Amino Acid Analysis

Amino Acid Analysis UNIT 11.
9
1 2
Shane M. Rutherfurd and G. Sarwar Gilani
1
Riddet Institute, Massey University, Palmerston North, New Zealand
2
Health Canada, Nutrition Research Division, Food Directorate, Health Products and Food
Branch, Ottawa, Ontario, Canada
ABSTRACT
Amino acid analysis is used to determine the amino acid content of amino acid, peptide-
and protein-containing samples. With minor exceptions, proteins are long linear polymers
of amino acids connected to each other via peptide bonds. The first step of amino
acid analysis involves hydrolyzing these peptide bonds. The liberated amino acids are
then separated, detected, and quantified. The method was first developed by Moore,
Stein and coworkers in the 1950s using HCl acid hydrolysis, and, despite considerable
effort by many workers, the basic methodology remains relatively unchanged. This unit
provides an overview and strategic planning for amino acid analysis, discussing a range
of methodologies and issues. In addition, several common methods used for analysis
of L-amino acids are described in detail, including: HCl acid hydrolysis, performic acid
oxidation for methionine and cysteine analysis, base hydrolysis for tryptophan analysis,
analysis of free amino acids, and analysis of reactive lysine. Curr. Protoc. Protein Sci.
58:11.9.1-11.9.37. C 2009 by John Wiley & Sons, Inc.
Keywords: amino acids r hydrolysis r derivatization r chromatography
INTRODUCTION
Amino acids are a chemically diverse set of compounds present in proteins and peptides.
Accurately analyzing amino acids is difficult and labor intensive, and, as a result, is
performed only in select laboratories. Amino acid analysis (AAA) was first developed by
Moore and coworkers (Moore and Stein, 1948, 1951) in the early 1950s to determine the
amino acid content of pure proteins. Their method used 6 M HCl acid hydrolysis in an
oxygen-free environment at 110 C for 22 hr to liberate amino acids from proteins. They
then separated the free amino acids using ion-exchange chromatography and detected
and quantified the eluted amino acids using post-column ninhydrin detection. Numerous
studies have been conducted with the aim of reducing the analysis time, simplifying the
methods, and reducing data variability. While considerable progress has been made with
respect to the chromatography, the hydrolysis step has largely remained unchanged.
Many amino acids are prone to chemical modification during hydrolysis, and numerous
strategies have been developed to minimize these reactions, although not all are effective.
In this unit, a number of these strategies, such as the use of performic acid oxidation
for cysteine and methionine analysis, the use of additives during hydrolysis to improve
tyrosine yield, and the use of multiple hydrolysis times are discussed. Amino acids are
also difficult to detect since they are not natural chromophores and consequently require
derivatization with a chromophore to permit detection. Several derivatizing methods
are discussed in this unit, as are methods of separating amino acids. A discussion of
approaches used to reduce data variability is also presented. Finally, protocols for acid
hydrolysis (sealed tube method; see Basic Protocol 1), performic acid oxidation (see
Basic Protocol 2), base hydrolysis (LiOH; see Basic Protocol 3), determination of free
amino acids (see Basic Protocol 4), and determination of reactive lysine (guanidination
method; see Basic Protocol 5) are described in detail. A Support Protocol is also provided
for preparation of samples to be subjected to amino acid analysis. Chemical
Analysis
Current Protocols in Protein Science 11.9.1-11.9.37, November 2009 11.9.1

Published online November 2009 in Wiley Interscience (www.interscience.wiley.com).
DOI: 10.1002/0471140864.ps1109s58 Supplement 58
Copyright C 2009 John Wiley & Sons, Inc.
STRATEGIC PLANNING
Types of Samples and Applications
AAA is used for determining the amino acid content of any amino acid, peptide-, or
protein-containing sample. However, depending on the nature of the samples, distinct
preparation methods must be adopted to ensure that the amino acid analysis is accurate.
Samples can consist of liquid solutions either with or without particulate matter, or dry
powders. Samples may also be adhered to membranes, for example PDVF, which may
be used for electroblotting proteins. Amino acid analysis may be conducted on foods or
biological samples such as blood, urine, digesta, saliva, or feces, and the analysis of these
is not limited by the amount of sample, but rather may be compromised by the presence of
significant amounts of nonprotein material. AAA can also be applied to purified proteins,
and for these the milligram or perhaps microgram amounts present can make analysis
difficult. The amino acid composition of synthetic peptides is sometimes determined as
a quality-control tool, although mass spectrometry has largely superceded amino acid
analysis in this capacity. Some of the different types of samples that commonly undergo
amino acid analysis and the problems associated with their analysis are discussed below.
Sampling and Sample Preparation

The first step of amino acid analysis, and indeed any laboratory test, involves obtaining
a representative sample. Samples may be solid or liquid, and, in each case, different
sampling and preparation strategies must be adopted.
Liquid samples
Liquid samples may be accurately aliquotted directly into hydrolysis tubes and then
dried using either a freeze dryer or a centrifugal concentrator, or an equivalent instrument.
Samples undergoing freeze drying generally cannot contain acids, since the stainless steel
present within the dryer can be corroded. Centrifugal concentrators are often designed
to dry samples containing more aggressive solvents and acids, and provide a suitable
alternative for samples containing volatile corrosive components. In addition, samples
can only be freeze dried if they are initially frozen solid, since unfrozen liquid samples
will often bubble vigorously under vacuum due to release of dissolved air, and the sample
can be lost from the container when this occurs. Freeze drying is more suitable for heat-
labile compounds, since the sample is always either frozen or maintained at relatively
low temperatures after drying. In contrast, the centrifugal concentrator can dry unfrozen
liquid samples, which is an advantage for samples such as HPLC fractions that contain
organic solvents, which may not freeze or will not remain frozen during drying, since
the centrifugal force keeps the sample from bubbling out of the tube. When the vacuum
is applied to the centrifugal chamber, evaporative cooling will lower the temperature of
the sample, often below its freezing point. However, once the sample is dry, the sample
temperature will increase to that of the centrifugal chamber, which can be as high as
40 C simply from heat produced by the system. If heat-labile samples are to be dried,
then care must be taken to remove the samples from the concentrator as soon as possible
after they are dry.
Some liquid samples contain particulate matter which may include precipitated protein.
Such samples must be dried and further treated as described below for dry samples. In
contrast, if the particulate matter is known to be protein-free, then it can be filtered out
and the sample treated as described above for liquid samples.
Dry samples
Amino Acid
Dry samples must generally be ground and homogenized before being analyzed. The
Analysis fineness of the grind largely depends on the amount of sample to be weighed into the
11.9.2
Supplement 58 Current Protocols in Protein Science
hydrolysis tubes. Generally, the smaller the amount of sample to be weighed, the finer
the sample must be ground. As a general rule, a 1-mm grinding mesh is suitable for
most samples. Care must be taken, when grinding some samples, to avoid separation of
sample constituents and heating of the samples. This is discussed in more detail below
(see Food samples).
Pure proteins and peptides

These include proteins and peptides that have been purified by HPLC. Such samples are
relatively simple to prepare as liquid samples, aliquotted into a hydrolysis tube, and dried
using a centrifugal concentrator. The main problem with these types of samples is that
they generally contain only microgram quantities of protein.
Physiological samples
Physiological samples are derived from animals or humans and may include blood, urine,
digesta, saliva, and feces. Each type of sample must be prepared for amino acid analysis
in a different manner. Digesta and feces are usually freeze dried and finely ground.
Whole blood (prevented from clotting by collecting the blood into EDTA or heparin
Vacutainers), urine, and saliva may be aliquotted directly into the hydrolysis tube and
freeze dried or dried in a centrifugal concentrator. Plasma and serum can both be prepared
from whole blood by centrifuging and collecting the supernatant. For serum, the blood is
allowed to clot first so the clotting proteins are removed. For plasma, the blood is collected
into EDTA or heparin Vacutainers to prevent clotting. The red blood cells are then spun
down and discarded. For the determination of free amino acids in serum or plasma,
acid hydrolysis is not used, and the proteins are generally removed by precipitation
with either (1) solvents such as ethanol, methanol, acetone, or acetonitrile (Sedgwick
et al., 1991; Bruce et al., 2009), although there is evidence that some free amino acids
are precipitated as well (Sedgwick et al., 1991); or (2) acids such as 5-sulfosalicylic
acid (Bos et al., 2003), picric acid (Stein and Moore, 1954), or perchloric acid (Koros
et al., 2007), followed by centrifugation to remove the precipitated protein. Alternatively,
ultrafiltration using microconcentrator tubes can be used (Davey and Ersser, 1990). Free
amino acids in urine can often be analyzed directly by HPLC after simply filtering the
sample through a 0.22-m filter. Urine can also be filtered through decolorizing charcoal
to partially clean up urine samples prior to injection.
Food samples
The variety of food samples analyzed for amino acid content is extensive. Foods contain
a wide variety of nutrients besides protein, including carbohydrate, fat, vitamins, and
minerals. For foods that contain >10% fat, it may be necessary to defat the sample prior
to hydrolysis, particularly for tryptophan analysis. This can be carried out using solvent
extraction at room temperature or at elevated temperature using a Soxtec fat extractor
(a product of Foss; http://www.foss.dk/). Heated extraction tends to be more effective,
but generally it is wise to keep extraction temperatures as low as possible to preserve
heat-labile amino acids.
The next step is usually to freeze dry the samples and finely grind them before weighing
them into hydrolysis tubes. Homogenous liquids such as milk can be treated as described
above (see Liquid Samples). Food samples are often heterogeneous in nature, and this
can lead to problems when grinding; for example, for whole grains, the hull is often
difficult to grind through a 1-mm mesh, while the embryo passes through easily. This
can lead to separation of the kernel from the hull, and under these circumstances it
is important to ensure all the sample passes through the mesh and that the sample is
thoroughly reconstituted and mixed after grinding. In the case of breakfast cereals where Chemical
there might be grains and fruit pieces present, the same procedure applies. It is also Analysis
11.9.3
Current Protocols in Protein Science Supplement 58
important to ensure that the grinder does not get excessively hot while grinding difficult
samples, since amino acids such as lysine can be chemically modified when heated in the
presence of other compounds such as reducing sugars, and this leads to complications
during analysis. Foods that are high in sugars or fat can also be difficult to grind. Fats, for
example, will melt if the grinder heats up to any significant degree, and this will quickly
clog the mesh, while sugars can caramelize at higher temperatures.
The importance of sample homogeneity, particularly for food samples, cannot be overem-
phasized, and high sample heterogeneity can be a leading cause of variability of amino
acid composition data.
Sample storage
Most amino acids are relatively stable; however cysteine, methionine, and tryptophan
can all be oxidized, while lysine can react with other food compounds. These reactions
can be significant even when samples are stored under relatively mild conditions such as
at room temperature. As a general rule, it is good practice to store all samples at 20 C
or colder, and in the dark.
Protein Hydrolysis
The purpose of hydrolysis is to break the peptide bonds that hold amino acids together
within a protein. The standard method of hydrolysis is incubation in an oxygen-free
environment in constant-boiling 6 M HCl at 110 C for 18 to 24 hr, with 24-hr hydrolysis
being used most commonly (FAO/WHO, 1991; Ozols, 1990; Darragh and Moughan
2005; also see Basic Protocol 1). However, amino acids are a chemically diverse group
of compounds, and not all are stable under the hydrolysis conditions described above.
Of the 22 amino acids commonly present in proteins, only aspartic acid, glutamic acid,
proline, glycine, alanine, leucine, phenylalanine, histidine, arginine, and hydroxyproline
can be determined quantitatively during acid hydrolysis. The remaining amino acids are
discussed below.
Problematic amino acids during hydrolysis

Asparagine and glutamine
Asparagine and glutamine are amides and are deaminated during acid hydrolysis to
aspartic acid and glutamic acid, respectively. Consequently, determined aspartic acid
and glutamic acid values each represent the sum of the acid and amide derivatives.
Numerous methods have been proposed to determine asparagine and glutamine, and these
include esterification-reduction of carboxylic acids groups (Wilcox, 1967), carbodiimide
modification to the free carboxylic groups (Carraway and Koshland, 1972), enzymatic
hydrolysis (Tower, 1967), and converting the amide to the amine (Soby and Johnson
1981). However, few laboratories use the latter methods.
Serine and threonine

