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1.0 INTRODUCTION
A key component of the quality of pharmaceutical drugs is the
control of Impurities. It is important to identify and quantify the level of
impurities that may be present to provide safe, effective and well
controlled medicines. The identification and quantification of
impurities to today‘s standards presents significant challenges to the
analytical chemist. The development of modern quantitative methods
driven by these challenges and the rapid development of spectrometers
has provided increasing opportunity to identify the structure & therefore
the origin and safety potential, of such Impurities.
The pharmaceutical analytical chemistry is concerned with new
analytical techniques and the analytical chemist should consider various
principles related to interdisciplinary sciences such as chemistry,
physics, biology, engineering, computer science, etc. in developing
methods of analysis. For instance, the analytical instruments such as
mass spectrometer developed by physicists found to have great
applications in pharmaceutical analysis .
The DMF ( Drug Master File) holder or the ANDA (Abbrevated New
Drug Application) applicant should summarise those actual and
potential Impurities most likely to arise during synthesis, purification
and storage of the drug substance. This summary should be based on
sound scientific appraisal of both chemical reactions involved in the
synthesis and impurities associated with raw materials that could
2
contribute to the impurities profile of the drug substance and also about
possible degradation products. The studies (e.g.NMR,IR and MS)
conducted to charecterise the structure of actual Impurity or degradation
product present in the drug substance at the appearent level of 0.1% or
above (calculated using the response factor of drug substance) should be
described.
Hence, the bulk drug manufacturer should include authentic
documented evidence for degradation products and impurities with the
validated analytical method for quantification, along with structural
elucidation reports before getting the registration or marketing approval.
Analytical methods are required for a variety of reasons during
drug development process. The regulatory agencies expect that any
investigational new drug or new drug product contains what is stated on
the label and in the correct amount over the shelf-life of the API or
product. There are also further expectations about absence of any
harmful contaminants and has not been otherwise adulterated. ICH
(International Confrrence on Harmonisation) rules must ultimately be
complied with regarding Impurities and degradation products.
Pharmacopeia tests are also mandatory, even for investigational drugs
and drug products.
The regulatory authorities are aware of the scientific and technical
difficulties of developing such tests and allowed some discretion as to
3
when certain test methods and specifications should be applied. In
general terms, commercial kind of specifications are required for Phase-
III studies and beyond. Less stringent specifications and tests may be
acceptable for Phase-I and Phase-II stages of clinical trials of a drug
substance.
Thoroughly validated analytical methods are not required For
Phase-I manufacture (API or pharmaceutical product). However, there is
an expectation about the methods used will be appropriate for their
intended use and capable of distinguishing between acceptable and
unacceptable batches.
The qualification process of advanced intermediates and active
pharmaceutical ingredients (API) requires the precise information from
the following studies
a. Analytical method development
b. Synthesis/isolation and purification of Impurities, degradation
products and characterizations of Impurities.
c. Analytical method validation of developed analytical methods as
per harmonized guidelines such as ICH ( International Conference
on Harmonization).
4
To establish well qualified methods, the analytical scientist should
take the aid of sophisticated analytical instruments for various
applications such as
a. Preparapative HPLC, SFC (Super Fluid Chromatography) for
isolation of Impurities.
b. Advanced spectroscopic equipment such as FTIR, LC- MS/MS,
NMR (13C & 1H) for the characterization of Impurities, degradation
products and other chemical entities.
c. Analytical method development by chromatography (HPLC,GC…)
and validation of Stability Indicating Assay Methods (SIAM) for
qualification of developed analytical methods. Sometimes the
impurities can be enriched by forced degradation studies which
intern the part of the analytical method validations. The validation
of analytical procedures in compliance with international
regulatory guidelines assures the quality of the product for
regulatory purpose and can be easily marketed in highly
regulated markets like US, Europe and Japan.
5
1.1 Scope of the research work
The analytical chemist should strive to develop comprehensive
analytical methods to qualify the bulk drug substances and advanced
intermediates. Scope of this study includes providing the comprehensive
analytical method development studies for isolation, identification and
quantification of impurities in drug substances and intermediates, covers
the following topics
a. Identification, isolation and charecterisation of impurities of
drug substances.
b. Development and validation of analytical methods for advanced
drug intermediates.
c. Development of stability indicating LC methods and validation of
analytical methods for Active Pharmaceutical Ingradients such
as proton pump inhibotors (benzimadazole derivatives such as
Rabeprazole Sodium) and anti psychotic drugs (quinoline
derivatives such as Aripiprazole).
6
1.1.1 Proton pump inhibitors (PPI)
The discovery of the proton pump1 and elucidation of its
function2 led to the discovery of another antisecratory therapy. First
PPI, H83/88, is a benzimidazole derivative that non competitively
inhibited both receptor (HA)- mediated and non receptor (Camp)-
mediated acid secretion from isolated gastric mucosa2.
PPI acts by inhibiting the enzyme H+/K+-ATPase, which is located
on the luminal surface of gastric pareietal cells. PPI‘s are both more
potent and of longer duration than H2-receptor antagonists. PPI‘s are
substituted benzimidazole based compounds. These are inactive
prodrugs and activated in the acid environment of the gastric glands.
In 1973, AB Haessle has identified Timoprozole3(1) as one of the
first well defined inhibitors of gastric proton pump. It was subsequently
followed by the more potent derivatives, Picoprazole4(2) and
Omeprazole5(3) (Fig:1.1).
NH
NS
O
N NH
NS
O
N
H3C
OH3C
O
NH
NS
O
N
H3C
OH3C
CH3
OCH3
1 2
3
Fig:1.1 Chemical structures of Timoprozole(1), Picoprazole (2) and
Omeprazole5(3).
7
Omeprazole (4) is characterised by the presence of the substituted
pyridine ring, the substituted benzimidazole and the methylsulfinyl
linking group as key structural features.
