The aim of the coupling is to obtain an information-rich detection for both identification and quantification compared to that with a single analytical technique.

1. (GC-MS/MS, and
LC-MS/MS
Presented By
Prof.(Dr.) Dinesh Kr. Mehta
MM College of Pharmacy,
MM(DU), Mullana

2. Hyphenated techniques
 Hyphenated technique is a combination or coupling of two analytical
techniques with the help of proper interface.
 The hyphenated technique is developed from the coupling of a separation
technique and detection technique. The term “hyphenation” was first adapted
by Hirschfeld in 1980 to refer to the on-line combination of a separation
technique and one or more spectroscopic detection techniques.
 The aim of the coupling is to obtain an information-rich detection for both
identification and quantification compared to that with a single analytical
technique.

3. Advantages
For fast and accurate analysis
 A Higher degree of automation.
 Higher sample throughput.
 Better reproducibility.
 Reduction of contamination due to its closed system.
 Separation of quantification at the same time.

4. Types of hyphenated techniques
Double hyphenated techniques.
Triple hyphenated techniques.
Double hyphenated techniques
 LC-MS
 LC-NMR
 LC-IR
 CE-MS
 GC-IR
 GC-MS
 HPLC-DAD
 GC-FTIR

5. Hyphenated Technique(GC-MS/MS)
CONTENTS
 General Introduction
 Principle
 Instrumentation
 GC-MS Setup
 Types of Mass Spectrometer detector
 Application

6. General Introduction
 Gas Chromatography – Mass Spectrometry (GC-MS) is an analytical method
that combines the features of gas chromatography and mass spectrometry to
identify different substances with in a test sample.
 GC-MS separates chemical mixtures into individual components (using a gas
chromatograph) and identifies / quantifies the components at a molecular
level (using a MS detector).
 It is one of the most accurate and efficient tools for analyzing volatile organic
samples.
 Mass spectrometer (MS) is an instrument that serves for establishment of the
molecular weight and structure of both inorganic and organic compounds,
and the identification and determination of analytes in complex mixtures.

7. Principle
GC-MS instrument separates chemical mixtures (the GC component) and
identifies the components at a molecular level (the MS component). GC
works on the principle that a mixture will separate into individual substances
when heated. The heated gases are carried through a column with an inert
gas.
As the separated substances emerge from the column opening, they flow
into MS.
Mass spectrometry identifies compounds by the mass of the analyte
molecule.

8. Instrumentation

9. The GC-MS is
composed of two major
building blocks:
• Gas chromatograph
• Mass spectrometer

10.  The molecules are retained by the
capillary column and elute from the
column at different times.
 The Mass spectrometer capture, ionize,
accelerate, deflect and detect the
ionized molecules separately by
breaking each molecule into ionize
fragments and detecting these
fragments using their mass to charge
ratio.

11. Types of Mass spectrometer Detectors
1. Quadrupole Mass Spectrometer- most common
2. Ion Trap Mass Spectrometer
3. Magnetic Mass Spectrometer
Advantages of GC-MS
• Accurate identification of a particular molecule is possible.
• Differentiate between multiple molecules in the same amount of time.
• Identification carried out at a molecular level.
• Easy to Operate.

12. Application
Quantitation of pollutants in drinking and waste water.
Identification of unknown organic compounds in hazardous waste dumps and
reaction products by synthetic organic chemistry
Quantitation of drug in metabolites and urine is done for the pharmacological
and forensic use.
Used for drug analysis, pesticide and herbicide detection
Characterization of odour and flavor component of food
Law enforcement
Sports Anti- doping analysis
Medical Diagnosis
Security of Airports
Criminal Forensics

13. Liquid Chromatography-Mass Spectrometry (LC/MS)
General Introduction
 The coupling of MS with LC (LC-MS) was an obvious extension but
progress in this area was limited for many years due to the relative
incompatibility of existing MS ion sources with a continuous liquid stream.
 The reasons for choosing LC-MS over LC with conventional detectors are
essentially the same as with GC-MS, namely high specificity and the ability
to handle complex mixtures.
Analytes are separated by Liquid Chromatography (LC) prior to analysis
by Mass Spectrometry (MS)
 Provides enhanced specificity, based on retention times
 Reduces the number of molecules entering the MS ionization source at a
given time
 reduces the competition for charge

14. Principle
Liquid chromatography-mass spectrometry is the technique which performs
separation by liquid chromatography and mass analysis with the help of the
mass spectrometry.
Liquid chromatography tandem mass spectrometry (LC–MS/MS)
 Liquid Chromatography
 Separates mixture components
 Based on polarity
Tandem Mass Spectrometry
 Detector
 Identification & Quantification of components
 Based on compound mass

15. • It is now generally accepted as the preferred technique for quantitation of
small molecule drugs, metabolites in biological matrices (plasma, blood,
serum, urine, and tissue)
• • Electrospray needle is used as bridge to connect the liquid chromatography
with that of the mass.
• LC-MS is mainly separated into the three parts-
• Chromatography – In liquid chromatography separation is performed which
is detected with the help of Photo diode Array.
• These separated components then transferred to the interface.
• 2. Interface – In interface the liquid is volatilized and transferred to the MS.
• 3. Spectrometry – With the help of various ionization techniques the
compound is ionized and then it is analyzed by mass analyzer.

16. Instrumentation
Various mass analyzers are used viz. Quadrupoles, quadrupole ion traps, time-
to-flight (TOF), time-to-flight reflection (TOFR), and ion cyclotron resonance
(ICR) mass analyzers.
It is a method that combines separation power of HPLC with detection
power of Mass spectrometry.
 In LC-MS we remove the detector from the column of LC and fit the column
to interface of MS.
 In most of the cases the interface used in LC-MS are ionization source
 A liquid sample is introduced into the ionization source of the mass
spectrometer.
Example: Extracts from plasma, serum, whole blood, Urine, CSF, etc.

17. The liquid sample is usually delivered by an Liquid Chromatography (LC) system.

18. LC-MS System Components
• Mass spectrometers work by ionizing molecules and then sorting and identifying
the ions according to their mass- to-charge (m/z) ratios.
The mobile phase is the solvent that moves the solute throughout column.
Solvent strength and selectivity: - It is the ability of solvent to elute solutes from
a column.
COLUMN
The use of di-functional or tri-functional silanes to create bonded groups
with two or three attachment points leading to phases with higher stability in
low or higher pH and lower bleed for LC-MS
• Most widely used columns for LC-MS are: -
(1) fast LC column :- the use of short column.(15-50mm)
(2) Micro LC column :- the use of large column. (20-150mm)

20. Advantages of LC-MS/MS Accuracy and Precision
 Robustness
 Sensitivity
 Allows multi-analyte panels
 Requires less sample prep
 Compatible with generic sample prep
 Versatility, can easily add new compounds
 Lower cost-per-sample
 Speed

21. Applications
 LC-MS used to detect compounds from polyaromatic (non-polar) to peptide
and proteins.
 LC-MS used for compounds identification and purity.
 Used for determination of pesticides, herbicides & organic pollutant for
environmental monitoring.
 Proteome analysis is done by this technique.