Appendix III L. Amino Acid Analysis¹

(Ph. Eur. method 2.2.56)

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 organised 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 proteins 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 hydrolyse 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 derivatised 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 post-column derivatisation capability, unless the sample is analysed using precolumn derivatisation. The detector is usually an ultraviolet/visible or fluorescence detector depending on the derivatisation method used. A recording device (e.g., integrator) is used for transforming the analogue 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 analysed 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 analysing 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 analysed. 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.

4 to 6 amino acid standard levels are analysed to determine a response factor for each amino acid. The response factor is calculated as the average peak area or peak height per nanomole of amino acid present in the standard. A calibration file consisting of the response factor for each amino acid is prepared and 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 nanomoles 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 correspond 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 analysing many replicates (e.g., 6 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. The amino acid composition of a standard protein (e.g., bovine serum albumin) is often analysed 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 quality of reagents and/or 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 utilise post-column derivatisation of the amino acids are generally not affected by buffer components to the extent seen with pre-column derivatisation methods. It is desirable to limit the number of sample manipulations to reduce potential background contamination, to improve analyte recovery, and to reduce labour. Common techniques used to remove buffer components from protein samples include the following methods: (1) injecting the protein sample onto a reversed-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); (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 of the protein solution. Free amino acids, however, do not behave in the same way as protein-bound amino acids during hydrolysis, whose 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 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 α-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 h in 1 M hydrochloric acid or soak tubes in concentrated nitric acid or in a mixture of equal volumes of concentrated hydrochloric acid and nitric acid. 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 °C for 4 h may also be used to eliminate contamination from hydrolysis tubes. Adequate disposable laboratory material can also be used.

Acid hydrolysis is the most common method for hydrolysing 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 h, 48 h and 72 h) is often employed to analyse the starting concentration of amino acids that are partially destroyed or slow to cleave. By plotting the observed concentration of labile amino acids (e.g., 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 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 one 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.

During initial analyses of an unknown protein, experiments with various hydrolysis time and temperature conditions are conducted to determine the optimal conditions.

Acid hydrolysis using hydrochloric acid containing phenol is the most common procedure used for protein/peptide hydrolysis preceding amino acid analysis. The addition of phenol to the reaction prevents the halogenation of tyrosine.

Hydrolysis solution 6 M hydrochloric acid containing 0.1 per cent to 1.0 per cent of phenol.

Procedure

Liquid phase hydrolysis Place the protein or peptide sample in a hydrolysis tube, and dry (the sample is dried so that water in the sample will not dilute the acid used for the hydrolysis). Add 200 µL of hydrolysis solution per 500 µg of lyophilised protein. Freeze the sample tube in a dry ice-acetone bath, and flame seal in vacuo. Samples are typically hydrolysed at 110 °C for 24 h in vacuo or in an inert atmosphere to prevent oxidation. Longer hydrolysis times (e.g., 48 h and 72 h) are investigated if there is a concern that the protein is not completely hydrolysed.

Vapour phase hydrolysis This is one of the most common acid hydrolysis procedures, and it is preferred for microanalysis when only small amounts of the sample are available. Contamination of the sample from the acid reagent is also minimised by using vapour phase hydrolysis. Place vials containing the dried samples in a vessel that contains an appropriate amount of hydrolysis solution. The hydrolysis solution does not come in contact with the test sample. Apply an inert atmosphere or vacuum (less than 200 µm of mercury or 26.7 Pa) to the headspace of the vessel, and heat to about 110 °C for a 24 h hydrolysis time. Acid vapour hydrolyses the dried sample. Any condensation of the acid in the sample vials is to be minimised. After hydrolysis, dry the test sample in vacuo to remove any residual acid.

Tryptophan oxidation during hydrolysis is decreased by using mercaptoethanesulfonic acid as the reducing acid.

Hydrolysis solution 2.5 M mercaptoethanesulfonic acid solution.

Vapour phase hydrolysis Dry about 1 µg to 100 µg of the protein/peptide under test in a hydrolysis tube. Place the hydrolysis tube in a larger tube with about 200 µL of the hydrolysis solution. Seal the larger tube in vacuo (about 50 µm of mercury or 6.7 Pa) to vaporise the hydrolysis solution. Heat the hydrolysis tube to 170-185 °C for about 12.5 min. After hydrolysis, dry the hydrolysis tube in vacuo for 15 min to remove the residual acid.

