Nitrosation and analysis of amino acid derivatives by isocratic HPLC

Article abstract of DOI:10.1039/c5ra25854e

The objective of this study was to characterize the nitrosation of the classical amino acids by N₂O₃. Nitrosation of amino acids results in the formation of mainly α-hydroxy-acids that are suitable for isocratic HPLC analysis and subsequent quantification of amino acids in biological samples. The method is particularly suitable for detection of amino acids in e.g. fermentation media as the α-hydroxy-acids can be quantified in parallel to a variety of other organic substrates and products. The amino acids were transformed into their corresponding α-hydroxy-acids in acidic KNO₂ solutions. The reactions were terminated by NaOH addition and the α-hydroxy-acids separated by isocratic HPLC and quantified by refractive index or UV absorption detection. Nitrosation of 18 of the classical amino acids; glycine, l-alanine, l-valine, l-leucine, l-isoleucine, l-methionine, l-serine, l-threonine, l-asparagine, l-glutamine, l-aspartic acid, l-glutamic acid, l-proline, l-cysteine, l-phenylalanine, l-lysine, l-tyrosine, and l-tryptophane formed detectable nitrosation products. l-Lysine, however, needed incubation in 96 mM formic acid to produce a detectable product, while l-phenylalanine had to be incubated in 120 mM HNO₃ and 100 mM HCl. Optimal reaction conditions for most amino acids included 40 min of incubation of up to 5 g L^-1 amino acid in 160 mM KNO₂ in 100 mM HCl at 45 °C to maximize product yields.

Full text of DOI:10.1039/c5ra25854e

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Nitrosation and analysis of amino acid derivatives
by isocratic HPLC
Cite this: RSC Adv., 2016, 6, 13120
Songul Ulusoy,ab Halil Ibrahim Ulusoy, ac Daniel Pleissnerd and Niels Thomas Eriksena
*
¨
The objective of this study was to characterize the nitrosation of the classical amino acids by N2O3.
Nitrosation of amino acids results in the formation of mainly a-hydroxy-acids that are suitable for
isocratic HPLC analysis and subsequent quantification of amino acids in biological samples. The method
is particularly suitable for detection of amino acids in e.g. fermentation media as the a-hydroxy-acids
can be quantified in parallel to a variety of other organic substrates and products. The amino acids were
transformed into their corresponding a-hydroxy-acids in acidic KNO2 solutions. The reactions were
terminated by NaOH addition and the a-hydroxy-acids separated by isocratic HPLC and quantified by
refractive index or UV absorption detection. Nitrosation of 18 of the classical amino acids; glycine,
L-alanine, L-valine, L-leucine, L-isoleucine, L-methionine, L-serine, L-threonine, L-asparagine, L-glutamine,
L-aspartic acid, L-glutamic acid, L-proline, L-cysteine, L-phenylalanine, L-lysine, L-tyrosine, and
L-tryptophane formed detectable nitrosation products. L-Lysine, however, needed incubation in 96 mM
formic acid to produce a detectable product, while L-phenylalanine had to be incubated in 120 mM
HNO3 and 100 mM HCl. Optimal reaction conditions for most amino acids included 40 min of
incubation of up to 5 g Lꢀ1 amino acid in 160 mM KNO2 in 100 mM HCl at 45 ꢁC to maximize product yields.
Received 4th December 2015
Accepted 24th January 2016
DOI: 10.1039/c5ra25854e
www.rsc.org/advances
This is one of the most versatile analytical methods for quan-
tication of small biological molecules. On the same column,
1. Introduction
disaccharides, hexoses, pentoses, organic acids, ketones, alco-
hols, polyols and more different molecules can be separated
and simultaneously quantied in the same sample. The method
is well established in microbial biotechnology8 and has been
used to quantify substrate uptake and metabolite synthesis in
cultures of bacteria,9,10 lamentous fungi,11,12 and micro-
algae.13,14 The method has also been used to analyse metabolites
in e.g. foods,15 beverages,16,17 human body uids,18,19 and plant
extracts.20 The separation of individual molecules is, however,
restricted to uncharged molecules and therefore cannot be used
directly to analyse amino acids as these will be positively
charged in the acidic mobile phase. In contrast will a-hydroxy
acids be neutral at low pH. Nitrosation of samples prior to
analysis therefore enables simultaneous quantication of
amino acids along with other small molecules in one sample. So
far has this method6 been used to quantify the amino acids,
glycine, L-alanine, and L-glutamic acid aer transformed into
their corresponding a-hydroxy acids, glycolic acid, lactic acid, or
a-hydroxyglutaric acid, respectively, in parallel to glucose, acetic
acid, and phosphate (measured as phosphoric acid) in cultures
of the dinoagellate Crypthecodinium cohnii, a heterotrophic
microalga that depends on organic nitrogen sources.6,21
Primary amino acids can be transformed into their corre-
sponding a-hydroxy acids by nitrosation of their amino group
by dinitrogen trioxide, N2O3, formed from nitrite under acidic
conditions. The reaction progresses via the intermediate
formation of rstly a nitrosamine and secondly a diazonium
group.1–4 The diazonium group is released as N2 via an intra-
molecular lactone formation with a carboxyl group.5 The reac-
tion can be stopped by addition of NaOH and the lactones are
hydrolysed into a-hydroxy acids.6 The reaction is named aer
van Slyke who measured the release of N2 to quantify peptide
concentrations in biological samples.7
Pleissner et al. demonstrated that the a-hydroxy acids
formed by the van Slyke reaction could be used to quantify
individual amino acids in e.g. fermentation media.6 Aer
nitrosation with nitrite, the a-hydroxy acids were subjected to
isocratic separation on an Aminex (Bio-Rad) column under
acidic conditions followed by refractive index (RI) detection.
