Measurement of 15N enrichment of glutamine and urea cycle amino acids derivatized with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate using liquid chromatography-tandem quadrupole mass spectrometry

Article abstract of DOI:10.1016/j.ab.2015.02.002

Abstract 6-Aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) is an amino acid-specific derivatizing reagent that has been used for sensitive amino acid quantification by liquid chromatography-tandem quadrupole mass spectrometry (LC-MS/MS). In this study, we aimed to evaluate the ability of this method to measure the isotopic enrichment of amino acids and to determine the positional ¹⁵N enrichment of urea cycle amino acids (i.e., arginine, ornithine, and citrulline) and glutamine. The distribution of the M and M + 1 isotopomers of each natural AQC-amino acid was nearly identical to the theoretical distribution. The standard deviation of the (M + 1)/M ratio for each amino acid in repeated measurements was approximately 0.1%, and the ratios were stable regardless of the injected amounts. Linearity in the measurements of ¹⁵N enrichment was confirmed by measuring a series of ¹⁵N-labeled arginine standards. The positional ¹⁵N enrichment of urea cycle amino acids and glutamine was estimated from the isotopic distribution of unique fragment ions generated at different collision energies. This method was able to identify their positional ¹⁵N enrichment in the plasma of rats fed ¹⁵N-labeled glutamine. These results suggest the utility of LC-MS/MS detection of AQC-amino acids for the measurement of isotopic enrichment in ¹⁵N-labeled amino acids and indicate that this method is useful for the study of nitrogen metabolism in living organisms.

Full text of DOI:10.1016/j.ab.2015.02.002

Analytical Biochemistry 476 (2015) 67–77
Contents lists available at ScienceDirect
Analytical Biochemistry
journal homepage: www.elsevier.com/locate/yabio
Measurement of 15N enrichment of glutamine and urea cycle amino
acids derivatized with 6-aminoquinolyl-N-hydroxysuccinimidyl
carbamate using liquid chromatography–tandem quadrupole mass
spectrometry
Hidehiro Nakamura, Sachise Karakawa, Akiko Watanabe, Yasuko Kawamata, Tomomi Kuwahara,
⇑
Kazutaka Shimbo, Ryosei Sakai
Institute for Innovation, Ajinomoto, Kawasaki-ku, Kawasaki-shi 210-8681, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
6-Aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) is an amino acid-specific derivatizing reagent
that has been used for sensitive amino acid quantification by liquid chromatography–tandem quadrupole
mass spectrometry (LC–MS/MS). In this study, we aimed to evaluate the ability of this method to
measure the isotopic enrichment of amino acids and to determine the positional 15N enrichment of urea
cycle amino acids (i.e., arginine, ornithine, and citrulline) and glutamine. The distribution of the M and
M + 1 isotopomers of each natural AQC–amino acid was nearly identical to the theoretical distribution.
The standard deviation of the (M + 1)/M ratio for each amino acid in repeated measurements was
approximately 0.1%, and the ratios were stable regardless of the injected amounts. Linearity in the
measurements of 15N enrichment was confirmed by measuring a series of 15N-labeled arginine standards.
The positional 15N enrichment of urea cycle amino acids and glutamine was estimated from the isotopic
distribution of unique fragment ions generated at different collision energies. This method was able to
identify their positional 15N enrichment in the plasma of rats fed 15N-labeled glutamine. These results
suggest the utility of LC–MS/MS detection of AQC–amino acids for the measurement of isotopic
enrichment in 15N-labeled amino acids and indicate that this method is useful for the study of nitrogen
metabolism in living organisms.
Received 28 September 2014
Received in revised form 3 February 2015
Accepted 3 February 2015
Available online 11 February 2015
Keywords:
Urea cycle amino acids
LC–MS/MS
Isotopic enrichment
Ó 2015 Elsevier Inc. All rights reserved.
Among the precolumn derivatization procedures for amino acid
analysis, the 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate
(AQC)1 method is widely accepted. AQC reacts with primary and
secondary amines to produce highly stable fluorescent derivatives.
This derivatization reaction is straightforward and can be completed
within 10 min. The AQC derivatization method has been used in con-
junction with high-performance liquid chromatography (HPLC) [1,2]
or ultra-performance liquid chromatography (UPLC) [3,4] for the
measurement of amino acid concentrations.
Armenta and coworkers applied the AQC method together with
UPLC coupled with tandem quadrupole mass spectrometry (MS/
MS) for fast, selective, and sensitive amino acid quantification in
biological samples [5]. Because amino acids derivatized with AQC
generate the m/z 171 ion in MS/MS, this specific ion is selected
as the daughter ion in all selected reaction monitoring (SRM) tran-
sitions. Although it has also been used to measure the isotopic
enrichment of amino acids [6,7], insufficient data are available
regarding the accuracy and precision of the AQC method in
measuring the isotopic enrichment of amino acids.
Urea cycle amino acids such as arginine, ornithine, and citrul-
line have various biological functions. They play important roles
in not only synthesizing urea in the liver but also generating
bioactive substances such as nitric oxide, creatinine, polyamines,
and proline [8,9]. For the investigation of their metabolism
in vivo, tracer techniques using stable isotopes are essential, and
these techniques require sensitive analytical methods for deter-
mining isotopic enrichment. Although various methods to measure
⇑
Corresponding author. Fax: +81 44 210 5874.
E-mail address: ryosei_sakai@ajinomoto.com (R. Sakai).
Abbreviations used: AQC, 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate;
1
HPLC, high-performance liquid chromatography; UPLC, ultra-performance liquid
chromatography; MS/MS, tandem quadrupole mass spectrometry; SRM, selected
reaction monitoring; GC–MS, gas chromatography–mass spectrometry; N, nitrogen;
LC, liquid chromatography; ESI, electrospray ionization; SD, standard deviation; CV,
coefficient of variation.
http://dx.doi.org/10.1016/j.ab.2015.02.002
0003-2697/Ó 2015 Elsevier Inc. All rights reserved.

