3. Hexane–diethyl ether–glacial acetic acid 80:20:1 (v/v/v).
4. Silica gel coated TLC plates (10 Â 20 cm, 0.25 mm, Si G60).
5. 200 μL syringe for application of sample on TLC plates.
6. TLC heater.
7. Scanner and software for image processing to calculate optical
density.
2.5 Isolation of the
Micellar Fraction
1. Benchtop centrifuge, temperature 4
C and equipped with
rotors for low and high-speed centrifugation.
2. Centrifuge tubes of 15 mL and 50 mL capacity.
3. Nylon 25 mm (i.d.) Â 0.22 μm pore size filters.
2.6 Extraction and
Analysis of the
Carotenoid Fraction
from the Meal Puree
and the Micelles
Fraction
1. Benchtop centrifuge, temperature 4
C.
2. Centrifuge tubes of 50 mL capacity.
3. Diethyl ether, hexane, and NaCl (10%, w/v).
4. Nylon 13 mm (i.d.) Â 0.22 μm pore size filters.
5. LC-MS/MS platform with UV-visible detector and APCI
source coupled to UHR-TOF mass analyzer.
6. C30 analytical column (3 μm particle size, 25 Â 0.46 cm i.d.).
7. LC grade solvents for chromatographic separation: methyltert-butyl ether, methanol and water.
8. Standard stock solutions of astaxanthin, β-carotene,
β-cryptoxanthin, canthaxanthin, lutein, lycopene, and violaxanthin, which are prepared at a concentration of 25 mg L
À1
following the procedures described in [15]. Once the exact
concentration is determined, working stock solutions for external calibration curves are prepared at 5 concentration levels
ranging from 0.15 to 10.0 mg L
À1 .
3 Methods/Study Design
3.1 Standard Meals
1. Once the meal is ready to eat, grind it finely in an electric food
mixer until a puree is obtained.
2. Keep the puree at 40
C until the application of the in vitro
procedure. Take three portions (5 g) to determine the carotenoid content in the puree.
3.2 Three-Stage In
Vitro Digestion
Procedure and
Isolation of the
Micellar Fraction
1. The procedure is based on Minekus et al. [2]. Mix 30 g of the
puree with 21 mL of simulated salivary electrolyte stock solution, and place 3 aliquots (17 mL) of the puree in 100 mL
GL45 clear glass laboratory bottles (see Note 12) with a cross
type magnetic bar. Mix each aliquot with 1 mL of salivary
α-amylase stock solution (1500 U/mL), 50 μL of 0.3 M
380
Antonio Pe ´ rez-Ga ´ lvez and Javier Fontecha
4. Silica gel coated TLC plates (10 Â 20 cm, 0.25 mm, Si G60).
5. 200 μL syringe for application of sample on TLC plates.
6. TLC heater.
7. Scanner and software for image processing to calculate optical
density.
2.5 Isolation of the
Micellar Fraction
1. Benchtop centrifuge, temperature 4
C and equipped with
rotors for low and high-speed centrifugation.
2. Centrifuge tubes of 15 mL and 50 mL capacity.
3. Nylon 25 mm (i.d.) Â 0.22 μm pore size filters.
2.6 Extraction and
Analysis of the
Carotenoid Fraction
from the Meal Puree
and the Micelles
Fraction
1. Benchtop centrifuge, temperature 4
C.
2. Centrifuge tubes of 50 mL capacity.
3. Diethyl ether, hexane, and NaCl (10%, w/v).
4. Nylon 13 mm (i.d.) Â 0.22 μm pore size filters.
5. LC-MS/MS platform with UV-visible detector and APCI
source coupled to UHR-TOF mass analyzer.
6. C30 analytical column (3 μm particle size, 25 Â 0.46 cm i.d.).
7. LC grade solvents for chromatographic separation: methyltert-butyl ether, methanol and water.
8. Standard stock solutions of astaxanthin, β-carotene,
β-cryptoxanthin, canthaxanthin, lutein, lycopene, and violaxanthin, which are prepared at a concentration of 25 mg L
À1
following the procedures described in [15]. Once the exact
concentration is determined, working stock solutions for external calibration curves are prepared at 5 concentration levels
ranging from 0.15 to 10.0 mg L
À1 .
3 Methods/Study Design
3.1 Standard Meals
1. Once the meal is ready to eat, grind it finely in an electric food
mixer until a puree is obtained.
2. Keep the puree at 40
C until the application of the in vitro
procedure. Take three portions (5 g) to determine the carotenoid content in the puree.
3.2 Three-Stage In
Vitro Digestion
Procedure and
Isolation of the
Micellar Fraction
1. The procedure is based on Minekus et al. [2]. Mix 30 g of the
puree with 21 mL of simulated salivary electrolyte stock solution, and place 3 aliquots (17 mL) of the puree in 100 mL
GL45 clear glass laboratory bottles (see Note 12) with a cross
type magnetic bar. Mix each aliquot with 1 mL of salivary
α-amylase stock solution (1500 U/mL), 50 μL of 0.3 M
380
Antonio Pe ´ rez-Ga ´ lvez and Javier Fontecha