SOUJANYA SATHI REGISTRATION NO: 099051482 DRUG ...

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SOUJANYA SATHI

REGISTRATION NO: 099051482

DRUG DICOVERY AND DEVELOPMENT

UNIVERSITY OF SUNDERLAND





INTRODUCTION


USES IN PHARMACEUTICAL INDUSTRY


HPLC

has a considerable range of applications in

both clinical research an
d routine clinical analysis.
The
variety of molecules

is

commonly
analyzed

by this method.

Methods based on chroma
-

tography from small cation exchange columns as well

as various
electrophoresis methods have been used

successfully to resolve glycated from non
-
glycated

Hemoglobin’s
.

Cation

exchange HPLC
, however, can resolve all the
subtypes of

glycated haemoglobin both from
each other and

from the F, S, and C

forms. This form of assay show
excellent precision with rapid
separation, and many

manufacturers market HPLC systems dedicated

entirely to this purpose.

The ability of HPLC to resolve clos
ely related

molecules makes it the method of choice for detailed

investigation of many congenital metabolic disorders

or diseases.

For instance, though amino acids in

plasma or urine, or both, can be investigated with

paper
chromatography, thin layer chro
matography,

or high voltage electrophoresis, these methods give

relatively poor separation and results are difficult to

quantify.

Ion exchange HPLC methods

have been

much more successful in separating, identifying, and

quantifying the main amino acid spec
ies in plasma and

in urine.

A high resolution

technique like HPLC can differentiate between these

states with the same basic
method.

Other general types of molecule can also be separated

into individual smaller molecules by HPLC to
give

metabolic profile.

With ion exchange HPLC techniques

the biogenic amines can be
analyzed.

Simmonds et al recently described an anion exchange

HPLC procedure for the separation of the major

nucleotides and their corresponding deoxyderivatives.

Reverse phase HPLC

has been use
d successfully

f
or
identifying and quantifying individual urinary

porphyrins.

Many HPLC methods have been developed for the

study of vitamins and their metabolites.

One of the main areas in which HPLC is used is in

therapeutic drug monitoring like


W
hen

the therapeutic dose is close to the toxic dose, when

signs of toxicity are difficult to detect
clinically, when

the rate of metabolism varies widely between patients,

or when drug metabolism is
impaired owing to organ

dysfunction or altered by other drugs
.


Monitoring

when rates of metabolism
might vary is especially

important if the drug metabolite is the
therapeutically

active form or the toxic form.

Drugs that are commonly monitored by HPLC

include antiasthmatic drugs (theophylline and its

active metabo
lities, including caffeine), anticonvulsants

such as carbamazepine, phenobarbitone,

phenytoin, ethosuximide, primidone and valproate,

tricyclic antidepressants, and cardioactive drugs
such

as procainamide and propranolol. In many cases the

drug may be monitored by
radioimmunoassay, but

when no antibody to the drug (or metabolite) exists, or

if simultaneous
measurement of a mixture of drugs or

metabolites is required then HPLC is more effective.

[4]


Adsorption (normal phase) HPLC
-
separation
of molecules on the basis of their

solubility in water

Chiral molecules
-
those containing a carbon
atom to which is

bound four different

groups.

Ion exchange HPLC
-
separation of molecules on the basis of their net charge

Liquid phase
-
the solvent passing thr
ough the HPLC column

Reverse phase HPLC
-
separation of molecules on the basis of their solubility in

organic solvents

Size exclusion HPLC
-
separation

of molecules on the basis of their size
.
Solid phase
-
the surface of the
solid particles (in an HPLC column) w
ith which

molecules can interact

(2)

HPLCIMS holds great promise for measuring
drug
con
centrations

LC
-
MS

Liquid chromatography
-
mass spectrometry

(
LC
-
MS
, or alternatively
HPLC
-
MS
) is an
analytical
chemistry

technique that combines the physical separation capabilities of
liquid chromatography

(or
HPLC) with the ma
ss analysis capabilities of
mass spectrometry
. LC
-
MS is a powerful technique used
for many applications which has very high sensitivity and selectivity. Generally its application is
oriented to
wards the specific detection and potential identification of chemicals in the presence of
other ch
emicals.
[17]

Pharmacokinetics
:
LC
-
M
S is very commonly used in
pharmacokinetic

studies of pharmaceuticals and
is thus the most frequently used technique in the field of
bioanalysis
. These studies give information
about how quickly a drug will be cleared from the

hepatic blood flow, and organs of the body. MS is
used for this due to high sensitivity and exceptional specificity compared to UV (as long as the analyte
can be suitably ionised), and short analysis time.