68
Measurement of 15N enrichment in AQC–amino acids / H. Nakamura et al. / Anal. Biochem. 476 (2015) 67–77
isotopic enrichment with gas chromatography–mass spectrometry
(GC–MS) have been reported [10–12], they are not entirely
satisfactory because of complicated precolumn derivatization steps
or contaminant peaks, as reported previously [13]. Furthermore,
the measurement of the positional 15N enrichment of each nitro-
gen (N) in urea cycle amino acids is important for more precise
investigation of their N metabolism because one amino acid con-
tains multiple N atoms of distinct origins. Marini reported a
method to measure the positional 15N enrichment of glutamine
and citrulline derivatized with dansyl chloride [14]. However,
there has been no report of positional 15N enrichment using AQC
(ꢀ80% of the ad libitum intake) of an experimental diet in which
all glutamine was replaced with 15N-labeled glutamine (2-15N or
5-15N). Thus, hourly consumption rates of [2-15N]glutamine and
[5-15N]glutamine were 20 mg/h. Six hours after the initiation of
feeding, the rats were anesthetized with ether to collect blood from
the descending aorta. Blood containing ethylenediaminetetraacetic
acid (EDTA) as an anticoagulant was centrifuged at 2010g for
20 min at 4 °C to separate the plasma. The plasma samples were
stored at ꢁ80 °C until analysis. A 20
l
l aliquot of the pooled plasma
was deproteinized by adding 80 l of methanol. A 10
l
l
l aliquot of
the deproteinized supernatant was derivatized with AQC as
as
a derivatization reagent despite the advantages of this
described above.
derivatization method compared with others [15].
In the current study, we evaluated the accuracy of the AQC
method for measuring the 15N enrichment of amino acids and
developed new methods for determining the positional 15N enrich-
ment of urea cycle amino acids and glutamine. Furthermore, we
investigated the efficacy of these methods for analyzing biological
samples.
Instrumentation
An Agilent 1200 series LC system (Agilent Technologies,
Waldbrunn, Germany) with a binary pump, degasser, autosampler,
column compartment, and ultraviolet (UV) detector was used. The
system was coupled to a triple-quadrupole Agilent 6400 series
mass spectrometer (Agilent Technologies) equipped with
JetStream interface.
a
Materials and methods
Materials
LC–MS/MS selected reaction monitoring for quantitative analysis of
amino acid derivatives
A solution of 41 authentic amino acids was prepared from
commercially available
Wako Pure Chemical Industries, Osaka, Japan) [16] and crystallized
-glutamine, -tryptophan, and -asparagine (Wako Pure Chemical
Industries). 15N-labeled -arginine (U-15N4 and guanidino-15N2)
and 15N-labeled -glutamine (2-15N and 5-15N) (>98% 15N for all)
were purchased from Cambridge Isotope Laboratories (Andover,
MA, USA). The AccQ-Fluor Reagent Kit was purchased from
Waters (Milford, MA, USA). Acetic acid and acetonitrile were pur-
chased from Junsei Chemical (Tokyo, Japan).
L-amino acid solutions (type B and AN-II,
Amino acid derivatives were injected into an Ascentis Express
C18 HPLC column (2.7
particle size, 15 cm ꢂ 2.1 mm
l
m
L
L
L
length ꢂ inner diameter, Supelco, Bellefonte, PA, USA). The column
temperature was maintained at 40 °C. Mobile phase A was 25 mM
formic acid (pH 6.0 adjusted with aqueous ammonium), and
mobile phase B consisted of 60% acetonitrile in MilliQ water
(v/v). The gradient conditions (B%) were 0.00 min = 10%, 0.00 to
21.00 min = 10 to 20%, 21.00 to 21.01 min = 20 to 40%, 21.01
to 23.00 min = 40 to 80%, 23.00 to 23.01 min = 80 to 100%, 23.01
to 25.00 min = 100%, 25.00 to 25.01 = 100 to 10%. Reequilibration
was performed for 10 min at 10% of mobile phase B. The flow rate
was 0.2 ml/min. The autosampler was maintained at 4 °C. The elec-
trospray ionization (ESI) was operated in the positive mode at
4000 V. Other MS parameters were as follows: nebulizer pressure,
15 psi; nozzle voltage, 1500 V; drying gas temperature, 300 °C;
drying gas flow, 10 L/min; capillary voltage, 4000 V; sheath gas
temperature, 400 °C; sheath gas flow, 12 L/min; fragmentor volt-
age, 120 V. Quantitative analysis of the amino acids was performed
by SRM. MassHunter software (Agilent) was used for HPLC system
control, data acquisition, and data processing.
L
L
Derivatization of amino acids with AQC
AQC was dissolved in acetonitrile to a concentration of 10
mg/ml. A 10
mixed with 30
sealed 1.5 ml polypropylene microtube. A 10
AQC reagent was added to the above solutions, and the mixture
was heated at 55 °C for 10 min. A 200 l aliquot of 0.2% aqueous
l
l aliquot of the authentic amino acid solution was
l of 0.2 M sodium borate buffer at pH 8.8 in a
l aliquot of the
l
l
l
formic acid was then added to the reaction mixture. The samples
were stored in a tightly sealed container at 4 °C until liquid chro-
matography (LC)–MS/MS analysis.
Fragmentation
Preparation of blood samples
A collision energy of 25 eV generated the common product ion
of m/z 171 originating from the AQC molecule for all amino acids.
