Gas ChromatographyMass Spectrometry
Dr Erica Zarate
Auckland Science Analytical Services - Mass Spectrometry
12 June 2015
Gas chromatography Mass Spectrometry
Image: Gerstel
• Robust
• More reproducible than LC-MS
• Can be fully automated
– high throughput
• Cheaper than other mass spec
techniques
– $22 per sample if you do
prep and analysis (we
provide training)
– $42/sample if we do it for
you
– Full pricing on iLab
GC-MS available
Agilent
Thermo
How it works
• Samples are carried in a gas, not a liquid
– Helium, hydrogen, nitrogen, argon, or a combination of these
• Compounds are carried and separated in a column
– Typically capillary and 30– 100m for metabolomics work
• Separation is achieved by:
– column heating
– sample interaction with the stationery phase inside the column
– Many different columns for different applications
~ 1 hour
per
sample
Elution and Ionisation
•
•
•
•
Compounds arrive separated at the end of the column
They are ionised by electron bombardment and fragment
Fragments are conveyed to detector electromagnetically
The detector amplifies the fragment signal
Fragmentation pattern
Chromatogram
Spectrum
Sample
Identification
Library
Sample Introduction
• Samples must be injected VOLATILE
– They might already be volatile (eg: essential oils)
– If not, they can be made volatile (extraction into
volatile solvent, derivatisation, pyrolysis).
Extraction
Anything you can think of we can probably
develop an extraction method for.
Sample size limitations:
− 300uL liquids
− 200mg fresh tissue
•
Samples can be:
• Liquid
• Solid
• Swab samples
Examples
•
Honey, Yeast, Bacteria, Wine, Juices,
Fungi, Growth Media, Fruits and
Veggies, Feathers, Fish oil
•
Humans: Plasma, Serum, Urine,
Saliva, Sweat, Mucus, Lymph, Milk,
Hair, Faeces, Tissue, Amniotic fluid
•
Marine animals: sea urchins, sea
cucumbers, corals, mussels
Metabolomics methods
• Screening methods (discovery – hypothesis
generation)
− quick
− provide relative abundance
− only trends can be compared with published literature
− good for finding possible biomarkers
− Show response to treatment (eg:mode of action – new drugs)
− Eg. MCF and TMS methods
• Targeted methods (hypothesis testing)
− take time
− cost more
− provide absolute concentrations
− data easily compared with published literature
− required for validating biomarkers
− Eg: our Q-FAMEs method, isotopically labelled internal
standards
Derivatisation
Derivatisation is a chemical reaction that makes non-volatile
compounds volatile
•
Trimethylsilylation
– Good universal method
– Most derivatives in NIST library
TMS
– But derivatives not stable
•
Methylchloroformate derivatisation
– Good for amino acids and fatty acids
– But several derivatives formed
– Limited to in-house library
– Stable derivatives
•
Direct transesterification
– Fast
– Good for fatty acids
– Stable derivatives
QFAMEs
MCF
What compounds can be
detected?