aDepartment of Chemistry and Bioscience, Aalborg University, Fredrik Bajers Vej 7H,
DK-9220 Aalborg, Denmark. E-mail: hiulusoy@yahoo.com; Fax: +90 346 219 16 34;
Tel: +90 346 219 10 10/3905
bDepartment of Chemistry, Faculty of Science, Cumhuriyet University, TR-58140 Sivas,
Reactions between amino acids and nitrosating agents may
deviate from the course of reaction described above. Proline is
a secondary amine and forms a stable nitrosamine upon reac-
tion with N2O3.1 For some of the other amino acids more than
Turkey
cDepartment of Analytical Chemistry, Faculty of Pharmacy, Cumhuriyet University, TR-
58140 Sivas, Turkey
dDepartment of Bioengineering, Leibniz-Institute for Agricultural Engineering Potsdam-
Bornim e. V., Potsdam, Germany
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one product is formed in reactions with N2O3. Glycine reacts for where Y represents the yield of detectable product relative to the
example with nitrous acid via different parallel reactions22 amount of amino acid converted. Y is expected to have a value of
forming glycolic acid and one additional product.6 N2O3 may 0–1 and may depend on the amino acid, the product, and the
also cause nitration of the ring structure in aromatic amino reaction conditions. Before reactions are started by HCl addi-
acids.23 L-Tryptophane can be nitrated at several positions on tion, X ¼ 0. Reactions are completed when all amino acids are
the aromatic ring24 and 5 products from nitrosation of histidine consumed7 and the degree of conversion at this point, XN will
have previously been identied.25 In cysteine is also the sulf- approach a value of 1.
hydryl group a target for nitrosation in a reaction with NO+.26
In this study, we have characterized and evaluated the
The end product of the reaction between N2O3 and glutamic reactions between the 20 classical amino acids and nitrite in
acid is a-hydroxyglutaric acid under alkaline conditions while it hydrochloric acid, including reaction conditions, product
is a g-lactone under acidic additions.6,27 Similarly is aspartic yields, and their pseudo 1'order reactions kinetics. We have also
acid converted into malic acid at low pH, while probably modied the prior analytical protocols and increased the
a b-lactone is the end product under alkaline conditions.6 number of classical amino acids that provide detectable nitro-
Therefore have only 13 of the 20 classical amino acids so far sation products from 13 to a total of 18.
been transformed by nitrite into products that are detectable by
RI detection and applicable for amino acid quantication.6
Nitrosation of the different amino acids by nitrite is not yet
well characterised and kinetic models of the nitrosation reac-
2. Materials and methods
2.1. Reaction conditions
Reactions were, in most cases, carried out in 2.5 mL poly-
propylene tubes (Eppendorf) at 45 ꢁC for 40 min, containing
1 mL of amino acid solution (0–5 g Lꢀ1) mixed with 0.2 mL of
1 M KNO2. Reactions were started by addition of 0.04 mL 3 M
HCl and stopped by addition of 0.2 mL 2 M NaOH. The reaction
mixtures contained 0–66.6 mM amino acid, 160 mM KNO2, and
100 mM HCl. Deviations from this protocol are described in
Results and discussion.
tions that can be used to evaluate and optimise the reaction
conditions are still lacking. Spectrophotometric quantication
of N2O3 concentrations in reaction mixtures has demonstrated
that nitrosation of a number of amino acids are 3'order reac-
tions, being 1' and 2'order with respect to amino acid and nitrite
concentrations, respectively.28 If, however, the initial concen-
tration of nitrite is substantially higher than the amino acid
concentration, as would be desirable when the purpose of the
reaction is the rapid formation of a detectable nitrosation
product, the course of the nitrosation reaction can expectantly
be described by a pseudo 1'order expression
2.2. HPLC analysis of a-hydroxy acids and metabolites
All a-hydroxy acids and other products produced by reactions
with N2O3 were analysed using isocratic HPLC: 25 mL of reaction
mixture was added on an Aminex HPX-87H columnꢁ(Bio-Rad)
and eluted with 0.4 mL minꢀ1 of 5 mM H2SO4 at 27 C. Detec-
tion was performed by a Knauer K-2300 RI detector at room
temperature as well as by a Shimadzu SPD-10A UV/VIS detector.
