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Determination and removal of malondialdehyde and other 2-thiobarbituric acid reactive substances in waste cooking oil
Journal of Food Engineering 107 (2011) 379–384
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Journal of Food Engineering journal homepage: www.elsevier.com/locate/jfoodeng
Determination and removal of malondialdehyde and other 2-thiobarbituric acid reactive substances in waste cooking oil Zuojun Wei a,⇑, Xinghua Li a, Dilantha Thushara a, Yingxin Liu b a b
Department of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, PR China College of Pharmaceutical Science, Zhejiang University of Technology, Hangzhou 310032, PR China
a r t i c l e
i n f o
Article history: Received 16 December 2010 Received in revised form 8 May 2011 Accepted 22 June 2011 Available online 14 July 2011 Keywords: Malondialdehyde Waste cooking oil Adsorbent HPLC method 2-Thiobarbituric acid reactive substance
a b s t r a c t Waste cooking oil can be recovered and processed into animal feed additives after purification. Unfortunately, the traditional purification processes are insufficient for the removal of the harmful compounds formed during frying, mainly about malondialdehyde and other 2-thiobarbituric acid reactive substances. In the present paper, firstly, a simple and reliable HPLC method was developed to measure the content of malondialdehyde in purified waste cooking oil. The detection limit and the standard recovery of the analysis method are 1.20 105 g l1 and 96.5–99.2%, respectively, which is accurate and valid enough for the detection of malondialdehyde in waste cooking oil. Furthermore, the removal of malondialdehyde and other 2-thiobarbituric acid reactive substances from waste cooking oil was investigated using three methods, i.e., water extraction, physical adsorption, and chemical adsorption. Results show that among the three methods, chemical adsorption using lysine or monosodium glutamate as chemical adsorbent is the most effective, which can remove 80% of malondialdehyde and other 2-thiobarbituric acid reactive substances from waste cooking oil. Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction Edible oil (EO) is one of the indispensable nutrition components in our daily diet. While we consume, a part of EO turns into kitchen waste, which produces lots of waste cooking oil (WCO) after being refined. It is estimated that more than 10 million tons of WCO is generated over the world every year. Such waste cooking oil, if recovered, will obviously save resource, bring economic profit and relieve environmental pressure. One of the secure reutilization of WCO is to be proceeded into animal feed additives after purification (Awawdeh et al., 2009; Gatlin et al., 2002). The industrial procedure for WCO purification is quite similar to the standard process for EO purification, i.e., through degumming, bleaching, and deodorizing processes (Bailey, 2005), with the only difference that the former uses kitchen WCO as raw material while the latter uses crude plant oil. During the general industrial purification processes mentioned above, most of the impurities in WCO can be removed to meet the standard of the animal feed additives. Unfortunately, these processes are insufficient for the removal of the lipid peroxidation products, which is usually represented by malondialdehyde (MDA), a final decomposition product of lipid peroxidations (Torun et al., 1995). Since these lipid peroxidation products are harmful to the feeding animals and in the end, to
⇑ Corresponding author. Tel.: +86 13588810769. E-mail address: [email protected] (Z. Wei). 0260-8774/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.jfoodeng.2011.06.032
human beings (Torun et al., 1995). Therefore, an effective method for removing MDA and other peroxidation products from WCO should be developed in order to meet the standard of the animal feed additives. To remove MDA from WCO, it is firstly necessary to quantify it by using an accurate and valid method. Since the 1960s, many methods have been developed to quantify MDA in different sample sources such as fried food, meat, organism tissues, and EOs. Of all the measurement methods, the most classic one is the 2-thiobarbituric acid (TBA) test. This method involves the reaction of MDA with TBA at about 100 °C in an acidic media, forming a pink complex which can be spectrophotometrically detected at 532–538 nm (Kosugi et al., 1988; Pikul et al., 1989; Tarladgis et al., 1960). The TBA test method is simple and inexpensive, yet not accurate since other impurities such as carbohydrates, proteins, ketones, and other aldehydes may also react with TBA (also called TBA reactive substances (TBARSs)) and contribute to spectrophotometrical adsorption as well (Buttkus and Bose, 1972; Knight et al., 1988). Furthermore, the TBA test has been criticized for its lack of sensitivity since some lipid peroxidation products such as hydroperoxides and conjugated aldehydes interfere with TBA (Almandos et al., 1986; Shamberger et al., 1977; Squires, 1990). To overcome the lack of the specificity of MDA detection mentioned above, the HPLC method, which is viewed as a more advanced technique, has been developed. The general HPLC method normally utilizes derivatization reagents, such as TBA and various hydrazine compounds, to enhance the sensitivity to detect MDA
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(Bergamo et al., 1998; Grotto et al., 2007; Mendes et al., 2009; Rovellini, 2006). Based on the properties of the formed derivatives, a Vis/UV detector or a fluorometric detector is generally used (Cheng et al., 2008; Pilz et al., 2000; Templar et al., 1999). Both of the detectors are good at specificity, sensibility, and respect analytical performances, including baseline noise level, baseline drift and signal-to-noise ratio. However, the derivative procedure is rather time-consuming, and therefore this method is not practical for routine analysis. A rapid and convenient HPLC method without any pre-derivatization has been developed and successfully used for the determination of MDA in edible oils (Tsaknis et al., 1998). However, in this method, an additional distillation procedure is required to pre-separate MDA from edible oil with water steam before HPLC analysis, making the analysis process practically more complicated and also bringing new risk of the detection accuracy since MDA may not be completely distillated. Therefore, in the first stage of the present paper, we try to develop a rapid and accurate HPLC method to determine MDA in WCO. The removal of the lipid peroxidation products such as MDA and other TBARSs in EO refinery industry is related to the deodorization process, which is used for the removal of the disagreeable flavor and odor compounds as well as free fatty acids by steam distillation at high temperature and high vacuum (Dudrow, 1983; Gavin, 1978). Adding antioxidants can also delay the onset of lipid peroxidation for a period of time (Mosca et al., 2010). For EO, it itself contains vitamin E as a natural antioxidant, and adding synthetic antioxidants are also approved to prolong its shelf life. But for kitchen WCO, all the containing antioxidants may be completely decomposed, since it has been cooked at high temperature, exposed to oxygen and other severe conditions, which may be the reason why WCO is more liable to deserve oxidation and contains much more TBARSs (at the order of 1 lg g1 WCO) than EO (at the order of 0.1 lg g1 EO) during the industrial processing. As a result, the deodorization process for WCO refinery using the same method for EO refinery seems not sufficient for the removal of MDA and other TBARSs. Theoretically, increasing the temperature and the degree of vacuum in deodorization process may remove more amounts of MDA and other TBARSs, but may also accelerate the decomposition of the lipid to fatty acids, which thereby decreases the yield of WCO and increases the processing cost to a certain extent. Therefore, in the second stage of the present paper, we try to develop a more economic and efficient method to remove MDA and other TBARSs from WCO. 2. Materials and methods 2.1. Chemicals and reagents TBA, EDTA, trichloride acetic acid, hydrochloric acid, phosphoric acid (98%), activated carbon, alumina oxide, activate clay, HZSM-5 zeolite and urea were purchased from China National Medicines Co. Lysine and monosodium glutamate were purchased from Shanghai Kangjie Bio-Tech Co. 1,1,3,3-tetraethoxypropane (TEP) was purchased from Sigma Chemical Co. All the reagents were of analytical grade unless otherwise stated. Aqueous solutions were prepared with Milli-Q purified water. WCO used was supplied by Nongchen Feed Co. in China, purified using the conventional refinery process including degumming, bleaching and steaming deodorization, similar to the purification of crude vegetable oil (Bailey, 2005). 2.2. Preparation of MDA standard solutions A 10 ll of 1,1,3,3-tetraethoxypropane was accurately weighed and diluted to 10 ml with 0.1 mol l1 HCl in a screw-caped test
tube, heated for 5 min in a boiling water bath and then rapidly cooled down with tap water. 1 ml of the above solution was transferred to a 100 ml volumetric flask and diluted to volume with ultra pure water (corresponding to MDA content of 4.37 mg l1). Then 100 ml solution was further diluted to 2, 4, 6, 8, and 10 lg l1 for the calibration graphs in both TBA spectrophotometric method and HPLC method. 2.3. Preparation of WCO sample Ten grams of WCO was accurately weighed in a 100 ml Erlenmeyer flask and extracted with 50 ml of 0.1 mol l1 HCl for 30 min at 30–50 °C incubated in a thermostat shaker. The mixture was then filtered twice by two-layer filter paper (102#, GB/T19142007). The filtrate was used for both TBA spectrophotometric method and HPLC method. 2.4. Traditional TBA spectrophotometric method The traditional TBA spectrophotometric method was based on Chinese national standard GB/T 5009181-2003 with slight modifications. 5 ml of the filtered WCO sample prepared as Section 2.3, MDA standard solutions or blank (0.1 mol l1 HCl solution) was pipetted into a 25 ml cuvette, followed by adding 5 ml of 0.02 mol l1 TBA solution. The mixture was vigorously agitated in a votex for 40 min in 90 °C oil bath. After being cooled, the TBAMDA adduct was spectrophotometrically measured at 532 nm. 2.5. HPLC method HPLC measurements were performed on a Waters 1525 HPLC equipped with binary pumps and Waters 2487 dual k absorbance detector. Samples were analyzed on a Sunfire™ C18 column (250 4.6 mm i.d., 5 lm particles) at 30 °C with mobile phases consisting of 1% acetic acid in water (A) and acetonitrile (B). Gradient elution is programmed as follows: from 85% to 50% A in the first 20 min, and from 50% back to 85% A in the next 20 min. The flow rate was 0.6 ml min1 and the chromatographic data were acquired at 245 nm. The injection volume of sample was 10 ll. 2.6. Process for the removal of MDA from WCO Extraction method: 10 g of purified WCO was placed into a 100 ml Erlenmeyer flask, and a certain percentage of water adjusted at different pH was added to extract MDA at 30 °C for 30 min. Afterwards, the mixture was filtered by two-layer filter paper. MDA and other TBARSs contents in filtrate were then measured by both TBA spectrophotometry method and HPLC method. Adsorption method: 10 g of purified WCO was placed into a 100 ml Erlenmeyer flask, and a certain percentage of adsorbent was added to adsorb MDA at 50 °C for 30 min. The mixture was centrifuged and then measured by both TBA spectrophotometry and HPLC methods. 2.7. Statistical analysis Data determined by HPLC and spectrophotometry was repeated more than three times, and the results were expressed as average ± absolute deviation. For function y = f(x1, x2, . . ., xn), the propagation of absolute deviation is calculated as:
dev y
sffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi 2 2 2 @f @f @f 2 2 2 ¼ dev x1 þ dev x2 þ þ dev xn @x1 @x2 @xn
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newly developed HPLC method, and the content of TBARSs was measured by the traditional TBA method for comparison.
