Journal of Chromatography B, 976–977 (2015) 91–95
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Improved determination of malonaldehyde by high-performance liquid chromatography with UV detection as 2,3-diaminonaphthalene derivative Sara Panseri a , Luca Maria Chiesa a , Andrea Brizzolari b , Enzo Santaniello b , Elena Passerò c,1 , Pier Antonio Biondi c,∗ a
Department of Veterinary Sciences and Public Health, Università degli Studi di Milano, via Celoria 10, 20133 Milan, Italy Department of Health Sciences, Università degli Studi di Milano, Via Di Rudinì 8, 20142 Milan, Italy c Department of Health, Animal Science and Food Safety, Università degli Studi di Milano, via Celoria 10, 20133 Milan, Italy b
a r t i c l e
i n f o
Article history: Received 14 August 2014 Accepted 17 November 2014 Available online 29 November 2014 Keywords: Malonaldehyde HPLC-UV 2,3-Diaminonaphthalene Canned mackerel ﬁllet
a b s t r a c t A rapid, speciﬁc and simple procedure is proposed for the determination of free malonaldehyde (MA) contained in ﬁsh tissue. The method is the optimization of the reaction of MA with 2,3-diaminonaphthalene to afford a naphtodiazepinium ion that present a UV absorption at 311 nm, useful for MA determination by HPLC with UV detection. The reaction proceeds in the presence of 25% acetonitrile at 37 ◦ C in 20 min at pH 2 using 2,4-pentanedione as internal standard. The method has been applied to homogenized samples of canned mackerel ﬁllets that were treated with 2,3-diaminonaphthalene in an acidic aqueous:acetonitrile mixture. The produced naphtodiazepinium ion was extracted in acetonitrile by a salting-out homogeneous liquid–liquid extraction. A standard calibration was carried out in the range 0.625–10 nmol/g. The reliability of the procedure is demonstrated by linearity (r2 = 0.998), limit of detection (0.16 nmol/g), limit of quantiﬁcation (0.22 nmol/g), repeatibility (RSD 5.57%), and intermediate precision (RSD 8.92%). © 2014 Elsevier B.V. All rights reserved.
1. Introduction Malonaldehyde (propandial, MA) has been extensively used as a marker of polyunsaturated fatty acids oxidation [1–3]. Its determination is still based on spectrophotometric assays and, among these, the one that relies on UV-evaluation of the adduct formed between MA thiobarbituric acid (TBA) is very popular and frequently used for oxidative stress evaluation [4,5]. However, TBA test has been considered nonspeciﬁc for MA  and several procedures have been suggested in order to overcome interference problems . Furthermore, the sample preparation requires harsh experimental conditions that may cause the formation of several artefacts that can severely intefere with MA determination . Therefore, various methods have been introduced to produce MA derivative under mild conditions before its determination by GC or HPLC tecniques . For instance, HPLC analysis of MA with the UV detection 2,4-dinitrophenylhydrazine (DNPH) can be used as
∗ Corresponding author. Tel.: +39 02 50317930; fax: +39 02 50317941. E-mail address: [email protected]
(P.A. Biondi). 1 Present address: Department of Food, Environmental and Nutritional Sciences, Università degli Studi di Milano, via Luigi Mangiagalli 25, 20133 Milan, Italy. http://dx.doi.org/10.1016/j.jchromb.2014.11.017 1570-0232/© 2014 Elsevier B.V. All rights reserved.
