Sterols (ST) determination - Lembcke et al.

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Contents 1 Abstract 2 Analysed Matrices 3 Analytes 4 Sample Preparation 5 Analytical Method 5.1 HPLC-MS-MS 5.1.1 HPLC 5.1.2 MDS SCIEX API 3000 (Applied Biosystems) 6 Method Validation 6.1 Accuracy 6.2 Precision 6.3 LOD 6.4 LOQ 6.5 Lineartiy 7 Internal Standard 8 Reference

Abstract

A novel analytical platform based on liquid chromatography and tandem mass spectrometry using atmospheric pressure photoionization was applied for the simultaneous quantification of free and esterified ß-sitosterol, campesterol, brassicasterol, and stigmasterol. The total time for sample pretreatment and analysis could be reduced from ~3 h [gas chromatography-mass spectrometry (GC-MS)] to 15 min. The detection limits of the different phytosterols ranged between 0.25 and 0.68 µg/l. Linear ranges were between 1 and 1,000 µg/l. The within-run and between-run variabilities ranged between 1.4% and 9.9%. The analytical sensitivity was at least 150-fold higher compared with GC-MS. Our new method allows a rapid and simultaneous determination of free and esterified phytosterols in serum.

Analysed Matrices

human serum

Analytes

• 8 phytosterols (+ 1 IS)

free + esterified ß-sitosterol; campesterol; brassicasterol; stigmasterol

Sample Preparation

• mixing of 20 µl of calibrators, controls, and serum samples with 980 µl of internal standard solution (quantification of free and esterified phytosterols)
• centrifugation (10 min; 11,400 g)
• transfer of supernatant into a glass vial
• addition of 10 µl of internal standard (100 mg/l) and 20 µl of butylated hydroxyl toluene (1 g/l)to 20 µl of a cholesterol-cholesteryl stearate standard solution (51.7 µmol/l each) to the serum control (quantification of total sterol concentration)
• mixing with 2 ml of freshly prepared 1 M ethanolic NaOH
• hydrolysis for 1 h; 68°C
• addition of 1 ml of deionized water
• sample extraction twice with 3 ml of cyclohexane
• drying of extract (65°C; under a stream of nitrogen)
• reconstitution with 200 µl of isopropanol

Analytical Method

HPLC-MS-MS

HPLC

• series 200 autosampler, a column oven, and a binary micro pump system from Perkin-Elmer (Rotgau-Jügesheim, Germany)
• column: Chromolith SpeedRod RP-18e(50 x 4.6 mm) monolithic (Merck KGaA, Darmstadt,Germany)
• T(column): 40 °C
• injection volume: 25 µl
• equilibration: methanol-water (75:25, v/v)
• flow rate: 600 µl/min

After 1 min under these isocratic conditions, a linear gradient step to 100% isopropanol was performed in 1 min and kept for 3.5 min. After chromatographic separation, the system was reequilibrated with the starting solvent mixture for 4.5 min.

MDS SCIEX API 3000 (Applied Biosystems)

• triple quadrupole mass spectrometer
• with a Photospray™
• without solvent splitting
• flow rate: 600 µl/min
• T = 400°C
• positive ion mode
• lamp voltage: 1,700 V
• orifice voltage: 45 V
• ionization dopant: Toluene; flow rate: 90 µl/min (Perkin-Elmer series 200 binary pump)

Method Validation

Accuracy

Precision

LOD

0.24 - 0.68 µg/l

LOQ

n.d.

Lineartiy

1 - 1000 µg/l

Internal Standard

25,26,26,26,27,27,27-d7-cholesterol

Reference

• J. Lembcke, U. Ceglarek, G. M. Fiedler, S. Baumann, A. Leichtle, J. Thiery J. Lipid Res. 2005, 46, 21-26.

Institute of Laboratory Medicine, Clinical Chemistry, and Molecular Diagnostics; University Hospital; Leipzig; Germany.

e-mail: thiery@medizin.uni-leipzig.de