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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

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