The 15N enrichment of amino acids was estimated from the mass
distribution of their precursor ions using the common fragment
m/z 171 as a reporter ion, as shown in Table 1. In cases where
amino acids contained multiple N atoms in a single molecule, the
enrichments were the sum of those in all Ns because the precursor
ions contained all of the N atoms. For the measurement of posi-
tional 15N enrichments in urea cycle amino acids (arginine, citrul-
line, and ornithine) and glutamine, other fragment ions generated
from the same precursor ions were analyzed. The 15N enrichment
of the alpha-amino Ns of these amino acids was estimated using
smaller fragment ions (m/z 70 for urea cycle amino acids and m/z
84 for glutamine) generated at collision energies of 25 and 38 eV.
For the measurement of the guanidino N of arginine, m/z 158 gen-
erated at a collision energy of 25 eV, which lacks one of the two
All animal studies were reviewed and approved by the animal
care committee of Ajinomoto. Male 6-week-old Fischer (F344) rats
(Charles River Japan, Atsugi, Japan) were maintained under con-
trolled conditions with 12 h cycles of dark (10:00–22:00) and light
(22:00–10:00) at 23 1 °C and 55 10% humidity. The rats were
fed an amino acid-defined purified diet based on the AIN-93G com-
position [17]. The amino acid composition followed the recom-
mendations of the National Research Council (values in g/kg:
arginine, 4.3; histidine, 2.8; isoleucine, 6.2; leucine, 10.7; lysine
hydrochloride, 11.5; methionine, 6.5; cysteine, 3.3; phenylalanine,
6.8; tyrosine, 3.4; threonine, 6.2; tryptophan, 2.0; valine, 7.4; ala-
nine, 4.0; aspartic acid, 4.0; glycine, 6.0; proline, 4.0; serine, 4.0;
asparagine monohydrate, 4.6; glutamic acid, 20.0; glutamine,
20.0; Ajinomoto, Tokyo, Japan). The rats were adapted to these
conditions for 2 weeks and were then fed hourly with 1.0 g

Measurement of 15N enrichment in AQC–amino acids / H. Nakamura et al. / Anal. Biochem. 476 (2015) 67–77
69
Table 1
Mass spectral settings for each 15N measurement.
Data analysis
The data are presented as means standard deviations (SDs).
Significant differences between two groups were analyzed using
Student’s t-test.
Test amino acid
(positional N)
Collision
energy (eV)
Monitoring ions
(precursor ? reporter, m/z)
M
M + 1
M + 1 enrichment for whole molecule
Alanine
Arginine
25
25
25
25
25
25
25
25
25
25
25
25
30
25
25
25
25
25
25
25
260 ? 171
345 ? 171
303? 171
304 ? 171
346 ? 171
318 ? 171
317 ? 171
246 ? 171
326 ? 171
487 ? 171
320 ? 171
188 ? 171
473 ? 171
336 ? 171
286 ? 171
276 ? 171
290 ? 171
375 ? 171
352 ? 171
288 ? 171
261 ? 171
346 ? 171
304? 171
305 ? 171
347 ? 171
319 ? 171
318 ? 171
247 ? 171
327 ? 171
488 ? 171
321 ? 171
189 ? 171
474 ? 171
337 ? 171
287 ? 171
277 ? 171
291 ? 171
376 ? 171
353 ? 171
289 ? 171
Results
Asparagine
Aspartic acid
Citrulline
Glutamic acid
Glutamine
Glycine
Chromatograms and accuracy of isotopic ratio measurements
A mixture of 41 authentic amino acids was derivatized with
AQC and analyzed by LC–MS/MS. Monitoring of m/z 171 as an
AQC-specific product ion allowed the detection of AQC–amino acids
with high sensitivity (Fig. 1). Urea cycle amino acids such as
arginine, citrulline, and ornithine were detected as peaks with
precursor ions of m/z 345, 346, and 473, respectively. A larger m/z
of precursor ion for ornithine compared with arginine and citrulline
indicated that two molecules of AQC bind to a single ornithine. SRM
measurement of the mixture of authentic amino acids detected the
peaks of M and M + 1 isotopomers for each amino acid without any
interfering peaks (Fig. 2). The isotopic ratios of the M + 1 and M
areas were nearly identical to the theoretical values (Table 2). The
SDs of their (M + 1)/M ratios in repeated measurements (n = 6) of
authentic amino acids were generally approximately 0.1 mol%
(0.01–0.36 mol%), and the coefficients of variation (CVs) were less
than 4%. This method was also applicable to the measurements of
the M and M + 1 isotopic ratios of various amino acids in rat plasma
samples with similar high accuracy. The SDs of the ratios in
repeated measurements of plasma amino acids were also approxi-
mately 0.1 mol% (0.01–0.56 mol%), and the CVs were less than 4%
(Table 2).
Histidine
Lysinea
Methionine
NH3
Ornithinea
Phenylalanine
Proline
Serine
Threonine
Tryptophan
Tyrosine
Valine
M + 1 enrichment for positional N
Ornithine (amino N)
Citrulline (amino N)
Citrulline (ureido N)
Arginine (amino N)
Arginine (guanidino
N)b
38
38
8
38
25
473 ? 70
346 ? 70
346 ? 329
345 ? 70
345 ? 158
474 ? 71
347 ? 71
347 ? 329
346 ? 71
346 ? 158
Arginine (guanidino N2 38
345 ? 60
346 ? 61
and delta N)c
Glutamine (amino N)
38
317 ? 84
318 ? 85
Note. Sets of precursor and product ions for the measurement of 15N enrichment of
each amino acid are shown with MS/MS settings (collision energy). Targeted
positional Ns are shown in parentheses.
Dynamic range and linearity in measurement of 15N enrichment
a
Two moles of AQC are bound to lysine and ornithine.
b
The 15N enrichment of one of the two guanidino Ns was measured.
The influence of the injected dose of each urea cycle amino acid
on the isotopic enrichment was investigated (Fig. 3). The signal
intensity of m/z 171 increased nonproportionally with larger
injected doses of each urea cycle amino acid from 0.1 to 12 pmol.
However, the (M + 1)/M ratios for all amino acids tested remained
nearly constant up to 12 pmol per injection.