•
GC-MS is best for small molecules: ie: 0 - 800 amu
•
We have in-house mass spectral libraries (reference standards)
•
We can screen for unknowns using the NIST mass spectral library
(>300,000 compounds)
In-house libraries
MCF
10-Heptadecenoic acid
10-Pentadecenoic acid
11,14,17-Eicosatrienoic
acid
Amino acids, fatty acids and organic acids
3-Hydroxypropionic acid beta-Citryl-L-glutamic acid
3-Methyl-2-oxopentanoic
acid
beta-Methylamino-alanine
Glutamine
Methionine
Putrescine
Glutaric acid
Myristic acid
Pyroglutamic acid
3-Oxoadipic acid
Butylated hydroxytoluene
bishomo-gamma-Linolenic
acid
Glutathione
Myristoleic acid
Pyruvic acid
Glyceric acid
N-Acetylcysteine
Quinic acid
Caffeine
Glycerol
N-Acetylglutamic acid
S-Adenosylhomocysteine
cis-4-Hydroxyproline
Glycine
NADP_NADPH
S-Adenosylmethionine
cis-Aconitic acid
cis-Vaccenic acid
Glyoxylic acid
Gondoic acid
N-alpha-Acetyllysine
Nervonic acid
Salicylic acid
Sebacic acid
Citraconic acid
Citramalic acid
Heneicosanoic acid
Heptadecane
Nicotinamide
Nicotinic acid
Serine
Sinapic acid
Citric acid
Creatinine
Hexanoic acid
Hippuric acid
Nonacosane
Nonadecanoic acid
Stearic acid
Suberic acid
Cystathionine
Cysteine
Dibutyl phthalate
Decanoic acid
Histidine
Homocysteine
Indole-3-butyric acid
Isocitric acid
Norvaline
O-Acetylserine
Octanoic acid
Oleic acid
Succinic acid
Syringic acid
Tartaric acid
Thiamine
Isoleucine
Itaconic acid
Lactic acid
Leucine
Ornithine
Oxalic acid
Oxaloacetic acid
Palmitic acid
Threonine
trans-4-Hydroxyproline
trans-Cinnamic acid
Tricosane
Arachidonic acid
Docosahexaenoic acid
Dodecane
Dodecanoic acid
Docosapentaenoic acid
Ethylenediaminetetraacetic
acid
Levulinic acid
Palmitoleic acid
Tricosanoic acid
Asparagine
Aspartic acid
Azelaic acid
Behenic acid
Benzoic acid
beta-Alanine
Eicosapentaenoic acid
Erucic acid
Ferulic acid
Fumaric acid
gamma-Linolenic acid
Glutamic acid
Lignoceric acid
Linoleic acid
Lysine
Malic acid
Malonic acid
Margaric acid
para-Toluic acid
Pentadecane
Pentadecanoic acid
Phenethyl acetate
Phenylalanine
Pimelic acid
Proline
Tridecane
Tridecanoic acid
Tryptophan
Tyrosine
Undecanoic acid
Valine
Vanillic acid
11,14-Eicosadienoic
4-Aminobenzoic acid
4-Aminobutyric acid
13,16-Docosadienoic acid (GABA)
1-Aminocyclopropane-1carboxylic acid
4-Hydroxycinnamic acid
4-Hydroxyphenylacetic
1-Phenylethanol
acid
2,3-Butanediol
4-Hydroxyphenylethanol
4-Methyl-2-oxopentanoic
2,4-Diaminobutyric acid acid
2,6-Diaminopimelic acid 5-Hydroxy-L-lysine
5-Hydroxymethyl-22-Aminoadipic acid
furaldehyde
2-Aminophenylacetic acid 5-Methyltryptophan
5-Oxotetrahydrofuran-22-Hydroxybutyric acid
carboxylic acid
2-Hydroxycinnamic acid 9-Heptadecenoic acid
2-Hydroxyisobutyric acid Adipic acid
2-Isopropylmalic acid
Adrenic acid
2-Methyloctadecanoic acid Alanine
2-Oxoadipic acid
alpha-Linolenic acid
2-Oxobutyric acid
Anthranilic acid
2-Oxoglutaric acid
Arachidic acid
2-Oxovaleric acid
2-Phosphoenolpyruvic
acid
2-Phosphoglyceric acid
3,5-Diiodo-L-tyrosine
3-Hydroxybenzoic acid
3-Hydroxydecanoic acid
3-Hydroxyoctanoic acid
In-house libraries
QFAMEs
Fatty acids
9,12-trans-Octadecadienoic acid (E,E) C18:2(n6t)
7-trans-Nonadecenoic acid, (7E)- C19:1(n-12t)
