29

2.0 Introduction:

Chromatography in general and high performance liquid chromatography

(HPLC)in particular plays an important role in the separation/detection techniques.It is

widely used for separation, identification and estimation of both samples and complex

components present in the raw materials, intermediates, bulk drugs and their formulations.

Thin layer chromatography (TLC) is a powerful technique for rapid screening of unknown

materials in bulk drugs [60-61]. Gas liquid chromatography (GLC) has significant role in

analysis of pharmaceutical products [62-65]. HPLC, which was introduced in 1960s, is the

most common of pharmaceutical several chromatographic techniques employed in the

purity control of pharmaceuticals.Impurities in the bulk drug substances at levels of 0.1%

or even less can be detected by HPLC.

High performance liquid chromatography (HPLC) is an important tool for the

analysis of pharmaceutical drugs, for drug monitoring and for quality assurance. Gradient

elution, temperature programming and wavelength programming techniques provide

valuable information regarding the undetected components of a given drug.In the case of

UV detection where impurity components differ in their absorption maxima, a multiple

wavelength scanning program is capable of monitoring several wavelength

simultaneously. Photo diode Array (PDA) detectors are generally used not only to scan

the components throughout the entire UV range, but also record the spectra and

chromatograms of all the components in a drug. Grady et al has outlined different

approaches towards the establishment of impurity profiles of synthetic drugs [66-68].

These methods involve the prediction of likely impurities with in the synthetic process,

their isolation and identification by suitable analytical techniques.Several approaches for

the determination of impurities in a drug substance using HPLC have been reported [69-

73].

High performance liquid chromatography (HPLC) has proven to be the

predominant technology used in laboratories worldwide during the past 30-plus years.

Many laboratory experts have acknowledged that UPLC will ultimately supersede all the

conventional HPLC techniques. The perfect separation method of HPLC has many

advantages like robustness, ease of use, changeable sensitivity and selectivity but the

main limitation is lack of efficiency in comparison to UPLC [74,75].

30

2.1 UPLC an Introduction and overview.

In precedent decade, substantial technological advances have been done in

enhancing particle chemistry performance, improving detector design and in optimizing

the system,data processors and various controls of chromatographic techniques.When all

was blended together, it resulted in the outstanding performance via ultra-high

performance liquid chromatography (UPLC), which holds back the principle of HPLC

technique. This technique is considered as a new focal point in field of liquid

chromatographic studies.Ultra performance liquid chromatography (UPLC) takes

advantage of technological strides made in particle chemistry performance, system

optimization, detector design and data processing and control. This new category of

analytical separation science retains the practicality and principles of HPLC while creating

a stepfunction improvement in chromatographic performance.

UPLC is a derivative of HPLC whose underlying principle is that as column

packing particle size decreases, efficiency and thus resolution increases. One of the

primary drivers for the growth of this technique has been the evolution of packing

materials used to effect separations. The underlying principles of this evolution are

governed by the van Deemter equation, with which any student of chromatography is

intimately familiar [76]. The van Deemter equation is an empirical formula that describes

the relationship between linear velocity (flow rate) and plate height (HETP, or column

efficiency). And, since particle size is one of the variables, a van Deemter curve can be

used to investigate chromatographic performance.Over the many years, researchers have

looked at “fast LC” with accuracy as a way to speed up analyses [77,78]. The “need for

speed” has been elevated by the sheer numbers of samples in the laboratories

(specifically in pharmaceutical drug discovery) and the availability of affordable, easy to

use mass spectrometers. Smaller columns and faster flow rates (amongst other

parameters) have been used. Elevated temperature, having the dual advantages of

lowering viscosity, and increasing mass transfer by increasing the diffusivity of the

analytes, has also been investigated [79]. However, as illustrated in Figure 2.1.1.F1, as

the particle size decreases to less than 2.5 mm, not only is there a significant gain in

efficiency. By using smaller particles, speed and peak capacity (number of peaks resolved

31

per unit time) can be extended to new limits, termed Ultra Performance Liquid

Chromatography, or UPLC.

