Liquid Chromatography-Mass Spectrometry (LC-MS)

1. LIQUID CHROMATOGRAPHY- MASS SPECTROMETRY
Subject- Methods in Pharmaceutical Research
DEPARTMENT OF PHARMACEUTICAL SCIENCES
Dr. Hari Singh Gour Vishwavidyalaya
Sagar (M.P)- 470003, India
(A Central University)
Submitted By:
DEBASIS SEN
M.PHARM 1ST SEMESTER
Y22254008
Submitted To:
Prof. A. GAJBHIYA
(Professor, DOPS)
Prof. S. K. KASHAW
(Professor, DOPS)

2. CONTENT
• WHAT IS HYPHENATED TECHNIQUE
• INTRODUCTION TO LC-MS
• PRINCIPLE
• PROBLEMS IN COMBINING HPLC AND MS
• WORKING OF LC-MS
• INTERFACE
• MASS ANALYZER
• DETECTORS
• CONCLUSION
• REFERENCES

3. What is Hyphenated Technique?
Hyphenated Technique:
The term “hyphenation” was first adapted by Hirschfeld in 1980. The technique developed
from the coupling of a separation technique and spectroscopic detection technology is known
as hyphenated technique.
Advantages of hyphenated techniques:
• Fast and accurate analysis.
• Higher degree of automation.
• Reduction of contamination due to its closed system.
• Separation and quantification achieved at same time.

4. INTRODUCTION TO LC-MS
• Liquid Chromatography/Mass Spectrometry (LC/MS) is a powerful analytical technique that
combines the resolving power of liquid chromatography with the detection specificity of mass
spectrometry.
• Liquid chromatography (LC) separates the sample components and then introduce them to the
mass spectrometer (MS). The MS creates and detects charged ions.
• The LC/MS data may used to provide information about the molecular weight, structure,
identity and quantity of specific sample components.
• In LC-MS we remove the detector from the column of LC and fitting the column to interface
of MS.
• In the most of the cases the interface used in LC-MS are ionization source.

5. PRINCIPLE
• The LC-MS technology involves use of an HPLC, wherein the individual components in a
mixture are first separated followed by ionization and separation of the ions on the basis
of their mass/charge ration.
• The separated ions are then directed to a electron multiplier tube detector, which identifies
and quantifies each ion.
• The ion source is an important component in any MS analysis, as this basically aids in
efficient generation of ions for analysis.
• To ionize intact molecules, the ion source could be APCI (Atmospheric Pressure Chemical
Ionization), ESI (Electron Spray Ionization) etc.

6. PROBLEMS IN COMBINING HPLC AND MS
HPLC MS
Liquid phase operation Vacuum operation
20-50°C 200-300°C
No mass range limitation Up to 4000 Da for quadrupole MS
Inorganic buffer Requires volatile buffers
1ml/min eluent flow is equivalent to
500ml/min in gas
Accept 10ml/min gas flow

7. WORKING OF LC/MS
• LC-MS is mainly separated into the three parts- chromatography, interface and mass
spectrometry.
• In liquid chromatography, separation is performed and the separated components
transferred to the interface.
• In interface the liquid is volatilized and transferred to the MS.
• With the various ionization technique the compound is ionized and then analysed by mass
analyser.

9. INTERFACES
• LC-MS systems include a device for introducing samples such as HPLC, an interface for
connecting such device, an ion source that ionizes samples, an electrostatic lens that
efficiently introduces the generated ions, a mass analyzer unit that separates ions based on
their mass-to-charge (m/z) ratio, and a detector unit that detects the separated ions.
• In a LC-MS system, however, if the LC unit is simply connected directly to the MS unit,
the liquid mobile phase would vaporize, resulting in large amounts of gas being
introduced into the MS unit.
• This would decrease the vacuum level and prevent the target ions from reaching the
detector. So, the interfaces are to be used.

10. TYPES OF INTERFACES
• It is difficult to interface a liquid chromatography to a mass spectrometer cause of the
necessity to remove the solvent.
• The commonly used interfaces are-
1. Electrospray Ionization (ESI)
2. Atmospheric Pressure Chemical Ionization (APCI)
3. Atmospheric Pressure Photoionization (APPI)
4. Thermospray Ionization (TSI)

11. ELECTROSPRAY IONIZATION
• It is a type of evaporative ionization technique used to analyze the high molecular weight
biomolecules, labile and non-volatile compounds.
• In ESI, the sample is dissolved in a volatile solvent, typically a mixture of water and an
organic solvent such as methanol or acetonitrile.
• The sample solution is then introduced into a small-diameter capillary held at a high
voltage, typically 2-5 kV, which generates a strong electric field at the capillary tip.
• The electric field causes the solvent to evaporate, leaving behind a charged droplet
containing the sample molecules.
• As the droplet moves towards the mass analyzer, it becomes increasingly charged, causing
it to break up into smaller droplets.
• The smaller droplets carry some of the sample molecules, which become further ionized
by the electric field. This process is repeated until the droplets become small enough to
produce individual gas-phase ions.