Tryptophan oxidation during hydrolysis is prevented by using thioglycollic acid (TGA) as the reducing acid.

Hydrolysis solution 7 M hydrochloric acid containing 1 per cent of phenol, 10 per cent of trifluoroacetic acid and 20 per cent of thioglycollic acid.

Vapour phase hydrolysis Dry about 10 µg to 50 µg of the protein/peptide under test in a sample tube. Place the sample tube in a larger tube with about 200 µL of the hydrolysis solution. Seal the larger tube in vacuo (about 50 µm of mercury or 6.7 Pa) to vaporise the TGA. Heat the sample tube to 166 °C for about 15-30 min. After hydrolysis, dry the sample tube in vacuo for 5 min to remove the residual acid. Recovery of tryptophan by this method may be dependent on the amount of sample present.

Cysteine/cystine and methionine oxidation is performed with performic acid before the protein hydrolysis.

Oxidation solution Use performic acid freshly prepared by mixing 1 volume of hydrogen peroxide solution (30 per cent) and 9 volumes of anhydrous formic acid and incubating at room temperature for 1 h.

Procedure Dissolve the protein/peptide sample in 20 µL of anhydrous formic acid and heat at 50 °C for 5 min; then add 100 µL of the oxidation solution. Allow the oxidation to proceed for 10-30 min. In this reaction, cysteine is converted to cysteic acid and methionine is converted to methionine-sulfone. Remove the excess reagent from the sample in a vacuum centrifuge. The oxidised protein can then be acid hydrolysed using Method 1 or Method 2. This technique may cause modifications to tyrosine residues in the presence of halides.

Cysteine/cystine oxidation is accomplished during the liquid phase hydrolysis with sodium azide.

Hydrolysis solution To 6 M hydrochloric acid containing 0.2 per cent of phenol, add sodium azide to obtain a final concentration of 2 g/L. The added phenol prevents halogenation of tyrosine.

Liquid phase hydrolysis Conduct the protein/peptide hydrolysis at about 110 °C for 24 h. 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 2 oxidative products, methionine-sulfoxide and methionine-sulfone.

Cysteine/cystine oxidation is accomplished with dimethyl sulfoxide (DMSO).

Hydrolysis solution To 6 M hydrochloric acid containing 0.1 per cent to 1.0 per cent of phenol, add dimethyl sulfoxide to obtain a final concentration of 2 per cent V/V.

Vapour phase hydrolysis Conduct the protein/peptide hydrolysis at about 110 °C for 24 h. 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 compensate for partial destruction, it is recommended to evaluate the cysteic acid recovery from oxidative hydrolysis of standard proteins containing 1-8 mol of cysteine. The response factors from protein/peptide hydrolysates are typically about 30 per cent lower than those for non-hydrolysed cysteic acid standards. Because histidine, methionine, tyrosine, and tryptophan are also modified, a complete compositional analysis is not obtained with this technique.

Cysteine/cystine reduction and alkylation is accomplished by a vapour phase pyridylethylation reaction.

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 vacuo (about 50 µm of mercury or 6.7 Pa), and heat at about 100 °C for 5 min. Then remove the inner hydrolysis tube, and dry it in a vacuum desiccator for 15 min to remove residual reagents. The pyridylethylated sample can then be acid hydrolysed using previously described procedures. The pyridylethylation reaction is performed simultaneously with a protein standard sample containing 1-8 mol of cysteine to evaluate the pyridylethyl-cysteine recovery. Longer incubation times for the pyridylethylation reaction can cause modifications to the α-amino terminal group and the ∊-amino group of lysine in the protein.

Cysteine/cystine reduction and alkylation is accomplished by a liquid phase pyridylethylation reaction.

Stock solutions Prepare and filter 3 solutions: 1 M Tris-hydrochloride pH 8.5 containing 4 mM disodium edetate (stock solution A), 8 M guanidine hydrochloride (stock solution B), and 10 per cent of 2-mercaptoethanol (stock solution C).

Reducing solution Prepare a mixture of 1 volume of stock solution A and 3 volumes of stock solution B 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 C. Store under nitrogen or argon for 2 h 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 h at room temperature in the dark. Desalt the protein/peptide by collecting the protein/peptide fraction from a reversed-phase HPLC separation. The collected sample can be dried in a vacuum centrifuge before acid hydrolysis.