2.1.1.F1.Van Deemter plot,illustrating the evolution of mobile phase velocity vs

plate height (1) particle sizes over the last three decadess

2.2. Principle of small particle chemistry and UPLC

The Van Deemter plot is governed by the equation: H = A +B/v + Cv, where v is

linear velocity and A, B and C are constants. A is independent of velocity and represents

the „Eddy‟ mixing and when column particles are uniformly small, the value of A is the

lowest. B is axial diffusion or the natural diffusion tendency of molecules and this effect is

diminished at high flow rates, so this term is divided by v.C is due to kinetic resistance to

equilibrium in the separation process. The design and development of sub-2 mm

particles is a significant challenge, and researchers have been active in this area for

some time to materialise on their advantages [80-82]. Although high efficiency, non-

porous 1.5 mm particles are commercially available, they suffer from poor loading

capacity and retention due to low surface area.

To maintain retention and capacity similar to HPLC, UPLC must use novel porous

particles that can with stand high pressures. Polymeric columns can overcome pH

limitations, but they have their own issues,including low efficiencies and limited

capacities. In Figure 2.1.1.F1, Van Deemter plot demonstrates that as the particle size

decreases less than 2 μm there is a significant gain in the efficiency, and this condition is

32

well maintained even if there is an increase in flow rate or linear velocities.This plot

indicates that the usable flow range which shows good efficiency is much greater for

smaller particles as compared to the larger particles [83, 84]. UPLC required porous

particles, which can withstand the high pressure in order to maintain their retention and

capacity similar to that of HPLC [85, 86].

Silica particles possess good mechanical strength but their application was limited

by narrow pH application range and generally exhibit tailing during analysis of basic

analytes [87]. In 2000, the first generation hybrid chemistry utilizes the classical sol-gel

synthesis method to create durable columns that incorporated carbon in the form of

methyl groups [88-91]. These columns exhibit several advantages such as mechanical

strength, high efficiency and are operative over an extended pH range.

2.2.1 System Selectivity Retentivity efficiency of UPLC

Efficiency is the primary separation parameter on the UPLC since it relies on the

same selectivity and sensitivity as HPLC. As shown in the figure 2.1.1.F1 samller

particles provide not only efficiency but also the ability to work at increased linear velocity

without a loss of efficiency providing both resolution and speed. In the fundamental

resolution Rs equation

Rs = N

4 𝑎−1

𝑎𝛼

𝐾

𝐾+1

Resolution is proportional to the square root of N. But since N is inversely proportional to

the particle size(dp).

N 1

dp

As the particle size is lowered by a factor of three, from, for example, 5 mm (HPLC scale)

to 1.7 m (UPLC scale), N is increased by three and resolution by the square root of

three or 1.7.N is also inversely proportional to the square root of the peak width. Peak

hight is inversely proportional to the peak width. As the particle size decreases to

increase N and subsequently Rs an increase in sensitivity is obtained, since narrower

33

peaks are taller peaks. Narrower peaks also mean more peak capacity per unit time in

gradient seperations desirable for many applciaitons.

N 1

W2

Still another equation comes into paly when migrating towards smaller particles.

Fopt 1

dp

This relationship also is revealed from the Van deemter plot. As particle size

decreases the optimum flow to increase maximum N increases. When analysis is the

primary objective, efficiency is proportional to column length and inversely proportional to

the particle size. Using a flow rate three times higher due to the smaller particles and

shortening the column by one third(again due to smaller particles) the separation is

completed by 1/9 the time while maintaining resolution. UPLC provide improved

resolution, speed and sentivity with no compromises. UPLC is suitable for

chromatographic applications for developing appropriate new methods and improving

existing methods.

