Cholesterol / Cholesterylester - ESI-MS/MS - Liebisch et al.
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Contents |
Abstract
In order to allow parallel analysis of free cholesterol (FC) and cholesteryl ester (CE) using ESI-MS/MS Liebisch et al. developed an acetyl chlorid derivatization method to convert FC to cholesterylacetate (CE 2:0). This method shows a precision and detection limit sufficient for routine analysis and due to the short run time of 1,3 min paired with an automated data analysis it allows high-throughput analysis.
Analysed Matrices
EDTA-plasma
Analytes
CE 14:0; CE 15:0; CE 16:0; CE 16:1; CE 17:1; CE 18:3; CE 18:2; CE 18:1; CE 18:0; CE 19:0; CE 20:5; CE 20:4; CE 20:3; CE 20:0; CE 22:6; CE 22:5
Sample Preparation
- 100 μl of CHCl3-solution (containing 100 μg/ml of CE 17:0 and CE 22:0)
- evaporation of solvent
- lipid extraction of 20 μl EDTA-plasma according to Bligh and Dyer (1959)
- drying of CHCl3-phase
- derivatization by adding 200 μl acetyl chloride/CHCl3 (1/5), 60 min, room temperature
- vacuum centrifugation
- dissolvation of residue in NH4OAc(10 mM) in MeOH/CHCl3 (3/1) resulting in a 200-fold dilution corresponding to initial plasma volume
Analytical Method
ESI-MS-MS
- electrospray ion source
- positive ion mode
- triple quadrupole
- direct flow injection analysis
- injection volume: 20 μl
- solvent mixture: 10 mM ammonium acetate in MeOH/CHCl3 (3/1)
- gradient: 55 μl/min (0,1 min); 30 μl/min (1 min); 250 μl/min (0,2 min)
- capillary voltage: 3500 V
- cone voltage: 50 V
- collision energy: 13 V
- collision gas: Ar (1,0*10^-3 torr)
- scan modi: selected reaction monitoring (SRM) for FC m/z 446,4 -> 369,3 (CE 2:0); m/z 453,4 -> 376,3 (D7-CE 2:0)
fragment ion: m/z 372 (13C3-FC; 13C3-CE)
precursor ion scanning (PIS; m/z 369,3) for CE
- mass resolution above unit resolution
- data analysis: MassLynx software including NeoLynx tool (Micromass)
- data aquisition: 1,3 min
Method Validation
Accuracy
Precision
within-run: 3 %(major), 5-10 % (minor); total: 10 % (major), 15-20 % (minor)
