hplc pharma 25-1-09-numbered-all print

8/4/2019 HPLC Pharma 25-1-09-Numbered-All Print

1/19

Created by: Dr. Shula Levin Medtechnica January 26, 2009

[email protected] Page 1 of 19

High Performance Liquid Chromatography

(HPLC) in the pharmaceutical analysis

Shulamit Levin, Medtechnica; Efal St 5 Petach Tikva

49002

1 Types of HPLC: Reversed Phase chromatography..2 HPLC Theory: System Suitability parameters3 The Role of HPLC in Drug Analysis

3.1 Discovery: High throughput screening andbioanalysis

3.2 Development: Method development andvalidation

3.3 Manufacturing: Dissolution, content uniformity,assay, drug impurities and stability, in process

and cleaning verification.

4 Specialized HPLC Separations.

4.1 Ultra-Performance Liquid Chromatography.4.2 Preparative HPLC

4.3 Chiral Separations

Introduction

Drug manufacturing control requires high level andintensive analytical and chemical support of all stages to

ensure the drug's quality and safety (1). The

pharmacopeia constitutes a collection of recommended

procedures for analysis and specifications for the

determination of pharmaceutical substances, excipients,

and dosage forms that is intended to serve as sourcematerial for reference or adaptation by anyone wishing to

fulfill pharmaceutical requirements. The most important

analytical technique used during the various steps of

drug development and manufacturing is the separation

technique: High Performance Liquid Chromatography

(HPLC).

The key to a proper HPLC system operation is

knowledge of the principles of the chromatographic

process, as well as understanding the reasons behind the

choice of the components of the chromatographic

systems such as column, mobile phase and detectors. A

scheme of an HPLC system is shown in Figure 1. Ahigh pressure pump is required to force the mobile phase

through the column at typical flow rates of 0.5-2

ml/min. The sample to be separated is introduced into

the mobile phase by injection device, manual or

automatic, prior to the column. The detector usually

contains low volume cell through which the mobile

phase passes carrying the sample components eluting

from the column. There are books describing the

practicality of HPLC operation (2-11). It is expected ofany proper HPLC system that is used in the

pharmaceutical laboratories to produce highly accurate

and precise results, due to health related issues of

improper measurements. Every HPLC system must be

qualified to comply with the strict demands from health

authorities for high quantitative performance.

Quality standards in pharmaceutics require that all

instruments should be adequately designed, maintained,calibrated, and tested. The approach that has been

adopted in the environment of the analytical instrument

has become known as the "Four Qs": design qualification(DQ), installation qualification (IQ), operational

qualification (OQ), and performance qualification (PQ).Design qualification is performed at the vendors site,

and it is representative of the way an instrument is

developed and produced, usually governed by

International Organization for Standardization (ISO)

criteria.

The installation qualification (IQ) process can be divided

into two steps:pre-installationandphysical installation.

During pre-installation, all information relevant to the

proper installation, operation, and maintenance of the

instrument is checked. Workers confirm the site

requirements and the receipt of all of the parts, pieces,

and manuals necessary to perform the installation of the

specific HPLC unit. During physical installation, serial

numbers are recorded and all fluidic, electrical, and

communication connections are made for system

components. Documentation describing how the

instrument was installed, who performed the installation,

and other various details are archived.

HPLC Column

in OvenAuto

SamplerPump

)flows50-500 0L/min

Fraction

Collector

Waste

Detector&Control

DataProcessing

1.Fucose

2.Galactosami ne

3.Glucosamine

4.Galactose

5.Glucose

6.Mannose

20.00Minutes5.00

mV

0.00

300

12

34

5

6

a bc d

Or manual injector

Figure 1: A Scheme of an HPLC System

The operational qualification process ensures that the

separate modules of a system (pump, injector, and

detector) are operating according to the defined

specifications such as accuracy, linearity, and precision.

Specific tests are performed to verify parameters such asdetector wavelength accuracy, flow rate, or injector

precision.

The performance qualification (PQ) step verifies system

performance as a whole. Performance qualification

testing is conducted under real operating conditions inthe analytical laboratory that is going to be using the

instrument. In practice, sometimes operational and


2/19



performance qualification blend together, particularly for

linearity and precision (repeatability) tests, which can be

conducted more easily at the system level.

The performance qualification test of the HPLC systemuses a method with a well-characterized analyte mixture,

column, and mobile phase. It incorporates type of

measurements from the system suitability section of thegeneral chromatography chapter in the U.S.

Pharmacopeia (12). In the end of the process proper

documentation is archived.

1. Modes of HPLC

There are various modes of operation of HPLC. The

mechanism of interaction of the solutes with the

stationary phases determines the classification of themode of liquid chromatography. Table 1 summarizes

the variety of modes of liquid chromatography, of which

Reversed Phase stands out as the most widely used modein HPLC, therefore, the discussion will elaborate on this

mode.

Reversed Phase (13)

Reversed phase liquid chromatography (RPLC) is

considered as the method of choice for the analysis of

pharmaceutical compounds for several reasons, such as

its compatibility with aqueous and organic solutions aswell as with different detection systems and its high

consistency and repeatability. Sensitive and accurate

RPLC analysis, whether in the pharmaceutical or

bioanalytical field, necessitates the use of stationaryphases which give symmetrical and efficient peaks.Therefore, manufacturers of stationary phases are

continuously improving and introducing new RPLC

products, and the selection of various types of reversed

phase stationary phases is high. The needs for

consistency as well as the globalization of the

pharmaceutical companies require that the methods willbe transferred from site to site, using either the same

column brands or their equivalents. Therefore, an

extensive categorization or characterization of the rich

selection of stationary phases has been done in recent

years (14-21).

The stationary phase in the Reversed Phase

chromatographic columns is a hydrophobic support that

is consisted mainly of porous particles of silica gel in

various shapes (spheric or irregular) at various diameters

(1.8, 3, 5, 7, 10 M etc.) at various pore sizes (such as60, 100, 120, 300). The surface of these particles is

covered with various chemical entities, such as various

hydrocarbons (C1, C6, C4, C8, C18, etc) as can be seen

in Figure 2. There are also hydrophobic polymeric

supports that are used as stationary phases when there is

an extreme pH in the mobile phase. In most methods

used currently to separate medicinal materials, C18

columns are used, which sometimes are called ODS(octedecylsilane) or RP-18.

The more hydrophobic are the sample components the

longer they stay in the column thus they are separated.

The mobile phases are mixtures of water and organic

polar solvents mostly methanol and acetonitrile. These

mixtures contain frequently additives such as acetate,phosphate, citrate, and/or ion-pairing substances, which

are surface active substances such as alkylamines as ion-

pairng of anions or alkylsulfonates, ion-pairing ofcations. The purpose of using such additives is to

enhance efficiency and/or selectivity of the separation,

mostly due to control of their retention.

Reversed Phase - Stationary Phase Surface

O

Si

O

Si

O

Si

O

Si

O

Si

O

O

O

Si

O

Si

O

Si

O

Si

O O

Si

O

Si

OO

O

O

O

O

O

O O O O O O O

OH

Si

HO O

OH O

OHHO

O O

O

Si

O

O

OH

R

R

R

R R

R

NORMAL PHASE

R=H

REVERSED PHASE

. R= C18; C8; C4; CN; C1 and more

Si

Si

Figure 2: The surface of Reversed Phase stationary phases

The parameters that govern the retention in Reversed

Phase systems are the following:

A. The chemical nature of the stationary phase

surface(22-27).B. The type of solvents that compose the mobile

phase and their ratio(28)

C. The pH and ionic strength and additives of themobile phase(23, 29)

When the effect of these parameters on the retention of

the solutes is understood it is possible to manipulate

them to enhance selectivity.

A. The chemical nature of the stationary phase(16)

The surface of the stationary phase is described in Figure2. The chemical nature is determined by the size and

chemistry of hydrocarbon bonded on the silica gel

surface, its bonding density (units ofmole/m2), and thepurity and quality of the silica gel support. As a rule, the

more carbons in a bonded hydrocarbon the more itretains organic solutes (as long as similar % coverage is

compared). The higher the bonding density the longer

the organic solutes are retained. A column is considered

relatively hydrophobic if its bonding density exceeds 3

mole/m2.

Very important modifiers of the stationary phase's

surface are surface-active substances used as mobilephase's additives, acting as ion-pair reagents. These are


3/19



substances such as tri-ethylamine or tetrabutylamine or

hexyl, heptyl, octyl sulfonate. They are distributed

between the mobile phase and the hydrophobic surface

and cover it with either positive (alkylamines) or

negative (alkylsulfonates) charges. This change of thesurface into charged surface affects the retention

significantly, especially on charged species in the

sample(30).

B. Composition of the mobile phase

As a rule, the weakest solvent in Reversed Phase is the

most polar one, water. The other polar organic solvents

are considered stronger solvents, where the order of

solvent strength follows more or less their dielectric

properties, or polarity. The less polar the solvent added

to the mobile phase, the stronger it gets, shortening the

retention times.