2.2.2 Bridged ethane hybrid (BEH) technology

In the year 2000, a first generation hybrid chemistry that took advantage of the

best of both the silica and polymeric column world was introduced [92]. In order to

provide the kind of enhanced mechanical stability required for UPLC, a second

generation bridged ethane hybrid (BEH) technology was developed [93-95]. This

technology increases the mechanical stability of 1.7 μm particles by bridging the methyl

groups in the silica matrix as shown in Figure 2.2.1.F2. These small particles derive their

enhanced mechanical stability by bridging the methyl groups in the silica matrix.

Requirements include a smoother interior surface of the column hardware, and re-

designing the end frits to retain the small particles and resist clogging. In addition, at high

pressures, frictional heating of the mobile phase can be quite significant and must be

considered[96]. With column diameters typically used in HPLC (3.0 to 4.6 mm), a

consequence of frictional heating is the loss of performance due to temperature induced

34

non uniform flow.To minimize the effects of frictional heating, smaller diameter columns

(1–2.1 mm) are typically used for UPLC [97,98].

Instrument technology also had to keep pace to truly take advantage of the

increased speed,superior resolution, and sensitivity afforded by smaller particles.One-of-

a-kind systems,capable of delivering the pressures required to realize the potential of

UPLC have been reported in the literature and elsewhere [99–101]. By making use of

the smaller particles,the speed of analysis and peak capacity i.e.,number of peaks

resolved per unit time, can be prolonged to the maximum values and these values are

much better than the values achieved earlier by HPLC.

In early 2004(Figure 2.5.1), the first commercially available UPLC system that

embodied these requirements was described for the separation of various

pharmaceutical related small organic molecules, proteins,and peptides [102–104].

Furthermore to improve the efficiency of ultra performance chromatography

elevated temperature range should be employed, which will allow high flow rate of mobile

phase by reducing its viscosity and thus it will significantly reduce back pressure [105-

109].

Earlier, monolithic columns were introduced exclusively for organic-polymer-based

materials [110-112] including compressed soft gels, while short columns (about 1 mm;

monolithic disks) were introduced for rapid separation of proteins.These columns have

polymerized porous support structure that provides lower flow resistance than

conventional particle packed columns [113-117]. Using UPLC, it is now possible to take

full advantage of chromatographic principles to run separations using shorter columns,

and/or higher flow rates for increased speed, with superior resolution and sensitivity.

2.3 Fast and Rapid resolution LC(RRLC/UPLC) parameters

A major advantage of UPLC over conventional UPLC is its capability to increase

separation speed and/or efficiency. In comparison to traditional UPLC, UPLC can

achieve 5-to 10-fold faster separations while maintaining or increasing peak

resolution,thereby resulting inhigher throughput.UPLC enables high resolution

35

applications resulting in significant advantages in analytical performance over traditional

HPLC.

Due to sharper and more concentrated peaks, UPLC can often improve detection

sensitivity. All these analytical procedures can be improved by using RRLC, which can

speed up the analysis and, combined with time-of-flight, provide accurate mass

information in the initial phase of the investigation. UPLC can provide up to 20 times

faster analysis and 60% higher resolution than conventional High-performance liquid

chromatography (HPLC). This means that RRLC can process more than 2,000 samples

a day on a single system, in contrast to 100 samples per day with conventional LC.

UPLC is a technique which comprizes the above mentioned features and stands better

than HPLC in many ways as it shows better chromatographic resolution,performs more

sensitive analysis, consumes less time, reduces solvent consumption [118-121] and has

high analysis speed [122-123].

2.4 UPLC-Instrumentation

UPLC take advantage of technical strides made in particle chemistry

performance,system optimisation,detector design,data processing and control.When

taken together,these achievements have created a step function improvement in

chromatographic performance. Defined as UPLC this new category of separation science

retains the practicality and principles of HPLC while increasing the overall attributes of

speed, sensitivity and resolution.

The basic instrument in UPLC technique had to keep its tempo up in order to take

advantage of the enhanced speed, superior resolution and greater sensitivity provided by

small particles. A design with advanced technology in the pump, autosampler, detector,

data system, and service diagnostics was required to fulfil the purpose. The basic

instrumentation of UPLC is discussed below.

