Sterols (ST) determination - Lembcke et al.

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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


  • 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



  • 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




0.24 - 0.68 µg/l




1 - 1000 µg/l

Internal Standard



  • 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.


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