By James F. Lawrence (Eds.)
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17. HPLC of soy-base infant-formula products (after enzymatic hydrolysis) using systems similar to that shown in Fig. 16. Peak identification: (1) retinol; (2) δ-tocopherol; (3) 7-tocopherol; (4a) vitamin D ; (4b) vitamin D ; (5) α-tocopherol; (6) tocopheryl acetate; (7) phylloquinone; (8) cholesterol phenyl acetate; (9) retinyl palmitate. Retention times given in minutes. [With permission, from Barnett et al. (1980). Anal. Chem. 52, 610-614. ] 3 2 40 J. Ν. 01 20 30 40 MINUTES Fig. 18. Separation of phylloquinones, menaquinones, and triglycerides on 120-cm column HAPS in hexane (1 ml/min): (a) phylloquinones 3, 4, and 5; (b) menaquinones 1, 2, 4.
A fluorescence detector provides much greater sensitivity. 1 μg/ml. Jansson et al. (1980) reported that with a similar method, the minimum detectable level, which 37 Trace Analysis of Vitamins gave a response double that of the detector noise, was 40 ng/ml tocopherol. Detector response can be increased in some instruments by using a shorter excitation wavelength, such as 205 nm (Hatam and Kay den, 1979). This involves a marked decrease in specificity, however, and an aggravation of quenching effects.
Fluorescence measured at 290 nm excitation and 330 nm emission; absorbance measured at 295 nm. (c) HPLC of concentrated extract using absorbance detector; note presence of interfering substances. Identification of peaks: (1) α-tocopherol; (4) antioxidant BHA; (5) 7-tocopherol; (8) δ-tocopherol. [From Thompson and Hatina (1979), p. ] Trace Analysis of Vitamins 35 hexane and washing this solution with aqueous ethanol or acetone. These steps can be combined to give an extraction procedure which is applicable to a wide variety of foods (Thompson and Hatina, 1979).