EUROPEAN PHARMACOPOEIA 6.0

used that employ mathematical formulas, models, andpattern recognition. Such approaches are, for example, theautomated identification of compounds by IR spectroscopy
and the application of diode-array UV spectral analysis for
identification of peptides. These methods have limitations
due to inadequate resolutions, co-elution of fragments,
or absolute peak response differences between reference
substance and sample digest fragments.
The numerical comparison of the peak retention times
and peak areas or peak heights can be done for a selectedgroup of relevant peaks that have been correctly identified
in the peptide maps. Peak areas can be calculated using
1 peak showing relatively small variation as an internalreference, keeping in mind that peak area integration issensitive to baseline variation and likely to introduce error
in the analysis. Alternatively, the percentage of each peptide
peak height relative to the sum of all peak heights can becalculated for the sample under test. The percentage is thencompared to that of the corresponding peak of the reference
substance. The possibility of auto-hydrolysis of trypsin ismonitored by producing a blank peptide map, that is, thepeptide map obtained when a blank solution is treated withtrypsin.
The minimum requirement for the qualification of peptide
mapping is an approved test procedure that includes system
suitability as a test control. In general, early in the regulatory
process, qualification of peptide mapping for a protein issufficient. As the regulatory approval process for the protein
progresses, additional qualifications of the test can include
a partial validation of the analytical procedure to provide
assurance that the method will perform as intended in thedevelopment of a peptide map for the specified protein.
ANALYSIS AND IDENTIFICATION OF PEPTIDESThis section gives guidance on the use of peptide mapping
during development in support of regulatory applications.
The use of a peptide map as a qualitative tool does not
require the complete characterisation of the individual
peptide peaks. However, validation of peptide mappingin support of regulatory applications requires rigorous
characterisation of each of the individual peaks in the
peptide map. Methods to characterise peaks range fromN-terminal sequencing of each peak followed by amino acidanalysis to the use of mass spectroscopy (MS).
For characterisation purposes, when N-terminal sequencing
and amino acids analysis are used, the analytical separationis scaled up. Since scale-up might affect the resolution of
peptide peaks, it is necessary, using empirical data, to assurethat there is no loss of resolution due to scale-up. Eluatescorresponding to specific peptide peaks are collected,
vacuum-concentrated, and chromatographed again, if
necessary. Amino acid analysis of fragments may be limitedby the peptide size. If the N-terminus is blocked, it may need
to be cleared before sequencing. C-terminal sequencing
of proteins in combination with carboxypeptidase andmatrix-assisted laser desorption ionisation coupled totime-of-flight analyser (MALDI-TOF) can also be used forcharacterisation purposes.
The use of MS for characterisation of peptide fragments
is by direct infusion of isolated peptides or by the use of
on-line LC-MS for structure analysis. In general, it includes
electrospray and MALDI-TOF-MS, as well as fast-atombombardment (FAB). Tandem MS has also been used tosequence a modified protein and to determine the type ofamino acid modification that has occurred. The comparisonof mass spectra of the digests before and after reduction
provides a method to assign the disulphide bonds to thevarious sulphydryl-containing peptides.
General Notices (1) apply to all monographs and other texts

2.2.56. Amino acid analysis

If regions of the primary structure are not clearly
demonstrated by the peptide map, it might be necessary todevelop a secondary peptide map. The goal of a validated
method of characterisation of a protein through peptide
mapping is to reconcile and account for at least 95 per centof the theoretical composition of the protein structure.
01/2008:20256

2.2.56. AMINO ACID ANALYSIS
Amino acid analysis refers to the methodology used todetermine the amino acid composition or content ofproteins, peptides, and other pharmaceutical preparations.
Proteins and peptides are macromolecules consisting ofcovalently 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 proteinsor peptides based on their amino acid composition, tosupport 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 toits individual amino acid constituents before amino acidanalysis. Following protein/peptide hydrolysis, the aminoacid 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 typicallyderivatised for analysis.
APPARATUS
Methods used for amino acid analysis are usually based ona chromatographic separation of the amino acids present
in the test sample. Current techniques take advantage ofthe automated chromatographic instrumentation designed
for analytical methodologies. An amino acid analysis
instrument will typically be a low-pressure or high-pressureliquid chromatograph capable of generating mobile phasegradients that separate the amino acid analytes on achromatographic column. The instrument must havepost-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 preferredthat instrumentation be dedicated particularly for aminoacid analysis.
GENERAL PRECAUTIONS
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 cancontribute to glycine contamination). Analytical reagents arechanged routinely every few weeks using only high-pressureliquid 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 aminoacid analysis. Place the instrumentation in a low traffic areaof the laboratory. Keep the laboratory clean. Clean and89