Subsequently, benzimidazole group of Omeprazole has been
replaced by other heterocyles and its activity retained. For example,
methoxyimidazopyridine is the structural moity in Tenatoprazole6 (4).
Similarly, benzimidazole group of Omeprazole (3) is replaced by
thieno[3,4-d]imidazole in compounds such as Savaprazole7 (5) and S-
19248 (6) (Fig: 1.2).
NH
NS
O
N
H3C
OH3C
CH3
OCH2CF2CF2CF3
NH
NS S
O
N
OCH2CF2CF3
NH
NS S
O
N
H3C
4 5
6
Fig:1.2 Chemical structures of Tenatoprazole (4) Savaprazole (5)
and S-1924 (6).
Increasing the nucleophilic character of the pyridine ring, by the
incorporation of electron donating substituents, led to the recent
advanced prazoles such as, Lansoprazole9 (7), Pantoprazole10 (8) and
Rabeprazole 11 (9) (Fig: 1.3).
8
Fig:1.3 Chemical structures of Lansoprazole (7), Pantoprazole (8) and
Rabeprazole (9).
1.1.2 Anti psychotic drugs
The antipsychotic drugs were primarily developed for the
treatment of schizophrenia during last half of 20th century. Out of
various medications indicated, three basic classes of medications
(conventional, atypical and dopamine partial agonist antipsychotics),
act principally on dopamine systems.
1.1.2.1 Conventioal or First Generation Antipsychotic (FGA) agents
The common effect of FGAs is a high affinity for dopamine D2
receptors,12 and correlation observed between the therapeutic doses of
these drugs and their binding affinity for the D2 receptor14-19.
The other classes of FGAs includes ―Benzamides‖ such as
Amisulpride, is a highly selective anatagonist of D2 and D3 receptors
with little affinity for D1-like or nondopaminergic receptors 20-22
9
1.1.2.2 Atypical or Second Generation Antipsychotic (SGA) agents
The drug candidates like Quetiapine 10 (dibenzothiazepine
derivatives), Clozapine 11(dibenzodiazepine derivative), Olanzapine 12
(thienobenzodiazepinederivatives) and Risperidone 13 (benzisoxazole
derivatives)24-25 are few among the atypical or second-generation
antipsychotic (SGA) agents (Fig: 1.4). The mechanism of action has been
explained by ‗serotonin–dopamine (S2/D2) antagonism‘ promulgated by
Meltzer et.al,23.
S
NN
N OOH
H+
2
O
-O
O
O-
NH
NN
NCH3
Cl
NH
NN
NCH3
S CH3
N
N
O
CH3
N
ON
F
1110
12 13
Fig:1.4 Chemical structures of Quetiapine(10), Clozapine(11) Olanzapine
(12) and Risperidone (13).
10
1.1.2.3 The next generation psychotics - Partial dopamine agonists
Aripiprazole 14 (Fig:1.5) is the possible ‗next- generation
antipsychotics‘ with a mechanism of action that differs from currently
marketed FGAs and SGAs22, approved for clinical use in the US and
more recently in Europe. It is the first of a possible partial dopamine
agonist with a high affinity for D2 and D3 receptors23 and demonstrates
properties of a functional agonist and antagonist in animal models of
dopaminergic hypoactivity and hyperactivity, respectively. 25-28
N
ClCl
N O
NH
O
14
Fig:1.5 Chemical structure of Aripiprazole
11
1.2 Impurities in Drug substances and Application of
Chromatographic, Spectroscopic and Hyphenated techniques
for the Structure Elucidation of Impurities
Impurities of active pharmaceutical ingradients (API) fall in to three
main categories: process related Impurities, degradation products and
contaminent Impurities carried out from the reagents being used in the
synthesis31-34. Further, enantiomers and polymorphs may also be
considered as impurities under particular circumstances.
Impurities of API generally fall in to the following categories:
Organic Impurities (process- and drug-related)
Inorganic Impurities
Residual solvents
Organic Impurities may arise during the manufacturing process
and/or storage of the drug substances. They can be identified or
unidentified, volatile or non-volatile and include:
Starting materials
By-products
Intermediates
Degradation products
Reagents, ligands and catalysts
Residual solvents
12
Organic Impurities often called related, ordinary or synthesis
related Impurities can originate from various sources and various phases
of the synthesis of bulk drugs. It is very difficult to identify the
differences between process related impurities and degradation
impurities. Moreover, degradation products can be formed either during
the synthesis or isolation of the end product and even during storage
of the drug substance or product.
The origin of impurities of Rabeprazole sodium and others were
discussed in corresponding chapters.
1.3 Structural Elucidation of Impurities
The first spectroscopic data which are usually obtained in the drug
impurity profiling are the UV spectra of the impurities. Whenever
chromatographic technique such as HPLC, GC or one of the other
chromatographic techniques are used for the separation of impurities,
rapid scanning by using diode array detectors produce good quality
UV spectra. If the information obtained from UV spectrum is inadequate,
mass spectroscopic data may provide relavant information as a next step.
Sophisticated analytical techniques such as online GC/MS and
HPLC/MS facilities available in the sufficiently developed laboratories
dealing with impurity profiling29 and usage of these systems will be the
adavantageous way to carry out impurity profiling efficiently and data
can be obtained simultaneously on several impurities, down to the 0.01%
level. The GC-MS technique provides the reliable information for
13
molecular weight by using chemical ionization and the information on
fragmentation obtained by using the electron impact ionization, which is
necessary for the solution of more delicate structural elucidation
problems. Applicability of this technique is limited due to volatility and
thermal stability problems.
Infact, HPLC/MS technique is having great advantages such as
its general applicability and possibility of coupling it with diode array UV
detectors (HPLC/UV/MS). Though, the first generation instruments are
based on the soft ionization techniques usually give only molecular
weight information, the modern instruments are capable to provide the
important information on fragmentation. HPLC/MS/MS is the most
effective devise for impurity characterization, which can simultaneously
furnish all information discussed so far.