3. Results and discussion 3.1. Measurement of MDA
3.2. Removal of MDA and other TBARSs The analysis conditions of the HPLC method for the measurement of MDA by UV detector were carefully optimized after many trials to meet a certain accuracy and sensitivity, as mentioned in Section 2.5. Different from Tsaknis et al., the sample was not steam distillated before injection. Using the HPLC method developed in this study, the column pressure is 1000–1100 psi. The detection limit is 1.20 105 g l1 calculated by the three folds of signal/noise ratio, which is nearly 2-order higher than that of the steam-distillation-pretreatment method (Tsaknis et al., 1998). It is found that the content of MDA in WCO samples is in the order of 103 g l1, nearly 100 folds higher than the detection limit. Therefore, our method is accurate and valid enough for the current measurement of MDA in WCO samples. Typical chromatograms for the MDA standard and a WCO are shown in Fig. 1. The retention time of MDA is about 7.4 min. The plot of the standards against HPLC peak area shows a regression equation of y/(peak area) = 1.26 105x/(concentration of MDA, lg ml1) 6.25 103 with r2 = 0.9998. The recovery test was evaluated by adding known concentration of MDA standards. Both MDA standards and WCO samples were evaluated (Table 1). The recoveries for MDA standards are within the range of 96.5–99.2%, which are similar to those reported by Tsaknis et al. (1998) and most of the derivative HPLC methods (Antunes et al., 2008; Carbonneau et al., 1991; Cordis et al., 1995; Mateos et al., 2005; Mendes et al., 2009; Young and Trimble, 1991). The average recoveries for WCO sample are around 85%, which are much higher than those obtained by the traditional TBA method (Fenaille et al., 2001), and comparable to those obtained using some other HPLC methods (Mendes et al., 2009). So in the following study, the content of MDA was measured by our
0.08
0.12
0.06
Sample
0.08
0.04
Standard
0.02
0.06 Malondialdehyde RT: 7.43 min
0.04
0.00
0.02
-0.02
0.00 0
5
10
15
20
25
30
35
40
45
-0.04 50
Time (min) Fig. 1. Typical chromatograms for MDA standard and WCO sample.
A (AUFS)
A (AUFS)
0.10
As stated, the removal of MDA and other TBARSs from WCO to a certain low content is required before it is used as animal feed additives due to the toxicity of the TBARSs. For example, a maximum permitted TBARSs value of 6 lg g1 is executed by most of Chinese WCO refinery factories. Based on the properties of TBARSs and the cost considerations, three methods, i.e., water extraction, physical adsorption, and chemical adsorption were tried to remove MDA and other TBARSs. 3.2.1. Water extraction method Three content levels of water with/without acid were tried for the removal of MDA in WCO, as shown in Table 2. It can be seen that when using pure water as extractant, the removal efficiencies of MDA and TBARSs increase from 9.8% to 15.6% and from 9.6% to 16.7%, respectively, with the water/WCO ratio increasing from 1:9 to 1:1. Compared with pure water, the acidified water is more effective for the removal of MDA and TBARSs (Table 2). This may be due to the fact that MDA and TBARSs containing carbonyl groups tend to exist in enol forms (Guillen-Sans and Guzman-Chozas, 1998; Nair et al., 1981; Tuma et al., 2001) at acidic atmosphere, which are more hydrophilic and have higher water solubility than their keto forms. A remarkable drawback of the water extraction method is the emulsification of oil occurred during water extraction, which brings a big problem on the separation of water from WCO. From the viewpoint of industry, the emulsification of oil in water means the loss of WCO and the increase in the waste polluted water. So the water extraction method is not suitable for MDA removal from WCO in industry. 3.2.2. Physical adsorption method Four conventional adsorbents, i.e., activated carbon, alumina oxide, activated clay, and HZSM-5 zeolite were used for the removal of MDA from WCO. As shown in Table 3, 1% (w/w) activated carbon without H3PO4 shows the best adsorption behavior for MDA and other TBARS, with about 48% of MDA and 50% of TBARSs being removed from WCO, respectively. For physical adsorption, it is generally known that the nature of the surface functional groups will affect the interaction forces between adsorbate and adsorbent surface and therefore affect the adsorption capacity of an adsorbent (Thacker et al., 1984). Since the target adsorbates MDA and other TBARSs (mainly aldehydes and ketones) are of weak polarity, and the activated carbon, which is the most hydrophobic one among the tested adsorbents, may therefore attract more adsorbates onto its surface by different forces like unelectric force and van de wals force (Al-Degs et al., 2004; Toles et al., 1999). Contrarily, while WCO is acidified with H3PO4 for increasing the polarity of the adsorbents, the adsorption ability of activated carbon on MDA
Table 1 Recoveries for the determination of MDA by HPLC method. Concentration (lg ml1)
Added (lg ml1)
Found (lg ml1)
Recovery (%)
Average recovery (%)
1.31 ± 0.05
1.60 2.00 2.40
1.54 ± 0.01 1.97 ± 0.02 2.38 ± 0.02
96.3 ± 3.2 98.5 ± 2.7 99.2 ± 2.2
98.0 ± 2.7
10.31 ± 0.05b
1.60 2.00 2.40
1.45 ± 0.02 1.57 ± 0.01 1.91 ± 0.01
90.6 ± 3.4 78.5 ± 2.5 79.6 ± 2.1
82.9 ± 2.7
a
a
MDA standard solution. WCO sample. For WCO samples, different amounts of concentrated MDA standards were added respectively to 10 g of purified WCO, followed by HCl extraction procedure (see Section 2.3). b
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Table 2 Results of the removal of MDA and TBARSs from purified WCO sample by water extraction method. Water:WCO (v/v)
MDA (lg g1)
TBARSs (lg g1)
Initial
Final
Efficiency (%)
Initial
Final
Efficiency (%)
1:9a 1:4a 1:1a 1:9b 1:4b 1:1b
1.33 ± 0.05 1.33 ± 0.05 1.33 ± 0.05 1.33 ± 0.05 1.33 ± 0.05 1.33 ± 0.05
1.20 ± 0.03 1.17 ± 0.05 1.12 ± 0.04 1.04 ± 0.05 0.94 ± 0.03 0.89 ± 0.04
9.8 ± 4.1 12.0 ± 5.0 15.8 ± 4.4 21.8 ± 4.8 29.3 ± 3.5 33.1 ± 3.9
11.4 ± 0.2 11.4 ± 0.2 11.4 ± 0.2 11.4 ± 0.2 11.4 ± 0.2 11.4 ± 0.2
10.3 ± 0.1 9.9 ± 0.2 9.5 ± 0.1 8.8 ± 0.1 8.2 ± 0.2 7.8 ± 0.1
9.6 ± 1.8 13.2 ± 2.3 16.7 ± 1.7 22.8 ± 1.6 28.1 ± 2.2 31.6 ± 1.5
Note: Extraction was performed at 30 °C for 30 min under nitrogen atmosphere with continuous magnetic stirring; the amount of WCO sample was 10 g; centrifugation was done after each extraction for oil/water phase separation. a Pure water was used. b Water was adjusted to pH = 2 by H3PO4.