2.2.56. Amino acid analysis

EUROPEAN PHARMACOPOEIA 6.0

calibrate pipets according to a maintenance schedule. Keeppipet tips in a covered box ; the analysts may not handle pipet
tips with their hands. The analysts may wear powder-freelatex or equivalent gloves. Limit the number of times a test
sample vial is opened and closed because dust can contributeto elevated levels of glycine, serine, and alanine.
A well-maintained instrument is necessary for acceptableamino acid analysis results. If the instrument is used ona routine basis, it is to be checked daily for leaks, detector
and lamp stability, and the ability of the column to maintainresolution of the individual amino acids. Clean or replace all
instrument filters and other maintenance items on a routineschedule.
REFERENCE MATERIAL
Acceptable amino acid standards are commercially available
for amino acid analysis and typically consist of an aqueous
mixture of amino acids. When determining amino acidcomposition, protein or peptide standards are analysed withthe test material as a control to demonstrate the integrity ofthe entire procedure. Highly purified bovine serum albuminhas been used as a protein standard for this purpose.
CALIBRATION OF INSTRUMENTATION
Calibration of amino acid analysis instrumentation typicallyinvolves analysing the amino acid standard, which consistsof a mixture of amino acids at a number of concentrations, todetermine the response factor and range of analysis for eachamino acid. The concentration of each amino acid in thestandard is known. In the calibration procedure, the analyst
dilutes the amino acid standard to several different analytelevels within the expected linear range of the amino acidanalysis technique. Then, replicates at each of the different
analyte levels can be analysed. Peak areas obtained for eachamino acid are plotted versus the known concentration foreach of the amino acids in the standard dilution. These
results will allow the analyst to determine the range of aminoacid concentrations where the peak area of a given aminoacid is an approximately linear function of the amino acidconcentration. It is important that the analyst prepare thesamples for amino acid analysis so that they are within theanalytical limits (e.g., linear working range) of the techniqueemployed 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 pernanomole of amino acid present in the standard. A calibration
file consisting of the response factor for each amino acid isprepared and used to calculate the concentration of eachamino 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 togive the nanomoles of the amino acid. For routine analysis,a single-point calibration may be sufficient ; however, thecalibration file is updated frequently and tested by the
analysis of analytical controls to ensure its integrity.

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 andintegrated 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 fromstarting 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 analyticallimits to ensure that the analyses from the laboratory are
under control. It is desirable to establish the lowest practicalvariation 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 thevalidation work.
SAMPLE PREPARATION
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 theextent seen with pre-column derivatisation methods. It is
desirable to limit the number of sample manipulations to
reduce potential background contamination, to improveanalyte 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 theprotein 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 orwater ; (4) precipitating the protein from the buffer using anorganic solvent (e.g., acetone) ; (5) gel filtration.
INTERNAL STANDARDS

It is recommended that an internal standard be used tomonitor physical and chemical losses and variations during
amino acid analysis. An accurately known amount ofinternal 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 thesame 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 duringhydrolysis may give unreliable results. It will be necessary
to take this point into consideration when interpreting the
REPEATABILITY
results. Internal standards can also be added to the mixtureof amino acids after hydrolysis to correct for differences in
Consistent high quality amino acid analysis results from ananalytical laboratory require attention to the repeatability of sample application and changes in reagent stability andthe assay. During analysis of the chromatographic separation flow rates. Ideally, an internal standard is an unnaturallyof the amino acids or their derivatives, numerous peaks can occurring primary amino acid that is commercially available
and inexpensive. It should also be stable during hydrolysis,
be observed on the chromatogram that correspond to theamino acids. The large number of peaks makes it necessary its response factor should be linear with concentration,and it needs to elute with a unique retention time without
to have an amino acid analysis system that can repeatedly
overlapping other amino acids. Commonly used amino
identify the peaks based on retention time and integrateacid standards include norleucine, nitrotyrosine, andthe peak areas for quantitation. A typical repeatability
evaluation involves preparing a standard amino acid solution α-aminobutyric acid.
90