The major advantage MS has is the use of
tandem MS
-
MS
. The detector may be programmed to select
certain ions to fragment. The process is essentially a selection technique, but is in fact more complex
.
The measured quantity is the sum of molecule fragments chosen by the operator. As long as there are
no interferences or ion suppression, the LC separation can be quite quick. It is common now to have
analysis times of 1 min
ute or less by MS
-
MS detection,

compared to over 10 mins with UV
detection.
[3
]
[4
]
[5
]

Proteomics /
metabolomics
:
LC
-
MS is also used in the study of
proteomics

where again components
of a complex mixture must be detected and identified in some manner. The
bottom
-
up proteomics

LC
-
MS ap
proach to proteomics generally involves protease digestion and denaturation (usually
trypsin

as
a protease, urea to denature tertiary structure and iodoacetamide to cap cysteine residues) followed by
LC
-
MS with
peptide mass fingerprinting

or LC
-
MS/MS (
tandem MS
) to derive sequence of i
ndividual
peptides.
[6
]


LC
-
MS/MS is most commonly used for proteomic analysis of complex samples where peptide masses
may overlap even with a

high
-
resolution mass spectrometer. Samples of complex biological fluids like
human serum may be run in a modern LC
-
MS/MS system and result in over 1000 proteins being
identified, provided that the sample was first separated on an SDS
-
PAGE gel or HPLC
-
SCX.


Drug development
:

LC
-
MS is frequently used in drug development at many different stages
including Peptide Mapping, Glycoprotein Mapping, Natural Products Dereplication, Bioaffinity
Screening, In Vivo Drug Screening, Metabolic Stability Screening, Metabolite Identification, Impurity
Identification, Degradant Identification, Quantitative
Bio
analysis
, and Quality Control.
[7
]

Hplc/
Ms

holds a promise for measuring drug concentration in body
fluids, particularly

because many
drugs are large or labile molecules that do not lend themselves to analysis by GC/MS
.HPLC/MS has
two distinct areas of application in drug analysis: Qualitative
analysis of a drug or

quantification of a
drug.
[8]

LC
-
NMR

Hyphenated analytical techniques such as LC
-
MS, which combines liquid chromatography and mass
spectrometry, are well
-
developed laboratory tools now widely used in the
pharmaceutical industry.

Ho
wever, in most cases MS alone is insufficient for complete structural elucidation of unknown
compounds. Traditionally nuclear magnetic resonance (
NMR)

experiments are performed on more or
less pure samples, in which the signals of a single component domi
nate. Therefore, the structural
analysis of individual components of complex mixtures is normally time
-
consuming and less
economic. The combination of chromatographic separation techniques with
NMR

spectroscopy
offers advantages for the on
-
line separatio
n and structural elucidation of unknown compounds.
Mixtures such as crude reaction mixtures in drug discovery can be analyzed without prior separation.
Experiments in combining an HPLC with
NMR

on the study of mixtures were introduced to the
scientific community in the early 1980s. However,
LC
-
NMR

was not widely practical due to its low
sensitivity, approximately six orders in magnitude inferior to that of MS. Another challenge comes
from the
measurement of proton signals in mobile phase. However, recent developments in higher
magnetic field strength and electronics that improve the sensitivity of probes, together with advanced
solvent suppression techniques, have made
LC
-
NMR

measurement prac
tical.

[16]

During the last decade,
LC
-
NMR

has been fully commercialized and its application in both academia
and
industry

has been growing rapidly.
The emphasis is therefore on describing the experimental
design, the practical applications, and the re
cent developments in technology. With all the applications
to date,
LC
-
NMR

spectroscopy is still a relatively insensitive technique due to the poor mass
sensitivity of the
NMR

detection system. To this end, several other hyphenated
NMR

techniques
hav
e been developed to enhance the sensitivity of this technique.
LC
-
SPE
-
NMR

dramatically
increases the sensitivity up to a factor of four by utilizing a solid phase extraction device after the
LC

column. Capillary
LC
-
NMR

also significantly lowers the

detection limit to a low nanogram range
through integration of capillary
LC with NMR

detection. Other breakthroughs such as cryo
-
LC
-
probe technology combine the advantages of sample flow and the enhanced sensitivity from a
cryogenically cooled
NMR

probe.

