GB2120787A - Method of estimating feed additives - Google Patents

Abstract

There is described a method for estimating the quantity of an ionophore in a feedstuff, which comprises mixing the feedstuff with an alcoholic solvent, adding an acid having a pKa value of less than 4 and a colour-producing aryl aldehyde, both in solid form, to the resulting-solution phase, at a temperature sufficient to generate a colour reaction, and comparing the colour thus produced with a standard.

Description

SPECIFICATION Method of estimating feed additives This invention relates to a method of estimating additives in an animal feedstuff and to a test kit for use in such method.

It is common practice to incorporate feed additives in animal feedstuffs which contribute to the nutrient qualities of the feed or which fulfil the function of promoting growth. One such feed additive is monensin a product widely employed in both cattle and poultry feeds and disclosed in US Patent 3,501,568.

Other additives of a similar nature, referred to generically as ionophores, include, for instance, narasin and salinomycin.

We have discovered a convenient way in which the content of such feed additives can be estimated in the process of manufacture or under practical conditions of use.

The invention provides a method for estimating the quantity of an ionophore in a feedstuff, which comprises mixing the feedstuff with an alcoholic solvent, adding an acid having a pKa value of less than 4 and a colour-producing aryl aldehyde, both in solid form, to the resulting solution, at a temperature sufficient to generate a colour reaction, and comparing the colour thus produced with a standard.

The use of solid test ingredients, and in particular acid in solid form, in the test method has been found to give practical improvements. Examples of colour-producing aryl aldehydes include vanillin, ethyl vanillin, 2,4-dimethoxybenzaldehyde, 3,4-dimethoxybenzaldehyde, 2,4,5-trimethoxybenzaldehyde, 4-dimethylaminobenzaldehyde, cinnamaldehyde, and other aryl aldehydes with electron-donating substituents, the most preferred aldehyde being vanillin. The aldehyde need not be a solid, as such, provided that it is employed in solid form as for example when absorbed on a solid carrier.

Use of liquid ingredients in test for estimating ionophore, such as for example solutions of vanillin and of liquid acid required to catalyse the reaction of vanillin with ionophore, results in considerable inconvenience when a quick estimate needs to be made under practical conditions and has been found to give unreliable results. Surprisingly, the method of the present invention, as well as being more convenient, gives greater reliability. The method of the invention can also be employed to confirm the absence of ionophore in a feedstuff when for example it is intended to use the feed for animals such as a horse which react adversely to ionophores.

The method of the invention is applicable to the estimation of ionophores such as for example narasin and salinomycin and especially monensin. Solid acids that may be employed include for example 5-sulphosalicylic acid, sulphamic acid, hydroxylamine 0-sulphonic acid and p-toluene-sulphonic acid, and the preferred material is 5-sulphosalicylic acid.

The preferred solvent for mixing with the feedstuff is methanol but other alcoholic solvents such as for example ethanol can be used either alone or as an aqueous mixture comprising sufficient of the alcohol to extract the ionophore component efficiently.

The colour produced by the interaction of ionophore in the feedstuff with aryl aldehyde and acid is estimated by comparison with a standard which can be for example a colour solution prepared by mixing a known quantity of ionophore with aldehyde and acid, or by use of a calibrated colour chart. In order to produce a colour it is necessary to ensure that the reaction takes place at a suitable temperature which is preferably in the range from 50C to 70C, especially 60"C, applied for example by dipping a container holding the solution in a heated waterbath.

In practice it is often best to decant the solution formed from mixing the feedstuff with alcoholic solvent and then to mix the supernatant with and aldehyde and acid on a supporting medium such as for example a disc of filter material or, more preferably with tablets or aryl aldehyde and acid. Since the acid component and the aryl aldehyde may be difficult to formulate together in stable form it is preferred to employ two separate tablets each comprising one of the components. Both solid acid and aryl aldehyde can be readily prepared in tablet form. In case of liquid aldehydes the material may be absorbed on suitable carriers in the tablet formulation. Accordingly a preferred method of the invention is one which comprises the steps of (a) mixing a feedstuff containing a vanillinpositive ionophore with alcoholic solvent, (b) decanting a portion of the supernatant, (c) adding one or more tablets comprising a solid acid having a pKa of less than 4 and one or more tablets comprising vanillin to said portion of supernatant, at a temperature sufficient to generate a colour reaction, and (d) comparing the colour thus formed with a standard chosen according to the specific quantities and volumes of test materials employed.

It will be understood that the quantity of feedstuff, volume of solvent and other quantitites employed have to be carefully chosen so as to match the requirements of the standard.

