C: Food Chemistry
A Fast and Validated Method for the Determination of Malondialdehyde in Fish Liver Using High-Performance Liquid Chromatography with a Photodiode Array Detector Mohammad Faizan, Tuba Esatbeyoglu, Banu Bayram, and Gerald Rimbach
Malondialdehyde (MDA) is a biomarker of lipid peroxidation and is present in foods and biological samples such as plasma. A high-performance liquid chromatography (HPLC) method was applied to determine MDA in fish liver samples after derivatization with 2,4-dinitrophenylhydrazine (DNPH) using a ODS2 column (10 cm × 4.6 mm, 3 μm) and a photodiode array detector. The mobile phase consisted of 0.2% acetic acid (v/v) in distilled water and acetonitrile (42:58, v/v). The present method was validated in terms of linearity, lower limit of quantification, lower limit of detection, precision, accuracy, recovery, and stability of MDA according to U.S. Food and Drug Administration (FDA) guidelines. The limit of quantification of MDA was 0.39 μmol/L, which is comparable to other methods. The recovery of the spiked MDA liver samples was in the range of 92.4% to 104.2%. This newly modified HPLC method is specific, sensitive, and accurate and allows the analysis of MDA within 4 min in fish liver but also in other tissues and plasma.
Abstract:
Keywords: 2, 4-dinitrophenylhydrazine, HPLC, lipid peroxidation, malondialdehyde, polyunsaturated fatty acids
Malondialdehyde is an established biomarker of lipid peroxidation in foods. We developed a fast, simple, and sensitive HPLC method with photodiode array detection using DNPH for derivatization of malondialdehyde in fish liver.
Practical Application:
Introduction
high temperature (100 °C) required for derivatization (Volpi and Tarugi 1998; Sakai and others 1999). Furthermore, TBA reacts not only with MDA but also with other compounds such as amino acids, carbohydrates, pigments, and pyridines (Guill´en-Sans and Guzm´an-Chozas 1998; Seljeskog and others 2006; Mendes and others 2009), which may cause an overestimation of the actual lipid peroxidation (Mateos and others 2005). Instead of TBA, 2,4-dinitrophenylhydrazine (DNPH) can be used for the derivatization of MDA and subsequent highperformance liquid chromatography (HPLC) analysis. Importantly, derivatization of MDA with DNPH and conversion into pyrazole and hydrazone takes place at room temperature, thereby avoiding the production of analytical artifacts (Korchazhkina and others 2003; Sim and others 2003; Mateos and others 2004, 2005; Czauderna and others 2011). Run times seem to be crucial factors as far as HPLC methods are concerned (Bayram and others 2013). Newly developed core columns enable increased chromatographic efficiency as well as shorter analysis times (McCalley 2010; Bayram and others 2013). The spherisorb ODS2 column used in the present study is a silicabased, reversed-phase C18 column filled with a specified carbon load of 11.5%, enabling the fast analysis of MDA. Furthermore, we used a photodiode array detector, which exhibits multiple photodiode arrays to obtain information over a wide range of wavelengths simultaneously. Thus overlapping peaks at different wavelengths can be detected. MS 20131246 Submitted 9/4/2013, Accepted 1/23/2014. Authors are with In the current study, we developed a sensitive, specific, and fast Inst. of Human Nutrition and Food Science, Christian-Albrechts-Univ. of Kiel, Hermann-Rodewald St. 6, 24118, Kiel, Germany. Direct inquiries to author Rimbach HPLC method to detect MDA. We applied DNPH derivatization and used an ODS2 column with a photodiode array detector (E-mail: [email protected]). for MDA measurements in liver samples from Atlantic salmon
Malondialdehyde (MDA) is a product of lipid peroxidation of polyunsaturated fatty acids (PUFA) in foods (Angelo 1996) and biological samples (for example, plasma and tissues) (Kinter 1995; Tsaknis and others 1999; Mao and others 2006; Mendes and others 2009). Furthermore, MDA is a widely used biomarker of oxidative stress (Rimbach and others 1999). Lipid peroxidation causes food spoilage, rancidity, and deterioration particularly in oily foods including fish (Tsaknis and others 1999; Mendes and others 2009). Fish is an important source of long-chain PUFA (Strobel and others 2012). PUFA may be oxidized to produce odorless and tasteless hydroperoxides, which decay to secondary oxidation products, basically aldehydes including hexanal, 4–hydroxynonenal (HNE), and MDA. The latter can be used to assess the extent of lipid peroxidation (Frankel 2005; Papastergiadis and others 2012). MDA is often determined spectrophotometrically. In fact, the spectrophotometric measurement of the pink colored adduct of MDA with 2-thiobarbituric acid (TBA, maximum absorbance at 532 to 535 nm) is possibly the most widely used method for the determination of MDA in foods and biological samples because of its low cost and simplicity (Raharjo and Sofos 1993; Botsoglou and others 1994). However, most of the spectrophotometric methods have been criticized because of their lack of specificity and the
C484
Journal of Food Science r Vol. 79, Nr. 4, 2014
R C 2014 Institute of Food Technologists
doi: 10.1111/1750-3841.12412 Further reproduction without permission is prohibited
MDA analysis by HPLC in fish liver . . .
