Different behaviour of 3-nitrotyrosine and tyrosine toward perfluorinated reagents suitable for the one-step preparation of volatile derivatives

Article abstract of DOI:10.2298/JSC110304003P

With the view of developing a gas-chromatographic (GC) determination of the 3-nitrotyrosine (NY)/tyrosine (Y) ratio as a marker of nitro-oxidative stress, different reagents were tested with the objective of obtaining a single volatile fluorinated product for each amino acid in a one-step derivatisation procedure. The heptafluorobutyric anhydride (HFBA)/heptafluorobutanol (HFBOH) mixture proved unsuccessful for the simultaneous analysis of NY and Y. The reaction with different chloroformates, isobutyl chlorofomate (iBuCF) and ethyl chloroformate (EtCF) in the presence of different perfluorinated alcohols, such as trifluoroethanol (TFEOH) and HFBOH, was investigated. A combination EtCF/fluorinated alcohols yielded derivatives of NY and Y as single peaks suitable for the GC determination of the NY/Y ratio. The different behaviours of the two amino acids in the employed reaction mixtures and the parameters influencing the results are discussed. 2012 Copyright (CC) SCS.

Full text of DOI:10.2298/JSC110304003P

J. Serb. Chem. Soc. 77 (5) 667–683 (2012)
JSCS–4299
UDC 546.16–123+547.587.4+
547.56+547.466:543.544
Original scientific paper
Different behaviour of 3-nitrotyrosine and tyrosine toward
perfluorinated reagents suitable for the one-step
preparation of volatile derivatives
3
3
RADMILA PAVLOVIĆ1,2 , PIER ANTONIO BIONDI , LUCA MARIA CHIESA ,
*
NATAŠA TRUTIĆ2, MIRJANA ABRAMOVIĆ2 and ENZO SANTANIELLO1
1Department of Medicine, Surgery and Dentistry, School of Medicine c/o S. Paolo Hospital,
Università degli Studi di Milano, Italy, 2Department of Chemistry, Medical Faculty,
University of Niš, Serbia and 3Department of Veterinary Sciences and Technologies for
Food Safety, Faculty of Veterinary Medicine, Università degli Studi di Milano, Italy
(Received 4 March 2011, revised 10 January 2012)
Abstract: With the view of developing a gas-chromatographic (GC) determi-
nation of the 3-nitrotyrosine (NY)/tyrosine (Y) ratio as a marker of nitro-oxi-
dative stress, different reagents were tested with the objective of obtaining a
single volatile fluorinated product for each amino acid in a one-step deriva-
tisation procedure. The heptafluorobutyric anhydride (HFBA)/heptafluoro-
butanol (HFBOH) mixture proved unsuccessful for the simultaneous analysis
of NY and Y. The reaction with different chloroformates, isobutyl chlorofo-
mate (iBuCF) and ethyl chloroformate (EtCF) in the presence of different
perfluorinated alcohols, such as trifluoroethanol (TFEOH) and HFBOH, was
investigated. A combination EtCF/fluorinated alcohols yielded derivatives of
NY and Y as single peaks suitable for the GC determination of the NY/Y ratio.
The different behaviours of the two amino acids in the employed reaction mix-
tures and the parameters influencing the results are discussed.
Keywords: perfluorinated derivatives; tyrosine; 3-nitrotyrosine; GC–MS.
INTRODUCTION
Nitration of tyrosine Y (1) (Scheme 1), under particular conditions that are
generally termed “nitro-oxidative stress”, may impair cell function by altering the
protein conformation, solubility and susceptibility to aggregation, and could be
responsible for increased protein degradation. This is especially relevant if en-
1–3
zymes of patho-physiological significance are involved.
3-Nitrotyrosine (NY,
2) is formed under the above conditions and, therefore, its detection and quanti-
*Corresponding author. E-mail: radmila.pavlovic@medfak.ni.ac.rs
doi: 10.2298/JSC110304003P
667
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PAVLOVIĆ et al.
fication has been used as an indicator of the nitro-oxidative stress in biological
4–10
systems.
Scheme 1. Nitration of Y and structure of the derivative obtained from NY
by treatment with a HFBA/HFBOH mixture.
