The carotenoids lutein, zeaxanthin, β-cryptoxanthin, lycopene, and α-/β-carotene, α-/γ-tocopherol, and retinol in plasma were simultaneously determined by HPLC with UV and fluorescence detection as previously described [24 (link)]. In brief, plasma (40 µL) was extracted with ethanol/n-butanol (1:1, 200 µL) containing β-apo-8′-carotenal-methyloxime as an internal standard. After centrifugation (21,000× g, 15 min at 4 °C), 20 µL of the clear supernatant was analyzed on a Shimadzu Prominence HPLC (LC-20A) with chromatographic conditions, as previously described in detail [24 (link)]. Pure standard mixtures which were prepared and run as a sample were used for quantification. These standards were verified against serum pools with assigned values set against the Standard Reference Material (SRM 968c, NIST, Gaithersburg, MD, USA). For internal quality control, aliquots of a plasma pool run along within the > 30 batches gave inter-batch coefficients of variations (CVs) for carotenoids <8% (between 3.1% for α-carotene to 7.6% for lycopene), tocopherols <7% (4.1% for γ-tocopherol and 6.3% for α-tocopherol) and for retinol 3.7%. Serum cholesterol was analyzed by a standard enzymatic method at RIVM using an auto-analyzer (LX-20 Pro, Beckman-Coulter, Woerden, The Netherlands).
Free full text: Click here
