LCMS

LC-MS SOPHISTICATED INSTRUMENTATION

1. Principle & Applications of Liquid ChromatographyPrinciple & Applications of Liquid Chromatography
with Mass Spectrometry (LC-MS)with Mass Spectrometry (LC-MS)
PRESENTED BY:PRESENTED BY:
SANDHYA TALLASANDHYA TALLA
M.PHARM (PHARMACOLOGY)M.PHARM (PHARMACOLOGY)

2. Principle of MSPrinciple of MS

Bombardment of sample in vapor phase with Bombardment of sample in vapor phase with
high energy electron beam to converted into high energy electron beam to converted into
positively charged ion species which are positively charged ion species which are
separated on the basis of their mass to charge separated on the basis of their mass to charge
ratio.ratio.

14. ContentsContents

IntroductionIntroduction

Elements of LC-MSElements of LC-MS

Liquid ChromatographyLiquid Chromatography

InterfacesInterfaces

Mass spectrometryMass spectrometry

ApplicationsApplications

ReferencesReferences

15. Hyphen is a great funHyphen is a great fun
Hyphen is a lineHyphen is a line
which is used to joinwhich is used to join
the words of different designthe words of different design
Chromatography and spectroscopyChromatography and spectroscopy
Hyphened to give sensitivity and selectivityHyphened to give sensitivity and selectivity
GC-MS, LC-MS and GC or LC -MS-MSGC-MS, LC-MS and GC or LC -MS-MS

16. IntroductionIntroduction

History : Starts in early 1970’History : Starts in early 1970’s , in several laboratories the s , in several laboratories the
possibility of online LCMS were investigated .possibility of online LCMS were investigated .

LC-MS : Hyphenated techniqueLC-MS : Hyphenated technique

Basic concepts :Basic concepts :
LCLC –To resolve complex mixture of –To resolve complex mixture of
compoundscompounds
InterfaceInterface – To transport & desolvation of – To transport & desolvation of
sample into ion source of mass sample into ion source of mass
analyser.analyser.
MSMS – To ionise & analyse individual – To ionise & analyse individual
resolved components , on the basis of resolved components , on the basis of
their m/z ratio. their m/z ratio.

17. Liquid ChromatographyLiquid Chromatography

Principle – Partitioning of analyte between mobile Principle – Partitioning of analyte between mobile
(liquid) & stationary (solid) phase.(liquid) & stationary (solid) phase.

RPHPLC is most commonly used in LC-MS.RPHPLC is most commonly used in LC-MS.

Column is heart of LC. Column is heart of LC.
Length : 5 – 25 cmLength : 5 – 25 cm
ID : 3 – 5 mmID : 3 – 5 mm
Particle size : 3 or 5 micron Particle size : 3 or 5 micron
Material : Silica + long chain hydrocarbon.Material : Silica + long chain hydrocarbon.
Liquid flow rate : 0.1 – 1.5 ml/minLiquid flow rate : 0.1 – 1.5 ml/min
Column efficiency : 40,000 – 70,000 plates/meterColumn efficiency : 40,000 – 70,000 plates/meter

18. Mass SpectroscopyMass Spectroscopy

Mass spectrometerMass spectrometer produces a beam of ions, separates produces a beam of ions, separates
them according to m/z ratio of ions.them according to m/z ratio of ions.

The "heart" of the mass spectrometer is The "heart" of the mass spectrometer is
an analyzer.an analyzer.

Analyzer uses electrical or magnetic fields, or Analyzer uses electrical or magnetic fields, or
combination of both, to move the ions combination of both, to move the ions
from the region where they are produced from the region where they are produced
to a detector . to a detector .

19. Need of techniqueNeed of technique

Drawbacks :Drawbacks :
HPLC – On its own provide ambiguous HPLC – On its own provide ambiguous
confirmation of identity of analyte.confirmation of identity of analyte.
MS – Limitation in handling mixture of MS – Limitation in handling mixture of
compounds.compounds.

LC-MS – Benefited mutually, from high resolution LC-MS – Benefited mutually, from high resolution
separation capability of LC & high sensitive, structure separation capability of LC & high sensitive, structure
specific detection capacity of mass. specific detection capacity of mass.

