Spectrophotometric Measurement of Secondary Lipid Oxidation Products

The results from the TBA test, known in older literature as the TBA number, are usually expressed as mg malonaldehyde/kg sample for methods a to c cited above (note that results have also been reported as nmol malonaldehyge/kg or g sample) and as mg of malonaldehyde per unit of lipid for method d. Since it is known that malonaldehyde is not the only aldehyde present in the sample extract and because other aldehydes are capable of producing the same red pigment with TBA when the conditions are favorable, the TBA number/value is more appropriately expressed as the TBARS value, i.e., mg malonaldehyde equivalents/kg sample. To confuse the matter, the AOCS method, which is based on the protocol reported by Pokomy and Dieffenbacher (1989) and permits the direct determination of TBA value in oils and fats without preliminary isolation of secondary oxidation products, defines the TBA value as the increase of absorbance measured at 532 nm due to the reaction of the equivalent of 1 mg of sample per 1 ml volume with 

Generally, duplicate samples are mixed at the same time to speed up the process. The bottles containing the TCA reagent and distilled water are placed in plastic buckets containing ice, and a 50-ml Brinkmann dispensette is connected to each. 

Alternatively, a homogenizer can be used if a laboratory blender is not available. 

Add 50 ml ice-cold distilled water and mix the slurry with the blender for another minute. 

Place a glass or plastic funnel over the top of a 100-ml volumetric flask and line the funnel with Whatman no. 1 filter paper. Prewet the filter paper with 1 1 TCA reagent/water. 

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D2.4 Spectrophotometric Measurement of Secondary Lipid Oxidation Products... 

Analysis of the Decomposition Products of Hydroperoxides. Some authors have monitored formation of some of the decomposition products of the lipid hydroperoxides. Direct spectrophotometric measurements of the formation of oxo-octadecadienoic acids at 280 nm are possible , as are measurements of secondary oxidation products like a-diketones and unsaturated ketones at 268 nm. The formation of various aldehyde products of lipid peroxide decomposition can be monitored by reacting them with 2,4-dinitrophenylhydrazine and, after HPLC separation, measuring at 360-380 mn the DNPH derivatives formed , althongh the sensitivity of this particular technique makes it very susceptible to interference. 

Lipids are susceptible to oxidation and, as such, require analytical protocols to measure their quality. As described in vnitd2.i, autoxi-dation is one of the chief processes by which lipids degrade. The primary products from this reaction are hydroperoxides. These odorless and colorless transient species break down by various means to secondary products, which are generally odoriferous by nature. Being able to measure secondary oxidation products by simple spectrophotometric means is important for the food scientist so that he or she is able to characterize the extent of lipid oxidation. However, the researcher should be cautioned that one assay (e.g., TBA test) does not provide all the answers. To get a better picture of the story, both primary and secondary products of lipid oxidation should be assessed simultaneously by the different methods available (unitdu). 

Carbonyl compounds in oxidized lipids are the secondary oxidation products resulting from the decomposition of the hydroperoxides. They can be quantified by the reaction with 2,4-dinitrophenylhydrazine and the resulting colored hydrazones are measured spectrophotometrically at 430-460 nm. The carbonyl value is directly related to sensory evaluation, because many of the carbonyl molecules are those responsible for off-flavor in oxidized oil. The anisidine value is a measure of carbonyl compounds that have medium molecular weight and are less volatile (Frankel 1998). It can be used to discover something about the prior oxidation or processing history of an oil. 

Many methods have been developed to access the extent of oxidative deterioration, which are related to the measurement of the concentration of primary or secondary oxidation products or of both. The most commonly used are peroxide value (PV) that measures volumetrically the concentration of hydroperoxides, anisidine value (AV), spectrophotometric measurement in the UV region and gas chromatographic (GC) analysis for volatile compounds. Vibrational spectroscopy, because of its high content in molecular structure information, has also been considered to be useful for the fast measurement of lipid oxidation. In contrast to the time consuming chromatographic methods, modem techniques of IR and Raman spectrometry are rapid and do not require any sample preparation steps prior to analysis. These techniques have been used to monitor oil oxidation under moderate and accelerated conditions and the major band changes have been interpreted. ... 

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