International descriptive and interventional survey for oxycholesterol determination by gas- and liquid-chromatographic methods

Abstract

Increasing numbers of laboratories develop new methods based on gas-liquid and high-performance liquid chromatography to determine serum concentrations of oxygenated cholesterol metabolites such as 7α-, 24(S)-, and 27-hydroxycholesterol. We initiated a first international descriptive oxycholesterol (OCS) survey in 2013 and a second interventional survey 2014 in order to compare levels of OCS reported by different laboratories and to define possible sources of analytical errors. In 2013 a set of two lyophilized serum pools (A and B) was sent to nine laboratories in different countries for OCS measurement utilizing their own standard stock solutions. In 2014 eleven laboratories were requested to determine OCS concentrations in lyophilized pooled sera (C and D) utilizing the same provided standard stock solutions of OCS. The participating laboratories submitted results obtained after capillary gas-liquid chromatography-mass selective detection with either epicoprostanol or deuterium labelled sterols as internal standards and high-performance liquid chromatography with mass selective detection and deuterated OCS as internal standard. Each participant received a clear overview of the results in form of Youden-Plots and basic statistical evaluation in its used unit. The coefficients of variation of the concentrations obtained by all laboratories using their individual methods were 58.5-73.3% (survey 1), 56.8-60.3% (survey 2); 36.2-35.8% (survey 1), 56.6-59.8, (survey 2); 61.1-197.7% (survey 1), 47.2-74.2% (survey 2) for 24(S)-, 27-, and 7α-hydroxycholesterol, respectively. We are surprised by the very great differences between the laboratories, even under conditions when the same standards were used. The values of OCS's must be evaluated in relation to the analytical technique used, the efficiency of the ample separation and the nature of the internal standard used. Quantification of the calibration solution and inappropriate internal standards could be identified as major causes for the high variance in the reported results from the different laboratories. A harmonisation of analytical standard methods is highly needed.

Effect of industrial processing and storage procedures on oxysterols in milk and milk products

“…To a screw-capped vial sealed with a Teflon septum, 1 ml of milk or colostrum, 1ml of a 100 mg/mL distilled water suspended Table S1) using standard curves for the listed sterols. 32,33,34,35 Replicated analysis of 1 mL of whole-milk, standardized, pasteurized at standard parameters (n = 12) resulted in an interassay CV as 3.87% for 7βOHC, 3.97% for 7KC and 3.13% for 27OHC, respectively. Whole milk pasteurized at standard temperature was added with 25 ng of 7βOHC, 7KC and 27OHC, and analyzed as previously described (n = 3 independent experiments).…”

Standardizing and increasing the utility of lipidomics: a look to the next decade

“…A lipidomic study has been performed in which many different laboratories analysed the NIST SRM 1950 plasma sample using different MS methods [14] and further studies are planned targeting on specific lipid groups. Similar studies have already been performed with locally prepared plasma samples distributed to collaborating groups for sterol and oxysterol analysis [39,40]. While these "ring" trials will not necessarily lead to "true" quantitative values, they will lead to "consensus" values which are very important when comparing data sets from different laboratories.…”

Sterolomics: State of the art, developments, limitations and challenges

“…For example, in 2014 eleven laboratories measured the “total” 24S-HC content of two serum samples by MS methods, the mean ± standard deviation values for the two samples were 60.9 ± 36.7 ng/mL and 38.0 ± 21.6 ng/mL. The median values with the 16%–84% range given in brackets were 57.0 (38.5–78.9) ng/mL and 33.0 (23.7–48.6) ng/mL [17]. Although, most values clustered about the median, work still needs to be done to harmonise analytical methods.…”