This chapter provides guidance and procedures used for characterization of biotechnology-derived articles by amino acid analysis. This chapter is harmonized with the corresponding chapter in JP and EP. Other characterization tests, also harmonized, are shown in Biotechnology-Derived Articles—Capillary Electrophoresis 1053, Biotechnology-Derived Articles—Isoelectric Focusing 1054, Biotechnology-Derived Articles—Peptide Mapping 1055, Biotechnology-Derived Articles—Polyacrylamide Gel Electrophoresis 1056, and Biotechnology-Derived Articles—Total Protein Assay 1057.

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 derivatization capability, unless the sample is analyzed using precolumn derivatization. The detector is usually a UV-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.

Acceptable amino acid standards are commercially available* for amino acid analysis and typically consist of an aqueous mixture of amino acids. When 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 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 of the amino acid analysis technique. Then, replicates at each of the different analyte levels can be analyzed. Peak areas obtained for each amino acid are plotted versus the known concentration for each of the amino acids in the standard dilution. These results will allow the analyst to determine the range of amino acid concentrations where the peak area of a given amino acid is an approximately linear function of the amino acid concentration. It is important that the analyst prepare the samples for amino acid analysis so that they are within the analytical limits (e.g., linear working range) of the technique employed in order to obtain accurate and repeatable results.

Four to six amino acid standard levels are analyzed to determine a response factor for each amino acid. The response factor is calculated as the average peak area or peak height per nmol of amino acid present in the standard. A calibration file consisting of the response factor for each amino acid is prepared and is used to calculate the concentration of each amino acid present in the test sample. This calculation involves dividing the peak area corresponding to a given amino acid by the response factor for that amino acid to give the nmol of the amino acid. For routine analysis, a single-point calibration may be sufficient; however, the calibration file is updated frequently and tested by the analysis of analytical controls to ensure its integrity.

Consistent high quality amino acid analysis results from an analytical laboratory require attention to the repeatability of the assay. During analysis of the chromatographic separation of the amino acids or their derivatives, numerous peaks can be observed on the chromatogram that corresponds to the amino acids. The large number of peaks makes it necessary to have an amino acid analysis system that can repeatedly identify the peaks based on retention time and integrate the peak areas for quantitation. A typical repeatability evaluation involves preparing a standard amino acid solution and analyzing many replicates (i.e., six analyses or more) of the same standard solution. The relative standard deviation (RSD) is determined for the retention time and integrated peak area of each amino acid. An evaluation of the repeatability is expanded to include multiple assays conducted over several days by different analysts. Multiple assays include the preparation of standard dilutions from starting materials to determine the variation due to sample handling. Often, the amino acid composition of a standard protein (e.g., bovine serum albumin) is analyzed as part of the repeatability evaluation. By evaluating the replicate variation (i.e., RSD), the laboratory can establish analytical limits to ensure that the analyses from the laboratory are under control. It is desirable to establish the lowest practical variation limits to ensure the best results. Areas to focus on to lower the variability of the amino acid analysis include sample preparation, high background spectral interference due to the quality of reagents and/or to laboratory practices, instrument performance and maintenance, data analysis and interpretation, and analyst performance and habits. All parameters involved are fully investigated in the scope of the validation work.

Accurate results from amino acid analysis require purified protein and peptide samples. Buffer components (e.g., salts, urea, detergents) can interfere with the amino acid analysis and are removed from the sample before analysis. Methods that utilize postcolumn derivatization of the amino acids are generally not affected by buffer components to the extent seen with precolumn derivatization methods. It is desirable to limit the number of sample manipulations to reduce potential background contamination, to improve analyte recovery, and to reduce labor. Common techniques used to remove buffer components from protein samples include the following methods: (1) injecting the protein sample onto a reverse-phase HPLC system, removing the protein with a volatile solvent containing a sufficient organic component, and drying the sample in a vacuum centrifuge; (2) dialysis against a volatile buffer or water; (3) centrifugal ultrafiltration for buffer replacement with a volatile buffer or water; (4) precipitating the protein from the buffer using an organic solvent (e.g., acetone); and (5) gel filtration.

