General Information
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GENERAL INFORMATION use, keeping solvent reservoirs covered, and not placing ami- 1. AminoAcidAnalysis no acid analysis instrumentation in direct sunlight. Laboratory practices can determine the quality of the amino acid analysis. Place the instrumentation in a low tra‹c This test is harmonized with the European Pharmacopoeia area of the laboratory. Keep the laboratory clean. Clean and and the U.S. Pharmacopeia. calibrate pipets according to a maintenance schedule. Keep Amino acid analysis refers to the methodology used to de- pipet tips in a covered box; the analysts may not handle pipet termine the amino acid composition or content of proteins, tips with their hands. The analysts may wear powder-free la- peptides, and other pharmaceutical preparations. Proteins tex or equivalent gloves. Limit the number of times a test and peptides are macromolecules consisting of covalently sample vial is opened and closed because dust can contribute bonded amino acid residues organized as a linear polymer. to elevated levels of glycine, serine, and alanine. The sequence of the amino acids in a protein or peptide A well-maintained instrument is necessary for acceptable determines the properties of the molecule. Proteins are amino acid analysis results. If the instrument is used on a considered large molecules that commonly exist as folded routine basis, it is to be checked daily for leaks, detector and structures with a speciˆc conformation, while peptides are lamp stability, and the ability of the column to maintain reso- smaller and may consist of only a few amino acids. Amino lution of the individual amino acids. Clean or replace all acid analysis can be used to quantify protein and peptides, to instrument ˆlters and other maintenance items on a routine determine the identity of proteins or peptides based on their schedule. amino acid composition, to support protein and peptide structure analysis, to evaluate fragmentation strategies for Reference Standard Material peptide mapping, and to detect atypical amino acids that Acceptable amino acid standards are commercially might be present in a protein or peptide. It is necessary to available for amino acid analysis and typically consist of an hydrolyze a protein/peptide to its individual amino acid aqueous mixture of amino acids. When determining amino constituents before amino acid analysis. Following pro- acid composition, protein or peptide standards are analyzed tein/peptide hydrolysis, the amino acid analysis procedure with the test material as a control to demonstrate the integrity can be the same as that practiced for free amino acids in other of the entire procedure. Highly puriˆed bovine serum pharmaceutical preparations. The amino acid constituents of albumin has been used as a protein standard for this purpose. the test sample are typically derivatized for analysis. Calibration of Instrumentation Apparatus Calibration of amino acid analysis instrumentation Methods used for amino acid analysis are usually based on typically involves analyzing the amino acid standard, which a chromatographic separation of the amino acids present in consists of a mixture of amino acids at a number of concen- the test sample. Current techniques take advantage of the au- trations, to determine the response factor and range of tomated chromatographic instrumentation designed for ana- analysis for each amino acid. The concentration of each ami- lytical methodologies. An amino acid analysis instrument will no acid in the standard is known. In the calibration proce- typically be a low-pressure or high-pressure liquid chromato- dure, the analyst dilutes the amino acid standard to several graph capable of generating mobile phase gradients that diŠerent analyte levels within the expected linear range of the separate the amino acid analytes on a chromatographic amino acid analysis technique. Then, replicates at each of the column. The instrument must have postcolumn derivatiza- diŠerent analyte levels can be analyzed. Peak areas obtained tion capability, unless the sample is analyzed using for each amino acid are plotted versus the known concentra- precolumn derivatization. The detector is usually an ultrav- tion for each of the amino acids in the standard dilution. iolet-visible or ‰uorescence detector depending on the These results will allow the analyst to determine the range of derivatization method used. A recording device (e.g., amino acid concentrations where the peak area of a given integrator) is used for transforming the analog signal from amino acid is an approximately linear function of the amino the detector and for quantitation. It is preferred that acid concentration. It is important that the analyst prepare instrumentation be dedicated particularly for amino acid the samples for amino acid analysis so that they are within analysis. the analytical limits (e.g., linear working range) of the tech- nique employed in order to obtain accurate and repeatable General Precautions results. Background contamination is always a concern for the Four to six amino acid standard levels are analyzed to analyst in performing amino acid analysis. High purity determine a response factor for each amino acid. The reagents are necessary (e.g., low purity hydrochloric acid can response factor is calculated as the average peak area or peak contribute to glycine contamination). Analytical reagents are height per nmol of amino acid present in the standard. A changed routinely every few weeks using only high-pressure calibration ˆle consisting of the response factor for each ami- liquid chromatography (HPLC) grade solvents. Potential no acid is prepared and used to calculate the concentration of microbial contamination and foreign material that might be each amino acid present in the test sample. This calculation present in the solvents are reduced by ˆltering solvents before involves dividing the peak area corresponding to a given ami- 1655 1656 AminoAcidAnalysis/ General Information JP XV no acid by the response factor for that amino acid to give the standard can be added to a protein solution prior to hydroly- nmol of the amino acid. For routine analysis, a single-point sis. The recovery of the internal standard gives the general calibration may be su‹cient; however, the calibration ˆle is recovery of the amino acids of the protein solution. Free ami- updated frequently and tested by the analysis of analytical no acids, however, do not behave in the same way as protein- controls to ensure its integrity. bound amino acids during hydrolysis because their rates of release or destruction are variable. Therefore, the use of an Repeatability internal standard to correct for losses during hydrolysis may Consistent high quality amino acid analysis results from an give unreliable results. It will be necessary to take this point analytical laboratory require attention to the repeatability of under consideration when interpreting the results. Internal the assay. During analysis of the chromatographic separation standards can also be added to the mixture of amino acids af- of the amino acids or their derivatives, numerous peaks can ter hydrolysis to correct for diŠerences in sample application be observed on the chromatogram that correspond to the and changes in reagent stability and ‰ow rates. Ideally, an in- amino acids. The large number of peaks makes it necessary to ternal standard is an unnaturally occurring primary amino have an amino acid analysis system that can repeatedly iden- acid that is commercially available and inexpensive. It should tify the peaks based on retention time and integrate the peak also be stable during hydrolysis, its response factor should be areas for quantitation. A typical repeatability evaluation in- linear with concentration, and it needs to elute with a unique volves preparing a standard amino acid solution and analyz- retention time without overlapping other amino acids. Com- ing many replicates (i.e., six analyses or more) of the same monly used amino acid standards include norleucine, standard solution. The relative standard deviation (RSD) is nitrotyrosine, and a-aminobutyric acid. determined for the retention time and integrated peak area of each amino acid. An evaluation of the repeatability is ex- Protein Hydrolysis panded to include multiple assays conducted over several Hydrolysis of protein and peptide samples is necessary for days by diŠerent analysts. Multiple assays include the prepa- amino acid analysis of these molecules. The glassware used ration of standard dilutions from starting materials to deter- for hydrolysis must be very clean to avoid erroneous results. mine the variation due to sample handling. Often the amino Glove powders and ˆngerprints on hydrolysis tubes may acid composition of a standard protein (e.g., bovine serum cause contamination. To clean glass hydrolysis tubes, boil albumin) is analyzed as part of the repeatability evaluation. tubes for 1 hour in 1 mol/L hydrochloric acid or soak tubes By evaluating the replicate variation (i.e., RSD), the labora- in concentrated nitric acid or in a mixture of concentrated tory can establish analytical limits to ensure that the analyses hydrochloric acid and concentrated nitric acid (1:1). Clean from the laboratory are under control. It is desirable to estab- hydrolysis tubes are rinsed with high-purity water followed lish the lowest practical variation limits to ensure the best by a rinse with HPLC grade methanol, dried overnight in an results. Areas to focus on to lower the variability of the ami- oven, and stored