2.1 Pigment
Extraction
1. Optional for grinding of frozen leaf material: Ball mill with
stainless steel balls (3 mm diameter) and adaptors to fit microcentrifuge tubes (Retsch MM 300 or equivalent).
2. Liquid nitrogen.
3. Acetone: Precool a necessary amount in a glass bottle on ice.
4. 2 mL microcentrifuge tubes.
5. Refrigerated microcentrifuge (Eppendorf centrifuge 5415R or
equivalent).
6. 3 mL or 5 mL disposable syringes.
7. Nylon syringe filters (0.45 μm pore size, 3 mm diameter).
8. Amber glass vials (MS certified).
2.2 LC-PDA-MS
1. 500 mM ammonium acetate (NH 4 Ac) stock solution: Weigh
3.85 g NH 4 Ac in a 100 mL volumetric flask and make up to
100 mL with water (see Note 1).
2. Waters ACQUITY UPLC system (comprising a binary pump
and autosampler) or equivalent.
3. Waters PDA detector or equivalent.
4. Waters Xevo TQ-S triple quadrupole mass spectrometer or
equivalent.
5. Bischoff ProntoSil 200–3-C30 column (250 Â 4.6 mm, 3 μm
particle size) and a corresponding guard column
(10 Â 4.0 mm, 3 μm) or equivalent.
6. Elution solvent A: Mix 499 mL methanol (MeOH) and 1 mL
500 mM NH 4 Ac stock solution in a glass bottle to make 1 mM
NH 4 Ac in MeOH/water (99.8:0.2, v/v).
7. Elution solvent B: Methyl tert-butyl ether (MTBE).
8. Standards of plant photosynthetic carotenoids (Table 1) purchased from DHI (Hørsholm, Denmark) (see Note 2).
2.3 LC-DAD-FTICRMS
1. Agilent 1200 series HPLC system consisting of a binary pump,
autosampler, column oven and DAD or equivalent.
2. Hybrid linear ion trap FTICR mass spectrometer LTQ FT
Ultra (Thermo Fisher Scientific) equipped with a 7 T supraconducting magnet or equivalent.
3. For HPLC column, elution solvents and pigment standards, see
Subheading 2.2, items 5–8.
3 Methods
Carry out pigment extraction under a fume hood and wearing
safety glasses. Dim the light and keep samples on ice while extracting pigments to avoid degradation. Amber glass vials should be
used for LC-MS for protection against light.
Carotenoid Isotopologue Profiling
265
Extraction
1. Optional for grinding of frozen leaf material: Ball mill with
stainless steel balls (3 mm diameter) and adaptors to fit microcentrifuge tubes (Retsch MM 300 or equivalent).
2. Liquid nitrogen.
3. Acetone: Precool a necessary amount in a glass bottle on ice.
4. 2 mL microcentrifuge tubes.
5. Refrigerated microcentrifuge (Eppendorf centrifuge 5415R or
equivalent).
6. 3 mL or 5 mL disposable syringes.
7. Nylon syringe filters (0.45 μm pore size, 3 mm diameter).
8. Amber glass vials (MS certified).
2.2 LC-PDA-MS
1. 500 mM ammonium acetate (NH 4 Ac) stock solution: Weigh
3.85 g NH 4 Ac in a 100 mL volumetric flask and make up to
100 mL with water (see Note 1).
2. Waters ACQUITY UPLC system (comprising a binary pump
and autosampler) or equivalent.
3. Waters PDA detector or equivalent.
4. Waters Xevo TQ-S triple quadrupole mass spectrometer or
equivalent.
5. Bischoff ProntoSil 200–3-C30 column (250 Â 4.6 mm, 3 μm
particle size) and a corresponding guard column
(10 Â 4.0 mm, 3 μm) or equivalent.
6. Elution solvent A: Mix 499 mL methanol (MeOH) and 1 mL
500 mM NH 4 Ac stock solution in a glass bottle to make 1 mM
NH 4 Ac in MeOH/water (99.8:0.2, v/v).
7. Elution solvent B: Methyl tert-butyl ether (MTBE).
8. Standards of plant photosynthetic carotenoids (Table 1) purchased from DHI (Hørsholm, Denmark) (see Note 2).
2.3 LC-DAD-FTICRMS
1. Agilent 1200 series HPLC system consisting of a binary pump,
autosampler, column oven and DAD or equivalent.
2. Hybrid linear ion trap FTICR mass spectrometer LTQ FT
Ultra (Thermo Fisher Scientific) equipped with a 7 T supraconducting magnet or equivalent.
3. For HPLC column, elution solvents and pigment standards, see
Subheading 2.2, items 5–8.
3 Methods
Carry out pigment extraction under a fume hood and wearing
safety glasses. Dim the light and keep samples on ice while extracting pigments to avoid degradation. Amber glass vials should be
used for LC-MS for protection against light.
Carotenoid Isotopologue Profiling
265