2.4.1 Pumping devices

An ideal pump for UPLC has a capacity of delivering solvent at higher pressure

around 15000 psi for the optimum flow rate with maximum efficiency across 15 cm long

column packed with 1.7 μm particles [124, 125]. UPLC uses two serial pumps with

36

pressure limit of 1000 bar [126, 127] and have inbuilt solvent selector valves, which have

the capability to choose the accurate solvent ratio from up to four solvents [72].

2.4.F1 Types of Liquid Chromatography and development over the years.

37

2.4.F2 Working technology of HPLC /UPLC

2.4.2 Sample injector manager

The UPLC system with its flow through-needle design sample manager is shown

in figure 2.5.2.F1. When an injection is initiated, the inject valve diverts flow from the

needle in order to collect sample from the vial as shown in 2.5.2.F1. The needle is

pushed against the internal sealing surface of the port and the injection valve turns and

the sample is pushed down to the injection port. After injecting the sample, the needle is

washed for a specified amount of time to minimize sample carryover.Beside this, there

are also direct injections for biological substances mentioned in the literature [129,130].

38

2.4.3 Detectors

Two tuneable UV-visible photodiode array detectors namely PDA and PDA eλ

detectors are generally used for the routine analysis and method development, which

have the power of detecting and quantifying trace impurities.The data rates of these

detectors are up to 80 Hz having low noise specification i.e., 10 Au with wide range of

spectra analysis up to 500 nm (PDA detector)and 800 nm (eλ detector).

2.4.3.F1.The injection sequences of UPLC in both load and inject phase

39

2.4.3.F2 UPLC column chemistries

2.4.4 UPLC Cloumns

The UPLC columns are made up of small particles having size less than 2 μm.

The role played by small particle size in UPLC technique has been mentioned above.The

particles are bonded in matrix as the bonded stationary phase is required for providing

both retention and selectivity. Four bonded stationary phase (2.5.2.F2) columns

manufactured by Waters are available in the market,which can be used by UPLC

technique [131-132]. BEH C18 and C8 columns and These are straight alkyl chain, most

preferred UPLC columns as they can be used over wide pH range.

O

O

SiO

O

O

SiO

CH3

CH3

SiO OHN

O

O

O

SiO

B E H C18 Columns

B E H C8 Columns

B E H Shield RP 18 Columns

B E H Phenyl Columns

40

2.4.5 A general guide for choosing chromatography mode of separation by UPLC

The C18 column used in the UPLC have only 20-30% of the surface of silica

particles bonded with C18 phase and the remaining will be in the form of free

silanols.These silanols are acidic at moderate pH values.The cause of peak tailing in

UPLC is the secondary retention that occurs when an ion-exchange interaction takes

place between a positively charged analytes (basics compounds) and an acidic silanols

on the surface of silica particles of packed stationary phase (2.4.5.F1).

2.4.5.F1.The secondary ion exchange interaction between silanols of silica and

basic analytes.

41

2.5 UPLC and Applications

Before the introduction of this technique,chromatographers were compromising

with the factors like low speed and resolution. The UPLC technique has been successfully

applied to pharmaceutical analysis of numerous drugs, a few of them are enlisted in Table

2.5.T1 [133-139], with the improvement in the factors like retention time and mobile phase

consumption. The chromatographic separations of all the present drug candidates were

achieved on shorter runtimes when compared to HPLC. The applications of this technique

are discussed below. In 2004, the first commercial UPLC was available that fulfilled all the

requirements needed for the separation of various pharmaceutical products [140].

