Trials and Tribulations of Fat Soluble Vitamin Analysis by UHPLC-MS/MS

Separation Science, in collaboration with Biotage, offers an on-demand webinar that addresses work performed to develop a method for the analysis of fat-soluble vitamins in human serum.

Duration: Approximately 45 minutes

Alan Edgington (Biotage, UK)
Alan joined Biotage (GB) UK in Cardiff where he works as a Senior Applications Scientist to best utilize the wide range of the company’s sample preparation products. A large proportion of this application work has been spent developing methods of analysis of vitamins in human biological fluids. Alan also helps support the development of the automated platform Extrahera and is a trained internal auditor.






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This webinar summarizes the work performed to develop a method for the analysis of fat-soluble vitamins including vitamins A (Retinol and Beta Carotene), D (25 Hydroxy D3 and 25 Hydroxy D2), E (Alpha Tocopherol) and K (Phylloquinone and Menaquinone) in human serum.

The presentation goes into depth around some of the challenges involved with developing a combined analyte method including those related to analyte choice, solubility, sensitivity, stability, extent of protein binding and chromatography. It highlights the challenge of being able to measure endogenous analytes in samples and manually spiked standards in a stripped matrix in a consistent manner. The presentation includes highlights of selected method development steps of varying success and a detailed description of the final method. The quoted method performance is backed up by calibration line, analyte and phospholipid recovery data.

The final method involves an unconventional serum precipitation step followed by supported liquid extraction (SLE+) with LC-MS detection.

By viewing this presentation you will gain:

  • a general understanding of the challenges faced in setting up a method for multiple fat-soluble methods
  • knowledge in pre-treatment and loading techniques for the most popular fat-soluble vitamins.


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