It is recommended that an internal standard be used to monitor physical and chemical losses and variations during amino acid analysis. An accurately known amount of internal standard can be added to a protein solution prior to hydrolysis. The recovery of the internal standard gives the general recovery of the amino acids from the protein solution. Free amino acids, however, do not behave in the same way as protein-bound amino acids during hydrolysis because their rates of release or destruction are variable. Therefore, the use of an internal standard to correct for losses during hydrolysis may give unreliable results. It will be necessary to take this particular point into consideration when interpreting the results. Internal standards can also be added to the mixture of amino acids after hydrolysis to correct for differences in sample application and changes in reagent stability and flow rates. Ideally, an internal standard is an unnaturally occurring primary amino acid that is commercially available and inexpensive. It should also be stable during hydrolysis, its response factor should be linear with concentration, and it needs to elute with a unique retention time without overlapping other amino acids. Commonly used amino acid standards include norleucine, nitrotyrosine, and -aminobutyric acid.

Hydrolysis of protein and peptide samples is necessary for amino acid analysis of these molecules. The glassware used for hydrolysis must be very clean to avoid erroneous results. Glove powders and fingerprints on hydrolysis tubes may cause contamination. To clean glass hydrolysis tubes, boil tubes for 1 hour in 1 N hydrochloric acid or soak tubes in concentrated nitric acid or in a mixture of concentrated hydrochloric acid and concentrated nitric acid (1:1). Clean hydrolysis tubes are rinsed with high-purity water followed by a rinse with HPLC grade methanol, dried overnight in an oven, and stored covered until use. Alternatively, pyrolysis of clean glassware at 500 for 4 hours may be used to eliminate contamination from hydrolysis tubes. Adequate disposable laboratory material can also be used.

Acid hydrolysis is the most common method for hydrolyzing a protein sample before amino acid analysis. The acid hydrolysis technique can contribute to the variation of the analysis due to complete or partial destruction of several amino acids. Tryptophan is destroyed; serine and threonine are partially destroyed; methionine might undergo oxidation; and cysteine is typically recovered as cystine (but cystine recovery is usually poor because of partial destruction or reduction to cysteine). Application of adequate vacuum (less than 200 µm of mercury or 26.7 Pa) or introduction of an inert gas (argon) in the headspace of the reaction vessel can reduce the level of oxidative destruction. In peptide bonds involving isoleucine and valine, the amido bonds of Ile-Ile, Val-Val, Ile-Val, and Val-Ile are partially cleaved; and asparagine and glutamine are deamidated, resulting in aspartic acid and glutamic acid, respectively. The loss of tryptophan, asparagine, and glutamine during an acid hydrolysis limits quantitation to 17 amino acids. Some of the hydrolysis techniques described are used to address these concerns. Some of the hydrolysis techniques described (i.e., Methods 4–11) may cause modifications to other amino acids. Therefore, the benefits of using a given hydrolysis technique are weighed against the concerns with the technique and are tested adequately before employing a method other than acid hydrolysis.

A time-course study (i.e., amino acid analysis at acid hydrolysis times of 24, 48, and 72 hours) is often employed to analyze the starting concentration of amino acids that are partially destroyed or slow to cleave. By plotting the observed concentration of labile amino acids (i.e., serine and threonine) versus hydrolysis time, the line can be extrapolated to the origin to determine the starting concentration of these amino acids. Time-course hydrolysis studies are also used with amino acids that are slow to cleave (e.g., isoleucine and valine). During the hydrolysis time course, the analyst will observe a plateau in these residues. The level of this plateau is taken as the residue concentration. If the hydrolysis time is too long, the residue concentration of the sample will begin to decrease, indicating destruction by the hydrolysis conditions.

An acceptable alternative to the time-course study is to subject an amino acid calibration standard to the same hydrolysis conditions as the test sample. The amino acid in free form may not completely represent the rate of destruction of labile amino acids within a peptide or protein during the hydrolysis. This is especially true for peptide bonds that are slow to cleave (e.g., Ile-Val bonds). However, this technique will allow the analyst to account for some residue destruction. Microwave acid hydrolysis has been used and is rapid but it requires special equipment as well as special precautions. The optimal conditions for microwave hydrolysis must be investigated for each individual protein/peptide sample. The microwave hydrolysis technique typically requires only a few minutes, but even a deviation of 1 minute may give inadequate results (e.g., incomplete hydrolysis or destruction of labile amino acids). Complete proteolysis, using a mixture of proteases, has been used but can be complicated, requires the proper controls, and is typically more applicable to peptides than proteins. [note—During initial analyses of an unknown protein, experiments with various hydrolysis time and temperature conditions are conducted to determine the optimal conditions.]

