2. Intermediate stock internal standards: All intermediate internal
standards are prepared by diluting the commercial stock in
MeOH, and stored in dark vials at À20
C.
3. Apocarotenoid standards were also generated by oxidative
cleavage of the parent carotenoid by potassium permanganate
following the methodology reported by Rodriguez and
Rodriguez-Amaya [20]. A series of β-apo-carotenals were generated by oxidation of β-carotene, a series of apo-zeaxanthinals
and apo-capsorubinals were generated by oxidation of zeaxanthin and capsorubin, respectively, and a series of ε-apo-luteinals
were generated by oxidation of lutein. Briefly, in a round
bottom amber vial, 1.0 M equivalent of the parent carotenoid
was dissolved in 1 mL of CHCl 3 and treated with KMnO 4
(2.6 M equivalents) at room temperature for 12 h. When the
reaction was complete, the formed apocarotenoid mixture was
filtered through celite 535 coarse (Fluka) to remove MnO 2 and
the filtrate was washed several times with water, dried over
Na 2 SO 4 and concentrated in controlled vacuum.
4. Adsorbent powder “Miyazaki Hydro-Protect,” Patented in
Japan no. 3645552 (Shimadzu Corporation, Kyoto, Japan).
2.2 Instrumentation
for the SFE-SFC-MS
Analysis
The SFE-SFC-MS analyses were performed on a Shimadzu Nexera
UC system (Shimadzu, Japan), consisting of a CBM-20A controller, an SFE-30A module for supercritical fluid extraction, two
LC-20ADXR dual-plunger parallel-flow pumps, an LC-30ADSF
CO 2 pump, two SFC-30A back pressure regulator, a DGU degasser, a CTO-20 AC column oven, an SIL-30 AC autosampler, an
LCMS-8050 mass spectrometer equipped with an APCI source.
The entire system was controlled by the LabSolution ver. 5.8.
2.3 SFE-SFC-QqQ MS
Solutions
1. Mobile Phase A: CO 2 .
2. Mobile Phase B: MeOH.
3. Makeup Pump Mobile Phase: MeOH.
4. Wash 1: CO 2 .
5. Wash 2: MeOH.
2.4 Supplies
Ascentis
Express
fused-core
C30
column,
(150 mm  4.6 mm  2.7 μm d.p., Merck Life Science (Merck
KGaA, Darmstadt, Germany) or equivalent.
3 Methods
All procedures are carried out at room temperature; allow all standards and samples to equilibrate to room temperature before using.
Apocarotenoid Analysis by SFE-SFC-QqQ/MS
211
standards are prepared by diluting the commercial stock in
MeOH, and stored in dark vials at À20
C.
3. Apocarotenoid standards were also generated by oxidative
cleavage of the parent carotenoid by potassium permanganate
following the methodology reported by Rodriguez and
Rodriguez-Amaya [20]. A series of β-apo-carotenals were generated by oxidation of β-carotene, a series of apo-zeaxanthinals
and apo-capsorubinals were generated by oxidation of zeaxanthin and capsorubin, respectively, and a series of ε-apo-luteinals
were generated by oxidation of lutein. Briefly, in a round
bottom amber vial, 1.0 M equivalent of the parent carotenoid
was dissolved in 1 mL of CHCl 3 and treated with KMnO 4
(2.6 M equivalents) at room temperature for 12 h. When the
reaction was complete, the formed apocarotenoid mixture was
filtered through celite 535 coarse (Fluka) to remove MnO 2 and
the filtrate was washed several times with water, dried over
Na 2 SO 4 and concentrated in controlled vacuum.
4. Adsorbent powder “Miyazaki Hydro-Protect,” Patented in
Japan no. 3645552 (Shimadzu Corporation, Kyoto, Japan).
2.2 Instrumentation
for the SFE-SFC-MS
Analysis
The SFE-SFC-MS analyses were performed on a Shimadzu Nexera
UC system (Shimadzu, Japan), consisting of a CBM-20A controller, an SFE-30A module for supercritical fluid extraction, two
LC-20ADXR dual-plunger parallel-flow pumps, an LC-30ADSF
CO 2 pump, two SFC-30A back pressure regulator, a DGU degasser, a CTO-20 AC column oven, an SIL-30 AC autosampler, an
LCMS-8050 mass spectrometer equipped with an APCI source.
The entire system was controlled by the LabSolution ver. 5.8.
2.3 SFE-SFC-QqQ MS
Solutions
1. Mobile Phase A: CO 2 .
2. Mobile Phase B: MeOH.
3. Makeup Pump Mobile Phase: MeOH.
4. Wash 1: CO 2 .
5. Wash 2: MeOH.
2.4 Supplies
Ascentis
Express
fused-core
C30
column,
(150 mm  4.6 mm  2.7 μm d.p., Merck Life Science (Merck
KGaA, Darmstadt, Germany) or equivalent.
3 Methods
All procedures are carried out at room temperature; allow all standards and samples to equilibrate to room temperature before using.
Apocarotenoid Analysis by SFE-SFC-QqQ/MS
211