an alternative to TBA. In a recent study on the speciﬁcity of MA determination in ﬁsh, it has been shown that DNPH is preferred to TBA “for a ﬁner understanding of lipid peroxidation in ﬁsh” . Compared to the TBA assay, the derivatization procedure with DNPH involves a more favourable treatment at room temperature, but still requires a strongly acidic treatment. Another reagent, 2,3-diaminonaphthalene (DAN), originally introduced for the quantitative determination of ␤-diketones , has been recently applied to MA determination in plasma or serum by HPLC with UV detection . The reaction of DAN with MA affords a naphtodiazepine (MA-DAN) with a high UV response, very interesting in the view of free MA measurements in biological ﬂuids. However, the reported experimental protocol requires a long derivatization time (180 min), low pH (pH 0.8) and no internal standard is described. We have, therefore, carefully analyzed several aspects of the experimental conditions such as the sample treatment, the derivatization step and the use of the most suitable internal standard. Once these conditions have been optimized, we have compared UV responses of MA-DAN with the corresponding MA derivative with DNPH (MA-DNP). Finally, we have applied our protocol to the estimation of free MA in a ﬁsh product available on market, like canned mackerel ﬁllet.
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2. Materials and methods
Spectrophotometric measurements were performed on an UVIDEC-610 double beam instrument (Jasco).
2.1. Chemicals and samples MA–tetrabutylammonium salt, 3-oxobutanal diethylacetal, 2,4-pentanedione (acetylacetone), DAN, DNPH, heptaﬂuorbutyric acid (HFBA), acetonitrile (ACN) HPLC grade, organic solvents and inorganic reagents of analytical grade were obtained from Sigma–Aldrich (Buchs, Switzerland). 2-Methyl1,1,3,3-tetraethoxypropane (methylmalonaldehyde diethylacetal) was purchased from Seratec (Epinoy sure Seine, France) and deionized water was from a Milli-Q puriﬁcation system (Millipore, Milan, Italy). Stock (1 mg/ml) Solutions of MA and 2,4-pentanedione, as internal standard (IS) were prepared in a phosphate buffer 0.15 M pH 7.0 and stored at 4 ◦ C for a week. Working solutions (10 and 1 nmols/ml) were prepared every day. Concentrations of MA solutions were evaluated by UV absorbance using 31,800 M−1 cm−1 coefﬁcient . Samples of canned mackerel (Scomber scombrus) ﬁllets from different producers were purchased at local market within their shelf-life and analyzed immediately after opening.
2.2. Sample preparation A portion of a homogenized mackerel ﬁllet (5 g) was treated with chloroform (20 ml). After centrifugation at 4000 × g for 10 min at 4 ◦ C and carefull withdrawal of the organic phase, the residual aqueous suspension was extracted with 0.15 M phosphate buffer pH 7.0 (20 ml), containing BHT 0.05% (w/v) and EDTA 0.1% (w/v). After centrifugation, the supernatant was transferred in another test tube, added with IS (200 nmols) and brought to a ﬁnal volume of 25 ml. A portion of the resulting supernatant (2 ml, corresponding to the 0.4 g extract and containing 16 nmols of IS) was transferred in a glass screw capped tube equipped with a silicon/PTFE septum and added with 10 mM DAN solution in ACN (1 ml) and 0.3 M HCl aqueous solution (1 ml), to adjust pH to 2.0. After 20 min at 37 ◦ C the reaction mixture was saturated with magnesium sulphate and shaken; an aliquot (0.1 ml) of the supernatant ACN phase was withdrawn, diluted 1:3 with HPLC eluent and 20 L of the ﬁnal mixture were injected into the HPLC instruments.
2.3. MA-DAN and MA-DNP synthesis Excess of MA–tetrabutylammonium salt (20:1 ratio in moles with respect to DAN) was reacted with either 10 mM DAN solution in 0.01 M HCl or 10 mM DNPH solution in 2 M HCl in order to obtain precipitates of MA-DAN and MA-DNP derivatives, respectively. The solids were ﬁltered, washed with acidic aqueous solutions and kept under vacuum. MA-DAN was dissolved in the HPLC eluent described below, while MA-DNP was dissolved in a water:ACN: acetic acid mixture (55:45:0.2); these solutions were both analyzed by spectrophotometry in the 270–330 nm range.
