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Determination of Methionine in Feed Concentrates
Determination of Methionine in Feed Concentrates P. SLUMP and K. D. BOS Institute CIVO-Analysis TNO, P.O. Box 360, Zeist, The Netherlands (Received for publication May 7, 1984)
INTRODUCTION
A very important class of products for the feed industry is the poultry feed concentrates and super concentrates. These are mixed with basic feed constituents, such as cereals and soybean meal, for the production of poultry feeds. Several concentrates have a high content of minerals and synthetic methionine. To check the accuracy of the manufacturing process, the constituents of these concentrates must be determined by suitable analytical procedures. The content of synthetic methionine can be determined by an automated amino acid analyzer or by high performance liquid chromatography (HPLC) after an extraction procedure (Matesic, 1982). Poultry producers, however, are usually interested in the value of total methionine, which is the sum of synthetic and proteinbound methionine. This value is obtained after oxidation with performic acid, a subsequent hydrolysis of the sample, and a determination of the methionine as methionine sulfone by an amino acid analyzer (Friedman and Noma, 1975). We have recently been involved with the analyses of several of the constituents in these concentrates and have encountered severe
1 Benzstrasse 28, 8039 Puchheim, Germany (BRD). 2 2905 Stender Way, Santa Clara, CA 95051.
problems with determination of methionine in concentrates containing sodium chloride at up to 8%. The methionine content for these concentrates, determined as methionine sulfone using a modification of the performic acid procedure of Moore (1963), was much lower than the value that was calculated on the basis of the amounts of components added by the feed manufacturer. A first indication of the interference of the methionine determination by sodium chloride was found by Slump (1969). Hydrolysis of a casein containing sodium chloride and determination of the methionine in such by an amino acid analyzer gave a higher methionine value than obtained via the oxidation procedure according to Moore (1963). In the present paper, the effect of sodium chloride on the methionine determination is demonstrated, and, additionally, an improved analytical procedure for the determination of methionine is described in which the interference by the sodium chloride is eliminated. MATERIALS AND METHODS
Experimental Procedure. Experiments have been carried out with a commercially available super concentrate, containing 3.6% sodium chloride. ANRC casein was used as a reference substance and was obtained from Humko (Sheffield, NJ). The methionine content of this casein is 2.78%. Methionine was determined as methionine sulfone, using a performic acid oxidation procedure that is a variant of the method published by Moore (1963).
705
Downloaded from http://ps.oxfordjournals.org/ at Swinburne University of Technology on May 17, 2015
ABSTRACT The sodium chloride present in several broiler and layer concentrates is found to interfere with the determination of methionine after oxidation with performic acid. In the analytical procedure used by many laboratories, the performic acid is prepared from 88% formic acid and hydrogen peroxide. This results in low values for the methionine concentration if the feed also contains sodium chloride. It is shown that the chloride ions, rather than the sodium ions, are responsible for the variance in the results of the conventional analytical procedure. Addition of water to the performic acid reagent in a 3:1 ratio eliminates the interference by chloride ions. Unfortunately, the improved analytical procedure for the analysis of methionine cannot be used for the determination of cystine. (Key words: methionine, feed concentrates) 1985 Poultry Science 64:705-707
706
SLUMP AND BOS
RESULTS AND DISCUSSION
Effect of Increasing Amounts of Sodium Chloride on the Determination of Methionine. The results obtained with the performic acid procedure for the determination of methionine in samples of casein and of synthetic methionine containing increasing amounts of sodium chloride are shown in Figure 1. To obtain comparable results, the amount of casein was chosen so that it contained 3.0 mg of methionine, and the experiment with synthetic methionine was carried out using a quantity of 3.0 mg of the amino acid. In Figure 1, the amount of sodium chloride is expressed as milligrams of sodium chloride per 3.0 mg of methionine. For the casein sample, a lower value for the methionine content of up to 12% was found upon the addition of sodium chloride. The methionine value for the study with synthetic methionine was reduced further. With a ratio of 20 mg of sodium chloride to 3.0 mg of methio-
o 4
o DL METHIONINE 4 CASEIN
METHIONINE FOUND (% OF TOTAL CONTENT)
t AMOUNT OF NaCI (mg NaCI/3mg of methionine)
FIG. 1. Effect of sodium chloride on the determination of methionine in synthetic methionine and in casein.
