Antioxidant Measurement and Applications - American Chemical Society

Chapter 10

Downloaded by NORTH CAROLINA STATE UNIV on May 6, 2015 | http://pubs.acs.org Publication Date: March 12, 2007 | doi: 10.1021/bk-2007-0956.ch010

Method Development for Monitoring Seal Blubber Oil Oxidation Based on Propanal and Malondialdehyde Formation 1

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Chia-Pei Liang , Mingfu Wang , James E. Simon , Fereidoon Shahidi , and Chi-Tang Ho 4

1

1

Department of Food Science, Rutgers, The State University of New Jersey, New Brunswick, NJ 08901 Department of Botany, The University of Hong Kong, Pokfulam Road, Hong Kong Center for New Use Agriculture and Natural Plant Products, Department of Plant Biology and Pathology, Rutgers, The State University of New Jersey, New Brunswick, NJ 08901 Department of Biochemistry, Memorial University of Newfoundland, St. John's, Newfoundland A1B 3X9, Canada 2

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4

Omega-3 polyunsaturated fatty acids (PUFA) in fish oils are extremely susceptible to oxidation and rapid degradation. Many studies have shown the health benefits of omega-3 fatty acids for human beings thus lead to the effort in improving the stability of these oils. Propanal, which is specific formed from the oxidation of omega-3 P U F A , could be used as an indicator of the extent of seal blubber oxidation. Solid-phase microextraction (SPME) is a solvent-free volatile extraction method and has been widely used in volatile analysis. However, short-chain aldehydes like propanal do not bind to the fiber. We have developed a rapid method to determine the extent of lipid oxidation of seal blubber oil based on propanal and malondialdehyde formation. 0-(2,3,4,5,6-Pentafluorobenzyl) hydroxylamine hydrochloride (PFBHA) was used as derivatization agent to ensure the binding of short chain

© 2007 American Chemical Society In Antioxidant Measurement and Applications; Shahidi, F., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2007.

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aldehydes to S P M E fiber. Comparing with other traditional methods for measuring lipid oxidation, our method is convenient and reduces the amount of toxic solvent used. It targets the major end products produced by polyunsaturated omega-3 fatty acids oxidation, propanal and malondialdehyde.

Seal blubber oil is a good source of co-3 fatty acids; it contains approximately 20% of eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA) and docosahexaenoic acid (DHA) (7) (Table I). The source of long chain polyunsaturated fatty acids in marine oil is from microalgae (2). After consumed by the marine animals, those fatty acids, mainly E P A and D H A are accumulated in the food chain. Omega-3 fatty acids have aroused many interests because of its benefits on human health. The health benefits of omega-3 fatty acids have been thoroughly reviewed by many researchers (3-6) on cardiovascular disease (7-9), infant development (70,77), inflammatory and autoimmune disorders, neuronal and visual function.

Table 1: Major Fatty Acids from Seal Blubber Oil (7). Fatty Acid 14:0 14:1 16:0 16:1 18:1ω9

Weight % 3.7 1.1 6.0 18.0 20.8

Fatty Acid 18:2a>6 20:1ω9 20:5ω3 22:l(oll 22:5ω3

Weight % 1.5 12.2 6.4 2.0 4.7

18:1ω11

5.2

22:6(03

7.6

Omega-3 fatty acids are very susceptible to oxidation and generate an unpleasant odor due to their chemical structures. There are several commonly used indexes for monitoring lipid oxidation, such as measuring the conjugated dienes (72), peroxide value (75, 14), the oil stability index analysis method (OSI, A O C S official method Cd 12b-92) (16-17), gas chromatography method (13, 18), and 2-thiobarbituric acid (TBA) method. Malondialdehyde is usually found in the oxidation products from P U F A containing 3 or more double bonds. The T B A method has been widely used to quantify the malondialdehyde because its simplicity, however, not only

In Antioxidant Measurement and Applications; Shahidi, F., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2007.