ꢀr ¼ k0caa
(1)
where ꢀr is the reaction rate, k0 is a pseudo 1'order rate
constant, and caa is the concentration of amino acid. The
magnitude of k0 depends on the 3'order rate constant for the
particular amino acid and the concentration of nitrite. If reac-
tions are performed in the presence of nucleophilic species,
additional nitrosating agents may be formed. Chloride, for
example, forms nitrosyl chloride in presence of nitrite. These
nitrosating agents act as catalysts and increase the reaction
rates.29–31 In batch reactions, the concentration of amino acid
will then decrease exponentially
2.3. Determination of gas evolution
Gas formation from nitrosation of amino acids was measured
by adding 50 mL of 10 g Lꢀ1 amino acid solution into a 100 mL
glass ask placed on a magnetic stirrer. The ask was sealed by
a lid with 3 ports. Two ports tted with on-off valves were used
to add 10 mL of 1 M KNO2 as well as 0.5–2 mL of 3 M HCl to start
reactions and to equilibrate the pressure in the ask at the time
of HCl addition. Through the third port, a Teon tubing (i.d. ¼
2 mm) provided a connection to the head-space of a second,
250 mL lidded ask, lled with water. When pressure was build
up in the headspace, water was pushed out of the second ask
via a second Teon tubing and into a 100 mL glass measuring
cylinder where the volume of displaced water was read. Reac-
tions were carried out at room temperature and the gas equa-
tion was used to estimate the amount of produced gas from the
volume of displaced water.
0
caa ¼ c0eꢀk t
(2)
where c0 is the initial amino acid concentration and t is the time
of the reaction. The amino acid concentration, caa, is related to
the degree of conversion of the amino acid, X
caa ¼ c0(1 ꢀ X)
(3)
Since only the concentration of a-hydroxy acid (or other
detectable products), cp, is quantied experimentally,6 the right
hand terms of eqn. (2) and (3) are combined, X is isolated, and
cp is described by
2.4. Reaction analysis
For three amino acids, glycine, L-alanine, and L-aspartic acid
were their corresponding a-hydroxy acids, glycolic acid, lactic
acid, and malic acid, available in pure form and used to prepare
0
cp ¼ Yc0X ¼ Yc0(1 ꢀ eꢀk t
)
(4)
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standard solutions of known concentrations. The concentra- The yields of the different products depended on the reaction
tions of these a-hydroxy acids produced by nitrosation of conditions as well as the time of incubation.
glycine, L-alanine, or L-aspartic acid were quantied in reaction
Nitrosation of L-lysine, L-phenylalanine, L-arginine, and
mixtures by HPLC by comparison of their respective peak areas L-histidine in HCl and KNO2 did not give products that were
to those of the corresponding standard solutions. Yields of detectable by neither RI nor UV-absorption at wavelengths
these a-hydroxy acids, Y, were determined as
cp cp
c0XN c0
higher than 310 nm. However, when 120 mM HNO3 was
included in the reaction mixture, a product from L-phenylala-
nine was detectable by RI at 10.7 min. The product developed
a brownish colour suggesting that modications of the
aromatic part of the molecule had taken place. Most likely was
one or more nitro-groups added to the aromatic ring by HNO3
(ref. 32) in a parallel reaction to the nitrosation of the amino
group by N2O3. When HCl was replaced by formic acid,
a product from L-lysine was detectable by RI at 24.6 min. It is
possible that the amino group at the 3-position in lysine is
formylated33 and the amino group on the a-position then
undergoes nitrosation and produces a detectable product. In
the end, it was therefore only from L-arginine and L-histidine we
did not nd nitrosation products that were detectable by the
HPLC analysis.
Y ¼
¼
(5)
where cp was measured aer reactions had been completed
(XN z 1).
Since most of the a-hydroxy acids produced from nitrosation
of amino acids were not available in pure form, the course of
nitrosation reactions were described by a modied version of
eqn (4)
0
A ¼ YAc0(1 ꢀ eꢀk t
)
(6)
where product concentration is replaced by product peak area
in chromatograms, A, and product yield is replaced by the yield
in terms of product peak area, YA. Pseudo 1'order rate constants,
k0 for the nitrosation of the amino acids were estimated by
tting eqn (6) to experimentally determined peak areas by
simultaneous optimization of the values of k0 and YA in order to
obtain the smallest overall discrepancy (evaluated as root mean
square error) between eqn (6) and the measured peak areas.
The time needed for 90% conversion of amino acids into
their respective nitrosation products, s90 was calculated as
3.2. Acid concentration
The acidity of the reaction mixture affects the yield of products
from nitrosation of amino acids. In most experiments, we used
HCl to create low pH values and start the nitrosation reactions.
