First international descriptive and interventional survey for cholesterol and non-cholesterol sterol determination by gas- and liquid-chromatography-Urgent need for harmonisation of analytical methods.

Serum concentrations of lathosterol, the plant sterols campesterol and sitosterol and the cholesterol metabolite 5α-cholestanol are widely used as surrogate markers of cholesterol synthesis and absorption, respectively. Increasing numbers of laboratories utilize a broad spectrum of well-established and recently developed methods for the determination of cholesterol and non-cholesterol sterols (NCS). In order to evaluate the quality of these measurements and to identify possible sources of analytical errors our group initiated the first international survey for cholesterol and NCS. The cholesterol and NCS survey was structured as a two-part survey which took place in the years 2013 and 2014. The first survey part was designed as descriptive, providing information about the variation of reported results from different laboratories. A set of two lyophilized pooled sera (A and B) was sent to twenty laboratories specialized in chromatographic lipid analysis. The different sterols were quantified either by gas chromatography-flame ionization detection, gas chromatography- or liquid chromatography-mass selective detection. The participants were requested to determine cholesterol and NCS concentrations in the provided samples as part of their normal laboratory routine. The second part was designed as interventional survey. Twenty-two laboratories agreed to participate and received again two different lyophilized pooled sera (C and D). In contrast to the first international survey, each participant received standard stock solutions with defined concentrations of cholesterol and NCS. The participants were requested to use diluted calibration solutions from the provided standard stock solutions for quantification of cholesterol and NCS. In both surveys, each laboratory used its own internal standard (5α-cholestane, epicoprostanol or deuterium labelled sterols). Main outcome of the survey was, that unacceptably high interlaboratory variations for cholesterol and NCS concentrations are reported, even when the individual laboratories used the same calibration material. We discuss different sources of errors and recommend all laboratories analysing cholesterol and NCS to participate in regular quality control programs.

Development and validation of a comprehensive two-dimensional gas chromatography-mass spectrometry method for the analysis of phytosterol oxidation products in human plasma.

Phytosterol oxidation products (POPs) have been suggested to exert adverse biological effects similar to, although less severe than, their cholesterol counterparts. For that reason, their analysis in human plasma is highly relevant. Comprehensive two-dimensional gas chromatography (GC×GC) coupled with time-of-flight mass spectrometry (TOF-MS) has been proven to be an extremely powerful separation technique for the analysis of very low levels of target compounds in complex mixtures including human plasma. Thus, a GC×GC/TOF-MS method was developed and successfully validated for the simultaneous quantification of ten POPs in human plasma. The calibration curves for each compound showed correlation coefficients (R(2)) better than 0.99. The detection limits were below 0.1 ng mL(-1). The recovery data were between 71.0% and 98.6% (RSDs <10% for all compounds validated). Good results were obtained for within- and between-day repeatability, with most values being below 10%. In addition, non-targeted sterol metabolites were also identified with the method. The concentrations of POPs found in human plasma in the current study are between 0.3 and 4.5 ng mL(-1), i.e., 10-100 times lower than the typical values found for cholesterol oxidation products.

Validation of a gas chromatography-mass spectrometry method for the analysis of sterol oxidation products in serum.

A validated gas chromatography-mass spectrometry (GC-MS) detection method for the quantitative analysis of sterol oxidation products (SOPs) in serum is described. After a lipid extraction procedure with chloroform-methanol, a cold saponification and purification by solid phase extraction, oxysterols were derivatized to form trimethyl-sylil-ethers which were subjected to GC-MS analysis. Calibration curves for cholesterol oxidation products showed determination coefficient (R(2)) of 1.0, with low values for the coefficient of variation of the response factors (< 1%). Detection and quantification limits were below 5 ng/mL and 10 ng/mL, respectively. Recovery data were between 77.65% and 110.29% (CV < 10% for all compounds). Good results were obtained for within- and between-day repeatability, with values below 10%. In conclusion, the method performed is suitable for the determination and quantification of SOPs in serum.