C. PH and ionic strength of the mobile phase

When the samples contain solutes of ionizable functional

groups, such as amines, carboxyls, phosphates,

phosphonates, sulfates and sulfonates, it is possible to

control their ionization degree with the help of buffers in

the mobile phase. The carboxyl groups in the solutes

obtain more negative charge as pH increases above their

pKa. As a rule, the change of an ionizable molecule to

an ion makes it more polar and less available to the

stationary phase. For example, increasing the pH of the

mobile phase above 4-5, which is the typical pKa of

carboxyl groups, reduces the retention of carboxyl

containing compounds. On the other hand, substancesthat contain amines whose pKa is around 8 will retain

longer when the pH will be above 8. In most of the

traditional silica-gel based stationary phases it is not

possible to increase the mobile phases pH above 8 due

to hydrolysis of the silica gel. During the 2000s there

have been developed extended pH stationary phases (31,

32)

2. HPLC Theory: System Suitability

Parameters

High performance liquid chromatography is defined as a

separation of mixtures of compounds due to differencesin their distribution equilibrium between two phases, the

stationary phase packed inside columns and the mobile

phase, delivered through the columns by high pressure

pumps. Components whose distribution into the

stationary phase is higher, are retained longer, and get

separated from those with lower distribution into thestationary phase. The theoretical and practical

foundations of this method were laid down at the end of

1960s and at the beginning of 1970s. The theory of

chromatography has been used as the basis for System-

Suitability tests, which are set of quantitative criteria that

test the suitability of the chromatographic system to

identify and quantify drug related samples by HPLC atany step of the pharmaceutical analysis.

Retention Time (tR) and Capacity Factor k' and Relative

Retention Time (RRT)

The time elapsed between the injection of the samplecomponents into the column and their detection is known

as the Retention Time (tR). The retention time is longer

when the solute has higher affinity to the stationaryphase due to its chemical nature. For example, in reverse

phase chromatography, the more lypophilic compounds

are retained longer. Therefore, the retention time is a

property of the analyte that can be used for its

identification.

A non retained substance passes through the column at a

time t0, called the Void Time. TheRetention Factoror

Capacity Factor k' of an analyte is measured

experimentally as shown in Figure 3 and Eqn 1:

Eqn 1a

o

R

tttk 0' =

The Capacity Factordescribes the thermodynamic basis

of the separation and its definition is the ratio of the

amounts of the solute at the stationary and mobile phases

within the analyte band inside the chromatographic

column:

Eqn 1b =m

s

C

Ck'

Where Cs

is the concentration of the solute at the

stationary phase and Cm is its concentration at the mobile

phase and is the ratio of the stationary and mobilephase volumes all within the chromatographic band.

The Retention Factor(Eqn 1a) is used to compare the

retention of a solute between two chromatographic

systems, normalizing it to the column's geometry and

system flow rate. The need to determine the void time

can be tricky sometimes, due to the instability of the

elution time of the void time marker, t0, therefore, when

the chromatogram is complex in nature, and one known

component is always present at a certain retention time,

it can be used as a retention marker for other peaks. Insuch cases the ratio between the retention time of any

peak in the chromatogram and the retention time of themarker is used (tR (Peak)/ tR (Marker)) and referred to as the

Relative Retention Time (RRT). RRT is also used

instead of the capacity ratio for the identification of the

analyte as well as to compare its extent of retention in

two different chromatographic systems.


4/19



0

.1

7

6

0

.7

3

9

A

U

0.000

0.015

0.030

0.045

0.060

Minutes

0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45 0.50 0.55 0.60 0.65 0.70 0.75 0.80 0.85 0.90 0.95 1.00

Figure 3: Example of Capacity factor calculation in LC. In this

case: tR = 0.739, t0 = 0.17, therefore k' = (0.739-0.176)/0.176 = 3.20

Efficiency: Plate Count N and Peak Capacity Pc

Figure 4 describes a chromatogram with 4 peaks and a

detected void peak. The parameters of the System

Suitability are displayed in the inserted table. The

sharpness of a peak relative to its retention time is a

measure of the system's efficiency, calculated as N, platecount. Band-broadening phenomena in the column such

as eddy diffusion, molecular diffusion, mass-transfer

kinetics and extra-column effects reduce the efficiency

of the separation (33). The sharpness of a peak isrelevant to the limit of detection and limit of

quantification of the chromatographic system. Thesharper the peak for a specific area, the better is its

signal-to-noise; hence the system is capable of detecting

lower concentrations. Therefore, the efficiency of the

chromatographic system must be established by the

system suitability test before the analysis of low

concentrations that requires high sensitivity of thesystem, such as the analysis of drug impurities and

degradation products.

The efficiency of the separation is determined by the

Plate Count N when working at isocratic conditions,whereas it is usually measured by Peak Capacity Pc when

working at gradient conditions (34). The following

equation for the plate count is used by the United States

Pharmacopoeia (USP) to calculate N (See Figure 5):

Eqn. 22)(16

)(Baselinew

Rt

N =

Where w is measured from the baseline peak width

calculated using lines tangent to the peak width at 50 %

height (See Figure 5). European and Japanese

Pharmacopoeias use the peak width at 50% of the peakheight (See Figure 5), hence the equation becomes:

Eqn 32)(54.5

%)50(w

Rt

N =

Peak CapacityPc is defined as number of peaks that canbe separated within a retention window for a specific

pre-determined resolution. In other words, it is the

runtime measured in peak width units (34). It is

assumed that peaks occur over the gradient

chromatogram. Therefore, Peak Capacity can be

calculated from the peak widths w in the chromatogramas follows:

Eqn. 4:

+

n

g

wn

t

1

)/1(

1

Where n is the number of peaks at the segment of the

gradient selected for the calculation, tg. Thus peak

capacity can be simply the gradient run time divided by

the average peak width. The sharper the peaks the

higher is the peak capacity, hence the system should be

able to resolve more peaks at the selected run time as

well as detect lower concentrations.

Another measure of the column's chromatographic

efficiency is the Height Equivalent to Theoretical Plate

(HETP) which is calculated from the following equation:

Eqn 5: HETP = (L/N)

Where L is column length and N is the plate count.

HETP is measured in micrometer. For example, in the

peak of Figure 5 the column length was 100 mm (0.1 m)

therefore HETP was 8.8 m at the flow rate of theexperiment.

Sample: Resolution Solution

Void

Peak

1

Peak2

Peak

3

Peak4

AU

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

0.16

0.18

Minutes

0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50

1

2

3

4

5

Name RTArea

(V*sec)

Height

(V)k'

Signal

To Noise

USP

Plate Count

USP

Resolution

USP

Tailing

Void

Peak 1

Peak 2

Peak 3

Peak 4

0. 665

1. 279

2. 818

3. 606

5. 120

1780

459570

238940

41372

26102

725

153161

59382

8776

4277

0.00

0.92

3.24

4.42

6.70

32

6747

2616

387

188

1742

4191

11320

13266

15902

8.34

16.30

6.69

10.34

1.28

1.36

1.21

1.17

1.11

System Suitability Separation Results

Figure 4: Example of a System Suitability Runs Result


5/19



The behavior of HETP as function of linear velocity has

been described by various equations (35). It is

frequently called "The Van-Deemter curve", and it is

frequently used to describe and characterize various

chromatographic stationary phases' performance andcompare them to each other (36-39). The lower are the

values of HETP, the more efficient is the

chromatographic system, enabling the detection of lowerconcentrations due to the enhanced signal-to-noise ratio

of all the peaks in the chromatogram.

Peak:Peak 2

Peak2

-2.8

18

AU

0.000

0.015

0.030

0.045

0.060

Minutes2.72 2.74 2.76 2.78 2.80 2.82 2.84 2.86 2.88 2.90 2.92 2.94 2.96 2.98

Name Peak 2Retention Time: 2.818K Pr ime: 3.24Area: 238940Height 59382(V)USP Plate Count11320USP Resolution 16.30USP Tailing 1.21Signal To Noise 2616

W=Width at Tangent

W at 50%

A B

5% of Height

10% of Height

Figure 5: System Suitability measurements on a single peak

PeakAsymmetry Factor,Af and Tailing Factor T

The chromatographic peak is assumed to have a

Gaussian shape under ideal conditions, describingnormal distribution of the velocity of the molecules

populating the peak zone migrating through the

stationary phase inside the column. Any deviation from

the normal distribution indicates non-ideality of the

distribution and the migration process, therefore might

jeopardize the integrity of the peak's integration,reducing the accuracy of the quantitation. This is the

reason why USP Tailing is a peak's system suitability

parameter almost always measured in the system

suitability step of the analysis.

The deviation from symmetry is measured by the

Asymmetry Factor, Af or Tailing Factor T. The

calculation ofAsymmetry Factor, Af is described by the

following equation:

Eqn 6

)%10(

)%10(

h

h

fB

AA =

WhereA andB are sections in the horizontal line parallel

to the baseline, drawn at 10% of the peak height asshown in Figure 5.

The calculation of Tailing Factor T, which is more

widely used in the pharmaceutical industry, as it is

suggested by the pharmacopeias, is described by the

following equation:

Eqn 7

)%5(

)%5()%5(

2 h

hh

A

BAT

+=

Where A and B are sections in the horizontal line parallel

to the baseline, drawn at 5% of the peak height, as also

shown in Figure 5. The USP suggests that Tailing

Factorshould be in the range of 0.5 up to 2 to assure a

precise and accurate quantitative measurement. The

peaks in Figures 4 and 5 have a tailing factor within the

USP requirements.