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EUROPEAN PHARMACOPOEIA 6.0

2.2.56. Amino acid analysis

PROTEIN HYDROLYSIS
Hydrolysis of protein and peptide samples is necessary foramino 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 inconcentrated nitric acid or in a mixture of equal volumesof 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, pyrolysisof clean glassware at 500 °C for 4 h may also be used toeliminate 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 acidhydrolysis technique can contribute to the variation of theanalysis due to complete or partial destruction of severalamino 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 (lessthan 200 µm of mercury or 26.7 Pa) or introduction of aninert 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 bondsof 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 lossof tryptophan, asparagine, and glutamine during an acidhydrolysis limits quantitation to 17 amino acids. Some of thehydrolysis techniques described are used to address theseconcerns. 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 techniqueare weighed against the concerns with the technique and aretested adequately before employing a method other thanacid hydrolysis.
A time-course study (i.e., amino acid analysis at acidhydrolysis times of 24 h, 48 h and 72 h) is often employed
to analyse the starting concentration of amino acids thatare 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 canbe 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 tocleave (e.g., isoleucine and valine). During the hydrolysis
time course, the analyst will observe a plateau in theseresidues. 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 tosubject an amino acid calibration standard to the samehydrolysis conditions as the test sample. The amino acidin free form may not completely represent the rate ofdestruction of labile amino acids within a peptide orprotein 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 accountfor 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 eachindividual 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 beenused but can be complicated, requires the proper controls,
and is typically more applicable to peptides than proteins.
During initial analyses of an unknown protein, experimentswith various hydrolysis time and temperature conditions are
conducted to determine the optimal conditions.
METHOD 1
Acid hydrolysis using hydrochloric acid containing phenol
is the most common procedure used for protein/peptidehydrolysis preceding amino acid analysis. The addition ofphenol 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 driedso 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 concernthat the protein is not completely hydrolysed.
Vapour phase hydrolysis. This is one of the mostcommon acid hydrolysis procedures, and it is preferred for
microanalysis when only small amounts of the sample areavailable. Contamination of the sample from the acid reagent
is also minimised by using vapour phase hydrolysis. Placevials 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 µmof mercury or 26.7 Pa) to the headspace of the vessel, andheat to about 110 °C for a 24 h hydrolysis time. Acid vapourhydrolyses the dried sample. Any condensation of the acid in
the sample vials is to be minimised. After hydrolysis, dry thetest sample in vacuo to remove any residual acid.
METHOD 2
Tryptophan oxidation during hydrolysis is decreased byusing mercaptoethanesulfonic acid as the reducing acid.
Hydrolysis solution. 2.5 M mercaptoethanesulfonic acidsolution.
Vapour phase hydrolysis. Dry about 1 µg to 100 µg of theprotein/peptide under test in a hydrolysis tube. Place thehydrolysis tube in a larger tube with about 200 µl of thehydrolysis 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.
METHOD 3
Tryptophan oxidation during hydrolysis is prevented byusing thioglycollic acid (TGA) as the reducing acid.
Hydrolysis solution. 7 M hydrochloric acid containing 1 percent 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 ofthe protein/peptide under test in a sample tube. Placethe sample tube in a larger tube with about 200 µl of thehydrolysis solution. Seal the larger tube in vacuo (about
50 µm of mercury or 6.7 Pa) to vaporise the TGA. Heat theGeneral Notices (1) apply to all monographs and other texts

912.2.56. Amino acid analysis

EUROPEAN PHARMACOPOEIA 6.0

sample tube to 166 °C for about 15-30 min. After hydrolysis,
dry the sample tube in vacuo for 5 min to remove theresidual acid. Recovery of tryptophan by this method may be
dependent on the amount of sample present.
METHOD 4
Cysteine/cystine and methionine oxidation is performed
with performic acid before the protein hydrolysis.
Oxidation solution. Use performic acid freshly prepared bymixing 1 volume of hydrogen peroxide solution (30 per cent)and 9 volumes of anhydrous formic acid and incubating atroom 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 ; thenadd 100 µl of the oxidation solution. Allow the oxidation
to proceed for 10-30 min. In this reaction, cysteine isconverted to cysteic acid and methionine is converted tomethionine-sulphone. Remove the excess reagent from thesample in a vacuum centrifuge. The oxidised protein canthen be acid hydrolysed using Method 1 or Method 2. This
technique may cause modifications to tyrosine residues in
the presence of halides.
METHOD 5
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/peptidehydrolysis 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 thehydrolysis solution. This technique allows better tyrosine
recovery than Method 4, but it is not quantitative formethionine. Methionine is converted to a mixture of
the parent methionine and its 2 oxidative products,
methionine-sulphoxide and methionine-sulphone.
METHOD 6
Cysteine/cystine oxidation is accomplished with dimethyl
sulphoxide (DMSO).
Hydrolysis solution. To 6 M hydrochloric acid containing
0.1 per cent to 1.0 per cent of phenol, add dimethyl
sulphoxide to obtain a final concentration of 2 per cent V/V.
Vapour phase hydrolysis. Conduct the protein/peptidehydrolysis at about 110 °C for 24 h. During the hydrolysis,
the cysteine/cystine present in the sample is converted tocysteic 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 acidrecovery from oxidative hydrolysis of standard proteins
containing 1-8 mol of cysteine. The response factors fromprotein/peptide hydrolysates are typically about 30 per centlower 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.
METHOD 7
Cysteine/cystine reduction and alkylation is accomplishedby 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
92

