Gas Chromatography-Mass - PowerPoint Presentation

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Gas Chromatography-Mass Spectrometry

Dr

Erica

Zarate

Auckland Science Analytical Services - Mass Spectrometry

12 June 2015

Gas chromatography -

Mass Spectrometry

Robust

More reproducible than LC-MSCan be fully automatedhigh throughputCheaper than other mass spec techniques $22 per sample if you do prep and analysis (we provide training)$42/sample if we do it for youFull pricing on iLab

Image:

Gerstel

GC-MS available

Agilent

Thermo

Samples are carried in a gas, not a liquidHelium, 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 heatingsample interaction with the stationery phase inside the columnMany different columns for different applications

How it works

~ 1 hour per sample

Elution and Ionisation

C

ompounds arrive separated at the end of the column

They are ionised by electron bombardment and fragmentFragments are conveyed to detector electromagneticallyThe detector amplifies the fragment signal

Fragmentation pattern

Chromatogram

Spectrum

Identification

Sample

Library

Sample Introduction

Samples must be

injected

VOLATILEThey might already be volatile (eg: essential oils)If not, they can be made volatile (extraction into volatile solvent, derivatisation, pyrolysis).

Humans: Plasma, Serum, Urine, Saliva, Sweat, Mucus, Lymph, Milk, Hair, Faeces, Tissue, Amniotic fluid

Extraction

Marine animals: sea urchins, sea cucumbers, corals, mussels

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

Honey, Yeast, Bacteria, Wine, Juices, Fungi, Growth Media, Fruits and Veggies, Feathers, Fish oil

Examples

Metabolomics methods

Screening methods (discovery – hypothesis generation)

quick

provide relative abundanceonly trends can be compared with published literatureg

ood for finding possible biomarkersShow response to treatment (eg:mode

of action – new drugs)

Eg

. MCF and TMS methods

Targeted methods (hypothesis testing)

take timecost moreprovide absolute concentrationsdata easily compared with published literature

required for validating biomarkers

Eg: our Q-FAMEs method, isotopically labelled internal standards

Derivatisation

Trimethylsilylation

Good universal method

Most derivatives in NIST libraryBut derivatives not stableMethylchloroformate derivatisation

Good for amino acids and fatty acidsBut several derivatives formed

Limited to in-house library

Stable derivatives

Direct

transesterification

FastGood for fatty acidsStable derivatives

Derivatisation

is a chemical reaction that makes non-volatile compounds volatile

TMS

MCF

QFAMEs

What compounds can be detected?

GC-MS is best for small molecules:

ie

: 0 - 800 amuWe have in-house mass spectral libraries (reference standards)We can screen for unknowns using the NIST mass spectral library (>300,000 compounds)

10-Heptadecenoic acid

3-Hydroxypropionic acid

beta-Citryl-L-glutamic acid

Glutamine

Methionine

Putrescine

10-Pentadecenoic acid

3-Methyl-2-oxopentanoic acid

beta-Methylamino-alanine

Glutaric acid

Myristic acid

Pyroglutamic

acid

11,14,17-Eicosatrienoic acid

3-Oxoadipic acid

Butylated hydroxytoluene

Glutathione

Myristoleic acid

Pyruvic acid

11,14-Eicosadienoic

4-Aminobenzoic acid

bishomo-gamma-Linolenic acid

Glyceric acid

N-Acetylcysteine

Quinic acid

13,16-Docosadienoic acid

4-Aminobutyric acid (GABA)