2.4. Apparatus and chromatographic conditions Preliminary HPLC tests were carried out on a Shimadzu (Kyoto, Japan) instrument, composed by a LC-10AT pump, a Reodyne manual injector and a SPD-M10A diode array detector, in order to investigate the UV spectra of eluted compounds. The ﬁnal experiments were carried out on a Jasco (Easton, MD, USA) instrument composed by an AS-2057 Plus autosampler, a DU-2089 Plus pump, and an UV-2075 Plus detector set at 311 nm. Chromatographic separations were performed on a 25 cm x 4.6 mm column Luna C-18 (Phenomenex, Torrance, CA, USA), using a water:ACN:HFBA 82:18:0.15 mixture. The ﬂow rate was set at 1 ml min−1 .
2.5. Validation experiments A standard curve was made using MA–tetrabutylammonium solutions (2 ml) in 0.15 M phosphate buffer pH 7.0 containing increasing amounts of MA (0.25, 0.5, 1, 2, 4 nmol, corresponding to 0.625, 1.25, 2.50, 5.00, 10.00 nmol/g of sample) and a same amount of IS (16 nmol), treated according the procedure above described for the supernatant portions of the aqueous extract of samples (2.2). In order to verify the matrix effect a matrix-based calibration curve was obtained by adding to 2 ml aliquots of the supernatant from a mackerel sample (with a previously estimated MA content of 1.14 nmol/g) the same amounts of MA and IS above reported. The ratios between MA and IS derivatives areas (Ra) were reported against the MA contents (nmol/g) to verify a linear relationship. The MA content in samples was calculated from the corresponding Ra using the standard calibration curve. The precision of the method was expressed as the RSD estimated using the same sample treated according the described procedure: three sets of ﬁve replicates were analyzed in three different days in order to obtain the repeatibility (or intra-day precision) and the intermediate (or inter-days) precision values. According to Mendes et al. , the limit of detection (LD) and quantiﬁcation (LQ) were calculated as the mean of trace solution response plus 3 or 5 standard deviation of the same trace solution response (ten replicates). Trace solution content (0.07 nmol/g) was determined by diluting the lowest standard solution until the lowest concentration enabling a signal (3 signal to noise ratio) was attained. 3. Results and discussion 3.1. Improvement of the experimental protocol for MA-DAN preparation In order to overcome the limits of the procedure for the derivatization of MA with DAN , several modiﬁcations have been introduced. First of all, MA tetrabutylammonium salt has been used as standard MA instead of tetramethoxypropane (TMP), originally proposed as source of MA. The tetrabutylammonium salt of MA directly releases MA in water, whereas TMP, the dimethyl acetal of MA, requires apreliminary hydrolytic step in the presence of acids. Differently from the original procedure , we have dissolved DAN in a 1:3 ACN)/aqueous solution and observed that the reaction rate is not affected by the presence of ACN. As a ﬁrst result, following the time course of MA-DAN production at 311 nm, a fast pseudo ﬁrst order curve was obtained from the reaction of the DAN ACN/water solution with MA prepared from the corresponding tetrabutylammonium salt. On the contrary, a slow sigmoidal curve resulted when TMP was the substrate (data not shown), thus showing that a ﬁrst preliminary step occurred to produce MA before its reaction with DAN. Typical time courses of MA reaction with DAN are shown in Fig. 1, where the effect of other parameters such as pH and temperature are evidenced. The pH value 2.0 was selected on the ground of the observation that at a pH value lower than 2.0 MA-DAN derivative is formed more quickly but appeared unstable . On the other hand a pH value higher than 2.0 produces a less stable diazepinium ion, which could be neutralized and hydrolysed . The same effect was observed when the reaction was carried out at a temperature higher than 37 ◦ C. In conclusion, pH 2.0 and 25% ACN were established as optimal conditions for a stable preparation of the MA-DAN derivative at 37 ◦ C within 10 min of treatment. These conditions appear to be suitable to give a reliable value of the free MA content. In
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Fig. 3. MA-DAN versus MA-DNP UV absorbances. Overlapped partial UV spectra of equimolar solutions of MA-DAN and MA-DNP are shown.