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 acidhydrolysed using previously described procedures. The
pyridylethylation reaction is performed simultaneously witha 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 -aminogroup of lysine in the protein.
METHOD 8
Cysteine/cystine reduction and alkylation is accomplishedby 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 abuffered solution of 6 M guanidine hydrochloride in 0.25 M
tris-hydrochloride.
Procedure. Dissolve about 10 µg of the test sample in 50 µlof 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 bycollecting the protein/peptide fraction from a reversed-phase
HPLC separation. The collected sample can be dried in a
vacuum centrifuge before acid hydrolysis.
METHOD 9
Cysteine/cystine reduction and alkylation is accomplishedby a liquid phase carboxymethylation reaction.
Stock solutions. Prepare as directed for Method 8.
Carboxymethylation solution. Prepare a 100 g/l solutionof 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 temperaturein the dark. Add the carboxymethylation solution in aratio 1.5 fold per total theoretical content of thiols, andincubate 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 nmolof protein present. The reaction is stopped by adding
excess of 2-mercaptoethanol. Desalt the protein/peptide bycollecting the protein/peptide fraction from a reversed-phase
HPLC separation. The collected sample can be driedin a vacuum centrifuge before acid hydrolysis. The
S-carboxyamidomethyl-cysteine formed will be converted to
S-carboxymethyl-cysteine during acid hydrolysis.
METHOD 10
Cysteine/cystine is reacted with dithiodiglycolic acid or
dithiodipropionic acid to produce a mixed disulphide. The
choice of dithiodiglycolic acid or dithiodipropionic aciddepends on the required resolution of the amino acidanalysis method.
Reducing solution. A 10 g/l solution of dithiodiglycolic acid(or dithiodipropionic acid) in 0.2 M sodium hydroxide.

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EUROPEAN PHARMACOPOEIA 6.0

Procedure. Transfer about 20 µg of the test sample to ahydrolysis tube, and add 5 µl of the reducing solution.
Add 10 µl of isopropyl alcohol, and then remove all of thesample liquid by vacuum centrifugation. The sample is thenhydrolysed using Method 1. This method has the advantagethat other amino acid residues are not derivatised by side
reactions, and that the sample does not need to be desalted
prior to hydrolysis.
METHOD 11
Asparagine and glutamine are converted to aspartic acidand glutamic acid, respectively, during acid hydrolysis.
Asparagine and aspartic acid residues are added andrepresented by Asx, while glutamine and glutamic acidresidues 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 todiaminopropionic acid and diaminobutyric acid residues,
respectively, upon acid hydrolysis. These conversions allow
the analyst to determine the asparagine and glutaminecontent of a protein/peptide in the presence of aspartic acidand glutamic acid residues.

2.2.56. Amino acid analysis

reversed-phase HPLC. Pre-column derivatisation techniques
are very sensitive and usually require between 0.5 µg and1.0 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 thanpre-column derivatisation techniques.
The following methods may be used for quantitative
amino acid analysis. Instruments and reagents for theseprocedures 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 theexact equipment and procedure used. Many laboratories
will use more than one amino acid analysis technique toexploit the advantages offered by each. In each of thesemethods, the analogue signal is visualised by means of adata acquisition system, and the peak areas are integrated
for quantification purposes.

Reducing solutions. Prepare and filter 3 solutions : asolution of 10 mM trifluoroacetic acid (Solution A), asolution 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).