Method 1

Hydrolysis Solution: 6 N hydrochloric acid containing 0.1% to 1.0% of phenol.

Procedure—

Method 2

Hydrolysis Solution: 2.5 M MESA solution.

Vapor Phase Hydrolysis— About 1 to 100 µg of the protein/peptide under test is dried in a hydrolysis tube. The hydrolysis tube is placed in a larger tube with about 200 µL of the Hydrolysis Solution. The larger tube is sealed in vacuum (about 50 µm of mercury or 6.7 Pa) to vaporize the Hydrolysis Solution. The hydrolysis tube is heated to between 170 to 185 for about 12.5 minutes. After hydrolysis, the hydrolysis tube is dried in vacuum for 15 minutes to remove the residual acid.

Method 3

Hydrolysis Solution: a solution containing 7 M hydrochloric acid, 10% of trifluoroacetic acid, 20% of thioglycolic acid, and 1% of phenol.

Vapor Phase Hydrolysis— About 10 to 50 µg of the protein/peptide under test is dried in a sample tube. The sample tube is placed in a larger tube with about 200 µL of the Hydrolysis Solution. The larger tube is sealed in vacuum (about 50 µm of mercury or 6.7 Pa) to vaporize the TGA. The sample tube is heated to 166 for about 15 to 30 minutes. After hydrolysis, the sample tube is dried in vacuum for 5 minutes to remove the residual acid. Recovery of tryptophan by this method may be dependent on the amount of sample present.

Method 4

Oxidation Solution— The performic acid is prepared fresh by mixing formic acid and 30 percent hydrogen peroxide (9:1), and incubating at room temperature for 1 hour.

Procedure— The protein/peptide sample is dissolved in 20 µL of formic acid, and heated at 50 for 5 minutes; then 100 µL of the Oxidation Solution is added. In this reaction, cysteine is converted to cysteic acid and methionine is converted to methionine sulfone. The oxidation is allowed to proceed for 10 to 30 minutes. The excess reagent is removed from the sample in a vacuum centrifuge. This technique may cause modifications to tyrosine residues in the presence of halides. The oxidized protein can then be acid hydrolyzed using Method 1 or Method 2.

Method 5

Hydrolysis Solution: 6 N hydrochloric acid containing 0.2% of phenol, to which sodium azide is added to obtain a final concentration of 0.2% (w/v). The added phenol prevents halogenation of tyrosine.

Liquid Phase Hydrolysis— The protein/peptide hydrolysis is conducted at about 110 for 24 hours. During the hydrolysis, the cysteine-cystine present in the sample is converted to cysteic acid by the sodium azide present in the Hydrolysis Solution. This technique allows better tyrosine recovery than Method 4, but it is not quantitative for methionine. Methionine is converted to a mixture of the parent methionine and its two oxidative products, methionine sulfoxide and methionine sulfone.

Method 6

Hydrolysis Solution: 6 N hydrochloric acid containing 0.1% to 1.0% of phenol, to which DMSO is added to obtain a final concentration of 2% (v/v).

Vapor Phase Hydrolysis— The protein/peptide hydrolysis is conducted at about 110 for 24 hours. During the hydrolysis, the cysteine-cystine present in the sample is converted to cysteic acid by the DMSO present in the Hydrolysis Solution. As an approach to limit variability and to compensate for partial destruction, it is recommended to evaluate the cysteic acid recovery from oxidative hydrolyses of standard proteins containing 1 to 8 mol of cysteine. The response factors from protein/peptide hydrolysates are typically about 30% lower than those for nonhydrolyzed cysteic acid standards. Because histidine, methionine, tyrosine, and tryptophan are also modified, a complete compositional analysis is not obtained with this technique.

Method 7

Reducing Solution— Transfer 83.3 µL of pyridine, 16.7 µL of 4-vinylpyridine, 16.7 µL of tributylphosphine, and 83.3 µL of water to a suitable container, and mix.

Procedure— Add the protein/peptide (between 1 and 100 µg) to a hydrolysis tube, and place in a larger tube. Transfer the Reducing Solution to the large tube, seal in vacuum (about 50 µm of mercury or 6.7 Pa), and incubate at about 100 for 5 minutes. Then remove the inner hydrolysis tube, and dry it in a vacuum desiccator for 15 minutes to remove residual reagents. The pyridylethylated protein/peptide can then be acid hydrolyzed using previously described procedures. The pyridylethylation reaction is performed simultaneously with a protein standard sample containing 1 to 8 mol of cysteine to improve accuracy in the pyridylethyl-cysteine recovery. Longer incubation times for the pyridylethylation reaction can cause modifications to the -amino terminal group and the -amino group of lysine in the protein.