The sum of 15N enrichments in the two guanidino and one delta Ns was
c
measured.
guanidino Ns, was used. At a collision energy of 38 eV, a fragment
ion of m/z 60 is generated from AQC–arginine derivatives, and this
ion was used for the total 15N enrichments of the delta and the two
guanidino Ns of arginine. For the analysis of the ureido N of citrul-
line, the fragment ion m/z 329 generated at a collision energy of
8 eV, which is unique to the ureido N, was used. The collision
energy and precursor and reporter ions for each measurement of
positional 15N enrichments in the current study are also shown
in Table 1.
Next, linearity in the measurement of 15N enrichment was tested
using a series of [guanidino-15N2]arginine standards containing 0, 5,
10, 15, 20, 25, 30, 50, or 100 mol% of [guanidino-15N2]arginine in
place of natural arginine. The measured isotopic enrichment of the
M + 2 isotopomer for arginine calculated from the (M + 2)/M ratio
(i.e., the ratio of m/z 347 to 345 using m/z 171 as a reporter ion)
increased linearly and was nearly identical to the actual enrichment
levels (r2 = 0.9995, y = 1.0086x) (Fig. 4).
Calculation
Measurement of positional 15N enrichment of amino acids
The isotopic enrichment (IE; mole percentage excess, mol%) of
each amino acid was calculated from the tracer–tracee ratio
(TTR) as follows:
The applicability of the method to determine positional 15N
enrichment in glutamine was assessed using [2-15N]glutamine
and [5-15N]glutamine. A signal of m/z 85, rather than m/z 84,
appeared when fragment ions of [2-15N]glutamine were analyzed
(Fig. 5B). In contrast, no signal for m/z 85 was observed when
[5-15N]glutamine was analyzed, but the signal at m/z 84 was
observed (Fig. 5C). Thus, the positional 15N enrichment of the
amino N in glutamine can be determined by monitoring this frag-
ment ion. Besides, there was no detectable signal of ions containing
only amide N of glutamine. The accuracy of this method was fur-
ther tested by measuring standard mixtures of natural glutamine
and [2-15N]glutamine or [5-15N]glutamine (the theoretical 15N
enrichment for both is 19.6 mol%). Monitoring of the transitions
of m/z 317 to 84 and m/z 318 to 85 revealed that the 15N
TTR
1 þ TTR
IE ¼
ꢂ 100
TTR ¼ IRobs ꢁ IRbkd
;
where IRobs represents the observed isotopic ratio and IRbkd repre-
sents the isotopic ratio of the background.
The TTR of the M + 2 isotopomer for [guanidino-15N2]arginine
was corrected according to a previous report [18]. Theoretical val-
ues of the isotopic ratios for each amino acid were calculated using
MS-Isotope in Protein Prospector (available at http://prospector.
ucsf.edu/prospector/mshome.htm).

70
Measurement of 15N enrichment in AQC–amino acids / H. Nakamura et al. / Anal. Biochem. 476 (2015) 67–77
Fig.1. Chromatogram of the AQC–amino acids. (A) General cleavage pattern of AQC–amino acids in a tandem mass spectrometer. A common product ion m/z 171 is generated
from all amino acids derivatized with AQC. (B) Chromatogram of authentic amino acid mixture derivatized with AQC. AQC–amino acids were separated by reverse phase
HPLC, and m/z 171, a common product ion for AQC–amino acids, was detected in precursor ion scan mode. Peaks of representative amino acids are shown as follows: 1,
aspartic acid; 2, glutamic acid; 3, asparagine; 4, serine; 5, glutamine; 6, glycine; 7, alanine; 8, histidine; 9, citrulline; 10, threonine; 11, arginine; 12, ammonia; 13, proline; 14,
tyrosine; 15, cysteine; 16, valine; 17, methionine; 18, lysine; 19, ornithine; 20, phenylalanine; 21, tryptophan.
enrichment of the amino N of the [2-15N]glutamine standard
was 19.9 0.1 mol%, whereas the 15N enrichment of the
[5-15N]glutamine standard was 1.0 0.2 mol%. In contrast, the
urea cycle amino acids. An m/z 329 fragment was generated at
8 eV of collision energy in the analysis of citrulline (Fig. 7A). The
mass difference of 17 (corresponding to NH3) from the precursor
ion indicates that this fragment ion lacks the ureido N and that
its structure is likely [AQC-(NH)CH(COOH)C3H6(NH)CO]+.
15N enrichments of the amide
N
of [2-15N]glutamine and
[5-15N]glutamine standards were 0.0 0.2 and 18.1 0.2 mol%,
respectively (Table 3).
Next, the ability of the methods to determine positional 15N
enrichments in arginine was assessed using [U-15N4]arginine and
[guanidino-15N2]arginine standards. In the analysis of natural
arginine, the major fragment ions at 25 eV of collision energy were
m/z 60, 158, and 175 in addition to the common fragment
(m/z 171) for all amino acids. The structures of the fragments of
m/z 60, 70, 158, and 175 were predicted as [C(NH2)3]+, [C4NH8]+,
[C(NH2)NHC3H6CH(NH)COOH]+, and [(NH2)2C(NH)C3H6CH(NH2)
COOH]+, respectively, from their mass and the molecular structure
of the precursor ion as shown in Fig. 6 [19]. Based on these predic-
tions, the m/z 175 fragment contains all of the Ns of arginine, the
m/z 70 fragment contains only the amino N of arginine, and the
m/z 60 and 158 fragments lack the amino N and one of the two
guanidino Ns, respectively. Mass shifts from m/z 175 to 179, m/z
158 to 161, m/z 60 to 63, and m/z 70 to 71 in the analysis of
[U-15N4]arginine indicated that these fragment ions contain four,
three, three, and one N atoms, respectively. The lack of change in
the m/z 70 fragment in the analysis of [guanidino-15N2]arginine
indicated that this fragment does not contain any guanidino Ns.