42

2.5.T1 Parameters comparison of HPLC and UPLC

S.No. Parameter HPLC UPLC

Difference

1 Particle size 3-5 microns < 2 microns Reduction in analysis

time

2 Mobile phase

flow rate

More

consumption

Less

consumption

Lesser Mobile phase

consumption

3 Injection volume 5l(Std in 100%

methanol)

2l(Std in 100%

methanol)

Small traces of sample

in UPLC

4 Column ZORBAX

C18,C8

BEH C18 and

C8

Can withstand with

high pressures and

have high mechanical

stability and efficiency

5 Column

dimension

150x3-5

microns

150x2 microns Higher resolution

6 Column

temperature

30-350C 60-650C Lower solvent

viscosity, increased

selectivity and

increase in mass

transfer rate

7 Maximum back

pressure

35-40 Mpa 104 Mpa Speration is faster in

UPLC

43

2.5.1 Identification of metabolites by UPLC

When a new chemical entity (NCE) reaches the development stage, identification

of its metabolites becomes a perpetual process.The detection of all circulating

metabolites of a candidate drug is necessary.The identification of major metabolites is

done by performing in vitro discovery studies.The weak spots of metabolites of drug

candidate molecules are recognized and hence protected by altering the compound

structure. Plumb et al. have explored the application of UPLC-MS for analysis of

metabolites of candidate drug [141,142].Their studies revealed that higher resolution of

UPLC in terms of specificity and spectral quality diminishes the risk of missing any

detection of potentially important metabolites.

2.5.2 UPLC-application in Bioanalytical studies

The detection of drug in biological sample is very important to study the

pharmacokinetics,toxicity and bioequivalence of drug.Blood, plasma and urine can be

used as biological matrices which consist of sample drug in them. Previously LC-MS was

used for detecting drug in these biological matrices.But now a days UPLC-MS is used

as it has some advantages over LC-MS such as UPLC-MS provides unprecedented

performance and compliance support.It has excellent chromatographic resolution and

sensitivity.UPLC can accommodate more number of samples than HPLC so sample

throughput is enhanced.It gives not only high quality results [143].

2.5.3 Detection of impurities using UPLC

The detection of impurities in raw material as well as in final product is the most

vital phase of the drug development process.Earlier studies have accounted for excellent

detection of impurities by HPLC as it has sufficient resolution for the determination of the

lowest level of impurities with good reproducibility results, but due to the presence of

excipients, there is prolonged HPLC analysis so it becomes necessary to perform several

analytical runs to get the required data. This curb can be overcome by using UPLC

technique as it offers exact required data and is operational at alternate low and high

collision energies.The fast change of collision energy produces both precursor and

product ions of all analytes present in the sample, which allows rapid identification and

profiling of impurities. Lippert et al. [144] pointed that in some cases, the impurity and

44

compound are of the same molecular weight and have similar structures so they could

not be differentiated by MS or LC/MS necessitating higher chromatographic resolution

which can be provided by UPLC.

2.5.4 Identification of Matabolites and bioequivalence studies by UPLC

A wide variety of manufactured and natural chemicals such as drugs, industrial

chemicals, pollutants, pesticides, pyrolytic products from cooked food, secondary plant

metabolites (polyphenols,flavonoides, glycosides, terpenes, steroids, alkaloids,

antibiotics) and toxins from bacteria, molds, plants and animals can influence the vital

functions of an organism and they represent serious risks for its homeostasis, integrity

and health. These categories of chemical compounds are usually denoted as

xenobiotics, foreign compounds or exogenous compounds. The hyphenation approach to

the bioanalysis(the combination of HPLC with photodiode-array UV, fluorescence,

polarimetry, NMR and MS as detection techniques) offers the most comprehensive

analytical information involving the chromatographic behavior of individual analytesunder

study and the information from individual detectors.

2.6 Mass spectrometry

Mass spectrometry, with its reproducibility, specificity and sensitivity is an

indispensable tool in the field of structure elucidation and impurity profiling of

pharmaceutical products [145]. In majority of cases, the data obtained from mass spectra

(with other chemical information) is sufficient to propose a tentative structure of the

impurity. By the application of mass spectrometry after chromatographic separation, the

isolated compounds of identical structures can also be analyzed. This technique makes it

possible to differentiate between stereoisomers and positional isomers, which have

different fragmentation pathways.For example, the structure elucidation of impurities of

allylestrenol (1) after isolation by preparative HPLC followed by EI high-resolution

measurements was carried out [146]. Both the unknown impurities isolated by

preparative HPLC have molecular weights of 289, two mass units less than molecular

weight of allylestrenol indicating an additional double bond in the impurities (II,III).This

assumption was confirmed by different fragmentation patterns relating to the two

different structure.