Method 8

Stock Solutions— Prepare and filter three solutions: 1 M Tris hydrochloride (pH 8.5) containing 4 mM edetate disodium (Stock Solution 1), 8 M guanidine hydrochloride (Stock Solution 2), and 10% of 2-mercaptoethanol in water (Stock Solution 3).

Reducing Solution— Prepare a mixture of Stock Solution 2 and Stock Solution 1 (3:1) to obtain a buffered solution of 6 M guanidine hydrochloride in 0.25 M Tris hydrochloride.

Procedure— Dissolve about 10 µg of the test sample in 50 µL of the Reducing Solution, and add about 2.5 µL of Stock Solution 3. Store under nitrogen or argon for 2 hours at room temperature in the dark. To achieve the pyridylethylation reaction, add about 2 µL of 4-vinylpyridine to the protein solution, and incubate for an additional 2 hours at room temperature in the dark. The protein/peptide is desalted by collecting the protein/peptide fraction from a reverse-phase HPLC separation. The collected sample can be dried in a vacuum centrifuge before acid hydrolysis.

Method 9

Stock Solutions— Prepare as directed for Method 8.

Carboxymethylation Solution— Prepare a solution containing 100 mg of iodoacetamide per mL of alcohol.

Buffer Solution— Use the Reducing Solution, prepared as directed for Method 8.

Procedure— Dissolve the test sample in 50 µL of the Buffer Solution, and add about 2.5 µL of Stock Solution 3. Store under nitrogen or argon for 2 hours at room temperature in the dark. Add the Carboxymethylation Solution in a 1.5 fold ratio per total theoretical content of thiols, and incubate for an additional 30 minutes at room temperature in the dark. [note—If the thiol content of the protein is unknown, then add 5 µL of 100 mM iodoacetamide for every 20 nmol of protein present.] The reaction is stopped by adding excess of 2-mercaptoethanol. The protein/peptide is desalted by collecting the protein/peptide fraction from a reverse-phase HPLC separation. The collected sample can be dried in a vacuum centrifuge before acid hydrolysis. The S-carboxyamidomethylcysteine formed will be converted to S-carboxymethyl-cysteine during acid hydrolysis.

Method 10

Reducing Solution: a solution containing 10 mg of dithiodiglycolic acid (or dithiodipropionic acid) per mL of 0.2 M sodium hydroxide.

Procedure— Transfer about 20 µg of the test sample to a hydrolysis tube, and add 5 µL of the Reducing Solution. Add 10 µL of isopropyl alcohol, and then remove all of the sample liquid by vacuum centrifugation. The sample is then hydrolyzed using Method 1. This method has the advantage that other amino acid residues are not derivatized by side reactions, and the sample does not need to be desalted prior to hydrolysis.

Method 11

Reducing Solutions— Prepare and filter three solutions: a solution of 10 mM trifluoroacetic acid (Solution 1), a solution of 5 M guanidine hydrochloride and 10 mM trifluoroacetic acid (Solution 2), and a freshly prepared solution of dimethylformamide containing 36 mg of BTI per mL (Solution 3).

Procedure— In a clean hydrolysis tube, transfer about 200 µg of the test sample, and add 2 mL of Solution 1 or Solution 2 and 2 mL of Solution 3. Seal the hydrolysis tube in vacuum. Heat the sample at 60 for 4 hours in the dark. The sample is then dialyzed with water to remove the excess reagents. Extract the dialyzed sample three times with equal volumes of n-butyl acetate, and then lyophilize. The protein can then be acid hydrolyzed using previously described procedures. The -, -diaminopropionic and -, -diaminobutyric acid residues do not typically resolve from the lysine residues upon ion-exchange chromatography based on amino acid analysis. Therefore, when using ion-exchange as the mode of amino acid separation, the asparagine and glutamine contents are the quantitative difference in the aspartic acid and glutamic acid assayed contents with un-derivatized and BTI-derivatized acid hydrolysis. [note—The threonine, methionine, cysteine, tyrosine, and histidine assayed content can be altered by BTI derivatization; a hydrolysis without BTI will have to be performed if the analyst is interested in the protein/peptide content of these other amino acid residues.]

