Total Amino Acid parameters
Back to AusScan Online products and prices
Parameters:
- Alanine
- Arginine
- Aspartic acid
- Cysteine
- Glutamic acid
- Glycine
- Histidine
- Isoleucine
- Leucine
- Lysine
- Methionine
- Phenyalanine
- Proline
- Serine
- Threonine
- Tryptophan
- Tyrosine
- Valine
Wet chemistry reference methods:
Reference methods used are as outlined in Premium Grains For Livestock Program. Component 1: Co-ordination. An overview of outcomes from PGLP 1 & 2. Final report. September 2008. Prepared by John Black.
1. For all amino acids except methionine, cystine and tryptophan the sample is hydrolysed in 6M HCl for 24 hours in screw cap tubes under an atmosphere of nitrogen (Rayner, 1985). After hydrolysis, the solution is diluted, filtered and the HCl removed under reduced pressure. The residue is taken up in a sodium citrate buffer solution. Quantitation is carried out on a cation exchange column with post column ninhydrin derivatisation and detection at 546nm (and 436nm for proline).
2. For methionine and cystine the samples are pre-oxidised with performic acid at 0°C for 16 hours by 6M HCl hydrolysis for 24 hours, and then following the procedure after hydrolysis as above.
3. For tryptophan, which is destroyed during acid hydrolysis, hydrolysis is carried out with barium hydroxide. After hydrolysis the solution's pH is adjusted and allowed to clarify (and filtered) and tryptophan is quantitated on a reverse phase column with detection at 280nm. An internal standard is used to compensate for any losses during hydrolysis. The method is based on Landry and Delhaye (1992).
The above methods were based on the method recommended by Petterson et al. (Australian Journal of Agricultural Research, 50: 775-789) which is described below:
Amino acid analysis of feeds can be undertaken by a variety of methods. The first step in the analysis involves hydrolysing the protein with 6M HCl for 24 hours to free the amino acids. This can be achieved by either; reflux hydrolysis (AOAC, 994.12); evacuation of oxygen under reduced pressure in sealed glass tubes (Rowan et al., 1992); or under an atmosphere of nitrogen in screw cap tubes (Rayner, 1985; Gehrke et al., 1985). After hydrolysis the solution is diluted, filtered and the HCl removed under reduced pressure or by neutralisation with sodium hydroxide (Llames and Fontaine, 1994). All amino acids except methionine, cystine and tryptophan, which are partially destroyed in 6M HCl, can be hydrolysed with the above procedure. However during analysis at 110°C for 24 hours threonine and serine are partially degraded and valine and isoleucine are incompletely hydrolysed from the protein (Blackburn, 1978). For this reason recovery factors are used to account for the degradation or incomplete hydrolysis of these amino acids. These are determined on different matrices by carrying out hydrolysis for different times and either extrapolating results back to zero time (threonine and serine) or to 72 hours for valine and isoleucine (Rayner, 1985; Davies et al., 1992).
Since amino acids have low absorptivity in the UV/V range, the analysis involves pre-column or post-column derivatisation to form compounds which have high absorption in the UV or are fluorescent and therefore can be determined by HPLC. Alternatively GLC analysis can be employed, although none of the amino acids are volatile enough for direct determination, and therefore it is necessary to produce derivatives e.g. by esterification with acidified alcohols followed by N-acylation using any of a number of acid anhydrides (Baxter, 1996). As a result of the large range of pre-column derivatisation procedures for HPLC, GLC procedures for amino acids have lost some popularity, although its major strength is its high resolution.
The most common pre-column derivatisation procedures for HPLC include Picotag (Hagan et al., 1989), AQC Tag (Liu et al., 1995) OPA and FMOC (Lai et al., 1991). Pre-column procedures, unlike post-column derivatisation, have the advantage that they are conducted on common HPLC equipment using gradient systems with C18 columns, Ninhydrin (AOAC, 1995) and OPA (Ashworth, 1987) are commonly used for post-column derivatisation using cation exchange columns with a post column reaction system consisting of additional pumps, oven and reaction coil. This system has higher equipment costs, long residence time between column and detector because of the reaction coil and therefore poorer resolution resulting in broader peaks. The major advantage of post column derivatisation is that it is not matrix-sensitive and therefore it is a more robust procedure. Although these systems are still in extensive use in Australia they are slowly being phased out in favour of the pre-column approach.
The most common procedur for methionine and cystine plus cysteine is pre-oxidation with performic acid at 0°C for 16 hours followed by 6M HCl hydrolysis (AOAC, 994.12). Methionine is converted to methionine sulphone and cystine and cysteine are converted to cysteic acid. These oxidised acids are then determined by HPLC using either the pre- or post-column derivatisation procedures. As well as measuring methionine and cystine this procedure can determine all amino acids except tryptophan and tyrosine (AOAC, 994.12).
For all amino acids except tryptophan we recommen the method of Llames and Fontaine (1994).
Since tryptophan is destroyed during acid hydrolysis alkaline conditions using lithium, sodium or barium hydroxide has been successfully used to carry out the hydrolysis. Landry and Delhaye (1992) described a reliable method based on barium hydroxide hydrolysis using 5-methyl tryptophan as an internal standard. After hydrolysis free tryptophan was analysed using a reverse phase column and fluorescence. The AOAC method 988.15 (1995) describes a procedure using 4.2M NaOH for hydrolysis in evacuated micro Kjeldahl flasks. After pH adjustment and clarification, tryptophan is separated by ion exhange chromatography with measurement by post column derivatisation with ninhydrin or by reverse phase HPLC with UV detection at 280nm. Both methods are recommended.
As well as running secondary reference materials, a good quality control procedure for amino acid analysis is to determine the amino acid nitrogen as a percentage of the total nitrogen (Kjeldahl or Dumas method), for all samples analysed. For most samples the recovery of amino acid nitrogen should be 85-105%. Samples with high levels of non-protein nitrogen e.g. ileal digesta will have lower recoveries.
Amino acids can be expressed as g/100g sample, mg/g N or g/16g N, either on an as received or dry matter basis. To compare different feed ingredients with a range of protein contents, g/16g N which is equivalent to g/100g protein, is normally used. These units enable the amino acid profile of different protein supplements to be easily compared so that the amino acid balance that is the level of essential amino acidds in each protein supplement can be directly compared.