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Samples were analyzed in single ion monitoring mode using characteristic fragment ions of analytes and corresponding internal standards.
Analyses were carried out in multiple reaction monitoring mode, using two specific transitions for each analyte and the detector was fixed at maximum Extended Dynamic Range with peak mass widths of 2 and 1.5 amu for the first and third quadrupoles, respectively.
The detector was operated in the multiple-reaction monitoring mode using the transitions from the protonated molecules at m/z 392.1 to 152.1 for tiotropium.
MS quantitation was performed in multiple reaction monitoring mode using an ESI+ ion source.
The mass spectrometer was operated in multiple reaction monitoring mode using positive ion electrospray (ESI).
The mass spectrometer was operated in selected ion monitoring mode, using electron capture negative ionization with methane (99.97% purity) as the reagent gas.
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MS detection was carried out in the selected ion-monitoring mode using m/z values of 356 and 348 (molecular ion +1 for deuterated and undeuterated anandamide), 384.35 and 379.35 (molecular ion +1 for deuterated and undeuterated 2-AG), 304 and 300 (molecular ion +1 for deuterated and undeuterated PEA) and 330 and 326 (molecular ion +1 for deuterated and undeuterated OEA).
Analyses were performed by GC/MS in selected-ion-monitoring mode, using a 30 m ZB-50 (0.25 mm × 0.25 μm) column with injection volumes of 1-3 μL.
Further support for the formation of pentadecanal and 2-hydroxy-16:0 from 16:0 was provided by GC-MS analyses run in the selected-ion-monitoring mode using synthetically prepared trideuterated standards (Additional file 2).
The TOF mass analyzer covers the entire mass range, e.g., m/ z 100 1000 shown here, and not only a few channels as in the selected or multiple reaction monitoring modes used on tandem quadrupole instruments for ultrasensitive detection.
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