RAPID AND HIGH-THROUGHPUT ANALYSIS OF STEROLS/STANOLS OR DERIVATIVES THEREOF

1 . A rapid, high-throughput process for analyzing one or more sterols/stanols or derivatives thereof in a plurality of samples, comprising:

introducing a plurality of samples containing one or more sterols/stanols or derivatives thereof into individual vessels in a multi-vessel plate;

cleaving the one or more sterols/stanols or derivatives thereof of each sample in the multi-vessel plate to form sterols/stanols;

extracting the free sterols/stanols of each sample by solid phase extraction; and

detecting the level of the extracted free sterols/stanols in each sample by liquid chromatography tandem mass spectrometry,

wherein the free sterols/stanols do not undergo an additional derivitization step of adding a functional group to the free sterols/stanols prior to the detecting step.

2 . The process of claim 1 , wherein each vessel is wide and tall enough to allow for adequate mixing, and thin enough to allow the multi-vessel plate to fit in an automated fluid handling station and/or an automated multi-vessel plate handling station.

3 . The process of claim 1 , wherein the step of cleaving comprises:

pipetting a cleaving agent into each sample in the multi-vessel plate;

vortexing the composition containing the sample and cleaving agent in each vessel; and

heating the multi-vessel plate to a desirable temperature.

4 . The process of claim 3 , wherein each vessel of the multi-vessel plate is sealed by a matching multi-cap mat.

5 . The process of claim 3 , wherein the temperature ranges from 40 to 50° C.

6 . The process of claim 1 , wherein the cleaving step involves hydrolyzing the sterols/stanols or derivatives thereof to form free sterols/stanols.

7 . The process of claim 6 , wherein the hydrolyzing step involves a saponification of the sterols/stanols derivatives.

8 . The process of claim 7 , wherein the saponification reaction takes place in presence of an alkali hydroxide or alkaline hydroxide catalyst.

9 . The process of claim 8 , wherein the catalyst is sodium hydroxide or potassium hydroxide.

10 . The process of claim 1 , wherein the extracting step comprises:

transferring each sample in the multi-vessel plate to a multi-vessel solid phase extraction plate after the cleaving step; and

eluting the free sterols of each sample from the multi-vessel solid phase extraction plate into a multi-vessel collecting plate.

11 . The process of claim 10 , wherein the eluting is carried out with dicholoride methane.

12 . The process of claim 10 , wherein the extracting step further comprises:

drying the eluted free sterols/stanols of each sample in a multi-vessel collecting plate; and

adding a reconstitution solution to the dried free sterols/stanols in the multi-vessel collecting plate to reconstitute the free sterols/stanols.

13 . The process of claim 12 , wherein the reconstitution solution is a methanol/isopropanol/formic acid solution.

14 . The process of claim 1 , further comprising:

adding an internal standard to each sample in the multi-vessel plate.

15 . The process of claim 14 , wherein the internal standard is a deuterated internal standard.

16 . The process of claim 1 , further comprising:

labeling the plurality of samples in the multi-vessel plate; and

detecting the labeled samples for a sequential processing.

17 . The process of claim 16 , wherein the labeling step is carried out by an automated bar-coding equipment, and the detecting is carried out by an automated bar code detector.

18 . The process of claim 1 , wherein the sample is a blood component selected from the group consisting of plasma, serum, red blood cells, whole blood, platelets, white blood cells, sterol/stanol esters, free sterols/stanols, and mixtures thereof.

19 . The process of claim 1 , wherein the sterols/stanols comprise at least one of desmosterol, campesterol, cholestanol, and sitosterol.

20 . The process of claim 1 , wherein the sterols/stanols comprise desmosterol, campesterol, cholestanol, and sitosterol.

21 . The process of claim 1 , wherein the entire process is carried out in less than about 7 minutes for the detecting step.