Teunissen et al.

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Contents 1 Abstract 2 Analysed Matrices 3 Analytes 4 Sample Preparation 5 Analytical Method 5.1 GC/GC-MS 5.1.1 GC HP6890 5.1.2 GC-MS HP5890/HP5972 6 Method Validation 6.1 Accuracy 6.2 Precision 6.3 LOD 6.4 LOQ 6.5 Linearity 7 Internal Standard 8 Reference

Abstract

Teunissen et al. presented a sensitive GC-MS method for the quantitation of human serum cholesterol and cholesterol precursors lathosterol, lanosterol, and desmosterol as well as the oxidized cholesterol metabolites 24-S-OH-cholesterol and 27-OH-cholesterol.

Analysed Matrices

human serum

Analytes

• 6 sterols (+ 4 IS)

cholesterol; lathosterol; lanosterol; desmosterol; 24-S-OH-cholesterol; 27-OH-cholesterol

Sample Preparation

Sterols and oxysterols were extracted from serum by cyclohexane after saponification. Fifty microgram 5α-cholestane, 1 µg epicoprostanol, 200 ng racemic [23,23,24,25-2H4]24S-OH-cholesterol and [2H5]25R-27-OH-cholesterol were added as internal standards. The solvents were evaporated and the hydroxy groups of the sterols and oxysterols were trimethylsilylated.

Analytical Method

GC/GC-MS

GC HP6890

• column: HP1 (Methyl Siloxane, crosslinked) fused silica capillary column (10m × 0.1mm i.d., 0.4 µm phase thickness)
• temperature program: 150/1.5-59-290/8 (initially kept at 150 °C for 1.5 min, increased at 59 °C/min to a final temperature of 290 °C for 8 min9
• injection volume: 1.0 µl (automated injector (HP)); 280 °C
• pulsed splitless mode
• carrier gas: H2
• initial flow: 1.0 ml/min
• flame ionization detection (280 °C)

The concentration of cholesterol was calculated from the ratio of the peak area of cholesterol to the area of 5α-cholestane multiplied by the amount of internal standard (50 µg) added to a defined serum volume.

GC-MS HP5890/HP5972

• (GC–MS–SIM)
• column: DB-XLB column (30m×0.25mm i.d. × 0.25 µm film thickness, Alltech)
• injection volume: 1.0 µl; T = 280 °C
• splitless mode
• carrier gas: He
• gas-flow: 1.0 ml/min
• temperature program: 150/1-30-290/30.33 (initial oven temperature was kept at 150 °C for 1 min, increased at a rate of 30 °C/min to 290 °C and kept for 30.33 min)
• electron impact ionization: 70 eV
• T (transfer line): 280 °C.

• TMSi-ether of epicoprostanol: m/z 370 (M+ − OTMSi)
• lathosterol: m/z 458 (M+)
• desmosterol: m/z 456 (M+)
• lanosterol: m/z 393 (M+ − CH3 − OTMSi)
• campesterol: m/z 472 (M+)
• sitosterol: m/z 488 (M+)
• authentic and deuterated 24S-OH-Chol: m/z 413 (M+ −OTMSi −CH(CH3)2); m/z 416 (M+ −OTMSi − CH(CD3)2),
• authentic and deuterated 27-OH-Chol: m/z 456 (M+ − 90) and 461 (M+ − 90)

Method Validation

Accuracy

Precision

LOD

n.d.

LOQ

n.d.

Linearity

n.d.

Internal Standard

• 5α-cholestane
• epicoprostanol
• racemic [23,23,24,25-2H4]24S-OH-cholesterol
• [2H5]25R-27-OH-cholesterol

Reference

• C. E. Teunissen, J. De Vente, K. von Bergmann, H. Bosna, M. P. J. van Boxtel, C. de Bruijn, J. Jolles, H. W. M. Steinbusch, D. Lütjohann Neurobiol. Aging 2003, 24, 147-155.

Department of Molecular Biology; Vrije Universiteit Amsterdam; Van der Boechorststraat 7; 1081 BT Amsterdam; The Netherlands.

Tel.: +31-20-4448063; Fax: +31-20-4448081; e-mail: c.teunissen.cell@med.vu.nl