10-trans-Nonadecenoic acid, (10E)- (C19_1nDecanoic acid (C10_0)
10t)
Undecanoic acid (C11_0)
9,12-cis-Octadecadienoic acid (Z,Z) (C18_2n-6c)
Dodecanoic acid (C12_0)
Eicosanoic acid (C20_0)
6,9,12-cis-Octadecatrienoic acid, (6Z,9Z,12Z)Tridecanoic acid (C13_0)
(C18_3n-6c)
Tetradecanoic acid (C14_0)
11-trans-Eicosenoic acid, (11E)- C20:1(n-9t)
9,12,15-cis-Octadecatrienoic acid, (9Z,12Z,15Z)9-trans-Tetradecenoic acid (C14_1n-5t)
C18:3(n-3c)
9-cis-Tetradecenoic acid (C14_1n-5c)
11-cis-Eicosenoic acid, (11Z)- C20:1(n-9c)
Pentadecanoic acid (C15_0)
Heneicosanoic acid (C21_0)
10-trans-Pentadecenoic acid (C15_1n-5t)
11,14-cis-Eicosadienoic C20:2(n-6c)
10-cis-Pentadecenoic acid (C15_1n-5c)
Docosanoic acid (C22_0)
8,11,14-cis-Eicosatrienoic acid, (8Z,11Z,14Z)Hexadecanoic acid (C16_0)
C20:3(n-6c)
9-trans-Hexadecenoic acid (C16_1n-7t)
13-trans-Docosenoic acid, (13E)- (C22_1n-9t)
9-cis-Hexadecenoic acid (C16_1n-7c)
11,14,17-cis-Eicosatrienoic acid C20:3(n-3c)
Heptadecanoic acid (C17_0)
13-cis-Docosenoic acid, (13Z)- (C22_1n-9c)
10-trans-Heptadecenoic acid, (10E) (C17_1n-7t) 5,8,11,14-cis-Eicosatetraenoic acid (C20_4n-6c)
10-cis-Heptadecenoic acid, (10Z)- (C17_1n-7c) Tricosanoic acid (C23_0)
Octadecanoic acid (C18_0)
13,16-cis-Docosadienoic acid (C22_2n-6c)
5,8,11,14,17-cis-Eicosapentaenoic acid,
6-trans-Octadecenoic acid, (E)- C18:1(n-12t)
(5Z,8Z,11Z,14Z,17Z)- C20:5(n-3)
9-trans-Octadecenoic acid, (9E)- C18:1(n-9t)
Tetracosanoic acid (C24_0)
11-trans-Octadecenoic acid, (E)- C18:1(n-7t)
15-cis-Tetracosenoic acid, (15Z)-(C24_1n-9c)
7,10,13,16-cis-Docosatetraenoic acid,
6-cis-Octadecenoic acid, (Z)- C18:1(n-12c)
(7Z,10Z,13Z,16Z)- C22:4(n-6c)
4,7,10,13,16-cis-Docosapentaenoic acid,
9-cis-Octadecenoic acid (9Z)- (C18_1n-9c)
(4Z,7Z,10Z,13Z,16Z) C22:5(n-6c)
7,10,13,16,19-cis-Docosapentaenoic acid,
11-cis-Octadecenoic acid, (Z)- C18:1(n-7c)
(7Z,10Z,13Z,16Z,19Z)-C22:5(n-3c)
4,7,10,13,16,19-cis-Docosahexaenoic acid,
Nonadecanoic acid (C19_0)
(4Z,7Z,10Z,13Z,16Z,19Z) C22:6(n-3c)
Hexanoic acid (C6_0)
Octanoic acid (C8_0)
TMS
ducitol
fructose
myoinositol
glucose
glycerol
mannitol
sorbitol
fucitol
ribitol
galactose
mannose
rhamnose
sorbose
arabinose
ribose
trehalose
xylose
lactose
maltose
Sugars
Metabolomics methods
Same sample extract, different derivatisation method
(mussel gill tissue)
MCF (~100 compounds)
TMS (~300 compounds)
Metabolomics methods
Same sample, different extraction and derivatisation method
(human plasma)
MCF (~100 compounds)
QFAMEs (~60 compounds)
Automated Data Processing
Two options
MSOmics (Han)
Metab (Aggio)
higher false positive,
fewer zero values
lower false positive,
higher missing values
Both in GUI-R developed by Morgan
Han
They use R – XCMS package
Data processing
Figure: Morgan Han
•
Big data – eg. 1000 samples each
with 10-20MB datafile
•
Need to be processed batchwise so
that a data matrix is generated,
enabling sample comparison for
each compound
Data matrix
Samples
Compounds
Data analysis
Help with data analysis:
• Silas Villas Boas and Morgan Han (Metabolomics Lab)
• Katya Ruggiero and Kevin Chang (Statistics Consulting
Centre)
Current UoA research