Product yields in terms of peak areas from 18 amino acids aer
40 min of reaction at 45 ꢁC with 100 mM KNO2 in different
concentrations of HCl (except L-lysine in formic acid and
L-phenylalanine in HCl and HNO3) are seen in Fig. 1. For all the
amino acids, 50–100 mM acid was needed to maximize product
ꢀ
ꢁ
YAc0XN ꢀ 0:9YAc0
YAc0XN
ln
ln 0:1
ꢀk0
s90
¼
¼
(7)
ꢀk0
as XN z 1. Reactions were subsequently carried out for periods yields. HCl concentration higher than 100–200 mM had
of time longer than s90 to allow the formation of maximal a negative effect on the product yields from glycine, L-alanine,
concentrations of detectable products in the reaction mixtures. L-valine, L-leucine, L-proline, the aromatic amino acids,
L-phenylalanine, L-tyrosine, and L-tryptophane, and L-lysine.
Product formation from the remaining amino acids, all carrying
different functional groups that may favour the release of N2
and formation of a-hydroxy acids, a hydroxyl group in L-serine
3. Results and discussion
3.1. Detection of amino acid derivatives
We optimized reaction conditions for nitrosation of amino and L-threonine, a carboxylic acid group in L-aspartic acid and
acids in solutions of HCl and KNO2, and were able to reproduce L-glutamic acid, a carboxamide group in L-asparagine and
RI detectable products from the same 13 amino acids as L-glutamine, a thiol group in L-cysteine, and a thio-ether group
Pleissner et al.,6 i.e. glycine, L-alanine, L-valine, L-leucine, in L-methionine, was unaffected by high concentrations of HCl.
L-isoleucine, L-methionine, L-serine, L-threonine, L-asparagine, Subsequent nitrosation reactions were therefore carried out in
L-glutamine, L-aspartic acid, L-glutamic acid, and L-proline. Also 100 mM HCl (L-lysine in 96 mM formic acid, L-phenylalanine in
nitrosation of L-cysteine resulted in a product that was detected 100 mM HCl and 120 mM HNO3) where product yields from all
by RI at 28.7 min.
the amino acids were close to maximal. This concentration is
Nitrosation of the aromatic amino acids, L-tyrosine and 4 times below the HCl concentration of 400 mM used by
L-tryptophane resulted in brownish products as they were Pleissner et al.6 The lower HCl concentration had no apparent
nitrated at the aromatic ring.23 At least 3–4 products were inuence on which products that were formed, but resulted in
formed from both amino acids, as would also be expected.24 higher yields of the detectable products used in the analysis and
Products from nitrosation of L-tyrosine eluted aer 9.9, 20.2, with retention times shown in Table 1.
25.5, and 34.7 min, respectively and were detectable by UV
Nitrosation of amino acids have previously been carried out
absorption at 310–410 nm although their absorption maxima in different acid solutions. The original nitrosation procedure
differed. Only the products eluting at 9.9 and 34.7 min were also used by van Slyke7 used acetic acid while other authors used
seen in RI chromatograms. Three products from L-tryptophane nitrous acid,34 sulphuric acid,32 perchloric acid,26,35 butyric
were seen in UV chromatograms recorded at 310 nm aer 4.3, acid,6 or HCl.6,36 We used HCl. Organic acids may interfere with
26.1, and 30.3 min, respectively, but not in RI chromatograms. the HPLC analysis as their retention times may be similar to
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Fig. 1 Product peak areas of nitrosation products from 18 amino acids after 40 min of reaction with 160 mM KNO2 at 45 ꢁC in different acid
concentrations, i.e. HCl if other acids are not specified. Initial amino acid concentrations were 2 g Lꢀ1. (A) glycine ,, L-alanine B, and L-valine >.
(B) L-Leucine ,, L-isoleucine B, and L-serine >. (C) L-Threonine ,, L-methionine B, and L-cysteine >. (D) L-Aspartic acid ,, L-glutamic acid
B, and L-asparagine >, L-glutamine D. (E) L-Proline ,, L-phenylalanine in 100 mM HCl and 120 mM HNO3 B, and L-tyrosine >. (F) L-Tryp-
tophane ,, L-lysine in 96 mM formic acid B.
some of the a-hydroxy acids or other products formed from kinetics to describe reaction rates (eqn (1)) and product
nitrosation of the amino acids. Chloride ions from HCl are not concentrations in the reaction mixtures (eqn (4)). Product yields
retained by the column and will therefore not elute at the same from 18 amino acids aer 40 min of incubation at 45 ꢁC in
time as the nitrosation products. The chloride ions may also 100 mM HCl (96 mM formic acid for L-lysine, 100 mM HCl and
form nitrosyl chloride and speed up reaction rates29–31 allowing 120 mM HNO3 for L-phenylalanine) and 0 to 194 mM KNO2 are
for shorter incubation times.
shown in (Fig. 2). Nitrosation products were detected at initial
KNO2 concentrations above 23–46 mM. Product yields
increased at increasing KNO2 concentrations up to 88–125 mM.
