Single determination of a-ketoglutaric acid and pyruvic acid in beer by HPLC

with UV detection
Patrcia Montenegro, In^es Maria Valente, Lus Moreira Goncalves, Jose Antonio Rodrigues
and Aquiles Araujo Barros
*
Received 3rd November 2010, Accepted 15th February 2011
DOI: 10.1039/c0ay00669f
The present work describes a simple and straightforward methodology for the determination of a-
ketoglutaric acid (KA) and pyruvic acid (PA) in beer. This work involves three basic steps: (a) cleaning
of the sample by solid phase extraction (SPE); (b) a-ketoacids derivatization with o-phenylenediamine
(OPDA) to form quinoxalines; and (c) separation and detection of the quinoxalines by high-
performance liquid chromatography with spectrophotometric detection (HPLC-UV). The identity of
the eluted compounds was also conrmed by high-performance liquid chromatography-electrospray
ionization tandem mass spectrometry (HPLC-ESI-MS/MS) in the positive ion mode. The developed
methodology showed good repeatability (ca. 4%) and linearity as well as good limits of detection (0.091
and 0.055 mg L
1
for KA and PA, respectively) and quantication (0.30 and 0.18 mg L
1
for KA and
PA, respectively). To sum up, the goal of this technical note is to develop a single HPLC procedure with
previous SPE clean up suitable to use in routine analysis laboratories.
1. Introduction
a-Ketoacids (compounds that have a carbonyl group adjacent to
a carboxylic group), including pyruvic acid (PA) and a-keto-
glutaric acid (KA), play an important role in living beings.
a-Ketoacids are intermediates in several key biological processes,
such as glycolysis, the Krebs cycle, the metabolism of amino
acids and others.
1
There is a great interest in the analytical
determination of a-ketoacids in samples of biological origin,
such as urine, serum or blood.
25
The analysis of these
compounds in body uids can diagnose some irregularities that
occur when several biological processes do take place abnor-
mally, per example, they can help diagnose deciencies in
enzymes, particularly those involved in the metabolism of amino
acids.
4
In most microorganisms, KA and PA are the most
important regulators of the balance between carbon and nitrogen
ux.
6
Still today, scientists are discovering new crucial roles of
ketoacids in numerous life processes.
79
In food chemistry the
presence of a-ketoacids is normally not a problem in terms of
organoleptic properties, however, their control may be important
in fermented foods, such as wine and beer, to better understand
fermentation and to develop new yeast strains.
10
In wine, the
presence of a-ketoacids can interfere with sulte addition due to
their tendency to form adducts. Moreover, the level of KA and
PA can be correlated with the grape maturity at the harvest.
11
Ketoacids are the main non-volatile organic acids found in beer,
they are synthesized from amino acid metabolism by a trans-
amination reaction, and are products of the incomplete Krebs
cycle during fermentation, like succinic acid, KA and malic
acid.
12,13
Furthermore, PA can also be considered as a marker for
yeast growth as the maximum PA concentration is reached just
before its maximal growth; then it is taken up by the yeast and
converted into acetate.
14
The rst analytical methods for determination of a-ketoacids
arose nearly 50 years ago with the development of gas chroma-
tography (GC).
15,16
Later, methodologies using uorescence
detection were also published (either using derivatization
agents
2,3,17
or using an enzymatic mechanism
18,19
) and, more
recently, using capillary electrophoresis.
20
Polarography has also
been applied in the determination of KA and PA; though very
sensitive the signal distinction between KA and PA is almost
impossible.
11
Of all published papers, common problems are:
sample clean-up since biological and food samples are often too
dirty for direct instrumental measurement, avoiding matrix
effects since samples are often quite complex; and analyte detec-
tion since, for example, most a-ketoacids are not UV detectable
due to the absence of a signicant chromophore. In this work all
these problems were solved by the combination of a solid phase
extraction (SPE) clean up step, then derivatization with o-phe-
nylenediamine (OPDA) and subsequent high-performance liquid
chromatography with spectrophotometric detection (HPLC-UV)
as schematized in Fig. 1. The goal of this technical note is to
Requimte, Departamento de Qumica e Bioqumica, Faculdade de Ci^ encias,
Universidade do Porto, Rua do Campo Alegre, s/n, 4169-007 Porto,
Portugal. E-mail: ajbarros@fc.up.pt; Fax: +351 220 402 659; Tel: +351
220 402 639
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develop a single HPLC procedure with previous SPE clean up
suitable to use in routine analysis laboratories.