Table 3 Results of the removal of MDA and TBARSs from purified WCO sample by physical adsorption method. MDA (lg g1)
Adsorbents
Activated carbon Activated carbon + 0.1% H3PO4 Alumina oxide Activate clay HZSM-5
Efficiency (%)
Initial
Final
2.59 ± 0.03 2.59 ± 0.03 2.59 ± 0.03 2.59 ± 0.03 2.59 ± 0.03
1.34 ± 0.02 1.84 ± 0.03 2.50 ± 0.01 4.45 ± 0.03 1.76 ± 0.02
48.3 ± 1.0 29.0 ± 1.4 3.50 ± 1.2 71.8 ± 2.3 32.0 ± 1.1
TBARSs (lg g1)
Efficiency (%)
Initial
Final
17 ± 0.5 17 ± 0.5 17 ± 0.5 17 ± 0.5 17 ± 0.5
8.48 ± 0.08 11.89 ± 0.17 16.25 ± 0.15 29.75 ± 0.25 11.90 ± 0.15
50.1 ± 1.5 30.1 ± 2.3 4.40 ± 2.9 75.0 ± 5.4 30.0 ± 2.2
Note: Adsorption was performed at 50 °C for 30 min under nitrogen atmosphere with continuous magnetic stirring; the amount of WCO sample was 10 g; the amount of the adsorbent was 0.1 g.
and other TBARSs decreases by about 40%. Alumina oxide has only slight adsorption capacity for MDA and other TBARSs due to its higher polarity. The second favorable adsorbent is HZSM-5 zeolite, which removes about 32% and 30% of MDA and TBARSs from WCO, respectively. The positive effect of HZSM-5 zeolite on MDA and other TBARSs adsorption may be partly ascribed to the isomerization of aldehydes and ketones (MDA and TBARSs) into more hydrophilic enol forms, which may attract more MDA and other TBARSs onto the surface of HZSM-5 zeolite with hydrophilic nature. While activated clay was used as adsorbent, both the contents of MDA and other TBARSs increase obviously (by 72% and 75%, respectively). It may be due to the fact that the metal impurities like Cu or Fe in clay can accelerate the peroxidation of lipid as catalysts, generating more amounts of MDA and other TBARSs (Guillen-Sans and Guzman-Chozas, 1998; van Breemen et al., 2002). 3.2.3. Chemical adsorption method Based on the fact that the carbonyl group can covalently bond and cross-link a variety of biological macromolecules as well as the above-mentioned derivatization reagents having amino groups (Nair et al., 1981; Tuma et al., 2001) through a Skiff base reaction (Fig. 2), adding amino group-containing substances may be a good idea to remove MDA and other TBARSs from WCO. In this study, three commercially available amino-containing materials, i.e., lysine, monosodium glutamate, and urea, were under investigation. Due to their strong hydrophilicity, the three materials are not soluble but only suspended in WCO. MDA and other TBARSs in WCO
δ+
δ+
R1 C
R2
O δ- H + NH δ-
R3
H+
O H R1 C R2
N R3
H
-H2O
R1 R2
C
N
R3
Schif f Base
Fig. 2. Schiff reaction pathways for aldehydes or ketones. R1, R2 and R3 are organic functional groups.
are then supposed to react with and chemically adsorbed on the surface of the suspended particles, which is regarded as a chemical adsorption process. Results are shown in Table 4. As expected, around 80% of MDA and other TBARSs are effectively removed from WCO by each amino-containing material in the presence of 0.1% H3PO4. Such high removal efficiency is quite satisfactory for industrial use since the maximum permitted TBARSs is 6 lg g1, higher than those of all our acidic adsorbents treated products which contains only less than 2.5 lg g1 of TBARSs. It can be seen from Table 4 that moderate acid seems necessary for the removal of MDA and other TBARSs, as it is found that all of the three adsorbents have only slight reactive with MDA and other TBARSs without the addition of H3PO4. An explanation for the inactivation is that the carbonyl group is more liable to be protonized under weak or middle acidic environment which increases its nucleophilicity. Theoretically, Schiff-base reaction is more liable to occur under alkaline condition as the proton is released from the amine ion which increases the effective concentration of amino (Ge and Lee, 1997). However, it is not feasible in our case since the alkaline condition will lead to the saponification of lipids. 3.3. Discussion Compared with the widely used derivative HPLC method (Bergamo et al., 1998; Grotto et al., 2007; Mendes et al., 2009; Rovellini, 2006), direct measurement of MDA through UV detector is only limited to 1 or 2 reports (Karatas et al., 2002; Tsaknis et al., 1998), and has not been widely adopted yet. Three reasons may contribute to this fact. Firstly, MDA exists in aqueous phase as a mixture of ketonic and enolic tautomers, which have different functional groups and molecular polarity. As a result, the direct determination of MDA by UV detector in HPLC may appear two peaks corresponding to the two tautomers, which may bring difficulty in the quantification of MDA; whereas in the derivation method, MDA is fully transformed to one stable adduct. Secondly, the maximum response signal of MDA is much less than that of MDA derivatives at the same concentration, which may decrease
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Z. Wei et al. / Journal of Food Engineering 107 (2011) 379–384 Table 4 Results of the removal of MDA and TBARSs from purified WCO sample by the chemical adsorption method. Adsorbents
MDA (lg g1) Initial
Final
Lysine + H3PO4 Monosodium glutamate + H3PO4 Urea + H3PO4 Lysine Monosodium glutamate Urea
1.33 ± 0.05 1.33 ± 0.05 1.33 ± 0.05 1.33 ± 0.05 1.33 ± 0.05 1.33 ± 0.05
0.27 ± 0.03 0.26 ± 0.04 0.30 ± 0.05 1.27 ± 0.05 1.26 ± 0.03 1.26 ± 0.05
Efficiency (%)
TBARS (lg g1) Initial
Final
79.7 ± 2.4 80.5 ± 3.1 77.4 ± 3.9 4.5 ± 5.2 5.3 ± 4.2 5.3 ± 5.2
11.4 ± 0.2 11.4 ± 0.2 11.4 ± 0.2 11.4 ± 0.2 11.4 ± 0.2 11.4 ± 0.2
2.35 ± 0.2 2.09 ± 0.1 2.15 ± 0.2 10.9 ± 0.1 10.7 ± 0.2 11.0 ± 0.2
Efficiency (%)
79.4 ± 1.8 81.7 ± 0.9 81.1 ± 1.8 4.4 ± 1.9 6.1 ± 2.4 3.5 ± 2.4
Note: Extraction was performed at 50 °C for 30 min under nitrogen atmosphere with continuous magnetic stirring; the amount of WCO sample was 10 g; the amount of amino acid was 0.01 g, while urea was 0.1 g.
the analysis sensitivity of the direct detection method. Finally, the impurities in the sample usually disturb the chromatogram in the direct HPLC method; while in the derivative method, MDA is specifically derivated and has much higher response signal than that of impurities. In our method, the isomerization of MDA is inhibited by adjusting the pH of the sample to 3, at which most of MDA exists as the style of enolic tautomer, and the separation of MDA from other impurities is accomplished by the optimization of the mobile phase components and the flow rate. The only drawback of our method is the lower detection limit. But to our routine use for MDA measurement in WCO samples, the content of MDA is always rather high. We therefore regard that our HPLC method has enough accuracy for MDA measurement in WCO samples, as well as the advantage of simplicity and convenience. A comparative evaluation for the measurement of MDA in WCO and supermarket-available EO was made between the traditional spectrophotometric procedure and our HPLC method (Fig. 3). As expected, the measured MDA values between the two methods for both WCO and EO samples are quite different. The discrepancy is also frequently appeared between the traditional spectrophotometric procedure and other HPLC or GC methods. The high MDA value by traditional spectrophotometric procedure (TBA method) was generally explained by artefactual formation of MDA during the acid heating step required for the TBA derivatization at 90 °C (de las Heras et al., 2003; Mendes et al., 2009). However, such discrepancy reported being as high as 6–30 folds (Behrens and Madere, 1991; Fenaille et al., 2001; Karatas et al., 2002; Tsaknis et al., 1999, 1998; Yeo et al., 1994) could not be only ascribed to the ‘online’ formation of MDA. Since the HPLC method can very
precisely discriminate MDA, while TBA is quite unspecific for MDA, which is reasonable to deduce that ca 16.7–3.3% (corresponding to 6 and 30 folds of the discrepancy, respectively) of TBA reactive peroxidation products is MDA. That is, the discrepancy of the two methods mainly derives from MDA content in the peroxidation products rather than the ‘online’ formation of MDA. Different source of the samples has different MDA content, leading to different discrepancy folds between the two methods, which is 9.78 (slop in Fig 3) for WCO sample and 6.49 for EO sample in our experiment. In WCO industry, the TBARSs content in purified WCO is frequently in excess of 10 lg g1, which is much higher than Chinese national standard limitation of 6 lg g1 for animal feed additives. As stated above, both physical and chemical adsorbents are capable of removing considerable MDA or other TBARSs from WCO to meet such standard. However, taking into account of the processing costs and the environmental impact, the commercially available amino acids (or salt) such as lysine and monosodium glutamate are the best choice, based on the following three facts: (1) the prices of lysine and monosodium glutamate are of the same order as the prices of activated carbon and HZSM-5 zeolite, while their dosages are only 10 percent of the latter (0.1% (w/w) vs. 1% (w/w)) comparing with the removal efficiency of about 80% (see Table 4) and 50% (see Table 3), respectively; (2) when amino acids are separated from WCO after adsorbing MDA and other TBARSs, they can be reused as organic fertilizers, while the adsorbents like activated carbon or HZSM-5 zeolite can only be discarded as wastes; (3) of all the absorbents, urea is found to be the cheapest and the highest efficient for the removal of MDA and TBARSs. However, the toxicity at the concentration level of its solubility in WCO is not clear yet, which limits the application of urea.
30
25
MDA by TBA (μg·ml-1)
4. Conclusion
y = 6.49x R² = 0.97
In summary, a simple and reliable HPLC method is developed to measure the MDA content in purified WCO, which is expected to be more convenient, compared with the derivative HPLC methods as well as the steam distillation HPLC method. Three methods were used to remove MDA and other TBARSs in purified WCO. Among them, the chemical adsorption method using lysine and monosodium glutamate as adsorbents is the best choice.
20
15
10
References y = 9.78x R² = 0.93
5
0
0
1
2
3
4
5
MDA by HPLC (μg·ml-1) Fig. 3. MDA levels measured by HPLC method and spectrophotometric TBA method. 4 for WCO and s for EO.