127 malondialdehyde could react with thiobarbituric acid to form the chromogen, there are many other compounds, called thiobarbituric acid reactive substances (TBAR) that can react with thiobarbituric acid. The concept of T B A method is shown in Figure 1 ; thiobarbituric acid can react with the malondialdehyde in the oil sample and form the colored T B A chromagen (maximum U V absorbance at 532 nm). The methodology of this method has been reviewed thoroughly (7927). Nonetheless, there are many drawbacks of T B A method, such as the use of toxic materials, 2-thiobarbituric acid, the reaction need to be conducted in hazardous solvent (1-butanol), time consuming procedures, and possible interference with many other aldehydes (22). Malondialdehyde can also be quantified by H P L C or G C analysis; however, further derivatization is required to generate the stable malondialdehyde derivatives (20). Solid phase microextraction (SPME) is sample extraction technique, and it extracts the analytes without solvent. S P M E was first invented by Arthur and

(A) .SH

OH

OH

Maloncfialdhyde

TBA

(B)

OH

TBA chromagen

ο

OH

OH

Ο

Figure 1. (A) The formation pathway of TBA chromogen proposed by Sinnhuber et al. (23). (Β) Two tautomeric TBA chromogen proposed by Nair and Turner (modifiedfrom 24).


128 Pawliszyn in 1990 (25), after it became commercially available from Supelco in 1993; this technique was widely used because of its convenience, sensitivity, and omission of solvent. We applied this sample extraction technique to monitor the major oxidation end product from ω-3 fatty acids. The formation pathway of propanal and malondialdehyde is shown in Figures 2 and 3.


ο

Figure 2. Formation ofpropanal from ω-3 eicosapentaenoic acid (EPA) oxidation.

The challenge in using S P M E to sample propanal is that the binding affinity of short chain aldehydes to S P M E fiber is low, and the derivatization reagent is required to ensure the binding of propanal to fiber. 0-(2,3,4,5,6Pentafluorobenzyl) hydroxylamine hydrochloride (PFBHA) is a derivatization reagent widely used for the analysis of carbonyl-containing compounds in variety of environmental and biological studies. It was first synthesized in 1975 for the analysis of keto steroids (26). Marios and Pawliszyn (27) have developed the on-fiber derivatization method to monitor formaldehyde in the environment using P F B H A as derivatization reagent. The theory of on-fiber derivatization is shown in Figure 4. The aromatic group on P F B H A has good binding affinity to the fiber while the hydroxylamine group can freely react with the approaching carbonyl group and form oximes on the fiber. Our objective was to develop a rapid method to monitor seal blubber oil oxidation based on propanal and malondialdehyde formation using on-fiber derivatization SPME.



129

Malondialdehyde

Figure 3. The conversion of methyl linolenate to malondialdehyde

F

F

PFBHA

Carbonyl group

PFBHA carbonyl oxime isomers

Figure 4. Reaction mechanism of PFBHA with carbonyl compounds (modifiedfrom 27).


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Materials and Methods Propanal, corn oil, 0-(2,3,4,5,6-pentafluorobenzyl)hydroxylamine hydrochloride (PFBHA), ether and 1,1,3,3-tetramethoxypropane were purchased from Sigma-Aldrich (St. Louis, MO). Malondialdehyde tetrabutylammonium salt was purchased from Fluka (Milwaukee, WI). Poly (dimethylsiloxane)/ divinylbenzene solid-phase microextraction (SPME) fiber, fiber holder, and crimp top amber glass vials (10 mL) were all purchased from Supelco (Bellefonte, PA). Crude seal blubber oil was rendered and subsequently subjected to refining, bleaching and deodorization.

Sample Preparation The oil (100 mL) was added into separatory funnel, and free fatty acids were then removed by mixing oil with suitable amount of sodium hydroxide solution. Rinse the oil with 80 mL χ 3 distilled water. Dried the oil layer with anhydrous sodium sulfate. Seal blubber oil was further purified by passing through silica gel column. Vacuum was applied to accelerate the oil collecting process. The purified oil was transferred in an amber glass bottle, flushed the headspace with nitrogen, and stored the oil at -21 °C for further analysis.

Fish O i l Aging Study Aging studies were conducted at 60 °C. Five milliliters purified seal oil were put into a 10 mL crimp top amber glass vial with a T F E starburst stirring head (diameter 9.5 mm). Sampling of propanal and malondialdehyde was conducted every 24 hours.

Loading S P M E Fiber with P F B H A One milliliter of P F B H A solution (17 mg/mL) was put into 4 mL amber Teflon-capped vials with a 1-cm stir bar. S P M E fiber was inserted to the headspace of vial for 2 min to adsorb the volatile P F B H A , the solution was stirred at 600 rpm. The fiber was then inserted to the headspace of seal blubber oil containing vial. After 5 min, the fiber was removed and inserted to G C .