2. Materials and methods
All reagents used were of analytical grade and were used without
further purication. All solutions were prepared with ultrapure
water from a Millipore water purication system of resistivity
not less than 18.2 MU cm at 298 K. Ammonium acetate,
methanol and OPDA were purchased from Merck. PA, KA,
2-hydroxyquinoxaline and 2-hydroxy-3-methylquinoxaline were
purchased from Sigma-Aldrich. The 2 g L
1
OPDA derivatiza-
tion solution was daily prepared by dissolution of this compound
in aqueous HCl solution (0.2 mol L
1
). This solution was kept in
the dark and handled carefully since OPDA is toxic and may
cause allergenic reactions. The commercial beer samples were
purchased in local markets.
The developed procedure is represented in Fig. 1. A SPE
Chromabond C
18
column, 200 mg, 3 mL (Macherey Nagel), was
conditioned with 3 mL + 3 mL of acetonitrile (ACN), followed
by 3 mL + 3 mL of water. After being degassed ultrasonically,
a volume of 1 mL of sample was introduced into the column and
eluted with 2 mL of ACN : water (50 : 50, v/v). The eluate was
derivatized for 30 minutes with 1 mL of 0.5 g L
1
OPDA, in 0.05
mol L
1
HCl, since the reaction has to occur at a very low pH.
21
The reaction occurred at a temperature of 35

C, a compromise
between increasing the reaction kinetics and avoiding OPDA
degradation, and made up to a nal volume (5 mL) with acetate
buffer (0.2 mol L
1
, pH 4) prior to the chromatographic analysis.
Fig. 1 Scheme of the sample preparation.
Fig. 2 Derivatization reaction between OPDA and a-ketoacids for PA:
R CH
3
; for KA: R C
2
H
4
COOH.
Fig. 3 Chromatograms obtained by the analysis of a beer and three additions of KA and PA. Peak KA* is 3-(3-hydroxyquinoxalin-2-yl)propanoic acid,
the reaction product between KA and OPDA; peak PA* is 3-methylquinoxalin-2-ol, the reaction product between PA and OPDA. Inlay: the standard
addition curves obtained.
Table 1 Recovery of KA and PA in the extraction process
Compound Addition/mg L
1
Variation of peak area
Recovery
(%)
Standard
additions
Standard
solutions
KA 1.46 2.31 3.40 68
2.19 3.64 5.16 71
2.92 5.05 6.92 73
PA 13.21 17.2 35.9 48
26.42 35.9 74.5 48
39.63 54.0 108 50
1208 | Anal. Methods, 2011, 3, 12071212 This journal is The Royal Society of Chemistry 2011
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The HPLC system (Jasco Corporation) consisted of a low
pressure quaternary gradient unit (model PU-2089 Plus), a
UV-Vis detector (model UV-2070 Plus) and a manual injector
(Rheodyne, model 7725i). Separations were achieved on a Varian
Nucleosil C
18
column (250 4.6 mm, 5 mm particle size) kept at
room temperature and in isocratic conditions, at 0.8 mL min
1
for 15 minutes. The mobile phase was composed of 55% acetate
buffer (0.04 mol L
1
, pH 4) and 45% methanol. An injection
volume of 20 mL was used while quinoxalines were detected at
340 nm. Analytes were identied by comparing their retention
times (8.5 min for KAquinoxaline and 10.5 min for PAqui-
noxaline) and UV-Vis spectra with standards.
Conrmation studies of the peaks obtained by HPLC-UV
were performed by HPLC coupled online with electrospray
ionization (ESI) mass spectrometry (MS). The HPLC system
(Thermo Electron Corporation, model Finnigan Surveyor Plus)
consisted of a low pressure quaternary pump and an auto-
sampler. Separations were achieved on a Thermo Scientic
Hypersil GOLD column (150 4.6 mm, 3 mm particle size) and
a guard column with the same characteristics at room tempera-
ture. The chromatographic conditions were the following: mobile
phase was the same as in the HPLC-UV experiments, ow rate
0.4 mL min
1
and sample injection volume of 25 mL. In these
conditions retention times for KAquinoxaline and PAqui-
noxaline were 8.8 and 10.6 minutes, respectively. A quadropole
ion trap mass spectrometer (Finnigan LCQ Deca Plus) equipped
with an ESI source in the positive ion mode and Xcalibur soft-
ware v. 1.4 (Finnigan) was used for data acquisition and pro-
cessing. The interface conditions were applied as follows: positive
mode; capillary temperature, 325

C; source voltage, 5.0 kV;
capillary voltage, 15.0 V; sheath gas (N
2
) ow at 80 arbitrary
units and auxiliary gas (N
2
) ow rate at 30 arbitrary units. The
mass detection was performed in the base peak mode, for m/z
between 120 and 500. The negative ion mode was also tested
without success.
3. Results and discussion
Several different classes of compounds can be derivatized with
OPDA.