Al-Degs, Y.S., El-Barghouthi, M.I., Khraisheh, M.A., Ahmad, M.N., Allen, S.J., 2004. Effect of surface area, micropores, secondary micropores, and mesopores volumes of activated carbons on reactive dyes adsorption from solution. Separation Science and Technology 39 (1), 97–111. Almandos, M.E., Giannini, D.H., Ciarlo, A.S., Boeri, R.L., 1986. Formaldehyde as an interference of the 2-thiobarbituric acid test. Journal of the Science of Food and Agriculture 37 (1), 54–58. Antunes, M.V., Lazzaretti, C., Gamaro, G.D., Linden, R., 2008. Preanalytical and validation studies for the determination of malondialdehyde in human plasma through high performance liquid chromatography after derivatization with 2,4-
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dinitrophenylhydrazine. Revista Brasileira De Ciencias Farmaceuticas 44 (2), 279–287. Awawdeh, M.S., Obeidat, B.S., Kridli, R.T., 2009. Yellow grease as an alternative energy source for nursing Awassi ewes and their suckling lambs. Animal Feed Science and Technology 152 (3–4), 165–174. Bailey, A.E., 2005. Bailey’s Industrial Oil and Fat Products. John Wiley & Sons, New York. Behrens, W.A., Madere, R., 1991. Malonaldehyde determination in tissues and biological-fluids by ion-pairing high-performance liquid-chromatography. Lipids 26 (3), 232–236. Bergamo, P., Fedele, E., Balestrieri, M., Abrescia, P., Ferrara, L., 1998. Measurement of malondialdehyde levels in food by high-performance liquid chromatography with fluorometric detection. Journal of Agricultural and Food Chemistry 46 (6), 2171–2176. Buttkus, H., Bose, R.J., 1972. Amine-malonaldehyde condensation products and their relative color contribution in thiobarbituric acid test. Journal of the American Oil Chemists Society 49 (7), 440–443. Carbonneau, M.A., Peuchant, E., Sess, D., Canioni, P., Clerc, M., 1991. Free and bound malondialdehyde measured as thiobarbituric acid adduct by hplc in serum and plasma. Clinical Chemistry 37 (8), 1423–1429. Cheng, G.W., Wu, H.L., Huang, Y.L., 2008. Simultaneous determination of malondialdehyde and ofloxacin in plasma using an isocratic highperformance liquid chromatography/fluorescence detection system. Analytica Chimica Acta 616 (2), 230–234. Cordis, G.A., Maulik, N., Das, D.K., 1995. Detection of oxidative stress in heart by estimating the dinitrophenylhydrazine derivative of malonaldehyde. Journal of Molecular and Cellular Cardiology 27 (8), 1645–1653. de las Heras, A., Schoch, A., Gibis, M., Fischer, A., 2003. Comparison of methods for determining malondialdehyde in dry sausage by HPLC and the classic TBA test. European Food Research and Technology 217 (2), 180–184. Dudrow, F.A., 1983. Deodorization of edible oil. Journal of the American Oil Chemists Society 60 (2), 272–274. Fenaille, F., Mottier, P., Turesky, R.J., Ali, S., Guy, P.A., 2001. Comparison of analytical techniques to quantify malondialdehyde in milk powders. Journal of Chromatography A 921 (2), 237–245. Gatlin, L.A., See, M.T., Larick, D.K., Lin, X., Odle, J., 2002. Conjugated linoleic acid in combination with supplemental dietary fat alters pork fat quality. Journal of Nutrition 132 (10), 3105–3112. Gavin, A.M., 1978. Edible oil deodorization. Journal of the American Oil Chemists Society 55 (11), 783–791. Ge, S.-J., Lee, T.-C., 1997. Kinetic significance of the schiff base reversion in the earlystage Maillard reaction of a phenylalanine–glucose aqueous model system. Journal of Agricultural and Food Chemistry 45 (5), 1619–1623. Grotto, D., Santa Maria, L.D., Boeira, S., Valentini, J., Charao, M.E., Moro, A.M., Nascimento, P.C., Pomblum, V.J., Garcia, S.C., 2007. Rapid quantification of malondialdehyde in plasma by high performance liquid chromatographyvisible detection. Journal of Pharmaceutical and Biomedical Analysis 43 (2), 619–624. Guillen-Sans, R., Guzman-Chozas, M., 1998. The thiobarbituric acid (TBA) reaction in foods: a review. Critical Reviews in Food Science and Nutrition 38 (4), 315– 330. Karatas, F., Karatepe, M., Baysar, A., 2002. Determination of free malondialdehyde in human serum by high-performance liquid chromatography. Analytical Biochemistry 311 (1), 76–79. Knight, J.A., Pieper, R.K., Mcclellan, L., 1988. Specificity of the thiobarbituric acid reaction – its use in studies of lipid-peroxidation. Clinical Chemistry 34 (12), 2433–2438. Kosugi, H., Kato, T., Kikugawa, K., 1988. Formation of red pigment by a 2-step 2thiobarbituric acid reaction of alka-2,4-dienals – potential products of lipid oxidation. Lipids 23 (11), 1024–1031. 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 oxidative stress – application to a rat model for hypercholesterolemia and evaluation of the effect of diets rich in phenolic antioxidants from fruits. Journal of Chromatography B – Analytical Technologies in the Biomedical and Life Sciences 827 (1), 76–82. Mendes, R., Cardoso, C., Pestana, C., 2009. Measurement of malondialdehyde in fish: a comparison study between hplc methods and the traditional spectrophotometric test. Food Chemistry 112 (4), 1038–1045. Mosca, M., Ceglie, A., Ambrosone, L., 2010. Lipid oxidation in water-in-olive oil emulsions initiated by a lipophilic radical source. Journal of Physical Chemistry B 114 (10), 3550–3558. Nair, V., Vietti, D.E., Cooper, C.S., 1981. Degenerative chemistry of malondialdehyde. Structure, stereochemistry, and kinetics of formation of enaminals from reaction with amino-acids. Journal of the American Chemical Society 103 (11), 3030–3036. Pikul, J., Leszczynski, D.E., Kummerow, F.A., 1989. Evaluation of 3 modified tba methods for measuring lipid oxidation in chicken meat. Journal of Agricultural and Food Chemistry 37 (5), 1309–1313. Pilz, J., Meineke, I., Gleiter, C.H., 2000. Measurement of free and bound malondialdehyde in plasma by high-performance liquid chromatography as the 2,4-dinitrophenylhydrazine derivative. Journal of Chromatography B – Analytical Technologies in the Biomedical and Life Sciences 742 (2), 315–325. Rovellini, P., 2006. Malonaldehyde contents in vegetable oils and chips Liquid chromatography-apci-mass spectrometry method. Rivista Italiana Delle Sostanze Grasse 83 (5), 213–220. Shamberger, R.J., Shamberger, B.A., Willis, C.E., 1977. Malonaldehyde content of food. Journal of Nutrition 107 (8), 1404–1409. Squires, E.J., 1990. High-performance liquid-chromatographic analysis of the malondialdehyde content of chicken liver. Poultry Science 69 (8), 1371–1376. Tarladgis, B.G., Watts, B.M., Younathan, M.T., Dugan, L., 1960. A distillation method for the quantitative determination of malonaldehyde in rancid foods. Journal of the American Oil Chemists Society 37 (1), 44–48. Templar, J., Kon, S.P., Milligan, T.P., Newman, D.J., Raftery, M.J., 1999. Increased plasma malondialdehyde levels in glomerular disease as determined by a fully validated hplc method. Nephrology Dialysis Transplantation 14 (4), 946–951. Thacker, W.E., Crittenden, J.C., Snoeyink, V.L., 1984. Modeling of adsorber performance – variable influent concentration and comparison of adsorbents. Journal Water Pollution Control Federation 56 (3), 243–250. Toles, C.A., Marshall, W.E., Johns, M.M., 1999. Surface functional groups on acidactivated nutshell carbons. Carbon 37 (8), 1207–1214. Torun, M., Yardim, S., Gonenc, A., Sargin, H., Menevse, A., Simsek, B., 1995. Serum beta-carotene, vitamin-E, vitamin-C and malondialdehyde levels in several types of cancer. Journal of Clinical Pharmacy and Therapeutics 20 (5), 259–263. Tsaknis, J., Lalas, S., Hole, M., Smith, G., Tychopoulos, V., 1998. Rapid highperformance liquid chromatographic method of determining malondialdehyde for evaluation of rancidity in edible oils. Analyst 123 (2), 325–327. Tsaknis, J., Lalas, S., Evmorfopoulos, E., 1999. Determination of malondialdehyde in traditional fish products by HPLC. Analyst 124 (6), 843–845. Tuma, D.J., Kearley, M.L., Thiele, G.M., Worrall, S., Haver, A., Klassen, L.W., Sorrell, M.F., 2001. Elucidation of reaction scheme describing malondialdehyde– acetaldehyde–protein adduct formation. Chemical Research in Toxicology 14 (7), 822–832. van Breemen, R.B., Xu, X.Y., Viana, M.A., Chen, L.W., Stacewicz-Sapuntzakis, M., Duncan, C., Bowen, P.E., Sharifi, R., 2002. Liquid chromatography-mass spectrometry of cis- and all-trans-lycopene in human serum and prostate tissue after dietary supplementation with tomato sauce. Journal of Agricultural and Food Chemistry 50 (8), 2214–2219. Yeo, H.C., Helbock, H.J., Chyu, D.W., Ames, B.N., 1994. Assay of malondialdehyde in biological-fluids by gas-chromatography mass-spectrometry. Analytical Biochemistry 220 (2), 391–396. Young, I.S., Trimble, E.R., 1991. Measurement of malondialdehyde in plasma by high-performance liquid-chromatography with fluorometric detection. Annals of Clinical Biochemistry 28, 504–508.
