All solvents and NaCl were obtained from Fisher Scientific (Pittsburgh, PA, USA). Acetone and methyl tert-butyl ether (MTBE) were HPLC grade and methanol and water were Optima grade. Ammonium acetate was purchased from J.T. Baker (Phillipsburg, NJ, USA). Lycopene was isolated and crystallized from tomato paste as previously described [25 (link)]. Phytoene, phytofluene, ζ-carotene, neurosporene and tetra-cis-lycopene were isolated from tangerine tomato extracts using preparative HPLC. Identity and purity (>95%) was confirmed with HPLC/accurate mass before using as an external calibrant.
Carotenoids from tomato juices were analyzed using HPLC-DAD (Alliance 2695, 996 DAD, Waters Corporation, Milford, MA, USA) and TRL extracts were analyzed using HPLC-DAD-MS/MS (Agilent 1260, Santa Clara, CA, interfaced with an AB Sciex QTrap 5500 mass spectrometer, Foster City, CA, USA). Analytes were separated on a C30 column (4.6×250 mm, 3 μm, YMC Inc., Wilmington, NC, USA) at 35 °C using a gradient of A: 60% methanol, 35% MTBE, 3% water, 2% aqueous ammonium acetate (2% w/v), and B: 78% MTBE, 20% methanol, 2% aqueous ammonium acetate (2% w/v) flowing at 1.3 mL/min. A linear gradient was applied as follows: 0% B to 35.6% B over 9 min, to 100% B over the next 6.5 min, hold for 3.5 min at 100% B, and equilibrate for 3.5 min at initial conditions. Tomato juice extracts were re-dissolved in 2 mL of 1:1 MTBE:methanol, filtered using a 13 mm, 0.2 μm pore nylon filter, and 10 μL was injected. TRL extracts were re-dissolved in 200 μL 1:1 MTBE:methanol, centrifuged (model 5424, Eppendorf, Hamburg, Germany) at 21,130 × g for 2 min, and 20 μL of the supernatant was injected. Phytoene, phytofluene and ζ-carotene were quantified using DAD while neurosporene and all lycopene isomers were quantified using MS/MS. HPLC-DAD-MS/MS parameters are shown inTable 2 .
Carotenoids from tomato juices were analyzed using HPLC-DAD (Alliance 2695, 996 DAD, Waters Corporation, Milford, MA, USA) and TRL extracts were analyzed using HPLC-DAD-MS/MS (Agilent 1260, Santa Clara, CA, interfaced with an AB Sciex QTrap 5500 mass spectrometer, Foster City, CA, USA). Analytes were separated on a C30 column (4.6×250 mm, 3 μm, YMC Inc., Wilmington, NC, USA) at 35 °C using a gradient of A: 60% methanol, 35% MTBE, 3% water, 2% aqueous ammonium acetate (2% w/v), and B: 78% MTBE, 20% methanol, 2% aqueous ammonium acetate (2% w/v) flowing at 1.3 mL/min. A linear gradient was applied as follows: 0% B to 35.6% B over 9 min, to 100% B over the next 6.5 min, hold for 3.5 min at 100% B, and equilibrate for 3.5 min at initial conditions. Tomato juice extracts were re-dissolved in 2 mL of 1:1 MTBE:methanol, filtered using a 13 mm, 0.2 μm pore nylon filter, and 10 μL was injected. TRL extracts were re-dissolved in 200 μL 1:1 MTBE:methanol, centrifuged (model 5424, Eppendorf, Hamburg, Germany) at 21,130 × g for 2 min, and 20 μL of the supernatant was injected. Phytoene, phytofluene and ζ-carotene were quantified using DAD while neurosporene and all lycopene isomers were quantified using MS/MS. HPLC-DAD-MS/MS parameters are shown in