DISPLACEMENT CHROMATOGRAPHY

The basic principle of
displacement chromatography

is: A molecule with a high affinity for the
chromatography matrix (the displacer) will compete
effectively for binding sites, and thus displace all
molecules with lesser affinities.
[15
]
. Displacement chromatography has advantages over elution
chromatography in that components are resolved into consecutive zones of pure substances rather than
“peaks”. Because the process takes advantage of the nonlinearity of the isotherms, a larger colum
n
feed can be separated on a given column with the purified components recovered at significantly
higher concentrations.

[1]

HILLIC

Partition chromatography was the first kind of chromato
graphy that chemists developed.

Partition
chromatography uses a retai
ned solvent, on the surface or within the grains or fibres of an "inert" solid
supporting matrix as with
paper chromatography
; or takes advantage of some

coulombic

and/or
hydrogen donor

interaction with the solid support. Molecules equilibrate (partition) betwee
n a liquid
stationary phase and the eluent. Known as Hydrophilic Interaction Chromatography (HILIC) in HPLC,
this method separates analytes based on polar differences. HILIC most often uses a bonded polar
stationary phase

and a non
-
polar, water
miscible
, mobile phase. Partition HPLC has been used
historically on
unbounded

silica o
r alumina supports. Each works effectively for separating analytes
by relative polar differences, however, HILIC has the advantage of separating
acidic
,
basic

and neutral
solutes in a single chromatogram.

[1]

If you are trying to increase retention of hydrophilic molecules by RPC, there is a versatile, effective
alternative to consider: hydrophilic interaction chromatography (HILIC). A rival te
chnique to RPC for
separating polar peptides, HILIC is easy to use and works best where RPC works worst: with polar
solutes which aren't retained well on RPC. HILIC has been used successfully with phosphopeptides,
crude extracts, peptide digests, membrane
proteins, carbohydrates, histones,
oligonucleotides and their
anti
sense analogs, polar lipids and in preparative applications where changing the order of elution
affects isolation yields.
[20]

NORMAL PHASE
CHROMATOGRAPHY

Also known as normal
-
phase HPLC (NP
-
HPLC), or adsorption chromatography, this method separates
analytes based on adsorption to a stationary surface chemistry and by polarity. It was one of the first
kinds of HPLC that chemists developed. NP
-
HPLC uses a pol
ar stationary phase and a non
-
polar, non
-
aqueous mobile phase, and works effectively for separating analytes readily soluble in non
-
polar
solvents. The analyte associates with and is retained by the polar stationary phase. Adsorption
strengths increase wit
h increased analyte polarity, and the interaction between the polar analyte and
the polar stationary phase (relative to the mobile phase) increases the elution time. The interaction
strength depends not only on the functional groups in the analyte
molecule
, but also on steric factors.
The effect of sterics on interaction strength allows this method to resolve (separate)

structural isomers
.

Partition and NP
-
HPLC fell out of

favor in the 1970s with the development of

reversed
-
phase

HPLC
because of a lack of reproducibility of retention times as water or protic organic solvents changed the
hydration state of the silica or

alumina

chromatographic media. Recently it has become useful again
with the development of

HILI
C

bonded phase
s which improve reproducibility.
[1]

REVERSE PHASE HPLC

(RPC)


RPC is so commonly used that it is often incorrectly referred to as "HPLC" without further
specification. The pharmaceutical industry regularly employs RPC to qualify drugs before

their
release.

HPLC can readily be used to identify drug metabolites

that may be important in drug toxicity.

A role

for HPLC in both research and routine clinical analysis.

Reversed phase chromatography has proven itself t
o be an indispensable technique
in the
purification of biomolecules.
In recent years, with the
advent of high performance media and
instrumentation, reversed phase
chromatography has been applied to the purification of
bi
omolecules such as
peptides, proteins and oligonucleotides. Re
verse
d phase chromatography
has
proven so successful for biomolecule purification in
the research laboratory that it
is now
routinely applied for process scale purification of synthetic peptides
, and
recombinant peptides
and proteins.

[19]


Ion Pair Reverse
-
Pha
se Chromatography:


A Versatile Platform for the Analysis of RNA
.

Ion pair reverse
-
phase chromatography (IP RP HPLC) has been widely applied for the study of
nucleic acids, in particular DNA [9
-
10]. More recently, a number of studies have utilised IP RP
HPLC for the purification and analysis of RNA, demonstrating its versatility in a variety of
different applications; from the routine purification of synthetic oligoribonucleotides, through to
the analysis of complex RNA:RNA interactions. This research dem
onstrates that IP RP HPLC is
a versatile platform for the analysis of RNA. Careful selection of the chromatography conditions
including the ion pair reagent, temperature and additives to the mobile phase, facilitates the
operation of the IP RP HPLC under d
ifferent modes, enabling the study of a wide of range RNAs
and biological systems.