METHOD 1 - POST-COLUMN NINHYDRINDERIVATISATION
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 forProcedure. In a clean hydrolysis tube, transfer about 200 µg the analysis of the more complex physiological samples,of the test sample, and add 2 ml of Solution A or Solution B and the faster sodium-based cation-exchange system isused for the more simplistic amino acid mixtures obtainedand 2 ml of Solution C. Seal the hydrolysis tube in vacuo.
with protein hydrolysates (typically containing 17 amino
Heat the sample at 60 °C for 4 h in the dark. The sample
acid components). Separation of the amino acids on anis then dialysed with water to remove the excess reagents.
ion-exchange column is accomplished through a combination
Extract the dialysed sample 3 times with equal volumes ofof changes in pH and cation strength. A temperaturebutyl acetate, and then lyophilise. The protein can then beacid hydrolysed using previously described procedures. The gradient is often employed to enhance separation.
α,β-diaminopropionic and α,γ-diaminobutyric acid residues
When the amino acid reacts with ninhydrin, the reactant has
do not typically resolve from the lysine residues upona characteristic purple or yellow colour. Amino acids, except
ion-exchange chromatography based on amino acid analysis. imino acid, give a purple colour, and show an absorption
Therefore, when using ion-exchange as the mode of amino
maximum at 570 nm. The imino acids such as proline give aacid separation, the asparagine and glutamine contents are yellow colour, and show an absorption maximum at 440 nm.
the quantitative difference in the aspartic acid and glutamic The post-column reaction between ninhydrin and aminoacid content assayed with underivatised and BTI-derivatised acids eluted from the column is monitored at 440 nm andacid hydrolysis. The threonine, methionine, cysteine,
570 nm, and the chromatogram obtained is used for thetyrosine, and histidine assayed content can be altered bydetermination of amino acid composition.
BTI derivatisation ; a hydrolysis without BTI will have to be
The detection limit is considered to be 10 pmol for mostperformed if the analyst is interested in the composition ofof the amino acid derivatives, but 50 pmol for the proline
these other amino acid residues of the protein/peptide.
derivative. Response linearity is obtained in the range of
20-500 pmol with correlation coefficients exceeding 0.999.
METHODOLOGIES OF AMINO ACID ANALYSIS : GENERAL To obtain good composition data, samples larger than 1 µg
PRINCIPLES
before hydrolysis are best suited for this amino acid analysis
of protein/peptide.
Many amino acid analysis techniques exist, and the choiceof any one technique often depends on the sensitivity
METHOD 2 - POST-COLUMN OPA DERIVATISATION
required from the assay. In general, about one-half ofo-Phthalaldehyde (OPA) reacts with primary amines in thethe amino acid analysis techniques employed rely on
presence of thiol compound, to form highly fluorescent
the separation of the free amino acids by ion-exchange
isoindole products. This reaction is used for the post-column
chromatography followed by post-column derivatisation
derivatisation in analysis of amino acids by ion-exchange(e.g., with ninhydrin or o-phthalaldehyde). Post-column
chromatography. The rule of the separation is the same
derivatisation techniques can be used with samples thatas Method 1.
contain small amounts of buffer components, (such as
Although OPA does not react with secondary amines (imino
salts and urea) and generally require between 5 µg andacids such as proline) to form fluorescent substances, the
10 µg of protein sample per analysis. The remaining aminooxidation with sodium hypochlorite or chloramine T allows
acid techniques typically involve pre-column derivatisation
secondary amines to react with OPA. The procedure employsof the free amino acids (e.g., phenyl isothiocyanate ;a strongly acidic cation-exchange column for separation of
6-aminoquinolyl-N-hydroxysuccinimidyl carbamate or
free amino acids followed by post-column oxidation witho-phthalaldehyde ; (dimethylamino)azobenzenesulphonylsodium hypochlorite or chloramine T and post-column
chloride ; 9-fluorenylmethyl chloroformate ; andderivatisation using OPA and a thiol compound such as
7-fluoro-4-nitrobenzo-2-oxa-1,3-diazole) followed byGeneral Notices (1) apply to all monographs and other texts

