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, alpha-tocopherol, and beta-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 muL of a 1:1 (v/v) ethanol/ethyl acetate mixture added to 60 muL 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 muL) 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 alpha-tocopherol, whereas in the case of beta-carotene it was limited to 4 orders. Lower limits of quantitation were 1.7, 2.3, and 4.1 nM for retinol, alpha-tocopherol, and beta-carotene, respectively. Serum concentrations of retinol, alpha-tocopherol, and alpha+beta-carotene determined in a group of healthy volunteers were 2.48, 38.07, and 0.50 muM, respectively, in samples collected in winter (n=122) and 2.69, 45.88, and 0.90 muM during summer (n=66).