Selectivity Factor andResolution Factor Rs

The separation is a function of the thermodynamics ofthe system. Substances are separated in a

chromatographic column when their rate of migration

differs, due to their different distribution between the

stationary and mobile phases. The Selectivity Factor a

and Resolution Factor Rs measure the extent of

separation between two adjacent peaks. Selectivity

Factor accounts only for the ratio of the retentionfactors k' of the two peaks (k'2/k'1), whereas the

Resolution Factor Rs accounts for the difference

between the retention times of the two peaks relative totheir width.

The equation that describes the experimentalmeasurement of theResolution Factor Rs is as follows:

Eqn 8)(5.0 12

)1()2(

ww

ttRs

RR

+

=

Where tR are the retention times of peak 1 and 2

respectively and w is their respective peak widths at the

tangents' baseline (see Figure 5). According to the

pharmacopeias Rs should be above 2 for an accurate

quantitative measurement. Figure 4 shows that theresolution measured between every two adjacent peaks

in the chromatogram was above 2, therefore, it wasabove the minimum required.

The resolution is a critical value when working with

complex samples such as drug impurities anddegradation products, or when the formulation is

complex and excipients might interfere with the

quantitative measurements. Therefore, it is an essential

part of the system suitability measurement stage before

the quantitative work of these type of samples.

The sample used for the measurements ofRs during the

system suitability runs is sometimes called Resolution

Solution, as can be seen in Figure 4 It usually containsthe components that are the most difficult to resolve.


6/19



The theoretical description of the Resolution Factor Rs

equation is shown in Equation 9. It includes some of the

above parameters, the plate countN, the selectivity a and

the average of the two peaks' capacity factors k':

Eqn 9 )1'')(1(

4 +=

ave

ave

kkNRs

It can be clearly seen from this equation that the plate

count is the most effecting parameter in the increase of

the chromatographic resolution. Since the plate count

increases with the reduction in particle diameter, the

reason behind the reduction in particle diameter of the

stationary phase material during the last 3 decades of

HPLC can be understood. This is also the rational

behind the recent trend in HPLC, the use of sub 2 micron

particle columns and the development of a specially

design of ultra performance HPLC systems toaccommodate such columns (36, 40-42).

3. The Role of HPLC in Drug Analysis

The most characteristic feature of the development in the

methodology of pharmaceutical and biomedical analysis

during the past 25 years is that HPLC becameundoubtedly the most important analytical method for

identification and quantification of drugs, either in their

active pharmaceutical ingredient or in their formulations

during the process of their discovery, development and

manufacturing (43-50).

Figure 6: Steps in a drug development and manufacturing in a

pharmaceutical company

Figure 6 summarizes the various stages of the

pharmaceutical streamline of drug discovery,

development and manufacturing. Drug development

starts with the discovery of a molecule with a therapeutic

value (51, 52). This can be done by high throughput

screening during which separations by fast or ultra-fast

HPLC are performed (53). At the discovery stage there

can be also characterizing synthetic or natural products

(54-57). DMPK is the step where the candidate

compounds for drug are tested for their metabolism and

pharmacokinetics. The studies involve use of LC-MS or

LC-MS/MS (58-62).

The next stage is the development stage, where HPLC is

used to characterize the products of the chemicalsynthesis, by analyzing the active pharmaceutical

ingredients (API), their impurities (63, 64) and/or

degradation products generated by accelerated aging (65,

66). The development of formulation requires also

studies of the dissolution properties of solid dosage

forms as well as assays of the pharmaceutical

formulations (67). Method for the verification of

system's cleanliness during the manufacturing process

are developed and used at this stage (68). All the HPLC

methods that have been finalized at the developmental

stage are validated and transferred to the manufacturing

laboratories for a quality control analysis(69-76).

3.1 The Discovery Stage

The goal in the discovery stage of drug development is to

discover a new, safe and active chemical entity (NCE)

that will become medication for diseases. During the last

decade parallel synthesis of potential lead compounds,

using combinatorial chemistry, has been done (51, 77-

79). The large numbers of products created by the

combinatorial chemistry are then identified by fast LC-

MS methods and screened by in-vitro bioassays and/or

pharmacological or chemical tests to allow a selection of

a few chosen drug candidates (53, 59, 61, 80, 81).

When the lead compounds are selected, the next steps

involve pharmacological studies. Due to its high

sensitivity and selectivity, HPLC coupled with tandem

mass spectrometry, HPLC-MS/MS, has become the

predominant method in bioassays, and pharmacokinetic

and metabolic studies (58-62, 82). In addition, another

new development in this field has been the introduction

of column's packing with ultra-fine particles (


7/19



simpler and more automated and usually involves protein

precipitation and/or solid phase extraction techniques

(92).

The chromatographic method is typically a very rapidgradient or isocratic, using small columns' dimensions,

such as 2.1x50 mm or less. A trace of a typically stable

daughter ion signal, obtained from the fragmentation of aparent analyte is used for the detection and quantitation.

Such a mode is called MRM (multiple reaction

monitoring) or SRM (selective reaction monitoring),

which is more stable, sensitive and selective than either

the fluorescence or electrochemical signal. Samples are

usually ordered in 96 microplates for higher throughput

and minimizing variability of the sample vials (93). An

internal standard is used typically to minimize assay

variability, due to the clean-up stage and MS/MS

response, which is still less stable than UV response

(94).

3.2 The Development Stage

The development stage is the part during which some of

the HPLC methods that will be used during the

subsequent manufacturing stages are developed,

validated and then transferred. The methods involve

identification of the drug substance, its assay, impurities,

stability, the dosage form content uniformity,

dissolution, etc. The methods are validated first, and then

transferred to the quality control laboratories. Details

are given in 3.3.

All HPLC methods used for the development ofpharmaceuticals and for the determination of theirquality have to be validated. In cases wherebymethods from the Pharmacopoeia's are used, it is not

necessary to evaluate their suitability, provided that

the analyses are conducted strictly according to themethods' intended use. In most other cases, especially

in cases of modification of the drug composition, the

scheme of synthesis, or the analytical procedure, it isnecessary to re-evaluate the suitability of the HPLC

method to its original intended use (95).

The parameters tested throughout the method validation

as defined by the ICH, USP and FDA and other healthorganizations (95-97) are the following: Specificity,

selectivity, precision (repeatability, intermediate

precision, reproducibility or ruggedness), accuracy or

trueness or bias, linearity range, limit of detection, limit

of quantitation and robustness.

The terms selectivity and specificity are often used

interchangeably. The USP monograph defines selectivity

of an analytical method as its ability to measure

accurately an analyte in the presence of interference,

such as synthetic precursors, excipients, enantiomers and

known (or likely) degradation products that might bepresent in the sample matrix. A method whose

selectivity is verified is a "Stability Indicating Method",

for details please see section 3.3.

Precision of a method is measured by injecting a series

of standards and measuring the variability of the

quantitative results. The measured standard deviation can

be subdivided into three categories: repeatability,

intermediate precision, and reproducibility (or

ruggedness):

Repeatability is obtained when one operatorusing one system over a relatively short time-

span carries out the analysis in one laboratory.

At least 5 or 6 determinations of three different

matrices at two or three different concentrations

should be done and the relative standard

deviation calculated.

Intermediate precision is a term that has beendefined by ICH (96) as the long-term variability

of the measurement process and is determinedby comparing the results of a method run within

a single laboratory over a number of weeks. A

methods intermediate precision may reflect

discrepancies in results obtained by different

operators, from different instruments, with

standards and reagents from different suppliers,with columns from different batches or a

combination of these. Objective of intermediate

precision validation is to verify that in the same

laboratory the method will provide the same

results once the development phase is over.

Reproducibility (or reggedness), as defined by

ICH represents the precision obtained betweenlaboratories. Objective is to verify that the

method will provide the same results in

different laboratories, preparing it for the

transfer to other sites.

Typical variations affecting a methods reproducibility

are:

Differences in room temperature and humidity;

Operators with different experience andthoroughness;

Equipment with different characteristics, suchas delay volume of an HPLC system;

Variations in material and instrumentconditions, for example different protocols of

the mobile phases preparation; changes in

composition, pH, flow rate of mobile phase;

Equipment and consumables of different ages;

Columns from different suppliers or differentbatches;

Solvents, reagents and other material withdifferent quality

Accuracy of an analytical method is the extent to which

test results are close to their true value. It is measured

from the result of a quantitative determination of a well


8/19



characterized known sample. The amount measured is

compared to the known amount.

Linearity of an analytical method is determined by a

series of three to six injections of five or more standards

whose concentrations span 80-120 percent of the

expected concentration range. The response should be

(directly or by means of a well-defined mathematical

calculation) proportional to the concentrations of the

analytes. A linear regression equation, applied to the

results, should have an intercept not significantly

different from zero. If a significant nonzero intercept is

obtained, it should be demonstrated that there is no effect

on the accuracy of the method. The range of

concentrations that an analytical method can be

implemented on is the interval between the upper and

lower levels (including these levels) that have been

demonstrated to have the appropriate precision, accuracy

and linearity. The range is normally expressed in the

same units of the test results (e.g. percentage, parts permillion) obtained by the analytical method.

