Cholesterol / Cholesterylester - ESI-MS/MS - Liebisch et al.

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== Sample preparation  ==
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==== ESI-MS-MS ====
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*[[Lipid extraction according to Bligh and Dyer|Lipid extraction according to Bligh and Dyer]]
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*internal standards are added prior lipid extraction
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*to avoid the loss of lipids, extraction is performed in glassware
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*dried samples are derivatized by addition of 200 μl of acetyl chloride/chloroform = 1/5 (v/v) for 60 min at room temperature
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*derivatization reagents are removed by vacuum centrifugation
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*samples are dissolved in methanol containing 10 mM ammonium acetate/chloroform (3/1 = v/v)
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* electrospray ion source
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{| class="wikitable" style="text-align: center;"
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* positive ion mode
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|-
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* triple quadrupole
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! Material  
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* direct flow injection analysis
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! Material used  
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* injection volume: 20 μl
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! Internal Standard(s)  
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* solvent mixture: 10 mM ammonium acetate in MeOH/CHCl3 (3/1)
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! Internal Standard(s) added
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* gradient: 55 μl/min (0,1 min); 30 μl/min (1 min); 250 μl/min (0,2 min)
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* capillary voltage: 3500 V
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* cone voltage: 50 V
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* collision energy: 13 V
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* collision gas: Ar (1,0*10^-3 torr)
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* 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)
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fragment ion: m/z 372 (13C3-FC; 13C3-CE)
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precursor ion scanning (PIS; m/z 369,3) for CE
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* mass resolution above unit resolution
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* data analysis: MassLynx software including NeoLynx tool (Micromass)
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* data aquisition: 1,3 min
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== Method Validation ==
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=== Accuracy ===
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=== Precision ===
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within-run: 3 %(major), 5-10 % (minor); total: 10 % (major), 15-20 % (minor)
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=== LOD ===
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» 0,5 μM
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=== LOQ ===
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=== Recovery ===
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== Internal Standard ==
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CE 17:0 ; CE 22:0; D7-FC
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== Reference ==
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* G.Liebisch, M. Binder, R. Schifferer, T. Langmann, B. Schulz, G. Schmitz ''Biochemica et Biophysica Acta'' '''2006''', ''1761'', 121-128.
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-
 
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== Sample preparation ==
+
-
 
+
-
* [[Lipid extraction according to Bligh and Dyer]]
+
-
* internal standards are added prior lipid extraction
+
-
* to avoid the loss of lipids, extraction was performed in glassware
+
-
* samples are dissolved in methanol containing 10 mM ammonium acetate/chloroform (3/1 = v/v)
+
-
 
+
-
{| class="wikitable" style="text-align:center"
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! Material !! Material used !! Internal Standard(s) !! Internal Standard(s) added
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|-
|-
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! Cultured cells
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! Cultured cells  
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| 100µg protein || LPC 13:0, LPC 19:0 || 50ng each
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| 100µg protein  
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| D7-FC, CE 17:0, CE 22:0  
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| 3000ng D7-FC; 500ng each CE 17:0, CE 22:0
|-
|-
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! Human plasma
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! Human plasma  
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| 20µl ||  LPC 13:0, LPC 19:0 || 1500ng each
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| 20µl  
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| D7-FC, CE 17:0, CE 22:0  
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| 10µg each D7-FC, CE 17:0, CE 22:0
|}
|}
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* Ionization mode: ESI positive
* Ionization mode: ESI positive
* Ionization voltage: 3500 V
* Ionization voltage: 3500 V
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* Source temperature: 300°C
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* Source temperature: 220°C
* Collision gas: Argon
* Collision gas: Argon
* Collision gas pressure: 1.0 10-3 Torr
* Collision gas pressure: 1.0 10-3 Torr
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* Collision energy: 24 V
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* Collision energy: 13 V
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* MS/MS-mode: precursor ion scan of m/z 184.1
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* MS/MS-mode:  
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** selected reaction monitoring (SRM) for acetylated free cholesterol (FC) and D7-FC
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** precursor ion scan ''m/z'' 369.3 for cholesteryl ester (CE species)
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{| border="1" class="wikitable" style="text-align:center"
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! Analyte !! Precursor [m/z] !! Precursor [m/z] !! Collision energy [eV]
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|-
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! CE 2:0 (acetylated FC)
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| 446.4 || 369.3 ||13 V
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|-
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! D7-CE 2:0 (acetylated D7-FC) (IS)
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| 453.4 || 376.3 ||13 V
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|-
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! CE species
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| Scan 610 - 730|| 369.3 ||13 V
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|-
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|}
== Data analysis and quantification ==
== Data analysis and quantification ==
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! Species!! Cultured cells !! Human plasma  
! Species!! Cultured cells !! Human plasma  
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|-
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! LPC 16:0
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! FC
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| 0 - 100 pmol || 0 - 1000 pmol
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| 0 - 3 nmol || 0 - 10 nmol
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|-
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! LPC 18:0
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! CE 16:0
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| 0 - 100 pmol || 0 - 1000 pmol
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| 0 - 500 pmol || 0 - 10 nmol
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|-
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! LPC 18:1
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! CE 18:0
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| 0 - 100 pmol || 0 - 1000 pmol
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| 0 - 500 pmol || 0 - 10 nmol
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|-
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! CE 18:1
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| 0 - 500 pmol || 0 - 10 nmol
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|-
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! CE 18:2
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| 0 - 500 pmol || 0 - 10 nmol
|}
|}
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=== LOD ===
=== LOD ===
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* 0.6 - 0.8 µM in plasma
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* 0.5 µM in plasma
== Sample data ==
== Sample data ==
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=== Mass spectra ===
=== Mass spectra ===
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== Reference ==
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== Reference ==
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* [http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=16458590 Biochim Biophys Acta. 2006 Jan;1761(1):121-8.]
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*[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.]
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[[Category:Flow injection analysis]]
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[[Category:Flow_injection_analysis]] [[Category:ESI]] [[Category:Triple_quadrupole]] [[Category:Sterols_(ST01)]]
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[[Category:Triple quadrupole]]
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[[Category:Sterols (ST)]]
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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

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

Sample data

Mass spectra

Reference

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