Materials and Reagents

1. Citrus fruit tissue (ground frozen flavedo or pulp tissue)
   
2. Metaphosphoric acid (MPA) (65%) (Sigma-Adrich, catalog number: 79615 )
   
3. Orthophosphoric acid (Sigma-Adrich, catalog number: W290017 )
   
4. Tris base (Sigma-Adrich, catalog number: T1503 )
   
5. Methanol HPLC-grade (Sigma-Adrich, catalog number: 34860 )
   
6. Ascorbic acid (Sigma-Adrich, catalog number: A902902 )
   
7. C18 Sep Pak (Waters, catalog number: WAT091139 )
   
8. DTT (Sigma-Aldrich, catalog number: D0632 )
   
9. Sterile Mili-Q water
   
10. 0.1% MPA (see Recipes)
    
11. 2% MPA (see Recipes)
    
12. 8.5% orthophosphoric acid (see Recipes)
    
13. 400 mM Tris base (see Recipes)
    
14. 200 mM DTT in 400 mM Tris base (see Recipes)
    
15. Methanol:Milli-Q water (15:85, v/v) (see Recipes)
    
16. Ascorbic acid stock solution I for standard curve (see Recipes)
    

Equipment

1. Disposable 15 ml plastic tubes
   
2. 0.45 μm nylon filter (25 mm diameter) (Análisis Vínicos, catalog number: E0036 )
   
3. Refrigerated centrifuge (15 ml tubes)
   
4. Polytron (Kinematica AG, model: PT-1035 GT; http://www.kinematica.ch/en.html)
   
5. HPLC system with a photodiode array detector (PDA, Dionex)
   
6. Ultrabase C₁₈ column (100 x 4.6 mm, 2.5 μm)
   

Software

1. Chromeleon version 6.80 (Dionex)

Procedure