Limit of detection: It is the lowest concentration of

analyte in a sample that can be detected but not

necessarily quantified. In chromatography the detection

limit is the injected amount that results in a peak with aheight at least twice or three times as high as the baseline

noise level.

Limit of quantitation: It is the minimum injected

amount that gives precise measurements. Inchromatography it typically requires peak heights of 10

to 20 times higher than baseline noise.

Robustness of analytical method is a measure of its

capacity to remain unaffected by small but deliberate

variations in method parameters and provides an

indication of its reliability during normal usage.

Once validated, the methods are ready for transfer to themanufacturing quality control laboratories. Method

transfer is the last stage of the validation, whereby results

are tested on both the development and the

manufacturing sites. Technology transfer is especially

important in the era of increasing globalization of thepharmaceutical companies (69, 74, 98).

3.3 The Manufacturing Stage

Identification

The identification method by HPLC is aimed to confirmthe identity of the active pharmaceutical ingredient

inside the samples of either drug substance or drug

product. Two independent tests are needed, for example,

one chromatographic and one spectroscopic. Therefore,

a chromatographic run with a diode-array or MS detector

will provide both parameters, the retention time in thechromatogram and the spectrum UV or MS of the eluting

peak, matched against a known standard (99-103)

Assay and Content Uniformity

An assay of a sample containing either drug substance or

drug product quantitatively measures the actual amountof the active ingredient compared to that expected in the

drug substance assay, whereas in drug products the

actual amount measured is verified against the labelclaim. The official assays are described in the

pharmacopeias, such as United State's (USP) (12).

When a solid dosage form such as tablet or capsule are

tested, it is usually requested to sample a composite of

10-20 units to avoid tablet-to-tablet variations. The API

is extracted from the dosage form in the simplest most

effective way to optimize recovery. A portion of the

composite group, equivalent to an average unit dosage

form weight is taken for the analysis (104, 105). Typical

specifications for most drug products are 90-110% of the

label claim.

A content uniformity test is similar to the drug product

assay but an individual solid dosage form is tested

instead of a pool of the tablets/capsules as in the assay

tests. 10 tablets are taken for the assay, and if the

specifications of uniformity are not met, more tables are

taken. The typical calculations are made according to a

scheme given by the USP (106).

Most assays and content uniformity methods involve the

use of UV detection, no extensive sample preparation is

needed, the standards are usually external, and highly

qualified(107), typical specifications can be 98-102%purity on dried basis. Specifications for system

suitability were determined by the pharmacopeia(108).

Dissolution

Drug absorption from a solid dosage form after oral

administration depends on its release from the drug

product, its dissolution or solubilization under

physiological conditions, and the permeability across the

gastrointestinal tract. Because of the critical nature of

the first two of these steps, in vitro dissolution may be

relevant to the prediction of in vivo performance. Based

on this general consideration, in vitro dissolution tests

for solid oral dosage forms, such as tablets and capsules,

are used to (1) assess the lot-to-lot quality of a drug

product; (2) guide the developers of new formulations

and (3) ensure continuing product quality and

performance after certain changes, such as changes in the

formulation, the manufacturing process, the site of

manufacture, and the scale-up of the manufacturing

process

Dissolution test in the pharmaceutical laboratorymeasures the release of the drug substance from its

dosage form into a dissolution bath under standardized

conditions, specified by the pharmacopeias (67, 109-116). The US Food and Drug Administration issued a

guidance for the industry on 1997 (95). The tests are


9/19



performed usually on two types of bath apparatus, Type I

is a basket method and Type II is a paddle method.

Since most drugs have absorption in the UV-VIS range,

their dissolution has been traditionally measured by UV-

VIS spectrometry and HPLC-UV. The advantage ofHPLC over UV spectrometry is its separation

capabilities, providing higher specificity and sensitivity

as well as its applicability in formulations with multipleAPI's or very low dose.

The method is typically isocratic and very short to

accommodate for the high throughput needed, therefore

the columns are typically very short. The sample is

collected from the dissolution apparatus at certain time

intervals, designed according to its release pattern, and

the released substance must follow strict release

specifications. A dissolution profile of Prednisone

tablets, used to calibrate a dissolution bath are shown in

Figure 7.

Dissolution Plot : Prednisone

Claimed Amount A: 10.00

Q FactorA ( 30.0 , 4

Amount

1.40

2.10

2.80

3.50

4.20

Transfer Time (min)

10.00 20.00 30.00

Figure 7: Dissolution profile of Prednisone tablets

Drug Impurities

Many potential impurities result from the APImanufacturing process, including starting materials,

isomers, intermediates, reagents, solvents, catalysts and

reaction by-products (117), therefore, impurity control in

is essential during the process of drug development and

manufacturing. HPLC has become the major techniquein this process (63, 64, 118-126).

The quality of the API starting material is tested by

HPLC to qualify it for the production of API that meets

its specifications, as its impurities can be carried through

directly or can participate in the reaction chemistry toproduce significant impurities in the final product, the

API. Starting materials for the manufacturing of drug

substances and drug products can be purchased from

external suppliers or produced by the firm that also

manufactures the API. It is likely that different

synthetic routes that produce different impurities may beused by different suppliers. In some cases, the synthetic

route is not disclosed to the API producer; therefore, a

thorough investigation of impurities in starting materials

must then be conducted. This investigation includes

method development, a search for potential impurities, as

well as identification of significant unknown impurities

and assessment of their impact on the API quality.Results of such an investigation are used to set

specifications for the starting material that will assure its

suitability for use in API production (117). Similarconsiderations apply to the drug product. Strict

international regulatory requirements have been enforced

for several years as outlined in the International

Conference on Harmonization (ICH) Guidelines

Q3A(R), Q3B(R) and Q3C (127-129).

A typical method for the measurement of impurities can

be rather complicated and difficult, as it requires high

resolution to distinguish between closely related

compounds, high sensitivity to detect low concentrations

of the impurities and wide dynamic range of the system

to be able to detect the major components as well as their

impurities and degradation products in one run. The

method should have the following important attributes:

First of all the sample preparation should be assimple as possible, to stay true to the original

composition of the sample, either a drug

substance or a drug product, not to lose or

create an impurity during the process.

The chromatographic separation should beoptimal, to resolve all the impurities from each

other and from the API's.

The quantitative protocol involves frequently acalibration, using well characterized impurities

standards, and/or calibration against a dilutedstandard of the main API, taking into account

their relative response factor. Sometimes the

impurities are just normalized against the main

peak using % area.

The main peak is frequently at overloadedconcentrations and if the normalization methodof % area is used, its inclusion within the linear

range should be established

According to the ICH guidelines and dependingon the daily dose, the quantification limits of

unspecified impurities can get as low as 0.05-0.1% of the main peaks. Specified known

impurities can be reported at higher levels,provided their toxicology was determined.

Method validation must include theestablishment of limit of detection (to be used

for rejection of small peaks) and limit ofquantitation (which is used as the minimum

value for linearity range specifications)

There is a substantial effort to ensure that themain peak does not hide a co-eluted impurity by

measuring peak purity (see next section).

Most impurities methods involve a gradient of the

mobile phase, to be able to resolve a complex mixture of

all compounds in one run, polar as well as non-polar. Inrecent years the mobile phase is programmed to be mass


10/19



spectrometry compatible, i.e., the buffers used are

volatile, so that impurities can be identified by LC-MS at

the developmental and verification stages of the analysis.

Drug Stability

Stability testing of drug substance or product is

performed to ensure that their quality does not vary withtime under the influence of a variety of environmental

factors such as temperature, humidity, and light (130).

In addition stability studies are typically done using

HPLC methods, to determine the re-test period of a drug

substance and/or the shelf life of the drug product as well

as its recommended storage conditions. Guidelines to

the industry are given by the ICH (129).

An HPLC method used for stability studies is termed:

Stability Indicating Method (SIM) (65, 66, 131-137).

According to the FDA guidelines, a SIM is defined as a

validated analytical procedure that accurately and

precisely measures active ingredients (drug substance or

drug product) free from potential interferences like

degradation products, process impurities, excipients, or

other potential impurities. FDA recommends that all

assay procedures for stability studies will be stability

indicating. A typical chromatogram of a drug substance

in a stability program is shown in Figure 8. The results

are shown in Table 2.

Imp1-3.4

64

Imp2-6.6

72

Imp3-8.2

07

API-10.2

40

Imp4-13.8

79

14.6

30

AU

0.00000

0.00024

0.00048

0.00072

0.00096

Minutes0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00

Figure 8: A chromatogram of an API sample at a stability program

The HPLC system used for stability indicating methods

development and validation must contain 3D data

capabilities such as diode-array detectors and/or mass

spectrometers, to be able to detect spectral non-

homogeneity within the chromatographic peaks (99-101,

138). Figures 9 and 10 present 3D data obtained by

photo-diode array and mass detectors respectively. The

modern UV-VIS diode-array technology is capable of

evaluating specificity of the method to the analyte in

question by collecting spectrum at each acquisition data

point across a peak, and through software manipulations

each such spectrum is compared to a reference spectrum,to determine spectral homogeneity, hence determine

peak purity. Figures 11 and 12 demonstrate graphically

how the software compares spectra collected from

various points across the peaks to determine their

similarity. We can see in this example that there is a

minor interference in peaks 1 and 2, as their UV spectra

do not perfectly overlap. The mass spectrometer'sspectra in Figure 12 confirm the suspicion and indicate

the co-eluting interference in each of these two peaks.