Higher KNO2 concentrations resulted in no further increases in
product yields. Based on the results in Fig. 2, subsequent
nitrosation reactions were carried out at an initial KNO2
3.3. Initial KNO2 concentration
A surplus KNO2 concentration is needed for complete trans-
formation of amino acids into a-hydroxy acids or other nitro-
sation products and is a prerequisite for using pseudo 1'order
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Table 1 The 20 classical amino acids. Principle for detection of corresponding a-hydroxy acids (refractive index, RI, UV absorption, UV),
retention time, RT, pseudo 1'order rate constant, k0, time needed for 90% conversion of amino acids into their respective nitrosation products,
s
90, yield of a-hydroxy acid, Y, pH of reaction mixtures, gas yield, Ygas, initial amino acid concentrations resulting in confirmed linear standard
curves from nitrosation reactions, slopes of standard curves, YA, coefficient of determination for standard curves, r2. Reactions were carried out at
45 ꢁC in 160 mM KNO2 and 100 mM HCl
Amino acid
Detector
RT, min
k0, minꢀ1
s90, min
Y
pH
Ygas
co, g Lꢀ1
YA, L gꢀ1
r2
Glycine
L-Alanine
L-Valine
L-Leucine
L-Isoleucine
L-Serine
L-Threonine
L-Methionine
L-Cysteine
RI
RI
RI
RI
RI
RI
RI
RI
RI
RI
RI
RI
RI
RI
RI
UV
UV
RI
—
—
12.4
12.4
18.2
26.7
25.5
10.0
10.7
22.0
28.7
8.4
10.0
11.8
10.0
20.2
10.6
20.0
30.2
24.4
—
0.34
0.10
0.21
0.17
0.25
0.25
0.38
0.29
0.47
0.32
0.30
0.81
0.36
0.14
0.10
0.03
0.02
0.07
—
6.9
23.9
11.0
13.8
9.1
9.4
6.1
7.8
4.9
7.2
7.6
2.9
7.1
16.5
22.2
82.6
102.2
34.6
—
0.78
0.87
—
—
—
—
—
—
—
0.76
—
—
—
—
—
—
—
—
2.7
2.8
2.8
2.7
2.7
2.7
2.7
2.7
2.1
2.7
2.1
2.7
2.7
2.7
2.7
2.5
2.7
3.8
3.2
3.3
1.03
0.89
0.99
1.12
1.06
1.13
1.18
1.11
1.16
0.92
1.25
1.23
1.11
0.00
1.18
0.00
1.03
0.73
0.33
1.31
0–5
0–5
0–5
0–5
0–5
0–5
0–5
0–5
0–5
0–5
0–5
0–5
0–5
0–5
0–5
0–5
0–2
0–5
—
213.2
239.3
264.3
301.2
315.7
261.5
255.3
176.0
82.4
0.9996
0.9998
0.9987
0.9837
0.9975
0.9977
0.9991
0.9945
0.9987
0.9969
0.9954
0.9993
0.9959
0.9987
0.9656
0.9761
0.9954
0.9920
—
L-Aspartic acid
L-Glutamic acid
L-Aspargine
L-Glutamine
L-Proline
279.6
98.6
297.2
120.6
247.4
313.3
40.9
119.2
55.2
—
L-Phenylalaninea
L-Tyrosine
L-Tryptophane
L-Lysineb
L-Arginine
L-Histidine
—
—
—
—
—
—
—
—
a
Nitrosation of L-phenylalanine carried out in 100 mM HCl and 120 mM HNO3. b Nitrosation of L-lysine carried out in 96 mM formic acid.
ꢁ
concentration of 160 mM. The yields of all nitrosation products nitrosation of glycine and L-alanine at 45 C in 160 mM KNO2
detectable by RI were close to maximal at this initial KNO2 and 370 mM butyric acid the reactions were not completed aer
concentration.
120 min.6 Reactions therefore do progress at higher rates in the
Nitrite itself gave rise to a peak in the chromatograms at presence of chloride ions, as also expected.29–31
16.4 min, probably created by N2O3, reformed from unused
nitrite in the acidic mobile phase. When initial KNO2 concen-
3.5. Yields of a-hydroxy acids
trations had been above 25 mM, excess nitrite was still present
According to the van Slyke reaction,1–5 the nitrosation of glycine,
L-alanine, and L-aspartic acid will result in the formation of the
a-hydroxy acids, glycolic acid, lactic acid or malic acid, respec-
tively. Chromatograms of the same a-hydroxy acids in standard
in all reaction mixtures aer the reactions had been stopped.