Materials and Methods Chemicals and reagents Sodium hydroxide (NaOH) and hydrochloric acid (HCl; 37%) were purchased from Merck (Darmstadt, Germany). Perchloric acid (60%), DNPH (97%), acetonitrile (HPLC quality), and the MDA standard 1,1,3,3-tetraethoxypropane (TEP; 96%) (CAS nr. 122-31-6) were supplied by Sigma Aldrich (Steinheim, Germany). Sulfuric acid (95% to 97%) was purchased from J.T. Baker (Deventer, The Netherlands). Phosphate buffer saline (PBS) was obtained from PAA (Pasching, Austria). Double distilled water was used to prepare all aqueous solutions.
Optimization of the HPLC method In order to obtain a fast, simple, and sensitive method for MDA detection in biological samples, the HPLC methods by Mendes and others (2009) and Mateos and others (2005) have been modified herein. In comparison to Mateos and others (2005), the current method uses a Spherisorb ODS2 column (10 cm × 4.6 mm, 3 μm) instead of a Nucleosil 100 RP-18 column (125 mm × 4.0 mm, 5 μm). Furthermore, we modified the composition of the mobile phase by increasing the amount of acetonitrile from 38% to 58% which resulted in shorter retention times. We also increased the incubation time from 30 min to 1 h. The major aims of these modifications were to obtain a fast, simple, and sensitive method for MDA detection and to optimize previous experimental conditions, following the principle that sample preparation procedures should be kept as simple as possible to reduce errors and to maximize analyte recoveries (Mateos and others 2005).
Biological samples Fish liver, fillet, and plasma samples were supplied by Skretting ARC Stavanger, Norway. Atlantic salmon were sacrificed at ARC research trial station. Plasma, liver, and fillet samples were stored Selectivity at –80 °C until analysis. Succeeding the quantification of MDA, no interfering peaks were noticed. A clear separation of the MDA peak was obtained within 4.3 min. Standard solution preparation Standard solutions of MDA were prepared from 1,1,3,3 tetraethoxypropane (TEP) and stored at 4 °C in the dark. Standard Linearity, lower limit of detection (LOD), and lower limit of solutions were prepared in 1% sulfuric acid in PBS. quantification (LOQ) To establish the linear range of the detector response, 12 MDA Sample preparation of MDA-DNPH from biological samples standards were measured by triplicate injection on 3 different days Sample extraction was performed according to Mateos and oth- (Bayram and others 2013). Plotting the peak areas compared with ers (2005) with some modifications: 0.2 g of fish liver was homog- MDA concentrations resulted in linear curves for all 12 MDA stanenized in 1 mL of 1% sulfuric acid in PBS in 2 mL eppendorf for dards; the range of linearity for MDA detection in the standards 4 min using a tissue lyzer (Qiagen, Hilden Germany). Ho- was found to be between 0.39 and 200 μmol/L and the regresmogenates were centrifuged at 10000 rpm for 30 min at 4 °C. sion coefficients (R2 ) for all compounds were higher than 0.99 Subsequently, supernatants were collected. (Figure 1). Two hundred and fifty microliters of the supernatant were transThe LOQ and LOD of MDA were 0.39 and 0.195 μmol/L, ferred into a 1.5 mL eppendorf with 50 μL of 6 M NaOH. This respectively, which is comparable with the methods employed by mixture was incubated for 30 min at 60 °C in a water bath. Pro- Czauderna and others (2011), Mendes and others (2009), Mao tein was precipitated by adding 125 μL of 35% perchloric acid and others (2006), and Mateos and others (2005). and centrifuging at 2800 rpm for 10 min. Two hundred and fifty microliters of the supernatant were transferred into a new 1.5 mL Precision, accuracy, and recovery eppendorf and 50 μL of DNPH was added prior to incubation Intraday and interday precision was in the range of 6% to 12% in the dark at room temperature for 1 h. Following incubation, (Table 1), which is lower than the FDA’s established limits (ࣘ20% 20 μL sample was injected into the HPLC system. for LOQ and ࣘ15% for other concentration levels). HPLC analysis of MDA in biological samples HPLC analysis of MDA was performed on a Jasco system (Jasco GmbH Deutschland, Gross-Umstadt, Germany) equipped with an autosampler (Jasco AS-2057), pump (PU-2080), ternary gradient unit (LG-2080-02), 3 line degasser (DG-2080-53), and photodiode array detector. MDA was separated using a Supelco INC water spherisorb ODS2 column (10 cm × 4.6 mm, 3 μm) with a guard column, then eluted in isocratic mode with a mobile phase consisting of 0.2% acetic acid (v/v) in double distilled water/acetonitrile (42:58, v/v). The flow rate was set at 0.6 mL/min and the autosampler thermostat at 4 °C. MDA was analyzed at 310 nm. Method validation The present HPLC method was validated according to the FDA guidelines (U.S. FDA 2001) as previously reported (Bayram and Figure 1–Linearity of the detector responses for malondialdehyde (MDA) others 2013). in the range of 0.39 to 200 μmol/L. Vol. 79, Nr. 4, 2014 r Journal of Food Science C485
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containing high amounts of PUFA (Strobel and others 2012) prone Results and Discussion to lipid peroxidation.