nine, the value was 20% lower. In order to trace the factors influencing the methionine determination, sodium sulphate was added to a feed concentrate containing no chloride, and the methionine was analyzed according to the usual procedure. No reduction of the methionine value was observed. It may be concluded that the chloride ions, rather than the sodium ions, affect the methionine determination. Elimination of the Interference by Chloride. To eliminate interference by the sodium chloride on the methionine determination, several modifications of the performic acid procedure were tried. One of these — the addition of water to the oxidizing agent — resulted in a great improvement of the methionine determination (Figure 2). A complication
100 METHIONINE FOUND (% OF TOTAL
90
/r
80" 70
o—o A
DL
METHIONINE")
A LAYER CONCENTRATE")
60
t ML OF WATER ADDED
FIG. 2. Effect of addition of water on the methionine value found in samples containing sodium chloride. *In this experiment 3.0 mg of DL-methionine and 10 mg of sodium chloride were used. """This layer concentrate contains 3.6% of sodium chloride.
Downloaded from http://ps.oxfordjournals.org/ at Swinburne University of Technology on May 17, 2015
Standard Procedure. The performic acid is prepared by addition of 2 ml of 30% hydrogen peroxide to 18 ml of formic acid and the mixture is kept at 30 C for 1 hr. A feed sample (200 to 700 mg, containing about 3 mg of methionine) is stirred with 20 ml of the cooled performic acid reagent for 15 min and the mixture is kept at 0 C for 16 hr. The excess performic acid is destroyed by the addition of 3 ml of hydrobromic acid (containing 47% HBr) and the mixture is evaporated until dry. Hydrolysis of the residue is performed by refluxing with 120 ml of hydrochloric acid for 22 hr. After evaporation of the hydrochloric acid, the methionine sulfone is determined by ion-exchange chromatography using norleucine as an internal standard. Modified Procedure. A cooled mixture (0 C) of 20 ml of the performic acid reagent and 50 ml of water is added to a feed sample (200 to 700 mg, containing about 3 mg of methionine). The mixture is stirred for 15 min and then kept at 0 C for 16 hr. The excess performic acid is destroyed by the addition of hydrobromic acid (see Standard Procedure). Apparatus. The amino acid concentration of the hydrolysates were measured by the ionexchange column chromatographic method using a Biotronik 1 LC 6001 E. The calculations were performed by a Spectra Physics 2 4100 data system.
METHIONINE IN FEED
REFERENCES Friedman, M., and A. T. Noma, 1975. Ch. 27 in Protein
Nutritional Quality of Foods and Feeds. Part 1. M. Friedman, ed. Marcel Dekker, New York, NY. Mason, V. C , S. Bech-Andersen, and M. Rudemo, 1980. Hydrolysate preparation for amino acid determinations in feed constituents. 8. Studies of oxidation conditions for streamlined procedures. Z. Tierphysiol. Tierernahr. Futtermittelkd. 43: 146-164. Matesic, D., 1982. High pressure liquid chromatographic determination of supplemental methionine in poultry premix. J. Assoc. Offic. Anal. Chem. 6 5 : 6 2 - 6 5 . Moore, S., 1963. On the determination of cystine as cysteic acid. J. Biol. Chem. 238:235-237. Slump, P., 1969. Page 40 in Characterization of the nutritive value of food proteins by amino acid composition and the effect of heat and alkali treatment on the availability of amino acids. Thesis, Free Univ. Amsterdam, The Netherlands.
Downloaded from http://ps.oxfordjournals.org/ at Swinburne University of Technology on May 17, 2015
of the changed reaction conditions of the performic acid procedure is that the values found for cystine are too low. This is in accordance with the results reported by Mason et al. (1980). The reason for these reduced values is not yet known. Preliminary experiments have shown that application of our improved method for the analysis of methionine in other basic feed constituents such as meat meal tankage, which also contains chloride ions, results in higher methionine values than found earlier using the traditional method of Moore (1963).
