Principle & Applications of Liquid Chromatography

with Mass Spectrometry (LC-MS)

 Principle of MS
 Bombardment of sample in vapor phase with
high energy electron beam to converted into
positively charged ion species which are
separated on the basis of their mass to charge
ratio.
 Contents
 Introduction
 Elements of LC-MS
 Liquid Chromatography
 Interfaces
 Mass spectrometry
 Applications
 References
Hyphen is a great fun
Hyphen is a line
which is used to join
the words of different design
Chromatography and spectroscopy
Hyphened to give sensitivity and selectivity
GC-MS, LC-MS and GC or LC -MS-MS
 Introduction
 History : Starts in early 1970’s , in several laboratories the
possibility of online LCMS were investigated .
 LC-MS : Hyphenated technique
 Basic concepts :
LC –To resolve complex mixture of
compounds
Interface – To transport & desolvation of
sample into ion source of mass
analyser.
MS – To ionise & analyse individual
resolved components , on the basis of
their m/z ratio.
 Liquid Chromatography

 Principle – Partitioning of analyte between mobile

(liquid) & stationary (solid) phase.
 RPHPLC is most commonly used in LC-MS.
 Column is heart of LC.
Length : 5 – 25 cm
ID : 3 – 5 mm
Particle size : 3 or 5 micron
Material : Silica + long chain hydrocarbon.
Liquid flow rate : 0.1 – 1.5 ml/min
Column efficiency : 40,000 – 70,000 plates/meter
 Mass Spectroscopy
 Mass spectrometer produces a beam of ions, separates
them according to m/z ratio of ions.
 The "heart" of the mass spectrometer is
an analyzer.
 Analyzer uses electrical or magnetic fields, or
combination of both, to move the ions
from the region where they are produced
to a detector .
 Need of technique
 Drawbacks :
HPLC – On its own provide ambiguous
confirmation of identity of analyte.
MS – Limitation in handling mixture of
compounds.
 LC-MS – Benefited mutually, from high resolution
separation capability of LC & high sensitive, structure
specific detection capacity of mass.
 Elements of LC-MS

Working of an LC-MS system :

 Interfacing problem
 Problem in coupling LC-MS
- Enormous mismatch between large solvent volumes from
LC & vacuum requirement of MS So for solving this
problem interface is used.
 Problem while interfacing
 Necessity to remove solvent.

 Difficulty in vaporising nonvolatile solvent.

 Degradation of thermally labile compound.

 Interfaces Used in LC-MS
 Moving belt
 Thermospray interface
 Particle beam interface
 Atmospheric pressure ionisation (API)
- with ESI
- with APCI
 Capillary inlet
 Continuous flow – fast atom bombardment
 Direct liquid introduction
 Moving belt interface

 Important reason for it’s success is compatibility with wide

range of chromatographic conditions ; also EI, CI & FAB
methods employed easily.
 Limitations :1) Complex mechanical device
2) Renewal of belt & belt memory are
troublesome.
Thermospray interface
 Particle beam interface

 Column effluent is nebulised pneumatically into near

atmospheric pressure desolvation chamber
 Role of momentum separator
Momentum separator
 Electrospray Ionisation

 Unique feature – Desolvation & ionization process occur together in the

ion source at atmospheric pressure .
 Nebulisation is done by using strong electric potential difference of 3kv &
N2 flow.
 Nebulisation occur at only flow rate of below 10 microlitre/min.
 Electro Spray Ionization (ESI)

 3 stages of ESI :
- Formation of charged droplet
- Solvent evaporation & droplet fission
- Formation of gas phase ion
 Atmospheric Pressure Chemical
Ionisation (APCI)

 It’s based on solvent mediated CI by ion-molecule reactions

initiated by electron produced in the Corona discharge needle.
 Solvent vapor act as reagent gas.
 APCI

30
 Mass Analyser
 Linear quadrupole mass analyser
 Quadrupole – ion trap mass analyser
 Time of flight
 Quadrupole - time of flight
 FT –ICR - MS
 Linear Quadrupole Mass Analyser

 Mass scanning is done by varying RF & DC frequencies &

keeping ratio constant .
 Advantages :- easy in use.
- electric voltage easy & rapidly varied.
 Quadrupole – Ion Trap Mass
Analyser

 Ions are introduced to the trap in a pulsed mode & stabilised

by the He gas
 Function of ion trap is to retain unsorted ion temporarily then
they are released to detector sequentially scanning the electric
field.
 Applications
 Quantitative bioanalysis
 Clinical application – TDM
 Pharmacokinetic study
 Drug metabolism
 Proteomics
 Analysis of pestisides
 Drug discovery & drug develpoment
 Analysis of steroids
 Food safety analysis
 Stability testing & impurity profiling
 Quantitative Bioanalysis
 Methods for quantitative measurement of a drug,
drug metabolites or chemicals in biological fluids.
 Determination of :
 Resperine (by ESI or APCI )

