Simultaneous Determination of Vitamins A and E in Infant Formula by UPC2 with PDA Detection

In this technology brief, vitamin A (retinyl acetate and retinyl palmitate) and vitamin E (alpha-tocopheryl acetate and alpha-tocopherol) were extracted from infant formula samples by a simple liquid-liquid extraction, then analyzed on a Waters ACQUITY UPC² System coupled with a UPC² PDA Detector. The separation of the analytes was achieved on a single Viridis HSS C₁₈ SB 3.0 x 100 mm, 1.8 μm Column. UPC² provides a faster, simpler, and “greener” approach for the simultaneous determination of vitamins A and E in infant formulas.

Benefits

• UPC² provides a faster, simpler, and “greener” approach for the simultaneous determination  of vitamins A and E in infant formulas.
• Simultaneous determination of vitamins A and E in infant formulas by UtraPerformance Convergence Chromatography  (UPC2).

Determination of fat-soluble vitamins in food products typically involves lengthy sample preparation (such as saponification and extraction), followed by high performance liquid chromatography (HPLC) with UV or fluorescence detection.¹ Vitamins A and E are often determined in the same analysis because of their similar sample preparation requirements.¹ Recently, a simple sample preparation procedure without saponification was proposed in a simultaneous determination of vitamins A and E in infant formula (IF) method, where various forms of vitamins A and E were separated and quantified in a single injection.² The elimination of saponification greatly increased the throughput of the analysis; however, the chromatography time was still lengthy (25 minutes), and the resolution of cis- and trans- forms of vitamin A were not evaluated in the paper. 

Waters UltraPerformance Convergence Chromatography (UPC²) leverages the unique properties of supercritical carbon dioxide, including low viscosity, high diffusivity, and liquid-like solvation power. It provides an alternative approach to normal phase LC, reversed phase LC, and gas chromatography (GC)  for a wide range of analytical challenges.^3,4 To investigate the performance of UPC2 for the simultaneous determination of vitamins A and E, a feasibility study on commercial IF samples  was performed. 

In this feasibility study, vitamin A (retinyl acetate and retinyl palmitate) and vitamin E (alpha-tocopheryl acetate and alpha-tocopherol) were extracted from IF samples by a simple liquid-liquid extraction, then analyzed on an ACQUITY UPC² System coupled with an ACQUITY UPC² PDA Detector. The separation of the analytes was achieved on a single ACQUITY UPC² HSS C₁₈ SB 3.0 x 100 mm, 1.8 µm Column under a gradient elution of a carbon dioxide and methanol mixture (3% to 10% methanol). The chromatograms for these compounds were extracted from the PDA data at their maximum absorbance wavelength of 320 nm, 283 nm, 293 nm for retinol esters, alpha-tocopheryl acetate, and alpha-tocopherol, respectively, as shown in Figure 1.

UPC² provides a fast and well-resolved separation of vitamins A and E. All of the compounds, including the cis- and trans- forms of the retinol esters, were separated from each other, and were eluted before sample matrix peaks. The total cycle time, including column equilibration, was eight minutes per injection, which was at least three times faster than the typical run time (25 minutes) using other methods. Details of the method’s analytical performance in linearity, sensitivity, repeatability, and recovery are shown in Tables 1 and 2. Although the sample extract can be injected directly onto the system, the LOQ result indicated that evaporation  or concentration of the extract may be needed for some compounds, depending on their content levels in IF samples. In this study, the sample extracts  were concentrated ten-fold by evaporation for the vitamin E determination. 

UPC² is an environmentally friendly, or “green” technology. The source of the primary mobile phase, carbon dioxide, is recaptured carbon dioxide that is released from other industries, so the use of carbon dioxide does not generate any new greenhouse gases. The co-solvent (methanol) consumption was only 0.9 mL for each injection by UPC² compared to 10 mL of hexane used in the method in reference 2. This corresponds to at least a 90% reduction in solvent consumption.

Simultaneous determination of cis- and transretinyl palmitate, cis- and trans- retinyl acetate, alpha-tocopheryl acetate, and alpha-tocopherol in commercial IF samples was achieved in a single injection using a single Viridis Column on Waters’ ACQUITY UPC² System with PDA detection.

The sample analysis time took eight minutes, three times faster than a typical analysis time, and the solvent consumption for each injection was 0.9 mL, one tenth of that in a normal phase LC method. This approach provides promising results in resolution, linearity, sensitivity, precision, and accuracy. UPC² has great potential to become a practical solution for the routine determination of vitamins A and E in infant formula products.

1. DeVries JW, Silvera KR. Determination of vitamins A (retinol) and E (alpha-tocopherol) in foods by liquid chromatography: collaborative study. J. AOAC International. 2002; 85: 424.
2. Chavez-Servin JL, Castellote AI, Lopez-Sabater MC. Simultaneous analysis of Vitamins A and E in infant milk-based formulae by normal-phase high-performance liquid chromatography-diode array detection using a short narrow-bore column. J. Chromatogr. A. 2006; 1122: 138.
3. Aubin A. Analysis of Fat-Soluble Vitamin Capsules Using UltraPerformance Convergence Chromatography UPC² Waters Application Note 720004394en. 2012 June.
4. ACQUITY UPC² System brochure. 2012. p/n 720004225en. 2012 March.