Materials and Reagents

1. Horseradish tissue (hypogeous and epigeous portion) (see Note 1)
   
2. Methanol (MeOH) (LC/MS grade) (Carlo Erba Reagents, catalog number: 414831 )
   
3. Acetonitrile (ACN) (LC/MS grade) (Carlo Erba Reagents, catalog number: 412342 )
   
4. Ultrapure Milli-Q water
   
5. Liquid nitrogen
   
6. Sinigrin hydrate from horseradish (99%) (Sigma-Aldrich, catalog number: S1647 )
   
7. Rapeseed ERM certified Reference Material containing gluconapin, 4-hydroxyglucobrassicin, glucobrassicanapin, glucobrassicin and gluconaturtiin (Sigma-Aldrich, catalog number: ERMBC367 )
   
8. Glucoiberin (C2 Bioengineering, catalog number: 10-JS 12-05-02 )
   
9. Glucobarbarin (C2 Bioengineering, catalog number: 18-DM 19-10-99 )
   
10. Glucotrapeolin (C2 Bioengineering, catalog number: 16-PM 19-10-99 )
    
11. 70% MeOH (see Recipes)
    

In addition only for intact GLS determination

12. Formic acid (gradient grade) (Sigma-Aldrich, catalog number: F0507 )
    
13. 0.1% Formic acid (HCOOH) (see Recipes)
    

In addition only for desulfo-method

14. DEAE-Sephadex A-25 (formiate form) obtained by using DEAE-Sephadex A-25 (chloride form) (Sigma-Aldrich, catalog number: A25120 ) and imidazole (Sigma-Aldrich, catalog number: 56750 )
    
15. Sulfatase type H-1 (Sigma-Aldrich, catalog number: S-9626 )
    
16. Sulfatase type H-1 (1 to 2.5) (see Recipes)
    
17. DEAE-Sephadex A-25 (formiate form) (see Recipes)
    

Equipment

1. Freeze Dry Systems (e.g. Labconco, model: Freezone 4.5 )
   
2. Laboratory mill
   
3. Disposable 50 ml and 15 ml polypropylene tubes
   
4. 2 ml sample vials
   
5. Water bath: beaker filled with water and placed on a heating device (electric hotplate or similar device).
   
6. Thermometer
   
7. Vortex mixer
   
8. Refrigerated centrifuge (50 ml tubes) (e.g. Heraeus, model: Varifuge F )
   
9. Glass Pasteur pipettes
   
10. 0.22 µm nylon filter (Whatman)
    
11. HPLC system with a photodiode array detector (e.g. Agilent, model: Agilent 1200 HPLC Liquid Chromatrography System)
    
12. HPLC 2 ml glass vials (Phenomenex, model: AR1-3910-12 ) with caps (Phenomenex, model: AR0-8959-13-B )
    
13. Liquid chromatography (LC) coupled with electrospray ionization (ESI) and high resolution mass spectrometry (MS) (e.g. Thermo Fisher Scientific, model: LC-ESI-FTICR MS )
    
14. Intact glucosinolates Discovery C₁₈ column, 250 x 4.6 mm, 5 µm particle size (pore size, 180Å) (Sigma-Aldrich, catalog number: 504971 ), with a Discovery C₁₈ 20 x 4 mm security guard cartridge (Sigma-Aldrich, catalog number: 505129 )
    
15. Desulfoglucosinolates (Nucleodur C₁₈ column, 125 mm x 3 mm) (MACHEREY-NAGEL, catalog number: MN760051.30 )
    

Procedure