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

0.16

0.18

0.20

0.22

0.24

AU

1.15 1.20 1.25 1.30 1.35 1.40 1.45 1.50 1.55 1.60 1.65 1.70 1.75 1.80 1.85

Minutes

260.00280.00

300.00

320.00

340.00

360.00

380.00

Wavelength

(nm)

Figure 9: 3 dimentional plot of a chromatographic run using a diode-

array detector

Since 2006 it has been recommended that a peak purity

test, based upon photo-diode-array (PDA) detection, will

also be based on mass spectrometry (MS) to be more

definite (99, 101, 138). As already was noticed inFigures 9-12 and discussed in the previous section, the

spectroscopic data obtained by the MS detector is much

more reliable in evaluating the interferences co-elutingwith the major chromatogram's peaks. Moreover, the

UV-VIS diode-array detectors might be limited in

evaluating peak purity in cases whereby the UV-VISspectra of the main peak and its impurities are similar

and/or the noise in the system is relatively high(raising the detection limit of the impurities).

0.000e+000

1.000e+007

2.000e+007

3.000e+007

4.000e+007

5.000e+007

6.000e+007

7.000e+007

8.000e+007

9.000e+007

1.000e+008

1.100e+008

Intensity

1.00 1.05 1.10 1.15 1.20 1.25 1.30 1.35 1.40 1.45 1.50 1.55 1.60 1.65 1.70 1.75 1.80

Minutes

200.00

400.00

600.00

800.00

1000.00

m/z

Figure 10: 3 dimentional plot of a chromatographic runusing a mass spectrometer detector


11/19



Three conditions must be fulfilled for the major

components and their related compounds to be effective

in the peak purity determination by diode-arraydetectors:

They must have a UV chromophore, or someabsorbance in the wavelength range selected

There should be a minimal degree ofchromatographic resolution between the main

peak and its co-eluting impurities

(Chromatographic resolution between 0.3 to

0.7).

There must be some degree of spectraldifference between the analyte peak and the co-

eluting impurity.

Stress testing is carried out on a single batch of the drug

substance and/or the drug product, using forceddegradation of the drug substance (65). The results of

such studies can help identify the likely degradation

products, which can in turn help establish the

degradation pathways and the chemical stability of the

molecule and to validate the stability indicatingcapabilities of the analytical procedures used.

nm250.00

300.00 350.00

1.19

1.19

1.20

1.18

1.21

Peak1

nm250.00

300.00 350.00

1.32

1.31

1.33

1.31

1.34

Peak 2

nm250.00

300.00 350.00

1.75

1.74

1.76

1.74

1.77

Peak 3

AU

0.00

0.05

0.10

0.15

0.20

0.25

Minutes

1.00 1.10 1.20 1.30 1.40 1.50 1.60 1.70 1.80 1.90 2.00

AU

0.00

0.05

0.10

0.15

0.20

0.25

Minutes

1.00 1.20 1.40 1.60 1.80 2.00

Peak1 Peak 2 Peak 3

Figure 11: Chromatogram of three sulfa drugs at 270 nm and a plot

of UV Spectra collected from each peak's Apex,

Inflection and two offset points, to demonstrate

comparison of spectra collected at various points across

the peak.

The nature of the stress testing will depend on the

individual drug substance and the type of drug product

involved. It usually includes the effect of temperatures,

humidity, oxidation, and photolysis on the drug

substance. The testing also evaluates the vulnerability ofthe drug substance to hydrolysis across a wide range of

pH values when in solution or suspension. Photo-

stability testing is also an integral part of a stress testing(65, 66, 124, 129, 131, 135, 137).

SIR Chromatogram

265.1

279.1

311.1

Intensity

0.0

2.0x106

4.0x106

6.0x106

8.0x106

1.0x107

1.2x107

Intensity

0

1x107

2x107

3x107

4x107

5x107

Intensity

0

2x107

4x107

6x107

8x107

Minutes

1 .1 0 1. 20 1. 30 1 .4 0 1 .5 0 1 .6 0 1 .7 0 1 .8 0

Peak 1

Peak 2

Peak 3

Peak 1

265.1

266.1267.1 529.2

530.1

Intensity

0.0

5.0x106

1.0x107

1.5x107

279.1

280.2

281.1 557.2

Intensity

0

2x107

4x107

6x107

311.1

312.2313.1

Intensity

0

2x107

4x107

6x107

8x107

m/z

20 0. 00 3 00 .0 0 40 0. 00 500 .00 6 00. 00

Peak 2

Peak 3

MS Spectra

Figure 12: SIR chromatograms of the same sulfa drugs as in Figure

6 and their corresponding mass spectra, obtained by

combining spectra across the entire peak.

Cleaning Validation

Cleaning validation tests are performed to assure the

cleanliness of the pharmaceutical manufacturing

equipment, such as blender, tablet press, etc. There are

strict specifications for the maximum allowable amounts

of the residues, according to their toxicology and daily

dosage (139). The analytical methods must be extremely

sensitive, to be able to track the residues of the analytes,

their degradants or impurities, other contaminations orthe cleaning reagents themselves (85, 140-147). The

methods must be very sensitive and short, as the entire

manufacturing process depends on the approval of the

cleanliness of apparatus. Due to the enhanced sensitivity

as well as speed of analysis, the use of ultra performance

liquid chromatography has immediately adopted in the

cleaning validation tests (85).

In-Process Control

In-process control method monitors the progress in the

manufacturing of an active pharmaceutical ingredient or

its formulation. High performance liquidchromatography is frequently employed for the in-

process assay of active pharmaceutical ingredient

synthesis (68, 72, 148, 149). The results signal the

production chemist/pharmacist whether to proceed with a

subsequent unit operation. The decision about whether touse an in-process control test in a manufacturing is

established during process development and is based on

scientific judgment. In recent years the FDA issued a

recommendations for process analytical technology

(PAT) (150). The guidance intended to describe a

regulatory framework that will encourage the voluntarydevelopment and implementation of innovative

pharmaceutical development, manufacturing, and qualityassurance. The new LC ultra performance/rapid


12/19



technologies are emerging as the technology of choice

for the in-process control assay due to their extended

efficiency, sensitivity and speed of the analysis (68,

151).

4. Special Topics

4.1 Ultra High Performance Liquid Chromatography

In order to enhance chromatographic performances in

terms of efficiency and speed, LC has recently evolvedthanks to the development of short columns packed with

small particles (sub-2 m), working at high pressures(Above 1000 bar) (40, 152-155). This approach has

been described 30 years ago according to the

fundamental chromatographic equations (156). However,

systems and columns compatible with such high

pressures have been introduced into the market during

2004 only. Advantages of small particles working at high

pressure have been vividly discussed in the literature, interms of sensitivity, efficiency, resolution, and analysis

time, showing how systems working at a maximum

pressure of 1000 bar give reliable and reproducible

results. The implementation of the new technology in thepharmaceutical and life sciences is soaring and it

becomes the next benchmark technology for the near

future (41, 42, 84, 85, 152, 157).

There are important experimental parameters oneshould be aware of while reducing the particle size

(158). First and foremost is the column pressure.

Equation 10 shows the dependence of column head

pressure on a number of experimental parametersincluding the particle size. As can be seen in Eqn 10,the pressure is inversely proportional to the square of

the particle size.

Eqn 10:2100 pd

LP

=

Where P is the pressure drop, is the flow resistance

parameter, is viscosity (mPa/s), L iscolumn length(mm), - is the linear velocity (mm/s) and dpparticle

size (m). So when the particle size is halved, thepressure goes up by a factor of four. However, oftenfor fast separations, the column length is also reduced,

so the pressure increase is not nearly as high as onewould expect, because pressure is proportional tocolumn length.

If longer columns such as 100 or 150 mm are required

to achieve higher resolution and sensitivity, then

higher pressure pumps are required. Currently, thereare commercial HPLC systems with upper pressure

limits as high as 15,000 psi. It should be noted that the

total pressure that the HPLC system experiences is thesum of the column and the instrument backpressures.The latter results when small internal diameter

capillaries are used in the flow paths to reduce extra

column effects and minimize the gradient delayvolume (33). As the flow rate increases, the back

pressure due to these capillaries increasesproportionally.

An experimental parameter that requires the mostcareful consideration when reducing the columnlength and especially internal diameter is the

extracolumn band broadening. Some of the modernultra-fast LC columns are only 1.5-cm long with an

internal diameter of 2.1 mm. Such a column has a

total void volume of around 33 L. Manyconventional HPLC instruments were developed for

typical 15 and 25 cm x 4.6 mm analytical columns,where the total column void volumes are 1.6 and 2.6

mL, respectively. A 1.5 cm x 2.1 mm column packedwith 1.8-m particles often produces a few microliterspeaks, which requires that extra-column band

broadening will be minimized to accommodate for thetrue advantages of these small columns. Somemodern liquid chromatographs have been designed or

modified to minimize the extracolumn effects (157).For others, upgrade kits are available to modify some

older HPLC instruments to work satisfactorily with

sub-2-m columns.