G C Conditions An Agilent 6850 Gas Chromatography equipped with a flame ionization detector and a HP-1 methylsiloxane column (30.0 m χ 250 μπι χ 0.25 μπι) was


131 used. The inlet port and the detector were kept at 250 and 300 °C, respectively. The gas flow was controlled as follows: hydrogen flow at 30.0 mL/min and air flow at 300 mL/min. The oven temperature was hold at 45 °C for 1 min then increased to 200 °C at 10 °C/min and held at 200 °C for 8.5 min.


GC-MS Analysis of PFBHA Oximes A n Agilent 6890 Gas Chromatograph equipped with a Agilent 5973 Mass detector and HP-5 M S 5% phenylmethyl siloxane column (30.0 m χ 250 μπι χ 0.25 μπι) was used. The injection port was kept at 250 °C. The gas flow was controlled as follows: hydrogen flow at 30.0 mL/min and air flow at 300 ml/min. The oven temperature was hold at 45 °C for lmin then increased to 200 °C at 10 °C/min and held at 200 °C for 8.5 min. The mass detector was operated at the electronic ionization mode. The ionization voltage was held at 70 eV and ion temperature was at 280 °C.

Quantification of Propanal Corn oil was used as a propanal free medium in our study. Different concentrations of propanal were added to 5 mL of corn oil and the S P M E sampling procedure remained the same as describing above. The calibration curve was used to quantify the propanal concentration in seal blubber oil headspace.

Quantification of Malondialdehyde in Seal Blubber Oil Malondialdehyde solution (10 mM) was prepared by dissolving Malondialdehyde tetrabutylammonium salt in 0.1 M pH 2.5 citrate buffer. 15 mL corn oil were used to extract the malondialdehyde from 15 mL 10 m M malondialdehyde solution. In order to test the efficiency for extraction of malondialdehyde from citrate buffer to corn oil, the standard curve was conducted to determine the amount of malondialdehyde remaining in the citrate buffer after oil extraction. Malondialdehyde solution (5 mL) (4, 6, 8, and 10 mM) was mix with 5 mL 30 m M P F B H A , extracted with ether (10 mL χ 3) and then subjected for the G C analysis. Octanol was added as internal standard. The malondialdehyde containing oil was then used for conducting external standard curve in order to quantify the malondialdehyde existed in the seal oil blubber oil sample.


132 Synthesis of Malondialdehyde Oximes


Tetrametoxypropane (160 μΐ) was added into 5 mL 1M HC1 and heated at 95°C water bath for 3 min. The solution was slowly added into 5 mL P F B H A solution (34 mg/mL). The mixture was then shaken vigorously and put into ice bath. After centrifugation for 10 min, the precipitates were collected for L C - M S analysis.

L C - M S Analysis of Malondialdehyde Oximes Analytical H P L C analyses were performed on a Hewlett-Pachard 1100 modular system equipped with an auto-sampler, a quaternary pump system, a photodiode array detector, and a HP Chemstation data system. A Luna C18 (2) analytical column (Phenomenex), 250 χ 4.60 mm (5 μ particle size, 00G-4252E0) was used. The mobile phase consists of 70% acetonitrile and 30% of 1% formic acid in water. The analyses were conducted at isocratic condition. Negative and positive ESI-mass spectra were measured on Agilent 1100 L C M S D system (Agilent Technologies, Wilmington, DE) equipped with an electrospray source, Bruker Daltonics 4.0, and Data analysis 4.0 software. A l l the organic solvents are H P L C grade and obtained from Fisher Scientific Inc.

Results and Discussion S P M E Sampling A typical gas chromatogram of seal blubber oil incubated at 60 °C for 6 days is shown in Figure 5. Five aldehydes were identified based on the comparison of retention time and G C - M S spectrum with authentic compounds. Since there is no commercially available malondialdehyde, it was prepared by the hydrolysis of 1,1,3,3-tetrametoxypropane. Un-reacted P F B H A (peak 1) was also thermally desorbed from S P M E fiber and the retention time is 8.7 min. Propyl-oxime isomers have retention time at 10.1 and 10.2 min, respectively. Propenyl-oxime isomers have retention time at 10.1 and 10.3 mins, which overlap with the propyl-oxime at 10.1 min; therefore, the quantification of propanal was based on the peak area at 10.2 min. Malondialdehyde has two carbonyl groups which can react with 2 molecule of P F B H A and formed 4 isomers (E, E-, E, Z-, Z, Z-, and Ζ, E-). The malondialdehyde oximes have retention time at 21.2 and 22.8 min in the ratio of 1:3.