Many amino acid analysis techniques exist, and the choice of any one technique often depends on the sensitivity required from the assay. In general, about one-half of the amino acid analysis techniques employed rely on the separation of the free amino acids by ion-exchange chromatography followed by postcolumn derivatization (e.g., with ninhydrin or o-phthalaldehyde). Postcolumn detection techniques can be used with samples that contain small amounts of buffer components, such as salts and urea, and generally require between 5 and 10 µg of protein sample per analysis. The remaining amino acid techniques typically involve precolumn derivatization of the free amino acids (e.g., phenyl isothiocyanate; 6-aminoquinolyl-N-hydroxysuccinimidyl carbonate; (dimethylamino)azobenzenesulfonyl chloride; 9-fluorenyl-methylchloroformate; and 7-fluoro-4-nitrobenzo-2-oxa-1,3-diazole) followed by reverse-phase HPLC. Precolumn derivatization techniques are very sensitive and usually require between 0.5 and 1.0 µg of protein sample per analysis but may be influenced by buffer salts in the samples. Precolumn derivatization techniques may also result in multiple derivatives of a given amino acid, which complicates the result interpretation. Postcolumn derivatization techniques are generally influenced less by performance variation of the assay than precolumn derivatization techniques.

The following Methods may be used for quantitative amino acid analysis. Instruments and reagents for these procedures are available commercially. Furthermore, many modifications of these methodologies exist with different reagent preparations, reaction procedures, and chromatographic systems. Specific parameters may vary according to the exact equipment and procedure used. Many laboratories will utilize more than one amino acid analysis technique to exploit the advantages offered by each. In each of these Methods, the analog signal is visualized by means of a data acquisition system, and the peak areas are integrated for quantification purposes.

Method 1—Postcolumn Ninhydrin Detection

Mobile Phase Preparation—

Postcolumn Reagent— Transfer about 18 g of ninhydrin and 0.7 g of hydrindantin to 900 mL of a solution containing 76.7% of dimethyl sulfoxide, 0.7% of dihydrate lithium acetate, and 0.1% of acetic acid, and mix for at least 3 hours under inert gas, such as nitrogen. [note—This reagent is stable for 30 days if kept between 2 and 8 under inert gas.]

Buffer Solution— Prepare a solution containing 2% of anhydrous sodium citrate, 1% of hydrochloric acid, 0.5% of thiodiglycol, and 0.1% of benzoic acid in water, and adjust to a pH of 2.

Chromatographic System— The liquid chromatograph is equipped with a detector with appropriate interference filters at 440, 570, or 690 nm and a 4.0-mm × 120-mm column that contains 7.5-µm sulfonated styrene-divinylbenzene copolymer packing. The flow rate is about 14 mL per hour. The system is programmed as follows. Initially equilibrate the column with Solution A; at 25 minutes, the composition of the Mobile Phase is changed to 100% Solution B; and at 37 minutes, the composition is changed to 100% Solution C. At 75 minutes into the run, the last amino acid has been eluted from the column, and the column is regenerated with Column Regeneration Solution for 1 minute. The column is then equilibrated with Solution A for 11 minutes before the next injection. The column temperature is programmed as follows. The initial temperature is 48; after 11.5 minutes, the temperature is increased to 65 at a rate of 3 per minute; at about 35 minutes, the temperature is increased to 77 at a rate of 3 per minute; and finally at about 52 minutes, the temperature is decreased to 48 at a rate of 3 per minute.

Procedure and Postcolumn Reaction— Reconstitute the lyophilized protein/peptide hydrolysate in the Buffer Solution, inject an appropriate amount into the chromatograph, and proceed as directed for Chromatographic System. As the amino acids are eluted from the column, they are mixed with the Postcolumn Reagent, which is delivered at a flow rate of 7 mL per hour, through a tee. After mixing, the column effluent and the Postcolumn Reagent pass through a tubular reactor at a temperature of 135, where a characteristic purple or yellow color is developed. From the reactor, the liquid passes through a colorimeter with a 12-mm flow-through cuvette. The light emerging from the cuvette is split into three beams for analysis by the detector with interference filters at 440, 570, or 690 nm. The 690-nm signal may be electronically subtracted from the other signals for improved signal-to-noise ratios. The 440-nm (imino acids) and the 570-nm (amino acids) signals may be added in order to simplify data handling.