Cysteine/cystine reduction and alkylation is accomplished by a liquid phase carboxymethylation reaction.

Stock solutions Prepare as directed for Method 8.

Carboxymethylation solution Prepare a 100 g/L solution of iodoacetamide in alcohol.

Buffer solution Use the reducing solution, prepared as described for Method 8.

Procedure Dissolve the test sample in 50 µL of the buffer solution, and add about 2.5 µL of stock solution C. Store under nitrogen or argon for 2 h at room temperature in the dark. Add the carboxymethylation solution in a ratio 1.5 fold per total theoretical content of thiols, and incubate for an additional 30 min at room temperature in the dark. 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. Desalt the protein/peptide by collecting the protein/peptide fraction from a reversed-phase HPLC separation. The collected sample can be dried in a vacuum centrifuge before acid hydrolysis. The S-carboxyamidomethyl-cysteine formed will be converted to S-carboxymethyl-cysteine during acid hydrolysis.

Cysteine/cystine is reacted with dithiodiglycolic acid or dithiodipropionic acid to produce a mixed disulfide. The choice of dithiodiglycolic acid or dithiodipropionic acid depends on the required resolution of the amino acid analysis method.

Reducing solution A 10 g/L solution of dithiodiglycolic acid (or dithiodipropionic acid) in 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 hydrolysed using Method 1. This method has the advantage that other amino acid residues are not derivatised by side reactions, and that the sample does not need to be desalted prior to hydrolysis.

Asparagine and glutamine are converted to aspartic acid and glutamic acid, respectively, during acid hydrolysis. Asparagine and aspartic acid residues are added and represented by Asx, while glutamine and glutamic acid residues are added and represented by Glx. Proteins/peptides can be reacted with bis(1,1-trifluoroacetoxy)iodobenzene (BTI) to convert the asparagine and glutamine residues to diaminopropionic acid and diaminobutyric acid residues, respectively, upon acid hydrolysis. These conversions allow the analyst to determine the asparagine and glutamine content of a protein/peptide in the presence of aspartic acid and glutamic acid residues.

Reducing solutions Prepare and filter 3 solutions: a solution of 10 mM trifluoroacetic acid (Solution A), a solution of 5 M guanidine hydrochloride and 10 mM trifluoroacetic acid (Solution B), and a freshly prepared solution of dimethylformamide containing 36 mg of BTI per millilitre (Solution C).

Procedure In a clean hydrolysis tube, transfer about 200 µg of the test sample, and add 2 mL of Solution A or Solution B and 2 mL of Solution C. Seal the hydrolysis tube in vacuo. Heat the sample at 60 °C for 4 h in the dark. The sample is then dialysed with water to remove the excess reagents. Extract the dialysed sample 3 times with equal volumes of butyl acetate, and then lyophilise. The protein can then be acid hydrolysed using previously described procedures. The α,β-diaminopropionic and α,γ-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 content assayed with underivatised and BTI-derivatised acid hydrolysis. The threonine, methionine, cysteine, tyrosine, and histidine assayed content can be altered by BTI derivatisation; a hydrolysis without BTI will have to be performed if the analyst is interested in the composition of these other amino acid residues of the protein/peptide.

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 post-column derivatisation (e.g., with ninhydrin or o-phthalaldehyde). Post-column derivatisation techniques can be used with samples that contain small amounts of buffer components, (such as salts and urea) and generally require between 5 µg and 10 µg of protein sample per analysis. The remaining amino acid techniques typically involve pre-column derivatisation of the free amino acids (e.g., phenyl isothiocyanate; 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate or o-phthalaldehyde; (dimethylamino)azobenzenesulfonyl chloride; 9-fluorenylmethyl chloroformate; and 7-fluoro-4-nitrobenzo-2-oxa-1,3-diazole) followed by reversed-phase HPLC. Pre-column derivatisation techniques are very sensitive and usually require between 0.5 µg and 1.0 µg of protein sample per analysis but may be influenced by buffer salts in the samples. Pre-column derivatisation techniques may also result in multiple derivatives of a given amino acid, which complicates the result interpretation. Post-column derivatisation techniques are generally influenced less by performance variation of the assay than pre-column derivatisation 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, chromatographic systems, etc. Specific parameters may vary according to the exact equipment and procedure used. Many laboratories will use more than one amino acid analysis technique to exploit the advantages offered by each. In each of these methods, the analogue signal is visualised by means of a data acquisition system, and the peak areas are integrated for quantification purposes.

