Supplemented or free amino acids and taurine

Virtually all compound feeds are now supplemented with amino acids and determination of this additive is an important quality assurance tool in the feed industry. Fahnenstich and Tanner (1973) developed a method for extracting free amino acids from a few grams of finely ground feed using diluted cold 0.1 M hydrochloric acid. Mason et al. (1980a) described similar extraction conditions but with the addition of thiodiglyeol as stabilizer for methionine and protein precipitation in the extract prior to chromatography. Virtually identical extraction conditions were incorporated in the official EU method for the determination of supplemented amino acids as a result of the Commission Directive (1998). In the EU collaborative trial, where this extraction method was tested on a premix, excellent repeatability within laboratory CVr and reproducibility between laboratories CVR are achieved (Table 2.2). Protein hydrolysis does not occur with diluted hydrochloric acid. It is not possible, however, to distinguish supplemented from natural, non-protein-bound amino acids out of the feed raw materials. All that needs to be done prior to performing cation exchange chromatography is to adjust the pH to 2.20 and no special cleaning of the extracts is necessary according to our experi ence. Extractions of various compound feeds with the addition of norleucine as internal standard gave a very good intralaboratory repeatability CVr of 1-1.5% in our laboratory (Fontaine, 2002). Foulon et al. (1990), who compared extraction of methionine and lysine with water, 85% ethanol and buffer solution with the acid extraction described above, achieved the highest recovery with 0.1 M hydrochloric acid. Saurina and Hemandez-Cassou (1993) described a flow-injection spee-trophotometrie determination specialized for supplemented lysine in commercial feeds. Bech-Andersen (1997) developed special alkaline/acid extraction conditions for mineral premixes with high contents of heavy metals in order to obtain extracts without heavy metal ions as the latter cause considerable interference in the cation exchanger. Fontaine and Eudaimon (2000) gave a detailed description of an acid extraction method with subsequent determination in an amino acid analyser, specially designed for assays of commercial amino acid products and concentrated premixes, which they tested in an international collaborative trial with 17 laboratories. They achieved a between-laboratory reproducibility CVR of only 1.5-2.6% and a recovery of the amino acids in the accurately produced premixes of 97.5-102.8%. This method has been adopted as the Official First Action by AO AC International.

The effect of amino acids in the diet on free amino acid concentrations in blood plasma, muscles or liver, etc., has also been the subject of many animal experiments. These matrices require deproteinization by precipitation (addition of acids or solvents) or by means of physical techniques (ultrafiltration etc.). Walker and Mills (1995) wrote in a review article that in human clinical diagnostic work precipitation is performed predominantly with 4-5% final concentration of 5-sulphosalieylie acid, as also described by De Jonge and Breuer (1994) for porcine plasma and Hagen et al. (1993) for bovine plasma. Sedgwick et al. (1991) compared protein precipitation by means of acetone or acetonitrile with that performed using perchloric acid or trichloroacetic acid in ovine plasma and bovine serum albumin solutions. Acid precipitation achieved good amino acid recoveries

Table 2.2. Precision of amino acid analysis. Published results of collaborative trials on the analysis of total amino acid contents in mixed feed and ingredients and of supplemented amino acids in premixes and pure amino acid trade products.

Liâmes and Fontaine (1994) Commission Directive (1998) Bütikofer et al. (1992)

Whey protein

Broiler finisher Poultry meal Fish meal Mixed pig feed Broiler compound concentrate Feed

CVr

CVR

cvr

CVR

cvr

CVR

cvr

CVR

cvr

CVR

cvr

CVR

cvr

CVR

Methionine (total)

1.1

7.6

2.1

12.0

1.9

9.7

3.4

7.0

3.1

10.9

3.1

13.4

5.9

16.0

Cystine (total)

3.1

11.3

4.6

17.7

4.0

19.0

3.3

9.9

2.8

8.8

5.5

44.8

13.1

77.2

Lysine (total)

3.5

9.0

3.1

9.9

2.8

7.9

2.8

3.2

2.1

5.4

2.3

5.5

5.3

13.2

Threonine (total)

