Sport and Exercise Sciences Research Institute
Standard Operating Procedure
High Performance Liquid Chromatography (HPLC)
(SS/SOP0019)
Measurement of Lipid Soluble Antioxidants in Plasma or Serum
HPLC is used to separate the components in a mixture, and to identify and quantify
each component.
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Please see relevant COSHH forms and sign that these have been read.
Wear relevant protective clothing, and use fume cupboard as instructed
Use pipettes to dispense liquids
Standards, including an Internal Standard (IS) are made up as stated in the
schedule by the technician
The mobile phase is made by mixing methanol, acetonitrile and
dichloromethane in the following ratios 47:47:12 respectively
A 300 µl aliquot of plasma/serum (refer to Risk Assessment
SS0005)/standard mixture is added to a glass tube. Include three QCs
(pooled plasma) for every batch of samples run
Add 250 µl of the internal standard is added to each tube above
Heptane (500 µl) is added to tubes above
Vortex all the tubes vigorously for 60 seconds
Set the standard vials to one side
Centrifuge (refer to SS/SOP0017) all the sample vials for 5 minutes at 3000
rpm
The resulting heptane layer (350 µl) is added to an identically labelled glass
tube
Add a further 500 µl to the tubes and repeat the above from this step
The combined heptane layers (700 µl) are evaporated to dryness in a
centrifugal evaporator under vacuum
Add 150 µl of mobile phase to each tube and vortex
Pipette above into HPLC vials
Load vials into the carousel
Switch on HPLC and PDA system about an hour before using
Check that degasser is on
Perform a dry and/or wet prime
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Set the flow rate to 0.4 ml/min
Set up method including shutdown
Press run
Quantify samples