Did you use this reference protocol for the assay?

Eberhardt, M. V., Lee, C. Y., Liu, R. H. (2000). Antioxidant activity of fresh apples. Nature, 405, 903–904.

See extract procedures and the possebilities of NMR detection: Anal. Chem., 2003, 75 (22), pp 6288–6294 and Journal of Proteome Research 2009, 8 (4), 1694-1703.

http://ac.els-cdn.com/S0039914011010848/1-s2.0-S0039914011010848-main.pdf?_tid=0185e3b6-2e68-11e2-ab71-00000aab0f6c&acdnat=1352903681_26d5f411e23d2e817204d2a92e94ff65

Did you try SPE extraction and purification to eliminate organic acids that may alter your color? you can try it with some RP cartridge and wash away the water soluble phase. Then you can elute with an organic solvent to recover your analytes. Don't forget to measure the recovery rate of this technique to correct your data.

You may need to remove fat soluble components before extracting flavonoids to reduce the interfers of those compounds on total flavonoid determination.

R.L. Rouseff, S.F. Martin, C.O. Youtesy Quantitative survey of narirutin, naringin, hesperidin and neohesperidin in citrus J. Agric. Food Chem., 35 (1987), pp. 1027–1030

in case of yours if you are having standards you should go for gas chromatography or HPLC for detailed analysis ,

First, I would like to thank everyone who tried to help!

Donato Angelino:

We are using the same protocol as Nature's paper. It's Zhisen, J. , Mengcheng, T. & Jianming, W. Food Chem. 64, 555– 559 (1999).

The only difference is in the extraction, we use methanol 80% and in Natures' they use Acetone 80%. Next week, we are going to try using Acetone 80%, but i don't think it would give such a big difference... we'll see.

Henk As:

In Anal. Chem.'s paper the matrix was oregano's leaf, so they gound and extracted with acetone in Soxhlet apparatus for 6h. I don't think it's necessary to use the same procedure since our matrix is orange juice and our compounds are dissolved.

Thanks for suggesting the NMR detection, but now we are trying to establish a simple method to quantify total flavonoids in orange juice, maybe in a next step would be interesting to use the detection by NMR.

Ram Uckoo:

AlCl3 10% and NaOH 4%, we also use NaNO2 5% before adding AlCl3 solution.

We used Rutin because our lab didn't had hesperidin, but we already acquired hesperidin.

Giuseppe Genova and Aldo Todaro:

SPE extraction and purification will be our next try! Thanks for the idea.

Anant Atakari:

At the moment, we are just trying to establish a simple method to quantify total flavonoids in orange juice, maybe in a next step HPLC would be interesting.

Next week we are going to try an extraction with acetone 80%, and if still have problems with color interference we will try SPE extraction and purification.

use AlCl3 method.

The major flavonoids of orange juice are hesperetin and naringenin. It would be better to quantify these flavonoids using a specific HPLC method

Chang et al., 2002 method is the best with measured equivalent to rutin.

Using FC reagent you will identify total phenolic content and Total flavanoid by DPPH method

DPPH is a good method that will suffice you. Furthermore, You can try Folin ciocalteau method

Hi, I think, you have to read this: Robards, K., Antolovich, M., 1997. Analytical chemistry of fruit bioflavonoids. Analyst 122, 11–34.

There is described a method for total flavonoids content. This is simple colorimetric method with vanillic reagent. Good luck ;)

You can also improve this method by using of SPE method for sample praparation.

I think SPE coupled with Folin-Ciocalteau method could be best choice. For SPE, you can choose commercially available material, to say OASIS cartridges from Waters Company. For pre-treatment of the orange juice sample, you can also try above Ram Uckoo's mentioned method.

The major citrus flavonoids (flavanones) are the glycosides of hesperetin (hesperidin), naringenin (naringin) and eriodictyol. Good papers on (the analysis of) the flavonoid content of citrus fruits are: Tomás-Barberán & Clifford, Science of Food and Agriculture 80(7): 1073-1080, 2000; and: Yáñez et al., Biopharmaceutics and Drug Disposition 29(2): 63-82, 2008. The latter also adresses the stereochemistry of these compounds, a property of flavanones which is often forgotten. I am sure you can acquire them via Researchgate. Hopefully these can help you ahead!

I agree with Walter Brand's suggestion and I would suggest checking for more literature data on the most comprehensive database on food polyphenols that is available online and for free: http://www.phenol-explorer.eu/

Data are reported for different analytical methods of determination (chromatography before or after hydrolysis, Folin-Ciocalteu, etc.).

In order to increase the solubility of the flavanones, you may want to change the solvent (you could try with DMSO) and/or applying SPE before determination of the total flavonoid content with a colorimetric method (AlCl3), as suggested by Dayun Zhao.

Ascorbic acid and reducing sugars also react with Folin-Ciocalteau reagent. If you use the Folin method to analyse total phenolics you should not forget to correct their influence in the final result.

I agree that HPLC for flavonoids or other phenolics quantification is the best option for this sample.

HPLC method is better to quantify flavonides also you can use GCMS method you can find out all flavonides at singal analysis extraction losses can be minimized

You could use an enzymatic method, specific for phenolic compounds, and that solves the interferences of sugar in the sample: J. Agric. Food Chem. 2004, 52, 6287−6293. This method was used to measure the phenolics in our sampkes of grape juice and it showed as very convenient, reproducible and easy to perform it.

Some of my colleagues which are working with orange juice have used Tousi et al. 2011. J. Sci. Food Agric., 9, 142-151.