707
INTRODUCTION
A very important class of products for the feed industry is the poultry feed concentrates and super concentrates. These are mixed with basic feed constituents, such as cereals and soybean meal, for the production of poultry feeds. Several concentrates have a high content of minerals and synthetic methionine. To check the accuracy of the manufacturing process, the constituents of these concentrates must be determined by suitable analytical procedures. The content of synthetic methionine can be determined by an automated amino acid analyzer or by high performance liquid chromatography (HPLC) after an extraction procedure (Matesic, 1982). Poultry producers, however, are usually interested in the value of total methionine, which is the sum of synthetic and proteinbound methionine. This value is obtained after oxidation with performic acid, a subsequent hydrolysis of the sample, and a determination of the methionine as methionine sulfone by an amino acid analyzer (Friedman and Noma, 1975). We have recently been involved with the analyses of several of the constituents in these concentrates and have encountered severe
1 Benzstrasse 28, 8039 Puchheim, Germany (BRD). 2 2905 Stender Way, Santa Clara, CA 95051.
problems with determination of methionine in concentrates containing sodium chloride at up to 8%. The methionine content for these concentrates, determined as methionine sulfone using a modification of the performic acid procedure of Moore (1963), was much lower than the value that was calculated on the basis of the amounts of components added by the feed manufacturer. A first indication of the interference of the methionine determination by sodium chloride was found by Slump (1969). Hydrolysis of a casein containing sodium chloride and determination of the methionine in such by an amino acid analyzer gave a higher methionine value than obtained via the oxidation procedure according to Moore (1963). In the present paper, the effect of sodium chloride on the methionine determination is demonstrated, and, additionally, an improved analytical procedure for the determination of methionine is described in which the interference by the sodium chloride is eliminated. MATERIALS AND METHODS
Experimental Procedure. Experiments have been carried out with a commercially available super concentrate, containing 3.6% sodium chloride. ANRC casein was used as a reference substance and was obtained from Humko (Sheffield, NJ). The methionine content of this casein is 2.78%. Methionine was determined as methionine sulfone, using a performic acid oxidation procedure that is a variant of the method published by Moore (1963).
705
Downloaded from http://ps.oxfordjournals.org/ at Swinburne University of Technology on May 17, 2015
ABSTRACT The sodium chloride present in several broiler and layer concentrates is found to interfere with the determination of methionine after oxidation with performic acid. In the analytical procedure used by many laboratories, the performic acid is prepared from 88% formic acid and hydrogen peroxide. This results in low values for the methionine concentration if the feed also contains sodium chloride. It is shown that the chloride ions, rather than the sodium ions, are responsible for the variance in the results of the conventional analytical procedure. Addition of water to the performic acid reagent in a 3:1 ratio eliminates the interference by chloride ions. Unfortunately, the improved analytical procedure for the analysis of methionine cannot be used for the determination of cystine. (Key words: methionine, feed concentrates) 1985 Poultry Science 64:705-707
706
SLUMP AND BOS
RESULTS AND DISCUSSION
Effect of Increasing Amounts of Sodium Chloride on the Determination of Methionine. The results obtained with the performic acid procedure for the determination of methionine in samples of casein and of synthetic methionine containing increasing amounts of sodium chloride are shown in Figure 1. To obtain comparable results, the amount of casein was chosen so that it contained 3.0 mg of methionine, and the experiment with synthetic methionine was carried out using a quantity of 3.0 mg of the amino acid. In Figure 1, the amount of sodium chloride is expressed as milligrams of sodium chloride per 3.0 mg of methionine. For the casein sample, a lower value for the methionine content of up to 12% was found upon the addition of sodium chloride. The methionine value for the study with synthetic methionine was reduced further. With a ratio of 20 mg of sodium chloride to 3.0 mg of methio-
o 4
o DL METHIONINE 4 CASEIN
METHIONINE FOUND (% OF TOTAL CONTENT)
t AMOUNT OF NaCI (mg NaCI/3mg of methionine)
FIG. 1. Effect of sodium chloride on the determination of methionine in synthetic methionine and in casein.