Caffeine

Glycerol

N-Acetylglutamic acid

S-

Adenosylhomocysteine

1-Aminocyclopropane-1-carboxylic acid

4-Hydroxycinnamic acid

cis-4-Hydroxyproline

Glycine

NADP_NADPH

S-Adenosylmethionine

1-Phenylethanol

4-Hydroxyphenylacetic acid

cis-Aconitic acid

Glyoxylic acid

N-alpha-Acetyllysine

Salicylic acid

2,3-Butanediol

4-Hydroxyphenylethanol

cis-Vaccenic acid

Gondoic acid

Nervonic acid

Sebacic acid

2,4-Diaminobutyric acid

4-Methyl-2-oxopentanoic acid

Citraconic acid

Heneicosanoic acid

Nicotinamide

Serine

2,6-Diaminopimelic acid

5-Hydroxy-L-lysine

Citramalic acid

Heptadecane

Nicotinic acid

Sinapic acid

2-Aminoadipic acid5-Hydroxymethyl-2-furaldehydeCitric acidHexanoic acidNonacosaneStearic acid2-Aminophenylacetic acid5-MethyltryptophanCreatinineHippuric acidNonadecanoic acidSuberic acid2-Hydroxybutyric acid5-Oxotetrahydrofuran-2-carboxylic acidCystathionineHistidineNorvalineSuccinic acid2-Hydroxycinnamic acid9-Heptadecenoic acidCysteineHomocysteineO-AcetylserineSyringic acid2-Hydroxyisobutyric acidAdipic acidDibutyl phthalateIndole-3-butyric acidOctanoic acidTartaric acid2-Isopropylmalic acidAdrenic acidDecanoic acidIsocitric acidOleic acidThiamine2-Methyloctadecanoic acidAlanineDocosahexaenoic acidIsoleucineOrnithineThreonine2-Oxoadipic acidalpha-Linolenic acidDodecaneItaconic acidOxalic acidtrans-4-Hydroxyproline2-Oxobutyric acidAnthranilic acidDodecanoic acidLactic acidOxaloacetic acidtrans-Cinnamic acid2-Oxoglutaric acidArachidic acidDocosapentaenoic acidLeucinePalmitic acidTricosane2-Oxovaleric acidArachidonic acidEthylenediaminetetraacetic acidLevulinic acidPalmitoleic acidTricosanoic acid2-Phosphoenolpyruvic acidAsparagineEicosapentaenoic acidLignoceric acidpara-Toluic acidTridecane2-Phosphoglyceric acidAspartic acidErucic acidLinoleic acidPentadecaneTridecanoic acid3,5-Diiodo-L-tyrosineAzelaic acidFerulic acidLysinePentadecanoic acidTryptophan3-Hydroxybenzoic acidBehenic acidFumaric acidMalic acidPhenethyl acetateTyrosine3-Hydroxydecanoic acidBenzoic acidgamma-Linolenic acidMalonic acidPhenylalanineUndecanoic acid3-Hydroxyoctanoic acidbeta-AlanineGlutamic acidMargaric acidPimelic acidValineProlineVanillic acid

MCF

Amino acids, fatty acids and organic acids

In-house libraries

Fatty acids

Hexanoic

acid (C6_0)

9,12-trans-Octadecadienoic acid (E,E) C18:2(n-6t)

Octanoic acid (C8_0)

7-trans-Nonadecenoic acid, (7E)- C19:1(n-12t)

Decanoic acid (C10_0)

10-trans-Nonadecenoic acid, (10E)- (C19_1n-10t)

Undecanoic acid (C11_0)

9,12-cis-Octadecadienoic acid (Z,Z) (C18_2n-6c)

Dodecanoic

acid (C12_0)

Eicosanoic acid (C20_0)

Tridecanoic acid (C13_0)

6,9,12-cis-Octadecatrienoic acid, (6Z,9Z,12Z)- (C18_3n-6c)

Tetradecanoic acid (C14_0)

11-trans-Eicosenoic acid, (11E)- C20:1(n-9t)

9-trans-Tetradecenoic acid (C14_1n-5t)

9,12,15-cis-Octadecatrienoic acid, (9Z,12Z,15Z)- 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)

Hexadecanoic acid (C16_0)

8,11,14-cis-Eicosatrienoic acid, (8Z,11Z,14Z)-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)

6-trans-Octadecenoic acid, (E)- C18:1(n-12t)

5,8,11,14,17-cis-Eicosapentaenoic acid, (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)

6-cis-Octadecenoic acid, (Z)- C18:1(n-12c)

7,10,13,16-cis-Docosatetraenoic acid, (7Z,10Z,13Z,16Z)- C22:4(n-6c)

9-cis-Octadecenoic acid (9Z)- (C18_1n-9c)

4,7,10,13,16-cis-Docosapentaenoic acid, (4Z,7Z,10Z,13Z,16Z) C22:5(n-6c)

11-cis-Octadecenoic acid, (Z)- C18:1(n-7c)

7,10,13,16,19-cis-Docosapentaenoic acid, (7Z,10Z,13Z,16Z,19Z)-C22:5(n-3c)

Nonadecanoic

acid (C19_0)

4,7,10,13,16,19-cis-Docosahexaenoic acid, (4Z,7Z,10Z,13Z,16Z,19Z) C22:6(n-3c)

ducitol

fructose

myoinositol

glucose

glycerol

mannitol

sorbitolfucitolribitolgalactosemannoserhamnosesorbosearabinoseribosetrehalosexyloselactosemaltoseQFAMEsTMSIn-house librariesSugars

Metabolomics methods

TMS (~

3

00 compounds)

MCF (~100 compounds)

Same sample extract, different

derivatisation

method (mussel gill tissue)

Metabolomics methods

QFAMEs (~60 compounds)

MCF (~100 compounds)

Same sample, different extraction and

derivatisation

method (human plasma)

Automated Data Processing

Both in GUI-R developed by Morgan Han

Metab

(

Aggio

)

lower false positive, higher missing values

MSOmics

(Han)

higher false positive, fewer zero values

They use R – XCMS package

Two options

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

d

ata analysis:Silas Villas Boas and Morgan Han (Metabolomics Lab)Katya Ruggiero and Kevin Chang (Statistics Consulting Centre)

Current UoA

research