Serine and threonine are partially destroyed during acid hydrolysis, and losses of 10% to
15% for serine and 5% to 10% for threonine are not uncommon (Ozols, 1990; Darragh
et al., 1996). Hydrolysis for less than 24 hr has been used to obtain more quantitative
yields of these amino acids in foods and physiological samples (Rowan et al., 1992).
Using multiple hydrolysis times and extrapolating amino acid yields back to time 0 has
also been used. Correction factors have been developed to correct hydrolytic losses of
these amino acids. However, given that the latter losses may differ from laboratory to
laboratory, universal correction factors may not be appropriate. A suitable approach is
Amino Acid to report the determined serine and threonine values and to educate the client as to the
Analysis accuracy of the data and the method used to obtain them.
11.9.4
Cysteine and methionine
Cysteine can be destroyed during acid hydrolysis, and methionine can be oxidized to
methionine sulfoxide and methionine sulfone if care has not been taken to remove
all the oxygen from the hydrolysis tube. These amino acids are often determined by
quantitatively oxidizing cysteine to cysteic acid and methionine to methionine sulfone
using performic acid prior to acid hydrolysis (see Basic Protocol 2). After oxidation, the
unreacted performic acid can be reduced to formic acid using sodium metabisulfite or
HBr. The latter reaction produces bromine gas (very hazardous; handle in fume hood)
and water. The oxidized derivatives are generally stable during acid hydrolysis even
in complex foodstuffs (Rutherfurd et al., 2007a), although significant losses of cysteic
acid during analysis have been reported (Darragh et al., 1996). Most amino acids that
are generally determined using HCl acid hydrolysis can also be determined in performic
acidoxidized samples. However, tyrosine and tryptophan are destroyed during performic
acid oxidation and cannot be determined using this method.
In processed foods, cysteine can be readily oxidized to cysteic acid and methionine
to methionine sulfoxide, and even methionine sulfone, under more extreme processing
conditions (Todd et al., 1984; Hayashi and Suzuki, 1985). Consequently, performic acid
oxidation may not always be suitable for determining cysteine and methionine content in
such foods. To complicate matters further, methionine sulfoxide can generally be utilized
by animals and humans, while methionine sulfone and cysteic acid cannot (Anderson
et al., 1976; Friedman and Gumbmann, 1988). Ideally cysteine, methionine, and methio-
nine sulfoxide should be determined separately from cysteic acid and methionine sulfone
for processed foods. Methods that do not involve oxidation have been used to determine
methionine. These include reaction with CNBr and quantitation of methyl thiocyanate
(Ellinger and Duncan, 1976) or CNBr reaction and quantitation as homoserine (Elias
et al., 2005). For cysteine, iodoacetylation and vinylpyridation have been used.
Methionine sulfoxide is unstable during HCl acid hydrolysis, where it can be reduced to
methionine or oxidized to methionine sulfone (Rutherfurd and Moughan, 2008). While
these two reactions appear to be contradictory, their relative flux may be related to the
degree of oxygen removal from the hydrolysis tubes. There have been several methods
published and reported to be suitable for hydrolyzing protein containing methionine sul-
foxide. These include the use of methanesulfonic acid (Chiou and Wang, 1988; Puchala
et al., 1994; Sochaski et al., 2001), p-toluenesulfonic acid (Hayashi and Suzuki, 1985),
and alkaline hydrolysis (Gjen and Njaa, 1977; Sjgerg and Bostrm, 1977; Cuq et al.,
1978; Nakamura et al., 1983; Todd et al., 1984). However, recoveries of methionine sul-
foxide in fishmeal after hydrolysis with methanesulfonic acid have also been reported to
be incomplete when compared to the actual methionine sulfoxide amount determined us-
ing nonlinear least-squares regression (Rutherfurd and Moughan, 2008). A more detailed
discussion of the analysis of cysteine, methionine, and methionine sulfoxide, and their
nutritional availability, has been provided by Rutherfurd and Moughan (2008). These
workers concluded that more work is required in this area.
Tyrosine
Tyrosine can undergo halogenation during HCl hydrolysis, and phenol is often added to
the 6 M HCl to prevent this (Nissen, 1992). If iron or copper ions are present, tyrosine
recoveries can also be low, although this problem can largely be overcome by using
constant-boiling HCl (Finley, 1985). High fat content (>5%) can also lead to reduced
tyrosine yields (Finley, 1985); however, defatting samples prior to acid hydrolysis will
overcome this problem.
Chemical
Analysis
11.9.5
Valine and isoleucine
Peptide bonds involving two very hydrophobic residues, particularly valine and
isoleucine, are exceptionally difficult to cleave with acid, and observed quantities for
these amino acids are often low when the sample is hydrolyzed with HCl for 24 hr at
110 C. However, higher yields of these amino acids can be obtained when hydrolysis
times are increased to 72 hr (Rayner, 1985).
Tryptophan
Tryptophan can be destroyed during acid hydrolysis, particularly if >5% carbohydrates,
either starch or sugars, are present (Finley, 1985). For pure proteins, tryptophan recovery
can be increased with the addition of thiols such as -mercaptoethanol, but generally
recoveries are less than complete (Ng et al., 1987). Molnar-Perl and Khalifa (1992)
reported good recoveries of tryptophan after hydrolysis of lysozyme, human albumin,
and -chymotrypsin in HCl containing tryptamine. The reduction of tryptophan to di-
hydrotryptophan using pyridineborane prior to hydrolysis has also been used with some
success (Wong et al., 1984). The use of sulfonic acids gives good recoveries of trypto-
phan in pure proteins, but not in foods (Weiss et al., 1998). Davies and Thomas (1973)
reported good recoveries of tryptophan in pure proteins using p-toluenesulfonic acid, but
not in samples containing carbohydrates. Yamada et al. (1991) used S-pyridylethylation
before hydrolysis to determine tryptophan and cysteine in microquantities of protein.
Garcia and Baxter (1992) used pronase (a relatively nonspecific protease) to hydrolyze
protein in order to determine tryptophan in infant formulas, and reported slightly higher
recoveries of tryptophan compared to using NaOH hydrolysis.
Despite the numerous methods developed to quantitatively hydrolyze tryptophan, the

most commonly used approach is based on alkaline hydrolysis. A considerable number
of studies have been conducted to examine the efficacy of base hydrolysis, and these have
been thoroughly reviewed (Molnar-Perl, 1997; Fountoulakis and Lahm, 1998). Overall,
the most commonly used alkalis for tryptophan hydrolysis are sodium hydroxide (Hanko
and Rohrer, 2002; Nagaraja et al., 2003), lithium hydroxide (Landry et al., 1992; see
Basic Protocol 3), and barium hydroxide (Landry and Delhaye, 1992), and each has been
used with varying degrees of success. Overall, there appears to be no clear advantage to
the use of one alkali over another, and it may be that the type of samples being analyzed,
the method of degassing, and the laboratory conducting the analysis possibly have more
of an effect on tryptophan recovery than the alkali itself. It is of note however, that
barium hydroxide forms precipitates over time as barium carbonate, or, more rapidly, as
barium sulfate if sulfates are present. Unless these precipitates are removed, they can be
problematic during the chromatography step.
Teflon or polypropylene containers are often used in place of glass when conducting
base hydrolysis, reportedly to reduce compounds, such as heavy metals that may de-
stroy tryptophan, being leached from the glass (Steinhart, 1984). Rowan et al. (1989)
reported that when using 4.3 M LiOH hydrolysis, the recovery of tryptophan was higher
when conducted using Teflon screw-cap containers where the sample was sparged with
nitrogen, in comparison with using sealed, evacuated glass tubes.
Since tryptophan absorbs at 280 nm and is also a fluorophore ( excitation 280 nm,
emission 348 nm), it can be detected without derivatization. In addition, it is relatively
hydrophobic, so it lends itself to separation from the other amino acids using reversed-
phase chromatography.
There are also a number of methods described that determine tryptophan in protein
Amino Acid without the need for hydrolysis. One example uses the azo-coupling reaction with
Analysis p-phenylenediamine dihydrochloride to produce a product with an absorbance maximum
11.9.6
at 520 nm (Nagaraja et al., 2003). The main drawback with such methods is that the pro-
teins must be soluble in the reaction medium, and this is often not the case with many
food proteins.
The internal standard 5-methyltryptophan is often added to samples prior to hydrolysis

as a means of correcting for hydrolytic losses of tryptophan. It is assumed that the rates
of degradation of tryptophan and 5-methyltryptophan are similar, and, consequently,
tryptophan content is often mathematically corrected based on the recovery of the internal
standard. The validity of using an internal standard to correct for hydrolytic losses is
debatable; however, it is the view of the authors that given the structural similarity of the
tryptophan and 5-methyltryptophan, the approach is acceptable.
Lysine
Lysine and hydroxylysine are stable under standard acid hydrolysis conditions. In pure
proteins, physiological samples, and foods that have not undergone heat processing, these
amino acids can be determined readily using HCl acid hydrolysis. However, when foods
are processed, particularly heat processed, the side-chain amino group of lysine can react
with other compounds in the food, such as reducing sugars, to produce Maillard products
(Hurrell and Carpenter, 1981). Some of these Maillard products are acid labile and will
revert back to lysine (Mauron et al., 1955) and other compounds (Finot et al., 1968). This
reverted lysine interferes with the chromatographic determination of the native lysine
present in the food, resulting in an overestimate of the unmodified lysine present. This
unmodified lysine is often referred to as reactive lysine since it has a side chain amino
group that is capable of reacting with other compounds (Rutherfurd and Moughan, 2007;
see Basic Protocol 5).
Rutherfurd and Moughan (2007) have discussed the analysis of unmodified lysine as
well as the determination of bioavailable lysine in depth, and it is not the purpose
of this overview to repeat that here. However, brief discussion of this topic is war-
ranted. There have been many methods developed to determine reactive lysine, including
the fluorodinitrobenzene (FDNB) method, FDNB-difference method (Rao et al., 1963),
trinitrobenzenesulfonic acid (TNBS) method (Carpenter and Bjarnason, 1968), sodium
borohydride method (Hurrell and Carpenter, 1974), furosine method (Desrosiers et al.,
1989), dye-binding method (Hendriks et al., 1994), ninhydrin-reactive lysine method
(Friedman et al., 1984), o-phthaldialdehyde-reactive lysine method (Vigo et al., 1992),
and guanidination method (Mauron and Bujard, 1964; Mao et al., 1993; Rutherfurd
et al., 1997; Torbatinejad et al., 2005). Each of these methods has their own advantages
and disadvantages, but, among them, the two most commonly used are the FDNB and
guanidination methods.
The FDNB method converts reactive lysine to dinitrophenyl lysine, which can then be
extracted and determined either spectrophotometrically or by reversed-phase HPLC.
The drawback to this method is that DNP-lysine is not completely stable during HCl
acid hydrolysis, thus necessitating the use of correction factors (Booth, 1971). The
guanidination method involves the reaction of O-methylisourea (OMIU) and lysine to
form acid-stable homoarginine. The protein is then HCl acidhydrolyzed and the liberated
homoarginine quantified along with the other acid stable amino acids. The guanidination
method relies on complete conversion of lysine to homoarginine. Complete conversion
can be observed in unprocessed protein by the absence of any lysine peak, but, in a
processed protein source, it is not possible to know with certainty whether the presence of
lysine is due to reverted lysine or whether guanidination was incomplete. Consequently, a
number of quality-control measures should be undertaken to ensure that the determination
of reactive lysine is accurate. These include running pure unprocessed standard proteins Chemical
alongside the unknowns, grinding insoluble samples to at least 0.5 mm, and ensuring the Analysis
11.9.7
pH of the OMIU solution is above 12 (Moughan and Rutherfurd, 1996) after preparation,
since the pH is an indicator of the conversion of the OMIU hemisulfate to OMIU free
base.
Homoarginine can most easily be separated and quantified using ion-exchange HPLC,
where it elutes late in the chromatogram, following arginine. Most other amino acids that
can be determined using HCl hydrolysis in unguanidinated samples can be determined in
guanidinated samples. The main exception is arginine, which coelutes with the extremely
large ammonia peak derived from the hydrolysis of excess OMIU.
Ensuring an oxygen-free environment during hydrolysis