932.2.56. Amino acid analysis

EUROPEAN PHARMACOPOEIA 6.0

N-acetyl-L-cysteine or 2-mercaptoethanol. The derivatisationof primary amino acids is not noticeably affected by thecontinuous 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 derivatisationof eluted amino acids with OPA, the reactant passes
through the fluorometric detector. Fluorescence intensityof OPA-derivatised amino acids are monitored with an
excitation wavelength of 348 nm and an emission wavelengthof 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 toa few tens of nanomole level. To obtain good compositionaldata, samples larger than 500 ng of protein/peptide beforehydrolysis are recommended.
METHOD 3 - PRE-COLUMN PITC DERIVATISATION
Phenylisothiocyanate (PITC) reacts with amino acids to form
phenylthiocarbamyl (PTC) derivatives which can be detectedwith high sensitivity at 254 nm. Therefore, pre-column
derivatisation of amino acids with PITC followed by areversed-phase HPLC separation with UV detection is used
to analyse the amino acid composition.
After the reagent is removed under vacuum, the derivatisedamino 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 accomplishedthrough a combination of changes in concentrations ofacetonitrile 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 mostof the PTC-amino acids. Response linearity is obtainedin the range of 20-500 pmol with correlation coefficientsexceeding 0.999. To obtain good compositional data,
samples larger than 500 ng of protein/peptide beforehydrolysis are recommended.
METHOD 4 - PRE-COLUMN AQC DERIVITISATION
Pre-column derivatisation of amino acids with
6-aminoquinolyl-N-hydroxysuccinimidyl carbamate(AQC) followed by reversed-phase HPLC separation withfluorometric detection is used.
AQC reacts with amino acids to form stable, fluorescentunsymmetric 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 withfluorimetric detection is used to analyse the amino acidcomposition.
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 andbuffer ionic strengh. Selective fluorescence detection of thederivatives with an excitation wavelength at 250 nm and anemission wavelength at 395 nm allows for the direct injectionof the reaction mixture with no significant interference
from the only major fluorescent reagent by-product,
6-aminoquinoline. Excess reagent is rapidly hydrolysed(t1/2<15 s) to yield 6-aminoquinoline, N-hydroxysuccinimide
and carbon dioxide, and after 1 min no further derivatisation
can take place.
94

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 ofderivatised protein hydrolysates derived from as little as
30 ng of protein/peptide.
METHOD 5 - PRE-COLUMN OPA DERIVATISATION
Pre-column derivatisation of amino acids witho-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 primaryamine groups to form highly fluorescent isoindole products.
2-Mercaptoethanol or 3-mercaptopropionic acid can be usedas 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 automatedderivatisation and analysis using an autosampler to mix thesample with the reagent. However, lack of reactivity withsecondary 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 thisdrawback, this technique may be combined with anothertechnique described in Method 7 or Method 8.
Pre-column derivatisation of amino acids with OPA isfollowed by a reversed-phase HPLC separation. Becauseof the instability of the OPA-amino acid derivative, HPLC
separation and analysis are performed immediately following
derivatisation. The liquid chromatograph is equipped with afluorometric detector for the detection of derivatised aminoacids. Fluorescence intensity of OPA-derivatised amino acids
is monitored with an excitation wavelength of 348 nm andan emission wavelength of 450 nm.
Detection limits as low as 50 fmol via fluorescence have beenreported, although the practical limit of analysis remains
at 1 pmol.
METHOD 6 - PRE-COLUMN DABS-Cl DERIVATISATION
Pre-column derivatisation of amino acids with(dimethylamino)azobenzenesulphonyl chloride (DABS-Cl)
followed by reversed-phase HPLC separation with visible
light detection is used.
DABS-Cl is a chromophoric reagent employed for thelabelling 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 acidderivatives, can be separated on an ODS column of areversed-phase HPLC by employing gradient systems
consisting of acetonitrile and aqueous buffer mixture.
Separated DABS-amino acids eluted from the column aredetected 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 ofsensitivity, DABS-Cl derivatisation method permits thesimultaneous quantification of tryptophan residues
by previous hydrolysis of the protein/peptide withsulphonic acids such as mercaptoethanesulphonic acid,p-toluenesulphonic acid or methanesulphonic aciddescribed in Method 2 under Protein hydrolysis. The other