Recently, the preparation of derivative (3), obtained with a heptafluorobu-
tyric anhydride (HFBA)/heptafluorobutanol (HFBOH) mixture, was described
for the evaluation of the NY content in the plasma of human volunteers by gas
11
chromatography with an electron capture detector (GC–ECD). This method
12
was considered as a significant contribution to 3-nitrotyrosine quantification.
11
In the above paper, a combination of HFBA/HFBOH was selected in order
to prepare in a one-step procedure a derivative with the highest possible content
of fluorine atoms, since GC–ECD sensitivity is strongly influenced by the fluo-
13
rine content of the derivative to be analyzed*.
*Another procedure has been reported for the preparation of a volatile derivative for GC/
/negative chemical ionization (NCI) MS/MS analysis containing fourteen fluorine atoms.14
However, it should be mentioned that this derivative was obtained in three steps, which in-
cluded the reduction of NY to the corresponding amino derivative and the use of trimethylsilyl
diazomethane for the methylation step.
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DIFFERENT BEHAVIOR OF 3-NITROTYROSINE AND TYROSINE TOWARD PERFLUORINATED REAGENTS
669
During the experimental work, several conditions were tried in order to
achieve the formation of the tris-substituted derivative 4 (Scheme 2), but with no
success. This could be explained by the presence of an electron-withdrawing
group at position 3 in NY that should significantly influence the reactivity of the
phenolic OH and, consequently, the chemical properties of the NY derivatives
that could be prepared.
Scheme 2. Structure of NY heptafluorobutyl ester reacted with HFBA
at both amino and phenol moieties.
The main aim of the present work was to propose a one-step procedure
suitable for obtaining volatile derivatives for the simultaneous determination of Y
and NY as a useful approach to evaluate protein nitro-oxidative stress. Major ef-
forts were related to the possibility of obtaining the same fluorinated derivative
for both compounds, taking into account the different reactivity of NY and Y
under the derivatisation conditions.
All procedures described in the present paper were realised on the micromo-
lar scale under conditions that should simulate the analysis of biological samples.
Due to potential application as an analytical method, special attention was de-
voted to the achievement of the completeness of the derivatisation in a simple,
one-step procedure to give a single product. After performing preliminary studies
with the GC–FID analytical technique, all structures of the obtained derivatives
were identified by MS analysis and the fragmentation spectra are presented here-
in as proof of the chemical structures.
MATERIALS AND METHODS
Chemicals
Y (1) and NY (2) were purchased from Sigma–Aldrich Italia, as well as derivatisation
reagents (trifluoroacetic anhydride (TFAA), heptafluorobutyric anhydride (HFBA), ethyl
chloroformate (EtCF), iso-butyl chloroformate (iBuCF) 2,2,2-trifluoroethanol (TFEOH), hep-
tafluorobutanol (HFBOH), ethanol (EtOH), iso-butanol (iBuOH), N,O-bis(trimethylsilyl) tri-
fluoroacetamide (BSTFA) and N-methyl-N-(t-butyldimethylsilyl)trifluoroacetamide (MTBSTFA)).
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PAVLOVIĆ et al.
Preparation of N-perfluoroacyl perfluoroalkyl esters and their silyl derivatives
Y and NY were derivatised by conversion to their N- or N,O-perfluoroacyl perfluoro-
alkyl esters according to a previously reported procedure.15 Briefly, the two derivatising
agents (50 μL perfluoroalcohol and 100 μL of perfluoroalkyl anhydride) were added to the dry
amino acids (0.1 μmol each) and heated at 80 °C for 30 min. The alcohols used in the present
experiments were TFEOH and HFBOH, while the anhydrides were TFAA and HFBA. Excess
reagents were removed under a stream of nitrogen and the residues were dissolved in ethyl
acetate. The organic extract was both directly injected or treated with 100 μL of BSTFA or
MTBSTFA and heated at 80 °C for 30 min. After cooling to room temperature, 1µL of the
resulting mixture was injected into the GC–MS system.
Preparation of N(O)-alkoxycarbonyl alkyl esters
Y and NY were derivatised to their N(O)-alkoxycarbonyl alkyl esters and analyzed fol-
lowing the protocol previously described by Husek16 and modified by Wang et al.17 In brief,
amino acids (0.10 μmol each) were dissolved 100 μL of H2O and treated with 100 μL of al-
cohol/pyridine in a volume ratio 80:20. The alcohols used in the present experiments were:
EtOH, iBuOH, TFEOH and HFBOH. Then 12.5 μL of EtCF or iBuCF were added and mixed
by shaking gently for 30s. The derivatives were extracted with 150 μL of hexane containing 2 %
chloroformate. A 1 µL aliquot was taken from the organic layer and injected.