Mass shifts from m/z 158 to 159 and m/z 60 to 62 in this analysis
indicated that these fragment ions contain one and two guanidino
Ns, respectively. These results were consistent with the predicted
structures of the fragment ions described above. An increase in
the collision energy from 25 to 38 eV changed the fragmentation
pattern of the same precursor ion in the analysis of arginine. The
signal intensity at m/z 70 was tripled by the increase in collision
energy, whereas some of the other fragments such as m/z 158
and 175 disappeared in this condition (Fig. 6D–F).
Animal experiments
The methods for the measurement of the 15N enrichment of
amino acids were applied to a rat tracer study using 15N-labeled
amino acids (Table 4). Rats were fed hourly on small diets
containing [2-15N]glutamine (experiment 1) or [5-15N]glutamine
(experiment 2) for 6 h, and their plasma samples were collected.
The isotopic enrichment of [2-15N]glutamine (amino-15N;
16.9 0.6 mol%) was much higher than that of [5-15N]glutamine
(amide-15N; 2.6 0.3 mol%) in rats fed [2-15N]glutamine (experi-
ment 1), whereas the amide N was the primary site labeled with
15N (22.3 2.0 mol%) in rats fed [5-15N]glutamine (experiment 2).
The 15N enrichments of amino Ns in various amino acids such as
glutamic acid (9.3 0.3 mol%) and aspartic acid (7.6 0.3 mol%)
were greater in experiment 1 than in experiment 2 (4.7 0.4 and
3.4 0.3 mol%, respectively), whereas the 15N enrichment of
ammonia in experiment 1 (0.6 0.1 mol%) was lower than that in
experiment 2 (2.4 0.1 mol%). The 15N enrichment of the alpha-N
of ornithine, originating from glutamic acid, was 4.8 0.6 mol% in
experiment 1 but was only 0.2 0.2 mol% in experiment 2. The
15N enrichment of the amino N of citrulline, which is derived from
the alpha-N of ornithine, was also higher in experiment
1
(8.6 0.1 mol%) than in experiment 2 (0.9 0.3 mol%). The enrich-
ment differences between experiments 1 and 2 were also evident
in the amino N of arginine (2.2 0.2 vs. 0.3 0.1 mol%, respec-
tively). In addition, the 15N enrichment of the ureido N of citrulline,
which is derived from ammonia, was as high as 22.6 1.2 mol% in
experiment 2, whereas it was only 3.0 0.3 mol% in experiment 1.
The 15N enrichment of the guanidino N of arginine, which is
derived from the ureido N of citrulline or the amino N of aspartic
In the analysis of citrulline, the m/z 70 fragment ion was
detected at 25 and 38 eV of collision energy as in the analysis of
arginine (Fig. 7A and B). The m/z 70 fragment was also observed
in the analysis of ornithine at 38 eV of collision energy. [C4NH8]+
is the only plausible structure for this fragment ion common to
acid, was labeled to
(2.5 0.3 mol%) than in experiment 1 (1.8 0.1 mol%).
a greater extent in experiment 2

Measurement of 15N enrichment in AQC–amino acids / H. Nakamura et al. / Anal. Biochem. 476 (2015) 67–77
71
Fig.2. SRM measurement of M and M + 1 isotopomers of AQC–amino acids. An authentic amino acid mixture derivatized with AQC was analyzed in SRM mode, as shown in
Table 1. Each amino acid was detected without any interfering peaks for both M (blue) and M + 1 (red) isotopomers.
method was shown to determine the positional 15N enrichment
Discussion
of glutamine and urea cycle amino acids such as ornithine, citrul-
line, and arginine.
AQC is a specific derivatization reagent for the amino moiety
that enables selective measurement of amino acids through
In the current study, we applied the conventional LC–MS/MS
analytical method for amino acids to measure their isotopic enrich-
ment and evaluated the accuracy of the measurements. The

72
Measurement of 15N enrichment in AQC–amino acids / H. Nakamura et al. / Anal. Biochem. 476 (2015) 67–77
Table 2
Accuracy of the measurement of (M + 1)/M isotope ratios.
Theoretical
Measured (M + 1)/M ratio (%)
(M + 1)/M ratio (%)
Authentic amino acids
Plasma amino acids
Average
Average
SD
CV
SD
CV
Arginine
Citrulline
Ornithine
Alanine
Asparagine
Aspartic acid
Glutamine
Glutamic acid
Glycine
Histidine
Lysine
Methionine
NH3
Phenylalanine
Proline
Serine
Threonine
Tryptophan
Tyrosine
Valine
8.47
8.13
18.64
3.91
5.45
5.10
6.59
6.25
2.76
8.03
19.78
6.99
0.41
10.68
6.17
3.94
5.09
13.3
10.72
6.20
8.08
8.26
18.48
3.99
5.43
4.97
6.57
6.18
2.74
7.93
19.25
6.97
0.48
10.72
6.15
3.94
5.08
13.45
10.57
6.21
0.30
0.08
0.16
0.10
0.17
0.10
0.06
0.05
0.02
0.22
0.36
0.04
0.01
0.15
0.06
0.06
0.06
0.14
0.05
0.03
3.68
1.03
0.88
2.43
3.08
1.95
0.91
0.87
0.90
3.37
1.89
0.60
1.35
1.4
0.91
1.42
1.13
1.04
0.47
0.45
8.09
8.36
18.08
3.85
5.60
5.11
6.52
6.21
2.77
8.00
18.97
6.86
0.48
10.37
6.16
3.98
5.01
13.13
10.41
6.14
0.22
0.11
0.56
0.03
0.09
0.20
0.06
0.05
0.04
0.16
0.25
0.10
0.01
0.06
0.07
0.02
0.04
0.17
0.12
0.05
1.91
1.28
3.09
0.78
1.55
3.95
0.97
0.76
1.60
2.01
1.30
1.40
2.81
0.58
1.19
0.55
0.86
1.32
1.12
0.45
Note. Mixtures of authentic amino acid solutions and deproteinized rat plasma samples were mixed with AQC reagent and incubated as indicated in Materials and Methods to
generate AQC–amino acids. The natural abundance of the M + 1 isotopomer of each AQC–amino acid [(M + 1)/M ratio] was repeatedly measured by LC–MS/MS (n = 6 for
authentic amino acids and n = 8 for plasma amino acids).
