JOURNAL OF LIQUID CHROMATOGRAPHY & RELATED TECHNOLOGIESw
Vol. 27, No. 17, pp. 2733–2742, 2004
Analysis of Malonyldialdehyde Using Ion
Exclusion Chromatography
Bronislaw K. Głód1 and C. Kowalski2, *
1
Medical Research Center, Polish Academy of Sciences, Laboratory of
Experimental Pharmacology, Warszawa, Poland
2
Department of Pharmacology, Faculty of Veterinary Medicine,
Academy of Agriculture, Lublin, Poland
ABSTRACT
Malonyldialdehyde (MDA) plays an important role as a marker of the
cascade of free radical reactions with lipids. It is also responsible for
their toxicity. In this paper, preliminary results of the analysis of MDA
by means of ion exclusion chromatography (IEC) is presented. Two
methods based on IEC with photometric detection are discussed. The
first one is based on the derivatization of MDA with thiobarbituric acid
(TBA). In the second direct separation is obtained using ion-pairing
reagent added to the mobile phase. Better separation is obtained for the
second method although, for the first one, lower detection limit is achieved.
*Correspondence: C. Kowalski, Department of Pharmacology, Faculty of Veterinary
Medicine, Academy of Agriculture, Akademicka 12, 20-033 Lublin, Poland; E-mail:
cezaryk@agros.ar.lublin.pl.
2733
DOI: 10.1081/JLC-200029303
Copyright # 2004 by Marcel Dekker, Inc.
1082-6076 (Print); 1520-572X (Online)
www.dekker.com
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Głód and Kowalski
It is shown that MDA peak broadening is strongly affected by column
temperature, which is probably caused by kinetic effects.
Key Words: Malonyldialdehyde; Ion exclusion chromatography.
INTRODUCTION
Free radicals interact with all cell components and compounds they are
built of. Lipids are most strongly affected by free radicals, since the number
of other radicals and stable lipid hydrogen peroxides,[1] which are usually
more reactive and toxic than substrates, are formed as a product of reaction
with radicals. Lipid radicals are also formed by non-metal enzymatic reactions. This metabolically uncoupled lipid peroxidation plays a crucial role
in the degeneration associated with aging, and in the pathogenesis of a
number of diseases.[2,3]
Degradation of cell membranes during reaction of free radicals with
polyunsaturated fatty acids (PUFA) is observed. This process yields lipid
hydroperoxides, aldehydes, and ketones, which are relatively stable and cytotoxic.[1] They interact with the cell and membrane components, showing
strong chemical affinity. When formed in blood (endothelium, fibroblasts,
heart muscle, etc.), they are responsible for arteriosclerosis. Additionally,
they affect the change of electric charge, hydrophobicity, and “liquidity” of
membranes, decreasing their mobility. New electric charges on the membrane
surface indicate that new carboxylic groups are formed during peroxidation.
The membrane becomes more hydrophilic, which results in a change in its
structure (with respect to transport and receptors).
Estimation of oxygen radical damage in biological systems is usually
done by the determination of malonyldialdehyde (MDA).[4,5] The thiobarbituric acid (TBA) test is most frequently used. This assay has been favored
because of its simplicity and sensitivity.[6] It is still in use despite the fact
that the TBA assay is intrinsically non-specific, and is generally poor when
applied to biological samples. Positive response is obtained with sugars,
some amino, and bile acids, alkenals, alkadienals, etc. Certain improvement
can be obtained using RP-HPLC separation followed by fluorometric[7,8] or
photometric detection at 535 nm.[9 – 13] In this case, TBA influences both,
the retention (separation) as well as detection conditions. However, this
method requires a derivatization step. Additionally, the results are questionable because identical adducts can be produced from different substances.
Similar method is based on the derivatization with 2,4-dinitrophenylhydrazine[14 – 16] or diaminonaphthalene[17] followed by photometric detection at
310 nm. Simplification of this method (no derivatization required and easy
Analysis of MDA Using Ion Exclusion Chromatography
2735
sample preparation) was achieved by application of the reversed phase ion pair
chromatography.[18,19] The detection, accomplished by monitoring absorbance at 267 nm, enabled analysis of MDA in the presence of two antioxidants, namely ascorbic acid (AA) and uric acids (UA). Unfortunately, very
small and broad peaks of MDA are obtained with this method.