GC–FID
A Trace GC Ultra (Thermo) (Milan, Italy) fitted with an FID was used in the control ex-
periments. This chromatograph was equipped with a capillary column Optima 1701 (15 m×
×0.25 mm, 0.25 μm) (Macherey-Nagel, Duren, Germany). The temperatures of the injector
and detector were 250 and 280 °C, respectively. The split injection technique was used with a
1:10 ratio. The column temperature was programmed from 140 to 290 °C at 10 °C min-1.
Helium at a flow rate of 0.9 ml min-1 was used as the carrier gas.
GC/MS
GC/MS Analyses were performed on a 5972A mass selective detector interfaced to a
5890 gas chromatograph (Agilent, Milano, Italy). The instrument was operated in the electron
ionisation (EI) mode with an electron energy of 70 eV. The GC separation was accomplished
on a HP-5 MS (30 m×0.25 mm, 0.25 μm) column. The transfer line temperature was 310 °C
and the injector temperature was set at 300 °C. The injector was operated in the splitless
mode. The column temperature was held at 50 °C for 5 min, then programmed to increase at a
rate of 20 °C min-1 to 300 °C and finally held for 15 min at the end temperature.
RESULTS AND DISCUSSION
As the objective of this work was the simultaneous derivatisation of Y and
NY, the optimal reaction conditions (80 °C, 30 min) that afforded the NY bis-de-
11
rivative 3 with a HFBAA/HFBOH mixture in a previous study were repeated
on the parent proteinogenic amino acid Y. However, the reaction of Y afforded a
mixture of tris- and bis-derivatives (Scheme 3, compounds 5a and 5b) as shown
in Fig. 1.
As a possible explanation of these results, it should be considered that a
strong electron-withdrawing group, such as a nitro group, increases the acidity of
the phenolic hydroxyl moiety. Consequently, the intermediate O-heptafluoro bu-
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DIFFERENT BEHAVIOR OF 3-NITROTYROSINE AND TYROSINE TOWARD PERFLUORINATED REAGENTS
671
tanoate could be more susceptible to in situ hydrolysis, constantly producing free
OH. Interestingly, an internal hydrogen bond between the phenolic OH and the
18
nitro group in NY could lower the OH reactivity.
Since a reliable, quantitative analysis is generally based on unique peaks of
the analytes, the simultaneous determination of Y and NY would not be possible
using the HFBA/HFBOH mixture.
Scheme 3. Derivatisation of tyrosine by the perfluroacylation/perfluoroesterification
procedure.
Fig. 1. GC–FID Chromatogram of the products obtained by treatment of Y with HFBA and
HFBOH, tris- (first peak) and bis-substituted (second peak) derivatives.
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PAVLOVIĆ et al.
Therefore, the derivatives obtained using the TFAA/TFEOH combination
were examined. Both Y and NY afforded unique products, but while Y gave the
tris-derivative 6, NY afforded only the bis-derivative with a free phenolic OH
(Scheme 4, compound 7). The mass spectrometric data of the obtained deriva-
tives are given in Table I. Some results are in the agreement with previously re-
19,20
ported information,
while those regarding 5b are presented here for the first
time. In particular, the fragmentation pattern of the NY derivatives was espe-
cially investigated. The main peak for both derivatives was the 4-methylene-2-
-nitro-cyclohexa-2,5-dienylidene-oxonium cation (3-nitro-4-hydroxybenzyl cat-
ion) (m/z = 152). Important ions were present in both fluorinated derivatives 7
(Fig. 2a) and 3, such as the ones originating from the cleavage of the C–C bond
next to the ester group, with the formation of peaks at m/z = 277 (M–127) for the
TFAA–TFEOH derivative and m/z = 377 (M–227) for the one obtained with
HFBA–HFBOH. Moreover, other peaks in both cases were derived from the
fragment produced by the loss of CF CONH , m/z = 291 (M–113) for the
3
2
TFAA–TFEOH derivative or CF (CF ) CONH , m/z = 391 (M–213) for the one
3
2 2
2
obtained from HFBA–HFBOH.
