ENDOCANNABINOID CEREBROSPINAL FLUID LEVELS IN MIGRAINE 79 4 variation. Participants were asked to refrain from eating or drinking, apart from water, for at least 8 h prior to sampling. Sampling occurred interictally with migraine individuals being attack-free for a minimum of 3 days. Participants were urged to refrain from the use of prophylactic or any other chronic medication (other than oral contraceptives) in the four weeks preceding the LP. The LP and subsequent CSF collection were performed in a standardized matter to limit inter-individual variation. Two mL CSF was transferred to a 12-mL polypropylene Falcon tube (Cat. No. 160172; Greiner Bio-One B.V., Alphen aan den Rijn, the Netherlands) for routine diagnostics (cell count, glucose and total protein levels). Next, for endocannabinoid measurements, 3 mL CSF was sampled directly in a 15-mL polypropylene Falcon tube (Cat. No. 188271; Greiner Bio-One B.V.) that already contained 6 mL of ice-cold ethanol (to instantly stop any metabolic activity). The tube was immediately put back on ice after CSF collection. After gently shaking the tube, the CSF was divided in aliquots of 1.5 mL in 1.8-mL cryotubes (Art. No. 368632; NUNC Brand, SigmaAldrich, Zwijndrecht, the Netherlands). The cryotubes were placed on dry ice within 30 minutes of sampling and transferred to a -80°C freezer as soon as possible. All CSF samples remained at -80°C until sample preparation for analysis. Analysis of eCBs using micro-liquid chromatography – tandem mass spectrometry quantification The analysis of AEA, 2-AG and DHEA levels in CSF was performed using a validated method.31 Sample preparation, except the evaporation step, was carried out under ice-cold conditions. Analytes were extracted using liquid-liquid extraction. In brief, 750 µL of CSF samples (CSF:ethanol = 1:2, v/v) was evaporated to approximately 100 µL using a SpeedVac (Thermo Fisher Scientific, Waltham, MA, USA). Then, 10 µL of isotopically labelled internal standard solution and 50 µL of a buffer solution (0.1 M ammonium acetate solution, adjusted to pH 4 with formic acid) were added to each sample. One milliliter of methyl-tert-butylether (MTBE) was then added prior to agitation for 10 min using a bullet blender and subsequent centrifugation for 5 min at 16,000 g. The organic supernatant (900 µL) was collected and evaporated to dryness using the SpeedVac. The dry residues were then reconstituted in 20 µL of an ice-cold solution of water and acetonitrile (ACN) 50:50 (v/v), prior to agitation (20 min) and centrifugation at 16,000 g for 50 min. Finally, 15 µL of the supernatant was transferred into a glass vial and 3 µL was injected into a nanoAcquity micro-LC system (Waters Corporation, Mildford, MA, USA) hyphenated with a Shimadzu 8060 triple quadrupole mass spectrometer (Shimadzu Corporation, Kyoto, Japan). Separation was performed using a micro C18 column (0.3 × 150 mm, 2.6 μm) from Phenomenex (Torrance, CA, USA) maintained at 45°C. The mobile phase was composed of 2 mM ammonium formate and 10 mM formic acid in water (A), and acetonitrile (B). The flow rate was 4 μL/min. Ionization of the compounds was performed using a customized micro-electrospray ionization (micro-ESI) source in positive mode. Selected Reaction Mode (SRM) was used for MS/MS acquisition. SRM
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