3.4. Reaction rates and incubation time
Product formation from nitrosation of 18 amino acids in reac- solutions conrmed their identities as products in the reaction
ꢁ
tion mixtures incubated 0–110 min at 45 C in 160 mM KNO2 mixtures and were used to determine their concentrations.
and 100 mM HCl (96 mM formic acid for L-lysine, 100 mM HCl Fig. 4 shows concentrations of glycolicꢁacid, lactic acid or malic
and 120 mM HNO3 for L-phenylalanine) is shown in Fig. 3. Eqn acid aer 40 min of nitrosation at 45 C in 160 mM KNO2 and
(6) was tted to these data in order to model the progression of 100 mM HCl as function of the initial concentrations of glycine,
the reactions and estimate k0 and s90 (Table 1). For 16 amino L-lactic acid or L-aspartic acid, respectively. At this point, these
acids, s90 was between 3 and 24 min while longer incubation reactions have almost been completed (Fig. 3, XN z 1), and the
periods were needed to maximise the degree of conversion of slopes of the regression curves (eqn (5)) will represent the yields
the aromatic amino acids, L-tyrosine and L-tryptophane. Some of of the a-hydroxy acids, Y. Nitrosation of these 3 amino acids
the nitrosation products from the aromatic amino acids were resulted in 76–87% conversion into their corresponding
not stable, but rather intermediate products (Fig. 3C), and a-hydroxy acids when performed in 100 mM HCl. L-Alanine was
therefore less suitable for use as a measure of the initial amino transformed into lactic acid with a yield of 87% which is
acid concentration.
considerably higher than the 73% yield previously obtained in
With the exception of L-tyrosine and L-tryptophane, the 400 mM HCl.6 The higher yield is a result of the lower HCl
nitrosation of all amino acids were almost completed (X well concentration (Fig. 1A).
above 0.9) aer 30 min of incubation. Subsequent nitrosation
We also measured the evolution of gas aer 60 min of
reactions were therefore carried out for 40 min before stopped incubation (incubation times were prolonged to better allow
by the addition of NaOH to ensure almost complete conversion time for transfer of dissolved gases into the gas phase) of 10 g
of the amino acids. When Pleissner et al. followed the Lꢀ1 L-alanine, glycine, L-glutamic acid, or L-valine in 160 mM
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Fig. 2 Product peak areas of nitrosation products from 18 amino acids after 40 min of reaction at 45 ꢁC in 100 mM HCl if other acids are not
specified, and in different concentrations of KNO2. Initial amino acid concentrations were 2 g Lꢀ1. (A) glycine ,, L-alanine B, and L-valine >. (B)
L-Leucine ,, L-isoleucine B, and L-serine >. (C) L-Threonine ,, L-methionine B, and L-cysteine >. (D) L-Aspartic acid ,, L-glutamic acid B,
and L-asparagine >, L-glutamine D. (E) L-Proline ,, L-phenylalanine in 100 mM HCl and 120 mM HNO3 B, and L-tyrosine >. (F) L-Tryptophane
,, L-lysine in 96 mM formic acid B.
KNO2 and 30, 120 or 480 mM HCl (Fig. 5). Highest gas yields nitrosation products (Table 1). Nitrosation of L-arginine resul-
(0.82–1.15 moles of gas per mole of amino acid) evolved from ted in a gas yield of only 0.31 moles of gas per mole arginine,
nitrosation reactions taken place in 120 mM HCl. This obser- while the gas yield from nitrosation of L-histidine was as high as
vation seems to conrm the results in Fig. 4, namely that HCl 1.31 moles of gas per mole histidine, possibly because also its
concentration close to 100 mM favours the van Slyke reaction guanidinium group is a potential target for nitrosation.37
with the predominant synthesis of a-hydroxy acids and N2.
Nevertheless, both amino acids did react with N2O3, but if their
All 20 classical amino acids were subsequently screened for gas products remained charged at low pH, they will not be retained
evolution during nitrosation reactions in 100 mM KNO2 and 120 by the column and cannot be separated from other charged
mM HCl. Nitrosation of L-proline and L-tyrosine did not result in species in the reaction mixtures.
gas formation (Table 1). L-Proline forms a stable nitrosamine
Nitrosation of the remaining 16 amino acids resulted in gas
without releasing N12 and L-tyrosine is transformed to nitrotyrosine yields of 0.73–1.26 mole of gas per mole amino acid. It is not
while its primary amino group is apparently not reacting.23
new that nitrosation of some amino acids may result in gas
Quit different gas yields, Ygas, were seen from the two amino yields greater than 1 mole N2 per mole amino acid.34 NO from
acids, L-arginine and L-histidine that did not produce detectable decomposition of nitrous acid may also contribute to the total
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Fig. 3 Product peak areas of nitrosation products from 18 amino acids after reaction with 160 mM KNO2 at 45 ꢁC in 100 mM HCl if other acids are
not specified, after different times of incubation. Initial amino acid concentrations were 2 g Lꢀ1. (A) glycine ,, L-alanine B, and L-valine >. (B) L-
Leucine ,, L-isoleucine B, and L-serine >. (C) L-Threonine ,, L-methionine B, and L-cysteine >. (D) L-Aspartic acid ,, L-glutamic acid B,
and L-asparagine >, L-glutamine D. (E) L-Proline ,, L-phenylalanine in 100 mM HCl and 120 mM HNO3 B, and L-tyrosine >. (F) L-tryptophane
product peak with RT ¼ 30.2 min , and intermediate product peak with RT ¼ 26.2 min D, L-lysine in 96 mM formic acid B.