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Journal of Food Engineering journal homepage: www.elsevier.com/locate/jfoodeng
Determination and removal of malondialdehyde and other 2-thiobarbituric acid reactive substances in waste cooking oil Zuojun Wei a,⇑, Xinghua Li a, Dilantha Thushara a, Yingxin Liu b a b
Department of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, PR China College of Pharmaceutical Science, Zhejiang University of Technology, Hangzhou 310032, PR China
a r t i c l e
i n f o
Article history: Received 16 December 2010 Received in revised form 8 May 2011 Accepted 22 June 2011 Available online 14 July 2011 Keywords: Malondialdehyde Waste cooking oil Adsorbent HPLC method 2-Thiobarbituric acid reactive substance
a b s t r a c t Waste cooking oil can be recovered and processed into animal feed additives after purification. Unfortunately, the traditional purification processes are insufficient for the removal of the harmful compounds formed during frying, mainly about malondialdehyde and other 2-thiobarbituric acid reactive substances. In the present paper, firstly, a simple and reliable HPLC method was developed to measure the content of malondialdehyde in purified waste cooking oil. The detection limit and the standard recovery of the analysis method are 1.20 105 g l1 and 96.5–99.2%, respectively, which is accurate and valid enough for the detection of malondialdehyde in waste cooking oil. Furthermore, the removal of malondialdehyde and other 2-thiobarbituric acid reactive substances from waste cooking oil was investigated using three methods, i.e., water extraction, physical adsorption, and chemical adsorption. Results show that among the three methods, chemical adsorption using lysine or monosodium glutamate as chemical adsorbent is the most effective, which can remove 80% of malondialdehyde and other 2-thiobarbituric acid reactive substances from waste cooking oil. Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction Edible oil (EO) is one of the indispensable nutrition components in our daily diet. While we consume, a part of EO turns into kitchen waste, which produces lots of waste cooking oil (WCO) after being refined. It is estimated that more than 10 million tons of WCO is generated over the world every year. Such waste cooking oil, if recovered, will obviously save resource, bring economic profit and relieve environmental pressure. One of the secure reutilization of WCO is to be proceeded into animal feed additives after purification (Awawdeh et al., 2009; Gatlin et al., 2002). The industrial procedure for WCO purification is quite similar to the standard process for EO purification, i.e., through degumming, bleaching, and deodorizing processes (Bailey, 2005), with the only difference that the former uses kitchen WCO as raw material while the latter uses crude plant oil. During the general industrial purification processes mentioned above, most of the impurities in WCO can be removed to meet the standard of the animal feed additives. Unfortunately, these processes are insufficient for the removal of the lipid peroxidation products, which is usually represented by malondialdehyde (MDA), a final decomposition product of lipid peroxidations (Torun et al., 1995). Since these lipid peroxidation products are harmful to the feeding animals and in the end, to
⇑ Corresponding author. Tel.: +86 13588810769. E-mail address: [email protected] (Z. Wei). 0260-8774/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.jfoodeng.2011.06.032
human beings (Torun et al., 1995). Therefore, an effective method for removing MDA and other peroxidation products from WCO should be developed in order to meet the standard of the animal feed additives. To remove MDA from WCO, it is firstly necessary to quantify it by using an accurate and valid method. Since the 1960s, many methods have been developed to quantify MDA in different sample sources such as fried food, meat, organism tissues, and EOs. Of all the measurement methods, the most classic one is the 2-thiobarbituric acid (TBA) test. This method involves the reaction of MDA with TBA at about 100 °C in an acidic media, forming a pink complex which can be spectrophotometrically detected at 532–538 nm (Kosugi et al., 1988; Pikul et al., 1989; Tarladgis et al., 1960). The TBA test method is simple and inexpensive, yet not accurate since other impurities such as carbohydrates, proteins, ketones, and other aldehydes may also react with TBA (also called TBA reactive substances (TBARSs)) and contribute to spectrophotometrical adsorption as well (Buttkus and Bose, 1972; Knight et al., 1988). Furthermore, the TBA test has been criticized for its lack of sensitivity since some lipid peroxidation products such as hydroperoxides and conjugated aldehydes interfere with TBA (Almandos et al., 1986; Shamberger et al., 1977; Squires, 1990). To overcome the lack of the specificity of MDA detection mentioned above, the HPLC method, which is viewed as a more advanced technique, has been developed. The general HPLC method normally utilizes derivatization reagents, such as TBA and various hydrazine compounds, to enhance the sensitivity to detect MDA
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(Bergamo et al., 1998; Grotto et al., 2007; Mendes et al., 2009; Rovellini, 2006). Based on the properties of the formed derivatives, a Vis/UV detector or a fluorometric detector is generally used (Cheng et al., 2008; Pilz et al., 2000; Templar et al., 1999). Both of the detectors are good at specificity, sensibility, and respect analytical performances, including baseline noise level, baseline drift and signal-to-noise ratio. However, the derivative procedure is rather time-consuming, and therefore this method is not practical for routine analysis. A rapid and convenient HPLC method without any pre-derivatization has been developed and successfully used for the determination of MDA in edible oils (Tsaknis et al., 1998). However, in this method, an additional distillation procedure is required to pre-separate MDA from edible oil with water steam before HPLC analysis, making the analysis process practically more complicated and also bringing new risk of the detection accuracy since MDA may not be completely distillated. Therefore, in the first stage of the present paper, we try to develop a rapid and accurate HPLC method to determine MDA in WCO. The removal of the lipid peroxidation products such as MDA and other TBARSs in EO refinery industry is related to the deodorization process, which is used for the removal of the disagreeable flavor and odor compounds as well as free fatty acids by steam distillation at high temperature and high vacuum (Dudrow, 1983; Gavin, 1978). Adding antioxidants can also delay the onset of lipid peroxidation for a period of time (Mosca et al., 2010). For EO, it itself contains vitamin E as a natural antioxidant, and adding synthetic antioxidants are also approved to prolong its shelf life. But for kitchen WCO, all the containing antioxidants may be completely decomposed, since it has been cooked at high temperature, exposed to oxygen and other severe conditions, which may be the reason why WCO is more liable to deserve oxidation and contains much more TBARSs (at the order of 1 lg g1 WCO) than EO (at the order of 0.1 lg g1 EO) during the industrial processing. As a result, the deodorization process for WCO refinery using the same method for EO refinery seems not sufficient for the removal of MDA and other TBARSs. Theoretically, increasing the temperature and the degree of vacuum in deodorization process may remove more amounts of MDA and other TBARSs, but may also accelerate the decomposition of the lipid to fatty acids, which thereby decreases the yield of WCO and increases the processing cost to a certain extent. Therefore, in the second stage of the present paper, we try to develop a more economic and efficient method to remove MDA and other TBARSs from WCO. 2. Materials and methods 2.1. Chemicals and reagents TBA, EDTA, trichloride acetic acid, hydrochloric acid, phosphoric acid (98%), activated carbon, alumina oxide, activate clay, HZSM-5 zeolite and urea were purchased from China National Medicines Co. Lysine and monosodium glutamate were purchased from Shanghai Kangjie Bio-Tech Co. 1,1,3,3-tetraethoxypropane (TEP) was purchased from Sigma Chemical Co. All the reagents were of analytical grade unless otherwise stated. Aqueous solutions were prepared with Milli-Q purified water. WCO used was supplied by Nongchen Feed Co. in China, purified using the conventional refinery process including degumming, bleaching and steaming deodorization, similar to the purification of crude vegetable oil (Bailey, 2005). 2.2. Preparation of MDA standard solutions A 10 ll of 1,1,3,3-tetraethoxypropane was accurately weighed and diluted to 10 ml with 0.1 mol l1 HCl in a screw-caped test
tube, heated for 5 min in a boiling water bath and then rapidly cooled down with tap water. 1 ml of the above solution was transferred to a 100 ml volumetric flask and diluted to volume with ultra pure water (corresponding to MDA content of 4.37 mg l1). Then 100 ml solution was further diluted to 2, 4, 6, 8, and 10 lg l1 for the calibration graphs in both TBA spectrophotometric method and HPLC method. 2.3. Preparation of WCO sample Ten grams of WCO was accurately weighed in a 100 ml Erlenmeyer flask and extracted with 50 ml of 0.1 mol l1 HCl for 30 min at 30–50 °C incubated in a thermostat shaker. The mixture was then filtered twice by two-layer filter paper (102#, GB/T19142007). The filtrate was used for both TBA spectrophotometric method and HPLC method. 2.4. Traditional TBA spectrophotometric method The traditional TBA spectrophotometric method was based on Chinese national standard GB/T 5009181-2003 with slight modifications. 5 ml of the filtered WCO sample prepared as Section 2.3, MDA standard solutions or blank (0.1 mol l1 HCl solution) was pipetted into a 25 ml cuvette, followed by adding 5 ml of 0.02 mol l1 TBA solution. The mixture was vigorously agitated in a votex for 40 min in 90 °C oil bath. After being cooled, the TBAMDA adduct was spectrophotometrically measured at 532 nm. 2.5. HPLC method HPLC measurements were performed on a Waters 1525 HPLC equipped with binary pumps and Waters 2487 dual k absorbance detector. Samples were analyzed on a Sunfire™ C18 column (250 4.6 mm i.d., 5 lm particles) at 30 °C with mobile phases consisting of 1% acetic acid in water (A) and acetonitrile (B). Gradient elution is programmed as follows: from 85% to 50% A in the first 20 min, and from 50% back to 85% A in the next 20 min. The flow rate was 0.6 ml min1 and the chromatographic data were acquired at 245 nm. The injection volume of sample was 10 ll. 2.6. Process for the removal of MDA from WCO Extraction method: 10 g of purified WCO was placed into a 100 ml Erlenmeyer flask, and a certain percentage of water adjusted at different pH was added to extract MDA at 30 °C for 30 min. Afterwards, the mixture was filtered by two-layer filter paper. MDA and other TBARSs contents in filtrate were then measured by both TBA spectrophotometry method and HPLC method. Adsorption method: 10 g of purified WCO was placed into a 100 ml Erlenmeyer flask, and a certain percentage of adsorbent was added to adsorb MDA at 50 °C for 30 min. The mixture was centrifuged and then measured by both TBA spectrophotometry and HPLC methods. 2.7. Statistical analysis Data determined by HPLC and spectrophotometry was repeated more than three times, and the results were expressed as average ± absolute deviation. For function y = f(x1, x2, . . ., xn), the propagation of absolute deviation is calculated as:
dev y
sffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi 2 2 2 @f @f @f 2 2 2 ¼ dev x1 þ dev x2 þ þ dev xn @x1 @x2 @xn
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newly developed HPLC method, and the content of TBARSs was measured by the traditional TBA method for comparison.
