Amino acid analysis sample preparation

There are two major categories for amino acid analysis (a) free amino acid analysis and (b) determination of total amino acid content. The total amino acid content includes contributions from the free amino acids and the amino acids that are originally protein bound. These protein-bound amino acids must first be liberated before chromatographic analysis. This necessitates a more extensive, and problematic, sample preparation. Because the sample preparation procedures are so disparate, it is convenient to address these two categories of amino acid analyses separately. It should be noted that while the sample preparations for these analyses are quite different, both utilize essentially the same chromatographic techniques for the second stage of amino acid analysis. 

Sulfosalicylic acid has most commonly been used to precipitate proteins prior to ion-exchange amino acid analysis (11). In this mode, SSA allows for a very simple sample preparation that requires only centrifugation of the precipitated sample and then direct injection of the resulting supernatant solution. The supernatant solution is already at an appropriate pH for direct injection. Also, the SSA does not interfere chromatographically since it elutes essentially in the void volume of the column. It has been noted that, if an excessive amount of SSA is employed, resolution of the serine/threonine critical pair can suffer (12). The use of SSA prior to reversed-phase HPLC can be more problematic, since its presence can interfere with precolumn deriva-tization. For example, Cohen and Strydom (13) recommend the separation of the amino acids from the SSA solution on a cation-exchange resin prior to derivatization with phenylisothiocya-nate (PITC). 

Brief mention needs to be made regarding the employment of internal standards. While it is desirable to employ an internal standard for procedures that involve significant sample preparation procedures, there is no ideal choice for an internal standard for amino acid analysis. This fact is due to the wide spectrum of chemistries exhibited by the various amino acids. If one is analyzing for a single amino acid (or class, e.g., the hydrophobic amino acids), it is possible to choose an internal standard that mimics the chemistry of that particular amino acid very well. However, for the overall amino acid profile, an internal standard will do nothing more than allow the analyst to make nonvolumetric solution transfers and correct for variability of the injection volume by the HPLC injector. Unfortunately, the employment of an internal standard can actually skew the apparent recoveries for the overall amino acid profile. 

The earliest approach to amino acid analysis involved postcolumn reaction. This scheme offers several advantages compared to precolumn reaction. First, it simplifies the sample preparation necessary. Often, precolumn derivatizations require sample cleanup steps to eliminate sample... 

RW Hubbard, JG Chambers, A Sanchez, R Slocum, P Lee. Amino acid analysis of plasma studies in sample preparation. J Chromatogr Biomed Appl 431 163-169, 1988. 

Sulfosalicylic acid Used to prepare samples from amino acid analysis Fox (1989), Kuchroo and Fox (1982a), and Ramos et al. (1987)... 

With the exception of studies on bovine serum albumin (BSA) and human transferrin, all other digests were carried out on Coomassie Blue-stained gel bands that had been excised from SDS polyacrylamide gels and submitted in eppendorf tubes to the internal protein sequencing service of the HHMI Biopolymer Laboratory/W.M. Keck Foundation Biotechnology Resource Laboratory at Yale University (5). The BSA and transferrin samples were subjected to SDS-PAGE in the Keck Facility and were otherwise prepared as described (5). Proteins were quantified by subjecting 10-15% aliquots of all gel slices to hydrolysis and ion exchange amino acid analysis (5). 

Sample preparation is the key step in an accurate and reproducible amino acid analysis. In principle, physiological samples should be processed as quickly as possible to avoid contamination by sample handling or by the reagents being used. In addition, metabolic activities may lead to false results. 

To analyze free amino acids in plasma or tissue homogenates, it is necessary to remove proteins and peptides present in solution. The most widely used deproteinization method is precipitation with 5-sulfosalicylic acid followed by centrifugation for separating the precipitate. In comparison to other precipitation agents such as trichloroacetic acid, perchloric acid, picrinic acid, or acetonitrile, the best results with respect to completeness of precipitation are obtained with 5-sulfosalicylic acid [39]. Other deproteinization methods comprise ultrafiltration and ultracentrifugation [40], which have only recently been considered as sample preparation methods for amino acid analysis. 

Cyst(e)ine occurs in biological tissues and fluids, both in the free sulfhydryl form, cysteine, and in the disulfide form, cystine In the methods of sample preparation described above, cysteine is oxidized to cystine, and the proportion of the two forms in a sample cannot be determined. A method was devised to accomplish this, involving immediate addition to the sample of iodoacetate, which rapidly reacts with cysteine, converting it quantitatively to the stable S-carboxymethyl derivative (Brigham et al., 1960). Cystine is unaffected by iodoacetate. Both cystine and S-carboxymethylcysteine can be quantified by amino acid analysis. This same strategy can be used with other sulfhydryl compounds, such as homocyst(e)ine. 

The application of high performance liquid chromatography (HPLC) to the analysis of amino acids forms the subject matter of another chapter HPLC is one of the more recent developments in ammo acid determinations, and the use of precolumn fluorogenic denvatization in combination with reversed-phase liquid chromatography has simplified and improved the separation of amino acids. This system combines simplicity of sample preparation with high sensitivity and speed of analysis. Not surprisingly, HPLC is the system of choice for amino acid analysis by an ever-mcreasmg number of laboratories. 

Precolumn derivatization methods are not free from disadvantages. One is that side-products from the reaction can sometimes be difficult to separate from the peaks of interest. Also with precolumn derivatization, as its name implies, each sample must be derivatized prior to injection. This can add to the total analysis time. However, instrumentation is now available to derivatize several samples simultaneously in a closed system, which not only solves the sample preparation time problem but also results in highly reproducible chemistry. Since the reaction may not go to 100%, an internal standard is frequently necessary. Finally, the addition of a similar tag to all the amino acids may make them more difficult to separate. Table 1 summarizes the properties of several pre- and postcolumn derivatizing reagents employed for amino acid analysis. 

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