Sample preparation techniques for GC-MS and LC-MS

1. Presented by,
Latha M
2017601306
Department of Nano Science and
Technology

2.  Gas chromatography mass spectrometric sample preparation is performed
on smaller and more volatile samples including environmental pollutants,
industrial byproduct, food contaminants, pesticides and metabolites of illicit
and designer drugs.
 These molecules are more challenging to ionize and separate using liquid
chromatography mass spectrometry (LC-MS) and so are subjected to GC-
MS instead.
 Head space sampling
 Pyrolysis
 Solid phase extraction
 Automated solid phase extraction
 Solvent extraction
 Accelerated solvent extraction
 Solid phase micro extraction

3.  In this technique, the liquid or solid sample is added to a glass vial
until it stabilization.
 Some of the analytes vaporize from the liquid or solid and enter the
headspace above the sample.
 If this gas phase is then directly injected into the gas chromatograph
for separation, the method is referred to as static headspace
sampling.
 If an inert gas passed into the sample and the vaporized analyte
accumulate on an absorbant surface or cryogenic trap, the method is
termed dynamic head space sampling.
 This method of analyze is often used with blood, cosmetics, plastics,
solids and materials with high water content .

4. Pyrolysis:
 This step is performed prior to GC-MS and involves heating samples to
120 c in either an inert environment or in air, resulting in their
breakdown to smaller fragments.
 Samples that typically undergo pyrolysis include plastics, paints, dyes,
resins, cellulose, wood, oil and rubber as well as larger samples that
might be heavily cross-linked or insoluble.
 Because pyrolysis negates the need to use solvents, it is useful for
identification of solvents and additives.
Solid phase extraction (SPE):
 This manual extraction technique typically involves the use of solid
packaging material, often contained within a cartridge to separate
sample components.
 The sample itself will typically be in liquid form.
 Samples frequently extracted via SPE includes biological samples such
as urine, saliva and plasma, environmental samples such as water and
food product such as beverages.

5. Automated solid phase extraction (ASPE):
 It can process many Solid phase extraction samples in just a few
hours enabling quick analysis of compound of interest including
pesticides, flame retardant, semi volatiles and steroids.
Solvent extraction:
 Many analytes, such as pesticides are highly polar and therefore
amenable to extraction with solvents such as magnesium sulfate.
 This is the guiding principle behind the technology.
Accelerated solvent extraction (ASE):
 Automated batch samples processing of solids and semi-solid
samples including pesticides, oils, nutritional supplements and
biofuels is frequently accomplished using this method.
 By this way compounds are quickly extracted from the samples
using a minimum amount of time, solvent and overall cost.

6. Sample preparation for GC-MS analysis
1. Prepare a standard curve each time samples are being analyzed. Set up 9 tubes
containing 1ml of methanol and the following:
Tube 1 = Spike 1: 12μL (0.05 mg/L)
Tube 2 = Spike 2: 30μL (0.125mg/L)
Tube 3 = Spike 3: 60μL (0.250mg/L)
Tube 4 = Spike 4: 120μL (0.5mg/L)
Tube 5 = Spike 5: 300μL (1.25mg/L)
Tube 6 = Spike 6: 600μL (2.50mg/L)
Tube 7 = Spike 7: 1.2mL (5mg/L)
Tube 8 = LQ: 48μL (0.20mg/L – From a 0.5mg/L LQ stock)
Tube 9 = HQ: 48μL (4mg/L - From a 10mg/L HQ stock)
Note: All volumes of THC used come from a standard stock with a concentration of
0.5mg/L (Please refer to appendix for making the THC stock).
2. Add 200μL of the internal standard to all tubes. Vortex all tubes and centrifuge
them at 43°C for 1hr.
3. Make up a mixture of Hexane:BSTFA (4:1) and add 120μL to all tube. Vortex
and cap all tubes.
4. Incubate all tubes for 20 minutes in a 70°C oil bath and then allow the samples to
cool down at 4°C for 5 minutes.
5. Transfer all the samples to GC analysis vials and load them onto the GC-MS.
Appendix
Before each analysis, the following must be available:
1. 0.5mg/L stock of THC and 0.5mg/L stock of D3 – THC (internal std). To make
up stock add 50μL of THC or D3 – THC into 9.95mL of methanol.
2. 0.5mg/L LQ and 10mg/L HQ stock of THC which is to be made up in methanol
and water (1:1).
3. Hexane, Methanol (both being HPLC grade) and BSTFA.

7.  LC/MS sample containing non volatile or large compounds are
typically prepared for mass spectrometry (MS) analysis first
undergoing liquid chromatography (LC) separation.
 This is a necessary step in the eventual isolation and identification of
the target analyte from among hundreds, if not thousands, of the
other compounds and contaminants.
 Solid phase extraction (SPE)
 Liquid-Liquid extraction (LLE)
 Protein precipitation extraction (PPE)
 Desalting
 Isoelectric point precipitation
 Organic solvent extraction (OSE)

8. Solid phase extraction (SPE):
 This separation technique is often used to separate compounds (eg:pesticides) dissolved
in solutions (eg:Water).
 A cartridge is packed with an octodecyl (c-18) stationary phase, which may be silica-
based and through this column the solution is passed.
 Alternatively, a polymer –based cartridge may be utilized if the sample is highly acidic.
 Elution of individual compounds occurs over time, and one SPE column eventually
elute several compounds.
 Eluents can then be concentrated to dryness, diluted with 50:50 methanol/water and
injected into the mass spectrometer.
 Alternately, SPE can be performed in high-throughput 96-well plates, such as Solo SPE
plate.
Liquid-Liquid extraction (LLE):
 In LLE, the analyte of interest is first partitioned by a solvent, after which it is
extracted, concentrated and diluted`
 Phenol-chloroform extraction is one of the example of an LLE that, has historically
been used to separate not only protein but nucleic acids as well.
 A specific analyte such as testosterone is quantitated in samples by first adding the
organic solvent methyl-tertiary-butyl-ether followed by vortexing.
 The supernatant is then removed and transferred to a heating block.
 Following evaporation the residue is reconstructed with a 50:50 (methanol:water)
solution containing ammonium acetate and formic acid

9. Protein precipitation extraction (PPE):
 Several method of protein precipitation exist, including
desalting of protein, isoelectric point precipitation, organic
solvent extraction and ion exchange.
 The specific chromatography method used depends on sample
type (cell, tissue or fluid), location, abundance and size of the
target protein, matrix effect etc.,
Desalting:
 Also called ammonium sulfate precipitation, this protein
precipitation technique relies on increasing the concentration of
ammonium sulfate until the protein is salted out.
 This process occurs as the salt eventually outcompetes the
protein for available water.

10.  Organic solvent extraction (OSE):
 Protein solubility often depends on an environment having
a large dielectric constant, which is present in solvents such
as water.
 Organic solvents such as acetone and methanol, on the
other hand, have small dielectric constants and so promote
the aggregation of protein for mass spec analysis`
 During organic solvents extraction, a target protein is
precipitated due to its unequal solubility in water and the
organic solvent.
 Following precipitation and concentration, a buffer
solution is added to optimize pH.
 The target protein is then extracted using solvent such as
ether or chloroform.