Some other experimental parameters that must betaken into account when running fast and ultrafastseparations in LC are as follows (86, 159):

Detector time constant: Peaks can be only a secondor two wide when short narrow-bore columns run at

high flow rates. A time constant that is too slowwill make the peaks artificially broad because the

detector cannot keep up with the rapidly changingsignal.

Data sampling (acquisition) rate: Data systemneeds enough data points, minimum of 20 across apeak to define the peak as proper for integration of

area, determine retention time, etc.

Autosampler cycle time: Separations whose runtime is less than a minute or two, throughput can be

slowed down by a slow autosampler.Gradient delay volume: If the volume between the

points, where the gradient forms (mixer), to the

column head is too large, gradient profiles will becompromised because gradient has not reached

column in time.

When transferring methods from HPLC to UPLC there is

a scale-down process of the sample load, flow-rate, and

gradient times (86, 159). Sample load must be reduced

proportionally to the columns volumes, a typical factor

can be from ~1.5 mL (4.6x150 mm column) to ~0.15 mL(2.1x100 mm column). Equation 11 describes the

calculations required for such a scale-down.


13/19



Eqn 11:

HPLC

Uplc

HPLC

Uplc

HPLCUplcID

ID

L

LMM =

Where M is the sample load in mg, L is column's length

and ID is internal column's diameter.

It should be noted that the separation mechanisms inUPLC compared to HPLC are still the same;

chromatographic principles are maintained while speed,

sensitivity and resolution is improved. This all supports

easier method transfer from HPLC to UPLC and its

revalidation. The main advantages are significant

reduction of analysis time, which means also reduction

in solvent consumption, enhanced peak capacity and

sensitivity, which means that complex analytical

determination of pharmaceutical preparations can be

accomplished much easier within a reasonable time.

Moreover, time spent with new method development,

optimization and validation is significantly saved.

A typical chromatogram obtained in the UPLC system is

shown in Figure 13. A mixture of alkylphonone is

chromatographed on a 2.1x50 mm Acquity BEH C18

column packed with 1.7M particles at roomtemperature using a gradient of 5% MeCN to 95%Acetonitril within 3 minutes at flow rate of 0.5 mL/min .

2-acetylfuran-0.6

19

acetanilide

-0.7

37

acetophenone-1.1

45

propiophenone-1.5

04

butylparaben

-1.7

05

benzophenone-1.9

12

valerophenone

-2.0

67

AU

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

0.45

0.50

Minutes0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20

Figure 13: Mixture of alkylphenones injected into WatersAcquity UPLC using Acquity BEH C18 2.1x50 mm

1.7 M

4.2 Preparative Chromatography

Preparative chromatography is used to isolate and purify

compounds (6, 73, 160-166). Typically this type of

separations requires larger columns and the

chromatograms generally display broader peaks, due to

the increased column load. The separation is optimizedfor higher throughput (weight/time), then sample load is

maximized on small analytical columns, and finallyquantities are scaled up to the preparative column

according to the desired purity and recovery.

The parameters that will govern the efficiency of the

preparative separation are the plate count, the capacity

factor k' and the selectivity between the solutes (see

section 2). As selectivity, a, between two adjacentpeaks of interest is improved, a better separation between

them at higher sample loads is obtained, getting better

product purity. Mobile phase changes cause this changein selectivity, as well as selecting the appropriate

stationary phase.

Peak shape is no longer a concern, only the collected

product's purity. The scaling up of the mass load can be

done according to the column's volume ratio as is shown

in Eqn 11, but instead of ratio between UPLC and

HPLC, the ratio is between the analytical column and the

preparative one.

A rational approach to scaling up HPLC runs, using

adsorption isotherms measurements and calculations has

been described (167). In the pharmaceutical work

preparative chromatography is used mostly to isolate

drug impurities for the purpose of their identification

and/or using them as working standards for impurities

determination (168). Purification of compounds during

drug development and manufacturing has been done in

recent years using UV in line with mass spectrometer, to

achieve a mass-directed purification, whereby the mass

spectrometer is used to determine the cutoff of the

purified compound and direct the pure compound into its

collection vessel (169).

4.3 Chiral Chromatography

The biological activity of chiral substances often

depends upon their stereochemistry, since the living

body is a highly chiral environment (170-172). A large

percentage of commercial and investigationalpharmaceutical compounds are enantiomers, and many

of them show significant enantioselective differences in

their pharmacokinetics and pharmacodynamics. The

importance of chirality of drugs has been long

recognized, and the consequence of using them as

racemates or as enantiomers has been frequentlydiscussed in the pharmaceutical literature (173-175).

With increasing evidence of problems related tostereoselectivity in drug action, chiral chromatography

has become the major tool in their analysis (171, 173-

184). Great efforts have been devoted to the

development of better methodology for enantioselectivechromatography during the past two decades, and have

resulted in variety of chiral stationary phases. Chiral

agents were derivatized and immobilized on the surface

of the support (silica gel mostly), and served as the in

situ chiral discriminators during the chromatographicprocess.

The chiral stationary phases can be categorized into the

following classes:


14/19



A. Chiral affinity by proteins (serum albumin, a1-acid glycoprotein, ovomucoid and

chymotrypsin) (185).

B. Stereoselective access to helical chiral polymers(derivatized or free polysaccharides) (186, 187).

C. Steric interactions between p-Donor p-acceptortype of chiral aromatic amide groups (Pirkle)

(188).D. Host-guest interactions inside chiral cavities

(cyclodextrins, crown ethers, macrocyclic

antibiotics and imprinted polymers) (189-192).

Major developments in chiral stationary phases have led

to dramatic advances in enantioselective chromatography

(175). Chromatographic enantioseparation on chiral

stationary phases results from energy differences

between transient diastereomeric complexes formed by

the solute with the chiral agent on the surface of the

chiral stationary phase interactions. These differential

interactions are directly influenced by the mobile phaseenvironment. The solvents in the mobile phase can be

either normal phase, or aqueous reversed phase's (193) or

polar organic (187).

During recent years the trend in the pharmaceutical

manufacturing of drugs containing chiral centers is todevelop them as single enantiomers rather than

racemates due to the complexity of the tests andresearch involved in the use of racemates as API's

(64).

Conclusion

HPLC technology has matured to the extent that almost

any existing organic compound can be analyzed by an

existing method that can be found in the analytical

literature, such as professional journals, protocol books

such as Pharmacopeia's or AOAC manuals. The mostremarkable change in the pharmacopoeias in the past

25 years has been the increasing importance of HPLCtechnology in the analysis of all aspects of drug

development and manufacturing.

References1. Velagaleti, R., Burns, P., and Gill, M. (2003)

Drug Information Journal37, 407-438.2. Dong, M. W. (2006) Modern HPLC for

practicing scientists, John Wiley & Sons, New

Jersey.

3. Rochet, J. C. (2006)American Journal of

Pharmaceutical Education70.

4. Katz, E. (1998) Handbook of HPLC, CRCPress.

5. Farb, D., Luttrell, A., and Kirsch, R. (2005)

Pharmaceutical Quality Control Lab Guide

book, University Of Health Care.

6. Wellings, D. (2006) A Practical Handbook ofPreparative HPLC, Elsevier Science.

7. Winslow, P., and Meyer, R. (2004) Compliance

Handbook for Pharmaceuticals, Medical

Devices, and Biologics.

8. Hage, D. S. (2006) Handbook of Affinity

Chromatography, Taylor & Francis Group.9. Walker, J. M. (2002) The Protein Protocols

Handbook, Humana Pr Inc.

10. Ahuja, S., and Dong, M. W. (2005) Handbookof pharmaceutical analysis by HPLC, Elsevier

Academic Press.

11. Ahuja, S., and Scypinski, S. (2001) Handbook

of Modern Pharmaceutical Analysis, Academic

Press.

12. USP-NF (2005) United State Pharmacopeia

National Formulary, United State

Pharmacopeial Convention, Inc.

13. Levin, S. (2004)Journal of Liquid

Chromatography & Related Technologies27,

1353-1376.

14. Stella, C., Rudaz, S., Gauvrit, J. Y., Lantri, P.,

Huteau, A., Tchapla, A., and Veuthey, J. L.

(2007)Journal of Pharmaceutical andBiomedical Analysis43, 89-98.

15. Claessens, H. A., and van Straten, M. A. (2004)

Journal of Chromatography A1060, 23-41.

16. Marchand, D. H., Snyder, L. R., and Dolan, J.

W. (2008)Journal of Chromatography A1191,

2-20.

17. Luo, H., Ma, L., Zhang, Y., and Carr, P. W.

(2007)Journal of Chromatography A.

18. Euerby, M. R., and Petersson, P. (2003)Journal

of Chromatography A994, 13-36.

19. Haghedooren, E., Diana, J., Noszal, B.,Hoogmartens, J., and Adams, E. (2007) Talanta

71, 31-37.

20. Van Gyseghem, E., Jimidar, M., Sneyers, R.,

Redlich, D., Verhoeven, E., Massart, D. L., and

Vander Heyden, Y. (2004)Journal of

Chromatography A1042, 69-80.