Conducting hydrolysis in an oxygen-free environment enhances the recovery of several
amino acids, most notably methionine. There are several means of achieving an oxygen-
free environment, including using the reflux method for hydrolysis (FAO/WHO, 1991;
Kroll et al., 1998), sparging with an inert gas, sealing hydrolysis tubes under vacuum
(FAO/WHO, 1991; Yamada et al., 1991; Weiss et al., 1998), and both sparging with an
inert gas and sealing tubes under vacuum. There are also a range of methods for sealing
tubes, including using screw-cap lids, screw-down stopcocks, and melting glass tubes or
ampules.
Hydrolysis using the reflux method is suitable for removing oxygen from the sample-
HCl mixture. However, only a small number of samples can be hydrolyzed at one time,
making this method impractical for most laboratories (Rayner, 1985). This method is
unsuitable when sample quantity is low, but suitable for very heterogeneous samples
when large sample sizes are required.
The second approach involves sparging the sample with an inert gas, usually nitrogen.
Using a Pasteur pipet attached to an inert gas source, the gas is gently bubbled through
the sample. The main drawback of this approach is that sample can be lost on the tip of
the pipet. Sparging can also be conducted by flushing the headspace rather than bubbling
gas through the sample. However, sparging the headspace alone will not remove oxygen
present in the sample. Inert gas can also be bubbled through the hydrolyzing solution
before adding it to the sample. If the latter step is followed by headspace flushing, this
is a reasonable alternative to bubbling gas through the sample itself. Often, insufficient
methodological detail is published to allow the reader to ascertain which type of sparging
is used (Malmer and Schroeder, 1990; Rodrguez et al., 2007).
The third approach involves sparging with an inert gas and degassing under vacuum,
and requires specialized hydrolysis tubes. The tubes have a side arm or similar device
that is connected to an inert gas source and a screw-cap lid or screw-down stopcock.
The vacuum is applied, and when bubbling stops, the tube is flushed with inert gas.
The cycle can be repeated as often as required. The tubes can be sealed under vacuum
but do not have to be. This method is arguably the best at producing an oxygen-free
environment, and is particularly suitable for small sample numbers. However, with large
sample numbers this method may not be suitable, as the specialized hydrolysis tubes can
be very expensive. Furthermore, since the tubes are sealed only with a cap or stopcock,
air leakage can occur during hydrolysis.
The fourth approach involves connecting a tube or lyophilization ampule to a vacuum

pump. The glass tube usually needs to be stretched first to produce a narrow section near
the top of the tube. The vacuum is slowly applied until the air in the sample and HCl
bubbles out. Caution must be taken to avoid violent bubbling or bumping, as sample can
be lost from the tube. When bubbling ceases, the narrow section of the tube or ampule is
Amino Acid melted shut to seal the tube.
Analysis
11.9.8
Rutherfurd (2008) derived the actual amino acid contents (except cysteine, methionine
and tryptophan) of five complex feedstuffs using least-squares nonlinear regression and
compared these values to those determined using the sealed-tube method to remove
oxygen followed by 24-hr HCl acid hydrolysis. Overall, across all the amino acids, there
was only 2.5% difference between the actual amino acid content and the 24-hr hydrolysis
values. Furthermore, for labile amino acids like serine and threonine, the losses observed
using the sealed tube method were only 3% and 4.4%, respectively, much lower than the
5% to 10% often reported (Ozols, 1990; Darragh et al., 1996). Rutherfurd et al. (2007a)
have also reported that 103% of the methionine present in five complex feedstuffs
was recovered using this evacuated sealed tube approach when compared to the actual
methionine content determined using least-squares nonlinear regression. The sealed-tube
approach is an effective technique for removing oxygen from hydrolysis tubes, permits
high sample throughput, and is relatively inexpensive. The method is described in detail
in Basic Protocol 1.
Hydrolyzing acids
HCl is by far the most commonly used hydrolyzing agent, although others have been
tried. Methanesulfonic acid (MSA) has been used for amino acid analysis, particularly
tryptophan, methionine, and cysteine. The main disadvantage with MSA is that it is not
volatile, so the acid cannot be evaporated after hydrolysis. In addition, MSA cannot
be used for vapor-phase hydrolysis. Malmer and Schroeder (1990) used 4 M MSA to
determine the amino acid content of cytochrome c and reported recoveries slightly lower
than those commonly obtained using HCl hydrolysis. Weiss et al. (1998) used two pure
proteins of known sequence and reported that MSA hydrolysis gave data slightly more
accurate than conventional hydrolysis based on the identification of the proteins from the
amino acid data. They did not however, present any actual amino acid recovery data for
the MSA hydrolysis method.
Hydrolysis time and temperature

HCl acid hydrolysis is usually carried out at 110 C for 20 to 24 hr (FAO/WHO, 1991;
Ozols, 1990; AOAC, 1995; Darragh and Moughan, 2005). However, a number of studies
have been conducted exploring hydrolysis at higher temperatures and reduced hydrolysis
times in order to reduce amino acid losses, particularly for cysteine, methionine (Gehrke
et al., 1985; McDonald et al., 1985), and tryptophan (Muramoto and Kamiya, 1990;
Molnar-Perl et al., 1993).
Rogers et al. (1954) conducted HCl acid hydrolysis at 145 C in a sealed tube and achieved
complete hydrolysis of gelatin in 1.5 hr. Yamada et al. (1991) carried out vapor-phase
hydrolysis with 2.5 M mercaptoethanesulfonic acid at 176 C in vacuo for 12.5 and
25 min, and obtained 85% to 92% recoveries of tryptophan. The recoveries of the acid-
stable amino acids in lysozyme and myoglobin were in excess of 94%, with the exception
of serine and threonine, which were 85% and 89%, respectively. Nakazawa and Manabe
(1992) used a similar hydrolysis method to hydrolyze a range of pure proteins on PVDF,
and reported that the determined amino acid amounts were very similar to the theoretical
amounts. However, they drew this conclusion based on the relative amounts of amino
acids present, rather than the absolute amounts. The overall yields were actually low
(26% to 40%), although how much of this was due to incomplete transfer of the proteins
from the SDS-PAGE gels to the PVDF membrane and how much was due to hydrolytic
losses is unclear. Nakagawa and Fukuda (1989) also attempted to carry out amino acid
analysis of protein bands on PVDF. They used 5.7 M HCl acid hydrolysis at 110 C for
24 hr in vacuo, and reported poor recoveries of tryptophan.
Despite these and other studies, 24 hr at 110 C remains the most used hydrolysis time Chemical
Analysis
and temperature for amino acid analysis.
11.9.9
Microwave hydrolysis
Microwave radiation has been used to replace conventional convection as a means of
providing the energy necessary to hydrolyze proteins. The main advantage is the consid-
erably reduced hydrolysis time (as little as 4 min in Weiss et al., 1998). Kroll et al. (1998)
conducted a study investigating the use of microwave radiation for HCl acid hydrolysis of
proteins. They found that for a selection of pure proteins, incubation for at least 2 hr was
necessary for complete hydrolysis. They reported excellent recoveries (>95%) for all
amino acids except tryptophan, methionine, and cysteine. These workers also compared
microwave hydrolysis with conventional acid hydrolysis and found excellent agreement
between the two methods, although methionine was not examined. Messia et al. (2008)
used a 20-min microwave digestion in 6 M HCl to determine the hydroxyproline con-
tent of a range of meat-based foods, and found that these values compared well with
hydroxyproline determined using conventional acid hydrolysis (in vacuo in a stoppered
tube).
A number of reports have shown that for microwave hydrolysis, the hydrolysis time is
important if quantitative yields of amino acids are to be obtained. Weiss et el. (1998)
showed that for several proteins 20-min and 45-min hydrolysis resulted in amino acid
yields significantly lower than the theoretical yield, while 30-min hydrolysis resulted in
satisfactory yields. Given that there was only a 10- to 15-min difference between good
amino acid yields and poor yields, the hydrolysis time would need to be selected with
care. Weiss et al. (1998) also showed that for human interferon and BSA, microwave
hydrolysis (liquid and vapor phase) produced similar amino acid yields to conventional
(liquid and vapor phase) hydrolysis, with the exception perhaps of threonine, which
was particularly low with the microwave-liquid hydrolysis method. Weiss et al. (1998)
also examined the use of HCl gas-phase/microwave hydrolysis of PVDF-bound protein.
Compared to conventional gas-phase hydrolysis, the recovery of some amino acids
(lysine, phenylalanine, methionine, arginine, threonine, histidine, serine, and glutamic
acid) was poor.
Overall there appears to be considerable promise with the use of microwave hydrolysis
for amino acid analysis; however, a number of factors need to be taken into account.
The radiation within the oven needs to be spatially uniform throughout the chamber;
otherwise, hydrolyses will differ depending on the tube placement within the oven. The
amount of liquid within the oven needs to be similar from run to run, since smaller
volumes will heat more quickly than larger volumes. It is also important to use ovens for
which the power output is known accurately, since different ovens, even with the same
power rating, may have differing power outputs.
Antioxidants and scavenging agents

Some amino acids cannot always be recovered completely during acid hydrolysis, and
antioxidants and oxygen scavengers are sometimes added to the hydrolyzing acid to
improve amino acid yields. All these additives probably act as free-radical scavengers
(Molnar-Perl, 1997). Some common additives include phenol (Muramoto and Kamiya,
1990; Rowan et al., 1992; Weiss et al., 1998; Rutherfurd, 2008), thioglycollic acid
(Meltzer et al., 1987; Nakagawa and Fukuda, 1989; Shindo et al., 1998), and tryptamine
(Molnar-Perl and Khalifa, 1992) for HCl acid hydrolysis, although not all laboratories
use additives (Kroll et al., 1998; Messia et al., 2008).
Vapor-phase hydrolysis
Vapor-phase hydrolysis involves hydrolysis of a sample in a sealed, evacuated container
also containing the hydrolyzing acid (usually HCl), but where the sample is not placed
Amino Acid
Analysis directly in the acid. The vacuum reduces the vapor pressure of the HCl, and the resulting
11.9.10
vapor envelops the sample. This method is useful if there are impurities, for example
heavy metals, in the hydrolyzing acid. Meltzer et al. (1987) carried out vapor-phase
HCl acid hydrolysis by placing hydrolysis tubes containing human serum albumin in an
evacuated desiccator containing a beaker of constant-boiling 6 M HCl. They reported
amino acid recoveries similar to those observed using liquid hydrolysis for all amino
acids except serine, for which the recovery was superior with gas-phase hydrolysis.
Higbee et al. (2003) described an automated system for gas-phase hydrolysis based on
the Waters Pico Tag workstation. They reported amino acid recoveries for bovine serum
albumin within 2.7% of the expected values. Higbee et al. (2003) commented that the
system requires operator skill and time to evacuate the specialized sample containers
without losing sample material through bumping, and that oxygen removal is not always
complete.
Vapor-phase hydrolysis is often used to hydrolyze proteins that have been blotted onto
PVDF membranes using HCl and MSA (Nakagawa and Fukuda, 1989; Nakazawa and
Manabe, 1992). Since proteins derived from gels are usually present only in low pico-
molar amounts, contamination from hands, chemicals, or equipment must be stringently
minimized.
Golaz et al. (1996) compared the amino acid composition of three purified proteins on
PVDF using vapor-phase HCl acid hydrolysis. While the determined and theoretical
proportions of some amino acids within the protein were similar, there were many which
compared poorly for some of the proteins tested, particularly lysine, tyrosine, and valine.
The reproducibility of the method was also relatively poor. Shindo et al. (1998) used
vapor-phase hydrolysis of PVDF-bound bovine serum albumin, and reported relative
proportions of amino acids compared to theoretical proportions. They found relative
recoveries ranging from 54% for aspartic acid to 188% for phenylalanine, but made no
comment about the rather poor amino acid recovery data. Absolute amino acid recoveries
were not given in any of the work described above, probably because protein transfer
losses from gels to PVDF membranes cannot easily be overcome.
Least-Squares Nonlinear Regression Analysis