Method 2—Postcolumn OPA Fluorometric Derivatization

Mobile Phase Preparation—

Postcolumn Reagent Preparation—

Chromatographic System— The liquid chromatograph is equipped with a fluorometric detector set to an excitation wavelength of 348 nm and an emission wavelength of 450 nm and a 4.0-mm × 150-mm column that contains 7.5-µm packing L17. The flow rate is about 0.3 mL per minute, and the column temperature is set at 50. The system is programmed as follows. The column is equilibrated with Solution A; over the next 20 minutes, the composition of the Mobile Phase is changed linearly to 85% Solution A and 15% Solution B; then there is a step change to 40% Solution A and 60% Solution B; over the next 18 minutes, the composition is changed linearly to 100% Solution B and held for 7 minutes; then there is a step change to 100% Solution C, and this is held for 6 minutes; then there is a step change to Solution A, and this composition is maintained for the next 8 minutes.

Procedure and Postcolumn Reaction— Inject about 1.0 nmol of each amino acid under test into the chromatograph, and proceed as directed for Chromatographic System. As the effluent leaves the column, it is mixed with the Hypochlorite Reagent. The mixture passes through the first postcolumn reactor which consists of stainless steel 0.5-mm × 2-m tubing. A second postcolumn reactor of similar design is placed immediately downstream from the first postcolumn reactor and is used for the OPA postcolumn reaction. The flow rates for both the Hypochlorite Reagent and the OPA Reagent are 0.2 mL per minute, resulting in a total flow rate (i.e., Hypochlorite Reagent, OPA Reagent, and column effluent) of 0.7 mL per minute exiting from the postcolumn reactors. Postcolumn reactions are conducted at 55. This results in a residence time of about 33 seconds in the OPA postcolumn reactor. After postcolumn derivatization, the column effluent passes through the fluorometric detector.

Method 3—Precolumn Determination

Mobile Phase Preparation—

Derivatization Reagent Preparation—

Sample Derivatization Procedure— Dissolve the lyophilized test sample in 100 µL of the Coupling Buffer, and then dry in a vacuum centrifuge to remove any hydrochloride if a protein hydrolysis step was used. Dissolve the test sample in 100 µL of Coupling Buffer, add 5 µL of PITC, and incubate at room temperature for 5 minutes. The test sample is again dried in a vacuum centrifuge, and is dissolved in 250 µL of Sample Solvent.

Chromatographic System— The liquid chromatograph is equipped with a 254-nm detector and a 4.6-mm × 250-mm column that contains 5-µm packing L1. The flow rate is about 1 mL per minute, and the column temperature is maintained at 52. The system is programmed as follows. The column is equilibrated with Solution A; over the next 15 minutes, the composition of the Mobile Phase is changed linearly to 85% Solution A and 15% Solution B; over the next 15 minutes, the composition is changed linearly to 50% Solution A and 50% Solution B; then there is a step change to 100% Solution C, and this is held for 10 minutes; then there is a step change to 100% Solution A, and the column is allowed to equilibrate before the next injection.

Procedure— Inject about 1.0 nmol of each PITC-amino acid under test (10-µL sample in Sample Solvent) into the chromatograph, and proceed as directed for Chromatographic System.

Method 4—Precolumn AQC Derivatization

Mobile Phase Preparation—

Sample Derivatization Procedure— Dissolve about 2 µg of the test sample in 20 µL of 15 mM hydrochloric acid, and dilute with 0.2 M borate buffer (pH 8.8) to 80 µL. The derivatization is initiated by the addition of 20 µL of 10 mM AQC in acetonitrile, and allowed to proceed for 10 minutes at room temperature.

Chromatographic System— The liquid chromatograph is equipped with a fluorometric detector set at an excitation wavelength of 250 nm and an emission wavelength of 395 nm and a 3.9-mm × 150-mm column that contains 4-µm packing L1. The flow rate is about 1 mL per minute, and the column temperature is maintained at 37. The system is programmed as follows. The column is equilibrated with Solution A; over the next 0.5 minute, the composition of the Mobile Phase is changed linearly to 98% Solution A and 2% Solution B; then over the next 14.5 minutes to 93% Solution A and 7% Solution B; then over the next 4 minutes to 87% Solution A and 13% Solution B; over the next 14 minutes to 68% Solution A and 32% Solution B; then there is a step change to 100% Solution B for a 5-minute wash; over the next 10 minutes, there is a step change to 100% Solution A; and the column is allowed to equilibrate before the next injection.