2.7

8.2

3.2

9.1

3.6

10.7

1.9

4.1

2.1

5.3

1.5

5.2

3.1

8.4

Arginine (total)

2.3

8.6

3.3

9.7

2.3

7.2

3.0

8.8

4.2

7.8

Isoleucine (total)

3.2

6.8

2.7

11.7

2.1

10.3

4.2

9.4

3.1

18.7

Leucine (total)

2.7

6.3

2.5

7.6

1.9

6.8

2.5

6.4

2.3

7.8

Valine (total)

3.8

12.7

3.2

12.8

2.3

11.2

2.9

10.2

6.1

16.4

Phenylalanine (total)

4.4

14.6

3.5

9.2

1.6

7.7

4.2

11.5

4.7

12.3

Commission

Commission Directive (2000)

Dir. (1998)

Fontaine and Eudaimon (2000)

Feed concentrate

for pigs

Soybean meal

Premixture

No. 3 Premix

No. 5 Premix

Biolys

TM

DL-Methionine

CVr

CVR

CVr

CVR

CVr

CVR

CVr

CVR

CVr

CVR

CVr

CVR

CVr

CVR

Tryptophan (total)

1.9

2.2

1.3

4.1

Methionine (suppl.)

2.4

6.9

1.2

1.8

0.5

2.6

0.9

1.5

Lysine (suppl.)

2.1

6.7

1.3

2.5

0.7

1.9

0.8

2.3

Threonine (suppl.)

2.2

4.3

0.7

1.9

0.8

1.9

CVr, coefficient of variation (%) for within laboratory standard deviation Sr, repeatability CVr; CVR, coefficient of variation (%) for between laboratory standard deviation SR, reproducibility CVR.

CVr, coefficient of variation (%) for within laboratory standard deviation Sr, repeatability CVr; CVR, coefficient of variation (%) for between laboratory standard deviation SR, reproducibility CVR.

over a wide concentration range of the dissolved protein (4-16%). The solvents, however, included free amino acids in the precipitate so that the recovery fell to only 50% with increasing protein content. Aristoy and Toldra (1991) on the other hand obtained equally satisfactory results in the determination of free amino acids in fresh pork muscle and dry-cured ham either by protein precipitation with perchloric acid, trichloroacetic acid, picric acid and acetonitrile or by ultrafiltration with a 10 kDa exclusion limit, whereas sulphosalieylie acid or ultrafiltration with 1 kDa gave a poorer recovery. Peter et al. (1999) also used trichloroacetic acid for protein precipitation in cell extracts of algae and Bugueno et al. (1999) used acetonitrile for deproteinization of raw salmon muscle. Antoine et al. (1999) used 75% methanol in the analysis of free amino acids in fish samples. Urine sample preparation on the other hand was done by Hara et al. (1999) with 30% sulphosalieylie acid. Van de Merbel et al. (1995) described online amino acid monitoring in fermentation processes employing ultrafiltration for protein separation and ion exchange chromatography. There are thus many suitable variants for the removal of unwanted protein and the recovery of supplemented amino acids should always be tested in preliminary experiments.

Taurine, a metabolic derivative of cysteine, is essential for cats but it also plays a role in infant nutrition. It occurs in the non-protein-bound form and can, in principle, be analysed in the same way as free amino acids. Balsehukat and Kunesch (1989) described its determination in fish meal, pet food and animal raw materials following extraction with 0.1 M hydrochloric acid in an amino acid analyser. Nicolas et al. (1990) measured taurine concentrations in infant formulas and human milk following ultrafiltration also by ion-exchange chromatography. McCarthy et al. (2001) tested a taurine assay in pet food after acid hydrolysis (6 M HC1, 110°C, 16 h) and precolumn derivatization with dansyl chloride in a collaborative trial. In six samples of wet and dry cat and dog food interlaboratory reproducibility CVRs of 6.1-13.3% were achieved, and the procedure was adopted as the official method by AO AC International.

Porter et al. (1988), Amiss et al. (1990), Sakai and Nagasawa (1992) and Messing and Sturmann (1993) described the determination of taurine in feline and other blood and plasma by HPLC separation following precolumn derivatization with different reagents.

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