Jinchuan Yang
Waters Technology (Shanghai) Co., Ltd.
purpose
The feasibility of simultaneous determination of vitamin A and E in infant formula was determined using UtraPerformance Convergence ChromatographyTM (UPC 2 TM).
background
Traditional steps for determining fat-soluble vitamins in foods include first performing cumbersome sample preparation (such as saponification and extraction) followed by high performance liquid chromatography (HPLC) through UV or fluorescence detectors. 1 Since the sample preparation requirements for vitamins A and E are similar, the analysis of both is usually performed simultaneously. 1 Recently, a simplified sample preparation method for the saponification-free reaction was proposed to be applied to the simultaneous determination of infant formula (IF) vitamin A and E. This method requires only one injection of different forms of vitamin A. Separation and quantification with E. 2 Omission of the saponification process greatly increases the throughput of the assay, however, the chromatographic analysis time is still very long (25 min) and the resolution of cis and trans vitamin A is not evaluated in writing. Waters® Ultra High Performance Convergence Chromatography (UPC 2 ) is the third addition to LC and GC with the unique properties of supercritical carbon dioxide such as low viscosity, high diffusivity and liquid-like solvency. A separation method that can cope with a wider range of analytical challenges. 3,4 To study the performance of UPC 2 in the simultaneous determination of vitamin A and E, we conducted a feasibility analysis experiment on IF samples purchased in the market.
UPC 2 technology provides a quick and easy "green" method for simultaneous determination of vitamins A and E in infant formula.
solution
In the feasibility study of this paper, vitamin A (retinyl acetate and retinyl palmitate) and vitamin E (α-tocopherol acetate) in IF samples were first extracted by liquid-liquid extraction. And alpha-tocopherol); analysis was then performed using an ACQUITY UPC 2 system equipped with a PDA detector. The column used was ACQUITY UPC 2 HSS C18 SB 3.0 x 100 mm, 1.8 μm, with a gradient elution of the mobile phase with a mixture of carbon dioxide and methanol (the methanol ratio increased from 3% to 10%). Chromatograms of these compounds were extracted from PDA data. The maximum absorption wavelengths of retinol ester, alpha-tocopherol acetate, and alpha-tocopherol were 320 nm, 283 nm, and 293 nm, respectively, as shown in FIG.
Figure 1. Typical chromatogram of vitamins A and E obtained using UPC 2 / PDA analysis. (A) Standard; (B) Infant formula samples. Peaks: 1 cis-retinyl acetate, 2 all-trans-retinyl acetate, 3 cis-retinyl palmitate, 4 all-trans-retinyl palmitate, 5 Alpha-tocopherol acetate and 6 alpha-tocopherol.
Quick and perfect separation of vitamins A and E with UPC 2 . All compounds, including cis and trans retinol esters, were separated from each other and eluted out before the peak of the sample matrix. The total run time for each injection is 8 min, including column equilibration time, which is at least 3 times faster than the run time required for other methods (typically 25 min). The linearity, sensitivity, repeatability, and recovery results of the analytical methods are shown in Tables 1 and 2. Although the sample extract can be injected directly, the LOQ results indicate that some compounds need to be evaporated and concentrated, depending on the level of compound in the IF sample. In this study, the sample extract was concentrated 10 times by evaporation when measuring vitamin E. UPC 2 is an environmentally friendly “green†technology. The main mobile phase carbon dioxide used in the analysis comes from the recovery of carbon dioxide released by other industries, so the use of carbon dioxide in the test will not generate new greenhouse gases. When analyzed by UPC 2 , the modifier (methanol) consumed per injection was only 0.9 mL, which reduced the solvent consumption by at least 90% compared to the consumption of 10 mL of hexane as described in Reference 2.
Table 1. Linear and estimated LOQ for the analysis using the UPC 2 /PDA system. a This concentration is the number of μg of analyte per mL of solution. b Y, peak area; x, concentration (μg/mL).
Table 2. Repeatability and recovery results for spiked infant formula samples. a This value is the μg of vitamins per gram of infant formula.
to sum up
This experiment uses the Waters ACQUITY UPC 2 /PDA system with a UPC 2 column to simultaneously analyze cis and trans retinyl palmitate in commercial IF samples in one injection. And trans-retinyl acetate, alpha-tocopheryl acetate and alpha-tocopherol.
The sample analysis time is 8 min, which is 3 times faster than the traditional analysis time; the solvent consumption per injection is 0.9 mL, which is 1/10 of the solvent consumption of the normal phase LC method. The analytical methods obtained in this paper have good resolution, linearity, sensitivity, precision and accuracy. Therefore, UPC 2 technology has a very large potential advantage in becoming a practical solution for the routine determination of vitamins A and E in infant formula products.
references
1. DeVries JW, Silvera K R. Determination of vitamins A (retinol) and E (alpha-tocopherol) in foods by liquid c hromatography:collaborative study. J. AOAC International. 2002; 85: 424. 2. C havez-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. 2006; 1122: 138. 3. Aubin A. Analysis of Fat-Solu ble Vitamin C a psules Using Ultra Performance Convergenc e Chromatography UP C.2 Waters Application Note 720004394en.2012 June. 4. ACQUITY UPC 2 System broc hure.2012 . p/n 720004225en.2012 Marc h.
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