20. Elements of LC-MSElements of LC-MS
Working of an LC-MS systemWorking of an LC-MS system :

21. Interfacing problemInterfacing problem

Problem in coupling LC-MSProblem in coupling LC-MS
- Enormous mismatch between large solvent volumes from - Enormous mismatch between large solvent volumes from
LC & vacuum requirement of MS So for solving this LC & vacuum requirement of MS So for solving this
problem interface is used.problem interface is used.

Problem while interfacingProblem while interfacing

Necessity to remove solvent. Necessity to remove solvent.

Difficulty in vaporising nonvolatile solvent. Difficulty in vaporising nonvolatile solvent.

Degradation of thermally labile compound. Degradation of thermally labile compound.

22. Interfaces Used in LC-MSInterfaces Used in LC-MS

Moving beltMoving belt

Thermospray interfaceThermospray interface

Particle beam interfaceParticle beam interface

Atmospheric pressure ionisation (API)Atmospheric pressure ionisation (API)
- with ESI- with ESI
- with APCI- with APCI

Capillary inletCapillary inlet

Continuous flow – fast atom bombardmentContinuous flow – fast atom bombardment

Direct liquid introductionDirect liquid introduction

23. Moving belt interfaceMoving belt interface

Important reason for it’s success is compatibility with wide Important reason for it’s success is compatibility with wide
range of chromatographic conditions ; also EI, CI & FAB range of chromatographic conditions ; also EI, CI & FAB
methods employed easily.methods employed easily.

Limitations :1) Complex mechanical device Limitations :1) Complex mechanical device
2) Renewal of belt & belt memory are 2) Renewal of belt & belt memory are
troublesome. troublesome.

24. Thermospray interfaceThermospray interface

25. Particle beam interfaceParticle beam interface

Column effluent is nebulised pneumatically into near Column effluent is nebulised pneumatically into near
atmospheric pressure desolvation chamber atmospheric pressure desolvation chamber

Role of momentum separator Role of momentum separator

26. Momentum separatorMomentum separator

27. Electrospray IonisationElectrospray Ionisation

Unique feature – Desolvation & ionization process occur together in the Unique feature – Desolvation & ionization process occur together in the
ion source at atmospheric pressure .ion source at atmospheric pressure .

Nebulisation is done by using strong electric potential difference of 3kv & Nebulisation is done by using strong electric potential difference of 3kv &
NN22 flow. flow.

Nebulisation occur at only flow rate of below 10 microlitre/min.Nebulisation occur at only flow rate of below 10 microlitre/min.

28. Electro Spray Ionization (ESI)Electro Spray Ionization (ESI)

3 stages of ESI : 3 stages of ESI :
- Formation of charged droplet - Formation of charged droplet
- Solvent evaporation & droplet fission - Solvent evaporation & droplet fission
- Formation of gas phase ion - Formation of gas phase ion

29. Atmospheric Pressure Chemical Atmospheric Pressure Chemical
Ionisation (APCI)Ionisation (APCI)

It’s based on solvent mediated CI by ion-molecule reactions It’s based on solvent mediated CI by ion-molecule reactions
initiated by electron produced in the Corona discharge needle.initiated by electron produced in the Corona discharge needle.

Solvent vapor act as reagent gas.Solvent vapor act as reagent gas.

30. 3030
A
P
C
I

31. Mass AnalyserMass Analyser

Linear quadrupole mass analyserLinear quadrupole mass analyser

Quadrupole – ion trap mass analyserQuadrupole – ion trap mass analyser

Time of flight Time of flight

Quadrupole - time of flight Quadrupole - time of flight

FT –ICR - MSFT –ICR - MS

32. Linear Quadrupole Mass AnalyserLinear Quadrupole Mass Analyser

Mass scanning is done by varying RF & DC frequencies & Mass scanning is done by varying RF & DC frequencies &
keeping ratio constant .keeping ratio constant .

Advantages :- easy in use.Advantages :- easy in use.
- electric voltage easy & rapidly varied.- electric voltage easy & rapidly varied.