Procedure— Inject about 0.05 nmol of each AQC-amino acid under test into the chromatograph, and proceed as directed for Chromatographic System.

Method 5—Precolumn OPA Derivatization

Mobile Phase Preparation—

Derivatization Reagent— Dissolve 50 mg of OPA in 1.25 mL of methanol (protein sequencing grade). Add 50 µL of 2-mercaptoethanol and 11.2 mL of 0.4 M sodium borate (pH 9.5), and mix. [note—This reagent is stable for 1 week.]

Sample Derivatization Procedure— Transfer about 5 µL of the test sample to an appropriate container, add 5 µL of the Derivatization Reagent, and mix. After 1 minute, add not less than 20 µL of 0.1 M sodium acetate (pH 7.0). Use 20 µL of this solution for analysis. [note—Use of an internal standard (e.g., norleucine) is recommended for quantitative analysis because of potential reagent volume variations in the sample derivatization. The sample derivatization is performed in an automated on-line fashion. Because of the instability of the OPA-amino acid derivative, HPLC separation and analysis are performed immediately following derivatization.]

Chromatographic System— The liquid chromatograph is equipped with a fluorometric detector set at an excitation wavelength of 348 nm and an emission wavelength of 450 nm and a 4.6-mm × 75-mm column that contains 3-µm packing L3. The flow rate is about 1.7 mL per minute, and the column temperature is maintained at 37. The system is programmed as follows. The column is equilibrated with 92% Solution A and 8% Solution B; over the next 2 minutes, the composition of the Mobile Phase is changed to 83% Solution A and 17% Solution B, and held for an additional 3 minutes; then changed to 54% Solution A and 46% Solution B over the next 5 minutes, and held for an additional 2 minutes; then changed to 34% Solution A and 66% Solution B over the next 2 minutes, and held for 1 minute; then over the next 0.3 minute changed to 20% Solution A and 80% Solution B, and held for an additional 2.6 minutes; and then finally over 0.6 minute changed to 92% Solution A and 8% Solution B, and held for an additional 0.6 minute.

Procedure— Inject about 0.02 nmol of each OPA-amino acid under test into the chromatograph, and proceed as directed for Chromatographic System.

Method 6—Postcolumn DABS-Cl Derivatization

Mobile Phase Preparation—

Derivatization Reagent Preparation—

Sample Derivatization Procedure— Dissolve the test sample in 20 µL of Sample Buffer, add 40 µL of Derivatization Reagent, and mix. The sample container is sealed with a silicon-rubber stopper, and heated to 70 for 10 minutes. During the sample heating, the mixture will become completely soluble. After the derivatization, dilute the test sample with an appropriate quantity of the Sample Dilution Buffer.

Chromatographic System— The liquid chromatograph is equipped with a 436-nm detector and a 4.6-mm × 250-mm column that contains packing L1. The flow rate is about 1 mL per minute, and the column temperature is maintained at 40. The system is programmed as follows. The column is equilibrated with 85% Solution A and 15% Solution B; over the next 20 minutes, the composition of the Mobile Phase is changed to 60% Solution A and 40% Solution B; over the next 12 minutes, the composition is changed to 30% Solution A and 70% Solution B, and held for an additional 2 minutes.

Procedure— Inject about 0.05 nmol of the DABS-amino acids into the chromatograph, and proceed as directed for Chromatographic System.

Method 7—Precolumn FMOC-Cl Derivatization

Mobile Phase Preparation—

Derivatization Reagent Preparation—

Sample Derivatization Procedure— To 0.4 mL of the test sample add 0.1 mL of Borate Buffer and 0.5 mL of FMOC-Cl Reagent. After about 40 seconds, extract the mixture with 2 mL of pentane, and then extract again with fresh pentane. The aqueous solution with amino acid derivatives is then ready for injection.

Chromatographic System— The liquid chromatograph is equipped with a fluorometric detector set at an excitation wavelength of 260 nm and an emission wavelength of 313 nm and a 4.6-mm × 125-mm column that contains 3-µm packing L1. The flow rate is about 1.3 mL per minute. The system is programmed as follows. The column is equilibrated with Solution A, and this composition is maintained for 3 minutes; over the next 9 minutes, it is changed to 100% Solution B; then over the next 0.5 minute, the flow rate is increased to 2 mL per minute, and held until the final FMOC-amino acid is eluted from the column. The total run time is about 20 minutes.

