Analyst, February, 1968 ,Vol. 93, $p. 107-110 107 A Method for the Determination of Vitamin A, a- and p- Carotene in Margarine, including the Results of a Collaborative Test BY C. D. USHER, D. J. FAVELL (Unilever Research Laboratory, Colworth House, Shavnbrook, Beds.) AND H. LAVERY ( Unilever Research Laboratory, The Frythe, Welwym, Herts.) The statutory method for the determination of total vitamin A in margarine has been modified to enable vitamin A, u- and p-carotene to be determined separately. A collaborative test by six laboratories has shown that the proposed method is more accurate than the statutory method, particularly for margarine coloured with synthetic /&carotene, and there is no loss of precision. THE colour of most margarines produced in Great Britain is caused mainly by their synthetic /3-carotene content.This has not always been so. Before about 1960 very little synthetic /I-carotene was available and red palm oil was used as the source of colour. The latter is a mixture of a-, /3- and inert carotenoids; a-carotene has only about one half the activity of /3-carotene as a pro-vitamin A. The vitamin A content of margarine and the method for its determination1 are governed by Act of Parliament. The total is made up from the actual vitamin A and the calculated vitamin A contribution of the /3-carotene. Because the present regulations were drafted when palm oil was widely used, a conversion factor of 358 (based on the average a- and 8- carotene contents) was quoted. Today, when synthetic ,%carotene, with a true conversion factor of 667, is used, analysts must still use the statutory conversion factor.It has, of course, been difficult to separate u- and /3-carotene from each other, and from vitamin A, and to recover all of them quantitatively. This paper gives details of a method of separation. It also provides evidence to show that the method is more accurate than, and as reliable as, the present statutory method. In addition, the method makes it relatively easy to determine whether red palm oil or p-carotene has been used in the manufacturing process. EXPERIMENTAL PRINCIPLE- The unsaponifiable matter extracted from a portion of margarine, after refluxing with ethanolic potassium hydroxide, is chromatographed on a column of neutral alumina. The yellow carotene eluate, which precedes vitamin A off the column, is chromatographed again on a column of magnesium oxide to separate the a- and /3-carotene.The colourless vitamin A eluate is passed through a column of alkaline alumina. The concentration of a- and p-carotene and vitamin A in each eluate is measured with a spectrophotometer. APPARATUS- The apparatus has been fully described in a previous paper.2 REAGENTS- The reagents are identical with those listed in a previous paper,2 with the following additions. Potassiwn hydroxide solution, 60 per cent. wlv, aqueous. Additional developing solvent-Light petrolcum containing 50 per cent. of diethyl ether. Cyclohexane-Spectroscopic grade. Magutesia-Heat magnesium oxide (heavy) at 100" C for 2 hours. Cool in a desiccator 0 SAC and Unilever Ltd.and set aside for 3 or 4 days in an air-tight bottle before use.108 PROCEDURE- USHER, FAVELL AND LAVERY: METHOD FOR THE DETERMINATION [Analyst, Vol. 93 The whole process to take place as quickly as possible in subdued light. Saponijcation and extraction-Weigh 10.0 g of margarine into a 250-ml flat-bottomed flask. Add 20 mg of quinol, 60 ml of ethanol, 10 ml of potassium hydroxide solution and finally 10 ml of light petroleum. Boil under reflux for 30 minutes, protecting the flask from light. (Use flasks covered with a shield of aluminium foil.) Cool, and add 80 ml of distilled water. Transfer the solution quantitatively into a 500-ml separating funnel and rinse out the flask with a further 80 ml of distilled water. Add the rinsings to the separating funnel.Extract the unsaponified material with 100ml and three 50-ml portions of diethyl ether. Combine the ether extracts and wash with four 50 ml-portions of distilled water; carry out the first washing by swirling and the following three by gentle shaking. Evaporate the unsaponifiable extract to dryness on a water-bath at 50" C, with a stream of inert gas. The last stages of the evaporation require full attention, because the residue in the flask must not be allowed to remain dry longer than is absolutely necessary. Immediately after all of the diethyl ether has been removed, add 2 ml of absolute ethanol and again evaporate to dryness in a current of inert gas; if the residue appears wet, repeat the addition of absolute ethanol and evaporation to dryness.Immediately dissolve the residue in 5ml of light petroleum and again evaporate to dryness in a current of inert gas. Repeat the dissolution in light petroleum and evaporation to dryness twice more. Finally, dissolve the residue in 2 to 3 ml of light petroleum for chromatography. Chromatographic separatioiz of vitami% A-The chromatographic separation and deter- mination of vitamin A has been fully described elsewhere.2 The fraction containing carotenes is chromatographed again on magnesia to separate a- and /3-carotene. Chromatographic separation of cc- and /3-carotene-Evaporate the fraction containing carotene to dryness on a water-bath at 50" C, with a stream of inert gas. Dissolve the extract in 2 ml of light petroleum before chromatography. Place a pledget of cotton-wool in the lower tip of the upper chromatographic tube.Pour in light petroleum to a level half-way up the centre section, and add 3 g of magnesia. Add the cc- and /3-carotene