## 2 Materials and Methods
### Physico-Chemical Analyses
This method is similar to the AOAC Official Method 953.17 and the AACC Method 86-80. Thiamine is oxidized to thiochrome by potassium ferricanide in a strongly alkaline solution. Thiochrome is then extracted into isobutyl alcohol, and its fluorescence is measured at 435 nm after excitation at 365 nm. Thiochrome fluorescence is directly proportional to the concentration of thiamine in the sample. To extract vitamin B2 from the samples, hydrolysis with hydrochloric acid, enzymatic treatment, and then extraction in acetone were performed. The quantification of vitamin B2 in extracts is traditionally based on spectrofluorimetric detection (Cary-50 UV-VIS Spectrophotometer). This method is based on the automated AOAC Official Method 981.15 and is similar to the AACC Method 86-70. All methods use measurement of riboflavin's natural fluorescence under controlled pH conditions. Fluorescence is measured at 510 nm after excitation at 450 nm. The quantification of vitamin B3 in samples is traditionally based on spectrofluorimetric detection (Cary-50 UV-VIS Spectrophotometer) and includes an initial extraction step in a 1:1 solution of sulfuric acid and water (through autoclaving at 121 \({}^{\circ}\)C for 45 min). This method is based on the automated AOAC Official Method 975.41 and is similar to the AACC Method 86-50A. Modifications have resulted in a safer method with equivalent responses from both niacin and niacinamide, eliminating the need for the conversion of niacinamide to niacin. Niacin and niacinamide react with cyanogen chloride created in situ to form an aldehyde intermediate. The subsequent addition of sulfanilic acid results in the formation of a polymethine dye absorbing light at 470 nm. The absorbance is directly proportional to the total niacin concentration. For the determination of retinol, an RP-HPLC method was used with a UV detector (\(\uplambda=325\) nm). The method, adapted from the European Commission Regulation 152/2009 [31], is based on the extraction of vitamin A in petroleum ether from the sample hydrolyzed with ethanolic potassium hydroxide solution. The solvent is removed via evaporation, and the residue is diluted in methanol and, if necessary, diluted to the required concentration. The chromatographic parameters are chosen so that there is no difference between all trans alcohol compounds of vitamin A and the cis isomers. Vitamin A was separated on a C18 reversed phase column, and an elution with the methanolic mobile phase was performed. The average peak height of several injections from the same sample solution and the average peak height of several injections of the calibration solutions were calculated. Alphatocopherol was analyzed using a Shimadzu VP Series liquid chromatograph with an FR-10 AXL fluorescence detector (Shimadzu, Kyoto, Japan), based on a method adapted from the European Commission Regulation 152/2009. An Alltima RP C-18 column (250 mm \(\times\) 4.6 mm, 5 \(\upmu\)m) at 40 \({}^{\circ}\)C was used for chromatographic separation. The mobile phase was a mixture of methanol and acetonitrile (50:50, \(v/v\)) with a flow rate of 1.5 mL/min. For the detection of alpha tocopherol, a 290 nm excitation wavelength and a 325 nm emission wavelength were set on the detector.