MDA analysis by HPLC in fish liver . . . Table 1–Intraday and interday precision, accuracy, and recovery To establish short-term stability, 5 aliquots of the MDA standard of MDA at low, medium, and high concentrations (0.4, 2, 10 and 5 aliquots of spiked (low, medium, and high) fish liver samples μmol/L) added to fish liver samples (n = 5).
were analyzed. Both the standards as well as the spiked samples
Concentration (μmol/L) Intraday
C: Food Chemistry
Interday
Low (0.4) Medium (2.0) High (10.0) Low (0.4) Medium (2.0) High (10.0)
Recovery (%)
Accuracy (% bias)
± ± ± ± ± ±
−0.4 −7.6 −2.1 3.9 −3.6 −2.3
99.7 92.4 97.9 104.2 96.7 97.9
12.2 7.4 6.6 3.9 3.8 6.1
Precision were analyzed after storage at RT, 4 °C, –20 °C, and –80 °C for (% CV) 24 h in order to compare obtained with initial values. The stability 11.7 7.5 6.9 10.7 6.2 6.1
Table 2–Short- and long-term stabilities (expressed as percentage degradation) of MDA standards with low, medium, or high concentrations. For short-term stability, the samples were stored at room temperature (RT), 4, –20, and –80 °C for 24 h (n = 5) and for long-term stability the samples were stored at –80 °C for 6 wk. Short-term stability (% degradation) Concentration (μmol/L) −80 °C −20 °C 4 °C RT Low (1.56) Medium (12.5) High (100)
8.4 0.3 0.8
−1.4 0.5 −1.4
2.6 0.6 1.3
Long-term stability (% degradation) −80 °C
−1.8 −2.4 1.4
1.8 1.6 0.7
Accuracy values were also satisfactory, ranging from 0.4 to 7.6% (Table 1), and were within the FDA’s established limits (ࣘ20% for LOQ and ࣘ15% for other concentration levels). Recovery values were also satisfactory. Intraday recovery was 92.4% to 99.7% and interday recovery was 96.7% to 104.2% (Table 1).
Stability In the current study, we determined the stability of MDA in both 1,1,3,3-tetraethoxypropane standards at low (1.56 μmol/L ࣓ 0.344 μg/mL wet weight), medium (12.5 μmol/L ࣓ 2.75 μg/mL wet weight), and high (100 μmol/L ࣓ 22.0 μg/mL wet weight) concentrations and in spiked fish liver samples (at low, medium, and high concentrations). The MDA standard solutions and spiked liver samples were stored at RT, 4 °C, –20 °C, or –80 °C for 24 h. The detector response after storage was compared with the initial measurement. Data are expressed as percentage degradation (Table 2 and 3). The MDA standard exhibiting the highest degradation (8.4%) was the lowest concentration standard stored at –80 °C (Table 2). The spiked liver sample, which showed most degradation (16.2%), was the high MDA concentration stored at RT (Table 3).
of MDA prepared as standard solutions was comparable with the MDA standard; the highest level of degradation (8.4%) occurred with the lowest concentration of MDA at –80 °C. Importantly, only negligible degradation of the MDA standards occurred during 24 h storage at RT, 4 °C, –20 °C, or –80 °C (Table 2). The degradation of MDA in the spiked fish liver samples ranged between 0.1% and 16.2% with the highest degradation occurring at the high MDA concentration stored at RT (Table 3). To assess long-term stability, 5 aliquots of the MDA standard (low, medium, and high concentrations) were analyzed after storage at –80 °C for 6 wk. In this study, the level of degradation ranged between 0.7% and 1.8%. From these results, it may be concluded that MDA standards are stable for a relatively long time at –80 °C after extraction (Table 2). The long-term stability of MDA in fish liver samples was determined from 5 aliquots prepared at low, medium, and high concentrations and stored at –20 °C and –80 °C for 8 wk. The level of degradation ranged between 5.4% and 8.6% at –80 °C and 14.6% to 19.5% at –20 °C (Table 3). According to these results, less degradation was observed at –80 °C. The freeze-thaw stability of MDA in spiked fish liver was examined after 3 freeze-thaw cycles. Under the conditions investigated, percentage degradation was slightly higher at the low (9.9%) and medium concentrations (11.4%) than at the high MDA concentration (5.3%). In general, after 3 free-thaw cycles, MDA in fish liver samples remained stable (Table 3). The stability of the processed samples, including the resident time in the autosampler, was determined with 5 aliquots of fish liver samples for each MDA concentration (low, medium, and high) and injected at 0, 6, 12, and 18 h from the same HPLC vial. MDA spiked liver samples were found to remain stable for 18 h in the autosampler (Table 3). We detected the highest degradation after 18 h (14.2% for the lower MDA concentration and 12.3% for the medium concentration, respectively).
