Cholesterol / Cholesterylester - ESI-MS/MS - Liebisch et al.
From LipidomicsWiki
(Difference between revisions)
(→Mass spectrometer) |
Current revision (18:07, 1 April 2009) (view source) |
||
| (2 intermediate revisions not shown.) | |||
| Line 1: | Line 1: | ||
| - | == Sample preparation == | + | == Sample preparation == |
| - | * [[Lipid extraction according to Bligh and Dyer]] | + | *[[Lipid extraction according to Bligh and Dyer|Lipid extraction according to Bligh and Dyer]] |
| - | * internal standards are added prior lipid extraction | + | *internal standards are added prior lipid extraction |
| - | * to avoid the loss of lipids, extraction | + | *to avoid the loss of lipids, extraction is performed in glassware |
| - | * samples are dissolved in methanol containing 10 mM ammonium acetate/chloroform (3/1 = v/v) | + | *dried samples are derivatized by addition of 200 μl of acetyl chloride/chloroform = 1/5 (v/v) for 60 min at room temperature |
| + | *derivatization reagents are removed by vacuum centrifugation | ||
| + | *samples are dissolved in methanol containing 10 mM ammonium acetate/chloroform (3/1 = v/v) | ||
| - | {| class="wikitable" style="text-align:center" | + | {| class="wikitable" style="text-align: center;" |
| - | ! Material | + | |- |
| + | ! Material | ||
| + | ! Material used | ||
| + | ! Internal Standard(s) | ||
| + | ! Internal Standard(s) added | ||
|- | |- | ||
| - | ! Cultured cells | + | ! Cultured cells |
| - | | 100µg protein | | + | | 100µg protein |
| + | | D7-FC, CE 17:0, CE 22:0 | ||
| + | | 3000ng D7-FC; 500ng each CE 17:0, CE 22:0 | ||
|- | |- | ||
| - | ! Human plasma | + | ! Human plasma |
| - | | 20µl | | + | | 20µl |
| + | | D7-FC, CE 17:0, CE 22:0 | ||
| + | | 10µg each D7-FC, CE 17:0, CE 22:0 | ||
|} | |} | ||
| Line 119: | Line 129: | ||
=== Mass spectra === | === Mass spectra === | ||
| - | == Reference == | + | == Reference == |
| - | * [http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=16458590 Liebisch et al. Biochim Biophys Acta. 2006 Jan;1761(1):121-8.] | + | |
| + | *[http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=16458590 Liebisch et al. Biochim Biophys Acta. 2006 Jan;1761(1):121-8.] | ||
| - | [[Category: | + | [[Category:Flow_injection_analysis]] [[Category:ESI]] [[Category:Triple_quadrupole]] [[Category:Sterols_(ST01)]] |
| - | [[Category: | + | |
| - | [[Category: | + | |
Current revision
Contents |
Sample preparation
- Lipid extraction according to Bligh and Dyer
- internal standards are added prior lipid extraction
- to avoid the loss of lipids, extraction is performed in glassware
- dried samples are derivatized by addition of 200 μl of acetyl chloride/chloroform = 1/5 (v/v) for 60 min at room temperature
- derivatization reagents are removed by vacuum centrifugation
- samples are dissolved in methanol containing 10 mM ammonium acetate/chloroform (3/1 = v/v)
| Material | Material used | Internal Standard(s) | Internal Standard(s) added |
|---|---|---|---|
| Cultured cells | 100µg protein | D7-FC, CE 17:0, CE 22:0 | 3000ng D7-FC; 500ng each CE 17:0, CE 22:0 |
| Human plasma | 20µl | D7-FC, CE 17:0, CE 22:0 | 10µg each D7-FC, CE 17:0, CE 22:0 |
Instrumentation and method
Pump
- Type: binary high pressure gradient (Agilent 1100)
- Mode: isocratic flow gradient
- Solvent(s): Methanol containing 10 mM ammonium acetate/chloroform (3/1 = v/v)
- Flow gradient:
| Time [min] | Flow [ml/min] | % Solvent A | % Solvent B |
|---|---|---|---|
| 0 | 0.05 | 100 | 0 |
| 0.1 | 0.03 | 100 | 0 |
| 1.1 | 0.2 | 100 | 0 |
| 1.3 | 0.05 | 100 | 0 |
Autosampler
- Type: CTC Pal
- Injection volume: 20µl
- Wash solvent: methanol/chloroform = 1/1
Mass spectrometer
- Type: Triple quadrupole (Micromass, Quattro Ultima)
- Ionization mode: ESI positive
- Ionization voltage: 3500 V
- Source temperature: 220°C
- Collision gas: Argon
- Collision gas pressure: 1.0 10-3 Torr
- Collision energy: 13 V
- MS/MS-mode:
- selected reaction monitoring (SRM) for acetylated free cholesterol (FC) and D7-FC
- precursor ion scan m/z 369.3 for cholesteryl ester (CE species)
| Analyte | Precursor [m/z] | Precursor [m/z] | Collision energy [eV] |
|---|---|---|---|
| CE 2:0 (acetylated FC) | 446.4 | 369.3 | 13 V |
| D7-CE 2:0 (acetylated D7-FC) (IS) | 453.4 | 376.3 | 13 V |
| CE species | Scan 610 - 730 | 369.3 | 13 V |
Data analysis and quantification
Data handling
- combine spectra above half peak height
- smooth combined spectrum (if necessary)
- centroid combined spectrum
- pick peak intensities
Isotope correction
- using Excel Macros correcting the peak intensities in a sequential algorithm starting from low mass species
- five isotope peaks including the monoisotopic were used
- the detailed algorithm is described in the appendix of Liebisch et al. Biochim Biophys Acta. 2004 Nov 8;1686(1-2):108-17.
Calibration and quantification
- calibration type: matrix calibration - addition of naturally occurring species
- species used for calibration
| Species | Cultured cells | Human plasma |
|---|---|---|
| FC | 0 - 3 nmol | 0 - 10 nmol |
| CE 16:0 | 0 - 500 pmol | 0 - 10 nmol |
| CE 18:0 | 0 - 500 pmol | 0 - 10 nmol |
| CE 18:1 | 0 - 500 pmol | 0 - 10 nmol |
| CE 18:2 | 0 - 500 pmol | 0 - 10 nmol |
Method validation
Precision
- CV within-run: 3 %(major), 5-10 % (minor)
- CV total: 10 % (major), 15-20 % (minor)
LOD
- 0.5 µM in plasma