Scheme 4. Derivatisation of 3-nitrotyrosine by the perfluroacylation/perfluoroesterification
procedure.
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TABLE I. Characteristic ion peaks in the mass spectra of N,(O)-perfluoroacyl perfluoroalkyl
tyrosine and 3-nitrotyrosine esters
Main
m/z peak,
m/z
Characteristic
peaks, m/z
Amino acid
Tyrosine
Anhydride/alcohol
Derivative
TFAA/TFEOH N,O-trifluoracetyl tyrosine 455a
trifluorethyl ester 6
HFBA/HFBOH
203
231, 243,
328, 342
275, 303,
343
N,O-heptafluorobutyryl 755a
tyrosine heptafluorobutyl
ester 5a
542
N-heptafluorobutyryl
tyrosine heptafluorobutyl
ester 5b
N-trifluoracetyl
3-nitrotyrosine trifluor-
ethyl ester 7
559
404a
604
107
152
152
332, 346
3-Nitrotyrosine TFAA/TFEOH
HFBA/HFBOH
106, 277,
291, 386
N-heptafluorobutyryl
3-nitrotyrosine
106, 377,
391
heptafluorobutyl ester 3
aNo molecular ion identified
Fig. 2. Mass spectra of NY, N-trifluoracetyl trifluorethyl ester and its silyl derivatives:
a) compound 6.
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PAVLOVIĆ et al.
Fig. 2. (Continued) Mass spectra of NY, N-trifluoracetyl trifluorethyl ester and its silyl
derivatives: b) compound 8a and c) compound 8b.
Moreover, additional studies are reported that were performed during a
11
reappraisal of the NY protocol. Namely, it was observed that after numerous
injections of standard and biological samples into the same column, the peak
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675
shape of the NY derivatives showed an increasing tailing. It was reasoned that
the presence of the free phenolic group could influence the reliability of the
21
analytical procedure, as already observed for other 2-nitrophenols. In order to
demonstrate that a derivative of NY with a protected phenolic OH is more
suitable for gas-chromatographic analysis, silylation of compound 7 with
different silylating agents was performed. Trimethylsilyl (TMS) and t-
butyldimethylsilyl (TBDMS) derivatives 8a and 8b were prepared (Scheme 5)
and the corresponding MS data are given in Table II and shown in Figs. 2b and
2c. It is of interest to observe that this derivatisation technique offers the
advantage of a sufficient abundance of high molecular mass fragments that should
facilitate the identification of the compound.
Scheme 5. Structures of the silyl derivatives of NY pre-
viously reacted with TFA/TFEOH mixture.
TABLE II. Characteristic ion peaks in the mass spectra of NY silyl derivatives of N-trifluor-
ethyl esters
Derivative
m/z
Main peak, m/z Characteristic peaks, m/z
TMS derivate of NY
473
461
179, 224, 348
N-trifluoracetyl trifluorethyl ester (8a)
TBDMS derivate of NY
518a
461
179, 210, 445,503
N-trifluoracetyl trifluorethyl ester (8b)
aNo molecular ion identified
GC analysis of the silyl derivatives 8a and 8b showed that, as opposed to
derivatives 3 and 7, the peaks remained constantly sharp after many injections
onto the same column; thus confirming that a tris-derivative of NY would be
highly desirable.
In any event, the impossibility of obtaining the same derivatives for both NY
and Y by an anhydride/alcohol mixture, led to the exploration for alternative me-
16,22
thods. The Husek method
is a validated procedure for proteinogenic amino
acids that offers distinct advantages due to its simplicity and rapidity. According
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PAVLOVIĆ et al.
to this procedure, aqueous solutions of practically all natural amino acids react
with alkyl chloroformates and alcohols in pyridine to form N(O)-alkoxycarbonyl
alkyl esters suitable for GC analysis. A combination of alkyl chloroformates and
17,15,23–26
fluoroalkanols has already been used for amino acids analysis,
but, to
the best of our knowledge, has not been exploited for the purpose of NY determi-
17
nation. In accordance with previous studies, the reaction of the carboxylic
group with a chloroformate and an alcohol leads to the formation of an ester, the
alkyl moiety of which is provided by the alcohol. The same reaction with an
amine group leads to an N-alkoxycarbonyl derivative bearing the alkyl group of
the chloroformate. In the case of an amino acid, such as Y or NY, tris-substituted
N,O-alkoxycarbonyl derivatives are possible, due to the presence of the phenolic
OH. The main experiments in this work were addressed to the study of the dif-
ferent behaviour of NY with respect to Y, of which some Husek derivatives have
already been reported. The purpose was the careful monitoring of the influence
of the nitro group on the outcome of the derivatisation procedures performed
with different commercial chloroformate reagents and perfluoroalcohols.
