Haschke et al.

From LipidomicsWiki

Abstract

Haschke et al. reported the development and validation of an HPLC-MS/MS assay for the quantification of 15-F2t-IsoP with automated online extraction. The manual sample preparation is limited to a simple protein precipitation step.

Analysed Matrices

human plasma, human urine

Analytes

8-iso-PGF2a
PGF2a
15(R)-PGF2a
15(R)-F2t-IsoP; 15-F2t-IsoP-d4
9b,11a-15-F2t-IsoP
5-trans-PGF2a
9b,11a -PGF2; 9a,11b -PGF2
5-trans-9b,11a-PGF2

Sample Preparation

addition of 500 μl protein precipitation solution (MeOH/0.2 M ZnSO4; 7/3) containing 4 μg/l IS to 500 μl urine or plasma
vortexing (1 min) and centrifugation (13000 g, 10 min, 4 °C)
supernatant for HPLC analysis

Analytical Method

HPLC-MS/MS

HPLC (Agilent 1100 series)

6-port Rheodyne column switching valve

injection volume: 500 μl

extraction column: Eclipse XDB-C8 (4,6*12,5 mm, 5 μm, Agilent)

mobile phase: 30 % MeOH, 70 % HCOOH (0,1 %)

flow rate: 5 ml/ min

after 1 min back-flushing of analystes from the extraction column via switching valve onto the analytical column

analytical column: Synergi Hydro-RP (3,0*250 mm, 4 μm, Phenomenex)

mobile phase: MeOH, HCOOH (0,1 %)

flow rate: 0,6 ml/min

gradient: 63 % MeOH (0-1 min); 63-98 % MeOH (1.1-10 min); 98 % MeOH (10-11 min); 63 % MeOH reequillibration

column switching into the extraction position after 9 min
total run time between injections: 13 min
MS (API 4000, API 5000)

triple quadrupole; atmospheric pressure chemical ionization (APCI) source

CAD gas: N2

negative multiple reaction monitoring (MRM) mode

DP: -70 V; EP: -5 V; interface: 400 °C; CE: 36 V

m/z 353 -> 193 (15-F2t-IsoP)

m/z 357 -> 197(15-F2t-IsoP-d4)

Analyst software (Applied Biosystems) for examination

Method Validation

Accuracy

Precision

</= 15 % (for intraday and interday variability)

LOD

LLOQ

0,025 μg/l (API 5000, urine); 0,04 μg/l (API 4000, urine)

0,0025 μg/l (API 5000, plasma); 0,01 (API 4000, plasma)

Linearity

urine: 0,025 μg/l - 80 μg/l (API 5000); 0,04 μg/l - 80 μg/l (API 4000)

plasma: 0,0025 μg/l - 80 μg/l (API 5000); 0,01 - 80 μg/l (API 4000)

Internal Standard (IS)

15-F2t-IsoP-d4

Reference

M. Haschke, Y. L. Zhang, C. Kahle, J. Klawitter, M. Korecka, L. Shaw, U. Christians

Clinical Chemistry 2007, 53:3, 489-497.

Clinical Research and Development, Department of Anesthesiology, University of Colorado Health Sciences Center, 4200 East Ninth Ave., Room UH-2122, Campus Box B-113, Denver, Colorado 80262; Fax: 303-315-1858; e-mail: uwe.christians@uchsc.edu.