[18]

SIZE
-
EXCLUSION
CHROMATOGRAPHY

Size
-
exclusion chromatography (SEC), also known as

gel permeation chromatography

or

gel filtration
chromatography

separates particles on the basis of size. It is generally a low resolution
chromatography and thus it is often reserved for the final, "polishing" step of
purification
. It is also
useful for determining the

tertiary structure

and

quaternary structure

of purified proteins. SEC is used
primarily for the analysis of large molecules
such as proteins or polymers. SEC works by trapping
these smaller molecules in the pores of a particle. The larger molecules simply pass by the pores as
they are too large to enter the pores. Larger molecules therefore flow through the column quicker than
smaller molecules, that is, the smaller the molecule, the longer the retention time.

T
his technique is widely used for the molecular weight determination of polysaccharides. SEC is the
official technique (suggested by European pharmacopeia) for the
molecular weight comparison of
different commercially available low
-
molecular weight

heparins
.

The main analytical application is to determine molecular weight of single macromolecules and f
or
determination
of molecular

weight distributions
of polydispersed

polymers.

This method is less used

for quantitative determinations of specific macro molecules
and in that case reverse phase or ion
exchange is

preferred.

[9]

ION
-
EXCHANGE

CHROMATOGRAPHY


In ion
-
exchange chromatography, retention is based on the attraction between solute ions and charged
sites bound to the stationary phase. Ions of the same charge are excluded.
Types of ion exchangers
include:



Polystyrene resins



Cellulose and

dextran

ion exchangers (gels)



Controlled
-
pore glass or porous silica

This form of chromatography is widely used in the following applications: water purification,
preconcentration of trace components, lig
and
-
exchange chr
omatography, ion
-
exchange
chrom
atography

of proteins, high
-
pH anion
-
exchange chromatography of carbohydrates and
oligosaccharides, and others.


A
nd it’s a method of choice for analysis of inorganic ions and preferable to reverse phase for
analysis
of small organic ions.
[9]

R
apid ion exchange chromatography of plasma amino acids has been achieved on polystyrene
-
based
cation exchange resins.

[10]

A
QUEOUS NORMAL
-
PHASE CHROMATOGRAPHY

Aqueous normal
-
phase chromatography (ANP) is a
chromatographic technique which encompasses
the mobile phase region between reversed
-
phase chromatography (RP) and organic normal phase
chromatography (ONP). This technique is used to achieve unique selectivity for hydrophilic
compounds, showing normal pha
se eluti
on using reverse
-
phase solve
.


UPLC

This technique may therefore enhance the sensitivity and expand the scope of analysis to smaller cell
populations and possibly to single mammalian cells.

The “ultra” HPLC approach offers a wide range of applicati
ons, and is highly attractive for those
situations which require ultra
-
high sensitivity such as those done with limited sample quantities
(Lindon
et al
., 2003). The limitations of this approach are that it requires special high pressure
pumping equipment a
nd that its peak capacity is still too low to adequately resolve complex mixtures
often encountered in proteomics.

The HPLC separation takes in excess of 12min while the UPLC accomplishes the same separation in
under 30seconds. UPLC can also be used to sig
nificantly improve the success of the d
rug discovery
process.
By the mid
-
1990s, high performance liquid chromatography (HPLC) directly coupled to mass
spectrometry (MS) was in routine use in drug metabolism laboratories for these types of studies
(Mutton
e
t al
., 1998). Enhanced selectivity and sensitivity, and rapid, generic gradients made LC

MS
the predominate technology for both quantitative and qualitative analyses
.

At a time when many scientists have reached separation barriers with conventional HPLC,
UPLC
presents the possibility to extend and expand

the utility of chromatography.

UPLC
fulfills

the promise
of increased resolution, speed, and sensitivity predicted for liquid chromatography.
It might become
the gold standard for quantification in
clinical and forensic toxicology and doping control if the cost of
the equipment can be markedly reduced, if current disadvantages, for example irreproducibility of
fragmentation, reduction of ionization by matrix, etc., can be overcome, and, finally, if o
ne of the
increasing number of quite different techniques can become the apparatus standard.

[11]


LC
-
IR

The hyphenated technique developed from the coupling of an LC and detection method IR or FT
-
IR is
known as HPLC
-
IR.

And it is a useful t
echnique for identification of
organic compounds because in
the mid IR
region the
structure of organic
comp
o
unds has

many absorption bands that are
characteristics of particular functionalities.