gas production, especially at low pH.35 Still, the gas yields were amino acids.6 Although the need for UV detection in order to
close to 1 mole of gas per mole amino acid. This indicates that quantify nitrosation products from L-tyrosine and L-tryptophane
the degree of conversion of most of the amino acids into their reduces the possibility for simultaneous quantication of these
corresponding a-hydroxy acids have been high. The reaction two amino acids and other metabolites, UV chromatograms
conditions seem therefore to have been close to optimal for the were useful for the characterization of the reactions between
conversion of amino acids into their corresponding a-hydroxy N2O3 and these two amino acids. The relationships between
acids (or other nitrosation products) that can be separated and product peak areas and original amino acid concentrations
subsequently detected individually by the HPLC procedure.
from 0–5 g Lꢀ1 (0–2 g Lꢀ1 for tryptophane) were linear for all the
18 amino acids that were transformed into detectable products.
Regression analysis showed that the initial amino acid
concentrations explained 97–100% (r2 $ 0.97) of the recorded
peak areas. Since the slopes of the regression curves represent
product yields in terms of peak area, YA (see eqn (6)), these are
3.6. Standard curves
Quantication of amino acids from RI or UV chromatograms of
their corresponding a-hydroxy acids depends on standard
curves based on solutions of known concentrations of the
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and L-lysine formed nitrosation products that were retained and
could be used to quantify also these amino acids. Multiple
products from nitrosation of L-tyrosine and L-tryptophane could
be detected by UV absorption and used to quantify also these
two amino acids.
Versatile analytical procedures for quantication of amino
acids are essential. A number of derivatization procedures are
already well established, which add chromophores onto the
amino acids either before or aer chromatographic separation
allowing for UV absorbance or uorescence detection.38 Nitro-
sation of amino acids results in the formation of products that
can be separated by isocratic chromatographic principles
compatible with RI quantication and supplements thereby
existing analytical procedures. The established methodologies,
as well as novel chromatographic methods based on MS/MS
detection of either amino acids39 or amino acid derivatives40
may offer higher sensitivities than can be achieved by RI-
detection.41 Amino acids are, however, regularly present in more
than sufficient concentrations for RI quantication, e.g. in
microbial cultures where they in combination with other
organic molecules may be used as substrates.6,21 The nitrosation
of amino acids into their corresponding a-hydroxy acids and
subsequent HPLC analysis have so far therefore been a partic-
ularly useful analytical procedure in microbial cultivations for
simultaneous quantication of amino acids, additional organic
substrates, and metabolic products.
Fig. 4 Relationship between final concentrations of the a-hydroxy
acids, glycolic acid ,, lactic acid B, or malic acid >, and initial
concentrations of their corresponding amino acids, glycine, L-alanine,
and L-aspartic acid, respectively. Reactions were incubated for 40 min
at 45 ꢁC in 160 mM KNO2 and 100 mM HCl. Slopes of regression lines
correspond to yields, Y of a-hydroxy acid (Table 1).
References
1 M. L. Douglass, B. L. Kabacoff, G. A. Anderson and
M. C. Cheng, J. Soc. Cosmet. Chem., 1978, 29, 581.
´
2 J. M. Cachaza, J. Casado, A. Castro and M. A. Lopez Quintela,
Z. Krebsforsch., 1978, 91, 279.
´
3 J. Casado, A. Castro, J. R. Leis, M. A. Lopez Quintela and
Fig. 5 Gas yields from nitrosation of L-alanine, glycine, L-valine, and L-
glutamic acid after 60 min of incubation at 45 ꢁC in 160 mM KNO2 and
3 concentrations of HCl.
M. Mosquera, Monatsh. Chem., 1983, 114, 639.
´
4 M. P. Garcıa Santos, E. Calle and J. Casado, Polyhedron, 2003,
22, 1059.
´
5 M. P. Garcıa-Santos, E. Calle and J. Casado, J. Am. Chem. Soc.,
2001, 123, 7506.
6 D. Pleissner, R. Wimmer and N. T. Eriksen, Anal. Chem.,
2011, 83, 175.
7 D. D. van Slyke, J. Biol. Chem., 1911, 9, 185.
8 M. Gey, P. Kossler and U. Becker, Acta Biotechnol., 1990, 10,
459.
only comparable for nitrosation products detected by the same
detector. L-Isoleucine gave rise to the highest YA, 5.7 times
higher than YA for L-lysine and is therefore measured with
almost 6 times higher sensitivity than L-lysine.
9 M. L. Christensen and N. T. Eriksen, Enzyme Microb.
Technol., 2002, 31, 566.
4. Conclusion
We have characterized the nitrosation of amino acid by N2O3 in 10 N. T. Eriksen, M. L. Riis, N. K. Holm and N. Iversen,
acidic solutions. Quantitative analysis of amino acid derivatives Biotechnol. Lett., 2011, 33, 293.
aer reaction with N2O3 by isocratic HPLC and RI detection 11 B. J. Hidayat, N. T. Eriksen and M. G. Wiebe, FEMS Microbiol.
allowed quantication of 14 out of the 20 classical amino acids Lett., 2006, 254, 324.
when up to 5 g Lꢀ1 amino acid is reacted with 160 mM KNO2 in 12 B. J. Hidayat, M. G. Wiebe and N. T. Eriksen, Enzyme Microb.
