32.5.17 AOAC Official Method 938.

04 Carotenoids in Macaroni Products

Colorimetric Method First Action 1938 Final Action

(Applicable only to determination of carotenoids added for coloring purposes.)

A. Reagents

(a) Alcoholic potassium hydroxide solution.Dissolve 10 g KOH in 100 mL alcohol by warming on steam bath. (b) Methanol.92%. 8 mL H2O + 92 mL absolute methanol. (c) Adsorption mixture.Mix equal portions by volume of activated magnesia (Micron brand No. 2641, 2642, or Adsorptive Magnesia No. S-120, Fisher Scientific Co.) and diatomaceous earth (Hyflo Super-Cel, Celite Corp.). (d) Carotene standard solution.20 mg/L. Dissolve 100 mg natural mixture of a- and b-carotene in 56 mL CS2, add 3540 mL absolute alcohol, cool in refrigerator ca 1 h to ensure maximum crystallization, and filter on hard paper. Dissolve carotene crystals in 56 mL CS2, add 40 mL petroleum ether, refrigerate as before, filter on hard paper, and dry crystals in vacuum desiccator 1 h. Accurately weigh 20 mg purified crystals and wash with 20 mL absolute ether into 1 L glass-stoppered volumetric flask. Continue to wash with petroleum ether, and dilute to volume by adding petroleum ether as soon as carotene dissolves completely.
B. Preparation of Standard Curve

again with 25 mL ether. Discard lower layer and add the ether to original ether solution. Wash ether by pouring 50 mL H2O through it. After layers separate, withdraw aqueous layer and discard. Add 50 mL petroleum ether to ether solution, and wash with five 50 mL portions H2O, carefully inverting and rotating separator. Discard all aqueous layers (slight emulsions usually clear in few min but may be discarded, especially if there is no significant yellow tinge). Transfer etherpetroleum ether mixture to 250 mL distillation flask, rinsing separator with petroleum ether; place flask in beaker of H2O at 4550C. Stopper flask, connect side arm with centrifuge, and concentrate to ca 5 mL to remove ether. Filter through Allihn-type ad sorp tion tube with coarse fritted glass plate containing ca 3 mm layer anhydrous powdered Na2SO4, or through 5.57.0 cm paper half filled with the Na2SO4 (use small, long-stem funnel reaching through neck of flask) into 25 mL volumetric flask. Dilute to volume with petroleum ether used to rinse distillation flask and which has been passed portionwise through the filter containing Na2SO4, and mix by inverting few times. Transfer to 1 cm absorption cell and read A at 436 nm in spectrophotometer, making 3 readings. From average reading, calculate total carotenoid pigment in ppm (mg/g) from standard curve. If pure carotene is not available for standardization, multiply A by 64.2 for yolk, 13.05 for noodles, or 6.52 for semolina and macaroni.
E. Separation of Carotene from Xanthophylls

Dilute 0.0, 1.25, 2.50, 3.75, 5.00, 6.25, 7.5, 8.75, and 10.00 mL carotene standard solution to 250 mL with petroleum ether. (Concentrations 0.0, 0.10, 0.20, 0.30, 0.40, 0.50, 0.60, 0.70, and 0.80 mg/L.) Read solutions at 436 nm in spectrophotometer or in photometer. Obtain line of best fit for data by method of least squares, Definitions of Terms and Explanatory Notes. In applying this method, let x represent scale reading and y, concentration in mg/L. Standardize on same day stock solution is prepared.
C. Preparation of Test Sample

Grind macaroni and noodles to as near flour fineness as possible in ordinary coffee-type mill. (Products containing egg give no difficulty, but plain macaroni products require several grindings.) Take care not to set mill too tight, as enough heat may be generated to damage pigments.
D. Determination of Total Carotenoids and Carotene

Weigh 20 g flour, semolina, or macaroni, or 10 g egg noodles, or 2 g egg yolk into 125 mL Erlenmeyer. Add 50 mL alcoholic KOH solution and boil on steam bath 30 min under reflux condenser. Occasionally rotate flask but be as careful as possible to keep test portion from collecting on sides of flask. Remove flask and cool to room temperature. Filter through Bchner-type medium fritted glass filter into 250 mL suction flask, using suction, transferring most of material with few mL alcohol from wash bottle. Turn off suction, rinse flask with 25 mL ether, pour rinsing onto glass filter, and stir material with rod to let ether come in contact with all portions. Filter, and repeat this operation twice. Transfer filtrate to 250 mL glass-stoppered separator and rinse with ca 25 mL ether, disregarding soapy material in flask. Add 175 mL H2O, carefully invert, and rotate several times. When layers separate, remove lower aqueous-alcohol layer and extract this layer

(a) Carotene by phase separation.Quantitatively transfer all solution from cell and volumetric flask to 125 mL separator, rinse with petroleum ether, and dilute to ca 100 mL. Add 15 mL 92% methyl alcohol, shake moderately ca 2 min by hand or 10 min on mechanical shaker, and let separator stand in upright position ca 1 min until lay ers sepa rate. Decant lower layer con taining xanthophyll and repeat extractions 5 more times or until aqueous methanol layer is nearly colorless for semolina. (Eight extractions are generally enough for noodles but higher than normal egg content may require 10 extractions.) Examine final methanol layer recovered in test tube over white background to be sure solution is nearly colorless. Wash petroleum ether with 25 mL H2O, inverting separator several times; discard aqueous layer and repeat twice more. Filter petroleum ether through Allihn-type adsorption tube containing 6 mm layer of anhydrous powdered Na2SO4, or through 9 cm paper half filled with the Na2SO4, into 250 mL distillation flask, washing color from filter with petroleum ether. Concentrate to 5 mL by vacuum as in D and transfer to 10 mL volumetric flask, using very small portions of petroleum ether; dilute to volume, mix by inverting, and read A in spectrophotometer as in D. Calculate carotene in ppm (mg/g) from standard curve. If pure carotene is not available for standardization, multiply A by 5.22 for noodles, or by 2.61 for semolina. (b) Caro tene by chro mato graphic sep a ra tion.Pre pare column in adsorption tube ca 18 mm od 240 mm with ca 5 cm tip inserted through rubber stopper. Loosely plug with small pad of cotton, place in 250 mL suction flask, and turn on suction. Add adsorption mixture A(c), through funnel in small amounts from spatula to height of ca 11 cm; pack column by pressing down (only once af ter all this mix ture has been added) with cork stopper, just fit ting the tube, on end of rod. Place 12 cm anhy drous powdered Na2SO4 on top. Quantitatively transfer all solution from cell and volumetric flask to 250 mL distillation flask, and concentrate as in D to ca 5 mL,
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continuously applying suction to flask. Transfer to prepared column and rinse with 4 ca 5 mL portions petroleum ether to remove all color. Finally rinse down sides of tube with few mL petroleum ether. After few drops have come through column, change to another 250 mL suction flask. When nearly all petroleum ether is down to Na2SO4 layer, add 50 mL petroleum etheracetone mixture (9 + 1) to wash through carotene. When all this solvent has passed through Na2SO4, turn off suction. (Keep top of column covered with solvent during entire operation.) Transfer carotene solution (which should

be only few mL) to 10 mL volumetric flask, using very small portions of petroleum ether, dilute to volume with petroleum ether, and mix by inverting. Read as in (a) and calculate in ppm (mg/g). (These solutions should be read on same day as extraction.) References: JAOAC 21, 339(1938); 34, 275(1951). CAS-36-88-4 (carotene) CAS-977016-86-6 (carotenoids)

2005 AOAC INTERNATIONAL