Among N(O,S) alkoxycarbonyl alkyl esters of amino acids, isobutoxycarbo-
nyl isobutyl esters can be considered as an optimal solution to start with, from the
25
viewpoint of both sensitivity and stability. Therefore, the classical combination
iBuCF/iBuOH and bis-substituted derivatives of Y and NY was first investigated
and N-iso-butoxycarbonyl iso-butyl esters 9a and 9b were exclusively obtained
25,26
(Scheme 6). This result is in line with previously reported data for Y.
Scheme 6. Derivatisation of tyrosine and 3-nitrotyrosine by isobutyl
chloroformate/isobutanol combinations.
In this case, the reaction did not occur at the phenolic OH of either Y or NY,
which could be due to the presence of the apolar alcohol iBuOH in the reaction
mixture. This may affect some chemico–physical parameters of the derivatisation
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medium, with the effect of decreasing the reactivity of the phenolic OH toward
chloroformate.
Being as the aim was to prepare volatile fluorinated derivatives, TFEOH was
initially selected as the perfluoroalcohol in the Huseck procedure. The combi-
nation iBuCF/TFEOH afforded the tris-substituted derivative as the single pro-
duct for both Y and NY (Scheme 7, compounds 10a and 10b).
Scheme 7. Derivatisation of tyrosine and 3-nitrotyrosine by isobutyl chloroformate/TFEOH
combinations.
In a subsequent experiment, HFBOH was tested (Scheme 8). The encour-
aging result obtained with TFEOH was not confirmed by the combination of
iBuCF with the less hydrophilic HFBOH. In fact, Y afforded the tris-substituted
derivative 11a as a single product, whereas NY afforded two products (bis- and
tris-substituted 11b and 11c), as shown in Fig. 3. As the efficiency of the deri-
Scheme 8. Derivatisation of tyrosine and 3-nitrotyrosine by isobutyl
chloroformate/HFBOH combinations.
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PAVLOVIĆ et al.
Fig. 3. GC–FID Chromatogram of NY derivatives obtained by treatment
with iBuCF and HFBOH.
vatisation procedure may be attributed to different experimental conditions, an
attempt was made to eliminate the double products obtained with the iBuCF/
/HFBOH combination by varying the reaction time and the amounts of chlorofor-
mate, pyridine and alcohol in the reaction mixture. These changes, however, did
not completely lead to a single product. One possible explanation can be related
to the low solubility of iBuCF in the mixture H O/HFBOH/pyridine that might
2
27
cause incomplete derivatisation of NY. In this context, the high polarity of NY,
which renders this amino acid more hydrophilic than tyrosine itself, may also
have contributed to the incomplete derivatisation of NY when the iBuCF/HFBOH
combination was applied.
MS analysis of the derivatives obtained with iBuCF was used for the iden-
tification of the products. In Table III, the most significant peaks in the mass
spectra of derivatives are listed. The EI spectra could be interpreted with refe-
17
25
rence to that reported by Wang et al. and Sobolevsky et al. for tyrosine.
As a general comment, using iBuCF only in combination with TFEOH af-
forded a single tris-substituted derivative product for NY and Y (compounds 10a
and 10b). Although, the fluorine content was low, this result evidences that it is
possible to obtain tris-substituted products for both Y and NY by a suitable com-
bination of alkyl choroformates and alcohols.