Fig.3. Effect of injected amounts of each AQC–amino acid on the isotopic ratios. The relationships of the injected amounts of AQC–amino acids (ranging from 0.01 to 12 pmol)
with peak area responses for M isotopomers of arginine (A), citrulline (B), and ornithine (C), and with (M + 1)/M ratios (%) for arginine (D), citrulline (E), and ornithine (F), were
investigated. The data are expressed as means SDs (n = 3). Although the peak area of each amino acid (M) did not increase linearly in accordance with the increased
quantities injected, the (M + 1)/M ratios remained nearly constant within the range of 0.1 to 12 pmol per injection.

Measurement of 15N enrichment in AQC–amino acids / H. Nakamura et al. / Anal. Biochem. 476 (2015) 67–77
73
Table 3
Measurements of isotopic enrichment of positional 15N in glutamine.
Measured 15N enrichment (mol%)
Total 15N
2-15
N
5-15
N
[2-15N]glutamine
[5-15N]glutamine
19.9 0.1
19.1 0.1
19.9 0.1
1.0 0.2
ꢁ0.0 0.2
18.1 0.2
Note. Glutamine standards containing 20 mol% of [2-15N]glutamine or
[5-15N]glutamine were prepared to evaluate the method to measure the positional
15N enrichment in glutamine. The M + 1 isotopic enrichment of total [15N]glutamine
(including both [2-15N]glutamine and [5-15N]glutamine) and [2-15N]glutamine was
measured as described in Materials and Methods. Under the assumption that there
are no M + 2 isotopomers labeled with two 15Ns, the enrichment of
[5-15N]glutamine was determined as the difference in the isotopic enrichment
between total
[
15N]glutamine and [2-15N]glutamine. Values are reported as
means SDs (n = 3).
Fig.4. Linearity
in
the
measurement
of
M + 2
enrichment
for
[guanidino-15N2]arginine. Arginine standards containing 0, 5, 10, 15, 20, 25, 30,
50, and 100 mol% of [guanidino-15N2]arginine were prepared. After derivatization
with AQC, the M + 2 isotopic enrichments were calculated from the peak areas as
described in Materials and Methods. The peak area of the M + 2 isotopomer of
arginine was determined by monitoring the transitions of m/z 347 to 171.
Relationships between the actual and measured enrichments were plotted. The
regression line is y = 1.0086x (r2 = 0.9995).
as 0.1% with minor exceptions such as arginine, ornithine, and
lysine, which have multiple Ns in their molecules. The repro-
ducibility of the measurements of isotope ratios was tested in dif-
ferent analytical conditions, and hundred-fold differences in the
injection amount (0.1–10 pmol) gave nearly identical results in
the measurement of (M+1)/M ratios, although the peak areas of
their ions did not show linearity, presumably due to ion suppres-
sion [20]. Finally, the linearity and accuracy of the measurements
of isotopic enrichment were confirmed using a series of arginine
standards containing 0 to 100 mol% of [guanidino-15N2]arginine.
The measured values were nearly identical to the actual values
(slope = 1.0086), and the correlation coefficient was as high as
0.9995 (Fig. 3). Iwatani and coworkers already applied this method
to the measurement of the 13C isotopic enrichment of amino acids
to determine the metabolic flux of amino acids in Escherichia coli
but did not show any data for the accuracy of the measurements
[6]. Thus, this is the first study to evaluate the adequacy of this
method for the measurement of the isotopic enrichment of amino
acids derivatized with AQC.
monitoring of the common m/z 171 ion that is derived from AQC.
This analytical method also has a great advantage in evaluating
the isotopic distribution of each amino acid because of its
derivatization specificity. When M + 1 isotopomers of major amino
acids were analyzed, no interfering signals were detected, as
shown in Fig. 2, and this was also confirmed in rat plasma samples
(data not shown). The measured isotope ratios of M + 1 to M for all
of the amino acids were nearly identical to their theoretical ratios
in the measurements of both the standard amino acid mixture and
the rat plasma samples, as shown in Table 2. Furthermore, the
accuracy of the measurements of the isotopic ratios was as high
as that of the conventional methods using GC–MS; the SDs of the
(M+1)/M ratios of the natural amino acids were generally as low
Fig.5. Fragmentation patterns of glutamine. Natural glutamine (A), [2-15N]glutamine (B), and [5-15N]glutamine (C) derivatized with AQC were analyzed in a product ion scan
mode at 38 eV of collision energy. All three glutamine derivatives generated the m/z 171 ion, a fragment common to AQC–amino acids, whereas a fragment at m/z 84 was
shifted to m/z 85 in the analysis of [2-15N]glutamine, indicating that this fragment contains only the amino N of glutamine. Structures of fragments were predicted based on a
previous report [19].

74
Measurement of 15N enrichment in AQC–amino acids / H. Nakamura et al. / Anal. Biochem. 476 (2015) 67–77
Fig.6. Fragmentation patterns of AQC–arginine under different collision energy settings. Natural arginine (A, D), [U-15N4]arginine (B, E), and [guanidino-15N2]arginine (C, F)
derivatized with AQC were analyzed in a product ion scan mode at 25 eV (A–C) or 38 eV (D–F) of collision energy. Fragments of m/z 60, 70, 116, 158, 171, 175, and 201 were
primarily observed at 25 eV of collision energy in the analysis of natural arginine (A). Among these ions, mass numbers of fragments with m/z 60, 70, 116, 158, 175, and 201
were increased by 3, 1, 1, 3, 4, and 4, respectively, in the analysis of [U-15N4]arginine (B). In the analysis of [guanidino-15N2]arginine (C), fragments with m/z 60, 158, 175, and
201 were increased by 2, 1, 2, and 2, respectively. Signals for m/z 60 and 70 increased in magnitude at high collision energy (38 eV) (D–F). Structures of fragments were
predicted based on a previous report [19].