See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 6.0

2.2.56. Amino acid analysis

DATA CALCULATION AND ANALYSIS
When determining the amino acid content of aprotein/peptide hydrolysate, it should be noted that the acidhydrolysis step destroys tryptophan and cysteine. Serine andthreonine are partially destroyed by acid hydrolysis, while
The non-proteinogenic amino acid norleucine cannot be used isoleucine and valine residues may be only partially cleaved.
Methionine can undergo oxidation during acid hydrolysis,
as an internal standard in this method as this compoundand some amino acids (e.g., glycine and serine) are common
is eluted in a chromatographic region crowded with peaks
contaminants. Application of adequate vacuum (less thanof primary amino acids. Nitrotyrosine can be used as an
200 µm of mercury or 26.7 Pa) or introduction of inert
internal standard because it is eluted in a clean region.
gas (argon) in the headspace of the reaction vessel during
The detection limit of DABS-amino acid is about 1 pmol. As vapour phase hydrolysis can reduce the level of oxidative
little as 2-5 pmol of an individual DABS-amino acid can be
destruction. Therefore, the quantitative results obtained
quantitatively analysed with reliability, and only 10-30 ng
for cysteine, tryptophan, threonine, isoleucine, valine,of the dabsylated protein hydrolysate is required for eachmethionine, glycine, and serine from a protein/peptide
analysis.
hydrolysate may be variable and may warrant further
investigation and consideration.
METHOD 7 - PRE-COLUMN FMOC-Cl DERIVATISATION
Pre-column derivatisation of amino acids with
Amino Acid Mole Percent. This is the number of specific9-fluorenylmethyl chloroformate (FMOC-Cl) followed byamino acid residues per 100 residues in a protein. This result
reversed-phase HPLC separation with fluorometric detection may be useful for evaluating amino acid analysis data whenis used.
the molecular mass of the protein under investigation is
unknown. This information can be used to corroborate the
FMOC-Cl reacts with both primary and secondary amino
identity of a protein/peptide and has other applications.
acids to form highly fluorescent products. The reaction
Carefully identify and integrate the peaks obtained asproceeds under mild conditions in aqueous solution anddirected for each procedure. Calculate the mole percent foris completed in 30 s. The derivatives are stable, only theeach amino acid present in the test sample using the formula :histidine derivative showing any breakdown. Although
FMOC-Cl is fluorescent itself, the reagent excess andfluorescent side-products can be eliminated without loss of
FMOC-amino acids.
in which rU is the peak response, in nanomoles, of the amino
FMOC-amino acids are separated by a reversed-phase HPLC acid under test ; and r is the sum of peak responses, inusing an ODS column. The separation is carried out by
nanomoles, for all amino acids present in the test sample.
gradient elution varied linearly from a mixture of 10 volumes Comparison of the mole percent of the amino acids underof acetonitrile, 40 volumes of methanol and 50 volumes oftest to data from known proteins can help establish or
acetic acid buffer to a mixture of 50 volumes of acetonitrile corroborate the identity of the sample protein.
and 50 volumes of acetic acid buffer and 20 amino acidUnknown Protein Samples. This data analysis technique
derivatives are separated in 20 min. Each derivative eluted
can be used to estimate the protein concentration of anfrom the column is monitored by a fluorometric detector
unknown protein sample using the amino acid analysis data.
set at an excitation wavelength of 260 nm and an emission
Calculate the mass, in micrograms, of each recovered amino
wavelength of 313 nm.
acid using the formula :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.
acid-labile residues, asparagine and glutamine, can also be
analysed by previous conversion into diaminopropionicacid and diaminobutyric acid, respectively, by treatment ofprotein/peptide with BTI described in Method 11 underProtein hydrolysis.

METHOD 8 - PRE-COLUMN NBD-F DERIVATISATION
Pre-column derivatisation of amino acids with
7-fluoro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-F) followed byreversed-phase HPLC separation with fluorometric detection
is used.
NBD-F reacts with both primary and secondary aminoacids to form highly fluorescent products. Amino acids arederivatised with NBD-F by heating to 60 °C for 5 min.

in which m is the recovered quantity, in nanomoles, of theamino acid under test ; and Mr is the average molecular mass
for that amino acid, corrected for the mass of the watermolecule that was eliminated during peptide bond formation.
The sum of the masses of the recovered amino acids will givean 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 isavailable (i.e., by SDS-PAGE analysis or mass spectroscopy),
the amino acid composition of the unknown protein can bepredicted. Calculate the number of residues of each aminoacid using the formula :NBD-amino acid derivatives are separated on an ODS
column of a reversed-phase HPLC by employing a gradientelution 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.
in which m is the recovered quantity, in nanomoles, of theamino acid under test ; M is the total mass, in micrograms, of
The sensitivity of this method is almost the same as forthe protein ; and Mrt is the molecular mass of the unknown
the pre-column OPA derivatisation method (Method 5),
protein.
excluding proline to which OPA is not reactive, and might beadvantageous for NBD-F against OPA. The detection limit
Known protein samples. This data analysis technique can befor each amino acid is about 10 fmol. Profile analysis can be used to investigate the amino acid composition and protein
achieved with about 1.5 mg of protein hydrolysates in theconcentration of a protein sample of known molecular mass
pre-column reaction mixture.
and amino acid composition using the amino acid analysis
General Notices (1) apply to all monographs and other texts