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TABLE III. Characteristic ion peaks in the mass spectra of N-isobutoxycarbonyl(per-
fluoro)alkyl esters of tyrosine and 3-nitrotyrosine
Main
m/z peak,
m/z
Characteristic
peaks, m/z
Amino acid
Tyrosine
Chloroformate/alcohol
Derivative
iBuCF/iBuOH
iBuCF/TFEOH
N-isobutoxycarbonyl 337
isobutyl ester 9a
107
164, 220, 320
N,O-di-isobutoxycar- 463a
bonyl trifluoroethyl
ester 10a
107
207, 235,
246, 390
iBuCF/HFBOH
N,O-di-isobutoxycar- 563
bonyl heptafluorbutyl
ester 11a
107
57, 207,
256, 346
3-Nitrotyrosine
iBuCF/iBuOH
iBuCF/TFEOH
N-isobutoxycarbonyl 382a
isobutyl ester 9b
57
57
137, 152,
209,280
135, 152, 156,
291, 334, 435
N,O-di-isobutoxycar- 508
bonyl trifluoroethyl
ester 10b
iBuCF/HFBOH
N-isobutoxycarbonyl 508
heptafluorbutyl ester
11b
152
152
57, 234, 256,
281, 391, 434
N,O-isobutoxycar- 608a
bonyl heptafluorbutyl
ester 11c
57, 106, 256,
391, 462, 490,
508, 535
aNo molecular ion identified
This prompted the study of other combinations of reagents, and ethyl chloro-
formate (EtCF) was selected, which, although less stable than iBuCF, has already
17,19,23
been used in combination with different alcohols for Y derivatisation.
The
28
combination EtCF/EtOH that gave the tris-substituted derivative 12a with Y
was tested with NY. The non-proteinogenic amino acid NY also afforded the
N,O-diethoxycarbonyl ethyl ester 12b (Scheme 9).
It was gratifying to observe that both NY and Y reacted also with TFEOH or
HFBOH in the presence of EtCF and pyridine to give always the tris-derivatives
(compounds 12c–f).
The MS spectra of the derivatives obtained with EtCF are collected in Table
IV, in which the molecular ions and the most characteristic fragments of the mass
spectra of derivatives 12a–f are presented. The mass fragmentation pattern of the
NY tris-substituted derivative (12f) is shown in Fig. 4.
The results obtained with EtCF and fluorinated alcohols open several analy-
tical perspectives, since it has been demonstrated that by a suitable selection of
alkyl chloroformates and alcohols, NY and Y can be determined in the same run,
affording the corresponding tris-substituted derivative. This opens a new oppor-
tunity for the determination of their ratio as nitro-oxidative marker.
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PAVLOVIĆ et al.
Scheme 9. Derivatisation of tyrosine and 3-nitrotyrosine by ethyl
chloroformate/ethanol or perfluoro alcohols combinations.
TABLE IV. Characteristic ion peaks in the mass spectra of N,O-diethoxycarbonyl (per-
fluoro)alkyl esters of tyrosine and 3-nitrotyrosine
Main
m/z peak,
m/z
Characteristic
peaks, m/z
Amino acid
Tyrosine
Chloroformate/alcohol
Derivative
EtCF/EtOH
EtCF/TFEOH
EtCF/HFBOH
N,O-diethoxycarbonyl 353 107
ethyl ester 12a
N,O-diethoxycarbonyl 407 107 135, 179, 246,
trifluoroethyl ester 12c 280, 318
N,O-diethoxycarbonyl 507 107 135, 179, 280,
135, 192,
264, 280
heptafluorbutyl ester
346, 434
12e
3-Nitrotyrosine
EtCF/ EtOH
N,O-diethoxycarbonyl 398 102 106, 135, 152,
ethyl ester 12b
174, 237,
265, 235
EtCF/TFEOH
EtCF/HFBOH
N,O-diethoxycarbonyl 452 152 106, 135, 228,
trifluoroethyl ester 12d
N,O-diethoxycarbonyl 552 152
heptafluorbutyl ester
12f
291, 325, 362
135, 256,
391, 462
CONCLUSIONS
Several one-step preparations of fluorinated derivatives of Y and NY, which
would be suitable for GC analysis, were investigated. The simultaneous
determination of Y and NY could constitute a new, useful method for the eva-
luation of the nitro-oxidative stress of proteins; in this regard, the optimal deri-
vatisation method should afford only a single product in a one-step procedure
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DIFFERENT BEHAVIOR OF 3-NITROTYROSINE AND TYROSINE TOWARD PERFLUORINATED REAGENTS
681
Fig. 4. Mass spectrum of the N,O-diethoxycarbonyl heptafluorbutyl ester
derivative of NY (compound 12f).
with both amino acids. Actually, in addition to inductive effect reasons, the pre-
sence of an ortho-nitro group causes different availability of the phenolic OH
when bulky reagents are used, while small reagents are not influenced to the
same extent by groups located near the phenolic OH. In addition, the structure of
the alcohol appears to represent a critical parameter that could influence different
reaction courses with NY and Y. The chloroformate/perfluoroalcohol procedure
was evaluated by GC–FID and all the derivatives were identified by MS analysis.
