AMINO ACID ANALYSIS
Amino acid analysis refers to the methodology used to determine the amino acid composition or content of proteins, peptides, and other pharmaceutical preparations. Proteins and peptides are macromolecules consisting of covalently bonded amino acid residues organized as a linear polymer. The sequence of the amino acids in a protein or peptide determines the properties of the molecule. Proteins are considered large molecules that commonly exist as folded structures with a specific conformation, while peptides are smaller and may consist of only a few amino acids. Amino acid analysis can be used to quantify protein and peptides, to determine the identity of proteins or peptides based on their amino acid composition, to support protein and peptide structure analysis, to evaluate fragmentation strategies for peptide mapping, and to detect atypical amino acids that might be present in a protein or peptide. It is necessary to hydrolyze a protein/peptide to its individual amino acid constituents before amino acid analysis. Following protein/peptide hydrolysis, the amino acid analysis procedure can be the same as that practiced for free amino acids in other pharmaceutical preparations. The amino acid constituents of the test sample are typically derivatized for analysis.
Methods used for amino acid analysis are usually based on a chromatographic separation of the amino acids present in the test sample. Current techniques take advantage of the automated chromatographic instrumentation designed for analytical methodologies. An amino acid analysis instrument will typically be a low-pressure or high-pressure liquid chromatograph capable of generating mobile phase gradients that separate the amino acid analytes on a chromatographic column. The instrument must have postcolumn derivatization2
capability, unless the sample is analyzed using precolumn derivatization. The detector is usually an ultraviolet-visible or fluorescence detector depending on the derivatization method used. A recording device (e.g., integrator) is used for transforming the analog signal from the detector and for quantitation. It is preferred that instrumentation be dedicated particularly for amino acid analysis.
Background contamination is always a concern for the analyst in performing amino acid analysis. High purity reagents are necessary (e.g., low purity hydrochloric acid can contribute to glycine contamination). Analytical reagents are changed routinely every few weeks using only high-pressure liquid chromatography (HPLC) grade solvents. Potential microbial contamination and foreign material that might be present in the solvents are reduced by filtering solvents before use, keeping solvent reservoirs covered, and not placing amino acid analysis instrumentation in direct sunlight.
Laboratory practices can determine the quality of the amino acid analysis. Place the instrumentation in a low traffic area of the laboratory. Keep the laboratory clean. Clean and calibrate pipets according to a maintenance schedule. Keep pipet tips in a covered box; the analysts may not handle pipet tips with their hands. The analysts may wear powder-free latex or equivalent gloves. Limit the number of times a test sample vial is opened and closed because dust can contribute to elevated levels of glycine, serine, and alanine.
A well-maintained instrument is necessary for acceptable amino acid analysis results. If the instrument is used on a routine basis, it is to be checked daily for leaks, detector and lamp stability, and the ability of the column to maintain resolution of the individual amino acids. Clean or replace all instrument filters and other maintenance items on a routine schedule.
Reference Standard Material
Acceptable amino acid standards are commercially available for amino acid analysis and typically consist of an aqueous mixture of amino acids. When 3
determining amino acid composition, protein or peptide standards are analyzed with the test material as a control to demonstrate the integrity of the entire procedure. Highly purified bovine serum albumin has been used as a protein standard for this purpose.
Calibration of Instrumentation
Calibration of amino acid analysis instrumentation typically involves analyzing the amino acid standard, which consists of a mixture of amino acids at a number of concentrations, to determine the response factor and range of analysis for each amino acid. The concentration of each amino acid in the standard is known. In the calibration procedure, the analyst dilutes the amino acid standard to several different analyte levels within the expected linear range .................More Read....
Revised 2/21/02 with JP comments
TOTAL PROTEIN ASSAY
The following procedures are provided as illustrations of the determination of total protein content in pharmacopeial preparations. Other techniques, such as HPLC, are also acceptable if total protein recovery is demonstrated. Many of the total protein assay methods described below can be performed successfully using kits from commercial sources.
OTE—Where water is required, use distilled water.
