2.2 Reagents
1. For carotenoid extractions, the quality of the solvents should
reach HPLC-grade, while for MS analysis the solvents must be
LC-MS-grade (see Note 1).
2. Diethyl ether, sodium chloride 10% (w/v), anhydrous Na 2 SO 4 ,
acetone.
3. 0.22 μm PTFE syringe filter.
4. Calibration mix of synthetic polymer poly(ethylene glycol)
(PEG 400, 10 μg/mL).
3 Methods
3.1 Carotenoid
Extraction
The user should note that the extraction is the sum of simple steps,
but slight modifications can be introduced at any of them that
result in an improved success of the entire extraction procedure.
Hence, each sample material requires almost a tailored protocol, so
that the present description should be considered as a sequence of
criteria and description of critical points to diminish common drawbacks affecting the subsequent HPLC-MS analysis. Carotenoids are
usually stable in diminished light and avoiding excessive working
temperature and continuous contact with air (see Note 2). Extracts
are stored at À20
C for 1 month.
1. Mix 10 g of fresh tissue with 30 mL of extracting solvent and
break up the tissue with a homogenizer, filter the solution and
pour the solvent in a separating funnel. Repeat the operation
until no more color is extracted from the remaining tissue (see
Note 3).
2. Mix all the combined extracts with 100 mL of diethyl ether and
100 mL of 10% (w/v) sodium chloride. After gentle shaking let
the phases to separate (see Note 4).
3. Discard the water layer and filter the remaining organic phase
through a solid bed of anhydrous Na 2 SO 4 (25 g).
4. Evaporate the diethyl ether in a rotary evaporator and dissolve
the residue in 100 μL acetone.
5. Filter the extract through a 0.22 μm PTFE syringe filter before
chromatographic analysis.
3.2 LC–MS Data
Acquisition
1. Set up the LC-MS system with the isocratic or gradient conditions of the selected HPLC method. Once the system is equilibrated, analyze the sequence of samples in the autosample tray.
The following description is the practice routinely used in our
laboratory [11]. A binary solvent system consisting of methyltert-butyl ether–methanol–water 85:15:4 (solvent A) and
methyl-tert-butyl methyl–methanol–water 7:90:3 (solvent B)
is used at a flow rate of 1 mL min
À1 . Carotenoid separation is
Mass Spectrometry of Carotenoids
137
1. For carotenoid extractions, the quality of the solvents should
reach HPLC-grade, while for MS analysis the solvents must be
LC-MS-grade (see Note 1).
2. Diethyl ether, sodium chloride 10% (w/v), anhydrous Na 2 SO 4 ,
acetone.
3. 0.22 μm PTFE syringe filter.
4. Calibration mix of synthetic polymer poly(ethylene glycol)
(PEG 400, 10 μg/mL).
3 Methods
3.1 Carotenoid
Extraction
The user should note that the extraction is the sum of simple steps,
but slight modifications can be introduced at any of them that
result in an improved success of the entire extraction procedure.
Hence, each sample material requires almost a tailored protocol, so
that the present description should be considered as a sequence of
criteria and description of critical points to diminish common drawbacks affecting the subsequent HPLC-MS analysis. Carotenoids are
usually stable in diminished light and avoiding excessive working
temperature and continuous contact with air (see Note 2). Extracts
are stored at À20
C for 1 month.
1. Mix 10 g of fresh tissue with 30 mL of extracting solvent and
break up the tissue with a homogenizer, filter the solution and
pour the solvent in a separating funnel. Repeat the operation
until no more color is extracted from the remaining tissue (see
Note 3).
2. Mix all the combined extracts with 100 mL of diethyl ether and
100 mL of 10% (w/v) sodium chloride. After gentle shaking let
the phases to separate (see Note 4).
3. Discard the water layer and filter the remaining organic phase
through a solid bed of anhydrous Na 2 SO 4 (25 g).
4. Evaporate the diethyl ether in a rotary evaporator and dissolve
the residue in 100 μL acetone.
5. Filter the extract through a 0.22 μm PTFE syringe filter before
chromatographic analysis.
3.2 LC–MS Data
Acquisition
1. Set up the LC-MS system with the isocratic or gradient conditions of the selected HPLC method. Once the system is equilibrated, analyze the sequence of samples in the autosample tray.
The following description is the practice routinely used in our
laboratory [11]. A binary solvent system consisting of methyltert-butyl ether–methanol–water 85:15:4 (solvent A) and
methyl-tert-butyl methyl–methanol–water 7:90:3 (solvent B)
is used at a flow rate of 1 mL min
À1 . Carotenoid separation is
Mass Spectrometry of Carotenoids
137