In addition to the hyphenated techniques, mass spectra also
provides valuable information such as, first direct massspectroscopic
investigation of the sample without any preliminary chromatographic
separation is mentioned. As the mass spectrometry is highly sensitive, it
does not requires special instrumentation. The impurities scrapped off
and eluted after TLC separation and the quantity of the impurities in
fractions obtained from a sufficiently highly loaded analytical column
from HPLC separation is usually sufficient for MS investigation, unless
eluents contains inorganic salts and buffers. This methodology will be
14
beneficial to those laboratories where the hyphenated techniques are at
the disposal.
At this point in the complex procedure of elucidating the structure
of Impurity, being in possession of the information obtained from the
spectroscopic techniques such as UV-Vis spectrophotometry, IR
spectrophotometry and mass spectrophotometry, the drug analyst
should make a very important decision. Based on the data obtained
spectroscopic studies integrated with chromatographic data, it should
be decided weather the careful evaluation of this information and the
full knowledge of the chemistry of the synthesis make it possible to
suggest a structure for the Impurity in relation to the main component or
not.
The ultimate method for elucidating structure of impurity is NMR
spectroscopy. The sample quantity required to record quality NMR
spectra is much higher than that of mass spectroscopy. Also, to record
NMR spectrum of Impurities, It is usually not possible to take the NMR
after separation on ordinary plates or analytical HPLC columns: the
pure sample isolated from preparative scale separation is required to
obtain suitable sample quantity with desired purity which is required to
minimize the level of spectral background caused by ill defined artifacts.
Alternatively, the commercial availability of online HPLC/NMR,
moreover HPLC/NMR/MS instruments can be used. But due to the cost,
these techniques are available only in limited number of laboratories and
15
only a few publications are available on these techniques containing
initial results in structure elucidation of an Impurity and Impurity
profiling.
All the data so obtained from all the spectroscopic studies along
with the NMR spectrum, a structure can be proposed for the Impurity.
At this point it should be emphasized that the close team work between
spectroscopists, chromatographers and synthetic organic chemists is
required to elucidate the structure of an Impurity. The chromatographers
role is also very important to develop chromatographic methods for the
separation, detection of the Impurities. Also, for the generation of
spectra by hyphenated techniques along with carefull evaluation of the
TLC Rf values and HPLC or GC retention times (Rt) comparison of these
with those of the main component and other potential Impurities
provides useful data regarding the nature of the Impurity interms of
polarity and this information is also an Important source to propose the
probable structure for the Impurity. It is very Important to have organic
chemist as a team member along with analytical scientist who must be
familiar with the aspects of the synthesis of the drug in question whose
opinion should also be taken in to account in problematic cases to
interpret analytical data to solve the structure of the desired compound.
16
1.3.1 Synthesis, isolation and purification of impurities 36-39
It is very difficult to synthesise the Impurity with the proposed
structure when compared with the main component itself and the
synthesis of Impurity may involve multistep process and requires
several weeks of intensive work for this reason especially in the case of
complicated structures the proposal for the structure to be synthesized
should be made after extremely through and careful considerations.
After the successful synthesis and based on all the spectroscopic
and analytical investigation of the synthesized, the next item is to
compare the chromatographic and spectral data of material with the
Impurity found in the drug substance. The real Impurities can be
identified based on the chromatographic and on-line spectral matching
with known Impurities.
The quality of spectra obtained from the synthesized material is
usually better than that of those in the online mode or from small
isolated samples. After having proved their identity the spectra of better
quality can be used for regulatory registration and/or the research
publication purposes.
The Impurity standard can be designated from the gram scale
quantity of the synthesized Impurity with all the charecterisation details
obtained from spectral and chromatographic data. This means that in
possession of this it is possible to develop specific and selective
analytical methods for the quantitative estimation of the of the
17
Impurity. This Impurity standard has to be used routinely, when such a
specific analytical method becomes part of the analytical testing
procedure for every batch.
Sometimes, synthesis of the impurity can be almost impossible due
to its nature and the critical synthetic procedures involved, such as
explosive experimental conditions. In these exceptional cases, Impurity
standard can be prepared using preparative HPLC or by using SFC. Then
the synthesis can be omitted from the protocol of Impurity profiling and
the present preparative chromatographic methods are heavily reliant on
reversed phase chromatography. Based on the diverse nature of
Impurity and degradant characterization requirements, the reversed
phase chromatography might not be the method best suited for all
problems and normal phase chromatography, super fluid
chromatography may be the best choice for particular requirements.
1.3.2 Mass spectrophotometry in identification and structure
elucidation 40-69
Mass spectrometry with its reproducibility, specificity and
especially with its high sensitivity is an indispensible tool in the trace
analysis and structural elucidation of pharmaceutical compounds,40-48.
when ever analyzing Impurities is of prime Importance. Sensitivity
depends on the ionization methods applied. In electron ionization (EI)
mass spectra can be taken when a nanogram or more of substance is
available. FAB (Fast Atom Bombardment) mass spectra are
18
characteristically simple, often consisting only of protonated molecular
ions. Electrospray ionization (ESI) and atmospheric pressure chemical
ionization (APCI) techniques enable detection of compounds in the
pictogram range. Pico
Combined chromatography–mass spectrometry become a very
effective tool for the qualitative characterization of complex mixtures,
such as for trace and Impurity analysis by exploiting the resolving power
of chromatography and the strength of mass spectrometry in identifying
the spread compounds. Both combined techniques GC/MS and
HPLC/MS are capable of obtaining complete mass spectra of few
nanograms of each component.