Ion-exchange chromatography with post-column ninhydrin derivatisation is one of the most common methods employed for quantitative amino acid analysis. As a rule, a lithium-based cation-exchange system is employed for the analysis of the more complex physiological samples, and the faster sodium-based cation-exchange system is used for the more simplistic amino acid mixtures obtained with protein hydrolysates (typically containing 17 amino acid components). Separation of the amino acids on an ion-exchange column is accomplished through a combination of changes in pH and cation strength. A temperature gradient is often employed to enhance separation.

When the amino acid reacts with ninhydrin, the reactant has a characteristic purple or yellow colour. Amino acids, except imino acid, give a purple colour, and show an absorption maximum at 570 nm. The imino acids such as proline give a yellow colour, and show an absorption maximum at 440 nm. The post-column reaction between ninhydrin and amino acids eluted from the column is monitored at 440 nm and 570 nm, and the chromatogram obtained is used for the determination of amino acid composition.

The detection limit is considered to be 10 pmol for most of the amino acid derivatives, but 50 pmol for the proline derivative. Response linearity is obtained in the range of 20-500 pmol with correlation coefficients exceeding 0.999. To obtain good composition data, samples larger than 1 µg before hydrolysis are best suited for this amino acid analysis of protein/peptide.

o-Phthalaldehyde (OPA) reacts with primary amines in the presence of thiol compound, to form highly fluorescent isoindole products. This reaction is used for the post-column derivatisation in analysis of amino acids by ion-exchange chromatography. The rule of the separation is the same as Method 1.

Although OPA does not react with secondary amines (imino acids such as proline) to form fluorescent substances, the oxidation with sodium hypochlorite or chloramine T allows secondary amines to react with OPA. The procedure employs a strongly acidic cation-exchange column for separation of free amino acids followed by post-column oxidation with sodium hypochlorite or chloramine T and post-column derivatisation using OPA and a thiol compound such as N-acetyl-l-cysteine or 2-mercaptoethanol. The derivatisation of primary amino acids is not noticeably affected by the continuous supply of sodium hypochlorite or chloramine T.

Separation of the amino acids on an ion-exchange column is accomplished through a combination of changes in pH and cation strength. After post-column derivatisation of eluted amino acids with OPA, the reactant passes through the fluorometric detector. Fluorescence intensity of OPA-derivatised amino acids are monitored with an excitation wavelength of 348 nm and an emission wavelength of 450 nm.

The detection limit is considered to be a few tens of picomole level for most of the OPA-derivatised amino acids. Response linearity is obtained in the range of a few picomole level to a few tens of nanomole level. To obtain good compositional data, samples larger than 500 ng of protein/peptide before hydrolysis are recommended.

Phenylisothiocyanate (PITC) reacts with amino acids to form phenylthiocarbamyl (PTC) derivatives which can be detected with high sensitivity at 254 nm. Therefore, pre-column derivatisation of amino acids with PITC followed by a reversed-phase HPLC separation with UV detection is used to analyse the amino acid composition.

After the reagent is removed under vacuum, the derivatised amino acids can be stored dry and frozen for several weeks with no significant degradation. If the solution for injection is kept cold, no noticeable loss in chromatographic response occurs after 3 days.

Separation of the PTC-amino acids on a reversed-phase HPLC with an octadecylsilyl (ODS) column is accomplished through a combination of changes in concentrations of acetonitrile and buffer ionic strength. PTC-amino acids eluted from the column are monitored at 254 nm.

The detection limit is considered to be 1 pmol for most of the PTC-amino acids. Response linearity is obtained in the range of 20-500 pmol with correlation coefficients exceeding 0.999. To obtain good compositional data, samples larger than 500 ng of protein/peptide before hydrolysis are recommended.

Pre-column derivatisation of amino acids with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) followed by reversed-phase HPLC separation with fluorometric detection is used.

AQC reacts with amino acids to form stable, fluorescent unsymmetric urea derivatives (AQC-amino acids) which are readily amenable to analysis by reversed-phase HPLC. Therefore, pre-column derivatisation of amino acids with AQC followed by reversed-phase HPLC separation with fluorimetric detection is used to analyse the amino acid composition.