Protein in solution absorbs UV light at a wavelength of 280 nm, due to the presence of aromatic amino acids, mainly tyrosine and tryptophan. This property is the basis of this method. Protein determination at 280 nm is mainly a function of the tyrosine and tryptophan content of the protein. If the buffer used to dissolve the protein has a high absorbance relative to that of water, there is an interfering substance in the buffer. This interference can be compensated for when the spectrophotometer is adjusted to zero buffer absorbance. If the interference results in a large absorbance that challenges the limit of sensitivity of the spectrophotometer, the results may be compromised. Furthermore, at low concentrations protein can be absorbed onto the cuvette, thereby reducing the content in solution. This can be prevented by preparing samples at higher concentrations or by using a nonionic detergent in the preparation.
OTE—Keep the Test Solution, the Standard Solution, and the buffer at the same temperature during testing.
—Prepare a solution of the Reference Standard or Reference Material for the protein under test in the same buffer and at the same concentration as the Test Solution.2
—Dissolve a suitable quantity of the protein under test in the appropriate buffer to obtain a solution having a concentration of 0.2 to 2 mg per mL.
—Concomitantly determine the absorbances of the Standard Solution and the Test Solution in quartz cells at a wavelength of 280 nm, with a suitable spectrophotometer, using the buffer as the blank. To obtain accurate results, the response should be linear in the range of protein concentrations to be assayed.
—The accuracy of the UV spectroscopic determination of protein can be decreased by the scattering of light by the test specimen. If the proteins in solution exist as particles comparable in size to the wavelength of the measuring light (250 to 300 nm), scattering of the light beam results in an apparent increase in absorbance of the test specimen. To calculate the absorbance at 280 nm due to light-scattering, determine the absorbances of the .................More Read....
Nam Sun Wang
Department of Chemical & Biomolecular Engineering
University of Maryland
College Park, MD 20742-2111
The reaction between alpha-amino acid and ninhydrin involved in the development of color are described by the following five mechanistic steps:
alpha-amino acid + ninhydrin ---> reduced ninhydrin + alpha-amino acid + H2O alpha-amino acid + H2O ---> alpha-keto acid +NH3 alpha-keto acid + NH3 ---> aldehyde + CO2
Step (1) is an oxidative deamination reaction that removes two hydrogen from the alpha-amino acid to yield an alpha-imino acid. Simultaneously, the original ninhydrin is reduced and loses an oxygen atom with the formation of a water molecule. In Step (2), the NH group in the alpha-imino acid is rapidly hydrolyzed to form an alpha-keto acid with the production of an ammonia molecule. This alpha-keto acid further undergoes decarboxylation reaction of Step (3) under a heated condition to form an aldehyde that has one less carbon atom than the original amino acid. A carbon dioxide molecule is produced here. These first three steps produce the reduced ninhydrin and ammonia that are required for the production of color in the last two Steps (4) and (5). The overall reaction for the above reactions is simply (slightly inaccurately) expressed in Reaction (6) as follows:
alpha-amino acid + 2 ninhydrin ---> CO2 + aldehyde + final complex(BlUE) + 3H2O
In summary, ninhydrin, which is originally yellow, reacts with amino acid and turns deep purple. It is this purple color that is detected in this method.Ninhydrin will react with a free alpha-amino group, NH2-C-COOH. This group is contained in all amino acids, peptides, or proteins. Whereas, the decarboxylation reaction will proceed for a free amino acid, it will not happen for peptides and proteins. Thus, theoretically only amino acids will lead to the color development. However, one should always check out the possible interference from peptides and proteins by performing blank tests especially when such solutions are readily available. For example, one can simply add the ninhydrin reagent to a solution of only proteins and see if there is any color development. There is no excuse for failing to perform such a vital test when the sample mixture contains both proteins and amino acids. There are also reports that chemical compounds other than amino acids also yield positive results.
This test can be used routinely for the detection of glycine in the absence of other interfering species. Although this is a fast and sensitive test for the presence of alpha-amino acids, because of the nonselectivity, it cannot be used to analyze the relative individual contents of a mixture of different amino acids. Furthermore, the color intensity developed is dependent on the type of amino acid. Finally, it does not react with tertiary or aromatic amines.
Note that since ninhydrin is a strong oxidizing agent, proper caution should be exercised in handling this compound. It is especially potent at the elevated temperature under which the reaction is carried out. The ninhydrin reagent will stain the skin blue and cannot be immediately washed off completely if it comes in contact with the skin. However, as in any other stain on the skin, the color will gradually rub off after about a day.