showed a high absorption at 311 nm, due to the extensive resonance of the naphthodiazepinium ions 1 and 2, that is not possible in a structure like the di-imino cation 3. In order to conﬁrm the attractive feature of high UV response, MA-DAN was compared with MA-DNP. UV spectra of equimolar solutions in their respective HPLC eluents were measured (Fig. 3). MA-DAN response resulted 1.7 higher than that of MA-DNP at their maximum absorption wavelenghts (311 nm and 310 nm respectively) and, therefore, MADAN can be considered a suitable derivative for MA measurements by HPLC determination with UV detection.
Fig. 1. Kinetics of MA-DAN formation. Two different temperature (37 ◦ C and 45 ◦ C) and pH values (1.0 and 2.0) were tested.
comparison, TBA test used for MA determination requires high temperature (at least 90 ◦ C) and long reaction time (at least 30 min) at very low pH value. Treatment of MA with DNPH, although at lower temperature (37–50 ◦ C), is carried out in a concentrated HCl solution. 3.2. Salting-out homogeneous liquid–liquid extraction (SHLLE)
3.4. Internal standard
In order to increase the sensitivity of the method, a special attention was addressed to the extraction of the analyte from the reaction mixture that contained 25% ACN. In this view, we experimented that the highly hydrophobic diazepinium ion formed from MA-DAN can be extracted into organic solvents such as diethyl ether or ethyl acetate, provided that an ion-pair is formed with a hydrophobic anion as heptaﬂuorobutyrate. An improvement of the extraction was achieved, using the recently introduced technique, named salting-out homogeneous liquid–liquid extraction (SHLLE) [15,16]. Addition of magnesium sulphate to the solution increases its ionic strength and the aprotic ACN forms a distinct upper phase containing the MA-DAN product. Due to the ACN polar feature, the ion-pair of diazepinium ion MA-DAN product with chloride can be extracted, thus avoiding the addition of the hydrophobic heptaﬂuorobutyrate anion.
When low metabolite contents have to be estimated in biological samples the use of an internal standard (IS) is advisable to overcome precision problems. In this case a ␤-dicarbonylic compound very similar to MA is desirable and preliminary experiments were carried out to ﬁnd the best available one. According to previous reports [17,18] methylmalonaldehyde (MMA) was considered as IS, but, compared with MA, the reaction of MMA with DAN was slower. Furthermore, the UV spectrum of MMA derivative with DAN shows typical double peaks near 300 nm of the corresponding diazepinium moiety but a valley between them appears at just 311 nm (maximum absorption wavelenght for MA-DAN). Therefore, MMA could not be used as IS for the MA-DAN assay. In a second attempt, 3-oxobutanal (acetylacetaldehyde), a MA homologue, was tested. The compound forms a DAN derivative at the same reaction rate of MA-DAN with the same maximum absorption wavelenghts. Unfortunately, 3-oxobutanal is commercially available only as diethylacetal and its hydrolysis was expected to pose the same problems previously observed for tetramethoxypropane (TMP) as source of MA . Finally, 2,4-pentanedione (acetylacetone, AA) was tested as IS, although its molecular weight and hydrophobicity are higher than MA. Compared to MA, the reaction rate of AA with DAN was only slightly slower, since the complete formation of AA-DAN required 20 min. The UV spectrum of AA-DAN
3.3. Derivative structure and UV response of MA-DAN The reaction between MA and DAN is expected to afford a 1,5-naphthodiazepinium ion , whose mesomeric amino-imino structures 1 (1H form) and 2 (5 H form) are shown in Fig. 2, together with the less probable di-imino structure 3. The UV spectrum of MA-DAN in the solvent mixture selected for the HPLC analysis
Fig. 2. MA-DAN structure. Structures of mesomeric amino-imino forms (1 and 2) formed by reaction of MA with DAN (MA-DAN). Di-imino form (3) is also shown.