Tandem mass spectrophotometry (MS/MS)40,41,49-56 a widely
accepted method for the analysis of Impurities without chromatography,
involves separation by mass. A component of the mixture is separated by
selecting single ion (in most cases M+ or MH+ ) at one specific m/z value
with the first analyzer and fragment of this ion monitored by second
analyzer23-28. Impurity isolation and offline mass spectrophotometry have
often been used to confirm the identity of drug Impurities and degradates
by comparison, if possible to synthesized reference Impurities. The
general method is to isolate individual components by preparative or due
to the high sensitivity of mass specrophotometry-analytical HPLC or TLC
and the isolated fractions are subjected to mass spectrometric analysis.
19
In most of the cases, the data obtained from the mass spectra provides
sufficient information to propose a structure for the Impurity.
1.3.2.1 Principles of LC/MS
LC/MS is a hyphenated technique combining with separation
power of HPLC 57-60 with the detection power of mass spectrometry. Even
with a very sophisticated MS instrument, HPLC is still useful to remove
the interferences from the sample that would effect ionization.
Mass spectrometers work by ionizing molecules and then sorting
and identifying the ions according to their mass-to-charge (m/z)
ratios.Two key components in this process are the ion source, which
generates the ions, and the mass analyzer, which sorts the ions. Several
different types of ion sources are commonly used for LC/MS. Each one is
suitable for different classes of compounds. Several different types of
mass analyzers are also used. Each has advantages and disadvantages
depending on the type of information needed.
a) Ion Sources 61-64
Much of the advancement in LC/MS over the last ten years has
been in the developmentof ion sources and techniques that ionize the
analyte molecules and separate the resulting ions from the mobile phase.
Earlier LC/MS systems used interfaces that either did not separate the
mobile phase molecules from the analyte molecules (direct liquid inlet,
thermospray) or did so before ionization (particle beam). The analyte
molecules were then ionized in the mass spectrometer under vacuum,
20
often by traditional electron ionization. These approaches were
successful only for a very limited number of compounds. The
atmospheric pressure ionization (API) techniques is superior compared to
traditional electrion ionization and widely applied to the number of
compounds that can be analyzed by LC/MS. In atmospheric pressure
ionization, the ionosation of analyte molecules will occur at atmospheric
pressure. The analyte ions are then separated electrostatically from
neutral molecules. The common ionization techniques are :
Electrospray ionization (ESI)
Atmospheric pressure chemical ionization(APCI)
Atmospheric pressure photoionization (APPI)
Fig: 1.6 Applications of various LC/MS ionization techniques
21
b) Mass Analyzers:
Although in theory any type of mass analyzer could be used for
LC/MS, but in practice, four types used most often. Each has
advantages and disadvantages depending on the requirements of a
particular analysis. They are
• Quadrupole
• Time-of-flight (TOF)
• Ion trap
• Fourier transform-ion cyclotron resonance (FT-ICR or FT-MS)
22
a)
b)
Fig: 1.7 Mass spectrum of sulfamethazine [(a) acquired without collision-
induced dissociation exhibits little fragmentation and b) with collision-
induced dissociation exhibits more fragmentation thus more structural
information]
23
1.3.2.2 Applications 66-69
LC/MS is suitable for many applications, from pharmaceutical
development to environmental analysis. Its ability to detect a wide range
of compounds with great sensitivity and specificity has made it popular
in a variety of fields.
a) Molecular Weight Determination
One fundamental application of LC/MS is the determination of
molecular weights. This information is key to determine the identity of a
chemical compound.
b) Structural Determination
Another fundamental application of LC/MS is the determination of
information about molecular structure. This can be in addition to
molecular weight information or instead of molecular weight information
if the identity of the analyte is already known.
c) Pharmaceutical Applications
LC/MS has wide range of applications in determining molecular
weights to characterise impurities contaminated with an APIs for
regularity documentation purpose.
d) Rapid chromatography of benzodiazepines
The information available in a mass spectrum allows some
compounds to be separated even though they are chromatographically
unresolved. In this example, a series of benzodiazepines was analyzed
using both UV and MS detectors. The UV trace could not be used for
24
quantitation, but the extracted ion chromatograms from the MS could be
used.The mass spectral information provides additional confirmation of
identity. Chlorine has a characteristic pattern because of the relative
abundance of the two most abundant isotopes.
In Fig: 1.8, the triazolam spectrum shows that triazolam has two
chlorines and the diazepam spectrum shows that diazepam has only one.
Fig: 1.8 Mass spectrum of Triazolam
25
e) Identification of bile acidmetabolites
The MSn capabilities of the ion trap mass spectrometer make it a
powerful tool for the structural analysis of complex mixtures. Intelligent,
data-dependent acquisition techniques can increase ion trap
effectiveness and productivity. They permit the identification of minor
metabolites at very low abundances from a single analysis. One
application is the identification of metabolic products of drug candidates
(Fig: 1.9).
