The analysis of vitamin A and E was performed according to a modified method of Schaffer (151) and Göbel et al. (65) using an isocratic mobile phase consisting of acetonitril, tetrahydrofuran, methanol and 1 % ammonium acetate (684+220+68+28 by vol.) (73). For avoiding the formation of explosive peroxides from tetrahydrofuran, the mobile phase was prepared prior to use and degassed ultrasonically.
Preparation of the internal standard
For consideration of losses during the extraction process, as well as unsteady injection amounts into the HPLC system, tocol was added as internal standard to plasma and standard samples at the beginning of the analysis. To prepare the internal standard, the concentrate consisting of 250 mg tocol dissolved in 100 ml ethanol/BHT (0.0625 %) was diluted 1:1000 to a concentration of 2.5 mg/l to get an adequate peak height in the chromatogram. The internal standard was frozen at -80 °C until usage.
Prearrangements for calibration curves
For preparation of standard concentrates, the amounts of retinol and α-tocopherol (tab. 22) were dissolved in 10 ml ethanol each. From these standard concentrates, dilutions were prepared as described in table 22. Standard concentrates and dilutions were stored at -80°C until usage.
Tab. 22: Preparation of standard concentrates and dilutions
Substance Standard concentrate Standard dilution
Retinol 10 mg/10 ml ethanol 200 µl concentrate/10 ml ethanol (=0.02 g/l)
α-tocopherol 50 mg/10 ml ethanol 1000 µl concentrate/10 ml ethanol (=0.5 g/l)
Preparation of standard curves for vitamin A and E
For preparation of new standard curves, the standard dilutions were defrosted and their absorbance was measured on a spectrophotometer at 325 nm for retinol and at 292 nm for
α-tocopherol (tab. 23). Lambert-Beer Law was used to determine the exact concentration (c) from absorbance (A) (A1%
1cm, extinction coefficient of a compound at a certain wave length; d, thickness of cuvette).
Used formula: A = A1%1cm * c (g/dl) * d (cm) Calculation of the concentration: c (g/dl) = A / [A1%1cm * d (cm)]
Tab. 23: Extinction coefficients for retinol and α-tocopherol
Substance weight (g/mol) Molecular Wave length (nm) coefficient (AExtinction 1%
1cm) Source
Retinol 286.5 325 1780 (151)
α-tocopherol 430.7 292 75.8 (151)
Based on the photometrically determined concentrations, separate stock standards (which equal the highest concentrated standard of the standard curve
)
were obtained for each vitamin from standard dilutions. According to plasma levels in healthy adults, the following concentrations were chosen as the highest concentrated standards.Retinol ∼1.0 mg/l
α-tocopherol ∼20.0 mg/l
Exact concentrations of stock standards for retinol and alpha-tocopherol were determined photometrically as described above. For preparation of the 7-point calibration curve, the following volumes of the internal standard and each stock standard were pipetted into brown bottles:
Tab. 24: Preparation of the seven standard dilutions for the calibration curves
Std.7 Std.6 Std.5 Std.4 Std.3 Std.2 Std.1 Tocol (ISTD) 250 µl 250 µl 250 µl 250 µl 250 µl 250 µl 250 µl
Retinol 250 µl 210 µl 170 µl 130 µl 90 µl 50 µl 10 µl
α-tocopherol 250 µl 210 µl 170 µl 130 µl 90 µl 50 µl 10 µl
The concentrations of standards 1-7 were calculated according to their dilution factors as shown in the example for the seven standard dilutions for retinol and α-tocopherol calibration curves (mg/l):
Tab. 25: Calculation of the concentrations of the seven standard dilutions
Std. 7 Std. 6 Std. 5 Std. 4 Std. 3 Std. 2 Std.1 Dilution factor x1 x0.84 x0.68 x0.52 x0.36 x0.2 x0.04
Retinol 1.00* 0.84 0.68 0.52 0.36 0.20 0.04
α-tocopherol 20.0* 16.8 13.6 10.4 7.2 4.0 0.8
These seven standards were dried under a gentle stream of nitrogen. The dried extract was redissolved in 100 µl mobile phase, shaken mechanically for 10 min and transferred into microvials. For UV-Vis detection, 20 µl were injected into the HPLC system with an isocratic flow rate of 0.65 ml/min.
A 7-point standard curve was constructed for retinol and α-tocopherol using the software Microsoft Excel 97 SR-2 (Microsoft GmbH, Unterschleißheim) by plotting vitamin concentrations (tab. 24) against the peak-area ratios (vitamin/ IS, x-axis) (fig. A1). Subsequently, the equation of the regression line and the coefficient of determination (r2) were calculated.
Tab. 26: Program of the UV-Vis detector
Time (min) Wave length (nm) Detection of
0 325 Retinol
4 292 α-tocopherol
Extraction of plasma samples
For protein precipitation, 250 µl plasma, 250 µl internal standard and 500 µl ethanol/BHT (62.5 mg/dl) were pipetted into a 4 ml brown glass vial and vortexed for 15 seconds. Then, two extraction steps followed using 1 ml Hexan/BHT (5 mg/dl) and one further extraction step using 1 ml Hexan. After every extraction, the samples were vortexed for 30 sec each and centrifuged for 5 min (2.300 U/min). Thereafter, vials were placed on ice for 5 min. The upper phases were transferred into a 1.5 ml brown glass bottle and taken to dryness under a gentle stream of nitrogen. The dried extract was redissolved in 100 µl mobile phase, shaken mechanically for 10 min and transferred into microvials. For UV-Vis detection, 20 µl were injected into the HPLC system with an isocratic flow rate of 0.65 ml/min. The program of the UV-Vis detector is shown in table 26. Detector signals were evaluated with an automatic integrator (Chromato-Integrator D-2500, Merck-Hitachi, Darmstadt) and identified by comparison with the retention times of a standard mixture run previously (fig. 5).
For calculation of the vitamin concentrations, peak-area ratios (vitamin/ IS) were computed for retinol and α-tocopherol. The appropriate concentrations were calculated using the equations of the standard curves (see 7.2, fig. A1).
Intra-assay reproducibility was assessed by analyzing six pool samples in the same analytical run, whereas inter-assay reproducibilities of the used methods were assessed by analyzing nine pool samples during one week.
Fig. 5: Chromatogram of a standard mixture using UV-Vis detection*