Bile acids/-conjugates determination - Tagliacozzi et al.

From LipidomicsWiki

(Redirected from Tagliacozzi et al.)
Jump to: navigation, search

Contents

Abstract

A robust and sensitive method has been developed based on high performance liquid chromatography separation coupled to an electrospray triple-quadrupole mass spectrometer. Human plasma samples were analysed on a C18 reverse- phase column. The elution profiles were monitored in multiple reaction-monitoring mode, quantifying and identifying each analyte by its own unique precursor to product patterns. A linear correlation over a broad range of bile acid concentrations (0.1–100 μM) was observed. The average recovery period for all of the analysed bile acids was 98±3%. Intra-day and inter- day precision averages were 2% and 5.4%, respectively. The determination was achieved within a single chromatographic run for all unconjugated, glycineand taurine-conjugated isomeric forms of bile acids.

Analysed Matrices

human plasma

Analytes

  • 14 bile acids

TUDCA, TCA, GCA, TCDCA, TDCA, GCDCA, GDCA, UDCA, TLCA, CA, GLCA, CDCA, DCA, LCA

Sample Preparation

For protein precipitation, 250 μl of plasma sample were mixed with 800 μl of acetonitrile, followed by 1 min vortex-mixing. After 15 min centrifugation at 13000 g, 900 μl of the supernatant were transferred to an autosampler vial and blown to dryness with nitrogen. The residue was dissolved with 125 μl of methanol and 125 μl of water (final dilution factor = 1.17). 40 μl of this solution (corresponding to 34.2 μl of the original plasma) were injected.

Analytical Method

HPLC-MS-MS

HPLC

  • “LC-200” binary HPLC pump (Perkin-Elmer, Norwalk, CT, USA)
  • Series200 autosampler (Perkin-Elmer)
  • column: Pinnacle ODS reverse-phase C18 (3 μm, 100 × 4.6 mm); flow:1 ml/ min; split: 1:20
  • elution gradient was optimised for the simultaneous separation of either unconjugated, or glycine- and taurine-conjugated BA within a single run
  • binary gradient consisted of three steps;

solvent A: water solution containing 0.012% formic acid and ammonium acetate at 5 mM

solvent B: methanol containing 0.012% formic acid and ammonium acetate at 5 mM.

MS API 365 (Applied Biosystems-SCIEX)

  • triple quadrupole mass spectrometer
  • ion evaporation mode
  • ionspray ionisation probe (sprayer voltage of –4500 V)
  • data analysis: MassChrom 1.1 software including Multiview 1.4 (Applied Biosystems), for chromatographic and spectral interpretation, and MacQuan 1.6 (Applied Biosystems) for the quantitative processing
  • negative ion mode
  • orifice voltage: –61 to –74 V (as automatically optimised for each single compound by the “Auto-Tune” option)
  • Tandem mass spectrometry: collisionally activated dissociation (CAD)
  • collision cell: 8 mTorr pressure of nitrogen as collision gas
  • multiple reaction monitoring (MRM) mode
  • Collision energies were adjusted with specific optimum values for each BA (automatically done by the “AutoTune” option)

Method Validation

LOD

0.001 - 0.008 µM

LOQ

n.d.

Linearity

0.1 - 100 µM

Internal Standard (IS)

no IS used

Reference

  • D.Tagliacozzi, A. F.Mozzi, B. Casetta, P. Bertucci, S. Bernardini, C. Di Ilio, A. Urbani, G. Federici Clin. Chem. Lab. Med. 2003, 41,1633-1641.

Laboratorio di Biochimica Clinica; Policlinico di Tor Vergata; Università di Roma “Tor Vergata”; Sez. di Spettrometria di Massa; Viale Oxford 81, 00133 Roma, Italy.

Fax: +39-06-20902357; E-mail: giorgio.federici@uniroma2.it

Personal tools
Create a book