Eg:
-
OH,
-
COOH.

However the combination is difficult and
the h
yphenated technique is slow and it is sensitive.


BIO
-
AFFINITY CHROMATOGRAPHY

Bioaffinity chromatography is now the preferred choice for the purification, determination or removal
of many biologically active substances. This text includes information on bi
ologically active
substances with their affinants, solid supports and methods of coupling, summarized in tables covering
classical, high
-
performance liquid and large
-
scale bioaffinity chromatography. Optimization of the
preparation and the use of highly ac
tive and stable biospecific adsorbents.

Several applications of bioaffinity chromatography are there such as quantitative evaluation of
biospecific complexes and many uses in medicine and in the biotechnology industry.

[14]

POPLC



In Phase Optimized Liquid Chromatography (POPLC®), a technique developed by Bischoff
Chromatography, the stationary phase is first optimized for a given sample. Once you are assured of
having the optimum stationary phase, it is a much simpler task to adju
st the mobile phase for t
he
desired speed and resolution
, a technique developed by Bischoff Chromatography, the stationary phase
is first optimized for a given sample. Once you are assured of having the optimum stationary phase, it
is a much simpler task t
o adjust the mobile phase for the desired speed and resolution.

[12]

Stationary phase optimized liquid chromatography (POPLC) has been applied to the separation of
oligopeptides. Optimized conditions are maintained and all peptides could be separated in le
ss than
30

min with good resolution and peak symmetry.

[13]


REFERENCES:

1.
http://en.wikipedia.org/wiki/High
-
performance_liquid_chromatography

2
.
Department of
Biochemistry, University of Edinburgh, Edinburgh EH3


9YW


Ian M Bird, PHD, postdoctoral research fellow


Series edited by:


Drs Peter and John Hayes.Br MedJ 1989; 299:783
-
7


3.

Increasing

Speed and Throughput When Using HPLC
-
MS/MS Systems for Drug Metabolism and
Pharmacokinetic Screening, Y. Hsieh and W.A. Korfmacher, Current Drug Metabolism Volume 7,
Number 5, 2006, Pp. 479
-
489

4.

Covey TR, Lee ED, Henion JD. 1986. High
-
speed liquid chro
matography/tandem mass
spectrometry for the determination of drugs in biological samples. Anal Chem 58:2453
-
2460.


5.

Thermospray liquid chromatography/mass spectrometry determination of drugs and their
metabolites in biological fluids. Covey TR et al. An
al Chem. 1985 Feb; 57(2):474
-
81


6.
Wysocki VH, Resing KA, Zhang Q, Cheng G (March 2005). "Mass spectrometry of peptides and
proteins".
Methods

35

(3): 211

22.
Doi
:
10.1016/j.ymeth.2004.08.013
.
PMID

15722218
.



7.

LC/MS applications in drug development, Mass Spectrometry Reviews, Mike S. Lee, Edward H.
Kerns, Vol 18, 1999, pp 187
-
279


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. 7,1989


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.
W.J.Lough and I.W.Wainer (
1995)
.
High Performance Liquid Chromatography fundamental
principles and practice”
. 2
-
6 Boundary Row, London SE1 8HN, UK
: Chapman and Hall. p p205
-
269


10.
A.Pryde and M.T.Gilbert (1980).
Applications of High
Performance Liquid Chromatography”
.
USA: Chapman and Hall in association with Methuen
,
Inc
. p57
-
188,211
-
212.



11.
http://sunspace.sunderland.ac.uk/webct/urw/lc935970396001.tp935970418001//RelativeResourceManag
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http://www.mac
-
mod.com/pb/poplc
-
pb.html


13.Journal of Pharmaceutical and Biomedical Analysis Volume 51, Issue 3, 5 February 2010, Pages 764
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14.
http://www.amazon.com/Bioaffinity
-
Chromatography
-
Journal
-
Library/dp/0444890300


15.
http://www.sacheminc.com/industries/biotechnology/teaching
-
tools.html


l
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http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B75F9
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http://en.wikipedia.org/wiki/Liquid_chromatography
-
mass_spectrometry


18
.

M. J. Dickman. (march 2011). Ion Pair Reverse
-
Phase Chromatography: A Versatile
Platform for the Analysis of RNA4
Department of Chemical and
Biological Engineering,
ChELSI Institute, University of Sheffield, Sheffield, UK.


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.

http://teachline.ls.huji.ac.il/72682/Booklets/AmershamRPCManual.pdf

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http://www.nestgrp.com/protocols/polylc/hilic_op.shtml


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