Physicochemical Quantification of Abscisic Acid Levels in Plant Tissues with an Added Internal Standard by Ultra-Performance Liquid Chromatography

Materials and Reagents

1. Methanol (Merck Schuchardt OHG, catalog number: 603-001-00-X )
   
2. 2, 6-di-tert-butyl-4-methylphenol (BHT) (Sigma-Aldrich, catalog number: B1378 )
   
3. Liquid nitrogen (optional for manual homogenization method only)
   
4. Labelled or deuterated ABA (such as ¹³C labelled, [²H₆] ABA or other form with any number of deuterium atoms) (e.g. OlChemIm Ltd, catalog numbers: 03 2721-03 2723 ; catalog numbers: 35671-08-0 )
   
5. Acetic acid (Analytical grade, any supplier)
   
6. Diethyl ether (Analytical grade, any supplier)
   
7. Acetonitrile (for UPLC-MS) (Sigma-Aldrich, catalog number: 271004 )
   
8. Aluminium foil
9. 50 ml conical centrifuge tube (Greiner Bio-One GmbH, catalog number: 210261 )
10. 0.5 ml Eppendorf tubes (any brand will suffice)
11. 2 ml screw-cap tubes (Scientific Specialities, catalog number: 2330-00 ) with tethered screw tube caps (Scientific Specialities, catalog number: 2002-59 ) (optional for very small samples or samples that require long-distance freight)
12. Whatman no. 1 filter paper (optional for large samples only)
13. 1.5 ml Eppendorf tubes (any brand will suffice)
14. Centrifuge for 2 ml Eppendorf tubes
15. Filtered glass 150 mm Pasteur pipettes (Poulten & Graf GmbH, model: D810 )
16. Rubber nipple for Pasteur pipette
17. 10-100 µl pipette (any brand will suffice)
18. 100-1,000 µl pipette (any brand will suffice)
19. 5 ml pipette and tips (any brand will suffice)
    
20. 80% methanol in water (v/v) (see Recipes)
    
21. 2% acetic acid (v/v) (see Recipes)
    
22. 80% methanol in water (v/v) with added butylated hydroxytoluene (BHT) (see Recipes)
    
23. 80% methanol in water (v/v) (see Recipes)
    
24. 2% acetic acid (v/v) (see Recipes)
    
25. 5% methanol, 94% water and 1 % acetic acid (v/v) (see Recipes)
    
26. 1% acetic acid (v/v) (see Recipes)
    

Equipment

1. Balance (±0.001 g)
   
2. Scholander pressure chamber (e.g. PMS Instrument Company, model: 600 ) (optional for specialised experiments or quantification of ABA from xylem sap)
   
3. Stainless steel beads 7 mm (QIAGEN, catalog number: 69989 / 69990 ) (optional for very small samples only)
   
4. Modelling clay or Blu-tak (Bostik) (optional for very small samples only)
   
5. Glass beaker (variable size) (optional for large samples)
   
6. General purpose 21 cm scissors
   
7. Tissue homogeniser [either modified Ba-mix^® or Physcotron (Microtec Co., model: NS-7 )]
   
8. Cell lysis machine (e.g. QIAGEN, model: TissueLyser II ) (optional for very small samples only)
   
9. Conventional Ba-mix^® or similar hand-held blender (optional for large samples only)
   
10. Ceramic mortar and pestle (optional for manual homogenization method only)
    
11. Büchner funnel (optional for large samples only)
    
12. Vacuum flask (optional for large samples only)
    
13. Vacuum sample concentrator (or rotary evaporator with 100 ml round bottom flask, optional for large samples)
    
14. Fume hood
    
15. Heating block that can contain 0.5 ml Eppendorf tubes (gentle stream of nitrogen gas, passing through a low flow regulator, tubing and funnelled through a standard 100 µl pipette tip, if desired) (e.g. Ratek Instruments, model: DBH10DP )
    
16. A LCGC Certified Clear Glass 12 x 32 mm Screw Neck Vial, with Cap and PTFE/silicone Septum , 2 ml Volume, 100 /pkg [(Waters, catalog number: 186000272C ), Insert 150 µl with preinstalled plastic spring (Waters, catalog number: WAT094171 )]
    
17. Aerosol barrier pipette tips for both µl pipettes (these are important to prevent sample contamination of the pipette which can compromise future samples. They can be made by inserting a small amount of cotton wool into the barrel end of the pipette tip.) (any brand will suffice)
    
18. Ultra-performance liquid chromatograph and multiple reaction-monitoring tandem mass spectrometer [Water Acquity H-series UPLC coupled to a Waters Xervo triple quadrupole mass spectrometer, containing a Waters Acquity UPLC BEH C18 column (2.1 mm x 100 mm x 1.7 µm particles)]
    

Software

1. Waters MassLynx and TargetLynx software

Procedure