Schwedhelm et al.

From LipidomicsWiki

Abstract

Schwedhelm et al. developed two chromatographic procedures (TLC and HPLC) in combination with GC-tandem MS for the accurate quantification of dinor-dihydro F2- isoprostane metabolites in human urine investigating the formation and metabolism of 8-iso-PGF2a and other F2-isoprostanes as indicators of oxidative stress in vivo in humans.

Analysed Matrices

human urine

Analytes

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

Sample Preparation

urine samples containing 1mM HTMP (5-hydroxy-tempo) and 1 mM EDTA (ethylenedimine tetraacetic acid)
5 ml aliquots stored at -20°C until use
spectrophotometric determination of creatinine by an assay (based on alkaline picric acid reaction; automatic analyser)
extractions

addition of IS to 5 ml aliquots; 1 ng/ml and 2 ng/ml as final concentration; acidification to pH 3, applied to ODS cartridges preconditioned with 10 ml CH3OH and 3 ml HCOOH (0,05 M), washing of cartridges with 20 ml H2O and 2,5 ml heptane, elution with 2 mlethyl acetate, derivatization to PFB ester TMS ether derivatives using PFB bromide and BSTFA (100 μl)

TLC (TLC Applicator AS 30, DC-MAT, Desaga): reconstitution in EtOH (15 μl), chromatography on 20*20 cm silica plates; eluent: ethyl acetate/MeOH (98:2); reference compounds: PGF2a-4-fluorobenzyl ester for 8-iso-PGF2a-PBF derivative; PGF2a-methylester for ent-2,3-dinor-5,6-dihydro-8-iso-PGF2a-PFB derivatives

scrape off each 0,6-cm band from the TLC plate, extraction with 500 μl EtOH, centrifugation (4000 g, 10 min), decantation of supernatants, removing EtOH under nitrogen, converting of PFB esters to TMS ethers,

HPLC (HP 1050): column: 100-5C18 Nucleosil (250*4,6 mm, 5 μm, Macherey-Nagel); mobile phase: H2O/CH3CN (50:50); flow: isocratically, 2 ml/ min; detection wavelength: 235 nm; retention time: 6.02 ± 0.05 min (2,3-dinor-5,6-dihydro-8-iso-PGF2a-PFB); 12.08 ± 0.1 min (8-iso-PGF2a-PFB)

Analytical Method

GC-MS (Thermoquest GC Model Trace 2000, Thermoquest TSQ 7000)

triple-stage quadrupole, ionization: NICI
column: fused-silica capillary Optima 17 (30m*0,25 mm, 0,25 μm film thickness, Macherey-Nagel)
carrier gas: Helium
constant pressure: 55 kPa
p gas (NICI): 65 Pa Methane
p CAD gas: 0,15 Pa Argon
collision energy (CE): 25 eV
electron energy: 200 eV
emission current: 600 μA
T (injector): 280 °C; T (interface): 290 °C; T (ion source): 180 °C; T (column): 70/2-25-280-4-320
electron impact (EI): 70 eV

selected reaction monitoring (SRM) mode:

m/z 543→273 (2,3-dinor-5,6-dihydro-8-iso-PGF2a)

m/z 547→277 (18O-labelled analogue)

m/z 569→299 (8-iso-PGF2a)

GC-MS-MS (Thermoquest GC Model Trace 2000, Thermoquest TSQ 7000)

injection of aliquots (1 μl)
splitless mode

Method Validation

Accuracy

94±3 % (method A); 86 ±12 % (method B)

LOD

50 fg

LOQ

n.d.

Linearity

n.d.

Internal Standard (IS)

[3,3',4,4'-2H4]-8-iso-PGF2a (IS 1)
[1,1'-18O2]-ent-2,3-dinor-5,6-dihydro-8-iso-PGF2a (IS 2)

Reference

E. Schwedhelm, D. Triskas, T. Durand, F.-M. Gutzki, A. Guy, J.-C. Rossi, J. C. Frölich

Journal of Chromatography B 2000, 744, 99-112.

Institute of Clinical Pharmacology, Hannover Medical School, Carl-Neuberg Strasse 1, D- 30623 Hannover, Germany, Tel.: +49-511-5323-959; Fax: +49-511-5322-750; e-mail: tsikas.dimitros@mh-hannover.de