Fig: 1.9 Identification of metabolic components of drug candidate
26
1.3.3 NMR in identification and structure elucidation
NMR spectroscopy has been proven now in most cases to be the
most powerful technique in the structural elucidation or conformational
analysis of organic molecules provided that they are available in
adequate purity and quantity. For typical small organic molecules even
the normal one dimensional (1D)1H and 13C NMR spectra are profuse in
their information content: chemical shifts (Fig: 1.10), multiplicities,
coupling constants, peak areas obtained from the usually well resolved
resonances all provide abundant and easily accessible, geometry
dependent structural information. In many cases a key element in
utilizing such data rests on a comparison of the relevant spectral
parameters with reference data available from suitable analogues. In that
sense the assignment of the resonances and the process of structural
elucidation are based on relative approach. Secondly, the advent of a
host of multi dimensional particularly two dimensional (2D) and other
sophisticated 1D techniques have brought in to focus what is the most
profoundly Important aspect of modern NMR: it can identify through–
bond (scalar) and through space (di-polar) spin–spin connections.70-72
While scalar couplings give rise to readily observed multiplet structure of
resonances. Dipolar couplings can only observed indirectly and their
most extensively utilized manifestation is the famous nuclear
Overhausser Effect (NOE).73-74,78
27
For small organic molecules, it is noted that NMR structure
elucidation mostly involves 1H and 13C NMR spectroscopy. The 1H
spectrum is richer in dipolar and scalar coupling data (C-H connectivity),
much wider range and 13C chemical shifts can provides profoundly
Important information about a carbon which is not attached to proton,
the same is not available from 1H spectrum.75-76
In addition to basic spectral data (typically 1H and 13C chemical
shifts, intensities, multiplicities and some coupling constants) it is
possible to obtain, as will be exemplified below in one go the direct C-H
connectivity in a phase sensitive pulsed-field gradients –selected hetero
nuclear quantum coherence (HSQC) experiment, the H-H scalar 1H and
13 C connectivities (e.g. in a gradient – selected double quantum filtered
phase sensitive COrrelation SpectroscopY (COSY)77experiment, the long
range C-H connections (e.g. in a gradient-selected Heteronuclear Multiple
Bond Correlation (HMBC) and the NOE connections or with a sufficiently
large molecules a nuclear overhauser enhancement spectroscopy
(NOESY) experiment. DEPT (Distortionless Enhancement by Polarization
Transfer)79-84 experiment is a very useful method for determining the
presence of primary, secondary and tertiary carbon atoms. The DEPT
experiment differentiates between CH, CH2 and CH3 groups by variation
of the selection angle parameter (the tip angle of the final 1H pulse):
28
45° angle gives all carbons with attached protons (regardless
of number) in phase
90° angle gives only CH groups, the others being suppressed
135° angle gives all CH and CH3 in a phase opposite to CH2
Fig:1.10 Proton Chemical Shifts in 1H NMR spectroscopy
29
1.3.4 Vibrational spectroscopy
Contemporary approaches to chemical structure elucidation are
now heavily reliant on mass spectroscopy and NMR spectroscopy. The
high sensitive vibrational spectroscopic data from FT-IR and FT-Raman
requires only small amount of samples relative to the amount is
normally used in acquisition of NMR data. One of the strong points of
vibrational spectroscopic methods is in the area of characterization of
functional group analysis. As an example, consider the presence of
carbonyl groups in the structure of an Impurity or degradant. Carbonyl
moieties other than aldehydes are transparent in the proton reference,
COSY and multiplicity-edited HSQC experiments. It is thus conveniently
provide small aliquot of an isolated sample for interrogation by
vibrational methods in parallel with the the acquisition of the NMR and
MS spectroscopic data.
Overall, the vibrational spectroscopy data to be highly
complimentary to other spectral data amassed during the
characterization of the structure of a degradant or Impurity. As such,
given the relative ease of obtaining these data, it seems obivious that
they should be acquired and incorporated in to the structure elucidation
protocol used when Impurities and degradants of a pharmaceutical agent
are characterized.
30
1.4 Role of High Performance Liquid Chromatography in Analytical
Method Development and Analysis of Pharmaceutical
Compounds
High performance liquid chromatography is a separation technique
based on a solid stationary phase and a liquid mobile phase.
Separations are achived by partition, adsorption and ion exchange
processes depending on the type of stationary phase used.
1.4.1 Various Aspects of high performance liquid chromatography
1.4.1.1 Columns and column efficiency:
Columns are considered as heart in case of HPLC. It carries the
stationary phase within them. Systems with polar stationary phases
and non-polar mobile phases are called normal phases and those with
non-polar stationary phases and polar mobile phases are known as
reverse phases. The columns for HPLC are of two types , they are
Those used for analytical separations: Their diameter varies from 2-5 mm
Those used for preparative-chromatography: They have larger diameter.
Various columns are used in HPLC are :
C8 column: Octylsilane chemically bonded to totally porous silica
particles 3-10µm in diameter (L7 phase as per USP-32)
C18 column: Octadecyl silane chemically bonded to porous silica or
ceramic micro particles, 3-10µm in diameter. (L1 phase as per USP-32)
31
Table 1.1 Bonded stationary phases for HPLC
STATIONARY PHASE
FUNCTIONAL GROUP
APPLICATIONS
Silica Si-OH Normal phase material pesticides,
alkaloids
C18 Octadecyal Reverse phase material
Fatty acids, PAH, Vitamins
C8 Octyl Reverse phase and ion pair, peptides
proteins
C6H5 Phenyl Reverse phase
Polar aromatic fatty acids
CN Cyano
Normal and reverse phase, polar
compounds
No2 Nitro Normal and reverse phase, PHP,
Aromatic
NH2
Amino
Normal,Reverse,weak ion exchange
Carbohydrates, organic acids,
Chlorinated pesticides
OH Diol Normal, Reverse phase peptides,
proteins
SA Sulphonic acid Cation Exchange, séparation of cations
SB Quaternary
ammonium
Anion exchange, separation of anions
32
1.4.1.2 Mobile phase:
It acts as a carrier for sample solution. The chemical interactions
of mobile phase and sample with the column determine the degree of
migration and separation of components. The stronger the interaction,
faster is the elution and shorter is the retention time. Mobile phases are
of several types and they are of Isocratic and Gradient.
1.4.1.2 Stationary phase:
The solid support contained within the column over which the
mobile phase continuously flows is termed as the stationary phase.
1.4.1.3 Size-Exclusion:
It operates on the basis of molecules in solution are separated
based on their size, more correctly their hydrodynamic volume. This is
usually achieved with an apparatus called a column, which consists of a
hollow tube tightly packed with extremely small porous polymer beads
designed to have pores of different sizes. These pores may be depressions
on the surface or channels through the bead. As the solution travels
down the column some particles enter into the pores. The larger the
particles, the faster the elution.
1.4.1.4 Normal Phase:
It operates on the basis of hydrophilicity and lipophilicity by using
a polar stationary phase and a non-polar mobile phase. Thus,
hydrophobic compounds elute more quickly than do hydrophilic
compounds (Fig: 1.11).
