ANALYTICAL METHOD DEVELOPMENT FOR ACTIVE PHARMACEUTICAL INGREDIENTS (APIs)

1

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.

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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).

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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).

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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).

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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

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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).

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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

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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

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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

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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

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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

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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.

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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

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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

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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.

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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,

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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

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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)

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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]

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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

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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

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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

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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

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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.

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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

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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.