95

2.2.57. Inductively coupled plasma-atomic emission spectrometrydata. 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) andfree 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 theamount 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 thatamino acid to obtain the protein content based on eachwell-recovered amino acid. Average the protein content
results calculated. The protein content determined foreach 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 deviationfrom the mean. Typically greater than 5 per cent variationfrom the mean is considered unacceptable. Recalculate themean protein content from the remaining values to obtain
the protein content of the sample. Divide the content ofeach amino acid by the calculated mean protein content
to determine the amino acid composition of the sample byanalysis.
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 mS is the known residue value for that amino acid.
The average relative compositional error is the average ofthe 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 theamino acid composition between the protein sample and the
known composition can be used to corroborate the identityand purity of the protein in the sample.

EUROPEAN PHARMACOPOEIA 6.0

The plasma is formed by a tangential stream of support gas
through a ‘torch’, i.e. a system consisting of 3 concentric
quartz tubes. A metal coil (the load coil) surrounds the top
end of the torch and is connected to a radio-frequency (RF)generator. Power (usually 700-1500 W) is applied throughthe coil and an oscillating magnetic field corresponding
to the frequency of the generator (in most cases 27 MHz,
40 MHz) is formed. The plasma forms when the support
gas is made conductive by exposing it to an electric
discharge, which produces seed electrons and ions. Inside
the induced magnetic field, the charged particles (electrons
and ions) are forced to flow in a closed annular path. As
they meet resistance to their flow, heating takes place
producing additional ionisation. The process occurs almost
instantaneously, and the plasma expands to its full strengthand dimensions. The radio-frequency oscillation of thepower applied through the coil causes radio-frequency
electric and magnetic fields to be set up in the area at the
top of the torch. When a spark (produced by a Tesla tube
or some other seeding device) is applied to the support gas
flowing through the torch, some electrons are stripped fromthe support gas atoms. These electrons are then caught
up in the magnetic field and accelerated. Adding energy
to the electrons by the use of a coil is known as inductive
coupling. These high-energy electrons in turn collide withother support-gas atoms, stripping off still more electrons.
The collisional ionisation of the support gas continues in achain reaction, breaking down the gas into a physical plasma
consisting of support-gas atoms, electrons and support-gas
ions. The plasma is then sustained within the torch and loadcoil as radio-frequency energy is continually transferred to it
through the inductive coupling process.
The ICP appears as an intense, very bright, plume-shaped
plasma. At the base the plasma is toroidal, and this isreferred to as the induction region (IR), i.e. the region inwhich the inductive energy transfer from the load coil to theplasma takes place. The sample is introduced through theinduction region into the centre of the plasma.

APPARATUS
The apparatus consists essentially of the following elements :— sample-introduction system consisting of a peristaltic
pump delivering the solution at constant flow rate into
a nebuliser ;— radio-frequency (RF) generator ;— plasma torch ;— transfer optics focussing the image of the plasma at theentrance slit of the spectrometer ; radial viewing is better
01/2008:20257
for difficult matrices (alkalis, organics), whereas axial
viewing gives more intensity and better detection limits in
2.2.57. INDUCTIVELY COUPLED
simple matrices ;PLASMA-ATOMIC EMISSION— wavelength dispersive devices consisting of diffraction
gratings, prisms, filters or interferometers ;SPECTROMETRY— detectors converting radiant energy into electrical energy ;GENERAL PRINCIPLE— data-acquisition unit.
Inductively coupled plasma-atomic emission spectrometry
INTERFERENCE(ICP-AES) is an atomic emission spectrometry method thatuses an inductively coupled plasma (ICP) as the excitation
Interference is anything that causes the signal from ansource.
analyte in a sample to be different from the signal for thesame concentration of that analyte in a calibration solution.
An ICP is a highly ionised inert gas (usually argon) with
The well-known chemical interference that is encounteredequal numbers of electrons and ions sustained by ain flame atomic absorption spectrometry is usually weak inradio-frequency (RF) field. The high temperature reached
ICP-AES. In rare cases where interference occurs, it may
in the plasma successively desolvates, vaporises, excites
be necessary to increase the RF power or to reduce the- atomic emission spectrometry (AES) detection - andinner support-gas flow to eliminate it. The interference inionises - mass spectrometry (MS) detection - atoms from
ICP-AES can be of spectral origin or even the result of highthe sample. Detection limits are, generally, in the lower
nanogram (ICP-MS) to microgram (ICP-AES) per litre range. concentrations of certain elements or matrix compounds.
96

See the information section on general monographs (cover pages)