Cholesterol / Cholesterylester - ESI-MS/MS - Liebisch et al.
From LipidomicsWiki
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- | == | + | == Sample preparation == |
- | + | ||
- | + | ||
- | + | ||
- | + | ||
- | + | ||
- | + | ||
- | = | + | *[[Lipid extraction according to Bligh and Dyer|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) | ||
- | == | + | {| class="wikitable" style="text-align: center;" |
- | + | |- | |
- | CE | + | ! Material |
- | CE | + | ! Material used |
- | + | ! Internal Standard(s) | |
- | CE 17: | + | ! Internal Standard(s) added |
- | CE | + | |- |
- | + | ! Cultured cells | |
- | + | | 100µg protein | |
- | CE | + | | D7-FC, CE 17:0, CE 22:0 |
- | CE | + | | 3000ng D7-FC; 500ng each CE 17:0, CE 22:0 |
- | CE | + | |- |
- | + | ! Human plasma | |
- | + | | 20µl | |
- | + | | D7-FC, CE 17:0, CE 22:0 | |
- | CE 22: | + | | 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: | ||
+ | {| border="1" class="wikitable" style="text-align:center;" | ||
+ | ! 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) | ||
+ | |||
+ | {| border="1" class="wikitable" style="text-align:center" | ||
+ | ! 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 [http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=15522827 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 | ||
+ | {| class="wikitable" style="text-align:center" | ||
+ | ! 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 === | === Precision === | ||
+ | * CV within-run: 3 %(major), 5-10 % (minor) | ||
+ | * CV total: 10 % (major), 15-20 % (minor) | ||
=== LOD === | === LOD === | ||
+ | * 0.5 µM in plasma | ||
+ | |||
+ | == Sample data == | ||
- | === | + | === Mass spectra === |
- | == | + | == 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.] |
- | + | [[Category:Flow_injection_analysis]] [[Category:ESI]] [[Category:Triple_quadrupole]] [[Category:Sterols_(ST01)]] |
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