33
1.4.1.5 Reverse Phase:
It operates on the basis of hydrophilicity and lipophilicity. The
stationary phase consists of silica based packing‘s with n-alkyl chains
covalently bound i.e. the stationary phase is non-polar and the mobile
phase is polar. Thus, hydrophilic compounds elute more quickly than do
hydrophobic compounds (Fig: 1.11).
Fig:1.11 Selection of Normal phase and reverse phase for HPLC analysis
1.4.1.6 Ion exchange:
It operates on the basis of selective exchange of ions in the sample
with counter ions in the stationary phase. The sample is retained by
replacing the counter ions of the stationary phase with it‘s own ions. The
sample is eluted from the column by changing the properties of the
mobile phase do that the mobile phase will now displace the sample ions
from the stationary phase i.e., changing the pH.
34
1.4.2 Various Detectors and their detection limits:
The detector is positioned immediately posterior to the stationary
phase in order to detect the compounds as they elute from the column.
The bandwidth and height of the peaks may usually be adjusted using
the coarse and fine-tuning controls and the detection and sensitivity
parameters may also be controlled. Many types of detectors can be used
with HPLC
1.4.2.1 Refractive index detectors
They measure the ability of sample molecules to bend or refract
light. This property is called refractive index. Detection occurs when
light is bent due to samples eluting from the column and this is read as a
disparity between the two channels.
1.4.2.2 Ultra violet (UV) detectors
They measure the ability of samples to absorb light. This can be
established at one or several wavelengths.
Fixed wavelength: Measures at single wavelength, usually 254 nm
Variable wavelength: Measures one wave length at a time, but can
detect over a wide range of wavelengths.
Diode array: Measures a spectrum of wavelengths simultaneously.
35
1.4.2.3 Fluorescent detectors:
Each compound has a characteristic fluorescence and these
detectors measure the ability of a compound to absorb then re-emit light
at given wavelengths.
1.4.2.4 Radio chemical detectors
These detectors operates by detection of fluorescence along with
beta-particle ionization which involves the use of radio labeled material
usually tritium (3H) or carbon-14 (14C).
1.4.2.5 Electrochemical detectors:
Used in the analysis of compounds that undergo oxidation or
reduction reactions. They measure the difference in electrical potential
when the sample passes between the electrodes.
Table 1.2 UV Cut-off Wavelengths for Solvents
Solvent λ min (nm) SOLVENT λ min (nm)
Acetone 330 Dimethyl formamide 270
Acetonitrile 190 Ethanol 205
Chloroform 240 Ethyl acetate 260
Dichloromethane 230 n-Hexane 190
Diethyl ether 205 Methanol 205
Cyclohexane 200 Tetrahydrofuran 225
36
1.4.3 Applications of HPLC:
1.4.3.1 Preparative HPLC: It refers to the process of isolation and
purification of compounds. Important is the degree of solute purity and
the throughput, which is the amount of compound, produced per unit
time/operation.
1.4.3.2 HPLC for analytical determinations:
Helps us to obtain information about the sample compound, which
includes relative comparison, quantification and resolution of a
compound from the matrix that may present along with the main
component.
a. Separation of components based on their chemical properties:
This can be accomplished using HPLC by utilizing the fact that,
certain compounds have different migration rates given a particular
column and mobile phase. The extent or degree of separation is mostly
determined by the choice of stationary phase and mobile phase.
b. Identification:
For this purpose a clean peak of known sample assay has to be
observed from the chromatogram. Selection of column mobile phase and
flow rate matter to certain level in this process by comparing with
reference compound does identification and it can be assured by
combining two or more detection methods.
37
c. Quantification:
It is the analyte confirmation by using the known reference
standards. Quantification of known and unknown areas with respect to
the principal peak by various methods like - Area normalization method,
Internal standard method and External standard method.
1.5 Analytical Method Development by HPLC 85-100
1.5.1 Literature collection:
Method development starts with literature search. USP, EP, JP, IP,
Chromatography Journals, patents, etc., were referred for the same
molecule or for molecules having similar structure.
Short comes of the existing method was studied was checked.
Based on the synthetic scheme, selected the list of Impurities
that has to be checked. Raw materials used in the process,
degradation Impurities, Impurities generated during the
process, Impurities carried over from the penultimate stage were
also to be considered.
After deciding the Impurities that has to be monitored, API,
Impurity samples and standards should be collected.
38
1.5.2 Chemical structure:
The pH of the buffer, mobile phase, were selected based on the
structure of the compounds (API and Impurities).
1.5.3 Sample solution preparation:
For deciding the sample solution preparation, checked the
solubility of all the compounds in mobile phase, mobile phase - organic
solventmixtures, water-organic mixtures and mixtures of mineral acids
like perchloric acid, phosphoric acids, etc. Mobile phase was always
preferred to avoid base line noise and negative peaks.
1.5.4 Selection of stationary phase:
Depending on the nature of the compound, the stationary phase
was selected. Column parameters like internal diameter, particle
surface area, pore volume, commercial availability of the column were
also taken into account while selecting the stationary phase (Table 1.3).
Table 1.3 Selection of Chromatographic technique
Nature of the compound Chromatographic
technique
acids, bases and non-ionic samples reverse phase chromatography
ionic samples ion exchange chromatography
isomers, non-polar, non-ionic and chiral
samples
normal phase
chromatography
redox samples capillary electrophoresis
39
Fig: 1.12 FLOW CHART FOR HPLC COLUMN SELECTION
Normal phase, Bonded
Organic Solvent
Soluble
Methanol and
Methanol : Water
Soluble
Organic Solvent
Soluble
Molecular
Weight less than
2000
Sample Water
Soluble
Molecular
Weight greater
than 2000
Organic
Solvent Soluble
Water
Soluble
THF Soluble
Non-Ionic
Ionic
Reverse phase, Bonded
Small Molecule Gel Permeation
Chromatography (GPC).
