*4.4. Vitamin C Extraction and Analysis*

Apples were processed on the day of the harvest to determine their vitamin C content. The method used for the determination is compliant with standard SIST EN 14130:2003 (determination of vitamin C using HPLC (Agilent Technologies, Santa Clara, CA, USA). Apples were quickly peeled, chopped, and immersed in liquid nitrogen prior to being ground in a mortar, using ceramic knife and mortar. Twenty grams of flesh powder were extracted with the metaphosphoric acid (Merck) and placed on the shaker for 30 min. The further procedure was as described in the standard. HPLC (Agilent 1200) with the UV-VIS detector at 265 nm was used for detection. The Gemini C18 110A (250 × 4.6 mm, 5 µm) column with the flow rate 0.7 mL min−<sup>1</sup> was used. The results are in mg per kg of fresh sample.