The best results were obtained with iBuCF/TFEOH and EtCF/HFBOH combi-
nations. The N,O-diethoxycarbonyl heptafluorobutyl ester derivative 12f is a tris-
-substituted product endowed with good GC characteristics. This derivative can
be obtained in a single derivatisation step and could, therefore, be proposed as a
suitable derivative for the simultaneous determination of Y and NY by GC anal-
ysis coupled to detectors sensitive to the presence of atoms with a high electron
affinity, such as MS used in the negative ion chemical ionisation (NICI) mode.
Finally, the information presented herein could be useful from the viewpoint
of exploiting the potential availability of perfluoroalkylchloroformates recently
29
reported by Husek et al. The use of this reagent could allow the introduction of
a consistent number of fluorine atoms into the N,O-dialkoxycarbonyl part of the
final derivative.
Aknowledgements. We thank University of Milan for a PhD studentship to R. P. and the
financial support from the Italian Ministry for Education, University and Research, General
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2012 Copyright (CC) SCS

682
PAVLOVIĆ et al.
Management for International Research. Thanks are due to Dr. Federico M. Rubino (Depart-
ment of Medicine, Surgery and Dentistry, School of Medicine c/o S. Paolo Hospital, Univer-
sità degli Studi di Milano, Italy) for useful discussions related to the interpretation of the MS
data.
И З В О Д
РАЗЛИЧИТО ПОНАШАЊЕ 3-НИТРОТИРОЗИНА И ТИРОЗИНА У ОДНОСУ НА
ПЕРФЛУОРНЕ РЕАГЕНСЕ ПОГОДНЕ ЗА ЈЕДНОСТЕПЕНУ ПРИПРЕМУ
ИСПАРЉИВИХ ДЕРИВАТА
РАДМИЛА ПАВЛОВИЋ1,2, PIER ANTONIO BIONDI3, LUCA MARIA CHIESA3, НАТАША ТРУТИЋ2,
МИРЈАНА АБРАМОВИЋ2 и ENZO SANTANIELLO1
1Department of Medicine, Surgery and Dentistry, School of Medicine c/o S. Paolo Hospital, Università degli
Studi di Milano, Italy, 2Katedra za hemiju, Medicinski fakultet, Unoverzitet u Ni{u i 3Department of
Veterinary Sciences and Technologies for Food Safety, Faculty of Veterinary Medicine,
Università degli Studi di Milano, Italy
У циљу развијања гасно-хроматографске (GC) методе којом би се одредио однос 3-нит-
ротирозина (NY) и тирозина (Y) као маркера нитро-оксидативног стреса, тестирани су разли-
чити перфлуорни реагенси. Сврха овог рада је била добијање јединственог испариљивог
производа за сваку аминокиселину, и то у једностепеном дериватизационом поступаку. Про-
цедура са хептафлуоробутирил-анхидридом (HFBA)/хептафлуоробутанолом (HFBOH) пока-
зала се неуспешном за симултано одређивање NY и Y. Детаљно су проучаване реакције с
различитим хлорформијатима (изобутил-хлорформијат (iBuCF) и етил-хлорформијат (EtCF))
у присуству различитих перфлуорних алкохола (трифлуороетанол (TFEOH) и HFBOH). Ком-
бинација EtCF/перфлуорни алкохол даје деривате обе аминокиселине као јединствене пико-
ве који су погодни за одређивање NY/Y односа гасном хроматографском техником. Детаљно
је разматрано различито понашање две аминокиселине у испитиваним дериватизационим
поступцима, као што су разматрани и параметри који утичу на резултате одређивања.
(Примљено 4. маја 2011, ревидирано 10. јануара 2012)
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