SPIFAN Nutrients ERP Book_9-29-15

Carot-01 FOR ERP USE ONLY DO NOT DISTRIBUTE

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).