133

t;n-


on

Figure 5. The representative gas chromatogram of seal blubber oil stored at 60°C for 6 days. Peaks are identified based on the comparison of retention time and MS spectrum with authentic compounds. 1. PFBHA, 2&3: acetaldehyde, 4: co-eluent of propanal andpropenal, 5: propanal, 6: propenal, 7&8: malonadialdehyde

L C - M S Analysis of Malondialdehyde-PFBHA Adduct Malondialdehyde has two carbonyl group which can react with P F B H A to form oximes. Three major peaks were identified as malondialdehyde-PFBHA and PFBHA-malondialdehyde-PFBHA isomers with the molecular weight of 267 and 462 Da, respectively (Figure 6).

Quantification of Propanal The external standard curve of propanal is shown in Figure 7A, and the linear regression is observed (R = 0.99). Propanal concentrations in the headspace of seal blubber oil containing vials were quantified by the external standard curve. Rapid increase in propanal concentration was observed after 24 hours. After 6 days incubation at 60 °C, the propanal concentration reached about 1.6 ppm (Figure 7B). We've attempted to investigate the possibility of adding known amounts of different aldehydes into oil sample as internal standard, however, the competition of P F B H A between propanal and the additional aldehydes could lead to the misinterpretation of the data. (Data not shown). Thus, the external standard curve could give us a better quantification of propanal existed in the headspace of oil sample. 2


134


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Figure 6. (A) LC chromatogram of malondialdehyde oximes. Peak 1: MAPFBHA, peak 2 &3: PFBHA -MA -PFBHA. (B) LC-MS positive spectrum of MAPFBHA (C) LC-MS positive spectrum ofPFBHA-MA-PFBHA.



135

Figure 7. (A) Standard curve for propanal quantification. (B) Propanal formation of seal blubber oil incubated at 60 °C.



136

Figure 8. (A) Standard curve for determination of corn oil extraction efficiency. (B) Standard curve for the quantification of malondialdehyde.


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Quantification of Malondialdehyde In order to generated the external standard curve to quantify the malondialdehyde in seal blubber oil, different amount of malondialdehyde need to be added into corn oil. However, malondialdehyde is a very unstable compound, it's not commercially available and usually generated from acid hydrolysis of its stable derivatives tetramethoxylpropane (TMP) (28,29), tetraethoxylpropane (TEP) (30-32) or dissolving tetrabutylammonium salt in water under acidic condition (33). Water is a polar solvent that would affect binding affinity of aldehydes to P F B H A . Since malondialdehydes generated by the methods described above were dissolved in water solution; further extraction step was needed to transfer standard malondialdehyde into corn oil. The standard curve to quantify the efficiency for oil extraction is shown in Figure 8A. Malondialdehyde is a hydrophilic compound; only 2.34 out of 50 μπιοίε of malondialdehyde was extracted into 15 mL corn oil. Figure 8B shows the external standard curve to quantify the malondialdehyde in seal blubber oil. Malondialdehyde formed rapidly after 1 day incubation at 60 °C and reached about 4 ppm in seal blubber oil headspace (Figure 9).

time (day)

Figure 9. Formation of malondialdehyde from seal blubber oil oxidation.


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Conclusions A rapid method to monitor the oxidation of oil which rich in omega-3 fatty acids has been developed. It's very sensitive with the detection limit at ppb level. Comparing to the T B A method, this method is based on the measurement of major end products from omega-3 P U F A oxidation without the interference of T B A R S generated during heating step with T B A method. It also saves time and substantially reduces the quantity of organic solvent used. Because of its high sensitivity, this method may be used to determine the effectiveness of antioxidants on lipid oxidation in food or other complex biological systems.

References 1.

2.

3. 4. 5.

6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17.