The technique of using multiple hydrolysis times and extrapolating amino acid yields
back to 0-hr hydrolysis has been used to more accurately determine acid-labile amino
acids such as serine and threonine (Rowan et al., 1992). Moreover, hydrolysis times
greater than 24 hr have been used to determine amino acids such as isoleucine and valine
(Rowan et al., 1992). Often, hydrolysis times between 12 hr and 72 hr are used when
extrapolation to either 0 hr (serine and threonine) or hydrolysis for 72 to 96 hr (isoleucine
and valine) is required (Rayner, 1985; Rowan et al., 1992). This method assumes that
amino acid hydrolysis and degradation occur consecutively. In reality, however, when
a protein is hydrolyzed, the liberated residues for any given amino acid will begin to
degrade, while other residues of the same amino acid are still protein- or peptide-bound.
Robel and Crane (1972) recognized this, and were the first to use least-squares nonlinear
regression to estimate amino acid content assuming concurrent amino acid hydrolysis
and degradation. Multiple hydrolysis times are used to plot amino acid yield against
hydrolysis time, and the iterative process of least-squares nonlinear regression, using the
following equation, is then applied:
A h ( lt ht )
B (t ) = 0 e e + B0 (elt )
hl
where e is the exponential function, B(t) is the amino acid concentration at time t, B0 is Chemical
the free amino acid concentration prior to hydrolysis, h is the hydrolysis rate of the amino Analysis
11.9.11
acids (the rate at which the amino acids are released from the protein), l is the loss rate
of the amino acids (the rate at which the amino acids are lost after hydrolysis), and A0 is
the actual protein bound amino acid content. This method has been applied to lysozyme
(Robel and Crane; 1972; Darragh et al., 1996), human milk (Darragh and Moughan,
1998), cat hair (Hendriks et al., 1998), infant formulas (Rutherfurd et al., 2008), skim
milk powder, canola meal, corn meal, soybean meal, and meat and bone meal (Rutherfurd
et al., 2007a; Rutherfurd, 2008). While this method may not be suitable for routine use,
given that multiple hydrolysis intervals are required, it does have application where very
accurate amino acid composition data are required.
Chromatography
HPLC
Many different chromatographic techniques have been used to separate and quantify
amino acids, including paper chromatography, thin-layer chromatography, low-pressure
ion-exchange chromatography, ion-exchange HPLC, reversed-phase HPLC, gas chro-
matography, capillary electrophoresis, and mass spectrometry. Ion-exchange chromatog-
raphy of amino acids is an elegant technique, and was originally developed by Moore
and Stein (1951) in the early 1950s using a stepwise pH gradient to separate amino acids.
The original method was slow, taking up to 16 hr to complete one run. With the devel-
opment of HPLC, run times were greatly decreased, to 1 to 2 hr for ion-exchange and
20 to 40 min for reversed-phase chromatography. Recently, Ultra High Pressure Liquid
Chromatography (UPLC) systems have been developed, which can withstand back pres-
sure of up to 15,000 psi and permit the use of reversed-phase resins with a 1.8-m pore
size. While the lower pore size generates much higher back pressures compared to more
conventional reversed-phase columns, the increased resolution is dramatic, resulting in
run times for amino acid hydrolysates of <10 min.
Currently, the most commonly used chromatographic techniques for separating and quan-
tifying amino acids include ion-exchange chromatography using post-column derivati-
zation and reserved-phase chromatography using pre-column derivatization. For ion-
exchange chromatography a cation-exchange column is employed. The separation uti-
lizes the amino acids zwitterion properties where, due to the presence of amino and
carboxyl groups, the amino acids can be either negatively or positively charged, de-
pending on the pH of the environment. During analysis, the hydrolysate is loaded onto
the column in low-pH buffer (usually pH 2), and the positively charged amino acids
bind to the column. Amino acid elution is facilitated using an increasing pH gradient.
As the pH of the eluant reaches the isoelectric point of an amino acid, the amino acid
becomes neutrally charged and is eluted. Amino acids, with the exception of tryptophan,
tyrosine, and phenylalanine, cannot be easily detected using absorbance. Consequently,
for ion-exchange chromatography, the amino acids must be derivatized after elution from
the column. Ninhydrin and o-phthaldialdehyde (OPA) are the two most commonly used
derivatizing reagents.
The other common chromatographic method for separating amino acids is based on
reversed-phase chromatography, usually using a C18 column and an acetonitrile-based
gradient. Amino acids are not well resolved using reversed-phase chromatography, and
only the hydrophobic amino acids such as tryptophan, tyrosine, and phenylalanine are
separated with any degree of success. Consequently, amino acids must be derivatized prior
to reversed-phase chromatography. Derivatization has two functions: (1) to render amino
acids more hydrophobic and (2) to permit detection using absorbance or fluorescence.
The specific buffers and gradient conditions required for separation of derivatized amino
acids depend on the derivatizing agent being used. A vast number of papers have been
Amino Acid
Analysis published on the topic of amino acid derivatization, e.g., 1600 for OPA alone (Hanczko
11.9.12
et al., 2007) and it is certainly not the aim of this unit to discuss all the published data.
Instead, a discussion of the more salient points of the most common post-column and
pre-column derivatization methods is presented.
The advantages of pre- and post-column derivatization were succinctly described by

Bardelmeijer et al. (1998), and while their comments were made in relation to capillary
electrophoresis, they are applicable to HPLC as well. Briefly, the advantages of post-
column derivatization are: side reactions are unimportant, complete derivatization is not
required as long as the extent of derivatization is reproducible, and the derivative need
not be stable. The disadvantages include a limited choice of reaction conditions, the need
for reagent-delivery equipment, possible band broadening due to reagent introduction, a
longer flow path from column to detector, and possible reagent interference during de-
tection. The advantages of pre-column derivatization include a greater choice of reagents
and reaction conditions, no limit on reaction kinetics, and the possibility of multistep
derivatizations. The main disadvantage is possible chromatographic interference from
side products or unreacted derivatizing reagents.
Post-column derivatization
The ninhydrin reaction for post-chromatographically separated amino acids was first
conducted by Moore and Stein (1948). Ninhydrin reacts at elevated temperatures (100
to 120 C) at approximately pH 5 with primary amines such as the -amino group
of amino acids and the -amino group of lysine to produce diketohydrindylidene-
diketohydrindamine (Fig. 11.9.1A). This compound, known as Ruhemanns purple, has
an absorbance maximum at 570 nm. Ninhydrin will also react with secondary amines
such as the N-terminus of proline and hydroxyproline, but the sensitivity is less and the
absorbance maximum of the product is 440 nm. The detection limit of this method is
10 pmol (50 pmol for proline).
OPA is the other major post-column derivatizing reagent, and was first used as such by
Roth and Hampai (1973). OPA reacts with the -amino group of amino acids in the
presence of a thiol (often 2-mercaptoethanol) to produce a substituted isoindole ring
(Fig.11.9.1B) but, unlike ninhydrin, does not react with proline and only poorly with
cysteine. Proline can be derivatized with OPA, however, after oxidation with sodium
hypochlorite. A major advantage of OPA is that it is a fluorogenic compound, i.e.,
the OPA-amino acid derivative fluoresces strongly ( excitation 350 nm, emission
450 nm) while the unreacted OPA does not. Bensen and Hare (1975) compared the
sensitivity of OPA, ninhydrin, and the fluorescent reagent fluorescamine using post-
column derivatization. They reported that OPA was 5 to 10 times more sensitive than
either fluorescamine or ninhydrin.
The inability of OPA to derivatize proline has been utilized to determine felinine
(Hendriks et al., 2001; Rutherfurd et al., 2007b), a unique sulfur-containing amino acid
present in large quantities in the urine of uncastrated male cats. This amino acid coelutes
with proline when using ion-exchange chromatography, but after OPA derivatization,
proline is not detected, and as a result, the felinine peak can be integrated unobscured.
OPA can also be used for pre-column derivatization followed by reversed-phase HPLC,
and this is discussed below.
Pre-column derivatization
PITC
Phenylisothiocyanate (PITC) reacts with the N-terminal of amino acids, including pro-
line, to produce phenylthiocarbamyl (PTC)amino acids, which have an absorbance Chemical
Analysis
maximum at 254 nm (Fig. 11.9.1C). Precolumn derivatization with PITC for amino
11.9.13
A O O O
O
OH R OH N
OH NH2
O O O
ninhydrin amino acid Ruhemann's purple
B R
O S
O O
H R RSH
OH N
H OH
NH2 R
O
OPA amino acid OPAamino acid
H H O
C S O
C N CN
R OH
N OH
S R
NH2
PITC amino acid PTCamino acid
D
H H H O
O
N O O N N
N OH
R OH
O O R
N NH2 N
O
AQC amino acid AQCamino acid
E
O O R
O OH
O Cl R O N
OH
NH2 H O
FMOC amino acid FMOCamino acid
Figure 11.9.1 Derivatization reactions for amino acid analysis.
acid analysis was first described by Bidlingmeyer et al. (1984). Waters (http://www.
waters.com) developed the Pico-Tag amino acid analysis system to facilitate use of this
method for small sample amounts. The reaction is largely complete within 20 min, after
which the sample must be dried down to remove excess reagent. This dry-down step is
one of the main disadvantages of the method, making automation difficult. Bidlingmeyer
et al. (1984) reported that PTC derivatives are stable for several weeks if stored frozen,
and for 3 days if kept cold, while others have reported stability for 16 hr when stored at 5
to 8 C (Janssen et al., 1986). At room temperature, the amino acids are much less stable,
with stability varying markedly across amino acids (Guitart et al., 1991). PTC-amino
acids are usually separated using C18 reversed-phase chromatography, and the practical
detection limit of this method is about 1 pmol.
AQC
Derivatization of amino acids with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate
(AQC) produces highly fluorescent derivatives ( excitation 250 nm, emission 350 nm;
Amino Acid Fig. 11.9.1D). The reaction is rapid, taking only 1 min at room temperature to complete.
Analysis After derivatization, the mixture is heated to 55 C to convert the minor side product
11.9.14
of tyrosine to the major mono-derivatized compound (Cohen and Michaud, 1993) and
to hydrolyze the AQC reagent to 6-aminoquinoline, N-hydroxysuccinimide, and carbon
dioxide. The derivatives are stable at room temperature for up to a week, and the detection
limit of the method is about 40 to 800 fmol, depending on the amino acid (Cohen and
Michaud, 1993). The AQCamino acids can be separated using C18 reversed-phase chro-
matography using an acetonitrile-based gradient. Bosch et al. (2006) tested the accuracy
of the method when applied to infant formulas and pure proteins, and reported excellent
overall (interday and intraday) precision for the method (0.24% to 3.49%) across almost
all amino acids tested. Methionine sulfone was the exception, with an overall precision
of 6.74%. The accuracy of the derivatization method was assessed by determining the
recovery of spiked amino acids added to infant formula. The recoveries ranged from
86% for valine to 106% for glutamic acid. The authors deemed the method sufficiently
accurate to permit the determination of amino acids in infant formula; however, given
the relatively wide range in recoveries, that conclusion is debatable.
FMOC
9-Fluorenylmethyl chloroformate (FMOC) reacts with both primary and secondary amino
acids in a very rapid (30-sec to 1-min) reaction to produce fluorescent derivatives ( exci-
tation 260 nm, emission 313 nm; Einarsson et al., 1983; Fig. 11.9.1E). The derivatives
are stable at 4 C for at least a week, with only 3% losses in amino acid yields over that
time (Lozanov et al., 2004) and negligible losses at room temperature after 48 hr (Lozanov
et al., 2004). The detection limits for FMOC derivatives are in the low fmol range. One
drawback of FMOC derivatization is that the reagent can be hydrolyzed and decarboxy-
lated in the presence of water to produce a fluorescent alcohol which can coelute with
other amino acids (Bank et al., 1996). Malmer and Schroeder (1990) reported a strategy
to largely overcome this problem, whereby derivatization is conducted on-line and the
reaction mixture injected immediately onto the column, thereby minimizing the time
during which side reactions can occur. Lozanov et al. (2004) used a stronger-capacity
buffer and the reagent FMOC-O-succinimide instead of the usual FMOC-Cl to reduce
the reaction with hydroxyl groups, and therefore reduce the amount of FMOC alcohol
generated. Other workers have used quenching agents such as heptylamine (Kirschbaum
and Luckas, 1994) or other highly hydrophobic amines that react with excess FMOC but
elute remote from the FMOC-amino acid peaks (Melucci et al., 1999). Bank et al. (1996)
proposed a method by which FMOC derivatization is carried out in 0.1 M borate buffer
at pH 11.4 for 40 min followed by pentane extraction, which removes FMOC alcohol.
An additional disadvantage of FMOC derivatization is the production of multiple deriva-

