Determination Of Hydroxy-Octadecadienoic Acids(HODEs) In Cured Meat Products And Their Variations During Processing

In this paper,a HPLC-MS/MS method for determination of hydroxy-linoleic acids in cured meat products was developed.The contents of hydroxyoctadecadienoic acids during sausages making were monitored,and the contributions of enzymatic and non-enzymatic oxidation to lipid oxidation were analyzed according to the variations of hydroxyoctadecadienoic acids.Finally,effects of pH,ionic strength and temperature on the formations of hydroxyoctadecadienoic acids in linoleic acids oxidation systems were studied.1.Simultaneous Determination of Hydroxy-Linoleic Acids in Cured Meat Products by LC-MS/MS13-hydroxy-9,11-octadecadienoic acid(13-HODE),9-hydroxy-10,12-octadecadienoic acid(9-HODE),9,10-dihydroxy-12-octadecenoic acid(9,10-DHODE)and 9,10,13-trihydroxy-11-octadecenoic acid(9,10,13-THODE)in cured meat products were detected with high performance liquid chromatography tandem mass spectrometry(HPLC-MS/MS).The analytes,extracted with methanol and cleaned by solid phase extraction,were separated on an XBridge C18 column with a mobile phase consisting of 0.1%formic acid in water and acetonitrile,followed by ionization and quantification with an electrospray ionization in negative ion mode and multiple reaction monitoring mode(MRM).The results indicated that 13-HODE,9-HODE,9,10-DHODE and 9,10,13-THODE produced good linearity(R2>0.999)in the range of 0.05-2.0,0.02-2.0,0.01-0.5 and 0.01-0.5 ?g/mL,respectively.Limits of detection for 13-HODE,9-HODE,9,10-DHODE and 9,10,13-THODE were 0.120,0.024,0.008,0.016 pg/g,respectively,and the recoveries were 95.1%?95.9%?85.2%?86.2%,respectively.The method was successfully employed to detect the analytes in 26 cured meat products and the results shown that all samples contained 13-HODE,9-HODE,9,10-DHODE and 9,10,13-THODE ranging from 1.4?100.7,0.56?39.45,0.1?3.9 and 0.3?10.1 ?g/g,respectively.Since some isomers of the analytes were detected in samples as well,the real content of fatty acids with hydroxyl(s)from oxidation of LA might be much higher.2.Variations of HODEs during Sausages processingTwo kinds of methods,i.e.high-temperature hot-wind drying and low-temperature air-drying,were employed to make sausages.Variations of physicochemical indices,lipoxygenase(LOX)activity,peroxide value(POV),2-thiobarbituric acid-reactive substances value(TBA)and HODEs were observed during processing;and the contributions of LOX-catalyzed oxidation to the production of HODEs and lipid oxidation were analyzed.The values of moisture and salt in both end sausages differed insignificantly.pH value fluctuated during the process,and the value was lower in final low-temperature air-drying sausage(p<0.05).LOX activity increased unremarkably after curing,followed by declining,and the values in both final sausages differed insignificantly.POV and TBA values increased gradually during the whole processes,and both of them showed a significantly higher(p<0.05)values in final hot-wind drying sausage.HODEs increased consistently from the beginning to the end of process,and the values in final hot-wind drying sausage were significantly higher than that in air-drying sausage(p<0.05).The results indicated hot-wind drying at 45?50? resulted in more intense oxidation of lipids;LOX-catalyzed oxidation was being predominated at the early stage of curing and drying process,however it was overtaken by non-enzymatic oxidation gradually.3.Effect of Physicochemical Factors on the Generations of HODEs in Linoleic Acids Oxidation SystemsThe effects of pH,ionic strength and temperature on the formations of HODEs in linoleic acids oxidation system,e.g.non-enzymatic oxidation system(Fe2+-ascorbic acid/LA and Mb/LA)and enzymatic oxidation system(LOX/GSH/GSH-Px/LA),were studied.With the pH ranging from 5.5 to 8.0,contents of HODEs in Fe2+-ascorbic acid/LA system decreased and followed by increasing,while those in Mb/LA and LOX/GSH/GSH-Px/LA systems increased gradually.In the range of 0%?7.2%of salt content,the levels of HODEs in all systems showed a downward trend after the first rise.When the temperature varied from 4? to 50?,amount of HODEs in Fe2+-ascorbic acid/LA system increased continuously,whereas an increase followed decreasing was observed in both systems of Mb/LA and enzymatic.The formations of 13-HODE and 9-HODE differed insignificantly in the system of Fe2+-ascorbic acid/LA,while the contents of 13-HODE was much higher than 9-HODE in Mb/LA system(p<0.05),and in enzymatic oxidation system(p<0.01).The results provide a foundation for studies on the formation mechanism of HODEs and the regulation of harmful substances in processing of cured meat products.