Davies et al.

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Contents 1 Abstract 2 Analysed Matrices 3 Analytes 4 Pre-Analysis 4.1 Prearrangement 4.2 Sampling 4.3 Preservation and Storage 4.4 Sample Preparation 5 Analytical Method 5.1 HPLC-MS/MS 6 Method Validation 6.1 Accuracy 6.2 Precision 6.3 LOD 6.4 LOQ 6.5 Linearity 7 Internal Standard (IS) 8 Reference

Abstract

Davies et al. reported the development of a high-performance LC-MS/MS method to analyze all of the dinor, dihydro metabolites of the F2-IsoP isomeres. Compared to previous described GC-MS assays, this method provides an accurate and rapid assay to assess oxidative status in vivo being significantly less costly and time consuming.

Analysed Matrices

human urine

Analytes

• 8-iso-PGF2a
• 2,3-dinor-5,6-dihydro-8-iso-PGF2a

Pre-Analysis

Prearrangement

Sampling

Preservation and Storage

Sample Preparation

• addition of 10 ng IS to 3 ml H2O and then 0,25 ml urine samples
• acidification by HCl (1 N) to pH 3
• 1 ml aliquots urine aliquots (< 0,5 mg creatinine/ ml urine; Jaffe method)

SPE: C18 Sep-Pak cartridge (Waters)

equilibration: 5 ml MeOH, 5 ml H2O, pH 3

sample wash: 10 ml H2O (pH 3), 8 ml heptane

elution: 10 ml heptane/ ethyl acetate (1:1)

drying with Na2SO4

loading on Sep-Pak cartridge preequilibrated with 5 ml ethyl acetate

sample wash: 5 ml ethyl acetate

elution: 5 ml ethyl acetate/ MeOH (1:1)

sample drying under N2 and resuspended in 100 μl of NH4OAc (5mM) with 0,1 % acetic acid

Analytical Method

HPLC-MS/MS

• HPLC (ThermoFinnigan)

Surveyor MS pump, Surveyor autosampler

injection volume: 10 μl

mobile phase: A ( NH4OAc (5mM) with 0,1 % acetic acid); B (CH3CN/MeOH 95:5)

column: Magic C18 AQ (25 * 4 mm, 3 μm, Michrom BioResources)

flow rate: constant, 190 μl/min

linear gradient: 5 % B in A (0,00 min), 100 % B (0,25-1,75 min), 5 % B (2,5-5 min)

• MS (ThermoFinnigan TSQ)

triple quadrupole

electrospray ionization source (ESI)

N2 used as sheath (49) and auxiliary gas (21)

negative ion mode

electrospray needle: -4000 V

ion transfer tube: -35 V, 270 °C

tube lens voltage: -147 V

selected reaction monitoring (SRM) mode: m/z 327 -> 283, CE: 24 eV (F2-IsoP-M)

m/z 331 -> 287, CE: 24 eV ([18O2]15-F2t-IsoP-M)

examination: Xcaliber software (ThermoFinnigan) with 15-pint Gaussian smoothingand GraphPad Prism version 4.0 (Windows)

Method Validation

Accuracy

80%

Precision

± 5,0 %

LOD

20 pg

LOQ

n.d.

Linearity

1-100 ng

Internal Standard (IS)

• [18O2]15-F2t-IsoP-M

Reference

• S. S. Davies, W. Zackert, Y. Luo, C. C. Cunningham, M. Frisard, L. J. Roberts II

Analytical Biochemistry 2006, 348, 185-191.

Department of Pharmacology, Vanderbilt University, Nashville, TN 27232, USA; Fax: +1-615-322-3669; e-mail: sean.davies@vanderbilt.edu (S. S. Davies)