Hi,
I'm performing TBA assay to measure the degree of lipid oxidation of raw pork, using the steam distillation method with Kjeldahl distiller.
I collected 50 ml of distillate, and it didn't react with my TBA solution so didn't develop the reddish-pink color which can be absorbed at 530 nm.
The distillate should contain some amount of malondialdehyde, but I think my distillate contains only pure water without malondialdehyde.
If the heating temperature is high, distillate is collected very quickly, but it seems to contain only water. So I tried to decrease the heating temperature, then the distillate was just not collected at all.
Please help me, find anything wrong in my procedure...Here's my procedure.
10 g of pork + 97.5ml DW + 2.5ml 4N HCl ==> Blending, homogenizing
Put it into Kjeldahl flask+distiller
The collected distillate is reacted with 0.02 M TBA reagent dissolved in 90% acetic acid in 100'C for 40 min
The absorbance is measured at 530 nm
When the collected distillate is reacted with 0.02 M TBA reagent, try to add 1 mL of 10% tricloracetic acid.
Did you try to determine TBA in homogenate without distillation? It may help you better identify the problem.
In another point of view: we dissolve TBA in DMSO. Acidic condition (trichloroacetic acid in our experiments) is used because of purification from proteins. Consider the protocol. It is free available e.g. in:
Bratislava Medical Journal Vol.115, No.11, p.699-703, 2014
http://www.elis.sk/download_file.php?product_id=4056&session_id=u6hja1ham80379nr67ptcg7aa7
doi:10.4149/BLL_2014_135
Hope it is helpful for you.
Hi,
MDA dosage is not easy I know. But continue and you will catch it.
Change your acetic acid by Phosphoric
acid 400 µL 0.44 M vortex 5 min heating 1 hour 100◦ C
Hi there,
I dissolve TBA (0,02 M) with only DW and read at 532 nm. Maybe your raw pork has too few oxidation substances and is under the threshold of the spectrophotometer.
An ideal distillation should be performed at 400+°C, and the 50 ml distilled in about 15-25'. Check your glassware.
The following papers maybe help you:
TORRES, E. PEARSON, A. M.; GRAY, J. I.; KU, P. K. Lipid Oxidation in Charqui (Salted and Dried Beef *. Food Chemistry, v. 32, p. 257–268, 1989
TARLADGIS, B. G.; PEARSON, A. M.; DUGAN, L. R. Chemistry of TBA acid test for determination oxidative rancidity in foods II formation TBA MDA complex without Acid-Heat Treatment. Journal of Science and Food Agriculture, v. 15, n. 9, p. 602–607, 1964.
Hi,
The TBA assay contains some interfering: check the validity of reagent (TBA) - if it is in use for a long time the color formation is difficult.
In our laboratory we usually use extraction instead distillation. The extract can be obtained from the addition of TCA (trichloroacetic acid) and BHT in the sample following up with the buzz Turrax agitator, filtration and centrifugation (for meats). When in meat products, is necessary added sulfanilamide to avoid interference of sugars and nitrite. The curve can be prepared with TMP or TEP at the same wavelength (532 nm). Also check the temperature of the heating in this procedure - extended time at elevated temperature is a problem in the analysis. Opt 80 ° C for less time (30 min).
Hope it is helpful for you.
Hi,
I think your samples are low in fat (may also have too few substance reacting with TBA). Therefore, if you would like to examine the details of your procedure, you should try the method with samples that include high fat content. We use the distillation method described by Tarladgis et al. (1964) similar as you explained above. In the mean time if the problem still persists,I can share the details of our procedure.
Hope it is helpful for you.
Hi,
Can any one of you explain the standard distillation method for TBA value estimation
Follow this link
Article Modified thiobarbituric acid assay for measuring lipid oxida...
regards
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