21. Dehouck, P., Visky, D., Heyden, Y. V., Adams,

E., Kovacs, Z., Noszl, B., Massart, D. L., and

Hoogmartens, J. (2004)Journal of


22. O'Gara, J. E., Alden, B. A., Walter, T. H.,

Petersen, J. S., Niederlaender, C. L., and Neue,

U. D. (1995)Analytical Chemistry67, 3809-

3813.

23. Neue, U. D., Walter, T. H., Alden, B. A., Jiang,

Z., Fisk, R. P., Cook, J. T., Glose, K. H.,

Carmody, J. L., Grassi, J. M., and Cheng, Y.

(1999)AMERICAN LABORATORY 31, 36-39.

24. Neue, U. D., Tran, K. V., Mndez, A., and Carr,

P. W. (2005)Journal of Chromatography A

1063, 35-45.

25. Neue, U. D. (2007)JOURNAL OF

SEPARATION SCIENCE30, 1611.26. Neue, U. D., OGara, J. E., and Mndez, A.

(2006)Journal of Chromatography A1127,

161-174.27. Cheng, Y. F., Walter, T. H., Lu, Z., Iraneta, P.,

Alden, B. A., Gendreau, C., Neue, U. D.,


15/19



Grassi, J. M., Carmody, J. L., and O Gara, J. E.

(2000)LC GC18, 1162-1173.

28. Neue, U. D., Phoebe, C. H., Tran, K., Cheng, Y.

F., and Lu, Z. (2001)Journal of

Chromatography A925, 49-67.29. Neue, U. D., Mendez, A., Iran, K. V., and

Diehl, D. M. (2006)HPLC Made to Measure: A

Practical Handbook for Optimization.30. Gilar, M., Fountain, K. J., Budman, Y., Neue,

U. D., Yardley, K. R., Rainville, P. D., Russell

Ii, R. J., and Gebler, J. C. (2002)Journal of


31. Jenkins, K., Diehl, D., Morrison, D., and

Mazzeo, J. (2005)LC GC North America, 92-

93.

32. Fountain, K. J., Morrison, D., Diehl, D. M., and

Martin, J. (2007)LC GC North America, 64-65.

33. Kirkland, J. J., Yau, W. W., Stoklosa, H. J., and

Dilks Jr, C. H. (1977)J Chromatogr Sci15,

303-16.

34. Neue, U. D. (2005)Journal of Chromatography

A1079, 153-161.

35. Usher, K. M., Simmons, C. R., and Dorsey, J.

G. (2008)Journal of Chromatography A.

36. Wren, S. A. C., and Tchelitcheff, P. (2006)


37. Neue, U. D., and Kele, M. (2007)Journal of


38. Jones, M. D., and Plumb, R. S. (2006)J. Sep.

Sci29, 2409-2420.

39. Gritti, F., and Guiochon, G. (2006)Journal of


40. Swartz, M. (2005)Journal of Liquid


1253-1263.

41. Novakova, L., Matysova, L., and Solich, P.

(2006) Talanta68, 908-918.

42. King, S., Peter, J., Stoffolano, E. R., Eichhold,

T. E., Ii, S. H. H., Baker, T. R., Richardson, E.

C., and Wehmeyer, K. R. (2005)LC GC North

America, 36-39.

43. Schirmer, R. (1991) Modern Methods of

Pharmaceutical Analysis, CRC Press.

44. McGill, J., and Holly, D. (1996)Am J Pharm

Educ60, 370-4.

45. Tang, L., Fitch, W., Alexander, M., and Dolan,

J. (2000)Anal. Chem72, 5211-5218.

46. Lee, D., and Webb, M. (2003) Pharmaceutical

Analysis, Blackwell Science Ltd.

47. Lunn, G., and Wiley, J. (2005) HPLC methods

for recently approved pharmaceuticals, Wiley-

Interscience.

48. Gorog, S. (2006) Trends in Analytical

Chemistry25, 755-757.

49. Gorog, S. (2007) Trends in Analytical

Chemistry26, 12-17.50. Kazakevich, Y. V., and LoBrutto, R. (2007)

HPLC for Pharmaceutical Scientists, Wiley-

Interscience.

51. Bleicher, K., Bohm, H., Muller, K., and

Alanine, A. (2003)Nature Reviews Drug

Discovery2, 369-378.

52. Daniel, B. (2007)HPLC for Pharmaceutical

Scientists.53. Castro-Perez, J., Plumb, R., Granger, J. H.,

Beattie, I., Joncour, K., and Wright, A. (2005)

Rapid Commun. Mass Spectrom19, 843848.54. Butler, M. S. (2004)Journal of Natural

Products67, 2141-2153.

55. Smyth, W. F., McClean, S., Hack, C. J.,

Ramachandran, V. N., Doherty, B., Joyce, C.,

ODonnell, F., Smyth, T. J., OKane, E., and

Brooks, P. (2006) Trends in Analytical


56. Butler, M. S. (2005)Natural Product Reports22, 162-195.

57. Lam, K. S. (2007) Trends in Microbiology15,

279-289.

58. Chu, I., and Anomeir, A. (2006) Current Drug

Metabolism7, 467-477.

59. Kassel, D. B. (2005) Using Mass Spectrometryfor Drug Metabolism Studies.

60. Lappin, G., and Garner, R. C. (2005)Expert

Opin. Drug Metab. Toxicol.1, 23-31.

61. Kassel, D. B. (2004) Current Opinion in

Chemical Biology8, 339-345.

62. Wang, J., and Urban, L. (2004)DrugDiscovery, 73.

63. Ng, L., Lunn, G., and Faustino, P. (2007)

Analysis of Drug Impurities.

64. Argentine, M., Owens, P., and Olsen, B. (2007)

Advanced Drug Delivery Reviews59, 12-28.65. Bakshi, M., and Singh, S. (2002)Journal of

Pharmaceutical and Biomedical Analysis28,

1011-1040.

66. Consultant, P., and Raleigh, N. (2000)Drug

Stability: Principles and Practices.

67. Rosanske, T. W., Brown, C. K., and

Christopher, M. R. a. T. W. R. (1996) in

Progress in Pharmaceutical and Biomedical

Analysis, Vol. Volume 3, pp. 169, Elsevier.

68. Gomis, D. B., Nunez, N. S., Enguita, P. B.,

Abrodo, P. A., and Alvarez, M. D. G. (2006)

Journal of Liquid Chromatography & Related

Technologies29, 931-948.

69. Rozet, E., Dew, W., Morello, R., Chiap, P.,

Lecomte, F., Ziemons, E., Boos, K. S.,

Boulanger, B., Crommen, J., and Hubert, P.

(2008)Journal of Chromatography A1189, 32-

41.

70. Kataoka, H. (2003) Trends in Analytical


71. Koh, H. L., Yau, W. P., Ong, P. S., and Hegde,

A. (2003)Drug Discovery Today8, 889-897.

72. Wrezel, P. W., Chion, I., and Hussain, M. S.(2004)LC GC NORTH AMERICA22, 1006-

1009.

73. Brandt, A., AG, S., Berlin, G., and Kueppers, S.(2002)LC GC Europe March, 2.


16/19



74. Varma, S. (2001) The Quality Assurance

Journal5, 85-92.

75. Jimidar, M. I., and De Smet, M. (2007)HPLC

Method Development for Pharmaceuticals.

76. Gibson, M. (2005) Technology TrandferIntroduction and Objectives, PDA.

77. Gardner, C. R., Almarsson, O., Chen, H.,

Morissette, S., Peterson, M., Zhang, Z., Wang,S., Lemmo, A., Gonzalez-Zugasti, J., and

Monagle, J. (2004) Computers and ChemicalEngineering28, 943-953.

78. Maurer, H. (2000) Combinatorial Chemistry &

High Throughput Screening3, 467-480.

79. Schultz, L., Garr, C. D., Cameron, L. M., and

Bukowski, J. (1998)Bioorganic & Medicinal

Chemistry Letters8, 2409.

80. Corti, G., Maestrelli, F., Cirri, M., Zerrouk, N.,

and Mura, P. (2006)European Journal of

Pharmaceutical Sciences27, 354-362.

81. Alsenz, J., and Kansy, M. (2007)Advanced

Drug Delivery Reviews59, 546-567.

82. Korfmacher, W. A. (2005)Drug DiscoveryToday10, 1357-1367.

83. Plumb, R., Jones, M., Rainville, P., and

Nicholson, J. (2008)Journal of

Chromatographic Science46, 193-198.

84. Pedraglio, S., Rozio, M. G., Misiano, P., Reali,

V., Dondio, G., and Bigogno, C. (2007)Journalof Pharmaceutical and Biomedical Analysis44,

665-673.

85. Fountain, K. J., Wingerden, M., and Diehl, D.

M. (2007)LC GC Magazine-North America-

Solutions for Separation Scientists37, 66-67.86. de Villiers, A., Lestremau, F., Szucs, R.,

Glbart, S., David, F., and Sandra, P. (2006)


87. Nardo, C. (2005).

88. Johnson, K. A., and Plumb, R. (2005)Journal

of Pharmaceutical and Biomedical Analysis39,

805-810.

