CORRELATING RATIOS OF AMINES IN PLASMA AND CEREBROSPINAL FLUID 53 3 368632, Sigma-Aldrich, Saint Louis, MO, USA) that already contained 1.0 mL of cold ethanol (Prod. No.:8098, ethanol absolute, J.T.Baker, Phillipsburg, NJ, USA). The NuncTM cryotubes were inverted several times to thoroughly mix CSF and ethanol and were placed on dry ice within 30 min from sampling. Thereafter, aliquots were transferred to -80°C for storage within 60 min from sampling. Plasma samples were collected straight after lumbar puncture, while participants rested in supine position samples were drawn from the median cubital vein. Venous blood was collected in a EDTA plasma tube (Cat. No. 366643, BD Medical, Franklin Lakes, NJ, USA) and centrifuged at 4°C for 20 min (2,000 rpm, 747 g). The supernatant (plasma) was transferred to a new 15-mL polypropylene falcon tube, inverted several times, and divided in .5 mL aliquots into 1.0-mL NuncTM cryotubes (Cat. No. 366656, Sigma-Aldrich). Thereafter, all aliquots were transferred to -80°C for storage within 60 min from sampling. All CSF and plasma samples remained at -80°C until sample preparation. Amine measurements Both CSF and plasma samples were measured with an ultra-performance liquid chromatography mass spectrometry (UPLC-MS) method that was shown to reliably quantify 74 biogenic amines for mouse CSF samples 12. For details on sample preparation and amine measurements see Onderwater et al.13 In brief, quantitation of amino acids and biogenic amines utilized an AccQ-Tag derivatization strategy adapted from the protocol supplied by Waters. 5.0 µL of each sample was spiked with an internal standard solution. Proteins were precipitated by the addition of MeOH after which the samples were dried in a speedvac.The residue was reconstituted in a borate buffer (pH 8.8) with AQC reagent. LC-MS measurements were performed with an Acquity UPLC System (Waters, Milford, MA, USA) coupled to a QTRAP 6500 Triple-Quadruple MS System (Sciex, Framingham, MA, USA). For the chromatographic separation 1 µL of the sample was injected on an AccQ·Tag Ultra 100 x 2.1 mm column with a particle size of 1.7 µm (Waters, Milford, MA, USA) and chromatographic separation was achieved with the flow rate of .7 mL/ min. over an 11-min LC gradient program. The target analytes were detected in electrospray ionization (ESI) positive ion mode.The derivatized target metabolites and their internal standards were identified by their retention times and using their specific Multiple Reaction Monitoring (MRM) at nominal mass resolution. Data were pre-processed with MultiQuant Software for Quantitative Analysis v3.0.2 (Sciex, Framingham, MA, USA). Peak areas of target analytes relative to their corresponding internal standards were calculated as area ratios. All samples were measured in ten separate batches including calibration (injected at the start and the end), QC, blank, and randomized study samples. Plasma samples were measured in the first five batches, whereas CSF samples were measured later. QC samples were analyzed after every ten injections and used to monitor the intra-batch data quality and to correct for inter-batch analytical variance. To subtract the inter-batch analytical variance, all area ratios (study samples, calibration
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