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D. Preparation of Solutions
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.
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.
Ethanolic hydroxide 2M.
Dissolve 112 g of KOH in 100 ml water and fill to 1 L with EtOH. Prepare fresh daily.
Washing solution 5 % KOH.
Dissolve 50 g KOH in 1 L water.
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
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.
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.
Dimethyltocol (DMT) solution 1 g/L.
Dissolve circa 50 mg of DMT in 50 mL of EtOH. Store in the freezer.
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.
[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.
(6) Mobile phases and gradient. A. 7.5 g/L ammonium acetate in MeOH. B. ACN. C. IPA. D. ACN/water (1/1, V/V).
CAROT-01
FOR ERP/WORKING GROUP USE ONLY
DO NOT DISTRIBUTE