Hi Katarzyna,

You can contact Dr. Simon from the Department of Horticulture, University of Wisconsin-Madison for the recipe. His lab is dealing with carrots' nutrition, genomics, mapping and breeding. They have conducted the measuring of the carotenoids and anthocyanin (from purple carrots). This is the website with his and his lab's info:

http://www.nutrisci.wisc.edu/FACULTYPAGES/f_simon.html

Good luck with your research,

Hi, i'm working with Claudie Dhuique-meyer in CIRAD from France. We have a large experience on carotenoids determination in plants and fruits. You can send me an email at [email protected] if you want we talk more about this. 

Best regards

I suggest that first you try to use a methanol:water (1:1) solution to extract all the compounds in the plant tissues. And after this extraction, the use of several alcohols mixed with chloroform in different proportions to isolate the carotenoids and anthocyanins.

A known fresh weight from the leaves was cut in an ice-cold mortar, where some quartz sand was added as well as several milligrams of Na2CO3 used to reduce acidification. The leaves were ground with 1 ml of 80% acetone to squash. At the end of grinding, 3 ml of 80% acetone was added and the extract stirred in mortar, then the pigment solution was poured into a centrifuge tube, completed to 8 ml and the tube was covered with parafilm to prevent acetone evaporation during centrifugation.

The extract was centrifuged for 3 minutes at 1000 rpm. After centrifugation the color was measured within a short time or the extracts were stored closed in a cold dark place. The extract was measured (E) against a blank of pure 80% aqueous acetone at 3 wavelengths of 480, 644 and 663 nm using spectrophotometer. Taking into consideration the dilutions made, it was possible to determine the concentrations of the pigment fractions (Chl a, Chl b and carotenoids) as g ml-1 using the following equations:

Chl a = 10.3 E663 – 0.918 E644

Chl b = 19.7 E644 – 3.87 E663

Carotenoids = 5.02 E480

The fractions were then calculated as g g-1 dry weight of the differently-treated plants.

Anthocyanins were extracted from the oven-dried ground tissues by suspending in 10 ml of acidified methanol (methanol: water: HCl, 79: 20: 1, v/ v) and autoextracting at 0°C for 72 hours in dark with continuous shaking. The extracts were then centrifuged for 10 minutes at 5000 rpm and the absorbance was measured at 530 and 657 nm for each supernatant (Mirecki and Teramura, 1984). The absorbance readings at 530 nm (A530) were corrected for scattering using the absorbance readings at 657 nm (A657) using Rayleigh's formula as following:

Corrected A530 = A530 – 1/3 A657

Anthocyanins were calculated as the corrected absorbance (Lange et al., 1971; Mancinelli et al., 1975; Lindoo and Caldwell, 1978).

Just care with methanol and aceton extractions carotenoids are not fully soluble in thoses solvants and the extraction may be incomplete. Hexane is a lot better.

Hi Katarzyna,

I have an extraction protocol for anthocyanins in LC-MS analysis. If you have access to any liquid chromatography systems and would like to know more, please feel free to contact me.

Hello,

I have herewith attached a papers. 

The separation condition of anthocyanin by HPLC was also optimized. Several mobile phases have been described in the literatures for the analysis of anthocynin using the reversed phase column (Da Costa et al., 1998; Stintzing et al., 2002; Longo et al., 2007; Zhang et al., 2007). In this study, the organic

solvent selected for the preliminary experiments was methanol due to the solubility of anthocyanin. The mobile phases containing various percents of methanol

in aqueous acetic acid were then investigated. In order to evaluate the potential of the present method for quantitative analysis, linearity, LOD, LOQ, precision and recovery were investigated. According to this method, the calibration curve for the

determination of anthocyanin was constructed under optimum conditions using cyanin-3-glucoside as a standard compound. The anthocyanin content was

determined by comparison of the peak area obtained with the calibration curve of cyanin-3-glucoside. The results were expressed as μg of cyanin-3-glucoside

equivalents (CGE) per kg sample. Both LOD and LOQ of the present method were deduced based on the concentration of the analyte which produced a signal to noise ratio of three times and ten times, respectively. The repeatability and reproducibility were investigated in terms of relative standard

deviation (RSD) of both retention time and peak area. The repeatability was deduced from ten replicates within one day (intra-day precision, n = 10) and

reproducibility was calculated from the experiment in five consecutive days (inter-day precision, n = 5×3).

Sims & Gamon 2002. The best method!!!

Thank you everyone for answers !

Since anthocyanins and carotenoids have notably different polarity, choosing a common solvent for both these classes of compounds may be difficult. For a rough estimation however, MeOH will yield some carotenoids (but the extraction may not be complete) and most of the anthocyanins. You can determine carotenoids photometrically at the blue region (substracting for the participation of chlorophylls measured at the red region) and then acidify the extract to turn anthocyanins visible and measure again using the photometer at the green region of the spectrum (you also have to take account of the chlorophylls there).

See

Article Dihydroflavonol Reductase Activity in Relation to Differenti...

Article Determination of Total Carotenoids and Chlorophylls A and B ...

Estimation of anthocyanins:

Anthocyanins were extracted from the oven-dried ground tissues by suspending in 10 ml of acidified methanol (methanol: water: HCl, 79: 20: 1, v/ v) and autoextracting at 0°C for 72 hours in dark with continuous shaking. The extracts were then centrifuged for 10 minutes at 5000 rpm and the absorbance was measured at 530 and 657 nm for each supernatant (Mirecki and Teramura, 1984). The absorbance readings at 530 nm (A530) were corrected for scattering using the absorbance readings at 657 nm (A657) using Rayleigh's formula as following:

Corrected A530 = A530 – 1/3 A657

Anthocyanins were calculated as the corrected absorbance (Lange et al., 1971; Mancinelli et al., 1975; Lindoo and Caldwell, 1978).

Estimation of chlorophyll and carotenoids:

A known fresh weight from the leaves was cut in an ice-cold mortar, where some quartz sand was added as well as several milligrams of Na2CO3 used to reduce acidification. The leaves were ground with 1 ml of 80% acetone to squash. At the end of grinding, 3 ml of 80% acetone was added and the extract stirred in mortar, then the pigment solution was poured into a centrifuge tube, completed to 8 ml and the tube was covered with parafilm to prevent acetone evaporation during centrifugation.

The extract was centrifuged for 3 minutes at 1000 rpm. After centrifugation the color was measured within a short time or the extracts were stored closed in a cold dark place. The extract was measured (E) against a blank of pure 80% aqueous acetone at 3 wavelengths of 480, 644 and 663 nm using spectrophotometer. Taking into consideration the dilutions made, it was possible to determine the concentrations of the pigment fractions (Chl a, Chl b and carotenoids) as g ml-1 using the following equations:

Chl a = 10.3 E663 – 0.918 E644

Chl b = 19.7 E644 – 3.87 E663

Carotenoids = 5.02 E480

The fractions were then calculated as ug g-1 dry weight of the differently-treated plants.