RawData_TandemLCMS_PsilocybinPsilacetin_Psilocin_PlasmaConcentrations

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.

本研究评估了一种适用于定量分析裸盖菇碱浓度的液相色谱-串联质谱法，并应用于测定C57Bl6/J小鼠给予富马酸裸盖菇碱和裸盖菇碱后释放的裸盖菇碱血浆浓度。液相色谱-串联质谱法（LC-MS/MS）分析及定量在威斯康星大学-药学院分析仪器中心（AIC）进行。分析标准的制备中，裸盖菇碱（Usona Institute，Madison WI；纯度>99%）使用Optima LCMS级甲醇（Fisher Scientific，Hampton NH）配制。d10-裸盖菇碱溶液由Cerilliant（Round Rock TX）购买，用作内标（ITSD）。用于制备校准曲线和质量控制样品（QCs）的空白小鼠血浆由Innovative Research（Novi MI）购买。液相色谱的所有溶剂均为Optima LC/MS级（Fisher Scientific，Hampton NH）。LC/MS分析添加剂由Sigma Aldrich（St. Louis MO）购买。校准剂和QCs由活性药物成分的储备甲醇溶液制备，稀释至0.5至400 ng/ml的空白血浆中。样品、校准剂和QCs按照制造商的说明，使用Waters Sirocco板（Milford MA）进行蛋白质沉淀和过滤，制备LC-MS/MS样品。含有内标的标准沉淀混合物分装到96孔接收器的Sirocco板上。将样品、QCs和校准剂加入板中，孵育2分钟，然后通过正压分配器（Waters，Milford，MA）将板推过。处理后的样品在氮气下干燥，并在100 µl 98% A / 2% B溶剂中重新悬浮，然后进行LC/MS/MS分析。定量LC-MS/MS分析使用Waters Acquity I-Class二元泵（Waters Corp.，Milford MA）与Sciex QTrap 5500质谱仪（Sciex Corp.，Framingham MA）联用。样品在Kinetex Core-Shell苯基-己基2.1 x 100 mm柱（Phenomenex，Torrence CA）上使用3分钟梯度，流速为0.4 ml/min，柱温为28°C进行分离。初始条件为95%溶剂A（水中含有2.5 mM甲酸铵和0.1%甲酸）和5%溶剂B（含有0.1%甲酸的乙腈）。洗脱梯度从5% B开始，在1.8分钟内增加到8.6% B，然后迅速在0.15分钟内上升到95% B，保持0.6分钟，然后再次在0.15分钟内降至5% B，随后进行0.25分钟的再平衡。柱温保持在28°C，流速为0.4 ml/min。所有样品均以随机顺序重复注入三次，并使用这些注入的平均值进行分析。