the determination of thiamine in flour fortification mixtures

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312 Analyst, March, 1970, Vol. 95, pp. 312-315 The Determination of Thiamine in Flour Fortification Mixtures BY H. V. HART AND K. H. WILLIS (Flour Milling and Baking Research Association, Old London Road, St. A lbans, Hertfordshire) The reasons for the low recovery of thiamine from flour fortification mixtures containing ingredients used for the treatment of flour, in addition to iron, vitamins and chalk, are examined. The presence of potassium bromate and chalk is shown to be the cause of low recoveries, and methods for overcoming the interference of these substances with the analysis for thiamine content are described. To comply with the compositional requirement of the Bread and Flour Regulations 1963,l it is necessary for the flour miller to add to white flour (with certain exceptions), chalk and master-mix, the latter consisting of a concentrated mixture of nutrients, viz., iron, thiamine and nicotinic acid with flour. The additions of chalk and master-mix are usually made by separate feeders but, in the past, enquiries have been made as to whether these ingredients could be fed into the flour as a single admixture. The stability of such a mixture in respect of the thiamine content was examined by Ridyard2and found to be satisfactory. Analysis of this mixture for thiamine content by the method usually adopted for the determination of this vitamin in fl0ur,3 however, was found to be unsatisfactory and modifications to the method, principally in the amounts of hydrochloric acid required for the dissolution of the sample, were made. In addition to master-mix and chalk, small amounts of other materials may also be added to white flour, either for the purpose of improving its baking properties or for bleaching the slight yellow pigment. Treatment of flour with one pre-mix only, containing perhaps a mixture of ingredients such as master-mix, chalk, fungal amylase preparation , potassium bromate and benzoyl peroxide, would have some advantages and, in particular, would result in economies in feeding plant. When such a mixture was analysed for the content of thiamine by the Ridyard2 method, however, the amounts of vitamin recovered were found to be low. As it was expected that the mixture would be reasonably stable in the dry state, the low recoveries might be caused by losses occurring during the analysis. Investigation of the method of analysis revealed that the low recovery was attributable mostly to the presence of potassium bromate, which resulted in some destruction of thiamine during dissolution of the sample. A further slight loss of thiamine was also shown to be caused by the adsorption of thiamine on the insoluble matter remaining after native chalk is decomposed by hydrochloric acid. Modifications to the method of analysis are suggested to overcome these two causes of the low recovery of thiamine. EXPERIMENTAL A sample of a pre-mix containing 7.5 per cent. of master-mix, 74.5 per cent. of chalk, 5.3 per cent. of fungal amylase preparation, 0.54 per cent. of potassium bromate and 1-2 per cent. of benzoyl peroxide (the last two ingredients are incorporated in proprietary prepara- tions) when analysed by the Ridyard2 method gave a result of 0.0477 per cent. for thiamine instead of a theoretical figure of 0.0572 per cent., a recovery of only 83.4 per cent. By analysing mixtures of master-mix and chalk to which the other ingredients of the pre-mix were added individually, it was found that a substantial loss of thiamine occurred in the presence of potassium bromate, as Table I shows. 0 SAC and the authors. Published on 01 January 1970. Downloaded by Northeastern University on 23/10/2014 06:21:44. View Article Online / Journal Homepage / Table of Contents for this issue

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Page 1: The determination of thiamine in flour fortification mixtures

312 Analyst, March, 1970, Vol. 95, p p . 312-315

The Determination of Thiamine in Flour Fortification Mixtures

BY H. V. HART AND K. H. WILLIS (Flour Milling and Baking Research Association, Old London Road, St. A lbans, Hertfordshire)

The reasons for the low recovery of thiamine from flour fortification mixtures containing ingredients used for the treatment of flour, in addition to iron, vitamins and chalk, are examined. The presence of potassium bromate and chalk is shown to be the cause of low recoveries, and methods for overcoming the interference of these substances with the analysis for thiamine content are described.

To comply with the compositional requirement of the Bread and Flour Regulations 1963,l it is necessary for the flour miller to add to white flour (with certain exceptions), chalk and master-mix, the latter consisting of a concentrated mixture of nutrients, viz., iron, thiamine and nicotinic acid with flour.

The additions of chalk and master-mix are usually made by separate feeders but, in the past, enquiries have been made as to whether these ingredients could be fed into the flour as a single admixture. The stability of such a mixture in respect of the thiamine content was examined by Ridyard2 and found to be satisfactory. Analysis of this mixture for thiamine content by the method usually adopted for the determination of this vitamin in fl0ur,3 however, was found to be unsatisfactory and modifications to the method, principally in the amounts of hydrochloric acid required for the dissolution of the sample, were made.

In addition to master-mix and chalk, small amounts of other materials may also be added to white flour, either for the purpose of improving its baking properties or for bleaching the slight yellow pigment. Treatment of flour with one pre-mix only, containing perhaps a mixture of ingredients such as master-mix, chalk, fungal amylase preparation , potassium bromate and benzoyl peroxide, would have some advantages and, in particular, would result in economies in feeding plant.

