## 2 Materials and Methods
### Sample Preparation
#### 2.7.2 Lutein Extraction Method  
One of the first and widely used AOAC (Association of Official Analytical Chemists) methods [28] required extensive time and a large volume of chemicals. [PERSON] et al. [29] improved the AOAC method by simultaneously carrying out extraction and saponification under an inert atmosphere with the addition of a BHT or BHA (butyled hydroxyapatite) solution. Cold saponification was found to be better for extraction of xanthophylls. For separation of xanthophylls, the composition of the ternary solvent mixture was changed to hexane:acetone:methanol = 85:15:1 and the composition of adsorbent was adjusted to an activated MgO:diatomaceous earth = 1:1. Further improvement of the AOAC method was conducted by [PERSON] et al. [26]. The adapted AOAC method developed by [PERSON] et al. used hot saponification, and extraction and saponification was also conducted simultaneously but over a significantly shorter time. After extraction and saponification aliquot of the hexane layer was evaporated, diluted with a mixture of hexane:ethyl acetate = 65:35 and analyzed by HPLC. The method developed by [PERSON] et al. was adopted as the standard method for this investigation. A detailed description of the [PERSON] et al. method is as follows: 1 g of sample was weighed into 50 mL glass vials, then 15 mL of the extraction mixture of hexane:acetone:ethanol:toluene (10:7:6:7, \(v/v/v/v\)) and 1 mL of 0.2% methanolic BHT solution were added. The content was vortexed for 30 s, then 2 mL of ultrapure water and 2 mL of 40% methanolic KOH solution were added, and the mixture was vortexed again for 30 s. The vials were covered with aluminum foil to reduce the effect of light on the samples and heated in a water bath at 56 \({}^{\circ}\)C for 20 min. After heating, the vials were left in the dark for 1 h to cool down. Next, 15 mL of n-hexane and 5 mL of 10% aqueous Na\({}_{2}\)SO\({}_{4}\) solution were added to the mixture, after which the mixture was vigorously stirred. The mixture was again left in the dark for 1 h to allow the layers to separate. An aliquot of the upper hexane layer was filtered through a membrane filter with a 0.20 \(\upmu\)m pore size and 1 mL of the sample was evaporated on a rotary evaporator at 55 \({}^{\circ}\)C until dry. After evaporation, 1 mL of a hexane:ethyl acetate = 65:35 mixture was added to each sample. 1 mL of the prepared sample was transferred to an amber vial and the sample was analyzed by HPLC. The sample-to-extraction mixture ratio was 1:40. From this point forward, the method developed by [PERSON] et al. will be abbreviated as SM.  
To reduce the use of toxic solvents, guidelines from Pfzier's recommendation list [27] and relative solubility list published by [PERSON] and [PERSON]. [30] were followed. The procedure used in this study was as follows: 1 g of sample was weighed, then 15 mL of the solvent or solvent mixture, 2 mL of 0.2% BHT solution, and 3 mL of 40% KOH solution were added. The extraction was assisted by direct ultrasound for 3 min. After sonication, samples were left in the dark for 1 h, after which 1 mL of the upper layer was evaporated on a rotary evaporator at 55 \({}^{\circ}\)C until dry. Following evaporation, 1 mL of hexaneethy acetate (65:35, \(v/v\)) mixture was added to each sample, and the resulting sample was transferred to the amber vial. The sample was analyzed using HPLC. The ratio of the sample to the solvent or solvent mixture was 1:20. From this point forward, the ultrasound-assisted extraction method for solvent selection will be abbreviated as UAE-SOL.  
Modification of SM refers to changing the solvent composition in the extraction mixture. The mixture of hexane:acetone:ethanol:toluene (10:7:6:7, \(v/v\)) was replaced with a chosen solvent mixture. All other SM parameters remained unchanged. From this point forward, the modified SM will be abbreviated as MSM.  
All samples were analyzed the same day they were prepared, and measurements were performed on five parallel samples.