 Risperidone & 9-OH Risperidone ( by ESI )

 Lovastatin , Simvaststin .
 Clinical Application - TDM
 The ability to accurately measure drug levels in whole
blood is vital for effective quantification of drugs.
 With the help of MS-MS its easy to distinguish parent
drug molecule from metabolites by use of molecular mass .
 Determination of :
 Sirolimus , Tacrilimus

 Amphetamines (by LC-APCI-MS)

 LSD & Cocaine (by LC-ESI-MS)

 Pharmacokinetic Studies
 These studies tell us how quickly a drug will be
cleared from the hepatic blood flow, and organs of
the [Link] is done by detection of the drug in the
body matrices such as blood & urine.
 With the help of MS-MS we can program the detector
to select out certain ions to fragment .
 eg. Cefixime, Adefovir, Zolmitriptan
 Drug Metabolism
 Easier to characterize metabolites (previously
difficult task)
 Its done by comparing directly the spectra

of the drug with those of it’s metabolites.

 Common biotransformation (eg. oxidaton,

reduction,hydrolysis etc.) detected from molecular

mass of metabolite.
 eg. Zolpidem (by ESI )

Quercetin (by ESI)

 Proteomics
 Its the large-scale study of protein, particularly their
structures and functions .
 Important technique :
 peptide mass fingerprinting(by nano ESI-MS)

 Peptide sequence analysis – this is not possible by

MS, needs MS-MS.

 accurate mass,affinity or sequence tags-

(by FT-ICR-MS)
 Analysis of Pestisides
Suitability of LC-MS ionisation mode for various class
of pestisides
 Drug Discovery & Drug
Development
 Drug development steps : drug discovery, preclinical
development , clinical development , manufacturing .
 LC-MS needed in every step,
 It help in checking proper progress in the synthesis of
new chemical entity.
 Analysis of Steroids
 Doping analysis in sports
 Detection of Stanozolol , methandrostenolone in

equine & human urine by LC-ESI-MS.

 Detection of testosterone by LC-ESI-MS in

Human athletes .
 Food Safety Analysis
 LC-MS imp in quantification & confirmation of
identity of food contaminants (eg. Sea food)
 LC-MS of antibiotic & antibacterial compound:
 Stability Testing & Impurity
Profiling
 In stability testing : to determine the identity, chemical
structure of the API and quantification levels as well as
presence of degradants, excipients and impurities .
eg. determination of degradants in
-.Trimethoprim - Artesunate
 In impurity profiling : detection of minor components
in presence of major components.
eg. In identification of
Polar impurity in mosapride
 LC/MS in Clinical Labs
 Neonatal screening
 Specialist triple quard application
 Therapeutic drug monitoring
 To replace immuno assays
 Drugs of abuse
 LC/MS in the Environmental Field
 Water
 Identification and quantization of pollutants
 Pesticides, antibiotics
 Food
 chemical contaminants
 antibiotics
 natural toxins
 Animal feeds
 contaminants, illegal substances
 LC/MS in Other Industries
 Organometallics
 structure
 Detergents
 Quality Control, competitors products
 Polymers
 molecular weight, structure
 References
 Frank Settle. Handbook of Instrumental Techniques for Analytical
Chemistry 1st ed.647-659.
 Willard, Merritt, Dean, Settle. Instrumental Methods of Analysis .7th ed.

618-620.
 Skoog, Holler,Nieman. Principles of Instrumental

Analysis,5th ed.1992; 501-511:528-531:738-739.

 Christian Garry. Analytical Chemistry. 6th ed. 608-610

 Wilfried M. A. Niessen . Liquid Chromatography – Mass Spectroscopy .

3rd ed. 80-120 , 164- 180 .


J. Flarakos*, W. Luoa, M. Aman, D. Svinarov , Quantification of
risperidone and 9-hydroxyrisperidone in plasmaand saliva from adult and
pediatric patients by liquidchromatography–mass spectrometry .Journal of
Chromatography A, 1026 (2004) 175–183
 References
 N. Lindeg°ardh a,b*, A.M. Dondorp a,b, P.
Singhasivanon a, Validation and application of a liquid
chromatographic–mass spectrometric method for
determination of artesunate in pharmaceutical samples.
Journal of Pharmaceutical and Biomedical Analysis 45
(2007) 149–153.
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