Agilent 1200 series high performance liquid chromatograph

Fasting blood samples were collected by experienced personnel using standard venepuncture techniques. Upon collection, blood samples were processed immediately and centrifuged, with plasma collected and stored in aliquots at –80°C until analysis. The tubes containing plasma samples to be analysed for carotenoids were immediately wrapped in foil to minimise light exposure. Plasma carotenoid samples were sent to an external laboratory (University of Newcastle, Newcastle, NSW, Australia) for analysis. High Performance liquid chromatography methodology was used to determine β-carotene, α-carotene, β-cryptoxanthin, lycopene and lutein/zeaxanthin (combined) concentrations in plasma. Total carotenoid concentration was calculated from the addition of all measured plasma carotenoids. All extractions were carried out under red light in a darkened laboratory, using validated methodology as described in Wood et al.^{(30 (link))} Sample carotenoid peaks were identified and quantified using Agilent 1200 Series High Performance Liquid Chromatograph with Chemstations software (Agilent Corporation, Germany).^{(30 (link))} Separately, serum cholesterol was measured at a commercial laboratory (Dorevitch Pathology Pty Ltd, Heidelberg, VIC, Australia) using an automated blood analyser (ADVIA 2400 Chemistry System, Siemens).

¹H and ¹³C NMR spectra were recorded in CD₃OD on an AVANCE 600 spectrometer (Bruker, Rheinspettem, Germany) at 600 and 150 MHz, respectively, using tetramethylsilane as an internal standard, and chemical shifts are given in δ (ppm). UV spectra were obtained in methanol on a UVICON 933/934 spectrophotometer (Kontron, Milan, Italy), mass spectra on GMS-600 W or JMS-700 spectrometer (Jeol, Tokyo, Japan), and FT-IR spectra on a Nicolet Magna 550 series II spectrometer (Midac, Irvine, CA). Optical rotation was measured with an Autopol III polarimeter (Rudolph Research Analytical, Flanders, NJ). Silica gel 60 (0.063–0.2 mm) (Merck, Darmstadt, Germany) was used for column chromatography. Merck precoated silica gel plates (Kieselgel 60 F₂₅₄) were used for analytical thin-layer chromatography (TLC). Merck silica gel 60 RP-18 F_254S plates (for RP-TLC) and an Agilent 1200 series high-performance liquid chromatograph (HPLC) (Santa Clara, CA) were used for isolation of active principles.

The ¹H and ¹³C nuclear magnetic resonance (NMR) spectra were recorded in CDCl₃ on Varian NMR system spectrometers (Varian, Palo Alto, CA, USA), using tetramethylsilane as an internal standard. The chemical shifts are given in δ (ppm). The ultraviolet (UV) spectra were obtained in methanol on a UVICON 933/934 spectrophotometer (Kontron, Milan, Italy) and the mass spectra on a GSX 400 spectrometer (Jeol, Tokyo, Japan). Silica gel 60 (0.063–0.2 mm) (Merck, Darmstadt, Germany) and Sephadex LH-20 (Sigma-Aldrich, St. Louis, MO, USA) were used for column chromatography. Merck precoated silica gel plates (Kieselgel 60 F₂₅₄) were used for analytical thin-layer chromatography (TLC). An Agilent 1200 series high-performance liquid chromatograph (Agilent, Santa Clara, CA, USA) was used to isolate the active constituents.