035 0.018 6230 1.2

84

Calculations:

The percentage of anhydrous enalaprilat present in the portion of tablets is calculated by

the formula;

(492.52/348.39)(CV/N)(rU / rS)(100/L),

in which 492.52 and 348.39 are the molecular weights of enalapril maleate and

anhydrous enalaprilat, respectively; C is the concentration, in mg per mL, of USP

Enalaprilat RS in the Standard preparation; V is the nominal capacity, in mL, of the

volumetric flask containing the Test preparation; N is the number of Tablets taken for the

Test preparation; rU and rS are the enalaprilat peak responses obtained from the Test

preparation and the Standard preparation, respectively; and L is the labeled amount of

enalapril maleate in theTablet.

Substituting the values from the chromatogram we get,

percentage of anhydrous enalaprilat

= (492.52/348.39) X 0.002 X (500/10) X (9526/6733) X10

= 2 %

The percentage of enalapril diketopiperazine (as enalapril maleate) present in the portion

of Tablets is calculated from the formula,

(492.52/358.44)(C¢V/N)(rU /1.25 rS)(100/L),

in which 492.52 and 358.44 are the molecular weights of enalapril maleate and enalapril

diketopiperazine, respectively; C¢is the concentration, in mg per mL, of USP Enalapril

Maleate RS in the Related compounds standard solution; V is the nominal capacity, in

mL, of the volumetric flask containing the Test preparation; N is the number of Tablets

taken for the Test preparation; rU is the enalapril diketopiperazine peak response

obtained from the Test preparation; 1.25 is the response for enalapril diketopiperazine

relative to that for enalapril maleate; rS is the enalapril peak response obtained from the

Related compounds standard solution; and L is the labeled amount, in mg, of enalapril

maleate in the Tablet.

Substituting the values from the chromatogram we get,

The percentage of enalapril diketopiperazine

85

= (492.52/358.44) X 0.002 X (500/10) X 3246/(1.25 X 44602)

= 0.008 %

No other impurity was found in the tablet as seen from comparison of chromatograms of

blank & test preparation.

86

Objective 2:

To study separation pattern of Parabens mixture (methyl, ethyl, butyl

Parabens) using conventional HPLC and to transfer the method to UFLC

and study the separation pattern.

Separation pattern of parabens using conventional HPLC:

Separation pattern of parabens using UFLC:

87

Peak table (HPLC):

Peak table (UFLC):

88

Conclusion

Objective 1:

Complete analysis of Enalapril Maleate as per USP

Complete analysis of Enalapril Maleate was done using HPLC as per USP.

Results of the complete quality analysis are summarized in the following table:

Parameters Acceptance criteria as

per USP

Values obtained after

analysis of the tablets.

Conclusion

Dissolution testing Not less than 80% of

the labeled amount of

the tablet should be

dissolved in 30

minutes

Amt of enalapril

maleate released in 30

minutes = 9.835 mg =

98.35 % of the labeled

amount

Batch passes

Content uniformity The tablets should

contain not less than

90% and more than

110% of the labeled

amount of enalapril

maleate.

Amt of enalapril

maleate per tablet

= 9.999 mg

= 99.99% of the

labeled amount

Batch passes.

Assay The tablets should

contain not less than

90% and more than

110% of the labeled

amount of enalapril

maleate.

Amt of enalapril

maleate per tablet

= 9.980mg = 99.80 %

of the labeled amount

Batch passes.

Contd…

89

Related substances The sum of all the

related compounds is

not greater than 5.0 %

The percentage of

anhydrous enalaprilat

= 2 %

The percentage of

enalapril

diketopiperazine

= 0.008 %

Since no other

impurity is present the

sum of all the related

substance becomes

2.008 %

Batch passes.

Thus the actual procedure of quality control of pharmaceutical products using HPLC was

studied.

Objective 2:

To study separation pattern of parabens mixture (methyl, ethyl, butyl

parabens) using conventional HPLC and to transfer the method to UFLC

and study the separation pattern.

By comparing the separation patterns of parabens mixture using conventional HPLC and

UFLC it was found that the stationary phase chemistry affects the speed of separation and

it was seen that the decrease in the size of the stationary phase particles decreases the

time required for separation with improvement in resolution.

As the analysis time was reduced to 3.0 minutes with UFLC from 21.0 minutes in the

conventional HPLC, UFLC can be considered to be better than HPLC for pharmaceutical

analyses.

90

References

1. D. A. Skoog, J. J. Leary – “ Principles of instrumental analysis”, 6th edition,

Indian reprint 2007,copyright © 2007 by Thomson Brooks/Cole, a part of the

Thomson corporation.

2. Phyllis Brown, DeAntonois, Prentice Hall – “Handbook of Instrumental

techniques for analytical chemistry”, 1997.

3. L. R. Snyder and J. J. Kirkland – “Introduction to Modern Liquid

Chromatography”, 2nd edition. John Wiley & Sons, 1979.

4. LC-GC Magazine, Advanstar Communications.

5. Sal Emmanuel, Jean-Pierre Fontelle – “Quality Assurance and Quality control in

pharmaceutical Industry”, 4th edition, 2004.

6. Craig S. Young and Raymond J. Weigand – “An Efficient Approach to HPLC

Method Development “, 1999.

7. V P shah, guidance for industry - “Bioanalytical method development and

validation” May 2001.

8. J. Mark Green – “A Practical Guide to Analytical Method Validation” 1996, (68)

305A-309A Copyright © 1996 by the American Chemical Society

9. Dissolution testing of immediate release and sustained release solid dosage forms.

U.S. Department of Health and Human Services,

Food and Drug Administration,

91

Center for Drug Evaluation and Research (CDER),

August 1997.

10. General chapters - The United States Pharmacopoeia 29

11. General chapters - The Indian Pharmacopoeia

12. General chapters - British Pharmacopoeia

92