tives of tyrosine and histidine (mono- and disubstituted forms). Bank et al. (1996) reported
that the longer incubation time and high pH they used resulted in the presence of only the
monosubstituted tyrosine (N-FMOC-Tyr), since at high pH carbamates are not particu-
larly stable and the FMOC substituent on the phenol is particularly labile. Lysine is also
di-derivatized (on the - and -amino groups), but, since there are no reports of both the
mono- and disubstituted lysine being present after FMOC derivatization, it would appear
that lysine is quantitatively converted to the disubstituted form only.
OPA-FMOC
As discussed above, OPA is a commonly used derivatizing agent for the detection of
amino acids. However, its main drawback is its inability to react with secondary amino
acids. Recently, a new method has been proposed that uses a two-step derivatization
with both OPA and FMOC for biogenic amines as well as lysine and ornithine (Hanczko
et al., 2005) and all amino acids (Koros et al., 2008). This method has been reviewed
(Hanczko et al., 2007) in detail. Briefly, the initial reaction is conducted with OPA Chemical
so all primary amino acids are derivatized to OPA-amino acids. The second step uses Analysis
11.9.15
FMOC, which then derivatizes the secondary amino acids (proline and hydroxyproline).
Each derivatization is conducted at room temperature for 1 min, giving a total reaction
time of 2 min. FMOC, FMOCamino acid derivatives, and FMOC alcohol, are more
hydrophobic than the OPAamino acid derivatives, and thus do not interfere with the
elution of the OPA amino acids. Furthermore, FMOC-proline and FMOC-hydroxyproline
are the only FMOC amino acid derivatives present, and also elute more rapidly than
unreacted FMOC or FMOC alcohol. Consequently, the chromatographic interference
from FMOC or FMOC alcohol traditionally observed does not pose a problem with
this detection system. In addition, when FMOC derivatization is carried out, no free
tyrosine and histidine exist, so the problem of disubstituted tyrosine and histidine during
FMOC derivatization is overcome. Some workers have used ethanethiol as an additive
for OPA/FMOC derivatization (Koros et al., 2008) while others report that OPA amino
acid derivatives are more stable when 3-mercaptopropionic acid is used (Woodward
and Henderson, 2007). Butikofer et al. (1991) compared the pre-column OPA/FMOC
derivatization and reversed-phase chromatography with ion-exchange chromatography
for several feedstuffs. They found, first, good agreement between the two methods,
and, second, that the sensitivity of the OPA/FMOC method using florescence detection
was 5 to 20 times greater than for the ion-exchange method. Dorresteijn et al. (1996)
reported sensitivity down to 136 fmol for amino acids using pre-column derivatization and
reversed-phase chromatography. However, in practice, the limit of detection is probably
closer to 1 pmol.
The OPA/FMOC pre-column derivatization method does require an absorbance detector,

or preferably fluorescence detector, capable of switching wavelengths during the run,
since OPA and FMOCamino acids have different excitation and emission wavelength
maxima. Most modern detectors however, should be capable of performing this function.
The relatively simple and rapid nature of the derivatization has facilitated its adoption as
an on-line pre-column derivatization methodology, and published protocols are available
from some HPLC manufacturers. Agilent Technologies has reported a procedure for
OPA/FMOC detection which, with the advent of UPLC systems, results in run times of
10 min.
It is important to note that while the detection limit of a derivatization method is often
given, it may not reflect the minimum amino acid concentration that can be accurately
determined in a sample. If baseline resolution cannot be achieved between amino acids
during chromatography, then practical concentration limits for accurate quantitation may
be 10 to 20 times higher than the detection limit for a pure amino acid. In addition, sample
matrices can affect baseline noise, which will also impact on practical concentration limits
for accurate quantitation.
Detection of underivatized amino acids

A number of methods are available for detecting underivatized amino acids, and these
include low-wavelength UV absorbance, conductivity, nuclear magnetic resonance, re-
fractive index, evaporative light scattering, chemiluminescent nitrogen, and mass spec-
trometry. These methods have been well reviewed (Elfakir, 2005). In addition, Petritis
et al. (2002) has compared the methods listed above and concluded that tandem mass
spectrometry and evaporative light scattering along with chemiluminescent nitrogen were
the most promising methods of detection with higher sensitivity and greater selectivity.
Gas chromatography
Gas chromatography (GC) methods for determining amino acids have been available for
many years and have been reviewed (Peace and Gilani, 2005). GC amino acid analysis
Amino Acid is not used extensively in comparison with HPLC since impure samples, such as foods,
Analysis
11.9.16
require a clean-up step prior to GC chromatography and because amino acid composition
data generated using GC tend to be more variable than the equivalent data generated using
HPLC.
Capillary electrophoresis
Capillary electrophoresis is a relatively new method for amino acid separation and is
based on the use of an electric field to separate charged analytes such as amino acids.
The amino acid solution is loaded into one end of the fused-silica capillary filled with a
conducting buffer. An electric field is applied across the two ends of the capillary and the
amino acids migrate through the capillary at different rates based on their charge. The
amino acids are then detected using a range of detectors similar to those used for HPLC.
Capillary electrophoresis is not commonly used, but does offer superior resolution and
sensitivity compared to HPLC techniques. Column efficiencies of one million plates
are commonly reported and separation of compounds differing only in their isotopic
mass has even been reported (Yeung and Lucy, 1999), as have sensitivities down to a
few molecules. A thorough discussion of this method for amino acid analysis has been
published by Smith (1997).
Amino Acid Enantiomers

Common methods of analysis are not able to distinguish between D- and L-amino acid
enantiomers. L-amino acids are the predominant amino acid enantiomer present in na-
ture. Although D-amino acids are produced by microorganisms, they can be present in
foods as a result of heat processing at alkaline pH (Liardon and Hurrell, 1983). Amino
acid enantiomers can be determined directly by use of chiral columns, such as the Astec
HPLC column from Supelco or the Chirasil-L-Val capillary GC column (Chromtech;
http://www.chromtech.com), and indirectly using derivatization with a chiral-selective
reagent such as acetyl chloride-2-propanol in the presence of ethyl mercaptan (Liardon
and Hurrell, 1983), ethylchloroformate, and chloropropanol (Bertrand et al., 2008),
followed by separation on a nonchiral capillary GC column.
HPLC has also been used to separate derivatized amino acid enantiomers and
reagents such as N-isobutyryl-D-cysteine (Bruckner et al., 1994), N-isobutyryl-L-cysteine
(Bruckner et al., 1994), 1,5-difluoro-2,4-dinitrobenzene (Marfeys reagent; Marfey,
1984), and o-phthaldialdehyde/2,3,4,6-tetra-O-acetyl-1-thio--glucopyranoside (Einars-
son et al., 1987; Csapo et al., 2001). While there are a number of relatively simple indirect
derivatization methods reported for determining amino acid enantiomers, the chromato-
graphic separation is not straightforward, given the large number of compounds that must
be separated.
Use of Internal and External Standards

There are several types of standards that should be routinely used for amino acid analysis,
and these include calibration standards, internal standards, and external standards. A
calibration standard is a solution containing known concentrations of the amino acids of
interest. By comparing the retention time and the size of the peaks of the unknown sample
with those of the calibration standard, amino acids can be identified and quantified. A
common calibration standard is the Pierce Amino Acid H standard (Thermo Scientific),
which contains all the amino acids commonly present in a protein HCl acid hydrolysate.
Ideally, several different concentrations of calibration standard should be analyzed, and a
standard curve prepared. However, for ion-exchange chromatography, run times can be as
long as 1 to 2 hr, making it impractical to run more than one or two calibration standards
with each batch of samples. Calibration standards should be run at the beginning of
each sample batch and also periodically throughout a sample batch, for example, every Chemical
Analysis
11.9.17
10 samples. This provides a measure of repeatability for the analysis, which is important
for assessing the quality of amino acid composition data.
Amino acid analysis is a complex multistep procedure and can produce variable results
even within a given batch of samples. The use of internal and external standards is im-
portant in reducing data variability. Internal standards are added to samples and analyzed
along with the compounds of interest. The recovery of the internal standard is usually
then determined, and should approximate 100%. Internal standards should be compounds
that are freely available in pure form and stable under the conditions to which they will
be subjected, and that separate chromatographically from all other compounds and are
detectable using the chosen detection method. Some examples of internal standards used
for amino acid analysis include norleucine, norvaline, and -aminobutyric acid. Internal
standards can be included after hydrolysis (Davies and Thomas, 1973; Sarwar et al.,
1988; Malmer and Schroeder, 1990) and in this case would act as a quality control for the
addition of buffers and reagents, the HPLC injection, and the efficiency of derivatization.
However, internal standards can also be added prior to hydrolysis, which would correct
for physical losses during hydrolysis (van der Meer, 1990). However, given that amino
acids are chemically diverse with quite different tolerances to hydrolytic conditions, using
an internal standard to monitor hydrolytic losses may not be appropriate. The recovery
of the internal standard can be used to correct amino acid composition data for physical
losses, and this practice may be acceptable if recoveries are almost complete (i.e., losses
<10%). However, if losses are >10%, then steps should be taken to remedy the cause of
the variability and the samples run again.
External standards are usually pure proteins for which the amino acid composition has
been determined by a method other than amino acid analysis, for example, sequence anal-
ysis. Glycoproteins should be avoided, since the protein content cannot be known accu-
rately given the possible variation in oligosaccharide content (Rutherfurd and Moughan,
2000). External standards should be analyzed in conjunction with each batch of sam-
ples, and the recovery of each amino acid in the external standard determined. While
the recovery of most amino acids should approximate 100%, for the acid-labile amino
acids (serine and threonine) and those difficult to hydrolyze (valine and isoleucine), the
recoveries would be expected to be lower. Since the external standard should be a pure
protein, the matrix is likely to differ from that of some samples, particularly foods and
biological samples. Consequently, it is not appropriate to correct amino acid composition
data for the recoveries of amino acids in the external standard. In contrast to internal
standards, external standards provide an insight into the proficiency of the entire amino
acid analysis process.
Use of Bound Versus Free Amino Acid Molecular Weight for Calculating Amino
Acid Content
Amino acids are held together within a protein by peptide bonds. During hydrolysis,
the peptide bond is cleaved and a water molecule is added across it. Consequently, the
molecular weight of a free amino acid is 18 Da heavier than its in chain counterpart.
Often, amino acid composition data are presented as the weight of the amino acid present,
e.g., for foods, units of g of amino acid per 100 g of food are commonly used. To calculate
the amount of amino acid present, the correct amino acid molecular weight must be
applied. If amino acid composition data are to be used to calculate the amount of protein
present in a sample, then using free amino acid molecular weights is not appropriate and
will lead to an overestimate of the amount of protein present. For individual amino acids,
the use of free amino acid molecular weights, when in chain or residue molecular
weights should be used, can lead to overestimates of between 9.7% for tryptophan and
Amino Acid 32% for glycine. Most amino acid composition data for foods are calculated using free
Analysis amino acid molecular weights, which represent, therefore, the amount of each amino acid
11.9.18
the human (or animal) would receive assuming the protein is completely digested, rather
than the amount of each amino acid present within the food proteins. While either method
of calculation is defendable, it is important that the end users of amino acid composition
data understand how the data were derived. Rutherfurd and Moughan (1993) conducted
an informal survey of a number of Australasian laboratories and animal feed companies
and found that many groups, including some laboratories, were unaware that either free
or in chain molecular weights could be used. Clearly, it is important for amino acid
analysis laboratories to outline their method of calculating amino acid weights.
Reducing Variability in Amino Acid Analysis: Practical Tips

