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D. Preparation of Solutions
(a)
Ascorbate-sulfide solution.
Weigh 16 g of Na
2
S and 40 g of NaAsc into a flask and dissolve in 400 mL water.
Add 150 mL of glycerol and mix. Prepare fresh daily.
(b)
Dissolving solution
(ACN/MeOH/DCM, 70/15/20, V/V/V + 0.01 % BHT, m/V). Mix 700 mL of ACN, 150 mL of
MeOH and 200 mL of DCM. Add 0.1 g of BHT and dissolve. This solution can be stored at room temperature in
a closed container for 1 month.
(c)
Ethanolic hydroxide 2M.
Dissolve 112 g of KOH in 100 ml water and fill to 1 L with EtOH. Prepare fresh daily.
(d)
Washing solution 5 % KOH.
Dissolve 50 g KOH in 1 L water.
(e)
Washing solution phosphate buffer.
Dissolve NaH
2
PO
4
·H
2
O and Na
2
HPO
4
·2H
2
O in water (0.2 M, pH 6.7).
E. Preparation of Standards
(a) Carotenoids are sensitive to UV light and oxygen; conduct all operations using subdued yellow light (Osram
HE 28W/62 TL, Philips TLD 36W/16 TL or equivalent) or use amber glassware. Keep all solutions away from
direct light.
(b)
Beta-Carotene stock solution.
Into a 100 mL volumetric flask, weigh 10 mg beta-carotene and dissolve in 15
mL DCM. Make up to volume with n-hexane containing 0.1 % (m/V) BHT. Distribute into glass tubes and store
in the freezer. To determine the concentration, dilute 500 μL to 20 mL with n-hexane in a volumetric flask and
measure the extinction at 453 nm in triplicate in a 1 cm quarts cuvette. Use n-hexane as blank. Calculate the
beta-carotene concentration of the stock solution in mg/L as follows: (E x 20 x 10 x 1000) / (E1%453nm x 0.5)
where E = the average measured extinction and E1%453nm = 2592.
(c)
Dimethyltocol (DMT) solution 1 g/L.
Dissolve circa 50 mg of DMT in 50 mL of EtOH. Store in the freezer.
(d)
HPLC standard.
Into a glass tube, pipette 100 μL DMT solution, evaporate to dryness and re-dissolve in 2000
μL beta-carotene stock solution. This results in a 2.5 mg/L beta-carotene standard solution.
F. Preparation of Samples
(1) Weigh sample in duplicate into a 250 mL (Duran) glass bottle; for powder samples 6 and 10 g and for liquid
samples 16 and 20 ml.
(2) Add 15 mL of the ascorbate-sulfide solution and swirl to dissolve the sample.
(3) Add 50 mL of the ethanolic hydroxide solution, 500 μL of DMT solution and some carborundum.
(4) Place in a shaking water bath set at 85⁰C for 30 minutes.
(5) Cool to room temperature, add 100 ml of DIPE and extract the carotenoids by shaking for 5 minutes using a
shaker.
(6) Poor 30 mL of the DIPE extract into a 50 mL screw-cap (Greiner) tube, add 20 mL of 5% (m/V) KOH in water
and shake for 5 minutes using a shaker.
(7) Centrifuge at 1500 g for 5 minutes and remove the water phase (bottom layer) using the suction device.
(8) Add 20 mL of phosphate buffer and shake for 5 minutes using a shaker.
(9) Centrifuge at 1500 g for 5 minutes, pipette 5000 μL of the extract (top layer) in a glass tube, evaporate to
dryness and re-dissolve in 500 μL of ACN/MeOH/DCM (70/15/20 + 0.1 % BHT). Re-dissolve by vortex mixing
for 1 minute followed by ultrasonic vibration for 2 minutes and subsequently vortex mixing for 1 minute.
Transfer the extract to an HPLC vial.
(!) For samples containing a high amount of carotenoids (> 200 μg/100 g) the weigh-in should be lower and no
concentrating is required.
[This extract can also be used for the determination of vitamin A and E and (when vitamin D2 is added) also
for the determination of vitamin D3].
G. Analysis
(a)
Chromatographic conditions.
(1) Injection volume. 50 μL.
(2) Autosampler temperature. 10⁰C.
(3) Column temperature. 20⁰C.
(4) Flow rate. 1.5 mL/min.
(5) Run time. 41 min.
Carot-01 (February 2016)
FOR ERP USE ONLY
DO NOT DISTRIBUTE