CaCl 2 , and 1.95 mL of water. Incubate the mixture at 37
C
(20 mL) for 10 min or 2 min with magnetic stirring at
1000 rpm.
2. Mix each aliquot (oral bolus) of the preceding step with 15 mL
of simulated gastric electrolyte stock solution, 3.2 mL of porcine pepsin solution and 10 μL of 0.3 M CaCl 2 . Adjust the pH
of the mixture to 3.0 with 1 M HCl (see Note 13) and make the
final volume equal to 40 mL. Place the bottle in the incubator
chamber at 37
C for 2 h with magnetic stirring at 1000 rpm.
3. The gastric chyme is mixed with 22 mL of simulated intestinal
electrolyte stock solution, 10 mL of pancreatin solution, 5 mL
of fresh bile, and 80 μL of 0.3 M CaCl 2 . Adjust the pH of the
mixture to 7.0 with 1 M NaOH (see Note 13) and make the
final volume equal to 80 mL. Place the bottle in the incubator
chamber at 37
C for 2 h, with magnetic stirring at 1000 rpm.
4. The intestinal chyme of each bottle is divided in 40 mL aliquots
and transferred to 50 mL centrifuge tubes. Each bottle is
washed with two portions of 5 mL of water and added to the
centrifuge tubes to make the final volume equal to 50 mL.
Isolate the aqueous suspension from the undigested and solid
residues by low speed centrifugation at 4000 Â g for 20 min.
Transfer 10 mL aliquots of the aqueous suspension to 15 mL
centrifuge tubes and apply the high-speed centrifugation step
at 15000 Â g for 8 min. Isolate the micellar fraction by filtering
the clarified suspension through a 0.22 μm Nylon filter.
3.3 Extraction of
Carotenoids from the
Puree and Micelles
1. The extraction method is based on Rı ´os et al. [16]. Mix the
puree (5 g) or the micellar fraction (5 mL) with diethyl ether
(5 mL) and hexane (2 mL) and mix in a vortex for 2 min. Add
5 mL of NaCl (10%, w/v) and mix in a vortex for 2 min.
Centrifuge the sample at 4000 Â g for 5 min.
2. Isolate the organic layer in a rotatory flask and repeat the
extraction procedure (avoiding the addition of NaCl solution).
After centrifugation, isolate the organic layer again and evaporate the combined organic extracts to dryness in a rotatory
evaporator at 25
C.
3. Dissolve the extract in 0.5 mL of hexane and filter it through a
nylon 13 mm (i.d.) Â 0.22 μm pore size filter. Store the sample
at À20
C until HPLC-MS/MS analysis (within 1 week).
3.4 LC-MS/MS
Analysis
A great variety of LC systems, MS hardware configurations and
software tools is available for LC-MS/MS analysis of carotenoids,
including column, solvent composition, isocratic or gradient elution, flow rate and postcolumn operation, APCI ionization protocol, parameters and arrangement of the MS analyzer, and
conditions for MS/MS ionization and scan of product ions. The
Carotenoid In Vitro Digestion
381
C
(20 mL) for 10 min or 2 min with magnetic stirring at
1000 rpm.
2. Mix each aliquot (oral bolus) of the preceding step with 15 mL
of simulated gastric electrolyte stock solution, 3.2 mL of porcine pepsin solution and 10 μL of 0.3 M CaCl 2 . Adjust the pH
of the mixture to 3.0 with 1 M HCl (see Note 13) and make the
final volume equal to 40 mL. Place the bottle in the incubator
chamber at 37
C for 2 h with magnetic stirring at 1000 rpm.
3. The gastric chyme is mixed with 22 mL of simulated intestinal
electrolyte stock solution, 10 mL of pancreatin solution, 5 mL
of fresh bile, and 80 μL of 0.3 M CaCl 2 . Adjust the pH of the
mixture to 7.0 with 1 M NaOH (see Note 13) and make the
final volume equal to 80 mL. Place the bottle in the incubator
chamber at 37
C for 2 h, with magnetic stirring at 1000 rpm.
4. The intestinal chyme of each bottle is divided in 40 mL aliquots
and transferred to 50 mL centrifuge tubes. Each bottle is
washed with two portions of 5 mL of water and added to the
centrifuge tubes to make the final volume equal to 50 mL.
Isolate the aqueous suspension from the undigested and solid
residues by low speed centrifugation at 4000 Â g for 20 min.
Transfer 10 mL aliquots of the aqueous suspension to 15 mL
centrifuge tubes and apply the high-speed centrifugation step
at 15000 Â g for 8 min. Isolate the micellar fraction by filtering
the clarified suspension through a 0.22 μm Nylon filter.
3.3 Extraction of
Carotenoids from the
Puree and Micelles
1. The extraction method is based on Rı ´os et al. [16]. Mix the
puree (5 g) or the micellar fraction (5 mL) with diethyl ether
(5 mL) and hexane (2 mL) and mix in a vortex for 2 min. Add
5 mL of NaCl (10%, w/v) and mix in a vortex for 2 min.
Centrifuge the sample at 4000 Â g for 5 min.
2. Isolate the organic layer in a rotatory flask and repeat the
extraction procedure (avoiding the addition of NaCl solution).
After centrifugation, isolate the organic layer again and evaporate the combined organic extracts to dryness in a rotatory
evaporator at 25
C.
3. Dissolve the extract in 0.5 mL of hexane and filter it through a
nylon 13 mm (i.d.) Â 0.22 μm pore size filter. Store the sample
at À20
C until HPLC-MS/MS analysis (within 1 week).
3.4 LC-MS/MS
Analysis
A great variety of LC systems, MS hardware configurations and
software tools is available for LC-MS/MS analysis of carotenoids,
including column, solvent composition, isocratic or gradient elution, flow rate and postcolumn operation, APCI ionization protocol, parameters and arrangement of the MS analyzer, and
conditions for MS/MS ionization and scan of product ions. The
Carotenoid In Vitro Digestion
381