There have been some very good reviews published regarding reducing the variability
observed during amino acid analysis, including an excellent review by Finley (1985).
Some of the main factors that contribute to variability with amino acid analysis are the
many variations of amino acid analysis methods and the fact that no single method is used
by all laboratories (Sarwar et al., 1983). The use of one universal amino acid analysis
method would be a major step forward for reducing the variation in amino acid data
(Sarwar et al., 1983). The likelihood of such a development is low, and in the meantime
other more practical approaches to reduce the variability of amino acid composition data
can be adopted.
The primary means of detecting variation in amino acid composition data is to conduct
analyses in duplicate. While this would seem obvious, there are a significant number
of laboratories that do not conduct duplicate amino acid analysis unless specifically
requested to do so. This is exacerbated by the fact that it is often not made clear to
potential clients that only a single analysis is conducted. This is an area of heated debate
among analysts who must weigh the need to produce accurate and precise data against the
additional cost of duplicate analysis. At the very least, the client should be made aware
of the weakness of single analysis and be given the option of duplicate determinations.
Other measures for reducing analytical variation include ensuring hydrolysis tubes are
free of contaminating amino acids. This can be done by pyrolyzing tubes before use and
never touching the tops of the tubes unless gloves are worn.
Poor sample homogeneity, as is often observed with food samples, is a major contributing
factor to high analytical variation, and such samples must be prepared in a manner that
minimizes heterogeneity. Usually, the bulk sample is subsampled, dried if necessary,
finely ground, and mixed prior to analysis. Suitable subsampling techniques such as
quartering, where small samples are taken from each quarter of the bulk sample, need
to be used. Core sampling, in addition to surface sampling, may be necessary for larger
bulk samples. In order to obtain core samples from the center and lower parts of sample
containers, the use of specialized core-sampling equipment may be required. Sample
grinding is an important step for facilitating representative sampling, and this topic is
discussed in more detail above. Regardless of the difficulties, obtaining a representative
sample is vital to reducing analytical variability, and the time invested in the sample
preparation phase of the analysis will always be beneficial.
Sample weighing is the next step for most analyses. Scales should be in good working
order and calibrated regularly. Most modern balances have an internal calibration that
can easily be used by laboratory staff. However, periodically this calibration system must
also be checked. Ideally this should be done using certified agencies; however, if this
is not possible, then it should be carried out by trained staff using certified calibration
weights. Samples should be equilibrated at room temperature prior to weighing, to
prevent absorption of water from the atmosphere. For liquid samples, any pipets that
Chemical
are used should be calibrated regularly and the degree of variability of the dispensing Analysis
11.9.19
volume known. The accuracy of the pipet can be determined by dispensing a known
volume of deionized, degassed water and weighing the water. Since the density of the
water is 1.00 g/ml at 4 C and atmospheric pressure, the actual volume dispensed can
be calculated and compared with the intended volume. The precision of the pipet can
be determined by repeating the process multiple times.
Once the samples have been weighed into hydrolysis tubes and hydrolyzing acid added,
the samples are usually either degassed under vacuum or flushed with nitrogen. For
either approach, care must be taken to avoid physical losses. Blowing nitrogen into
samples under pressure can blow sample out of the tube if the nitrogen pressure is too
high. Furthermore, sample can stick to the nitrogen tube outlet if it is placed into the
hydrolyzing solution. When degassing under vacuum, care must be taken to avoid sample
bumping out of the tube or, if using flame sealing, onto the section of the tube that will
be flame sealed.
The next step of the amino acid analysis procedure is the hydrolysis step. This is usually
carried out in a forced air oven, but can also be done using a microwave oven. The
spatial temperature distribution of the oven should be checked periodically to ensure
that the temperature is constant at any position. This is best done by a certified agency,
but if one is not available, then it is best to always incubate tubes in the same place in
the oven. While for most amino acids small temperature differences during incubation
will have little impact on their amino acid yield, labile amino acids such as serine,
threonine, methionine, cysteine, and tryptophan, and amino acids that are more difficult
to hydrolyze, such as isoleucine and valine, may be affected.
Internal standards are often added after hydrolysis, and the accuracy of this step is critical.
The internal standard solution should be prepared with the utmost care by experienced
staff, making sure that the content of internal standard is known accurately. Weighing
must be conducted with extreme care, and it may also be necessary to correct for the
moisture content of the internal standard when calculating the final concentration. It
is also important that the same internal standard solution be added to samples and
calibration standard. This ensures that any inaccuracy in the concentration of internal
standard solution will not affect the calculation of the internal standard recovery in the
samples, since the same concentration will be applied to both the calibration standard
and unknown samples.
If the samples are to be derivatized prior to the chromatography step, then solution
addition needs to be accurate, as differences in derivatizing agent or buffer concentrations
may affect the extent of derivatization. Ideally, and if possible, it is better if this step
can be carried out using an automated system rather than manually. The extent of
derivatization can also affect the accuracy of amino acid composition data, particularly
if the derivatization is affected by the sample matrix. Post-column derivatization is
arguably more robust from a data-variability standpoint, since reagent addition, reaction
temperature, and time are strictly controlled. In addition, amino acids are derivatized in
the HPLC buffer rather than the sample matrix, and while this buffer will differ for each
amino acid depending on retention time and the buffer gradient, it will be similar for
each amino acid in both the standard and sample solutions.
The chromatography step is a complex part of amino acid analysisseparating 16 to

20 amino acids from each other and other interfering compounds is a demanding task.
Injection volumes need to be accurate, all buffers must be prepared with ultrapure water,
and the HPLC must be in optimal working order and well maintained. Baseline separation
of all amino acids should be the goal, and, where this is not possible, considerable
Amino Acid care must be taken when integrating amino acid peaks. A good understanding of the
Analysis chromatogram, its baseline, and how to deal with overlapping peaks is essential. Subtle
11.9.20
changes in the baseline, which can go unnoticed without careful inspection of each
chromatogram, can easily alter amino acid values by 5% or more. While the method of
integration may differ between amino acids, it should be consistent for each amino acid
between samples and standards.
Inter-laboratory proficiency programs are useful for evaluating the variability of amino
acid analysis between laboratories, and many papers have been published describing
such studies (Sarwar et al., 1985; Davies et al., 1992; Landry and Delhaye, 1994; Llames
and Fontaine, 1994). Sarwar et al. (1985) reported that intra-laboratory coefficients of
variation (CVs) for duplicate analyses of the essential amino acids in seven different
proteins ranged from 0.6% to 4.7%. However, inter-laboratory CVs for tryptophan, cys-
teine, and methionine were 23.7%, 17.6%, and 16.1%, respectively. Davies et al. (1992)
had three pure proteins and six feedstuffs analyzed in five laboratories, using different
analytical methods. For all amino acids excluding tryptophan (cysteine and methion-
ine were determined as cysteic acid and methionine sulfone), they reported within- and
between-laboratory CVs of 4.1% and 13.1% respectively. They also reported recoveries
of amino acids across proteins of 85% to 105% based on the nitrogen content. van der
Meer (1990) reported a study whereby 12 laboratories, all using the same method (acid
hydrolysis followed by ion exchange chromatography and post-column ninhydrin detec-
tion), analyzed 10 different samples ranging from feedstuffs to pig feces. They reported
intra- and inter-laboratory CV across all amino acids (cysteine and methionine were de-
termined as cysteic acid and methionine sulfone) to be 3% and 7%, respectively. Llames
and Fontaine (1994) had five feedstuffs or mixed poultry feeds tested in 28 laboratories
using performic acid oxidation followed by hydrochloric acid hydrolysis. These workers
reported intra-laboratory variation (relative standard error) of 1.1% to 12.7% across all
amino acids and all samples. The corresponding inter-laboratory variation ranged from
3.7% to 28.2%. It is clear that variation between laboratories is greater (approximately 2-
fold) than variation within laboratories. Amino acid data for animal feedstuffs were also
more variable than for pure proteins. Participation in inter-laboratory testing programs
is an important means of monitoring and perhaps reducing analytical variation in amino
acid analysis.
Conclusion
Amino acid analysis has changed very little over the last 50 years, despite the enor-
mous number of papers published on the topic. The variability inherent in the method is
still significant, and it is evident that there is a need to standardize amino acid analysis
methodologies in order to attempt to reduce this error (Gilani et al., 2008). However,
given the wide range of techniques available for each step of the amino acid analysis
procedure, standardization will be challenging. For clients requiring amino acid analysis,
it is important to choose a laboratory carefully. The laboratory should be using a gener-
ally accepted and commonly used methodology, have excellent laboratory practice, use
internal and external standards, and participate in a proficiency program. Once identified,
the client should then form a relationship with that laboratory and make every attempt to
fully understand the strengths and weaknesses on the method being used and the amino
acid data generated.
ACID HYDROLYSIS OF PROTEINS AND PEPTIDES FOR AMINO ACID BASIC

ANALYSIS PROTOCOL 1
This protocol describes a relatively simple sealed tube method for amino acid analysis
that permits good accuracy and precision, as well as high sample throughput. The method
can be applied to pure proteins, foods, feedstuffs, and biological samples.
Chemical
Analysis
11.9.21
Acid hydrolysis is commonly used to hydrolyze the peptide bonds that hold amino acids
together in the form of protein or peptides. Amino acids are highly diverse in their
chemistry and as such have variable stabilities during acid hydrolysis. Consequently, not
all amino acids can be accurately determined if only HCl hydrolysis is used. Notably,
glutamine, asparagine, tryptophan, and cysteine are largely modified or destroyed, while
yields of serine, threonine, isoleucine, valine, methionine, and, to some degree tyrosine,
are often less than 100% (see Strategic Planning). These points must be remembered
when examining amino acid composition data.
Materials
Sample (see Support Protocol)
Constant boiling 6 M HCl containing 0.1% phenol (see recipe)
Internal standard (norleucine) solution (see recipe)
Buffer suitable for downstream applications
Hydrolysis tubes: 100 12mm Schott Duran rimless test tubes
(http://www.us.schott.com)
Glass blowing torch (Wale Apparatus; http://www.waleapparatus.com/; if the
laboratory has a methane or natural gas tap, then use this and bottled oxygen to
provide the fuel mix)
Tweezers
Chemically resistant, oil-free PTFE diaphragm pump with a vacuum controller and
vacuum gauge (e.g., Buchi V-700 pump with V-850 controller)
Rubber vacuum hose to attach tube to vacuum pump
110 C forced air oven
Low-speed centrifuge (capable of 2600 g) with a rotor adapter that will
accommodate 100 mm 12 mm glass tubes
Glass-etching pen
Glass rod
Savant Speedvac (centrifugal concentrator) or equivalent that will accommodate
100 mm 12 mm glass tubes, resistant to acid
Sonication bath
0.22-m syringe filters (13-mm diameter)
1-ml luer-lock syringes
HPLC vials (2 ml or 4 ml depending on the HPLC autosampler)
Vortex mixer
Additional reagents and equipment to prepare samples for amino acid analysis (see
Support Protocol)
Prepare samples for hydrolysis
1. Prepare sample and weigh into a hydrolysis tube (see Support Protocol).
2. Using a calibrated dispenser, add 1 ml of constant-boiling 6 M HCl containing
0.1% phenol to each hydrolysis tube at room temperature.
The acid solution can be poured down the side of the tube to wash down any remaining
sample adhering to the side of the tube.
3. Light the glass blowing torch according to the manufacturers instructions. Adjust
the methane-to-oxygen ration to produce a flame that will render the hydrolysis tube
soft and pliable within 5 sec.
4. Hold the bottom of the tube with fingers and the top with tweezers (Fig. 11.9.2A).
5. Heat the tube with the torch 1 to 1.5 cm below the top of the tube until the glass
Amino Acid
becomes soft. Be sure to rotate the tube continuously to ensure even softening around
Analysis the tube (Fig. 11.9.2A).
11.9.22
A B C
Figure 11.9.2 Stretching the neck of the hydrolysis tube prior to degassing and sealing.
rubber
hose
hydrolysis
tube
vacuum
pump
Figure 11.9.3 Degassing the hydrolysis tube using a vacuum pump.
6. Pull the top of the tube with a pair of tweezers until the soft glass is stretched to
5 to 6 cm in length (Fig. 11.9.2B,C), and allow the tube to cool.
7. Turn on the vacuum pump and controller.
It is critical that the vacuum controller have a very fine vacuum control.
8. While the vacuum pump is at atmospheric pressure, attach the rubber hose to the
tube.
9. Turn on the vacuum very slowly until the sample starts to bubble, flicking the bottom
of the tube with a finger to remove the bubbles (Fig. 11.9.3).
10. Allow bubbling to subside, then turn the vacuum control knob 1/16 of a turn or until
bubbling starts again.
11. Repeat the last two steps until the vacuum has reached 5 mbar and the acid stops
bubbling.
If the vacuum is turned on too fast, sample will be sucked up into the narrowed section Chemical
Analysis
of the tube and will be destroyed when this section is melted later in the procedure.
11.9.23
This degassing step takes patience and practice, but once proficient most samples can be
degassed in less than 2 min.
12. Once there are no more bubbles in the sample and the vacuum has been fully applied
to the hydrolysis tube, use a Bunsen burner to melt the stretched part of the tube.
When the glass is soft, pull the bottom of the tube away from the top of the tube
until the two ends of the tube separate (the tube is now sealed). Melt any sharp glass
points on the tip of the melted section of the tube.
Perform the hydrolysis and collect the sample at the bottom of the tube
13. Allow the tube to cool so that the glass solidifies, and then place the sealed, evacuated
tube in a 110 C forced air oven for 24 hr.
14. After 24 hr, remove the tube from the oven and allow it to cool.
15. Centrifuge the tube in a low-speed tabletop centrifuge for 3 min at 2600 g, room
temperature, to force any acid that may have condensed on the side of the tube down
to the bottom of the tube.
Crack the hydrolysis tube open
16. Using a glass-etching pen, scratch a horizontal line (2 to 5 mm long) into the tube,
1 to 1.5 cm below the top shoulder of the tube.
17. Place a small drop of water onto the scratch.
18. Using the glass blowing torch, heat the end of a glass rod until it is white hot and
molten. Place the molten part of the rod firmly onto the glass tube just next to the
water droplet.
The tube should crack around its circumference.
19. If the tube does not crack, then repeat steps 17 to 18 until it does crack. If the tube
does not crack after three attempts, rescratch the tube and try again.
This step requires practice to become proficient.
20. Add the appropriate amount of internal standard to the amino acid hydrolysate
(50 l of 4 mM norleucine to tubes that contain <2 mg of protein, or 50 l of 40 mM
norleucine to tubes containing more than 2 mg of protein).
Process and store the hydrolyzed sample
21. Dry the hydrolysate using a Speedvac centrifugal concentrator or other suitable
device.
Make sure the device is HCl resistant.
22. Dissolve the sample in a suitable buffer.