Method application for the quantification of MDA-DNPH in biological samples MDA in fish liver samples was (range: 26.6 to 45.7 μmol/kg fresh matter) determined by using the validated HPLC method. HPLC-chromatogram of MDA in fish liver sample is depicted in Figure 2B. Applying our HPLC method, it is also feasible to analyze MDA in other tissues such as plasma and fillet, as shown in Figure 2A and 2C. For this method, we used a photodiode array detector enabling coelution. Furthermore, the ODS2 column
Table 3–Short-term, long-term, freeze-thaw, and postpreparative stabilities (expressed as percentage degradation) of MDA extracted from spiked (low, medium, or high concentrations) fish liver samples. For short-term stability, the samples were stored at room temperature (RT), 4, –20, and –80 °C for 24 h (n = 5) and for long-term stability the samples were stored at –20 and –80 °C for 8 wk. The samples were left in the autosampler at RT for up to 18 h for postpreparative stability. Short-term stability (% degradation)
Long-term stability (% degradation)
Concentration (μmol/L)
−80 °C
−20 °C
4°C
RT
−80 °C
Low (1.56) Medium (12.5) High (100)
5.4 9.1 −2.5
10.9 9.5 −0.2
8.4 −1.1 0.1
12.0 1.8 16.2
5.4 8.6 6.4
C486 Journal of Food Science r Vol. 79, Nr. 4, 2014
Postpreparative stability (% degradation)
−20 °C
Freeze-andthaw stability (% degradation)
6h
12 h
18 h
14.6 16.7 19.5
9.9 11.4 5.3
2.4 0.4 −0.7
3.5 3.0 3.2
14.2 12.3 2.0
MDA analysis by HPLC in fish liver . . .
C: Food Chemistry
Figure 2–Representative HPLC-chromatogram of MDA in fish plasma (A), liver (B), and fillet (C) after derivatization with DNPH (retention time 4.3 min); inset depicts a magnification of the MDA peak.
used in the present study allows a fast separation because of the short length of the column. The retention time of the MDA peak was around 4.3 min which is faster than previously published HPLC methods (Mateos and others 2004, 2005; Mendes and others 2009; Czauderna and others 2011). Recovery (ranging between 92.4% and 104.2%) and precision (ranging between 6.1% and 11.7%) were similar to values reported by Mateos and others (2005), Mendes and others (2009), and Czauderna and others (2011). Importantly, the measured MDA concentrations in our salmon liver samples are similar to values reported in the literature (Roig and others 2000; Alessio and others 2002; Yokozawa and others 2002; Mateos and others 2005). MDA was higher in liver (range: 26.6 to 45.7 μmol/kg fresh matter) as compared to fillet (range: 2.6 to 4.64 μmol/kg fresh matter) which is also in accordance with literature data (Khoschsorur and others 2000; Mateos and others 2005; Mendes and others 2009). Differences in MDA be-
tween liver and fillet samples may be related to differences in tissue vitamin E concentrations known to efficiently prevent lipid peroxidation (Rimbach and others 2002).
Conclusion In this paper, a modified HPLC method with photodiode array detection was validated for the rapid quantification of MDA in fish liver using an ODS2 column. The derivatization reaction of MDA with DNPH proceeds at low temperature, increasing specificity. The MDA-DNPH method is sensitive, fast, and simple. Furthermore, this method allows accurate, precise, and selective quantification of MDA.
Acknowledgments We are grateful to Dr. Ingunn Stubhaug and Dr. Wolfgang Koppe for providing Atlantic salmon samples.
Vol. 79, Nr. 4, 2014 r Journal of Food Science C487
MDA analysis by HPLC in fish liver . . .
Author Contributions M.F., T.E., and B.B. conducted the HPLC analyses. M.F., T.E., and G.R. wrote the manuscript. G.R. approved the final version of the manuscript.