Ion exclusion chromatography (IEC) is a technique widely applied to
separate ionic from non-ionic compounds, as well as mixtures of weak acids
(or bases).[20] The characteristic feature of IEC is the same sign of the electric
charge of the dissociated functional groups of the ion-exchange resin and
analyzed ionic compounds. It follows that samples of negatively charged ions,
e.g., dissociated acidic compounds, are separated on cation-exchange resins
with anionic functional groups. Usually, these are sulfonic acid groups. Similarly, samples containing positively charged species (bases) are separated on
the anion-exchange resin containing cationic functional groups. Usually, these
are tetraalkylammonium groups. The same columns can be used in both IEC
and in ion-exchange chromatography, although in the latter case, real ionexchange reactions are not involved. The usual supports are based on the
macro-porous styrene and divinylbenzene copolymers. For some compounds,
the observed retention is stronger than that provided by ion exclusion mechanism. This can be explained by hydrophobic as well as p-electron interaction
of analyte with the resin network.[20] Ion interaction reagents used in this technique strongly influence retention because of their interaction with the resin
functional groups, as well as the solute ions.[21]
The aim of the paper was to elaborate on a new, simple chromatographic
method of the MDA analysis. Because MDA is a medium strength acid
(pKa 4.5), it seems that IEC should be the preferable technique.[20]
EXPERIMENTAL
Apparatus
The chromatographic system consisted of P-580-A-LPG degasser and
pump, STH-585 column oven, UVD 170S four channel photometric detector
(Gynkotek, Germering, Germany), 2097 injector (Rheodyne, USA), and ion
exclusion column-strong cation exchanger based on PS/DVB [TSK-GEL
SCX (Hþ)] 5 mm, 300 7.8 mm2 I.D., . 4.2 meq/g, (TosoHaas, Japan).
The above system was controlled under ChromLeon (Gynkotek, Germering,
Germany) software, installed on IBM Pentium compatible computer
(Simens, Germany). Incubation was performed using TB-9414 (JWEelectronic, Warsaw, Poland) thermoblock.
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Głód and Kowalski
Materials
Acetonitrile (ACN), sulfuric acid (SA), and UA were purchased from
E. Merck (Darmstadt, Germany), AA from Avocado (Heysham, England),
and malonyldialdehyde bis(dimethyl acetal) from Sigma (St. Louis, USA).
All other reagents, being of analytical reagent grade, were used without
further purification. The Milli-Q (Millipore, Bedford, USA) water system
was used to prepare all solutions. The mobile phases were filtered through a
Millipore 0.22 membrane filter and degassed in an ultrasonic bath prior to use.
Procedure
The source of MDA was malonyldialdehyde bis(dimethyl acetal). A
10 mM stock solution was prepared by hydrolysis of acetal, using 1%
SA.[22] Reaction between TBA and MDA was carried out at pH 3, at 90–
1008C for 10– 15 min.[23]
The chromatographic experiments were performed with a flow rate
0.9 mL/min. The column was stabilized for 1 hr at the temperature of 208C
prior to the chromatographic measurements.
Solutions of 1 mM SA and 3 mM tetrabutylammonium bromide (TBABr)
in ACN were used as mobile phases. Stock solutions (10 mM) of the solutes
were prepared in Milli-Q water system and diluted to the required concentration before use. The solute solutions were injected into the chromatographic
system with a 100 mL syringe (Hamilton, Reno, USA) through the injection
port. The injected volumes were equal to 20 mL, and the signal output of
the dual channel photometric detector working at 267 and 535 nm was continuously monitored on the computer.