33. Quadrupole – Ion Trap Mass Quadrupole – Ion Trap Mass
AnalyserAnalyser

Ions are introduced to the trap in a pulsed mode & stabilised Ions are introduced to the trap in a pulsed mode & stabilised
by the He gas by the He gas

Function of ion trap is to retain unsorted ion temporarily then Function of ion trap is to retain unsorted ion temporarily then
they are released to detector sequentially scanning the electric they are released to detector sequentially scanning the electric
field.field.

34. ApplicationsApplications

Quantitative bioanalysisQuantitative bioanalysis

Clinical application – TDMClinical application – TDM

Pharmacokinetic studyPharmacokinetic study

Drug metabolismDrug metabolism

Proteomics Proteomics

Analysis of pestisidesAnalysis of pestisides

Drug discovery & drug develpomentDrug discovery & drug develpoment

Analysis of steroidsAnalysis of steroids

Food safety analysis Food safety analysis

Stability testing & impurity profilingStability testing & impurity profiling

35. Quantitative BioanalysisQuantitative Bioanalysis

Methods for quantitative measurement of a drug, Methods for quantitative measurement of a drug,
drug metabolites or chemicals in biological fluids.drug metabolites or chemicals in biological fluids.

Determination of : Determination of :

Resperine (by ESI or APCI )Resperine (by ESI or APCI )

Risperidone & 9-OH Risperidone ( by ESI ) Risperidone & 9-OH Risperidone ( by ESI )

Lovastatin , Simvaststin .Lovastatin , Simvaststin .

36. Clinical Application - TDMClinical Application - TDM

The ability to accurately measure drug levels in whole The ability to accurately measure drug levels in whole
blood is vital for effective quantification of drugs.blood is vital for effective quantification of drugs.

With the help of MS-MS its easy to distinguish parent With the help of MS-MS its easy to distinguish parent
drug molecule from metabolites by use of molecular drug molecule from metabolites by use of molecular
mass .mass .

Determination of :Determination of :

Sirolimus , TacrilimusSirolimus , Tacrilimus

Amphetamines (by LC-APCI-MS)Amphetamines (by LC-APCI-MS)

LSD & Cocaine (by LC-ESI-MS) LSD & Cocaine (by LC-ESI-MS)

37. Pharmacokinetic StudiesPharmacokinetic Studies

These studies tell us how quickly a drug will be These studies tell us how quickly a drug will be
cleared from the hepatic blood flow, and organs of cleared from the hepatic blood flow, and organs of
the body.This is done by detection of the drug in the the body.This is done by detection of the drug in the
body matrices such as blood & urine.body matrices such as blood & urine.

With the help of MS-MS we can program the detector With the help of MS-MS we can program the detector
to select out certain ions to fragment .to select out certain ions to fragment .

eg. Cefixime, Adefovir, Zolmitriptaneg. Cefixime, Adefovir, Zolmitriptan

38. Drug MetabolismDrug Metabolism

Easier to characterize metabolites (previously Easier to characterize metabolites (previously
difficult task)difficult task)

Its done by comparing directly the spectraIts done by comparing directly the spectra
of the drug with those of it’s metabolites.of the drug with those of it’s metabolites.

Common biotransformation (eg. oxidaton, Common biotransformation (eg. oxidaton,
reduction,hydrolysis etc.) detected from molecular reduction,hydrolysis etc.) detected from molecular
mass of metabolite. mass of metabolite.

eg. Zolpidem (by ESI ) eg. Zolpidem (by ESI )
Quercetin (by ESI) Quercetin (by ESI)

39. ProteomicsProteomics

Its the large-scale study of protein, particularly their Its the large-scale study of protein, particularly their
structures and functions .structures and functions .

Important technique :Important technique :

peptide mass fingerprinting(by nano ESI-MS)peptide mass fingerprinting(by nano ESI-MS)

Peptide sequence analysis – this is not possible by Peptide sequence analysis – this is not possible by
MS, needs MS-MS. MS, needs MS-MS.

accurate mass,affinity or sequence tags- accurate mass,affinity or sequence tags-
(by FT-ICR-MS)(by FT-ICR-MS)

40. Analysis of PestisidesAnalysis of Pestisides
Suitability of LC-MS ionisation mode for various class Suitability of LC-MS ionisation mode for various class
of pestisidesof pestisides

41. Drug Discovery & Drug Drug Discovery & Drug
DevelopmentDevelopment

Drug development steps : drug discovery, preclinical Drug development steps : drug discovery, preclinical
development , clinical development , manufacturing .development , clinical development , manufacturing .