3. Results and discussion 3.1. Measurement of MDA
3.2. Removal of MDA and other TBARSs The analysis conditions of the HPLC method for the measurement of MDA by UV detector were carefully optimized after many trials to meet a certain accuracy and sensitivity, as mentioned in Section 2.5. Different from Tsaknis et al., the sample was not steam distillated before injection. Using the HPLC method developed in this study, the column pressure is 1000–1100 psi. The detection limit is 1.20 105 g l1 calculated by the three folds of signal/noise ratio, which is nearly 2-order higher than that of the steam-distillation-pretreatment method (Tsaknis et al., 1998). It is found that the content of MDA in WCO samples is in the order of 103 g l1, nearly 100 folds higher than the detection limit. Therefore, our method is accurate and valid enough for the current measurement of MDA in WCO samples. Typical chromatograms for the MDA standard and a WCO are shown in Fig. 1. The retention time of MDA is about 7.4 min. The plot of the standards against HPLC peak area shows a regression equation of y/(peak area) = 1.26 105x/(concentration of MDA, lg ml1) 6.25 103 with r2 = 0.9998. The recovery test was evaluated by adding known concentration of MDA standards. Both MDA standards and WCO samples were evaluated (Table 1). The recoveries for MDA standards are within the range of 96.5–99.2%, which are similar to those reported by Tsaknis et al. (1998) and most of the derivative HPLC methods (Antunes et al., 2008; Carbonneau et al., 1991; Cordis et al., 1995; Mateos et al., 2005; Mendes et al., 2009; Young and Trimble, 1991). The average recoveries for WCO sample are around 85%, which are much higher than those obtained by the traditional TBA method (Fenaille et al., 2001), and comparable to those obtained using some other HPLC methods (Mendes et al., 2009). So in the following study, the content of MDA was measured by our
0.08
0.12
0.06
Sample
0.08
0.04
Standard
0.02
0.06 Malondialdehyde RT: 7.43 min
0.04
0.00
0.02
-0.02
0.00 0
5
10
15
20
25
30
35
40
45
-0.04 50
Time (min) Fig. 1. Typical chromatograms for MDA standard and WCO sample.
A (AUFS)
A (AUFS)
0.10
As stated, the removal of MDA and other TBARSs from WCO to a certain low content is required before it is used as animal feed additives due to the toxicity of the TBARSs. For example, a maximum permitted TBARSs value of 6 lg g1 is executed by most of Chinese WCO refinery factories. Based on the properties of TBARSs and the cost considerations, three methods, i.e., water extraction, physical adsorption, and chemical adsorption were tried to remove MDA and other TBARSs. 3.2.1. Water extraction method Three content levels of water with/without acid were tried for the removal of MDA in WCO, as shown in Table 2. It can be seen that when using pure water as extractant, the removal efficiencies of MDA and TBARSs increase from 9.8% to 15.6% and from 9.6% to 16.7%, respectively, with the water/WCO ratio increasing from 1:9 to 1:1. Compared with pure water, the acidified water is more effective for the removal of MDA and TBARSs (Table 2). This may be due to the fact that MDA and TBARSs containing carbonyl groups tend to exist in enol forms (Guillen-Sans and Guzman-Chozas, 1998; Nair et al., 1981; Tuma et al., 2001) at acidic atmosphere, which are more hydrophilic and have higher water solubility than their keto forms. A remarkable drawback of the water extraction method is the emulsification of oil occurred during water extraction, which brings a big problem on the separation of water from WCO. From the viewpoint of industry, the emulsification of oil in water means the loss of WCO and the increase in the waste polluted water. So the water extraction method is not suitable for MDA removal from WCO in industry. 3.2.2. Physical adsorption method Four conventional adsorbents, i.e., activated carbon, alumina oxide, activated clay, and HZSM-5 zeolite were used for the removal of MDA from WCO. As shown in Table 3, 1% (w/w) activated carbon without H3PO4 shows the best adsorption behavior for MDA and other TBARS, with about 48% of MDA and 50% of TBARSs being removed from WCO, respectively. For physical adsorption, it is generally known that the nature of the surface functional groups will affect the interaction forces between adsorbate and adsorbent surface and therefore affect the adsorption capacity of an adsorbent (Thacker et al., 1984). Since the target adsorbates MDA and other TBARSs (mainly aldehydes and ketones) are of weak polarity, and the activated carbon, which is the most hydrophobic one among the tested adsorbents, may therefore attract more adsorbates onto its surface by different forces like unelectric force and van de wals force (Al-Degs et al., 2004; Toles et al., 1999). Contrarily, while WCO is acidified with H3PO4 for increasing the polarity of the adsorbents, the adsorption ability of activated carbon on MDA
Table 1 Recoveries for the determination of MDA by HPLC method. Concentration (lg ml1)
Added (lg ml1)
Found (lg ml1)
Recovery (%)
Average recovery (%)
1.31 ± 0.05
1.60 2.00 2.40
1.54 ± 0.01 1.97 ± 0.02 2.38 ± 0.02
96.3 ± 3.2 98.5 ± 2.7 99.2 ± 2.2
98.0 ± 2.7
10.31 ± 0.05b
1.60 2.00 2.40
1.45 ± 0.02 1.57 ± 0.01 1.91 ± 0.01
90.6 ± 3.4 78.5 ± 2.5 79.6 ± 2.1
82.9 ± 2.7
a
a
MDA standard solution. WCO sample. For WCO samples, different amounts of concentrated MDA standards were added respectively to 10 g of purified WCO, followed by HCl extraction procedure (see Section 2.3). b
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Table 2 Results of the removal of MDA and TBARSs from purified WCO sample by water extraction method. Water:WCO (v/v)
MDA (lg g1)
TBARSs (lg g1)
Initial
Final
Efficiency (%)
Initial
Final
Efficiency (%)
1:9a 1:4a 1:1a 1:9b 1:4b 1:1b
1.33 ± 0.05 1.33 ± 0.05 1.33 ± 0.05 1.33 ± 0.05 1.33 ± 0.05 1.33 ± 0.05
1.20 ± 0.03 1.17 ± 0.05 1.12 ± 0.04 1.04 ± 0.05 0.94 ± 0.03 0.89 ± 0.04
9.8 ± 4.1 12.0 ± 5.0 15.8 ± 4.4 21.8 ± 4.8 29.3 ± 3.5 33.1 ± 3.9
11.4 ± 0.2 11.4 ± 0.2 11.4 ± 0.2 11.4 ± 0.2 11.4 ± 0.2 11.4 ± 0.2
10.3 ± 0.1 9.9 ± 0.2 9.5 ± 0.1 8.8 ± 0.1 8.2 ± 0.2 7.8 ± 0.1
9.6 ± 1.8 13.2 ± 2.3 16.7 ± 1.7 22.8 ± 1.6 28.1 ± 2.2 31.6 ± 1.5
Note: Extraction was performed at 30 °C for 30 min under nitrogen atmosphere with continuous magnetic stirring; the amount of WCO sample was 10 g; centrifugation was done after each extraction for oil/water phase separation. a Pure water was used. b Water was adjusted to pH = 2 by H3PO4.
