Rapid preparation of human urine and plasma samples for analysis of F 2 -isoprostanes by gas chromatography-mass spectrometry q Chung-Yung J. Lee, Andrew. M. Jenner, and Barry Halliwell * Department of Biochemistry, Faculty of Medicine, National University of Singapore, 8 Medical Drive, Singapore 117597, Singapore Received 31 May 2004 Available online 19 June 2004 Abstract Reliable MS-based methods have been developed for the measurement of free and esterified F 2 -isoprostanes. However, prior to sample analysis several steps of purification, including solid-phase extraction followed by TLC or HPLC, are usually required, making it tedious to analyze large sample numbers, e.g., for population studies. We report a quick sample purification method using anion exchange solid phase extraction (SPE), which is highly selective for acidic compounds. Urine and hydrolyzed plasma of healthy individuals were acidified before SPE extraction, washed with 4 different solvent mixtures and finally eluted with ethyl acetate. The eluted samples were first derivatized with pentafluorobenzyl bromide followed by a second derivatization with bis- (trimethylsilyl)trifluoroacetamide. F 2 -isoprostanes were analyzed by GC-MS-NCI. The method was highly sensitive; the limit of detection at 5:1 signal-to-noise ratio was 0.037 ng/ml and 0.007 ng/mg creatinine for plasma and urine, respectively. Anion exchange SPE extraction for F 2 -isoprostane showed recovery of 55–65% and high linearity for concentration 0–1.0 ng/ml for urine (CV ¼ 4.08%, r 2 ¼ 0.990) and 0–0.5 ng/ml for plasma (CV ¼ 4.07%, r 2 ¼ 0.998). Fasting for 6 h significantly increased plasma F 2 - isoprostanes levels, which has implications for the design of intervention studies using this biomarker. Ó 2004 Elsevier Inc. All rights reserved. Keywords: F 2 -isoprostane; 8-Iso-PGF 2a ; iPF 2a ; GC-MS-NCI; Oxidative stress; SPE F 2 -isoprostanes are a family of eicosanoids produced by non-enzymatic free radical oxidation of arachidonic acid esterified to phospholipids [1,2]. Several F 2 -iso- prostanes, including 8-iso-PGF 2a , are biologically ac- tive, e.g., 8-iso-PGF 2a is a potent vasoconstrictor in renal circulation and pulmonary artery [3]. In addition, higher levels of F 2 -isoprostanes in humans have been related to oxidative stress due to smoking [4], cardio- vascular disease [5], diabetes [6], and liver diseases [7]. F 2 -isoprostanes are produced in the esterified form in tissues and rapidly hydrolyzed to the free acid form, which later enters the circulatory system. F 2 -isopros- tanes are quickly removed from the circulation and they and their metabolites are excreted in the urine within 4 h [8]. Measurement of F 2 -isoprostanes and their metabo- lites is considered to be a reliable marker for assaying lipid peroxidation in vivo, recommended for studies of lipid peroxidation in human disease and for assessment of the in vivo effects of antioxidants [1,2,9,10]. It is also proposed as a risk marker for coronary heart disease [9]. Many methods have been developed previously such as radioimmunoassay [11], GC-MS [12], and LC-MS/MS [13] but the MS-based methods appear the most ro- bust (reviewed in [14]). Although these methods are valuable to quantitate F 2 -isoprostanes, sample prepa- ration requires numerous time-consuming purification steps, which makes it difficult to apply the method to q Abbreviations: SPE, solid phase extraction; MAX-SPE, mixed anion exchange solid phase extraction; GC, gas chromatography; MS, mass spectrometry; NCI, negative chemical ionization; HPLC, high- performance liquid chromatography; LC, liquid chromatography; TLC, thin layer chromatography; IAC, immunoaffinity column; 8-iso- PGF 2a ,9a, 11a, 15S-trihydroxy-(8b)-prosta-5Z, 13E-dien-1-oic-3,3,4,4 acid; iPF 2a -VI, (5S,6E,8a,9a, 11a, 12b, 14Z)-5,9,11-trihydroxyprosta- 6,14-dien-1-oic acid; BSFTA, N,O-bis(trimethylsilyl)trifluoroaceta- mide; TMCS, trimethylchlorosilane; PFBBr, pentafluorbenzylbro- mide; DIPEA, N,N-diisopropylethylamine; COX, cyclooxygenase. * Corresponding author. Fax: +65-6779-1453. E-mail address: bchbh@nus.edu.sg (B. Halliwell). 0006-291X/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2004.06.015 Biochemical and Biophysical Research Communications 320 (2004) 696–702 BBRC www.elsevier.com/locate/ybbrc