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

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== Abstract ==
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== Sample preparation  ==
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In order to allow parallel analysis of free cholesterol (FC) and cholesteryl ester (CE)
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using ESI-MS/MS Liebisch et al. developed an acetyl chlorid  derivatization method
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to convert FC to cholesterylacetate (CE 2:0).
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This method shows a precision and detection limit sufficient for routine analysis and
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due to the short run time of 1,3 min paired with an automated data analysis it allows
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high-throughput analysis.
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== Analysed Matrices ==
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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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== Analytes ==
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{| class="wikitable" style="text-align: center;"
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|-
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! Material
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! Material used
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! Internal Standard(s)
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! Internal Standard(s) added
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|-
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! Cultured cells
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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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|-
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! Human plasma
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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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|}
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== Sample Preparation ==
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== Instrumentation and  method ==
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== Analytical Method ==
 
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==== ESI-MS-MS ====
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=== Pump ===
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* Type: binary high pressure gradient (Agilent 1100)
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* Mode: isocratic flow gradient
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* Solvent(s): Methanol containing 10 mM ammonium acetate/chloroform (3/1 = v/v)
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* Flow gradient:
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{| border="1" class="wikitable" style="text-align:center;"
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! Time [min]!! Flow [ml/min]  !! % Solvent A !! % Solvent B
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|-
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! 0
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| 0.05 || 100 ||0
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|-
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! 0.1
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| 0.03 || 100 ||0
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|-
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! 1.1
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| 0.2 || 100 ||0
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|-
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! 1.3
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| 0.05 || 100 ||0
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|}
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== Method Validation ==
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=== Autosampler ===
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* Type: CTC Pal
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* Injection volume: 20µl
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* Wash solvent: methanol/chloroform = 1/1
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=== Accuracy ===
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=== Mass spectrometer ===
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* Type: Triple quadrupole (Micromass, Quattro Ultima)
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* Ionization mode: ESI positive
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* Ionization voltage: 3500 V
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* Source temperature: 220°C
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* Collision gas: Argon
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* Collision gas pressure: 1.0 10-3 Torr
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* Collision energy: 13 V
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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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|}
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== Data analysis and quantification ==
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=== Data handling ===
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* combine spectra above half peak height
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* smooth combined spectrum (if necessary)
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* centroid combined spectrum
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* pick peak intensities
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=== Isotope correction ===
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* using Excel Macros correcting the peak intensities in a sequential algorithm starting from low mass species
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* five isotope peaks including the monoisotopic were used
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* 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.]
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=== Calibration and quantification ===
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* calibration type: matrix calibration - addition of naturally occurring species
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* species used for calibration
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{| class="wikitable" style="text-align:center"
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! Species!! Cultured cells !! Human plasma
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|-
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! FC
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| 0 - 3 nmol || 0 - 10 nmol
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|-
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! CE 16:0
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| 0 - 500 pmol || 0 - 10 nmol
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|-
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! CE 18:0
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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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|}
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== Method validation ==
=== Precision ===
=== Precision ===
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* CV within-run: 3 %(major), 5-10 % (minor)
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* CV total: 10 % (major), 15-20 % (minor)
=== LOD ===
=== LOD ===
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* 0.5 µM in plasma
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== Sample data ==
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=== LOQ ===
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=== Mass spectra ===
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=== Recovery ===
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== Reference  ==
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== Internal Standard ==
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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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== Reference ==
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[[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

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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