Table 3 Results of the removal of MDA and TBARSs from purified WCO sample by physical adsorption method. MDA (lg g1)
Adsorbents
Activated carbon Activated carbon + 0.1% H3PO4 Alumina oxide Activate clay HZSM-5
Efficiency (%)
Initial
Final
2.59 ± 0.03 2.59 ± 0.03 2.59 ± 0.03 2.59 ± 0.03 2.59 ± 0.03
1.34 ± 0.02 1.84 ± 0.03 2.50 ± 0.01 4.45 ± 0.03 1.76 ± 0.02
48.3 ± 1.0 29.0 ± 1.4 3.50 ± 1.2 71.8 ± 2.3 32.0 ± 1.1
TBARSs (lg g1)
Efficiency (%)
Initial
Final
17 ± 0.5 17 ± 0.5 17 ± 0.5 17 ± 0.5 17 ± 0.5
8.48 ± 0.08 11.89 ± 0.17 16.25 ± 0.15 29.75 ± 0.25 11.90 ± 0.15
50.1 ± 1.5 30.1 ± 2.3 4.40 ± 2.9 75.0 ± 5.4 30.0 ± 2.2
Note: Adsorption was performed at 50 °C for 30 min under nitrogen atmosphere with continuous magnetic stirring; the amount of WCO sample was 10 g; the amount of the adsorbent was 0.1 g.
and other TBARSs decreases by about 40%. Alumina oxide has only slight adsorption capacity for MDA and other TBARSs due to its higher polarity. The second favorable adsorbent is HZSM-5 zeolite, which removes about 32% and 30% of MDA and TBARSs from WCO, respectively. The positive effect of HZSM-5 zeolite on MDA and other TBARSs adsorption may be partly ascribed to the isomerization of aldehydes and ketones (MDA and TBARSs) into more hydrophilic enol forms, which may attract more MDA and other TBARSs onto the surface of HZSM-5 zeolite with hydrophilic nature. While activated clay was used as adsorbent, both the contents of MDA and other TBARSs increase obviously (by 72% and 75%, respectively). It may be due to the fact that the metal impurities like Cu or Fe in clay can accelerate the peroxidation of lipid as catalysts, generating more amounts of MDA and other TBARSs (Guillen-Sans and Guzman-Chozas, 1998; van Breemen et al., 2002). 3.2.3. Chemical adsorption method Based on the fact that the carbonyl group can covalently bond and cross-link a variety of biological macromolecules as well as the above-mentioned derivatization reagents having amino groups (Nair et al., 1981; Tuma et al., 2001) through a Skiff base reaction (Fig. 2), adding amino group-containing substances may be a good idea to remove MDA and other TBARSs from WCO. In this study, three commercially available amino-containing materials, i.e., lysine, monosodium glutamate, and urea, were under investigation. Due to their strong hydrophilicity, the three materials are not soluble but only suspended in WCO. MDA and other TBARSs in WCO
δ+
δ+
R1 C
R2
O δ- H + NH δ-
R3
H+
O H R1 C R2
N R3
H
-H2O
R1 R2
C
N
R3
Schif f Base
Fig. 2. Schiff reaction pathways for aldehydes or ketones. R1, R2 and R3 are organic functional groups.
are then supposed to react with and chemically adsorbed on the surface of the suspended particles, which is regarded as a chemical adsorption process. Results are shown in Table 4. As expected, around 80% of MDA and other TBARSs are effectively removed from WCO by each amino-containing material in the presence of 0.1% H3PO4. Such high removal efficiency is quite satisfactory for industrial use since the maximum permitted TBARSs is 6 lg g1, higher than those of all our acidic adsorbents treated products which contains only less than 2.5 lg g1 of TBARSs. It can be seen from Table 4 that moderate acid seems necessary for the removal of MDA and other TBARSs, as it is found that all of the three adsorbents have only slight reactive with MDA and other TBARSs without the addition of H3PO4. An explanation for the inactivation is that the carbonyl group is more liable to be protonized under weak or middle acidic environment which increases its nucleophilicity. Theoretically, Schiff-base reaction is more liable to occur under alkaline condition as the proton is released from the amine ion which increases the effective concentration of amino (Ge and Lee, 1997). However, it is not feasible in our case since the alkaline condition will lead to the saponification of lipids. 3.3. Discussion Compared with the widely used derivative HPLC method (Bergamo et al., 1998; Grotto et al., 2007; Mendes et al., 2009; Rovellini, 2006), direct measurement of MDA through UV detector is only limited to 1 or 2 reports (Karatas et al., 2002; Tsaknis et al., 1998), and has not been widely adopted yet. Three reasons may contribute to this fact. Firstly, MDA exists in aqueous phase as a mixture of ketonic and enolic tautomers, which have different functional groups and molecular polarity. As a result, the direct determination of MDA by UV detector in HPLC may appear two peaks corresponding to the two tautomers, which may bring difficulty in the quantification of MDA; whereas in the derivation method, MDA is fully transformed to one stable adduct. Secondly, the maximum response signal of MDA is much less than that of MDA derivatives at the same concentration, which may decrease
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Z. Wei et al. / Journal of Food Engineering 107 (2011) 379–384 Table 4 Results of the removal of MDA and TBARSs from purified WCO sample by the chemical adsorption method. Adsorbents
MDA (lg g1) Initial
Final
Lysine + H3PO4 Monosodium glutamate + H3PO4 Urea + H3PO4 Lysine Monosodium glutamate Urea
1.33 ± 0.05 1.33 ± 0.05 1.33 ± 0.05 1.33 ± 0.05 1.33 ± 0.05 1.33 ± 0.05
0.27 ± 0.03 0.26 ± 0.04 0.30 ± 0.05 1.27 ± 0.05 1.26 ± 0.03 1.26 ± 0.05
Efficiency (%)
TBARS (lg g1) Initial
Final
79.7 ± 2.4 80.5 ± 3.1 77.4 ± 3.9 4.5 ± 5.2 5.3 ± 4.2 5.3 ± 5.2
11.4 ± 0.2 11.4 ± 0.2 11.4 ± 0.2 11.4 ± 0.2 11.4 ± 0.2 11.4 ± 0.2
2.35 ± 0.2 2.09 ± 0.1 2.15 ± 0.2 10.9 ± 0.1 10.7 ± 0.2 11.0 ± 0.2
Efficiency (%)
79.4 ± 1.8 81.7 ± 0.9 81.1 ± 1.8 4.4 ± 1.9 6.1 ± 2.4 3.5 ± 2.4
Note: Extraction was performed at 50 °C for 30 min under nitrogen atmosphere with continuous magnetic stirring; the amount of WCO sample was 10 g; the amount of amino acid was 0.01 g, while urea was 0.1 g.
the analysis sensitivity of the direct detection method. Finally, the impurities in the sample usually disturb the chromatogram in the direct HPLC method; while in the derivative method, MDA is specifically derivated and has much higher response signal than that of impurities. In our method, the isomerization of MDA is inhibited by adjusting the pH of the sample to 3, at which most of MDA exists as the style of enolic tautomer, and the separation of MDA from other impurities is accomplished by the optimization of the mobile phase components and the flow rate. The only drawback of our method is the lower detection limit. But to our routine use for MDA measurement in WCO samples, the content of MDA is always rather high. We therefore regard that our HPLC method has enough accuracy for MDA measurement in WCO samples, as well as the advantage of simplicity and convenience. A comparative evaluation for the measurement of MDA in WCO and supermarket-available EO was made between the traditional spectrophotometric procedure and our HPLC method (Fig. 3). As expected, the measured MDA values between the two methods for both WCO and EO samples are quite different. The discrepancy is also frequently appeared between the traditional spectrophotometric procedure and other HPLC or GC methods. The high MDA value by traditional spectrophotometric procedure (TBA method) was generally explained by artefactual formation of MDA during the acid heating step required for the TBA derivatization at 90 °C (de las Heras et al., 2003; Mendes et al., 2009). However, such discrepancy reported being as high as 6–30 folds (Behrens and Madere, 1991; Fenaille et al., 2001; Karatas et al., 2002; Tsaknis et al., 1999, 1998; Yeo et al., 1994) could not be only ascribed to the ‘online’ formation of MDA. Since the HPLC method can very
precisely discriminate MDA, while TBA is quite unspecific for MDA, which is reasonable to deduce that ca 16.7–3.3% (corresponding to 6 and 30 folds of the discrepancy, respectively) of TBA reactive peroxidation products is MDA. That is, the discrepancy of the two methods mainly derives from MDA content in the peroxidation products rather than the ‘online’ formation of MDA. Different source of the samples has different MDA content, leading to different discrepancy folds between the two methods, which is 9.78 (slop in Fig 3) for WCO sample and 6.49 for EO sample in our experiment. In WCO industry, the TBARSs content in purified WCO is frequently in excess of 10 lg g1, which is much higher than Chinese national standard limitation of 6 lg g1 for animal feed additives. As stated above, both physical and chemical adsorbents are capable of removing considerable MDA or other TBARSs from WCO to meet such standard. However, taking into account of the processing costs and the environmental impact, the commercially available amino acids (or salt) such as lysine and monosodium glutamate are the best choice, based on the following three facts: (1) the prices of lysine and monosodium glutamate are of the same order as the prices of activated carbon and HZSM-5 zeolite, while their dosages are only 10 percent of the latter (0.1% (w/w) vs. 1% (w/w)) comparing with the removal efficiency of about 80% (see Table 4) and 50% (see Table 3), respectively; (2) when amino acids are separated from WCO after adsorbing MDA and other TBARSs, they can be reused as organic fertilizers, while the adsorbents like activated carbon or HZSM-5 zeolite can only be discarded as wastes; (3) of all the absorbents, urea is found to be the cheapest and the highest efficient for the removal of MDA and TBARSs. However, the toxicity at the concentration level of its solubility in WCO is not clear yet, which limits the application of urea.
30
25
MDA by TBA (μg·ml-1)
4. Conclusion
y = 6.49x R² = 0.97
In summary, a simple and reliable HPLC method is developed to measure the MDA content in purified WCO, which is expected to be more convenient, compared with the derivative HPLC methods as well as the steam distillation HPLC method. Three methods were used to remove MDA and other TBARSs in purified WCO. Among them, the chemical adsorption method using lysine and monosodium glutamate as adsorbents is the best choice.
20
15
10
References y = 9.78x R² = 0.93
5
0
0
1
2
3
4
5
MDA by HPLC (μg·ml-1) Fig. 3. MDA levels measured by HPLC method and spectrophotometric TBA method. 4 for WCO and s for EO.