References
C: Food Chemistry
Alessio HM, Hagerman AE, Romanello M, Carando S, Threlkeld MS, Rogers J, Dimitrova Y, Muhammed S, Wiley RL. 2002. Consumption of green tea protects rats from exercise-induced oxidative stress in kidney and liver. Nutr Res 22:1177–88. Angelo AJ. 1996. Lipid oxidation on foods. Crit Rev Food Sci Nutr 36:175–224. Bayram B, Ozcelik B, Schultheiss G, Frank J, Rimbach G. 2013. A validated method for the determination of selected phenolics in oilve oil using high-performance liquid chromatography with coulometric electrochemical detection and a fused-core column. Food Chem 138:1663–9. Botsoglou NA, Fletouris DJ, Papageorgiou GE, Vassilopoulos VN, Mantis AJ, Trakatellis AG. 1994. Rapid, sensitive, and specific thiobarbituric acid method for measuring lipid peroxidation in animal tissue, food, and feedstuff samples. J Agric Food Chem 42:1931–7. Czauderna M, Kowalczyk J, Marounek M. 2011. The simple and sensitive measurement of malondialdehyde in selected specimens of biological origin and some feed by reversed phase high performance liquid chromatography. J Chromatogr B Analyt Technol Biomed Life Sci 879:2251–8. Frankel EN. 2005. Lipid oxidation. In: Frankel EN, editor. Lipid oxidation. Vol. 10, Bridgwater, U.K.: The Oily Press. xvi + 470 p. Guill´en-Sans R, Guzm´an-Chozas M. 1998. The thiobarbituric acid (TBA) reaction in foods: a review. Crit Rev Food Sci Nutr 38:315–30. Khoschsorur GA, Winklhofer-Roob BM, Rabl H, Auer T, Peng Z, Schaur RJ. 2000. Evaluation of a sensitive HPLC method for the determination of malondialdehyde, and application of the method to different biological materials. Chromatographia 52:181–4. Kinter M. 1995. Analytical technologies for lipid oxidation products analysis. J Chromatogr B Biomed Appl 671:223–36. Korchazhkina O, Exley C, Andrew Spencer S. 2003. Measurement by reversed-phase highperformance liquid chromatography of malondialdehyde in normal human urine following derivatisation with 2,4-dinitrophenylhydrazine. J Chromatogr B Analyt Technol Biomed Life Sci 794:353–62. Mao J, Zhang H, Luo J, Li L, Zhao R, Zhang R, Liu G. 2006. New method for HPLC separation and fluorescence detection of malonaldehyde in normal human plasma. J Chr B 832:103–8. Mateos R, Goya L, Bravo L. 2004. Determination of malondialdehyde by liquid chromatography as the 2,4-dinitrophenylhydrazone derivative: a marker for oxidative stress in cell cultures of human hepatoma HepG2. J Chromatogr B Analyt Technol Biomed Life Sci 805:33–9. Mateos R, Lecumberri E, Ramos S, Goya L, Bravo L. 2005. Determination of malondialdehyde (MDA) by high-performance liquid chromatography in serum and liver as a biomarker for
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A Fast and Validated Method for the Determination of Malondialdehyde in Fish Liver Using High-Performance Liquid Chromatography with a Photodiode Array Detector Mohammad Faizan, Tuba Esatbeyoglu, Banu Bayram, and Gerald Rimbach
Malondialdehyde (MDA) is a biomarker of lipid peroxidation and is present in foods and biological samples such as plasma. A high-performance liquid chromatography (HPLC) method was applied to determine MDA in fish liver samples after derivatization with 2,4-dinitrophenylhydrazine (DNPH) using a ODS2 column (10 cm × 4.6 mm, 3 μm) and a photodiode array detector. The mobile phase consisted of 0.2% acetic acid (v/v) in distilled water and acetonitrile (42:58, v/v). The present method was validated in terms of linearity, lower limit of quantification, lower limit of detection, precision, accuracy, recovery, and stability of MDA according to U.S. Food and Drug Administration (FDA) guidelines. The limit of quantification of MDA was 0.39 μmol/L, which is comparable to other methods. The recovery of the spiked MDA liver samples was in the range of 92.4% to 104.2%. This newly modified HPLC method is specific, sensitive, and accurate and allows the analysis of MDA within 4 min in fish liver but also in other tissues and plasma.
Abstract:
Keywords: 2, 4-dinitrophenylhydrazine, HPLC, lipid peroxidation, malondialdehyde, polyunsaturated fatty acids
Malondialdehyde is an established biomarker of lipid peroxidation in foods. We developed a fast, simple, and sensitive HPLC method with photodiode array detection using DNPH for derivatization of malondialdehyde in fish liver.
Practical Application:
Introduction
high temperature (100 °C) required for derivatization (Volpi and Tarugi 1998; Sakai and others 1999). Furthermore, TBA reacts not only with MDA but also with other compounds such as amino acids, carbohydrates, pigments, and pyridines (Guill´en-Sans and Guzm´an-Chozas 1998; Seljeskog and others 2006; Mendes and others 2009), which may cause an overestimation of the actual lipid peroxidation (Mateos and others 2005). Instead of TBA, 2,4-dinitrophenylhydrazine (DNPH) can be used for the derivatization of MDA and subsequent highperformance liquid chromatography (HPLC) analysis. Importantly, derivatization of MDA with DNPH and conversion into pyrazole and hydrazone takes place at room temperature, thereby avoiding the production of analytical artifacts (Korchazhkina and others 2003; Sim and others 2003; Mateos and others 2004, 2005; Czauderna and others 2011). Run times