3. Solvent B: Deionized water (HPLC-grade deionized ultrapure
water produced with a Milli-Q Advantage A10 system, Merck
Millipore, Madrid, Spain).
4. HPLC
column.
A
reversed-phase
C18
column
(200 mm  4.6 mm i.d., 3 μm, Mediterranea SEA18; Teknokroma, Barcelona, Spain), fitted with a guard column of the
same packing material, should be used (see Note 6).
2.5 Preparation
of Calibration Curves
1. UV/visible (UV/Vis) spectrophotometer. In our case the system consisted of a Hewlett-Packard UV/Vis diode array spectrophotometer model 8452A.
2. Quartz cuvette (0.5 mL).
3. 1 mL volumetric flasks.
4. Carotenoid standards (see Note 7).
5. Solvents (analytical-grade) for individual stock solutions of
carotenoids: petroleum ether, ethanol, n-hexane.
6. Acetone (HPLC-grade).
3 Methods
3.1 General
Precautions
Carotenoids are labile compounds which are prone to degradation
upon exposure to oxygen and light, thus precautions should be
taken to minimize the adverse effects of these factors. It is highly
recommended that all operations should be carried out under
dimmed light to prevent isomerization and photodegradation of
carotenoids. Follow “Good Laboratory Practice” procedures so
that all operations involving the manipulation of solvents are performed in a fume hood.
3.2 Extraction
of Carotenoids
(Standard Procedure)
1. Sample size. One to three grams taken from a homogeneous
and representative sample is generally sufficient to provide
accurate and reproducible data (see Note 8). In the case of
dehydrated samples the sample size can be scaled down by a
factor of 5–10 (see Note 9).
2. Place sample in a 50 mL centrifuge tube (Falcon tube or
similar) and extract it with 20 mL of acetone (containing
0.1% BHT) by using a homogenizer during 1 min (see Note
10). If an internal standard (IS) is used, this should be added at
this step (the IS amount should be adjusted to the expected
average carotenoid content) (see Note 11).
3. Separate extract from sample debris by centrifugation at
4500 Â g at 4
C during 5 min.
4. Repeat the extraction procedure 3–4 times until complete
extraction of color (see Note 12).
120
Da ´ maso Hornero-Me ´ ndez
water produced with a Milli-Q Advantage A10 system, Merck
Millipore, Madrid, Spain).
4. HPLC
column.
A
reversed-phase
C18
column
(200 mm  4.6 mm i.d., 3 μm, Mediterranea SEA18; Teknokroma, Barcelona, Spain), fitted with a guard column of the
same packing material, should be used (see Note 6).
2.5 Preparation
of Calibration Curves
1. UV/visible (UV/Vis) spectrophotometer. In our case the system consisted of a Hewlett-Packard UV/Vis diode array spectrophotometer model 8452A.
2. Quartz cuvette (0.5 mL).
3. 1 mL volumetric flasks.
4. Carotenoid standards (see Note 7).
5. Solvents (analytical-grade) for individual stock solutions of
carotenoids: petroleum ether, ethanol, n-hexane.
6. Acetone (HPLC-grade).
3 Methods
3.1 General
Precautions
Carotenoids are labile compounds which are prone to degradation
upon exposure to oxygen and light, thus precautions should be
taken to minimize the adverse effects of these factors. It is highly
recommended that all operations should be carried out under
dimmed light to prevent isomerization and photodegradation of
carotenoids. Follow “Good Laboratory Practice” procedures so
that all operations involving the manipulation of solvents are performed in a fume hood.
3.2 Extraction
of Carotenoids
(Standard Procedure)
1. Sample size. One to three grams taken from a homogeneous
and representative sample is generally sufficient to provide
accurate and reproducible data (see Note 8). In the case of
dehydrated samples the sample size can be scaled down by a
factor of 5–10 (see Note 9).
2. Place sample in a 50 mL centrifuge tube (Falcon tube or
similar) and extract it with 20 mL of acetone (containing
0.1% BHT) by using a homogenizer during 1 min (see Note
10). If an internal standard (IS) is used, this should be added at
this step (the IS amount should be adjusted to the expected
average carotenoid content) (see Note 11).
3. Separate extract from sample debris by centrifugation at
4500 Â g at 4
C during 5 min.
4. Repeat the extraction procedure 3–4 times until complete
extraction of color (see Note 12).
120
Da ´ maso Hornero-Me ´ ndez