The composition of the buffer will depend on the type of chromatographic technique to be
used to quantify the amino acids.
23. Break up any undissolved residue by sonicating the tube in a sonication bath and
mix the hydrolysate by vortexing.
24. If there is particulate matter present, filter the hydrolysate through a 0.22-m syringe
filter (13-mm diameter) into an HPLC sample vial using a 1-ml syringe.
25. Store the hydrolysate at 4 C if it is to be analyzed within 72 hr. Alternatively, store
samples at 20 C up to several months.
The sample is now ready for chromatographic separation and quantitation of the amino
Amino Acid acids. Since there are a multitude of methods, a detailed protocol is not supplied here (see
Analysis Strategic Planning).
11.9.24
PERFORMIC ACID OXIDATION OF PROTEINS FOR CYSTEINE AND BASIC
METHIONINE ANALYSIS PROTOCOL 2
Cysteine, and to some degree methionine, are oxidized during acid hydrolysis. In order
to quantitate these amino acids, they must be converted to acid-stable derivatives. This
protocol describes the performic acid oxidation method, which is carried out prior to
acid hydrolysis and followed by chromatography of the amino acid hydrolysate. During
this step, cysteine is oxidized to cysteic acid and methionine is oxidized to methionine
sulfone. Cysteic acid and methionine sulfone are then determined.
Materials
Performic acid (see recipe), freshly prepared and ice cold
48% hydrobromic acid, ice-cold
100-mm 12-mm glass tubes, resistant to formic acid
Additional reagents and equipment to prepare samples for amino acid analysis
(Support Protocol) and hydrolysis of proteins/peptides for amino acid analysis
(Basic Protocol 1)
1. Prepare and weigh sample as described in the Support Protocol.
However, note that smaller sample sizes can be used (0.5 to 1 mg of protein).
2. Cool the hydrolysis tube containing the sample to 2 C in an ice bath.

3. Add 1 ml of freshly prepared ice-cold performic acid to the tube.
4. Cover the tube with Parafilm.
5. Incubate in an ice bath in the refrigerator for 16 hr.
6. Slowly add 150 l of ice-cold 48% hydrobromic acid to the tube sitting in ice-cold
water, and allow the mixture to sit on ice for 10 to 30 min or until bubbling subsides
The hydrobromic acid reduces the performic acid to formic acid.
7. Dry down the sample in a Speedvac centrifugal concentrator or other suitable device.
8. Hydrolyze the sample as described in Basic Protocol 1.
BASE (LiOH) HYDROLYSIS OF PROTEINS FOR TRYPTOPHAN ANALYSIS BASIC

PROTOCOL 3
Tryptophan is not stable during HCl hydrolysis. Consequently, base hydrolysis is often
used to determine tryptophan (see Strategic Planning). This protocol describes a method
suitable for the hydrolysis of tryptophan in pure proteins, foods, feedstuffs, and biological
samples.
Materials
Lysozyme from chicken egg white (Sigma, cat. no. L 7651)
4.3 M lithium hydroxide (see recipe)
Internal standard (5-methyltryptophan) solution (see recipe)
Constant boiling 6 M HCl containing 0.1% phenol (see recipe)
HPLC buffer for LiOH hydrolysis (see recipe)
30-ml screw-cap Teflon containers (Nalge, cat. no. DS1630-0001), thoroughly
cleaned and rinsed with 18 M deionized water.
Balance (accurate to 5 decimal places) Chemical
Analysis
11.9.25
Nitrogen cylinder equipped with a flexible tube to which a Pasteur pipet can be
attached
110 C forced-air oven
25-ml volumetric flask
0.22-m syringe filters (13-mm diameter)
1-ml luer-lock syringes
HPLC vials (2-ml or 4-ml depending on the HPLC autosampler)
HPLC system with column heater
C8 column (4.6 150 mm)
Peak integration software for HPLC
Weigh out sample and external standard (lysozyme)
1. Weigh between 10 and 50 mg of protein (50 to 100 mg of sample depending on
the protein concentration) into a screw-cap Teflon container on a balance accurate
to 5 decimal places. Avoid, as much as possible, getting sample on the side of the
container when weighing.
Samples should be hydrolyzed at least in duplicate.
2. Similarly weigh out a duplicate 25-mg sample of lysozyme for hydrolysis as an

external control.
Prepare samples for hydrolysis
3. Sparge the 4.3 M lithium hydroxide with nitrogen gas for 10 min prior to use to
remove dissolved oxygen.
4. Aliquot 10 ml of the sparged 4.3 M lithium hydroxide solution and 100 l of 125 mM
5-methyltryptophan internal standard into the Teflon container.
5. Flush the headspace for 30 sec with the cap placed over the container, then quickly
seal the container.
6. Place the sealed container in a 110 C forced air oven for 20 hr.
7. After 20 hr, remove the container from the oven and allow it to cool.
8. Quantitatively transfer the contents to a 25-ml volumetric flask using 18 M deion-
ized water.
9. Adjust the pH to 7 with 6 M HCl and bring the volume to 25 ml with 18-M
deionized water.
10. Filter a portion of the sample through a 0.22-m syringe filter into a HPLC vial
using a 1-ml syringe.
11. Store the sample at 4 C if it is to be analyzed within 72 hr. Alternatively, store the
samples at 20 C for several months.
Perform chromatography of the tryptophan hydrolysate
Since tryptophan is a fluorophore ( excitation 280 nm, emission 348 nm) and is
hydrophobic, it can easily be analyzed by reversed-phase HPLC using fluorescence
detection. The method described below is taken from Garcia and Baxter (1992).
12. Attach a C8 column (4.6 150 mm) to the HPLC and set the column heater temper-
ature to 50 C.
13. Set the HPLC buffer flow rate to 1 ml/min.
Amino Acid
14. Set the fluorescence detector excitation wavelength to 295 nm and the emission
Analysis wavelength to 345 nm.
11.9.26
15. Condition the column until the baseline is stable.
16. Inject the calibration standard and sample hydrolysate.
Tryptophan should elute at 3 min and 5-methyltryptophan at 6 min. It should be noted
that retention times will vary depending on the type and brand of column, mobile phase
composition, column temperature, and flow rate.
17. Determine the peak area of both peaks using peak integration software. Compare
the retention time of peaks in the sample hydrolysate to those of tryptophan and
5-methyltryptophan in the calibration standard in order to identify these amino
acids. Compare the area under the peak for each amino acid in the sample to the area
of the same amino acid in the calibration standard in order to quantify the amount of
tryptophan and 5-methyltryptophan in the sample.
ANALYSIS OF FREE AMINO ACIDS BY HPLC BASIC

PROTOCOL 4
This protocol is suitable for liquid samples containing free amino acids in physiological
fluids such as plasma and urine.
Materials
Internal standard (norleucine) solution (see recipe)
2 HPLC loading buffer (see recipe)
Refrigerated microcentrifuge
Microcon tube (MWCO, 3000 Da; Millipore) or similar centrifugal ultrafiltration
device
HPLC vials (2 ml or 4 ml depending on the HPLC autosampler)
Additional reagents and equipment for pre-column derivitization (see Strategic
Planning)
1. Aliquot 1 ml of sample into a microcentrifuge tube.
2. Add a suitable amount of internal standard such that the amount of internal standard
is similar to the free amino acids present. Microcentrifuge the tube for 15 min at
approximately 10,000 g, 4 C, to remove particulate matter.
3. Aliquot 0.5 ml of the supernatant into a Microcon tube (MWCO, 3000) or similar
ultrafiltration device. Centrifuge the Microcon tube according to the manufacturers
instructions. If, after centrifugation, there is insufficient ultrafiltrate for HPLC anal-
ysis, recentrifuge until sufficient solution has passed through the filter.
4a. If the sample is to be analyzed using ion-exchange chromatography: Aliquot the
ultrafiltrate into an HPLC vial. Add an equal amount of 2 HPLC loading buffer
before injecting onto the HPLC column.
4b. If the sample is to be analyzed using reversed-phase HPLC: Derivatize sample prior
to loading on the HPLC column (see discussion of Pre-column derivatization in
Strategic Planning).
ANALYSIS OF REACTIVE LYSINE CONTENT IN FOOD SAMPLES BASIC

PROTOCOL 5
During the processing and storage of protein products and samples, the side-chain amino
acid group of lysine can react with other compounds present, resulting in modified lysine
derivatives. During acid hydrolysis, a portion of these derivatives can break down and
revert back to lysine. This leads to an overestimation of the amount of lysine present. This Chemical
method uses the guanidination reaction in which the lysine side-chain amino group is Analysis
11.9.27
reacted with O-methylisourea (OMIU) to convert chemically reactive lysine to the acid-
stable derivative homoarginine, which can be determined using amino acid analysis.
Materials
0.6 M OMIU (see recipe)
20 C shaking water bath
100-mm 12-mm glass tubes
Additional reagents and equipment to prepare samples for amino acid analysis
(Support Protocol) and hydrolysis of proteins/peptides for amino acid analysis
(Basic Protocol 1)
1. Prepare and weigh sample as described in Support Protocol; however, use smaller
sample sizes (0.5 to 1 mg of protein).
2. Estimate the approximate amount of lysine present in the sample.
If the lysine content of similar types of samples is known, then the amount of lysine in those
samples can be extrapolated to the samples of interest. Otherwise, use the calculation
below to estimate the lysine content of the sample.
a. It is assumed that the protein contains equimolar amounts of each amino acid,
i.e., 4.5% lysine on a molar basis assuming 22 amino acids are present on the
protein.
protein content of sample (g) 4.5

lysine present =
average AA "in chain" mol. wt. 100
b. In the above equation, the average AA in chain mol. wt. is calculated as the
mean in-chain mol. wt. of all 22 amino acids found in protein.
3. Assuming that a molar excess of OMIU:lysine of 1000 is required for the reaction
mixture, calculate the volume of 0.6 M OMIU to be added to the hydrolysis tube for
any given sample:
lysine present (mol) 1000 1000

volume of 0.6 M OMIU required (ml) =
0.6
The lysine present is multiplied by 1000 twice, firstly, since a 1000:1 ratio of OMIU to
lysine is used, and, secondly, to convert the amount of 0.6 M OMIU required from liters
to milliliters.
For ease of addition, the volume of 0.6 M OMIU can be rounded up to the nearest
0.05 ml.
4. Add the appropriate amount of freshly made 0.6 M OMIU to each hydrolysis tube
containing protein sample prepared as indicated in step 1.
5. Cover the tubes with Parafilm.
6. Incubate the tubes in a shaking water bath at 20 2 C for 3 days for pure proteins
and food samples, or 7 days for digesta samples.
7. Dry down the sample in a Speedvac centrifugal concentrator or other suitable device.
Amino Acid 8. Hydrolyze the sample as described in Basic Protocol 1.