seem to be crucial factors as far as HPLC methods are concerned (Bayram and others 2013). Newly developed core columns enable increased chromatographic efficiency as well as shorter analysis times (McCalley 2010; Bayram and others 2013). The spherisorb ODS2 column used in the present study is a silicabased, reversed-phase C18 column filled with a specified carbon load of 11.5%, enabling the fast analysis of MDA. Furthermore, we used a photodiode array detector, which exhibits multiple photodiode arrays to obtain information over a wide range of wavelengths simultaneously. Thus overlapping peaks at different wavelengths can be detected. MS 20131246 Submitted 9/4/2013, Accepted 1/23/2014. Authors are with In the current study, we developed a sensitive, specific, and fast Inst. of Human Nutrition and Food Science, Christian-Albrechts-Univ. of Kiel, Hermann-Rodewald St. 6, 24118, Kiel, Germany. Direct inquiries to author Rimbach HPLC method to detect MDA. We applied DNPH derivatization and used an ODS2 column with a photodiode array detector (E-mail: [email protected]). for MDA measurements in liver samples from Atlantic salmon
Malondialdehyde (MDA) is a product of lipid peroxidation of polyunsaturated fatty acids (PUFA) in foods (Angelo 1996) and biological samples (for example, plasma and tissues) (Kinter 1995; Tsaknis and others 1999; Mao and others 2006; Mendes and others 2009). Furthermore, MDA is a widely used biomarker of oxidative stress (Rimbach and others 1999). Lipid peroxidation causes food spoilage, rancidity, and deterioration particularly in oily foods including fish (Tsaknis and others 1999; Mendes and others 2009). Fish is an important source of long-chain PUFA (Strobel and others 2012). PUFA may be oxidized to produce odorless and tasteless hydroperoxides, which decay to secondary oxidation products, basically aldehydes including hexanal, 4–hydroxynonenal (HNE), and MDA. The latter can be used to assess the extent of lipid peroxidation (Frankel 2005; Papastergiadis and others 2012). MDA is often determined spectrophotometrically. In fact, the spectrophotometric measurement of the pink colored adduct of MDA with 2-thiobarbituric acid (TBA, maximum absorbance at 532 to 535 nm) is possibly the most widely used method for the determination of MDA in foods and biological samples because of its low cost and simplicity (Raharjo and Sofos 1993; Botsoglou and others 1994). However, most of the spectrophotometric methods have been criticized because of their lack of specificity and the
C484
Journal of Food Science r Vol. 79, Nr. 4, 2014
R C 2014 Institute of Food Technologists
doi: 10.1111/1750-3841.12412 Further reproduction without permission is prohibited
MDA analysis by HPLC in fish liver . . .
Materials and Methods Chemicals and reagents Sodium hydroxide (NaOH) and hydrochloric acid (HCl; 37%) were purchased from Merck (Darmstadt, Germany). Perchloric acid (60%), DNPH (97%), acetonitrile (HPLC quality), and the MDA standard 1,1,3,3-tetraethoxypropane (TEP; 96%) (CAS nr. 122-31-6) were supplied by Sigma Aldrich (Steinheim, Germany). Sulfuric acid (95% to 97%) was purchased from J.T. Baker (Deventer, The Netherlands). Phosphate buffer saline (PBS) was obtained from PAA (Pasching, Austria). Double distilled water was used to prepare all aqueous solutions.
Optimization of the HPLC method In order to obtain a fast, simple, and sensitive method for MDA detection in biological samples, the HPLC methods by Mendes and others (2009) and Mateos and others (2005) have been modified herein. In comparison to Mateos and others (2005), the current method uses a Spherisorb ODS2 column (10 cm × 4.6 mm, 3 μm) instead of a Nucleosil 100 RP-18 column (125 mm × 4.0 mm, 5 μm). Furthermore, we modified the composition of the mobile phase by increasing the amount of acetonitrile from 38% to 58% which resulted in shorter retention times. We also increased the incubation time from 30 min to 1 h. The major aims of these modifications were to obtain a fast, simple, and sensitive method for MDA detection and to optimize previous experimental conditions, following the principle that sample preparation procedures should be kept as simple as possible to reduce errors and to maximize analyte recoveries (Mateos and others 2005).
Biological samples Fish liver, fillet, and plasma samples were supplied by Skretting ARC Stavanger, Norway. Atlantic salmon were sacrificed at ARC research trial station. Plasma, liver, and fillet samples were stored Selectivity at –80 °C until analysis. Succeeding the quantification of MDA, no interfering peaks were noticed. A clear separation of the MDA peak was obtained within 4.3 min. Standard solution preparation Standard solutions of MDA were prepared from 1,1,3,3 tetraethoxypropane (TEP) and stored at 4 °C in the dark. Standard Linearity, lower limit of detection (LOD), and lower limit of solutions were prepared in 1% sulfuric acid in PBS. quantification (LOQ) To establish the linear range of the detector response, 12 MDA Sample preparation of MDA-DNPH from biological samples standards were measured by triplicate injection on 3 different days Sample extraction was performed according to Mateos and oth- (Bayram and others 2013). Plotting the peak