When such a mixture was analysed for the content of thiamine by the Ridyard2 method, however, the amounts of vitamin recovered were found to be low. As it was expected that the mixture would be reasonably stable in the dry state, the low recoveries might be caused by losses occurring during the analysis.

Investigation of the method of analysis revealed that the low recovery was attributable mostly to the presence of potassium bromate, which resulted in some destruction of thiamine during dissolution of the sample. A further slight loss of thiamine was also shown to be caused by the adsorption of thiamine on the insoluble matter remaining after native chalk is decomposed by hydrochloric acid.

Modifications to the method of analysis are suggested to overcome these two causes of the low recovery of thiamine.

EXPERIMENTAL A sample of a pre-mix containing 7.5 per cent. of master-mix, 74.5 per cent. of chalk,

5.3 per cent. of fungal amylase preparation, 0.54 per cent. of potassium bromate and 1-2 per cent. of benzoyl peroxide (the last two ingredients are incorporated in proprietary prepara- tions) when analysed by the Ridyard2 method gave a result of 0.0477 per cent. for thiamine instead of a theoretical figure of 0.0572 per cent., a recovery of only 83.4 per cent. By analysing mixtures of master-mix and chalk to which the other ingredients of the pre-mix were added individually, it was found that a substantial loss of thiamine occurred in the presence of potassium bromate, as Table I shows.

0 SAC and the authors.

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Page 2: The determination of thiamine in flour fortification mixtures

HART AND WILLIS 313 TABLE I

EFFECT OF PRE-MIX INGREDIENTS ON THE RECOVERY OF THIAMINE FROM MASTER-MIX Apparent thiamine content Recovery,

per cent. of master-mix, per cent. Master-mix and chalk only . . . . . . . . .. 0-745 97.0

Master-mix and chalk + potassium bromate . . . . 0.600 78.1

Master-mix + all ingredients (i.e., pre-mix) . . . . 0*641* 83.5 * Equivalent to 0.0478 per cent. of thiamine in pre-mix.

Master-mix and chalk + fungal amylase . . . . . . 0.752 97.9

Master-mix and chalk + benzoyl peroxide . . . . 0.752 97.9

Dissolution of the sample is accomplished in this method by gradually adding 5 g of the sample to 75 ml of concentrated hydrochloric acid (about 12 N), with constant shaking, followed by dilution to 500 ml with water. As potassium bromate and concentrated hydro- chloric acid react to liberate bromine and chlorine, it is likely that some decomposition of thiamine might occur a t this stage. Therefore, to counteract the effect of bromate at the high acidity, a reducing agent was added to the hydrochloric acid prior to adding the sample. Theoretical amounts of iron(I1) sulphate (0.30 g) and hydrazinium sulphate (0.035 g), sufficient just to react with the amount of bromate present, were dissolved in a few millilitres of water and added to the hydrochloric acid. Table I1 shows that the addition of a reducing agent to the hydrochloric acid results in a substantial improvement in the recovery of thiamine, and also shows the effect of adding increasing amounts of reducing agents.

TABLE I1 EFFECT OF INCREASING AMOUNTS OF REDUCING AGENTS ON THE RECOVERY

OF THIAMINE FROM A PRE-MIX

Reducing agent None . . . . . . Iron(I1) sulphate . . . .

Hydrazinium sulphate . .

Amount added, g

0.30 0.65 1.00 2.00 0.035 0-070 0.100

Thiamine, per cent. 0.0477 0.0559 0.0561 0.0556 0.0560 0-0552 0.0552 0-0549

Recovery, per cent. 83.4 97.7 98.0 97.2 97.9 96.5 96.5 95.9

Recovery of thiamine appeared to be better with iron(I1) sulphate than with hydra- zinium sulphate and it was therefore decided to use the former in future experiments.

Three further independent analyses of laboratory mixtures of all of the pre-mix in- gredients were carried out with 0-5 g of iron(I1) sulphate; the amounts of thiamine recovered were 97.3, 97-9 and 97.5 per cent., giving a mean of 97-6 per cent.

In our normal working method for determining thiamine in cereals we allow the extraction of the vitamin with dilute hydrochloric acid to continue overnight, followed by oxidation to thiochrome the next day. To determine whether allowing the extraction to stand overnight involved some loss of thiamine from the pre-mix solution, oxidation was carried out 1 hour after dissolving the sample and again after 24 hours. The mean values of duplicate deter- minations made on laboratory mixtures of all of the ingredients (98.1 and 97.9 per cent., respectively) were not significantly different.

The effect of iron(I1) sulphate and of an iron(II1) salt (which would be produced by reaction with bromate) on the recovery of thiamine from a mixture of chalk and master-mix was also determined. No difference in recovery of thiamine was detected in the presence or absence of these salts. ADSORPTION OF THIAMINE ON THE HYDROCHLORIC ACID INSOLUBLE MATTER FROM CHALK-

Although the use of iron(I1) sulphate improved the recovery of thiamine from pre-mix the amount recovered was still 2 or 3 per cent. below the theoretical amount. When analysing mixtures of master-mix and chalk, Ridyard2 had suggested the possibility of adsorption of thiamine on to the flocculent siliceous insoluble matter that remains after the chalk is dis- solved in hydrochloric acid, and as this might affect the present determination it was investigated further.