extract and rinse the flask with 2 ml of light petroleum. Develop the chroniatogram, under pressure if necessary, with light petroleum containing diethyl ether. The precise conditions required to produce the separation must be determined by experiment, and vary with different batches of magnesia. The optimum mixture lies within the range 4 to 12 per cent. of diethyl ether in light petroleum and gives a complete separation of a- and p-carotene, in sharp bands. If difficulty is experienced with the flow-rate, and with adequate separation of a- and /3-carotene, re-dry the magnesia immediately before use (24 hours).Collect the lower, pale yellow-coloured zone of a-carotene. After the elution of a-carotene, collect the deeper orange-coloured zone of p-carotene. Use 50 per cent. of diethyl ether in light petroleum to speed up the elution of p-carotene. Evaporate the two zones separately to dryness on a water-bath at 50" C, with a stream of inert gas. Dissolve the a- and /!-carotene in cyclohexane and dilute to 10 and 25m1, respectively, with cyclohexane. Measure the optical densities of these solutions in 1-cm glass cells against cyclohexane, at 2-mp intervals, from 440 to 456mp, repeating those at the peak for confirmation. CALCULATIONS- optical density x volume of carotene solution . 10 x 100 EiZ = a-Carotene potency = Et& x 334. /3-Carotene potency = E:,%, x 667.Total carotene potency (i.u. per g) = (Et:'; value of /3-carotene solution + $ Ei:& value, of a-carotene solution) x 667. Total carotene potency x 0.8 = vitamin A equivalent (i.u. per g). This vitamin A equivalent must be added to the vitamin A figure, already determined, to give the total vitamin A potency of the margarine. COLLABORATIVE TEST Two margarines, in sufficient amount for the whole test, were prepared in a conventional margarine plant.TABLE I COMPARISON OF THE RECOVERIES OF TOTAL VITAMIN A FROM TWO MARGARINES BY THE STATUTORY AND PRESENT METHODS ON THREE OCCASIONS Total vitamin A (all laboratories) 7 A , Method of analysis Statutory { { Present 1 week - Mean Mean Sample Actual, recovery, value, Standard number i.u.per g per cent. i.u. per g deviation 1 30.4 81 24.7 1-07 2 32-0 86 27.4 1.69 1 30.4 91 27.6 1.04 2 32.0 88 28.1 1.81 7 Mean recovery, per cent. 77 83 88 88 7 weeks Mean value, Standard i.n. per g deviation 23.5 1.63 26-6 0.76 __h______\ 26.7 1.94 28.0 0.80 7 Mean recovery, per cent. 75 84 85 88 14 weeks --h-----? Mean value, Standard i.u. per g deviation 22.9 0.85 264 0.94 25.9 0.83 28.1 1 -08 TABLE I1 COMPARISON OF THE SEPARATE RECOVERIES OF VITAMIN A AND CAROTENE FROM TWO MARGARINES BY THE STATUTORY AND PRESENT METHODS ON THREE OCCASIONS Vitamin A and “carotene” (all laboratories) 1 week 7 weeks 14 weeks P - w - 7 c A I Mean Mean Mean Mean Mean Mean Method of Sample Actual, recovery, value, Standard recovery, value, Standard recovery, value, Standard analysis number i.u.per g per cent. i.u. per g deviation per cent. i.u. per g deviation per cent. i.u. per g deviation Statutory { Present { 1 24.0 89 21.4 1.32 86 20.6 1.72 84 20.2 0.86 30.0 86 26.9 1.46 84 26.2 0.69 86 26.6 0.99 1 24.0 91 21.8 0.95 90 21.6 1.77 88 21.0 0-61 30.0 88 26.6 1.76 89 26-6 0.89 89 26-7 1.03 Vitamin A added 1 6.4 63 3.4 0.25 47 3.0 0.16 42 2.7 0.10 2.0 70 1-4 0.17 70 1.4 0.08 66 1.3 0.12 1 6.4 89 5.7 0.37 78 6.0 0.32 75 4.8 0-37 2.0 80 1.6 0.29 70 1.4 0.13 70 1.4 0.2 1 Statutory { Present { or red palm oil as vitamin ,4110 USHER, FAVELL AND LAVERY Sample 1 contained 24.0 i.u. per g of vitamin A and synthetic /3-carotene equivalent Sample 2 contained 30.0 i.u. per g of vitamin A and red palm oil equivalent to 2.0 i.u. Samples were despatched from a central depot in time for each of the six participating laboratories to analyse the margarines after 1, '7 and 14 weeks at 15" C.At each of the three stages, single determinations were made on each margarine on each of 3 consecutive days by the two methods of analysis (statutory method and the method described above). Appropriate precautions were taken to avoid sampling errors, and every sample was analysed under code. to 6.4 i.u. per g of vitamin A. per g of vitamin A. RESULTS AND DISCUSSION The results of the collaborative test are shown in Tables I and 11. The recovery of added vitamin A is 90 per cent. by each method, with no difference in the precision. The recovery of pro-vitamin A from margarine containing red palm oil is 70 per cent. by each method. Recovery from margarine containing synthetic IS-carotene is 80 per cent. by the present method and less than 50 per cent. by the statutory method. During the collaborative test some participants had difficulty with the flow-rate through magnesia, and with adequate separation of 01- and /3-carotene. To overcome this problem a minor modification has been included in the method, These difficulties do not appear to have reduced significantly the precision of recovery of pro-vitamin A, compared with the recovery of vitamin A added as such (see Table 11). The authors acknowledge the help and advice given by Mr. G. Walley during the preparation of the method, and by Mr. K. T. Boyd and Mr. A. Rook on the design of the experiment and the analysis of results. They also thank the Margarine and Shortening Manufacturers Association for its help in making possible the collaboration of several of its member companies, and the Directors of Unilever Limited for permission to publish this paper. REFERENCES 1. 2. The Food Standards (Margarine) Order, 1964, S.I. 19541613. Analytical Methods Committee, Analyst, 1964, 89, 7. Received January 16th, 1967