45

OHOH OH

Allylestrenol Impurity-II Impurity-III

2.6.1 Principles of Liquid Chromatography/Mass Spectrometry

LC–MS/MS is an increasingly important tool in therapeutic drug monitoring

as it offers increased sensitivity and specificity compared to other methods, and

may be the only viable method for quantifying drugs without natural chromophores or

fluorophores. LC–MS/MS is a more involved technique than automated immunoassays,

but technological advances such as the development of pipetting robots and online solid

phase extraction mean that LC–MS/MS is becoming an attractive and convenient method

for therapeutic drug monitoring in pharmaceutical analytical laboratories.

Mass spectrometers also generate three-dimensional data. In addition to signal

strength, they generate mass spectral data that can provide valuable information about

the molecular weight, structure, identity, quantity, and purity of a sample.For most

compounds, a mass spectrometer is more sensitive and far more specific than all other

LC detectors. It can analyze compounds that lack a suitable chromophore.It can also

identify components in unresolved chromatographic peaks, reducing the need for perfect

chromatography. Mass spectral data complements data from other LC detectors. The

two orthogonal sets of data can be used to confidently identify, confirm, and quantify

compounds. Some mass spectrometers have the ability to perform multiple steps of

mass spectrometry on a single sample.They can generate a mass spectrum, select a

specific ion from that spectrum, fragment the ion, and generate another mass

spectrum;repeating the entire cycle many times.Such mass spectrometers can literally

deconstruct a complex molecule piece by piece until its structure is determined.

46

2.7 LC/MS Instrumentation

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 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.

The most common detectors in use for triple quadrupole instruments. In general

terms instrument performance has improved significantly over the past 10 years and

there have been gains of up to 10-fold in instrument sensitivity. The most common

ionization techniques are electrospray ionization (ESI) and atmospheric chemical

ionization (APCI). ESI is an efficient method for converting analyte in solution into gas

phase ions suitable for analysis by the processes of desolvation and ion desorption.

The analyte ions are then mechanically and electrostatically separated from neutral

molecules.Common atmospheric pressure ionization techniques are Electrospray

ionization (ESI), Atmospheric pressure chemical ionization (APCI) and Atmospheric

pressure photoionization (APPI).

The triple quadrupole instrument consists of two mass analysis quadruples

separated by a collision cell. A variety of internal standards has been used including

structural analogues of the analyte or deuterated compounds. Analogue internal

standards are often considered inferior to deuterated compounds but were often the only

cost effective and practical alternative [147-148].

Much of the advancement in LC/MS over the last ten years has been in the

development of ion sources and techniques that ionize the analyte molecules and

separate the resulting ions from the mobile phase.

47

2.8 Mass Analyzers

Although in theory any type of mass analyzer could be used for LC/MS, four types

as follows. Quadrupole, Time-of-flight, Ion trap and Fourier transform-ion cyclotron

resonance (FT-ICR or FT-MS) are used most often. Each has advantages and

disadvantages depending on the requirements of a particular analysis.

2.8.1 Quadrupole

A quadrupole mass analyzer consists of four parallel rods arranged in a

square.The analyte ions are directed down the center of the square. Voltages applied to

the rods generate electromagnetic fields. These fields determine which mass-to-charge

ratio of ions can pass through the filter at a given time. Quadrupoles tend to be the

simplest and least expensive mass analyzers. Quadrupole mass analyzers can operate

in two modes i.e Scanning (scan) mode Selected ion monitoring (SIM) mode In scan

mode, the mass analyzer monitors a range of mass-to-charge ratios.

