hplc pharma 25-1-09-numbered-all print
TRANSCRIPT
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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
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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
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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.
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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
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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.
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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 (
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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
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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
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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
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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
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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
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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.
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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:
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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.
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