For practical requirements the quatity of feedstuff will preferably be set at a value of 3, 4 or 5 gm which may be measured by, for example, filling a container such as a bottle or tube up to a specified mark. Solvent of specified volume for example 8 ml to 12 ml may then be added and the mixture heated for a period of one to two minutes. A specified portion of the supernatant, for example 1 .5 ml to 2.5 ml, is then decanted into a second e container to which are added the tablets of acid and aryl aldehyde, generally one each of these two components. The tablets contain an appropriate quantity of the acid catalyst or vanillin so as to produce a quantifiable colour reaction when matched with the standard colour solution or calibrated colour chart. Preferably an excess of acid and vanillin is employed and for example the quantity per tablet of acid can be 40 to 60 mg and of vanillin 40 mg and 60 mg. The standard colour solution may be prepared by mixing a tablet comprising for example a specified quantity of ionophore 100, 200 or 300 mcg with an appropriate quantity of solvent.

As stated above the test must be carried out at a temperatul-e which is sufficient to generate a colour reaction between the components. We have found that the optimum temperature is 60"C and that it is most convenient to apply heat after the aryl aldehyde and acid have been added to the alcoholic solution. Alternatively however the solvent can first be warmed to 60 C before mixing with the feedstuff or subsequently warmed immediately before addition of acid and aldehyde.

The tablets employed can be made by conventional means in which the solid acid or aldehyde is mixed with an appropriate quantity of filler and surfactant. Typical formulations for such tablets are as follows: Tablet 1 5-sulphosalicylic acid 50 mg magnesium stearate 0.25 mg Aerosil 0.25 mg Tablet2 vanillin 50 mg magnesium stearate 0.25 mg Aerosil 0.25 mg The invention also includes a test kit adapted for use in the method of the invention, comprising an extraction vessel preferably graduated according to the quantity of feedstuff to be extracted, the volume of solvent to be added and the volume of supernatant to be decanted, a test vessel for observation of the colour of the supernatant, a vessel containing solvent, tablets of standard quantities of solid acid and colour-producing aryl aldehyde, and a colour chart or one or more tablets of standard quantity of ionophore.

The invention is further illustrated by the following Example.

EXAMPLE A standard colour solution was first prepared by mixing a tablet containing 100 mcg monensin with 2 ml of methanol in a test tube, adding 50 mg of 5-sulphosalicyclic acid and 50 mg vanillin in separate tablet form, and maintaining the mixture at 60C by immersion in a water bath. When the components dissolve a red colouration was produced.

For comparison with this standard solution, 5 gm of cattle feedstuff containing a known quantity of 50 parts per million of monensin was placed in an extraction bottle, 10 ml of methanol added to it, the mixture shaken and the supernatant tested for its colour reaction.

Some (2 ml) of the supernatant was poured into a test tube and 50 mg of 5-sulphosalicylic acid and 50 mg vanillin added in separate tablet form. The test tube was immersed in a water bath at 60'C and held at this temperature until both tablets had dissolved.

A red colouration developed and when compared with the standard colour solution it was noted that the colour was less intense. From this two conclusions could be drawn (a) that ionophore was present in the feedstuff and (b) that the ionophore was present to the extent of less than 100 parts per million.

Claims (7)

1. A method for estimating the quantity of an ionophore in a feedstuff, which comprises mixing the feedstuff with an alcoholic solvent, adding an acid having a pKa value of less than 4 and a colour-producing aryl aldehyde, both in solid form, to the resulting solution, at a temperature sufficient to generate a colour reaction, and comparing the colour thus produced with a standard.

2. A method according to claim 1 in which the colour-producing aryl aldehyde is vanillin.

3. A method according to either of claims 1 and 2 in which the acid is 5-sulphosalicylic acid.

4. A method according to claim 1 which comprises the steps of (a) mixing a feedstuff containing a vanillinpositive ionophore with an alcoholic solvent, (b) decanting a portion of the supernatant, (c) adding one or more tables comprising a solid acid having a pKa of less than 4 and one or more tablets comprising vanillin to said portion of supernatant, at a temperature sufficient to generate a colour reaction, and (d) comparing the colour thus formed with a standard related to the specific quantities and volumes of test materials employed.

5. A method according to any of the preceding claims for estimating the quantity of monensin in a feedstuff.

6. A test kit adapted for use in a method according to any of the preceding claims, which comprises an extraction vessel, a test vessel for observation of the colour of the supernatant, a vessel containing solvent, tablets of standard quantities of solid acid and colour-producing aryl aldehyde, and a colour chart or one or more tablets of standard quantity of ionophore.

7. A test kit according to claim 6 in which the extraction vessel is graduated according to the quantity of feedstuff to be extracted, the volume of solvent to be added and the vol ume of supernatant to be decanted.