12. • The ions produced by ESI are typically multiply charged, due to the presence of multiple
charged species in the droplet, and are often detected in the positive ion mode.
• The ions produced by ESI are typically analyzed by a mass analyzer, which separates the
ions based on their mass-to-charge ratio (m/z). The resulting mass spectrum provides
information about the mass and composition of the sample molecules.

13. ATMOSPHERIC PRESSURE CHEMICAL IONIZATION (APCI)
• APCI is a type of soft ionization technique based on the mechanism of evaporation and
carried out at atmospheric pressure.
• APCI vaporize solvent and sample molecules by spraying the sample solution into a
heater ( heated to about 400C) using a gas, such as N2.
• Solvent molecules are ionized by corona discharge to generate stable reaction ions.

14. ATMOSPHERIC PRESSURE PHOTOIONIZATION (APPI)
The LC eluent is vaporized using a heater at atmospheric pressure. The resulting gas is made
to pass through a beam of photons generated by a discharge lamp (UV lamp) which ionizes
the gas molecules.

15. THERMOSPRAY IONIZATION (TSI)
• It is a evaporative ionization method in which solvent molecules are removed from the
analyte by evaporation.
• Eluent sample coming from the column will be passed through the heated capillary tube.
• Heated capillary tube will nebulize the eluent, partially evaporate the solvent and form a
stream of fine spray containing analyte.
• Fine spray droplets will be partially charged by thermal energy and solvent gets evaporated
from spray.
• Ionized analytes will move towards the mass analyser for further analysis.

16. MASS ANALYZER
• They deflect ions down a curved tubes in a magnetic fields based on their kinetic energy
determined by the mass, charge and velocity.
• The magnetic field is scanned to measure different ions.
• Types of mass analyser-
1. Quadrupole mass filter
2. Time of flight
3. Ion trap

17. QUADRUPOLE MASS ANALYZER
• It consist of four cylindrical metal rods arranged in a square parallel to the direction of ion
beam and radio frequency (RF) or direct current (DC) voltage is applied.
• Ions will enter into the mass analyser and depending on the ratio of RF amplitude and DC
voltage oscillating electrostatic filed will be generated for ions.
• If RF>DC, then larger ion will hit the detector first
If RF<DC, then small ions will hit the detector first.
• Inappropriate m/z ratio of ions (other than 1-1000) will undergo unstable oscillation and
hit the rod, and ultimately will not reach the detector.

18. TIME OF FLIGHT (TOF) MASS ANALYZER
• ToF is based on the simple idea that the velocities of two ions varies depending on the
mass of the ion but the ions should have created at same constant and should have same
kinetic energy.
• Light ion will strike the detector first due to higher velocity.

19. ION TRAP MASS ANALYZER
• Ion trap mass analyser is a high resolution, high sensitivity and multiple product ion scan
capability.
• One end cap electrode has single small central aperture through which ions are introduced
into trap while other one has several apertures through which ions are passed to a detector.
• Helium bath gas is present in the trap to stabilize the ion trajectories.
• Collison takes place between helium bath gas and ions. Due to this motion of ions
increases the trapping efficiency of the analyser.
• The ions are ejected from the trap on the basis of m/z values to create mass spectrum.

20. DETECTORS
The detector is an important tool of mass spectrometer that produces current that is
proportional to the number of ions strike it. Once the ions are formed passed from analyser
they have to be detected and transformed into signal.
Commonly used detectors-
Detectors
Photographic
Plates
Faraday Cup
Electron
Multiplier
Channel
Electron
Multiplier

21. CONCLUSION
The LC-MS is a hyphenated technique used in combination with separation power of HPLC
with detection power of Mass spectrometry. It is widely used in pharmaceutical, chemical,
food, agrochemical industries, environmental and forensic applications. LC-MS is used for
qualitative and quantitative determination of drug substances and biological samples. Also it
is commonly used in drug research and quality control.

22. REFERENCES
• Alhendi AS. A review: Protein identification by LC-MS: Principles, instrumentation, and
applications. Iraqi J Sci. 2020;61(10):2428-66.
• Kumar PR, Dinesh SR, Rini R. LCMS—a review and a recent update. J Pharm Pharm
Sci. 2016 Mar 1;5:377-91.
• Korfmacher WA. Foundation review: Principles and applications of LC-MS in new drug
discovery. Drug discovery today. 2005 Oct 15;10(20):1357-67.

23. THANK YOU