Development of a simplified method for the simultaneous determination of retinol, α-tocopherol, and β-carotene in serum by liquid chromatography–tandem mass spectrometry with atmospheric pressure chemical ionization

A new and simple method for the determination of fat-soluble vitamins (retinol, α-tocopherol, and β-carotene) in human serum was developed and validated by using liquid chromatography–tandem mass spectrometry with atmospheric pressure chemical ionization (LC-APCI-MS-MS). Different solvent mixtures were tested to obtain deproteinization and extraction of the analytes from the matrix. As a result, a volume of 240 μL of a 1:1 (v/v) ethanol/ethyl acetate mixture added to 60 μL of serum was found to be suitable for both protein precipitation and antioxidants solubilization, giving the best recovery for all three analytes. Deproteinized samples (20 μL) were injected after dilution, without the need for concentration or evaporation to dryness and reconstruction of the sample. Vitamins were separated on a C-8 column using a 95:5 (v/v) methanol/dichloromethane mixture and ionized in the positive-ion mode; detection was performed in the selected-reaction monitoring mode. Linearity of the LC-APCI-MS-MS method was established over 5 orders of magnitude for retinol and α-tocopherol, whereas in the case of β-carotene it was limited to 4 orders. Lower limits of quantitation were 1.7, 2.3, and 4.1 nM for retinol, α-tocopherol, and β-carotene, respectively. Serum concentrations of retinol, α-tocopherol, and α+β-carotene determined in a group of healthy volunteers were 2.48, 38.07, and 0.50 μM, respectively, in samples collected in winter (n=122) and 2.69, 45.88, and 0.90 μM during summer (n=66).