Reverse phase
Bonded
Reverse phase /
Ionization Control
Reverse phase
Paired-Ion
Ion exchange
Gel Permeation
Chromatography (GPC).
GEL filtration
(Aqueous GPC)
Ion exchange
Reverse phase Bonded
Normal phase, Adsorption
40
1.5.5 Detector selection:
Detectors were selected based on the nature of the compound. If
the compound contains chromopheres, UV detectors were used. The
absorption maximum of the compound and the Impurities is the basis
for selecting the detector wavelength in UV detectors.
1.5.6 Mobile phase selection:
Based on the type of the column and the solubility of the
compound, the mobile phase was selected. Buffers (such as phosphate
buffers, acetate buffers, perchlorate buffer, borate buffers), ion pair
reagents (such as sodium lauryl sulphate, heptan sulphonic acid,
tetrabutyl ammonium hydroxide, tetrabutyl ammonium hydrogen
sulphate, sodium salts of butane, pentane or heptane sulphonic acids,
etc.,), organic modifiers (such as triethyl amine, diethyl amine, trifluoro
acetic acid, etc.,) were added to the mobile phase to get optimum
resolution.
In general, for normal phase HPLC, the following solvents were
used – methanol, acetonitrile, isopropy alcohol, ethanol, n-hexane,
chloroform, methylene chloride, chloroform, etc. For reverse phase
HPLC, solvents such as methanol, tetra hydro furan and acetonitrile
were used.
1.5.6.1 Chiral molecules:
Chiral columns were used to separate the enantiomers. For amino
acids and their derivatives Chirobiotec T or Chirobiotec V columns can
41
be used. Also cellulose, amilose based and cyclodextrin columns were
used for separating the positional and chiral enantiomers. Chiral
additives like cyclodextrins, inorganic transition metal salts, amino acids
and their derivatives can be used for increasing the resolution.
1.5.6.2 Elution pattern:
Isocratic elution pattern were used for the resolution of straight
chain compounds. Stronger isocratic mobile phase, ie., 100% organic
solvent (generally methanol) were used in the first run and then
successively the organic content can be reduced to study the resolution.
Gradient techniques were used when compounds having varying polarity
have to be resolved.
1.5.7 Column temperature:
Ambient column temperature is mostly preferred. Peaks will be
sharper at higher temperatures and elute earlier. Higher temperatures
will lead to a shorter column life time and some chiral columns may not
even be able to tolerate temperatures around 40°C. Column coolers can
also be used for getting better resolution.
1.5.8 Degradation studies:
After achieving the required separation, degradation studies shall
be carried out. Degradation studies were carried over by forcibly
degradation the compound by acid hydrolysis, base hydrolysis, water
hydrolysis, oxidation, thermally or by photo degradation. All the
42
degraded samples were analyzed as per the final method using photo
diode array detector to ensure that there is no co-elution of peak.
To sum up, the complete method development methodology for
HPLC can be pictorially represented as below.
Fig: 1.13 Schematic flow for HPLC method development
Literature search
Sample solubility
Column selection
Detector selection
(Wavelength selection)
Mobile phase selection
Degradation studies
43
1.6 Validation of Analytical Procedures
HPLC method validation is the process used to confirm that the
HPLC procedure employed for a specific test is suitable for its intended
use. Results from method validation can be used to judge the quality,
reliability and consistency of HPLC results and it is an integral part of
good analytical practice. Method validation has received considerable
attention in literature and from industrial committees and regulatory
agencies.
The objective of validation of an analytical procedure is to
demonstrate that it is suitable for its intended purpose. For
pharmaceutical methods, guidelines from the United States
Pharmacopoeia recommends 32
a. Identification tests
b. Quantitative tests for the active moiety and Impuritiy content
(limit tests for the control of Impurities)
1.6.1 Identification tests:
They are intended to ensure the identity of an analyte in a sample.
This is normally achieved by comparison of a property of the sample
(e.g., spectrum, chromatographic behavior, chemical reactivity, etc.) to
that of a reference standard.
44
1.6.2 Quantitative tests for Impurities (limit tests for the control of
Impurities):32-34
Either test is intended to accurately reflect the purity
characteristics of the sample. Different validation characteristics are
required for a quantitative test than for a limit test. Assay procedures
are intended to measure the major components present in a given drug
substance. While, assaying for the active or other selected components
of a drug product or for the associated assays with analytical procedures
(e.g., dissolution) the same validation characteristics are applied.
Typical validation characteristics, which should be considered, are
listed as follows:
Accuracy
Precision
Repeatability
Intermediate precision
Specificity
Detection limit
Quantification limit
Linearity
Robustness
System suitability
45
1.6.3 Validation of Analytical procedures:
Analytical methods should be validated to ensure reliability,
consistency and accuracy of analytical data. Method validation has been
a requirement of FDA and international regulations. The analytical
procedure is the detailed description of performing the analytical test
and it should describe the steps necessary to perform each analytical
test. Various aspects of analytical procedure may includes, but not
limited to the preparation of sample, the reference standard and the
reagents, description and utility of apparatus, generation of the
statistical data like formulae for the calculation and calibration curve.
1.6.3.1 Specificity:
An investigation of specificity should be conducted during the
validation of identification tests, the determination of Impurities and the
assay. The procedures used to demonstrate specificity will depend on the
intended objective of the analytical procedure.
Specificity can be defined as the ability to assess unequivocally the
analyte in the presence of components, which may be expected to be
present. Typically, the studies may include but not limited to
Impurities, degradents, matrix, etc. It is not always possible to
demonstrate that an analytical procedure is specific for a particular
analyte (complete discrimination). In this case a combination of two or
more analytical procedures is recommended to achieve the necessary
level of discrimination.
46
Specificity of a method also Implies that the method is capable of
Ensuring the identity of an analyte.
Ensuring that all the analytical procedure performed allow an
accurate statement of the content of Impurities
Ensuring to provide an exact result, which allows an accurate
statement on the content or potency of the analyte in a sample.