nine, the value was 20% lower. In order to trace the factors influencing the methionine determination, sodium sulphate was added to a feed concentrate containing no chloride, and the methionine was analyzed according to the usual procedure. No reduction of the methionine value was observed. It may be concluded that the chloride ions, rather than the sodium ions, affect the methionine determination. Elimination of the Interference by Chloride. To eliminate interference by the sodium chloride on the methionine determination, several modifications of the performic acid procedure were tried. One of these — the addition of water to the oxidizing agent — resulted in a great improvement of the methionine determination (Figure 2). A complication
100 METHIONINE FOUND (% OF TOTAL
90
/r
80" 70
o—o A
DL
METHIONINE")
A LAYER CONCENTRATE")
60
t ML OF WATER ADDED
FIG. 2. Effect of addition of water on the methionine value found in samples containing sodium chloride. *In this experiment 3.0 mg of DL-methionine and 10 mg of sodium chloride were used. """This layer concentrate contains 3.6% of sodium chloride.
Downloaded from http://ps.oxfordjournals.org/ at Swinburne University of Technology on May 17, 2015
Standard Procedure. The performic acid is prepared by addition of 2 ml of 30% hydrogen peroxide to 18 ml of formic acid and the mixture is kept at 30 C for 1 hr. A feed sample (200 to 700 mg, containing about 3 mg of methionine) is stirred with 20 ml of the cooled performic acid reagent for 15 min and the mixture is kept at 0 C for 16 hr. The excess performic acid is destroyed by the addition of 3 ml of hydrobromic acid (containing 47% HBr) and the mixture is evaporated until dry. Hydrolysis of the residue is performed by refluxing with 120 ml of hydrochloric acid for 22 hr. After evaporation of the hydrochloric acid, the methionine sulfone is determined by ion-exchange chromatography using norleucine as an internal standard. Modified Procedure. A cooled mixture (0 C) of 20 ml of the performic acid reagent and 50 ml of water is added to a feed sample (200 to 700 mg, containing about 3 mg of methionine). The mixture is stirred for 15 min and then kept at 0 C for 16 hr. The excess performic acid is destroyed by the addition of hydrobromic acid (see Standard Procedure). Apparatus. The amino acid concentration of the hydrolysates were measured by the ionexchange column chromatographic method using a Biotronik 1 LC 6001 E. The calculations were performed by a Spectra Physics 2 4100 data system.
METHIONINE IN FEED
REFERENCES Friedman, M., and A. T. Noma, 1975. Ch. 27 in Protein
Nutritional Quality of Foods and Feeds. Part 1. M. Friedman, ed. Marcel Dekker, New York, NY. Mason, V. C , S. Bech-Andersen, and M. Rudemo, 1980. Hydrolysate preparation for amino acid determinations in feed constituents. 8. Studies of oxidation conditions for streamlined procedures. Z. Tierphysiol. Tierernahr. Futtermittelkd. 43: 146-164. Matesic, D., 1982. High pressure liquid chromatographic determination of supplemental methionine in poultry premix. J. Assoc. Offic. Anal. Chem. 6 5 : 6 2 - 6 5 . Moore, S., 1963. On the determination of cystine as cysteic acid. J. Biol. Chem. 238:235-237. Slump, P., 1969. Page 40 in Characterization of the nutritive value of food proteins by amino acid composition and the effect of heat and alkali treatment on the availability of amino acids. Thesis, Free Univ. Amsterdam, The Netherlands.
Downloaded from http://ps.oxfordjournals.org/ at Swinburne University of Technology on May 17, 2015
of the changed reaction conditions of the performic acid procedure is that the values found for cystine are too low. This is in accordance with the results reported by Mason et al. (1980). The reason for these reduced values is not yet known. Preliminary experiments have shown that application of our improved method for the analysis of methionine in other basic feed constituents such as meat meal tankage, which also contains chloride ions, results in higher methionine values than found earlier using the traditional method of Moore (1963).
707