Separation of the AQC-amino acids on a reversed-phase HPLC with an ODS column is accomplished through a combination of changes in concentrations of acetonitrile and buffer ionic strengh. Selective fluorescence detection of the derivatives with an excitation wavelength at 250 nm and an emission wavelength at 395 nm allows for the direct injection of the reaction mixture with no significant interference from the only major fluorescent reagent by-product, 6-aminoquinoline. Excess reagent is rapidly hydrolysed (t_1/2<15 s) to yield 6-aminoquinoline, N-hydroxysuccinimide and carbon dioxide, and after 1 min no further derivatisation can take place.

Peak areas for AQC-amino acids are essentially unchanged for at least 1 week at room temperature. Therefore AQC-amino acids have more than sufficient stability to allow for overnight automated chromatographic analysis.

The detection limit is considered to range from about 40 fmol to 320 fmol for each amino acid, except for cystein. The detection limit for cystein is approximately 800 fmol. Response linearity is obtained in the range of 2.5-200 µM with correlation coefficients exceeding 0.999. Good compositional data can be obtained from the analysis of derivatised protein hydrolysates derived from as little as 30 ng of protein/peptide.

Pre-column derivatisation of amino acids with o-phthalaldehyde (OPA) followed by reversed-phase HPLC separation with fluorometric detection is used. This technique does not detect amino acids that exist as secondary amines (e.g., proline).

OPA in conjunction with a thiol reagent reacts with primary amine groups to form highly fluorescent isoindole products. 2-Mercaptoethanol or 3-mercaptopropionic acid can be used as the thiol. OPA itself does not fluoresce and consequently produces no interfering peaks. In addition, its solubility and stability in aqueous solution, along with the rapid kinetics for the reaction, make it amenable to automated derivatisation and analysis using an autosampler to mix the sample with the reagent. However, lack of reactivity with secondary amino acids has been a predominant drawback. This method does not detect amino acids that exist as secondary amines (e.g., proline). To compensate for this drawback, this technique may be combined with another technique described in Method 7 or Method 8.

Pre-column derivatisation of amino acids with OPA is followed by a reversed-phase HPLC separation. Because of the instability of the OPA-amino acid derivative, HPLC separation and analysis are performed immediately following derivatisation. The liquid chromatograph is equipped with a fluorometric detector for the detection of derivatised amino acids. Fluorescence intensity of OPA-derivatised amino acids is monitored with an excitation wavelength of 348 nm and an emission wavelength of 450 nm.

Detection limits as low as 50 fmol via fluorescence have been reported, although the practical limit of analysis remains at 1 pmol.

Pre-column derivatisation of amino acids with (dimethylamino)azobenzenesulfonyl chloride (DABS-Cl) followed by reversed-phase HPLC separation with visible light detection is used.

DABS-Cl is a chromophoric reagent employed for the labelling of amino acids. Amino acids labelled with DABS-Cl (DABS-amino acids) are highly stable and show an absorption maximum at 436 nm.

DABS-amino acids, all naturally occurring amino acid derivatives, can be separated on an ODS column of a reversed-phase HPLC by employing gradient systems consisting of acetonitrile and aqueous buffer mixture. Separated DABS-amino acids eluted from the column are detected at 436 nm in the visible region.

This method can analyse the imino acids such as proline together with the amino acids at the same degree of sensitivity, DABS-Cl derivatisation method permits the simultaneous quantification of tryptophan residues by previous hydrolysis of the protein/peptide with sulfonic acids such as mercaptoethanesulfonic acid, p-toluenesulfonic acid or methanesulfonic acid described in Method 2 under Protein hydrolysis. The other acid-labile residues, asparagine and glutamine, can also be analysed by previous conversion into diaminopropionic acid and diaminobutyric acid, respectively, by treatment of protein/peptide with BTI described in Method 11 under Protein hydrolysis.

The non-proteinogenic amino acid norleucine cannot be used as an internal standard in this method as this compound is eluted in a chromatographic region crowded with peaks of primary amino acids. Nitrotyrosine can be used as an internal standard because it is eluted in a clean region.

The detection limit of DABS-amino acid is about 1 pmol. As little as 2-5 pmol of an individual DABS-amino acid can be quantitatively analysed with reliability, and only 10-30 ng of the dabsylated protein hydrolysate is required for each analysis.

Pre-column derivatisation of amino acids with 9-fluorenylmethyl chloroformate (FMOC-Cl) followed by reversed-phase HPLC separation with fluorometric detection is used.