RESULTS AND DISCUSSION
In the biomedical practice MDA is usually analyzed using the TBA
assay.[6] It is based on the photometric measurement of the product of TBA
and MDA reactions. As it is well known, the TBA test is intrinsically nonspecific.[6] Positive response is obtained with other compounds, especially
those containing double bonds in their molecular structure. Ion exclusion
chromatograms of the products of the reaction of TBA with MDA, obtained
with photometric detection at 535 nm, are presented in Fig. 1. The appearance
of a number of peaks, as well as their broadening, suggest no stoichiometric
reaction between TBA and MDA. Certain improvement can be obtained by
adding an ion interaction reagent to the mobile phase. In this case, the ion
Analysis of MDA Using Ion Exclusion Chromatography
2737
Figure 1. IEC chromatograms: (1) AA, (2) UA, (3) MDA, and (30 ) MDA . TBA.
Chromatographic conditions: column—300 7.8 mm2 I.D., .4.2 meq/g, TSK-GEL
SCH(Hþ) (TosoHaas); detector UV-267/535. The mobile phase: (A) 1 mM H2SO4,
3 mM TBABr, ACN, temp. 408C; (B) 1 mM H2SO4, ACN, temp. 208C.
interaction reagent interacts with the analyte, as well as the functional group of
the resin. It enables detection of MDA in the presence of AA and UA without
derivatization steps. This can be accomplished by monitoring absorbance at
267 nm (cf. Fig. 1). However, very broad and small MDA peaks were obtained
at room temperature. It was found that the broadening is strongly dependent on
the temperature (cf. Figs. 2 and 3). It is not clear, at the moment, why temperature influences broadening of MDA peaks only. Probably, it is due to
kinetic effects that occur in the sample. According to our knowledge, it is
the first such observation of the influence of temperature on the separation
performance in IEC. This, and the application of the described method in
neurochemical investigations, will be subject of our next paper.
Performance Characteristic of the Proposed Method
Optimized chromatographic conditions were set and the following
analytical characteristics were evaluated: precision and accuracy, limit of
detection (LOD) and limit of quantification (LOQ), linearity.
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Głód and Kowalski
Figure 2. IEC chromatograms of AA, UA, and MDA obtained for different column
temperatures. Chromatographic conditions: column 300 7.8 mm2 I.D., TSK-GEL
SCH(Hþ) (TosoHaas); mobile phase: 1 mM H2SO4, 3 mM TBABr, ACN; detector
UV-267 nm; temp. 208C–408C.
Precision and Accuracy
The repeatability of the method was verified by replicate injections
(n ¼ 6) of appropriately diluted (with 1% SA) stock standard solutions:
100 mM for MDA and 25 mM for MDA . TBA. The standard deviation (SD,
in mM) and relative standard deviation (RSD, in %) are summarized below.
Sample
100 mM MDA
25 mM
MDA . TBA
Found + SD
RSD
104 + 5
23 + 2
5
8
LOD and LOQ
Working standard solutions in the concentration range of 1–100 mM were
prepared by appropriate dilution of stock standard solutions with 1% SA.
Analysis of MDA Using Ion Exclusion Chromatography
2739
Figure 3. The number of theoretical plates (N ) plotted against the temperature (T ) for
MDA. Chromatographic conditions as in Fig. 2.
LOD (in mM), the quantity yielding the detector response approximately three
times the size of the background noise, and LOQ (in mM) are summarized below.
Sample
MDA
MDA . TBA
LOD
LOQ
8
1.2
25
3.8
Linearity
It was estimated that the linearity of the calibration curve extends over
approximately four orders of magnitude in the concentration for MDA,
approximately from 10 mM to 100 mM.
CONCLUSIONS
It was shown that IEC can be applied to the analysis of MDA. Two
methods were compared. The first one was based on the derivatization of
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Głód and Kowalski
MDA with thiobarbituric acid; and the second one was based on direct separation with ion interaction reagent added to the mobile phase. Better separation
was obtained for the second method, although lower detection limit was
achieved with the first one. Peak broadening was strongly affected by
column temperature, which was probably caused by kinetic effects that
occur in the sample.
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Received March 15, 2004
Accepted April 30, 2004
Manuscript 6355
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