2.8.2 Time-of-flight

In a time-of-flight (TOF) mass analyzer, a uniform electromagnetic force is applied

to all ions at the same time, causing them to accelerate down a flight tube. Lighter ions

travel faster and arrive at the detector first, so the mass-to-charge ratios of the ions are

determined by their arrival times.Time-offlight mass analyzers have a wide mass range

and can be very accurate in their mass measurements.

2.8.3 Ion trap

An ion trap mass analyzer consists of a circular ring electrode plus two end caps

that together form a chamber. Ions entering the chamber are “trapped” there by

electromagnetic fields. Another field can be applied to selectively eject ions from he trap.

Ion traps have the advantage of being able to perform multiple stages of mass

spectrometry without additional mass analyzers.

2.8.4 Fourier transform-ion cyclotron resonance (FT-ICR)

Ions entering a chamber are trapped in circular orbits by powerful electrical and

magnetic fields. When excited by a radio-frequency (RF) electrical field, the ions

generate a timedependent current. This current is converted by Fourier transform into

48

orbital frequencies of the ions which correspond to their mass-tocharge ratios. Like ion

traps, FT-ICR mass analyzers can perform multiple stages of mass spectrometry without

additional mass analyzers.They also have a wide mass range and excellent mass

resolution. They are,however, the most expensive of the mass analyzers.

2.8.5 Collision-Induced Dissociation and Multiple-Stage MS

The atmospheric pressure ionization techniques discussed are all relatively “soft”

techniques. They generate primarily,

• Molecular ions M+ or M–

• Protonated molecules [M + H]+

• Simple adduct ions [M + Na]+

• Ions representing simple loss of a water [M + H – H2O]+

The resulting molecular weight information is very valuable, but complementary

structural information is often needed. To obtain structural information, analyte ions are

fragmented by colliding them with neutral molecules in a process known as collision

induced dissociation (CID) or collisional activated dissociation (CAD). Voltages are

applied to the analyte ions to add energy to the collisions and create more fragmentation.

2.8.6 CID in single-stage MS

CID is most often associated with multistage mass spectrometers where it takes

place between each stage of MS filtering, but CID can also be accomplished in single-

stage quadrupole or time-of-flight mass spectrometers. In single-stage mass

spectrometers, CID takes place in the ion source and is thus sometimes called source

CID or in-source CID. Analyte (precursor) ions are accelerated and collide with residual

neutral molecules to yield fragments called product ions.

2.8.7 CID and multiple-stage MS

Multiple-stage MS (also called tandem MS or MS/MS or MSn) is a powerful way to

obtain structural information.In triple-quadrupole or quadrupole/quadrupole/time-of-flight

instruments, the first quadrupole is used to select the precursor ion. CID takes place in

the second stage (quadrupole or octopole), which is called the collision cell. The third

stage (quadrupole or TOF) then generates a spectrum of the resulting product ions. It

49

can also perform selected ion monitoring of only a few product ions when quantitating

target compounds.

2.9 Adapting LC Methods

Early LC/MS systems were limited by fundamental issues like the amount of LC

eluent the mass spectrometer could accept. Significant changes to LC methods were

often required to adapt them to MS detectors. Modern LC/MS systems are more

versatile. Changes to LC methods required for modern LC/MS systems generally involve

changes in sample preparation and solution chemistry to ensure adequate analyte

concentration, maximize ionization through careful selection of solvents and buffers and

Minimize the presence of compounds that compete for ionization or suppress signal

through gas-phase reactions.

2.9.1 Molecular Weight Determination

One fundamental application of LC/MS is the determination of molecular

weights.This information is key to determining identity. The hyphenated techniques such

as GC-MS and LC-MS are widely used for Characterization of complex mixtures, several

strategies for drug impurity Profiling have been presented in the literature [148]. Once a

method was developed using UV detection for determination of impurities of interest, the

same could be Transferred (with some modifications) to LC-MS. However, a number of

factors should be kept in mind when setting up an efficient LC-MS method for regular

operation [149]. In the valuation of drug impurities with poor or no chromophores, the

usage of LC-MS is the most suitable method to study impurity profiles.Buffersalts like

phosphates, citrates and borates, which are non-volatile, should be avoided in LC-

MS.The most suitable buffer for LC-MS analysis is ammonium acetate [150]. Several

other techniques such as LC-MS-MS,infusion MS-MS high resolution MS etc.,are also

used in the impurity profiling studies of drugs [151-174].