1.6.3.2 Accuracy:
The accuracy of an analytical procedure expresses the closeness of
agreement between the value, which is accepted either as a conventional
true value or an acceptable reference value and the value found. This is
sometimes termed trueness. Accuracy can be assessed on samples
spiked with known amounts of Impurities and should be reported as
percent recovery (percent recovery is the area obtained by spiking the
Impurity in the sample)
1.6.3.3 Precision:
The precision of an analytical procedure expresses the closeness of
agreement between a series of measurements obtained from multiple
sampling of the same homogeneous sample under the prescribed
conditions. The precision of an analytical procedure is usually expressed
as the variance, standard deviation or coefficient of variation of a series
of measurements. The standard deviation formula used for measuring
the precision is
47
Where,
= Standard deviation
r = Mean
N = Number of degrees of freedom
xi, ..., xn = Real numbers
The precision of a method can be estimated by considering the following
parameters
Repeatability - Expresses the precision under the same operating
conditions over a short interval of time and is also termed as
intra-assay precision.
Intermediate precision - Expresses within-laboratories variations:
different days, different analysts, different equipment, etc.
Reproducibility - Expresses the precision between laboratories
(collaborative studies, usually applied for standardization of
methodology).
1.6.3.4 Limit of Detection
The limit of detection an individual analytical procedure is the
lowest amount of analyte in a sample, which can be detected but not
necessarily quantitated as an exact value. Several approaches for
determining the detection limit are possible, depending on whether the
48
procedure is a non-instrumental or instrumental. Approaches other than
those listed below may be acceptable. They are described as follows
a) Based on Visual Evaluation
Visual evaluation may be used for non-instrumental methods but
may also be used with instrumental methods. The detection limit is
determined by the analysis of samples with known concentrations of
analyte and by establishing the minimum level at which the analyte can
be reliably detected .
b) Based on Signal-to-Noise
This approach can only be applied to analytical procedures which
exhibit baseline noise.
Determination of the signal-to-noise ratio is performed by
comparing measured signals from samples with known low
concentrations of analyte with those of blank samples and establishing
the minimum concentration at which the analyte can be reliably
detected. A signal-to-noise ratio between 3 or 2:1 is generally considered
acceptable for estimating the detection limit.
c) Based on the Standard Deviation of the Response and the
Slope
The detection limit (DL) may be expressed as:
49
where
= the standard deviation of the response
S = the slope of the calibration curve
The slope S may be estimated from the calibration curve of the analyte
1.6.3.5 Limit of Quantitation or Quantitation Limit
The limit of quantitation of an individual analytical procedure is
the lowest amount of analyte in a sample, which can be quantitatively
determined with suitable precision and accuracy. The quantitation limit
is a parameter of quantitative assays for low levels of compounds in
sample matrices, and is used particularly for the determination of
Impurities and/or degradation products. Severalapproaches for
determining the quantitation limit are possible, depending on whether
the procedure is a non-instrumental or instrumental. Approaches other
than those listed below may be acceptable.
a) Based on Visual Evaluation
Visual evaluation may be used for non-instrumental methods but
may also be used with instrumental methods.
The quantitation limit is generally determined by the analysis of samples
with known concentrations of analyte and by establishing the minimum
level at which the analyte can be quantified with acceptable accuracy
and precision.
50
b) Based on Signal-to-Noise Approach
This approach can only be applied to analytical procedures that
exhibit baseline noise.
Determination of the signal-to-noise ratio is performed by
comparing measured signals from samples with known low
concentrations of analyte with those of blank samples and by
establishing the minimum concentration at which the analyte can be
reliably quantified.A typical signal-to-noise ratio is 10:1.
c) Based on the Standard Deviation of the Response and the
Slope
The quantitation limit (QL) may be expressed as:
where
= the standard deviation of the response
S = the slope of the calibration curve
The slope S may be estimated from the calibration curve of the analyte.
1.6.3.6 Linearity:
The linearity of an analytical procedure is its ability (within a given
range) to obtain test results, which are directly proportional to the
concentration (amount) of analyte in the sample.
51
The correlation coefficient r (at times also denoted by R) is then
defined by
Where
X = Concentration
Y = Area
n = Number of levels
1.6.3.7 Robustness:
The robustness of an analytical procedure 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.
1.6.3.8 System suitability:
System suitability tests are an integral part of gas and liquid
chromatographic methods. They are used to verify that the resolution
and reproducibility of the chromatographic system, those are adequate
for the analysis to be done. The tests are based on the concept that the
equipment, electronics, analytical operations, and the samples to be
analyzed constitute an integral system that can be evaluated as such.
The resolution, ‗R’, is a function of column efficiency, ‗N’, and is specified
to ensure that closely eluting compounds are resolved from each other,
to establish the general resolving power of the system, and to ensure that
r =
n XY - XY
{[ n X2 – (X)
2] [ n Y2
– (Y)2]}
52
internal standards are resolved from the drug. Column efficiency may be
specified also as a system suitability requirement, especially if there is
only one peak of interest in the chromatogram.Unless otherwise specified
in the individual monograph, data from five replicate injections of the
analyte are used to calculate the relative standard deviation, SR, if the
requirement is 2.0% or less; data from six replicate injections are used if
the relative standard deviation requirement is more than 2.0%.
T denotes the tailing factor, is a measure of peak symmetry, is
unity for perfectly symmetrical peaks and its value increases as tailing
becomes more pronounced.
The control preparation can be a standard preparation or a
solution containing a known amount of analyte and any additional
materials useful in their control of the analytical system, such as
excipients or Impurities. Whenever there is a significant change in
equipment or in a critical reagent, suitability testing should be performed
before the injection of samples. No sample analysis is acceptable unless
the requirements of system suitability have been met. Sample analysis
obtained, while the system fails, requirements are unacceptable.
Recommended