100 mM HCl at 45 ꢁC for 40 min, i.e. glycine, L-alanine, L-valine,
Technol., 2007, 40, 902.
L-leucine, L-isoleucine, L-methionine, L-serine, L-threonine, 13 R. A. Schmidt, M. G. Wiebe and N. T. Eriksen, Biotechnol.
L-asparagine, L-glutamine, L-aspartic acid, L-glutamic acid, Bioeng., 2005, 90, 77.
L-proline, and L-cysteine. When HCl was replaced by formic acid 14 J. K. Sloth, M. G. Wiebe and N. T. Eriksen, Enzyme Microb.
or HNO3 included in the reaction mixture, also L-phenylalanine
Technol., 2006, 38, 168.
This journal is © The Royal Society of Chemistry 2016
RSC Adv., 2016, 6, 13120–13128 | 13127

View Article Online
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Paper
´
15 G. Zeppa, L. Conterno and V. Gerbi, J. Agric. Food Chem., 28 M. P. Garcıa Santos, J. Am. Chem. Soc., 2001, 124, 2177.
2001, 49, 2722.
16 J. P. Yuan and F. Chen, Food Chem., 1999, 64, 423.
29 K. I. Hildrum, J. L. Williams and R. A. Scanlan, J. Agric. Food
Chem., 1975, 23, 439.
17 F. Chinnici, U. Spinabelli, C. Riponi and A. Amati, J. Food 30 G. da Silva, E. M. Kennedy and B. Z. Dlugogorski, Ind. Eng.
Compos. Anal., 2005, 18, 121.
Chem. Res., 2004, 43, 2296.
18 H. A. B. Linke and S. J. Moss, J. Food Sci., 1992, 31, 147.
19 E. Compagnon, E. Thiberville, N. Moore, C. Thuillez and
31 G. da Silva, E. M. Kennedy and B. Z. Dlugogorski, J. Am.
Chem. Soc., 2005, 43, 3664.
C. Lacroix, J. Chromatogr. B: Biomed. Sci. Appl., 1996, 677, 32 R. J. Block and D. Bolling, J. Biol. Chem., 1939, 129, 1.
380. 33 C. J. Gerack and L. McElwee-White, Molecules, 2014, 9, 7689.
20 Z. Chen and M. A. Adams, Anal. Chim. Acta, 1999, 386, 249. 34 C. A. Schmidt, J. Biol. Chem., 1929, 82, 587.
˜´
´
´
21 D. Pleissner and N. T. Eriksen, Biotechnol. Bioeng., 2012, 109, 35 J. Arenas-Valganon, R. Comez-Bombarelli, M. Gonzalez-
´
´
´
2005.
Perez, M. Gonzalez-Jimenez, E. Calle and J. Casado, Int. J.
22 G. Kainz, F. Kasler and H. Huber, Mikrochim. Acta, 1959, 6,
883.
23 S. A. Boerner, C. R. Isham, Y. K. Lee, J. D. Tibodeau,
Chem. Kinet., 2014, 46, 321.
36 I. Meier, S. E. Shephard and W. K. Lutz, Mutat. Res., 1990,
238, 193.
S. H. Kaufmann and K. C. Bible, The nitric acid method 37 L. Fernandez, P. Herves and M. Parajom, J. Phys. Org. Chem.,
for protein estimation in biological samples, in The protein 2008, 21, 713.
protocols handbook, ed. J. M. Walker, Humana Press, 3rd 38 P. G. Rigas, Instrum. Sci. Technol., 2012, 40, 161.
edn, 2009, pp. 35–45.
39 A. Le, T. Ng, K. Kwan, T. M. Cusmano-Ozog and A. Cowan, J.
Chromatogr. B: Anal. Technol. Biomed. Life Sci., 2014, 944,
166.
40 X. Chen, D. Gao, F. Liu, X. Gao, S. Wang, Y. Zhao, H. Liu and
Y. Jiang, Anal. Chim. Acta, 2014, 836, 61.
41 K. Petritis, C. Elfakir and M. Dreux, J. Chromatogr. A, 2002,
961, 9.
24 Y. Fumiyuki and K. Ikeda, Nitric Oxide, 2006, 14, 152.
25 G. Danno, K. Kanazawa, M. Toda, M. Mizuno, H. Ashida and
M. A. Natake, J. Agric. Food Chem., 1993, 41, 1090.
26 J. Casado, A. Castro, J. R. Leis, M. Mosquera and E. M. Pena,
J. Chem. Soc., Perkin Trans. 2, 1985, 1859.
27 D. Bal and A. Gryff-Keller, Magn. Reson. Chem., 2002, 40, 533.
˜
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