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Fecal samples, Fractionation, LC-MS protocol, Loss evaluation (GC-MS) -…

- - - - Extraction temperature
        
        4 °C
        
        37 °C
        
        Clean-up
        
        Ultrafiltration
        3 kDa molecular weight spin cut-off filter
        
        LC-MS
        
        Liquid-liquid extraction
        500nl n-hexane x3
- - - - Source:
  - - - GC-MS
      - Silylating reagent?
        
        100 nl BSTFA
  - - - 1 ml
  - - - Overnight
  - - - Why?
      - Test:
- - - - Are standards pure?
- - - - Transfer 100 nl of supernatant to 1.5 ml low-bind centrifugation tube with boil-proof cap
        Dilute by 0.1% aqueous formic acid (1:5 v/v)
        Filter
        
        500 mL n-hexane (x3)
        
        Dry up
        
        Dissolve in 0.1% aqueous formic acid
        
        Transfer to LC-MS vial
  - - - LC-MS
- - - - F1:
        Hexane
      - F2:
        Toluene
      - F3:
        Tol:MeOH
        3:1 v/v
      - F4:
        DCM:MeOH
        3:1 v/v
    - - F1:
        Hexane
      - F3:
        DCM:MeOH
        2:1 v/v
        
        Dry up
        
        Dissolve in 0.1% aqueous formic acid
        
        Transfer to LC-MS vial
      - F2:
        DCM/Acetone
        2:1 v/v
- - - - Evaporate
- - - - F1: DCM
      - F2:
        DCM/Acetone
        2:1 v/v
      - F3:
        DCM:MeOH 1:1 v/v
    - - F1:
        Hexane
      - F3:
        Tol:MeOH 3:1 v/v
      - F4:
        Tol:MeOH
        1:1 v/v
      - F2:
        Toluene
      - F5:
        MeOH
- - - - Silylation
        100 nl of BSTFA
        1 hr 80 C
        
        GC-MS