2224
When OPDA reacts with substances with two
vicinal carbonyl groups, bicyclic aromatic ring products are
formed named quinoxalines; this is the classical Hinsberg
reaction.
23,25
The Hinsberg reaction also occurs with PA and
KA, as can be seen in Fig. 2.
11,15
Quinoxalinesor to be more
precise for this case, hydroxyquinoxalinesare compounds
that can be easily separated by liquid chromatography and
detected by UV spectrophotometry.
24,26
When this derivatiza-
tion reaction is applied to real samples, usually the major
problem is nding an effective prior separation or clean up
step
3
since dirty samples like beer should not be directly
injected into modern chromatographic columns. While, for
volatile analytes, extraction prior to derivatization seems to be
the best alternative,
27,28
in the case of non-volatiles (like PA and
KA), a clean up seems to be preferable. Current literature
techniques include purication by means of a hydrazine gel
column and later extraction with ethyl acetate.
4
In this work it
was decided to use SPE cartridges of the same material of the
chromatographic column, i.e. octadecyl bonded silica (C
18
).
Basically, all compounds that could irreversibly bind to the
column (like tannins and other large molecules), in theory, will
bind to the SPE silica in the clean up step and will therefore not
become an issue in the HPLC analysis. Moreover, the
sample passage through the SPE cartridge can also remove the
majority of small particles present in beer. The assays per-
formed with standard solutions of PA and KA with and
without SPE clean up showed recoveries between 96% and
100% for both. Several types and volumes of washing solution
were tested, however, the best results in terms of peak height
and repeatability were achieved using 2 mL of ACN : water
(50 : 50, v/v) (data not shown).
After derivatization, the obtained extracts were injected into
the chromatographic system (Fig. 3). Recovery studies were
performed for three concentration levels on a beer sample.
Values obtained (Table 1) were around 71% for KA and 49% for
PA, showing that the beer matrix has signicant effects on the
analytical signal. Due to that standard additions should be used
for quantication. Figures of merit of the developed method-
ology are shown in Table 2; coefcients of variance (CV) lower
than 5% were obtained for both compounds, as well as good
linearity and suitable limits of detection (LOD) and quantica-
tion (LOQ).
Using mass spectrometry, the chromatographic peak at 8.8
minutes was tentatively identied as KAquinoxaline (Fig. 4)
with an intense protonated molecule [M + H]
+
with an m/z
value of 219. Two ionization sites are possible for this case as
the compound presents two hydroxyl groups. The fragmen-
tation of this precursor ion, in positive ion mode, resulted in
a major fragment with m/z 201 by the loss of 18 Da corre-
sponding to a molecule of water. The [M + H H
2
O]
+
product ion produced a MS
3
fragment of m/z 173, attributed
to a carbonyl group loss, proving that the initial ionization of
the hydroxyquinoxaline occurs in the OH of the acidic
group of the molecule. For PAquinoxaline, a molecular ion
[M + H]
+
with m/z 161 at 10.6 min. was identied (Fig. 5).
MS
2
of this precursor ion resulted in the loss of a water
molecule, giving a fragment of 143 m/z and a second fragment
of 133 m/z. MS
3
over this ion was performed but no product
ions were detected.
Table 2 Figures of merit of the developed methodology; n 4 for the CV and n 6 for the calibration curve (aqueous standards)
Compound CV (%) y mx + b
a
r
2
LOD/mg L
1
LOQ/mg L
1
Linear range/mg L
1
KA 0.2 y 2.41x 0.131 0.998 0.091 0.30 Up to 5.8
PA 4.1 y 2.72x 0.130 0.998 0.055 0.18 Up to 79
a
y, peak area in mV min; x, concentration in mg L
1
.
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Fig. 4 ESI mass spectra in positive ion mode of the KA derivative peak showing the precursor ion, the fragmentation of the major product ion
(m/z value of 219) and then again the fragmentation of product ion (m/z value of 201) obtained. Corresponding molecular structures of the obtained
fragments are shown in the inlay.
1210 | Anal. Methods, 2011, 3, 12071212 This journal is The Royal Society of Chemistry 2011
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4. Conclusions
This technical note reports a very simple and straightforward
methodology for the chromatographic analysis of KA and PA in
beer, after derivatization with OPDA. The methodology has the
potential to be applied to other foodstuffs such as wine and in
biological samples as well. Furthermore, the obtained chro-
matographic peaks were characterized by ESI-MS/MS reporting
extra information about the derivatives.
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Fig. 5 ESI mass spectra in positive ion mode of the PA derivative peak showing the parent ions and the fragmentation of the major product ion (m/z
value of 161). Corresponding molecular structures of the obtained fragments are shown in the inlay.
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