Prostaglandin quantification - GC-MS

From LipidomicsWiki

Contents 1 Sample preparation 2 Instrumentation and method 2.1 GC  2.2 Column 2.3 Autosampler 2.4 Mass spectrometer 3 Data analysis and quantification 3.1 Data handling 3.2 Calibration and quantification 4 Method validation 4.1 Accuracy 4.2 Precision 4.3 Limit of quantitation 5 Reference

Sample preparation

• 1ml of sample gets acidified with 0.5 ml formic acid 0.5% and then 50µl of an internal standard solution are added. Following that samples are extracted with 3.5 ml ethyl acetate and the organic layer is evaporated. PFB-esters are synthesized by adding 50µl of a PFBBr solution (3.4 % in acetonitrile) and 10µl of N,N-Diisopropyl ethanolamine and incubating for 10min at RT. After evaporation 50µl of MOX reagens are added for 2h at 75°C. Samples are extracted with 0.4ml water and 2.5ml n-hexane. The organic layer gets evaporated again and 50µl of MSTFA 1% in pyridine are added for 20min at RT. Finally after evaporation the samples are taken up in 50µl of n-hexane containing 1% BSTFA.
• sample sovlent: Hexane / 1%BSTFA

Material	Material used	Internal Standard(s)	Internal Standard(s) added
Lipid extracts	1ml	PGE2, PGD2, PGF2a, 6-keto-PGF1a, all d4	10ng each

Instrumentation and method

GC 

• Type: Thermo Trace GC
• Mode: Split/Splitless
• Injector Temperature: 260°C
• Carrier gas: Helium
• Temperature gradient:

Time [min]	Flow [ml/min]	Temperature [°C]
0	1	160
1.0	1	160
4.75	1	310
8.75	1	310

Column

TR5MS 30m 

Autosampler

• Type: Thermo TriPlus
• Injection volume: 2µl
• Wash solvent: Hexane

Mass spectrometer

• Type: Thermo DSQ
• Ionization mode: NICI
• Scan type: SIM
• Electron energy: 70eV
• Source temperature: 200°C
• CI gas: Methane
• Transfer Line: 310°C
• Mass table

Analyte	M-PFB [m/z]
PGE2	524
PGD2	524
PGF1a	569
6-keto-PGF1a	614
d4-PGE2	528
d4-PGD2	528
d4-PGF2a	573
d4-6-keto-PGF1a	618

Data analysis and quantification

Data handling

• Peak areas for the M-PFB anions and quantitative data are calculated by QuanBrowser.

Calibration and quantification

• calibration type: standard curve 
• Calibration concentrations: 0.313, 0.63, 1.25, 2.5, 5, 10 & 20 ng/ml 

Method validation

Accuracy

The accuracy of the method is between 100% and 120%

Precision

Method precision including sample preparation for 5 replicates is better than 10%. Instrument precision for 5 injections is better than 2%.

Limit of quantitation

The LLOQ is at 0.3125ng/ml

Reference

• Leis et al. Biomed Environ Mass Spectrom. 1987; 14(11):617-621