Sircar et al.

From LipidomicsWiki

Contents 1 Abstract 2 Analysed Matrices 3 Analytes 4 Sample Preparation 5 Analytical Method 5.1 HPLC-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

Isoprostanes have emerged as one of the most important noninvasive markers of oxidative stress associated with the development of several deseases (heart desease, cancer, Alzheimer). Sircar et al. developed a simple and sensitive HPLC-MS method for the quantification of Isoprostane F2a involving only one step sample preparation.

Analysed Matrices

human plasma, human urine

Analytes

• 8-iso-PGF2a
• 8-iso-PGF2a-d4

Sample Preparation

• extraction from plasma:

addition of 200 pg IS and 0.3 ml KOH (15 %) to 0,3 ml freshly thawed plasma

incubation of sample for 60 min at 40 °C

neutralization by 1 ml KH2PO4 (1M) to pH 7,2- 7,4

loading onto the isoprostane affinity column

washing: 5 ml phosphate buffer (0,1 M) and 5 ml ultrapure H2O

elution: 2 ml EtOH (95%)

evaporation to dryness under vacuum and redissolvation in 60 μl 20 % CH3CN in H2O containing (1 mg/ml acetic acid)

regeneration of affinity column by washing with 5l ultrapure H2O and 5 ml phosphate buffer(0,1 M) containing 0,05 % NaN3

• extraction from urine:

dilution of 0,3 ml urine with 2 ml KH2PO4 (0,1 M, pH 7,4)

addition of 200 pg IS

loading onto the isoprostane affinity column directly without KOH traetment

elution: 2 ml EtOH (95%)

Analytical Method

HPLC-MS

• HPLC (ThermoFinnigan)

injection volume: 20 μl

column: Altima HP C18 (2,1*100 mm, 3 μm, Alltech); guard column: 10 mm* 2,1 mm

linear gradient: CH3CN in H2O (20 % to 45 % in 25 min)

flow rate: 0,2 ml/ min

reequillibration: 20 % CH3CN in 20 min

total run time between injections: 45 min

T (column): 30 °C

T (sample tray): 4 °C

• MS (Surveyor MSQ)

single quadruplole analyser, ESI probe

negative ESI mode

drying gas flow (N2): 650 l/h

needle voltage: 2300 eV

probe temperature: 400 °C

cone voltage: 60 eV

detector voltage: 1953 V

selected ion monitoring (SIM) mode

m/z 353.2 ( 8-iso-PGF2a)

m/z 357.2 ( 8-iso-PGF2a-d4)

Xcaliber 1.4 software for Windows (Thermo Finnigan)

Method Validation

Accuracy

Precision

4,68 % (intraday)

3,88 % (interday)

LOD

0,5 ng/l

LOQ

30 ng/l

Linearity

1-5000 ng/l

Internal Standard (IS)

• 8-iso-PGF2a-d4

Reference

• D. Sircar, P. V. Subbaiah

Clinical Chemistry 2007, 53:2, 251-258.

Department of Medicine, 1819 West Polk St., M/C 797, Chicago, IL 60612; Fax: 312-413-0435; e-mail: psubbaia@uic.edu.