Simple quantification of phytosterols and tocopherols using fast chromatography -Tandem mass spectrometry.

Tocopherols and Phytosterols are highly abundant compounds in waste products resulting from Canola Oil production. They have significant antioxidant and cholesterol lowering properties, respectively. Since Canola is a major crop product in Canada, effective extraction of these metabolites has economical impact. Hence, there is a need for the development of a fast and easy quantification method of these active metabolites.
Our analytical strategy relied on the use of fast chromatography -Tandem mass spectrometry (FC-MS/MS). A guard column was used to achieve fast separation and the method is compared to use of conventional C18 column. The mobile phase consisted of acetonitrile:methanol (99:1) with 0.1% acetic acid. The compounds were ionized in the positive ion mode using atmospheric pressure chemical ionization (APCI). The following parameters were employed: source temperature 380°C, curtain gas 40 psi, nebulizer current 2.5 µA and ion source gas 1 30 psi. 5α-Cholestan-3β-ol and Rac-tocol were used as internal standards for phytosterols and tocopherols, respectively.
Four phytosterols and four tocopherols, namely Stigmasterol, β-Sitosterol, Brassicasterol, Campesterol, Alpha-tocopherol, Gamma-tocopherol, Beta-tocopherol and Delta-tocopherol were determined using FC-Multiple-Reaction-Monitoring (MRM). The run time was 2 minutes only, compare to 6.5 minutes with the column injection. Beta-tocopherol and Gamma-tocopherol couldn’t be resolved on the guard column nor on the C18 column. The FC-MS/MS methods addressed the issue of cross talks among the target analytes. For example, stigmasterol and β-sitosterol precursor ions observed as [M+H-H20]+ has the same m/z values for the ion designated as [M+H-4H]+ for campesterol and brassicasterol. In fact, such interferences prevented the full removal of the column (i.e. loop injection). Calibration curves were established and a good linearity was achieved (0.25-10 µg/ml) with R2 of 0.996 and 0.997 for tocopherols and phytosterols, respectively.
In conclusion, a fast and simple FC-MS/MS method for the simultaneous quantitation of phytosterols and tocopherols was successfully developed.