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Fig. 4. MA-DAN HPLC. Typical HPLC chromatographic proﬁles corresponding to a standard solution containing 2.50 nmols/g of MA (A) and to a canned mackerel sample containing 1.14 nmols/g of MA (B).
was superimposable with that of MA-DAN (data not shown) and AA was selected as IS. 3.5. Chromatographic conditions Preliminary esperiments carried out using HCOOH as organic modiﬁer added to ACN/water mixture, showed a too short retention time of MA-DAN and, therefore, HFBA was then used. In fact, due to the hydrophobic feature of the ion-pair that heptaﬂuorobutyrate anion formed with the naphtodiazepinium ion, the retention time of MDA-DAN on a reversed phase column was
satisfactorily increased. Additionally, the use of DAN deserves a note of caution, since available commercial samples contain many impurities that could not be completely eliminated, even after repeated crystallizations. Nevertheless, a satisfactory separation of MA-DAN peak from interferences was possible by properly adjusting the eluent composition. In these conditions, MA-DAN was clearly detected between two impurities of DAN side products of the reagent, while IS was completely separated at the end of the chromatographic run. In Fig. 4 typical HPLC proﬁles of a standard solution (A) and of a mackerel sample (B) are shown.
Table 1 Validation data for determination of MA in canned mackerel ﬁllets.
Linear range (nmols/g)
Repeatability (RSD, n = 5)
Intermediate precision (RSD, n = 5)
Present in the relationship: Ra = m × MA content (nmols/g) + q.
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3.6. Method validation In Table 1 the data of the partial validation experiments are shown. A matrix-based calibration curve was obtained by adding standard amounts of MA (from 0.625 to 10 nmols/g) to a sample extract containing MA (1.14 nmol/g). The resulting relationship was Ra = 0.0513 nmol/g + 0.0587 (r2 = 0.995). The slope of this calibration curve, very similar to that obtained using MA aqueous solutions (0.0538), showed a negligible matrix effect in the tested range. 3.7. MA content in canned mackerel ﬁllets Commercial canned mackerel ﬁllets from different producers were purchased from local markets and their MA content was quantiﬁed according to our optimized MA-DAN assay. The resulting contents were between 0.89 and 6.44 nmols/g, with a mean of 3.35 nmols/g. At present, data on free MA in canned mackerel ﬁllets are not available, whereas MA in fresh mackerel samples have been estimated only by TBA spectrophotometric test under different conditions. The recently reported contents were in a range 3–20 nmols/g, corresponding to 0.21–14 mg MA/kg [19–21]. The MA concentration in the canned samples tested by us in most cases was lower than that estimated by TBA test in fresh samples. Since the assay is carried out in 20 min at pH 2.0, only MA weakly bound to peptides should be hydrolised and added to the original free MA. Therefore, according to the consideration elsewhere reported , the calculated content could be presented as “not strongly bound” MA. In conclusion our optimized MA-DAN assay can be proposed as a rapid, simple and reliable alternative method for the estimation of MA content by HPLC-UV technique. Acknowledgment This work was supported by a grant from Italian Ministero dell’Istruzione, dell’Universita` e della Ricerca, MIUR (FIRST 2003) to P.A.B. References  E.N. Frankel, Lipid oxidation: mechanism, products and biological signiﬁcance, J. Am. Oil Chem. Soc. 61 (1984) 1908–1917.  B. Halliwell, S. Chirico, Lipid peroxidation: its mechanism, measurement and signiﬁcance, Am. J. Clin. Nutr. 57 (1993) 715S–725S.  T.W. Kwon, B.M. Watts, Malonaldehyde in aqueous solution and its role as a measure of lipid oxidation in foods, J. Food Sci. 29 (1964) 294–302.
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