LC-MS needed in every step, LC-MS needed in every step,

It help in checking proper progress in the synthesis of It help in checking proper progress in the synthesis of
new chemical entity.new chemical entity.

42. Analysis of SteroidsAnalysis of Steroids

Doping analysis in sports Doping analysis in sports

Detection of Stanozolol , methandrostenolone in Detection of Stanozolol , methandrostenolone in
equine & human urine by LC-ESI-MS. equine & human urine by LC-ESI-MS.

Detection of testosterone by LC-ESI-MS in Detection of testosterone by LC-ESI-MS in
Human athletes .Human athletes .

43. Food Safety AnalysisFood Safety Analysis

LC-MS imp in quantification & confirmation of LC-MS imp in quantification & confirmation of
identity of food contaminants (eg. Sea food)identity of food contaminants (eg. Sea food)

LC-MS of antibiotic & antibacterial compound:LC-MS of antibiotic & antibacterial compound:

44. Stability Testing & Impurity Stability Testing & Impurity
ProfilingProfiling

In stability testing : to determine the identity, chemical In stability testing : to determine the identity, chemical
structure of the API and quantification levels as well as structure of the API and quantification levels as well as
presence of degradants, excipients and impurities .presence of degradants, excipients and impurities .
eg. determination of degradants ineg. determination of degradants in
--.Trimethoprim - Artesunate.Trimethoprim - Artesunate

In impurity profiling : detection of minor components In impurity profiling : detection of minor components
in presence of major components.in presence of major components.
eg. In identification of eg. In identification of
Polar impurity in mosapridePolar impurity in mosapride

45. LC/MS in Clinical Labs LC/MS in Clinical Labs

Neonatal screeningNeonatal screening

Specialist triple quard applicationSpecialist triple quard application

Therapeutic drug monitoringTherapeutic drug monitoring

To replace immuno assaysTo replace immuno assays

Drugs of abuseDrugs of abuse

46. LC/MS in the Environmental FieldLC/MS in the Environmental Field

WaterWater

Identification and quantization of pollutantsIdentification and quantization of pollutants

Pesticides, antibioticsPesticides, antibiotics

Food Food

chemical contaminantschemical contaminants

antibioticsantibiotics

natural toxinsnatural toxins

Animal feedsAnimal feeds

contaminants, illegal substancescontaminants, illegal substances

47. LC/MS in Other Industries LC/MS in Other Industries

OrganometallicsOrganometallics

structure structure

DetergentsDetergents

Quality Control, competitors productsQuality Control, competitors products

PolymersPolymers

molecular weight, structuremolecular weight, structure

48. ReferencesReferences

Frank Settle. Handbook of Frank Settle. Handbook of Instrumental TechniquesInstrumental Techniques for Analytical for Analytical
ChemistryChemistry 1 1
stst
ed.647-659. ed.647-659.

Willard, Merritt, Dean, Settle. Willard, Merritt, Dean, Settle. Instrumental MethodsInstrumental Methods of Analysisof Analysis .7 .7
thth
ed. ed.
618-620.618-620.

Skoog, Holler,Nieman. Skoog, Holler,Nieman. Principles of InstrumentalPrinciples of Instrumental
AnalysisAnalysis,5,5
thth
ed.1992 ed.1992; ; 501-511:528-531:738-739.501-511:528-531:738-739.

Christian Garry. Christian Garry. Analytical ChemistryAnalytical Chemistry. 6. 6
thth
ed. 608-610 ed. 608-610

Wilfried M. A. Niessen . Liquid Chromatography – Mass Spectroscopy . 3Wilfried M. A. Niessen . Liquid Chromatography – Mass Spectroscopy . 3
rdrd

ed. 80-120 , 164- 180 . 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

49. ReferencesReferences
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.

www.pubmedcentral.nih.govwww.pubmedcentral.nih.gov

www.sciencedirect.comwww.sciencedirect.com

www.agilent.com/chemwww.agilent.com/chem

www.waters.comwww.waters.com

www.interscience.wiley.comwww.interscience.wiley.com