Al-Degs, Y.S., El-Barghouthi, M.I., Khraisheh, M.A., Ahmad, M.N., Allen, S.J., 2004. Effect of surface area, micropores, secondary micropores, and mesopores volumes of activated carbons on reactive dyes adsorption from solution. Separation Science and Technology 39 (1), 97–111. Almandos, M.E., Giannini, D.H., Ciarlo, A.S., Boeri, R.L., 1986. Formaldehyde as an interference of the 2-thiobarbituric acid test. Journal of the Science of Food and Agriculture 37 (1), 54–58. Antunes, M.V., Lazzaretti, C., Gamaro, G.D., Linden, R., 2008. Preanalytical and validation studies for the determination of malondialdehyde in human plasma through high performance liquid chromatography after derivatization with 2,4-
384
Z. Wei et al. / Journal of Food Engineering 107 (2011) 379–384
dinitrophenylhydrazine. Revista Brasileira De Ciencias Farmaceuticas 44 (2), 279–287. Awawdeh, M.S., Obeidat, B.S., Kridli, R.T., 2009. Yellow grease as an alternative energy source for nursing Awassi ewes and their suckling lambs. Animal Feed Science and Technology 152 (3–4), 165–174. Bailey, A.E., 2005. Bailey’s Industrial Oil and Fat Products. John Wiley & Sons, New York. Behrens, W.A., Madere, R., 1991. Malonaldehyde determination in tissues and biological-fluids by ion-pairing high-performance liquid-chromatography. Lipids 26 (3), 232–236. Bergamo, P., Fedele, E., Balestrieri, M., Abrescia, P., Ferrara, L., 1998. Measurement of malondialdehyde levels in food by high-performance liquid chromatography with fluorometric detection. Journal of Agricultural and Food Chemistry 46 (6), 2171–2176. Buttkus, H., Bose, R.J., 1972. Amine-malonaldehyde condensation products and their relative color contribution in thiobarbituric acid test. Journal of the American Oil Chemists Society 49 (7), 440–443. Carbonneau, M.A., Peuchant, E., Sess, D., Canioni, P., Clerc, M., 1991. Free and bound malondialdehyde measured as thiobarbituric acid adduct by hplc in serum and plasma. Clinical Chemistry 37 (8), 1423–1429. Cheng, G.W., Wu, H.L., Huang, Y.L., 2008. Simultaneous determination of malondialdehyde and ofloxacin in plasma using an isocratic highperformance liquid chromatography/fluorescence detection system. Analytica Chimica Acta 616 (2), 230–234. Cordis, G.A., Maulik, N., Das, D.K., 1995. Detection of oxidative stress in heart by estimating the dinitrophenylhydrazine derivative of malonaldehyde. Journal of Molecular and Cellular Cardiology 27 (8), 1645–1653. de las Heras, A., Schoch, A., Gibis, M., Fischer, A., 2003. Comparison of methods for determining malondialdehyde in dry sausage by HPLC and the classic TBA test. European Food Research and Technology 217 (2), 180–184. Dudrow, F.A., 1983. Deodorization of edible oil. Journal of the American Oil Chemists Society 60 (2), 272–274. Fenaille, F., Mottier, P., Turesky, R.J., Ali, S., Guy, P.A., 2001. Comparison of analytical techniques to quantify malondialdehyde in milk powders. Journal of Chromatography A 921 (2), 237–245. Gatlin, L.A., See, M.T., Larick, D.K., Lin, X., Odle, J., 2002. Conjugated linoleic acid in combination with supplemental dietary fat alters pork fat quality. Journal of Nutrition 132 (10), 3105–3112. Gavin, A.M., 1978. Edible oil deodorization. Journal of the American Oil Chemists Society 55 (11), 783–791. Ge, S.-J., Lee, T.-C., 1997. Kinetic significance of the schiff base reversion in the earlystage Maillard reaction of a phenylalanine–glucose aqueous model system. Journal of Agricultural and Food Chemistry 45 (5), 1619–1623. Grotto, D., Santa Maria, L.D., Boeira, S., Valentini, J., Charao, M.E., Moro, A.M., Nascimento, P.C., Pomblum, V.J., Garcia, S.C., 2007. Rapid quantification of malondialdehyde in plasma by high performance liquid chromatographyvisible detection. Journal of Pharmaceutical and Biomedical Analysis 43 (2), 619–624. Guillen-Sans, R., Guzman-Chozas, M., 1998. The thiobarbituric acid (TBA) reaction in foods: a review. Critical Reviews in Food Science and Nutrition 38 (4), 315– 330. Karatas, F., Karatepe, M., Baysar, A., 2002. Determination of free malondialdehyde in human serum by high-performance liquid chromatography. Analytical Biochemistry 311 (1), 76–79. Knight, J.A., Pieper, R.K., Mcclellan, L., 1988. Specificity of the thiobarbituric acid reaction – its use in studies of lipid-peroxidation. Clinical Chemistry 34 (12), 2433–2438. Kosugi, H., Kato, T., Kikugawa, K., 1988. Formation of red pigment by a 2-step 2thiobarbituric acid reaction of alka-2,4-dienals – potential products of lipid oxidation. Lipids 23 (11), 1024–1031. 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 oxidative stress – application to a rat model for hypercholesterolemia and evaluation of the effect of diets rich in phenolic antioxidants from fruits. Journal of Chromatography B – Analytical Technologies in the Biomedical and Life Sciences 827 (1), 76–82. Mendes, R., Cardoso, C., Pestana, C., 2009. Measurement of malondialdehyde in fish: a comparison study between hplc methods and the traditional spectrophotometric test. Food Chemistry 112 (4), 1038–1045. Mosca, M., Ceglie, A., Ambrosone, L., 2010. Lipid oxidation in water-in-olive oil emulsions initiated by a lipophilic radical source. Journal of Physical Chemistry B 114 (10), 3550–3558. Nair, V., Vietti, D.E., Cooper, C.S., 1981. Degenerative chemistry of malondialdehyde. Structure, stereochemistry, and kinetics of formation of enaminals from reaction with amino-acids. Journal of the American Chemical Society 103 (11), 3030–3036. Pikul, J., Leszczynski, D.E., Kummerow, F.A., 1989. Evaluation of 3 modified tba methods for measuring lipid oxidation in chicken meat. Journal of Agricultural and Food Chemistry 37 (5), 1309–1313. Pilz, J., Meineke, I., Gleiter, C.H., 2000. Measurement of free and bound malondialdehyde in plasma by high-performance liquid chromatography as the 2,4-dinitrophenylhydrazine derivative. Journal of Chromatography B – Analytical Technologies in the Biomedical and Life Sciences 742 (2), 315–325. Rovellini, P., 2006. Malonaldehyde contents in vegetable oils and chips Liquid chromatography-apci-mass spectrometry method. Rivista Italiana Delle Sostanze Grasse 83 (5), 213–220. Shamberger, R.J., Shamberger, B.A., Willis, C.E., 1977. Malonaldehyde content of food. Journal of Nutrition 107 (8), 1404–1409. Squires, E.J., 1990. High-performance liquid-chromatographic analysis of the malondialdehyde content of chicken liver. Poultry Science 69 (8), 1371–1376. Tarladgis, B.G., Watts, B.M., Younathan, M.T., Dugan, L., 1960. A distillation method for the quantitative determination of malonaldehyde in rancid foods. Journal of the American Oil Chemists Society 37 (1), 44–48. Templar, J., Kon, S.P., Milligan, T.P., Newman, D.J., Raftery, M.J., 1999. Increased plasma malondialdehyde levels in glomerular disease as determined by a fully validated hplc method. Nephrology Dialysis Transplantation 14 (4), 946–951. Thacker, W.E., Crittenden, J.C., Snoeyink, V.L., 1984. Modeling of adsorber performance – variable influent concentration and comparison of adsorbents. Journal Water Pollution Control Federation 56 (3), 243–250. Toles, C.A., Marshall, W.E., Johns, M.M., 1999. Surface functional groups on acidactivated nutshell carbons. Carbon 37 (8), 1207–1214. Torun, M., Yardim, S., Gonenc, A., Sargin, H., Menevse, A., Simsek, B., 1995. Serum beta-carotene, vitamin-E, vitamin-C and malondialdehyde levels in several types of cancer. Journal of Clinical Pharmacy and Therapeutics 20 (5), 259–263. Tsaknis, J., Lalas, S., Hole, M., Smith, G., Tychopoulos, V., 1998. Rapid highperformance liquid chromatographic method of determining malondialdehyde for evaluation of rancidity in edible oils. Analyst 123 (2), 325–327. Tsaknis, J., Lalas, S., Evmorfopoulos, E., 1999. Determination of malondialdehyde in traditional fish products by HPLC. Analyst 124 (6), 843–845. Tuma, D.J., Kearley, M.L., Thiele, G.M., Worrall, S., Haver, A., Klassen, L.W., Sorrell, M.F., 2001. Elucidation of reaction scheme describing malondialdehyde– acetaldehyde–protein adduct formation. Chemical Research in Toxicology 14 (7), 822–832. van Breemen, R.B., Xu, X.Y., Viana, M.A., Chen, L.W., Stacewicz-Sapuntzakis, M., Duncan, C., Bowen, P.E., Sharifi, R., 2002. Liquid chromatography-mass spectrometry of cis- and all-trans-lycopene in human serum and prostate tissue after dietary supplementation with tomato sauce. Journal of Agricultural and Food Chemistry 50 (8), 2214–2219. Yeo, H.C., Helbock, H.J., Chyu, D.W., Ames, B.N., 1994. Assay of malondialdehyde in biological-fluids by gas-chromatography mass-spectrometry. Analytical Biochemistry 220 (2), 391–396. Young, I.S., Trimble, E.R., 1991. Measurement of malondialdehyde in plasma by high-performance liquid-chromatography with fluorometric detection. Annals of Clinical Biochemistry 28, 504–508.