areas compared with ers (2005) with some modifications: 0.2 g of fish liver was homog- MDA concentrations resulted in linear curves for all 12 MDA stanenized in 1 mL of 1% sulfuric acid in PBS in 2 mL eppendorf for dards; the range of linearity for MDA detection in the standards 4 min using a tissue lyzer (Qiagen, Hilden Germany). Ho- was found to be between 0.39 and 200 μmol/L and the regresmogenates were centrifuged at 10000 rpm for 30 min at 4 °C. sion coefficients (R2 ) for all compounds were higher than 0.99 Subsequently, supernatants were collected. (Figure 1). Two hundred and fifty microliters of the supernatant were transThe LOQ and LOD of MDA were 0.39 and 0.195 μmol/L, ferred into a 1.5 mL eppendorf with 50 μL of 6 M NaOH. This respectively, which is comparable with the methods employed by mixture was incubated for 30 min at 60 °C in a water bath. Pro- Czauderna and others (2011), Mendes and others (2009), Mao tein was precipitated by adding 125 μL of 35% perchloric acid and others (2006), and Mateos and others (2005). and centrifuging at 2800 rpm for 10 min. Two hundred and fifty microliters of the supernatant were transferred into a new 1.5 mL Precision, accuracy, and recovery eppendorf and 50 μL of DNPH was added prior to incubation Intraday and interday precision was in the range of 6% to 12% in the dark at room temperature for 1 h. Following incubation, (Table 1), which is lower than the FDA’s established limits (ࣘ20% 20 μL sample was injected into the HPLC system. for LOQ and ࣘ15% for other concentration levels). HPLC analysis of MDA in biological samples HPLC analysis of MDA was performed on a Jasco system (Jasco GmbH Deutschland, Gross-Umstadt, Germany) equipped with an autosampler (Jasco AS-2057), pump (PU-2080), ternary gradient unit (LG-2080-02), 3 line degasser (DG-2080-53), and photodiode array detector. MDA was separated using a Supelco INC water spherisorb ODS2 column (10 cm × 4.6 mm, 3 μm) with a guard column, then eluted in isocratic mode with a mobile phase consisting of 0.2% acetic acid (v/v) in double distilled water/acetonitrile (42:58, v/v). The flow rate was set at 0.6 mL/min and the autosampler thermostat at 4 °C. MDA was analyzed at 310 nm. Method validation The present HPLC method was validated according to the FDA guidelines (U.S. FDA 2001) as previously reported (Bayram and Figure 1–Linearity of the detector responses for malondialdehyde (MDA) others 2013). in the range of 0.39 to 200 μmol/L. Vol. 79, Nr. 4, 2014 r Journal of Food Science C485
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containing high amounts of PUFA (Strobel and others 2012) prone Results and Discussion to lipid peroxidation.
MDA analysis by HPLC in fish liver . . . Table 1–Intraday and interday precision, accuracy, and recovery To establish short-term stability, 5 aliquots of the MDA standard of MDA at low, medium, and high concentrations (0.4, 2, 10 and 5 aliquots of spiked (low, medium, and high) fish liver samples μmol/L) added to fish liver samples (n = 5).
were analyzed. Both the standards as well as the spiked samples
Concentration (μmol/L) Intraday
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Interday
Low (0.4) Medium (2.0) High (10.0) Low (0.4) Medium (2.0) High (10.0)
Recovery (%)
Accuracy (% bias)
± ± ± ± ± ±
−0.4 −7.6 −2.1 3.9 −3.6 −2.3
99.7 92.4 97.9 104.2 96.7 97.9
12.2 7.4 6.6 3.9 3.8 6.1
Precision were analyzed after storage at RT, 4 °C, –20 °C, and –80 °C for (% CV) 24 h in order to compare obtained with initial values. The stability 11.7 7.5 6.9 10.7 6.2 6.1
Table 2–Short- and long-term stabilities (expressed as percentage degradation) of MDA standards with low, medium, or high concentrations. For short-term stability, the samples were stored at room temperature (RT), 4, –20, and –80 °C for 24 h (n = 5) and for long-term stability the samples were stored at –80 °C for 6 wk. Short-term stability (% degradation) Concentration (μmol/L) −80 °C −20 °C 4 °C RT Low (1.56) Medium (12.5) High (100)
8.4 0.3 0.8
−1.4 0.5 −1.4
2.6 0.6 1.3
Long-term stability (% degradation) −80 °C
−1.8 −2.4 1.4
1.8 1.6 0.7
Accuracy values were also satisfactory, ranging from 0.4 to 7.6% (Table 1), and were within the FDA’s established limits (ࣘ20% for LOQ and ࣘ15% for other concentration levels). Recovery values were also satisfactory. Intraday recovery was 92.4% to 99.7% and interday recovery was 96.7% to 104.2% (Table 1).
Stability In the current study, we determined the stability of MDA in both 1,1,3,3-tetraethoxypropane standards at low (1.56 μmol/L ࣓ 0.344 μg/mL wet weight), medium (12.5 μmol/L ࣓ 2.75 μg/mL wet weight), and high (100 μmol/L ࣓ 22.0 μg/mL wet weight) concentrations and in spiked fish liver samples (at low, medium, and high concentrations). The MDA standard solutions and spiked liver samples were stored at RT, 4 °C, –20 °C, or –80 °C for 24 h. The detector response after storage was compared with the initial measurement. Data are expressed as percentage degradation (Table 2 and 3). The MDA standard exhibiting the highest degradation (8.4%) was the lowest concentration standard stored at –80 °C (Table 2). The spiked liver sample, which showed most degradation (16.2%), was the high MDA concentration stored at RT (Table 3).