Shahidi, F.; Wanasundara, U . N . In Omega-3 Fatty Acids: Chemistry, Nutrition, and Health Effects; Shahidi, F.; Finley, J.W., Eds.; A C S Symp. Ser. 788, American Chemical Society: Washington, DC, 2001; pp. 143-150. Zeller, S.; Barclay, W.; Abril R. In Omega-3 Fatty Acids: Chemistry, Nutrition, and Health Effects. Shahidi, F.; Finley, J.W., Eds.; A C S Symp. Ser. 788, American Chemical Society: Washington, D C , 2001; pp. 108-124. Connor, W. E. Am. J. Clin. Nutri. 2000, 71 (Suppl.), 171S-175S. Uauy-Dagach, R.; Valenzuela, A . Nutr. Rev. 1996, 54, S102-S108. Newton, I. S. In Omega-3 Fatty Acids: Chemistry, Nutrition, and Health Effects. Shahidi, F.; Finley, J.W., Eds.; A C S Symp. Ser. 788, American Chemical Society: Washington, DC, 2001; pp. 14-27. Ruxton, C. Nursing Standard 2004,18, 38-42. Appel, L . J. American Family Physician 2004, 70(1), 34-25. Hu, F.B.; Bronner, L . ; Willett, W.C.; Stampfer, M . J . ; Rexrode, K . M . ; Albert, C.; Ismail, H . M . Frontiers in Bioscience 2005, 10(2), 1079-1088. Hunter, D.; Manson, J.E. J. Ameri. Med. Assoc. 2002, 287(14), 1815-1821. Helland, I.B; Smith, L . ; Saarem, K . ; Saugstad, O.D.; Drevon, C.A. Pediatrics 2003, 111(1), 39-44. Simopoulos, A. Am. J. Clin. Nutri. 1991, 54, 438-463. Hopia, A.I.; Huang, S-W.; Schwarz, K . ; German, J.B.; Frankel, E . N . J. Agric. Food Chem. 1996, 4, 2030-2036. Frankel, E.N. JAOCS, 1993, 70, 767-772 Nieto, S.; Garrido, Α.; Sanhueza, J.; Loyola, L . A . ; Morales, G.; Leighton, F.; Valenzuela, A . JAOCS, 1993, 70, 773-778. Bastos De Maria, C.A.; Moreira Santos, M . C . ; Lima Dias, U.J.D.; Marana, M . J. Agric. Food Chem. 2000, 48, 3935-3938. Nakatani, N.; Tachibana, Y . ; Kikuzaki, H . JAOCS, 2001, 78, 19-23 Silva, F . A . M . ; Borges, F.; Ferreira, M . A . J. Agric. Food Chem. 2001, 49, 3936-3941.


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18. 19. 20. 21.

P.A.

Rørbáek, K.; Jensen, B. JAOCS, 1997, 74, 1607-1609. Hoyland, D.V.; Taylor, A.J. Food Chem. 1991, 40, 271-291. Raharjo, S.; Sofos, J.N. Meat Sci. 1993, 35, 145-169. Guillén-Sans, R.; Guzmán-Chozas, M. Crit. Rev. Food Sci. Nutr. 1998, 38, 315-330. 22. Careche, M.; Tejada, M. J. Sci. Food Agric. 1988, 43, 49-57. 23. Sinnhuber, R.O.; Y u , T.C. Food Res. 1958, 23, 626-634. 24. Nair, V.; Turner, G.A. Lipids, 1984, 19, 804-805. 25. Arthur, C.L.; Pawliszyn, J.; Anal.Chem. 1990, 62, 2145-2148. 26. Cancilla, D.A.; Que Hee, S.S. J. Chromatogr. 1992, 627, 1-16. 27. Martos, P.A.; Pawliszyn, J. Anal. Chem 1998, 70, 2311-2320. 28. Crackel, R.L.; Gray, J.I.; Pearson, A.M.; Booren, A.M.; Buckley, D.J. Food Chem. 1988, 28, 187-196. 29. Tsaknis, J.; Lalas, S.; Evmorfopoulos, E. Analyst 1999, 124, 843-845. 30. Gutteridge, J.M.C. Anal. Biochem. 1975, 69, 518-526. 31. Marnett, L.J.; Bienkowski, M.J.; Raban, M.; Turtle, M.A. Anal. Biochem. 1979, 99, 458-463. 32. Stalikas, C.D.; Konidari, C.N. Anal. Biochem. 2001, 290, 108-115. 33. Sangalli, L.; Chiesa, L.M.; Passerò E.; Manzocchi, A.; Maffeo, G.; Biondi, J. Chromatogr. B. 2003, 796, 201-207.


Antioxidant Measurement and Applications - American Chemical Society

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