Procedure— Inject not less than 0.01 nmol of each FMOC-amino acid under test into the chromatograph, and proceed as directed for Chromatographic System. The FMOC-histidine derivative will generally give a lower response than the other derivatives.

Method 8—Precolumn NBD-F Derivatization

Mobile Phase Preparation—

Derivatization Reagent Preparation—

Sample Derivatization Procedure— Dissolve the test sample in 20 µL of Sample buffer, add 10 µL of Derivatization Reagent, and mix. The sample container is heated at 60 for 5 minutes. After the derivatization, dilute the test sample with 300 µL of Solution A.

Chromatographic System— The liquid chromatograph is equipped with a fluorometric detector set at an excitation wavelength of 480 nm and an emission wavelength of 530 nm and a 4.6-mm × 150-mm column that contains 5-µm particle size ODS silica packing. The flow rate is about 1.0 mL per minute, and the column temperature is maintained at 40. The system is programmed as follows. The column is equilibrated with 94% Solution A and 6% Solution B; over the next 16 minutes, the composition is changed linearly to 63% Solution A and 37% Solution B; over the next 5 minutes, the composition is changed linearly to 62% Solution A and 38% Solution B; over the next 9 minutes, the composition is changed linearly to 100% Solution B, and held for an additional 5 minutes; then finally over 2 minutes, the composition is changed linearly to 94% Solution A and 6% Solution B; and then the column is allowed to equilibrate before the next injection.

Procedure— Inject about 15 pmol of each NBD-amino acid under test into the chromatograph, and proceed as directed for Chromatographic System.

When determining the amino acid content of a protein/peptide hydrolysate, it should be noted that the acid hydrolysis step destroys tryptophan and cysteine. Serine and threonine are partially destroyed by acid hydrolysis, while isoleucine and valine residues may be only partially cleaved. Methionine can undergo oxidation during acid hydrolysis, and some amino acids (e.g., glycine and serine) are common contaminants. Application of adequate vacuum (less than 200 µm of mercury or 26.7 Pa) or introduction of inert gas (argon) in the headspace of the reaction vessel during vapor phase hydrolysis can reduce the level of oxidative destruction. Therefore, the quantitative results obtained for cysteine, tryptophan, threonine, isoleucine, valine, methionine, glycine, and serine from a protein/peptide hydrolysate may be variable and may warrant further investigation and consideration.

Calculations

Amino Acid Mole Percent— This is the number of specific amino acid residues per 100 residues in a protein. This result may be useful for evaluating amino acid analysis data when the molecular weight of the protein/peptide under investigation is unknown. This information can be used to corroborate the identity of a protein and has other applications. Carefully identify and integrate the peaks obtained as directed for each Procedure. Calculate the mole percent for each amino acid present in the test sample by the formula: in which rU is the peak response, in nmol, of the amino acid under test; and r is the sum of peak responses, in nmol, for all amino acids present in the test sample. Comparison of the mole percent of the amino acids under test to data from known proteins can help establish or corroborate the identity of the sample protein.

Unknown Protein Samples— This data analysis technique can be used to estimate the protein concentration of an unknown protein sample using the amino acid analysis data. Calculate the mass, in µg, of each recovered amino acid by the formula: in which m is the recovered quantity, in nmol, of the amino acid under test; and MW is the average molecular weight, in mg, for that amino acid, corrected for the weight of the water molecule that was eliminated during peptide bond formation. The sum of the masses of the recovered amino acids will give an estimate of the total mass of the protein analyzed after appropriate correction for partially and completely destroyed amino acids. If the molecular weight of the unknown protein is available (i.e., by SDS-PAGE analysis or mass spectroscopy), the amino acid composition of the unknown protein can be predicted. Calculate the number of residues of each amino acid by the formula: in which m is the recovered quantity, in nmol, of the amino acid under test; M is the total mass, in µg, of the protein; and MWT is the molecular weight, in mg, of the unknown protein.

Known Protein Samples— This data analysis technique can be used to investigate the amino acid composition and protein concentration of a protein sample of known molecular weight and amino acid composition using the amino acid analysis data. When the composition of the protein being analyzed is known, one can exploit the fact that some amino acids are recovered well, while other amino acid recoveries may be compromised because of complete or partial destruction (e.g., tryptophan, cysteine, threonine, serine, methionine), incomplete bond cleavage (i.e., for isoleucine and valine), and free amino acid contamination (i.e., by glycine and serine).