2.9.2 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.

50

2.9.2.T1 Details of LC/MS methods for the studies structural identification of

degradation impurities

Compound Sample

volume

Sample

preparation

Ionisation

mode

Column Mass

Range

m/z

Equipment

Degradant

impurity in

Gefitinib

200 l Liquid Liquid

extraction

ESI+ C18 1800-

1200

Agilent EV series

liquid

chromatog

raphy system

triple quadrapole

mass

spectrometer

equipped with

an Electro

Spray Ion source

Imatinib

degradation

Impurities

20 l Liquid Liquid

extraction

ESI+ C18 1800-

1200

Agilent EV series

liquid

chromatography

system triple

quadrapole mass

spectrometer

equipped with an

Electro Spray Ion

source

51

Compound Sample

volume

Sample

preparation

Ionisation

mode

Colum

n

Mass

Range

m/z

Equipment

Bortezomib

degradant

product

20 l Liquid Liquid

extraction

ESI+ C18 105-

1200

Agilent-

2010EV (LC-MS)

series triple

Quadra pole

liquid

chromatography

system

Eplerenone

degradation

product

5 l Liquid Liquid

extraction

ESI+ C18 105-

1200

Agilent 2010 EV

series liquid

chromatography

system coupled

with triple

quadrapole mass

spectrometer

Nilotinib HCl

degradation

product

5 l Liquid Liquid

extraction

ESI+ C18 80-1200 Waters–Micro

mass, Quattro

micro-API-ESCI

liquid

chromatography

mass

spectrometer.

2.10 NMR spectroscopy

NMR is the most widely used technique for structural elucidation of synthesized

organic molecules. It plays an important role in identifying even low level impurities in

bulk drug materials with or without preparative charactrization of drug impurities,

52

modern NMR offers various ranges of experiments [175]. Structural elucidation of

impurities in drug materials mostly involves the use of 1H and 13C experiments.The

information obtained from these experiments is sufficient to ascertain the structure of an

unknown impurity in a drug material. Two-dimensional experiments such as correlation

spectroscopy (COSY),heteronuclear multiple bond correlation (HMBC) etc., are very

useful.

2.10.1 Application of LC-NMR

The introduction of NMR probes especially for on-line coupling to HPLC [176]

greatly reduced the need for preparative isolation of impurities.Stop –flow [177] and on-

flow techniques are used to detect the analytes of interest. HPLC analysis is carried out

in reversed-phase mode using D2O/ buffer- acetonitrile based eluents with an injection

volume of 50-100UL. The major problem in using LC-NMR for characterization of

impurities of interest is the lack of sensitivity.Sensitivity is not a major issue if the

impurity of interest is present in large amounts (5-10%). Applications describing the

use of LC-NMR have been reported in literature in the field of pharmaceuticals,

natural products, environmental samples and drug metabolites [178].

2.13.2 Other techniques

UV-VIS spectroscopy for identification and determination of impurities in drug

substances with out chromatographic separation is of very little importance.Nevertheless

for determining certain impurities in bulk drugs, UV-vis spectrometric method is

recommended in pharmacopoeias. IR spectroscopy is generally used to ascertain the

functional group present in the impurities of interest after chromatographic separation.

Capillary electrophoresis (CE) has its ability to provide a different selectivity to

characterize the impurity content and profile in drug substances [179]. Future

approaches for studying impurity profiling of drugs may be coupling of LC-NMR-

MS,CE-NMR,SFC-NMR etc. Thus,each technique has its own unique identity and

importance in the impurity profile of drug materials.