FMOC-Cl reacts with both primary and secondary amino acids to form highly fluorescent products. The reaction proceeds under mild conditions in aqueous solution and is completed in 30 s. The derivatives are stable, only the histidine derivative showing any breakdown. Although FMOC-Cl is fluorescent itself, the reagent excess and fluorescent side-products can be eliminated without loss of FMOC-amino acids.

FMOC-amino acids are separated by a reversed-phase HPLC using an ODS column. The separation is carried out by gradient elution varied linearly from a mixture of 10 volumes of acetonitrile, 40 volumes of methanol and 50 volumes of acetic acid buffer to a mixture of 50 volumes of acetonitrile and 50 volumes of acetic acid buffer and 20 amino acid derivatives are separated in 20 min. Each derivative eluted from the column is monitored by a fluorometric detector set at an excitation wavelength of 260 nm and an emission wavelength of 313 nm.

The detection limit is in the low femtomole range. A linearity range of 0.1-50 µM is obtained for most of the amino acids.

Pre-column derivatisation of amino acids with 7-fluoro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-F) followed by reversed-phase HPLC separation with fluorometric detection is used.

NBD-F reacts with both primary and secondary amino acids to form highly fluorescent products. Amino acids are derivatised with NBD-F by heating to 60 °C for 5 min.

NBD-amino acid derivatives are separated on an ODS column of a reversed-phase HPLC by employing a gradient elution system consisting of acetonitrile and aqueous buffer mixture, and 17 amino acid derivatives are separated in 35 min. ∊-Aminocaproic acid can be used as an internal standard, because it is eluted in a clean chromatographic region. Each derivative eluted from the column is monitored by a fluorometric detector set at an excitation wavelength of 480 nm and an emission wavelength of 530 nm.

The sensitivity of this method is almost the same as for the pre-column OPA derivatisation method (Method 5), excluding proline to which OPA is not reactive, and might be advantageous for NBD-F against OPA. The detection limit for each amino acid is about 10 fmol. Profile analysis can be achieved with about 1.5 mg of protein hydrolysates in the pre-column reaction mixture.

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 vapour 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.

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 mass of the protein under investigation is unknown. This information can be used to corroborate the identity of a protein/peptide 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 using the formula:

in which r_U is the peak response, in nanomoles, of the amino acid under test; and r is the sum of peak responses, in nanomoles, 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 micrograms, of each recovered amino acid using the formula:

in which m is the recovered quantity, in nanomoles, of the amino acid under test; and M_r is the average molecular mass for that amino acid, corrected for the mass 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 analysed after appropriate correction for partially and completely destroyed amino acids. If the molecular mass 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 using the formula:

in which m is the recovered quantity, in nanomoles, of the amino acid under test; M is the total mass, in micrograms, of the protein; and M_rt is the molecular mass 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 mass and amino acid composition using the amino acid analysis data. When the composition of the protein being analysed 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).

Because those amino acids that are recovered best represent the protein, these amino acids are chosen to quantify the amount of protein. Well-recovered amino acids are, typically, aspartate-asparagine, glutamate-glutamine, alanine, leucine, phenylalanine, lysine, and arginine. This list can be modified based on experience with one's own analysis system. Divide the quantity, in nanomoles, of each of the well-recovered amino acids by the expected number of residues for that amino acid to obtain the protein content based on each well-recovered amino acid. Average the protein content results calculated. The protein content determined for each of the well-recovered amino acids should be evenly distributed about the mean. Discard protein content values for those amino acids that have an unacceptable deviation from the mean. Typically greater than 5 per cent variation from the mean is considered unacceptable. Recalculate the mean protein content from the remaining values to obtain the protein content of the sample. Divide the content of each amino acid by the calculated mean protein content to determine the amino acid composition of the sample by analysis.

Calculate the relative compositional error, in percentage, using the formula:

in which m is the experimentally determined quantity, in nanomoles per amino acid residue, of the amino acid under test; and m_S is the known residue value for that amino acid. The average relative compositional error is the average of the absolute values of the relative compositional errors of the individual amino acids, typically excluding tryptophan and cysteine from this calculation. The average relative compositional error can provide important information on the stability of analysis run over time. The agreement in the amino acid composition between the protein sample and the known composition can be used to corroborate the identity and purity of the protein in the sample.

¹ This chapter has undergone pharmacopoeial harmonisation. See chapter 5.8. Pharmacopoeial harmonisation.Amino Acid Analysis