title: A robust capillary liquid chromatography/tandem mass spectrometry method for quantitation of neuromodulatory endocannabinoids.
abstract: BACKGROUND
  Methods for quantifying anandamide (AEA) and 2-arachidonoyl glycerol (2-AG) are needed to support programs investigating molecular mechanisms of the central nervous system. Existing methods, while useful, are not well adapted to efficiently process large numbers of very small tissue samples. A unique challenge involves the disparity in endogenous levels of AEA (pmol/g tissue) and 2-AG (nmol/g tissue).
  
  
  METHODS
  A simplified one-step solvent extraction procedure was developed for recovering endocannabinoids from rat brain tissues, and combined with capillary liquid chromatography/tandem mass spectrometry (LC/MS/MS). Various multiple reaction monitoring (MRM)-based methods were evaluated for limit of detection (LOD) and robustness.
  
  
  RESULTS
  The optimized simultaneous quantitation method achieves an LOQ of 50 amol for AEA and 25 fmol for 2-AG, both with a linearity over 3 orders of magnitude, and elution times under 3 min. Accuracy, expressed as relative error (RE), is less than 12% for AEA and less than 6% for 2-AG. Precision, expressed as relative standard deviation (RSD), is less than 6% for AEA and less than 3% for 2-AG. Sample handling routines are sufficiently robust to support the automated analysis of thousands of samples from a range of tissue types.
  
  
  CONCLUSIONS
  The microscale method is a sensitive, economical and robust alternative to the larger scale LC/MS methods currently implemented for quantitation of AEA and 2-AG.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Calgary, Calgary, Alberta, Canada, T2N 4N1.
authors.forename: Ming
authors.initials: M
authors.lastname: Qi
authors.affiliation: Department of Cell Biology & Anatomy and Psychiatry, University of Calgary, Calgary, Alberta, Canada, T2N 4N1.|Hotchkiss Brain Institute, University of Calgary, Calgary, Alberta, Canada, T2N 4N1.
authors.forename: Maria
authors.initials: M
authors.lastname: Morena
authors.affiliation: Department of Neuroscience, University of Calgary, Calgary, Alberta, Canada, T2N 4N1.|Hotchkiss Brain Institute, University of Calgary, Calgary, Alberta, Canada, T2N 4N1.
authors.forename: Haley A
authors.initials: HA
authors.lastname: Vecchiarelli
authors.affiliation: Department of Cell Biology & Anatomy and Psychiatry, University of Calgary, Calgary, Alberta, Canada, T2N 4N1.|Hotchkiss Brain Institute, University of Calgary, Calgary, Alberta, Canada, T2N 4N1.
authors.forename: Matthew N
authors.initials: MN
authors.lastname: Hill
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Calgary, Calgary, Alberta, Canada, T2N 4N1.
authors.forename: David C
authors.initials: DC
authors.lastname: Schriemer
mesh_terms: D000818:Animals; D001095:Arachidonic Acids / Q000032:analysis*; D001923:Brain Chemistry; D002853:Chromatography, Liquid / Q000379:methods*; D063388:Endocannabinoids / Q000032:analysis*; D005989:Glycerides / Q000032:analysis*; D018377:Neurotransmitter Agents / Q000032:analysis*; D053284:Polyunsaturated Alkamides / Q000032:analysis*; D051381:Rats; D017207:Rats, Sprague-Dawley; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D001095:Arachidonic Acids; D063388:Endocannabinoids; D005989:Glycerides; D018377:Neurotransmitter Agents; D053284:Polyunsaturated Alkamides; C094503:glyceryl 2-arachidonate; C078814:anandamide