Materials and Reagents

1. Disposable 2 ml plastic microcentrifuge tubes
   
2. 1.5 ml glass vials (Agilent Technologies, catalog number: 5182-0715 ), screw top caps (Agilent Technologies, catalog number: 5182-0717 ) and 200 μl glass inserts (Agilent Technologies, catalog number: 5182-0549 )
   
3. AcroVac Filter Unit (Pall Life Sciences, catalog number: AVFP02L )
   
4. Chloroform (HPLC grade) (Thermo Fisher Scientific, catalog number: C607 )
   
5. L-Tryptophan (Sigma-Aldrich, catalog number: T0254 )
   
6. L-Kynurenine (Sigma-Aldrich, catalog number: K8625 )
   
7. 3-Nitro-L-tyrosine (nitrotyrosine) (Sigma-Aldrich, catalog number: N7389 )
   
8. Methanol (HPLC grade) (Thermo Fisher Scientific, catalog number: A452 )
   
9. Acetonitrile (HPLC grade) (Thermo Fisher Scientific, catalog number: A998 )
   
10. Potassium phosphate monobasic anhydrous (KH₂PO₄) (Thermo Fisher Scientific, catalog number: P285-500 )
    
11. Potassium phosphate dibasic anhydrous (K₂HPO₄) (Thermo Fisher Scientific, catalog number: P288-500 )
    
12. PIPES (Thermo Fisher Scientific, catalog number: BP292450 )
    
13. EDTA (Thermo Fisher Scientific, catalog number: S311-500 )
    
14. Kynurenic acid (Sigma-Aldrich, catalog number: K3375 )
    
15. Quinolinate (Sigma-Aldrich, catalog number: P63204 )
    
16. Phosphate buffered saline (PBS)
    
17. 50% methanol/50% 3 mM PIPES-3 mM EDTA (pH 7.4) (see Recipes)
    
18. Potassium phosphate (pH 6.4) with 2.7% (v/v) acetonitrile (see Recipes)
    
19. Standard mix (see Recipes)
    

Equipment

1. Refrigerated tabletop centrifuge
   
2. HPLC system with binary pump, autosampler and UV diode array detector (DAD) (Agilent Technologies, model: 1100 )
   
3. Hypersil GOLD C18 column (50 mm with 2.1 mm diameter) (Thermo Fisher Scientific, catalog number: 25003-052130 )
   

Procedure