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RawData_TandemLCMS_PsilocybinPsilacetin_Psilocin_PlasmaConcentrations

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posted on 2023-10-25, 14:09 authored by CODY WENTHURCODY WENTHUR, Nathan T. Jones, Laura Wagner, Cameron O. Scarlett, Molly C. Pellitteri-Hahn

A liquid chromatography-tandem mass spectrometry method suitable for quantitative analysis of psilocin concentrations is assessed for accuracy and applied to determine the plasma concentrations of liberated psilocin following the administration of psilacetin fumarate and psilocybin to C57Bl6/J mice.

Liquid Chromatography / Tandem mass spectrometry (LC-MS/MS)

LC-MS/MS analysis and quantitation occurred in the Analytical Instrumentation Center (AIC) at the UW-School of Pharmacy. For preparation of analytical standards, psilocin (Usona Institute, Madison WI; > 99% purity) was prepared in Optima LCMS grade methanol (Fisher Scientific, Hampton NH). d10-Psilocin solution was purchased from Cerilliant (Round Rock TX) for use as an internal standard (ITSD). Blank mouse plasma for preparing calibration curves and quality control samples (QCs) was purchased from Innovative Research (Novi MI). All solvents for liquid chromatography were Optima LC/MS grade (Fisher Scientific, Hampton NH). Additives for LC/MS analysis were purchased from Sigma Aldrich (St. Louis MO).

Calibrators and QCs were prepared from stock methanol solutions of active pharmaceutical ingredients diluted to between 0.5 and 400 ng/ml in blank plasma. Samples, calibrators, and QCs were prepared for LC-MS/MS by protein precipitation and filtration using Waters Sirocco plates (Milford MA) according to the manufacturer’s instructions. A precipitation mix containing the ISTD was prepared and aliquoted to a Sirocco plate mounted on a 96-well receiver. Samples, QCs, and calibrators were added to the plate, incubated for 2 minutes, and pushed through the plate using a positive pressure manifold (Waters, Milford, MA). Processed samples were then dried under nitrogen and resuspended in 100 µl 98% A / 2% B solvent prior to LC/MS/MS analysis.

Quantitative LC-MS/MS was performed using a Waters Acquity I-Class binary pump (Waters Corp., Milford MA) coupled to a Sciex QTrap 5500 mass spectrometer (Sciex Corp., Framingham MA). Samples were separated on a Kinetex Core-Shell phenyl-hexyl 2.1 x 100 mm column (Phenomenex, Torrence CA) using a 3-minute gradient with a flow rate of 0.4 ml/min and a column temperature of 28C. The initial conditions consisted of 95% solvent A (2.5 mM ammonium formate in water with 0.1% formic acid) and 5% solvent B (acetonitrile with 0.1% formic acid). The elution gradient began at 5% B, increased to 8.6% B over 1.8 minutes, then quickly rose to 95% B in 0.15 minutes. This was held for 0.6 minutes, then decreased to 5% B in another 0.15 minutes, followed by a 0.25-minute re-equilibration period. The column temperature was maintained at 28°C with a flow rate of 0.4 ml/min. All samples were injected in triplicate in randomized order, and the average of these injections was used for analysis.

Funding

Training Program In Molecular AND Cellular Pharmacology

National Institute of General Medical Sciences

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