Bile acids - LC-MS/MS - Scherer et al.

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== Reference  ==
== Reference  ==
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[http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=19325012 Scherer et al. Clin Chem. 2009 Jun;55(6):1218-22.]  
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*[http://www.ncbi.nlm.nih.gov/pubmed/19819765?ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum Scherer M, Gnewuch C, Schmitz G, Liebisch G. Rapid quantification of bile acids and their conjugates in serum by liquid chromatography-tandem mass spectrometry. J Chromatogr B Analyt Technol Biomed Life Sci. 2009 Nov 15;877(30):3920-5.]
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[[Category:LC-MS/MS]] [[Category:Triple_quadrupole]] [[Category:Sphingolipids_(SP)]] [[Category:Sphingoid_bases_(SP01)]] [[Category:ESI]] [[Category:HPLC]] [[Category:Glycerophospholipids_(GP)]]
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[[Category:LC-MS/MS]] [[Category:Triple_quadrupole]] [[Category:ESI]] [[Category:HPLC]] [[Category:Bile_acids_and_derivatives_(ST04)]]

Current revision

Contents

Sample preparation

  • 10µL of a methanolic IS-mixture were added to plasma
  • 30µL of 1mol/L HCl were added to serum/plasma
  • protein precipitation was performed with 1mL acetonitrile, followed by 1min vortex-mixing
  • after 15min of centrifugation (14,000×g), the supernatant was evaporated to dryness under reduced pressure
  • samples were re-dissolved in 100µL methanol/water (1/1, v/v, containing 10mmol/L ammonium acetate and 0.1% ammonium hydroxide)
  • after centrifugation the supernatant was used for analysis
Material Material used Internal Standard(s) Internal Standard(s) added
Human plasma or serum 100µl 2.4µmol/L D4-CA 10µl
2.6µmol/L D4-DCA
4µmol/L D4-CDCA
0.6µmol/L D4-LCA
2.6µmol/L D4-UDCA
4.6µmol/L D4-GCA
14µmol/L D4-GCDCA

Instrumentation and method

Pump

  • Type: binary and isocratic pump (Agilent 1200)
  • Mode: gradient elution
  • Solvent(s): Solvent A methanol/water (1/1, v/v) containing 0.1% ammoniumhydroxide (25%) and 10mmol/L ammonium acetate (pH 9); Solvent B methanol containing 0.1% ammoniumhydroxide (25%) and 10mmol/L ammonium acetate (pH 9)
  • column flow was directed only from 1.0 to 5.0min into the mass spectrometer; otherwise methanol with a flow rate of 250µL/min was delivered
  • Gradient:
Time [min] Flow [ml/min]  % Solvent A  % Solvent B
0 0.5 100 0
0.5 0.5 100 0
4.5 0.5 50 50
4.6 0.5 0 100
5.5 0.5 0 100
5.6 0.5 100 0
6.5 0.5 100 0

Autosampler

  • Type: CTC Pal
  • Injection volume: 5µl
  • Wash solvent 1: Isopropanol/MeOH (1/1; v/v), Wash solvent 2: MeOH

Column

  • Type: NUCLEODUR C18 Gravity Macherey-Nagel
  • length: 50 x 2.1 mm i.d., 0.5µm pre-column filter
  • Particle size: 1.8 µm
  • Column temperature: 50°C

Mass spectrometer

  • Type: Triple quadrupole (Applied Biosystems, 4000 QTRAP)
  • Ionization mode: ESI negative
  • Ionization voltage: -4500V
  • Source temperature: 450°C
  • Collision gas: Nitrogen
  • Collision gas pressure: medium
  • MS/MS-conditions: multiple reaction monitoring
Analyte Precursor [m/z] Product [m/z] Collision energy [eV]
CA 407.3
407.3
−30
CDCA 391.3
391.3
−30
DCA 391.3
391.3
−30
LCA 375.3
375.3
−30
UDCA 391.3
391.3
−30
HDCA 391.3
391.3
−30
GCA
464.3
74
−72
GCDCA
448.3
74
−70
GDCA
448.3
74
−70
GLCA
432.3
74
−64
GUDCA
448.3
74
−70
GHDCA
448.3
74
−70
TCA
514.3
80
−116
TCDCA
498.3
80
−116
TDCA
498.3
80
−116
TLCA
482.3
80
−108
TUDCA
498.3
80
−116
THDCA
498.3
80
−116

Data analysis and quantification

Software

  • Analyst 1.4.2.

Calibration and quantification

  • calibration type: matrix calibration - addition of naturally occurring species
  • species used for calibration
Analyte Human serum/plasma up to (μM)
CA 4.4
CDCA 8
DCA 5.2
LCA 0.4
UDCA 4.7
HDCA 4.7
GCA
9.4
GCDCA
28.2
GDCA
3.2
GLCA
0.2
GUDCA
0.32
GHDCA
0.32
TCA
2.8
TCDCA
9.2
TDCA
0.77
TLCA
0.2
TUDCA
0.32
THDCA
0.32

Method Validation

Accuracy

89 - 111 %

Precision

CV: 4 - 11 %

LOD

below 10 nmol/L

Recovery

above 80%

Sample data

Mass spectra

Chromatogram 


Reference

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