The current analytical method using LC–MS/MS and AQC
derivatives has advantages compared with conventional methods
using GC–MS. In general, GC–MS methods for amino acid detection
require complex precolumn derivatization steps. For example, two
reaction steps and drying steps are needed to form
heptafluorobutyl propyl ester or trifluoroacetyl methyl ester
derivatives of amino acids. Furthermore, in biological samples, par-
tial purification of amino acids is necessary to remove interfering
peaks. However, the current AQC method has more convenient
sample preparation. The current method requires only one

Measurement of 15N enrichment in AQC–amino acids / H. Nakamura et al. / Anal. Biochem. 476 (2015) 67–77
75
Fig.7. Fragmentation patterns of AQC–citrulline and AQC–ornithine under different collision energy settings. Citrulline (A) and ornithine (B) derivatized with AQC were
analyzed in a product ion scan mode. The higher collision energy at 25 and 38 eV produced a common fragment (m/z 70) in both amino acids and in arginine (see Fig. 6). The
predicted structure of the fragment ion with m/z 329 produced at lower collision energy (8 eV) in the analysis of citrulline lacks ureido N but contains the amino N and delta N
of citrulline.
10 min reaction step to form stable AQC–amino acid derivatives
without any drying steps and does not require any purification
steps before derivatization even in the measurements of biological
samples. Armenta and coworkers reported that AQC–amino acids
are stable for more than 1 month when stored at 4 °C [5], which
is another advantage of the current method.
any purification steps, whereas the conventional method using
GC–MS requires separation of glutamine and glutamic acid before
derivatization [23].
The positional 15N enrichment of urea cycle amino acids (i.e.,
arginine, ornithine, and citrulline) was also determined using frag-
ment ions containing Ns originating from these amino acids. In the
analysis of AQC–arginine, multiple fragment ions were generated at
Although the conventional methods using GC–MS have been
applied to measurements of isotopic enrichment in various amino
acids, GC–MS methods have not provided satisfactory results for
measuring urea cycle amino acids [10–12]. For example, a previous
report showed that the derivatization of arginine with N-methyl-
N-tert-butyldimethylsilyltrifluoroacetamide (MTBSTFA) produced
the same derivative as that of ornithine [21]. Smith and coworkers
reported that citrulline showed the same peak as the ornithine
derivative [13]. Furthermore, Amelung and Zhang reported that
ensuring the complete derivatization of arginine (N-pentaflu-
oropropionyl-arginine isopropyl esters) is difficult [22]. In contrast,
the current AQC method produced unique derivatives of ornithine,
citrulline, and arginine without any molecular cleavage, and these
derivatives were distinguishable from others both in chro-
matography and in mass detection (Figs. 2, 6 and 7). Thus, this
method was quite suitable for measuring the isotopic enrichment
of these amino acids, similarly to the other amino acids mentioned
above. The measured isotopic ratios agreed well with the actual
ratios in the series of arginine standards labeled with 15N (Fig. 2).
The current method was also applicable to the measurement of
the positional 15N enrichment of glutamine. Increasing the collision
energy from 25 to 38 eV in the analysis of AQC–glutamine was effec-
tive for detecting an m/z 84 product ion that contains only the amino
N and not the amide N. Using this fragment ion, the enrichment of
the amino N of glutamine was determined and the enrichment of
Table 4
Measurements of positional 15N enrichment of plasma amino acids in rats fed with
[2-15N]glutamine or [5-15N]glutamine.
Amino
acid
Position of
15N enrichment (mol%)
the target N
[2-15N]glutamine
feeding
[5-15N]glutamine
feeding
P
value
(experiment 1)
(experiment 2)
Glutamine Total
Amino N
Glutamate Amino N
Aspartate
NH3
19.4 0.9
16.9 0.6
9.3 0.3
7.6 0.3
0.6 0.1
10.2 1.0
4.8 0.6
18.0 0.4
8.6 0.1
3.0 0.3
6.9 1.0
2.2 0.2
23.1 2.0
0.7 0.0
4.7 0.4
3.4 0.3
2.4 0.1
0.8 0.1
0.2 0.2
23.3 1.3
0.9 0.3
22.6 1.2
7.5 0.9
0.3 0.1
2.5 0.3
7.7 1.0
<0.05
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.01
<0.001
<0.001
NS
Amino N
–
Total
Amino N
Total
Amino N
Ureido N
Total
Ornithine
Citrulline
Arginine
Amino N
<0.001
<0.05
<0.05
Guanidino N 1.8 0.1
Guanidino
N2+ delta N
Guanidino
N, amino N
and delta N
(M + 1)
5.4 0.7
5.8 0.9
5.7 0.6
NS
the amide N was calculated as the difference between the total 15
N
Note. Diets containing [2-15N]glutamine (experiment 1) or [5-15N]glutamine
(experiment 2) were fed to rats hourly, and blood was collected from the
descending aorta after 6 h of feeding. Deproteinized plasma samples were deriva-
tized with AQC and analyzed by LC–MS/MS. 15N enrichment of the positional Ns in
individual amino acids was measured as described in Materials and Methods. The
data are expressed as means SDs (n = 3). NS, not significant.