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314 [Analyst, Vol. 95 A mixture of 0.373 g of a master-mix and 3.725 g of chalk was dissolved in 75 ml of

concentrated hydrochloric acid and diluted to 500 ml with water. After mixing well to dis- perse the insoluble matter, two aliquots were removed: one of 30 ml (a), which was diluted to 200 ml for the determination of thiamine in the normal ~ a n n e r , ~ and the other 50 ml (b) . The latter was centrifuged and the supernatant liquid rejected. The residue was washed twice with 2 per cent. hydrochloric acid to remove adhering thiamine, and separated by centrifugation. The residue was then eluted with two 10-ml portions of 2 per cent. hydro- chloric acid saturated with potassium chloride, at room temperature, and the thiamine content of the combined eluates determined. Laboratory-mixed samples of pre-mix (as previously described) were also analysed in a similar manner, except that O G g of iron(I1) sulphate was added to the hydrochloric acid before dissolving the samples. Additional thiamine was recovered from the residues, as shown in Table 111. By eluting the residue (b)

HART AND WILLIS : DETERMINATION OF THIAMINE

TABLE I11 AMOUNTS OF THIAMINE RECOVERED FROM CHALK ADMIXTURES AND

CHALK-INSOLUBLE MATTER

Master-mix Pre-mix Thiamine content direct from (a), per cent. .. 0.743 0.0555 Additional thiamine from residue (b ) , per cent. .. 0.022 0.00 15 Total thiamine, per cent. .. . . .. .. 0.765 0.0570 Theoretical thiamine content, per cent. .. .. 0.768 0.0572 Recovery, per cent. .. . . .. .. . . 99.6 99.7

from pre-mix with a hot solution of 2 per cent. hydrochloric acid saturated with potassium chloride, the additional thiamine obtained was 0.0017 per cent., thus giving an over-all recovery of 100 per cent.

To avoid separating the insoluble matter, a high proportion of the adsorbed thiamine can be removed by adding potassium chloride to the hydrochloric acid solution before oxidation to thiochrome. With this solution containing 15 per cent. of potassium chloride, the laboratory sample of pre-mix gave a result of 0.0568 per cent. for thiamine, a recovery of 99.3 per cent.

It was considered, however, that a more practical method would be to add potassium chloride to the hydrochloric acid before dissolution of the sample, thus perhaps avoiding adsorption of the thiamine on to the insoluble matter at this stage. Amounts of potassium chloride varying from 11.4 g to nil were used in addition to iron(I1) sulphate for the analysis of a sample of laboratory-mixed pre-mix. The results in Table IV show that a 99-7 per cent. recovery of vitamin was achieved by this procedure over the range of 1 to 4 g of potassium chloride.

TABLE IV EFFECT OF USING VARYING AMOUNTS OF POTASSIUM CHLORIDE DURING THE

DISSOLUTION OF PRE-MIX IN HYDROCHLORIC ACID

Amount of potassium chloride, g Thiamine, per cent. Recovery, per cent. 11.4 0.0565 98.7

4 0.0568 99.7 2 0.0568 99.7 1 0.0568 99.7

Nil 0.0562 98.2

METHOD OF PREPARATION OF EXTRACT OF PRE-MIX FOR THIAMINE ANALYSIS-

The final preferred method of dissolution of a sample of pre-mix, which might contain master-mix, chalk, fungal amylase, potassium bromate and benzoyl peroxide, is as follows. REAGENTS-

Irort(I1) sulphate solution, 10 per certt.-Dissolve 10 g of FeS0,.7H,O in water containing 2 ml of 2 per cent. hydrochloric acid and dilute to 100 ml.

Potassium chloride. Hydrochloric acid, concentrated, sp.gr. 1 *18. All reagents should be of analytical-reagent grade.

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March, 19701 I N FLOUR FORTIFICATION MIXTURES 315

PROCEDURE- Add 3 g of potassium chloride to a mixture of 75 ml of hydrochloric acid and 5 ml of

iron(I1) sulphate solution contained in a 2-litre beaker. Add 2 drops of octan-2-01; this helps to reduce foaming. Transfer 5 g of sample gradually to the mixture, constantly agitating it. Wash the solution into a 500-ml graduated flask with the aid of a jet of water, completing the transfer by rubbing the sides of the beaker with a rubber-tipped glass rod. Make up to volume with water and mix thoroughly. Dilute 30 and 200 ml with water to give a suitable concentration of thiamine in 0.24 N hydrochloric acid for oxidation to thiochrome and complete the determination of thiamine by the method of R i d ~ a r d . ~

REFERENCES 1.

2. 3.

Statutory Instruments, 1963, No. 1435, The Bread and Flour Regulations 1963, H.M. Stationery Office, London, 1963.

Ridyard, H. N., Analyst, 1955, 80, 834. - , Ibid., 1949, 74, 18.

Received June 30tk, 1969 Accepted September 16th, 1969

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