89. Korfmacher, W. A., Cox, K. A., Bryant, M. S.,

Veals, J., Ng, K., Lin, C., and Watkins, R.

(1997)Drug Discovery Today2, 532-537.

90. Wickremsinhe, E. R., Singh, G., Ackermann, B.

L., Gillespie, T. A., and Chaudhary, A. K.

(2006) Current Drug Metabolism7, 913-928.

91. Kamel, A., and Prakash, C. (2006) Current

Drug Metabolism7, 837-852.

92. Alnouti, Y., Csanaky, I. L., and Klaassen, C. D.

(2008)Journal of Chromatography B873, 209.

93. Mallet, C. R., Lu, Z., Fisk, R., Mazzeo, J. R.,

and Neue, U. D. (2003)Rapid communications

in mass spectrometry17, 163-170.

94. Li, R., Dong, L., and Huang, J. (2005)Analytica

Chimica Acta546, 167-173.

95. (CDER), C. f. D. E. a. R. (1997), U.S.Department of Health and Human Services,

Food and Drug Administration.

96. ICH-Q2B (1996), Geneva.97. USP-26-NF21 (2003).

98. Gibson, D. V., and Williams, F. (1990)

Technology transfer, Sage Publications

Newbury Park, Calif.

99. Li, W., and Hu, C. (2008)Journal of

Chromatography A.100. Gilliard, J. A., and Ritter, C. (1997)Journal of


101. Lindholm, J., Westerlund, D., Karlsson, K.,Caldwell, K., and Fornstedt, T. (2003)Journal

of Chromatography A992, 85-100.

102. Swartz, M. E. (1996) CHIRALITY867, 76.

103. Rivier, L. (2006)Applications of LC-MS in

Toxicology.

104. Lambropoulos, J., Spanos, G. A., and Lazaridis,

N. V. (1999)Journal of Pharmaceutical and

Biomedical Analysis19, 793-802.

105. Berman, J., and Planchard, J. A. (1995)Drug

Development and Industrial Pharmacy21,

1257-1283.

106.

http://www.usp.org/USPNF/compendi

alTools.html.

107. Williams, R. L. (2006)Journal of

Pharmaceutical and Biomedical Analysis40, 3.

108. Renger, B. (2000)Journal of Chromatography

B: Biomedical Sciences and Applications745,

167.

109. Aiache, J. M. (1990)Journal of Pharmaceuticaland Biomedical Analysis8, 499.

110. Zahirul, M., and Khan, I. (1996)International

Journal of Pharmaceutics140, 131.

111. QURESHI, S. A. (1996)Drug Information

Journal30, 1055-1061.

112. SHIU, G. K. (1996)Drug Information Journal

30, 10451054.

113. Castellano, P., Vignaduzzo, S., Maggio, R., and

Kaufman, T. (2005)Analytical and

Bioanalytical Chemistry382, 1711-1714.

114. Bateman, S., Paul, W., Alan, T., and Colin, P.

(2005) in Encyclopedia of Analytical Science,

pp. 101, Elsevier, Oxford.

115. Azarmi, S., Roa, W., and Lbenberg, R. (2007)

International Journal of Pharmaceutics328,

12.

116. Deng, G., Ashley, A., Brown, W., Eaton, J.,

Hauck, W., Kikwai, L., Liddell, M., Manning,R., Munoz, J., and Nithyanandan, P. (2008)

Pharmaceutical Research25, 1100-1109.

117. Gavin, P. F., Olsen, B. A., Wirth, D. D., and

Lorenz, K. T. (2006)Journal of Pharmaceutical

and Biomedical Analysis41, 1251-1259.

118. Smith, R. J., and Webb, M. L. (2007) Analysis

of Drug Impurities, Blackwell Publishing.

119. Ahuja, S. (2007)Advanced Drug Delivery

Reviews59, 3-11.

120. Kovaleski, J., Kraut, B., Mattiuz, A.,

Giangiulio, M., Brobst, G., Cagno, W.,

Kulkarni, P., and Rauch, T. (2007)Advanced

Drug Delivery Reviews59, 56-63.121. Basak, A., Raw, A., Al Hakim, A., Furness, S.,

Samaan, N., Gill, D., Patel, H., Powers, R., and


17/19



Yu, L. (2007)Advanced Drug Delivery Reviews

59, 64-72.

122. Qiu, F. (2007)Journal of Liquid


877-935.123. Olsen, B., Castle, B., and Myers, D. (2006)

Trends in Analytical Chemistry25, 796-805.

124. Baertschi, S. (2006) Trends in AnalyticalChemistry25, 758-767.

125. Nageswara Rao, R., and Nagaraju, V. (2003)

Journal of Pharmaceutical and Biomedical

Analysis33, 335-377.

126. Roy, J. (2002)AAPS PharmSciTech3, 1-8.

127. ICH (2006)INTERNATIONAL CONFERENCE

ON HARMONISATION OF TECHNICAL

REQUIREMENTS FOR REGISTRATION OF

PHARMACEUTICALS FOR HUMAN USE.

128. ICH (2005)INTERNATIONAL CONFERENCE

ON HARMONISATION OF TECHNICAL



129. ICH (2003)INTERNATIONAL CONFERENCEON HARMONISATION OF TECHNICAL



130. Carstensen, J., and Rhodes, C. (2000) Drug

Stability: Principles and Practices, Informa

Healthcare.

131. Kumar, V., Bhutani, H., and Singh, S. (2007)

Journal of Pharmaceutical and Biomedical

Analysis43, 769-773.

132. Mohammadi, A., Mehramizi, A., Moghaddam,

F., jabarian, L., pourfarzib, M., and Kashani, H.(2007)Journal of Chromatography B854, 152-

157.

133. Shah, R., Bryant, A., Collier, J., Habib, M., and

Khan, M. (2008)International Journal of

Pharmaceutics360, 77-82.

134. Mohammadi, A., Rezanour, N., Ansari

Dogaheh, M., Ghorbani Bidkorbeh, F., Hashem,

M., and Walker, R. (2007)Journal of

Chromatography B846, 215-221.

135. Xu, Q. A., Trissel, L. A., and American

Pharmacists, A. (1999) Stability-indicating

HPLC Methods for Drug Analysis, American

Pharmaceutical Association.

136. Chan, E. C. Y., Wee, P. Y., and Ho, P. C.

(1999) Clinica Chimica Acta288, 4753.

137. Swartz, M., and Krull, I. (2005)

Chromatography Online.

138. Fourneron, J. D. (2001)Journal of

Chromatographic Science39.

139. APIC (1999), Active Pharmaceutical

Ingredients Committee.

140. Yang, P., Burson, K., Feder, D., and

Macdonald, F. (2005) PharmaceuticalTechnology.

141. Schmidt, A. H. (2006)JOURNAL OF LIQUID

CHROMATOGRAPHY AND RELATEDTECHNOLOGIES29, 1663.

142. Zayas, J., Coln, H., Garced, O., and Ramos, L.

M. (2006)Journal of Pharmaceutical and

Biomedical Analysis41, 589-593.

143. Forsatz, B., and Snow, N. (2007)LC GC

NORTH AMERICA25, 960.144. Kathiresan, K., Kannan, K., Valliappan, K.,

Karar, P. K., and Manavalan, R. (2007)ASIAN

JOURNAL OF CHEMISTRY19, 5034.145. Baran, P. A., Bielejewska, A., Glice, M. M.,

Beczkowicz, H., Maruszak, W., Kosmacinska,

B., and Golebiewski, P. (2007) PRZEMYSL

CHEMICZNY86, 747.

146. Kathiresan, K., Kannan, K., Kannappan, N.,

Venkatesan, P., Shenoy, K. R. P., and

Manavalan, R. (2007)ASIAN JOURNAL OF

CHEMISTRY19, 4438.

147. Resto, W., Hernndez, D., Rey, R., Coln, H.,

and Zayas, J. (2007)Journal of Pharmaceutical


148. Laviana, L., Llorente, I., Bayod, M., and

Blanco, D. (2003)Journal of Pharmaceutical


149. Moes, J. J., Ruijken, M. M., Gout, E., Frijlink,

H. W., and Ugwoke, M. I. (2008)International

Journal of Pharmaceutics357, 108.

150. Guidance, F. (2004) ((CDER), C. f. D. E. a. R.,

Ed.), U.S. Department of Health and Human

Services, Food and Drug Administration.

151. Anurag S. Rathore, M. Y. S. Y. A. S. (2008)

Biotechnology and Bioengineering100, 306-

316.

152. Swartz, M. E. (2005)LC-GC23, 8-14.

153. Neue, U. D., Smith, B., and Johnson, K. A.(2007)J. Sep. Sci30, 1158-1166.

154. Mazzeo, J. R., Neue, U. D., Kele, M., and

Plumb, R. S. (2005)Analytical

chemistry(Washington, DC)77, 460-467.

155. Diehl, D. M., Tran, K., Grumbach, E. S., Neue,

U. D., and Mazzeo, J. R. (2003)LC GC North

America, 67-68.

156. J.C. Giddings, P., (1965).

157. Jerkovich, A. D., LoBrutto, R., and Vivilecchia,

hplc pharma 25-1-09-numbered-all print

Documents