and the amino 15N enrichments. The relevance of this method was
confirmed by measuring [2-15N]glutamine and [5-15N]glutamine
standards (Table 3). It should be noted that the current method to
measure positional 15N enrichment of glutamine does not require

76
Measurement of 15N enrichment in AQC–amino acids / H. Nakamura et al. / Anal. Biochem. 476 (2015) 67–77
Fig.8. Schematic representation of N flow from glutamine to urea cycle amino acids. The amino N of glutamine is transferred to the amino N of ornithine via glutamic acid and
glutamate–semialdehyde (GSA), as is their carbon skeleton in the gut. The amino N of glutamine also supplies the delta N of ornithine via glutamic acid through ornithine
aminotransferase. Ammonia (NH3) released from glutamine via glutaminase is used for the synthesis of carbamoyl phosphate (CP), which is incorporated into the ureido N of
citrulline in the gut and liver and then into one of the guanidino Ns of arginine mainly in the kidney. Aspartic acid N, derived from glutamic acid N, is incorporated into the
other guanidino N of arginine. N derived from the amino N and the amide N of glutamine are shown in blue and red, respectively.
25 or 38 eV of collision energy in addition to the common m/z 171
fragment for AQC derivatives. Among these fragments, those with
m/z 60, 70, 116, 158, 175, and 201 contained one to three Ns origi-
nating from arginine. The predicted molecular structures indicated
that m/z 70 only contains the amino N of arginine and that m/z 60
and 158 lack the amino N and one of the guanidino Ns, respectively.
These predictions were confirmed by the mass shifts in the analysis
of [U-15N4]arginine and [guanidino-15N2]arginine. The m/z 70 frag-
ment ion was also observed in the analysis of AQC–citrulline and
AQC–ornithine, and it was used for the measurement of their amino
15N enrichment. The m/z 329 fragment ion generated in the analysis
of AQC–citrulline at 8 eV of collision energy was used for the mea-
surement of ureido 15N enrichment based on the predicted molecu-
lar structure of the fragment. Although we did not validate these
methods for the positional 15N analysis of ornithine and citrulline
using their 15N-labeled standards, no other plausible molecular
structures for fragments with m/z 70 and 329 were predicted.
An animal study using [2-15N]glutamine and [5-15N]glutamine
suggested the adequacy of these methods (Table 4). The amino N
of plasma ornithine was labeled with 15N in rats treated with
[2-15N]glutamine, which agrees with the stoichiometry of ornithine
synthesis from glutamine in the intestines as shown in Fig. 8. The
greatly decreased enrichment of the total 15N and the amino 15N
of plasma ornithine in rats given [5-15N]glutamine also agrees with
the general consensus that the amide N of glutamine is catabolized
to ammonia in the first metabolic process in the gut and does not
transfer to ornithine directly [24,25]. The amino N of plasma citrul-
line, presumably originating from the amino N of intestinal
ornithine, was also labeled with 15N in rats fed [2-15N]glutamine,
whereas the 15N enrichment was much lower in rats fed
[5-15N]glutamine. In contrast, the enrichment of the ureido 15N of
citrulline, which is derived from ammonia via carbamoyl–phos-
phate, was much higher in rats fed [5-15N]glutamine than in those
fed [2-15N]glutamine. These labeling patterns of citrulline also
labeled the amino N but not the guanidino N to a greater extent
than feeding of [5-15N]glutamine, which was consistent with the
labeling patterns of citrulline and ornithine. Thus, these results sug-
gest the adequacy of the current methods to analyze the positional
15N enrichment of urea cycle amino acids.
Conclusions
The current study evaluated the accuracy and sensitivity of the
LC–MS/MS method for determining the isotopic enrichment of
amino acids derivatized with AQC and demonstrated the relevance
of the method. Furthermore, this study developed methods for
estimating the positional 15N enrichment in glutamine and urea
cycle amino acids. These methods were all sufficiently applicable
to biological samples in a tracer study using 15N-labeled glutamine.
Because AQC derivatization is known to be a highly sensitive
method for MS detection, these methods will provide powerful
tools to measure isotopic enrichment, thereby enabling the
investigation of the N metabolism of amino acids in living
organisms.2
2
Cooper and coworkers used 13N (positron-emitter, t1/2 = 9.96 min) to study
ammonia metabolism in the rat liver [27]. In this study, the authors showed that
despite the need for five enzyme steps and two mitochondrial transport steps, the
conversion of ammonia to urea in the rat liver is remarkably fast. The authors
suggested that this finding is evidence that the urea cycle enzymes and transporters
form a metabolon. The authors also showed that N exchange among components of
the alanine and aspartate aminotransferase reactions is also very fast in rat liver [28].
In a separate study, Cooper and coworkers investigated the short-term metabolic fate
of 13N-labeled glutamate, alanine, and glutamine(amide) in rat liver [28]. The authors
verified the rapid
N exchange among components of the alanine and aspartate
aminotransferase reactions. They also showed that glutamine amide N is a source of
urea. However, although these studies were instrumental in showing the previously
unappreciated rapidity of N exchange among certain metabolites in rat liver, the use
of 13N is not suitable for studies of N flux over periods of more than 30 min or so.
Moreover, 13N label in the amide group of glutamine can only be distinguished from
13N label in the amine group of glutamine by enzymatic analysis with glutaminase.
The current procedure has the advantage that long-term studies over many hours are
feasible. Moreover, determination of isotopomer content of glutamine N does not
require analysis with glutaminase and can be determined directly.
agree with the stoichiometry of glutamine
N metabolism.
Regarding arginine that is synthesized from citrulline in kidney
and released into the circulation [26], feeding of [2-15N]glutamine

Measurement of 15N enrichment in AQC–amino acids / H. Nakamura et al. / Anal. Biochem. 476 (2015) 67–77
77
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Acknowledgment
[12] S.J. Gaskell, A.W. Pike, Gas chromatography mass spectrometry of
androstanolones as methyl oxime, tert-butyldimethylsilyl ether derivatives,
Biomed. Mass Spectrom. 8 (1981) 125–127.
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