of MDA prepared as standard solutions was comparable with the MDA standard; the highest level of degradation (8.4%) occurred with the lowest concentration of MDA at –80 °C. Importantly, only negligible degradation of the MDA standards occurred during 24 h storage at RT, 4 °C, –20 °C, or –80 °C (Table 2). The degradation of MDA in the spiked fish liver samples ranged between 0.1% and 16.2% with the highest degradation occurring at the high MDA concentration stored at RT (Table 3). To assess long-term stability, 5 aliquots of the MDA standard (low, medium, and high concentrations) were analyzed after storage at –80 °C for 6 wk. In this study, the level of degradation ranged between 0.7% and 1.8%. From these results, it may be concluded that MDA standards are stable for a relatively long time at –80 °C after extraction (Table 2). The long-term stability of MDA in fish liver samples was determined from 5 aliquots prepared at low, medium, and high concentrations and stored at –20 °C and –80 °C for 8 wk. The level of degradation ranged between 5.4% and 8.6% at –80 °C and 14.6% to 19.5% at –20 °C (Table 3). According to these results, less degradation was observed at –80 °C. The freeze-thaw stability of MDA in spiked fish liver was examined after 3 freeze-thaw cycles. Under the conditions investigated, percentage degradation was slightly higher at the low (9.9%) and medium concentrations (11.4%) than at the high MDA concentration (5.3%). In general, after 3 free-thaw cycles, MDA in fish liver samples remained stable (Table 3). The stability of the processed samples, including the resident time in the autosampler, was determined with 5 aliquots of fish liver samples for each MDA concentration (low, medium, and high) and injected at 0, 6, 12, and 18 h from the same HPLC vial. MDA spiked liver samples were found to remain stable for 18 h in the autosampler (Table 3). We detected the highest degradation after 18 h (14.2% for the lower MDA concentration and 12.3% for the medium concentration, respectively).
Method application for the quantification of MDA-DNPH in biological samples MDA in fish liver samples was (range: 26.6 to 45.7 μmol/kg fresh matter) determined by using the validated HPLC method. HPLC-chromatogram of MDA in fish liver sample is depicted in Figure 2B. Applying our HPLC method, it is also feasible to analyze MDA in other tissues such as plasma and fillet, as shown in Figure 2A and 2C. For this method, we used a photodiode array detector enabling coelution. Furthermore, the ODS2 column
Table 3–Short-term, long-term, freeze-thaw, and postpreparative stabilities (expressed as percentage degradation) of MDA extracted from spiked (low, medium, or high concentrations) fish liver samples. For short-term stability, the samples were stored at room temperature (RT), 4, –20, and –80 °C for 24 h (n = 5) and for long-term stability the samples were stored at –20 and –80 °C for 8 wk. The samples were left in the autosampler at RT for up to 18 h for postpreparative stability. Short-term stability (% degradation)
Long-term stability (% degradation)
Concentration (μmol/L)
−80 °C
−20 °C
4°C
RT
−80 °C
Low (1.56) Medium (12.5) High (100)
5.4 9.1 −2.5
10.9 9.5 −0.2
8.4 −1.1 0.1
12.0 1.8 16.2
5.4 8.6 6.4
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Postpreparative stability (% degradation)
−20 °C
Freeze-andthaw stability (% degradation)
6h
12 h
18 h
14.6 16.7 19.5
9.9 11.4 5.3
2.4 0.4 −0.7
3.5 3.0 3.2
14.2 12.3 2.0
MDA analysis by HPLC in fish liver . . .
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Figure 2–Representative HPLC-chromatogram of MDA in fish plasma (A), liver (B), and fillet (C) after derivatization with DNPH (retention time 4.3 min); inset depicts a magnification of the MDA peak.
used in the present study allows a fast separation because of the short length of the column. The retention time of the MDA peak was around 4.3 min which is faster than previously published HPLC methods (Mateos and others 2004, 2005; Mendes and others 2009; Czauderna and others 2011). Recovery (ranging between 92.4% and 104.2%) and precision (ranging between 6.1% and 11.7%) were similar to values reported by Mateos and others (2005), Mendes and others (2009), and Czauderna and others (2011). Importantly, the measured MDA concentrations in our salmon liver samples are similar to values reported in the literature (Roig and others 2000; Alessio and others 2002; Yokozawa and others 2002; Mateos and others 2005). MDA was higher in liver (range: 26.6 to 45.7 μmol/kg fresh matter) as compared to fillet (range: 2.6 to 4.64 μmol/kg fresh matter) which is also in accordance with literature data (Khoschsorur and others 2000; Mateos and others 2005; Mendes and others 2009). Differences in MDA be-
tween liver and fillet samples may be related to differences in tissue vitamin E concentrations known to efficiently prevent lipid peroxidation (Rimbach and others 2002).
Conclusion In this paper, a modified HPLC method with photodiode array detection was validated for the rapid quantification of MDA in fish liver using an ODS2 column. The derivatization reaction of MDA with DNPH proceeds at low temperature, increasing specificity. The MDA-DNPH method is sensitive, fast, and simple. Furthermore, this method allows accurate, precise, and selective quantification of MDA.
Acknowledgments We are grateful to Dr. Ingunn Stubhaug and Dr. Wolfgang Koppe for providing Atlantic salmon samples.
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MDA analysis by HPLC in fish liver . . .
Author Contributions M.F., T.E., and B.B. conducted the HPLC analyses. M.F., T.E., and G.R. wrote the manuscript. G.R. approved the final version of the manuscript.
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