Simultaneous Detection and Quantification of Water- and Fat-Soluble Vitamins with

Liquid Chromatography and Tandem Ion Trap-Mass Spectrometry

Maryam Khaksari1, Lynn R. Mazzoleni2, Chunhai Ruan3, Peng Song4, Neil D. Hershey4, Robert
T. Kennedy4, Mark A.Burns5, Adrienne R. Minerick1*

1Department of Chemical Engineering, 2Department of Chemistry, Michigan Technological University, Houghton, MI, 3Metabolomics core, BRCF, 4Department of Chemistry, 5Department of Chemical Engineering, University of Michigan, Ann Arbor, MI

Abstract

A method for simultaneous determination of water-soluble and fat-soluble vitamins is described. This method includes detection of eight water-soluble vitamins, specifically, , , (nicotinamide), , (pyridoxine), , , and four fat-soluble vitamins, A (retinol), , E (α-tocopherol) and . The method uses liquid chromatography (LC) with a C18 reversed-phase column and an ion trap mass spectrometry (MS) detector coupled with an electrospray ionization (ESI) probe. Using vitamin standard solutions, the LC operating conditions were optimized to minimize elution times. Total analysis time was 60 min using gradient elution with ternary mobile phases of water and acetonitrile containing 0.1% formic acid and methanol containing 5 mM ammonium formate. The formic acid and ammonium formate enhanced the ionization required for MS detection. Vitamins were quantified with an internal standard method under MS/MS condition. Using 25 μL injection volumes, the limits of detection (LODs) were in the range of 0.043-9.5 ng for all 12 vitamins with linear responses to water-soluble and polynomial responses to fat-soluble vitamins. The proposed method reduces material and solvent demands, while decreasing the preparation and instrument time in comparison to separate methods published for water-soluble and fat-soluble vitamins. Also, this method minimizes biological sample volumes, which has distinct advantages in many diagnostic applications with limited available fluids or sampling small subjects (e.g. rodents). This combined method was validated with human blood plasma and may provide substantial time savings for nutritional assessments from blood and other biofluids.