Analysis
11.9.28
PREPARATION OF SAMPLES TO BE SUBJECTED TO AMINO ACID SUPPORT
ANALYSIS PROTOCOL
This protocol is suitable for any samples to be subjected to amino acid analysis but has
particular application for dry samples, samples containing insoluble protein material, and
liquid samples containing only soluble proteins, peptides, or amino acids. It can be applied
to foods, feedstuffs and biological materials such tissue samples, feces, and digesta.
Materials
Sample for analysis
Hydrolysis tubes: 100 12mm Schott Duran rimless test tubes
(http://www.us.schott.com/)
Muffle furnace (capable of attaining 500 C)
Freeze dryer
Grinder with mesh pore size 1 mm
Balance (accurate to 5 decimal places)
100 12mm glass tubes
1. Pyrolyze hydrolysis tubes at 500 C in a muffle furnace for at least 2 hr prior to use.
After pyrolysis, store tubes in a sealed plastic bag.
Tubes may be stored for an indefinite period of time under these conditions.
2. Freeze-dry wet samples that contain particulate matter and grind through a mesh with
a pore size no greater than 1 mm. If samples are stored at temperatures below room
temperature, allow the samples to equilibrate to room temperature before weighing.
3a. If the samples are dry: Weigh a quantity of sample containing between 0.5 and 4 mg
of protein (the total amount of sample weighed should be between 5 and 15 mg) into
the hydrolysis tube on a balance capable of weighing to 0.01 mg.
3b. If the proteins or peptides are soluble and in solution: Using a pipet, add 0.5 to
4 mg of protein to the hydrolysis tube and dry the sample in the Savant Speedvac or
equivalent.
Samples should be hydrolyzed at least in duplicate.
Avoid, as much as possible, getting sample on the side of the tube when adding the sample.
Dry sample on the side of the tube can often be dislodged by tapping the tube gently on a
bench.
A duplicate sample of 2 to 5 mg lysozyme (external standard) or equivalent must also be
weighed out into duplicate hydrolysis tubes.
REAGENTS AND SOLUTIONS

Use 18-M Milli-Q-purified water or equivalent in all recipes and protocol steps. For common
stock solutions, see APPENDIX 2E; for suppliers, see SUPPLIERS APPENDIX.
Constant-boiling 6 M HCl containing 0.1% phenol

Constant-boiling 6 M HCl: Add concentrated HCl to an equal volume of 18-
M deionized water in a round-bottom flask (Fig. 11.9.4). Connect the flask to a
distillation condenser equipped with a thermometer inlet. Heat the mixture until it
reaches 109 C and then collect the distillate. When the temperature rises to above
110 C, check the concentration of the distillate by titration against 6 M NaOH using
a couple of drops of phenolphthalein as an indicator. If the concentration of the
HCl is below 5.8 M, then continue collection of the distillate until the concentration
Chemical
continued Analysis
11.9.29
thermometer
condenser
round-bottomed flask
collection
vessel
heating mantle
Figure 11.9.4 Apparatus for the preparation of constant-boiling HCl.
of the HCl distillate reaches 5.8 M. A final concentration of 5.8 to 6.0 M HCl is
acceptable.
Constant-boiling 6 M HCl containing 0.1% phenol: Dissolve 1 g of phenol in 1 liter

of constant boiling 6 M HCl prepared as described above.
Store the solution in the dark at room temperature for no more than 3 months.
Note that constant-boiling HCl can be obtained commercially from Thermo Scientific.
HPLC buffer for LiOH hydrolysis

Dissolve 13.8 g of monobasic sodium phosphate in 2 liters of 18-M deionized
water. Add 7 ml of 85% phosphoric acid with mixing. Add 500 ml of methanol and
mix thoroughly. Filter the buffer through a 0.22 m filter. Store up to 3 months at
room temperature.
Internal standard (5-methyltryptophan) solution

Weigh out 272.8 mg of 5-methyltryptophan (making sure to record the exact
weight) into a 100-ml volumetric flask. Bring to 100 ml with 18-M deionized
water and allow the 5-methyltryptophan to dissolve. The concentration will be
approximately 125 mM but it is necessary to calculate the exact concentration on
the basis of the exact amount weighed. Store the solution in 10-ml aliquots in the
freezer for no more than 12 months, and store the thawed working aliquots in the
Amino Acid refrigerator for no more than 3 months.
Analysis
11.9.30
Internal standard (norleucine) solution
High-concentration norleucine standard (40 mM) for samples where there is >2
mg of protein in the hydrolysis tube: Weigh out 524.8 mg of norleucine (making
sure to record the exact weight) into a 100-ml volumetric flask. Bring volume
to 100 ml with 18-M deionized water, and allow the norleucine to dissolve. The
concentration will be 40 mM but it is necessary to calculate the exact concentration
on the basis of the exact amount weighed. Store the solution as 10-ml aliquots in
the freezer for no more than 2 years and store the working aliquot in the refrigerator
for no more than 6 months.
Low-concentration norleucine standard (4 mM) for samples where there is <2 mg

of protein in the hydrolysis tube: Dilute the high-concentration (40 mM) norleucine
standard (see above) 1:10 with 18-M deionized water, using a recently calibrated
automatic pipettor. Store the solution as 10-ml aliquots in the freezer for no more
than 2 years and store the working aliquot in the refrigerator no more than 3 months.
Lithium hydroxide, 4.3 M

Weigh out 102.99 g of anhydrous lithium hydroxide (or 180.43 g of lithium hy-
droxide monohydrate) into a 1000-ml beaker. Add 900 ml of 18-M deionized
water and mix the solution until dissolved. Quantitatively transfer the contents to a
1000-ml volumetric flask and bring to 1000 ml with 18-M deionized water. Store
up to 6 months at room temperature.
OMIU, 0.6 M
Weigh 2 g of O-methylisourea hydrogen sulfate (OMIU; Sigma-Aldrich, cat. no.
455598-100G) in a 40-ml screw-cap centrifuge tube. Weigh 4 g of barium hydroxide
octahydrate into a beaker. Boil 30 ml of 18-M deionized water in a 50-ml beaker
until there are 18 to 20 ml remaining. This removes dissolved carbon dioxide in
the water which would react with the barium hydroxide. When the volume of the
water reaches 18 to 20 ml, quickly add the barium hydroxide to the water. Swirl
and heat the solution until it just starts to boil again (be very careful, as the solution
will boil over very quickly after it has started boiling). Quickly pour the solution
into the centrifuge tube containing the OMIU. Tightly cap the centrifuge tube and
invert it several times. Carefully loosen the cap to release any pressure buildup,
and repeat a couple more times. Let the solution cool for 30 min. Centrifuge the
OMIU-containing tube 10 min at 6400 g. Retain the supernatant and check the
pH. If the pH of the solution is <12, then it is assumed that conversion of the sulfate
salt to the free base is incomplete and the solution must be remade. However, if
the pH is >12, then the pH is adjusted to the appropriate pH for guanidination (pH
10.6 for pure proteins, foods and feedstuffs, 11.2 for digesta samples). Filter the
solution through a 0.22-m syringe filter (25 mm diameter) using a 20-ml syringe.
Use the solution immediately.
This procedure makes 20 ml of 0.6 M OMIU. The procedure can be scaled up or down
accordingly. This solution must be prepared fresh just prior to use.
Performic acid
Slowly add 1 vol of 30% hydrogen peroxide to 9 vol of 88% formic acid at room
temperature with good mixing (note that the formic acid can be explosive in the
presence of oxidizing agent). Mix the solution and then allow it to sit at room
temperature for 30 min. Cool the solution in an ice-bath for 30 min.
CAUTION: As a precaution against explosion, prepare solution in a fume hood, slowly
Chemical
adding the H2 O2 to the formic acid with constant stirring. Analysis
This solution should be prepared fresh just before use.
11.9.31
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Chemical
Analysis
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Amino Acid Analysis

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Amino Acid Analysis

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Amino Acid Analysis UNIT 11.

Keywords: amino acids r hydrolysis r derivatization r chromatography

Current Protocols in Protein Science 11.9.1-11.9.37, November 2009 11.9.1

Sampling and Sample Preparation

Pure proteins and peptides

Problematic amino acids during hydrolysis

Serine and threonine

Despite the numerous methods developed to quantitatively hydrolyze tryptophan, the

The internal standard 5-methyltryptophan is often added to samples prior to hydrolysis

Ensuring an oxygen-free environment during hydrolysis

The fourth approach involves connecting a tube or lyophilization ampule to a vacuum

Hydrolysis time and temperature

Antioxidants and scavenging agents

Least-Squares Nonlinear Regression Analysis

The advantages of pre- and post-column derivatization were succinctly described by

PITC amino acid PTCamino acid

FMOC amino acid FMOCamino acid

Figure 11.9.1 Derivatization reactions for amino acid analysis.

An additional disadvantage of FMOC derivatization is the production of multiple deriva-

The OPA/FMOC pre-column derivatization method does require an absorbance detector,

Detection of underivatized amino acids

Amino Acid Enantiomers

Use of Internal and External Standards

Reducing Variability in Amino Acid Analysis: Practical Tips

The chromatography step is a complex part of amino acid analysisseparating 16 to

ACID HYDROLYSIS OF PROTEINS AND PEPTIDES FOR AMINO ACID BASIC

Figure 11.9.3 Degassing the hydrolysis tube using a vacuum pump.

22. Dissolve the sample in a suitable buffer.

2. Cool the hydrolysis tube containing the sample to 2 C in an ice bath.

BASE (LiOH) HYDROLYSIS OF PROTEINS FOR TRYPTOPHAN ANALYSIS BASIC

2. Similarly weigh out a duplicate 25-mg sample of lysozyme for hydrolysis as an

ANALYSIS OF FREE AMINO ACIDS BY HPLC BASIC

ANALYSIS OF REACTIVE LYSINE CONTENT IN FOOD SAMPLES BASIC

protein content of sample (g) 4.5

lysine present (mol) 1000 1000

Amino Acid 8. Hydrolyze the sample as described in Basic Protocol 1.

REAGENTS AND SOLUTIONS

Constant-boiling 6 M HCl containing 0.1% phenol

Figure 11.9.4 Apparatus for the preparation of constant-boiling HCl.

Constant-boiling 6 M HCl containing 0.1% phenol: Dissolve 1 g of phenol in 1 liter

HPLC buffer for LiOH hydrolysis

Internal standard (5-methyltryptophan) solution

Low-concentration norleucine standard (4 mM) for samples where there is <2 mg

Lithium hydroxide, 4.3 M

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