Online, Absolute Quantitation of Propranolol from Spatially Distinct 20- and 40-µm Dissections of Brain, Liver, and Kidney Thin Tissue Sections by Laser Microdissection-Liquid Vortex Capture-Mass Spectrometry.

Spatial resolved quantitation of chemical species in thin tissue sections by mass spectrometric methods has been constrained by the need for matrix-matched standards or other arduous calibration protocols and procedures to mitigate matrix effects (e.g., spatially varying ionization suppression). Reported here is the use of laser "cut and drop" sampling with a laser microdissection-liquid vortex capture electrospray ionization tandem mass spectrometry (LMD-LVC/ESI-MS/MS) system for online and absolute quantitation of propranolol in mouse brain, kidney, and liver thin tissue sections of mice administered with the drug at a 7.5 mg/kg dose, intravenously. In this procedure either 20 µm × 20 µm or 40 µm × 40 µm tissue microdissections were cut and dropped into the flowing solvent of the capture probe. During transport to the ESI source drug related material was completely extracted from the tissue into the solvent, which contained a known concentration of propranolol-d7 as an internal standard. This allowed absolute quantitation to be achieved with an external calibration curve generated from standards containing the same fixed concentration of propranolol-d7 and varied concentrations of propranolol. Average propranolol concentrations determined with the laser "cut and drop" sampling method closely agreed with concentration values obtained from 2.3 mm diameter tissue punches from serial sections that were extracted and quantified by HPLC/ESI-MS/MS measurements. In addition, the relative abundance of hydroxypropranolol glucuronide metabolites were recorded and found to be consistent with previous findings.

Extraction efficiency and implications for absolute quantitation of propranolol in mouse brain, liver and kidney tissue sections using droplet-based liquid microjunction surface sampling high-performance liquid chromatography/electrospray ionization tandem mass spectrometry.

RATIONALE: Currently, the absolute quantitation aspects of droplet-based surface sampling for tissue analysis using a fully automated autosampler/high-performance liquid chromatography/electrospray ionization tandem mass spectrometry (HPLC/ESI-MS/MS) system have not been fully evaluated. Knowledge of extraction efficiency and its reproducibility is required to judge the potential of the method for absolute quantitation of analytes from tissue sections. METHODS: Adjacent tissue sections of propranolol-dosed mouse brain (10-µm-thick), kidney (10-µm-thick) and liver (8-, 10-, 16- and 24-µm-thick) were obtained. The absolute concentration of propranolol was determined in tissue punches from serial sections using standard bulk tissue extraction protocols and subsequent HPLC separations and MS/MS analysis. These values were used to determine propranolol extraction efficiency from the tissues with the droplet-based surface sampling approach. RESULTS: Extraction efficiency of propranolol using 10-µm-thick brain, kidney and liver tissues using droplet-based surface sampling varied between ~45 and 63%. The extraction efficiency decreased from ~65% to ~36% with liver thickness increasing from 8 µm to 24 µm. Selecting half of the samples as standards, the precision and accuracy of propranolol concentrations were determined for the other half of the samples that were employed as a quality control data set. The resulting precision (±15%) and accuracy (±3%) were within acceptable limits. CONCLUSIONS: Quantitation of adjacent mouse tissue sections of different organs and of various thicknesses by droplet-based surface sampling in comparison with bulk extraction of tissue punches showed that extraction efficiency was incomplete using the former method, and that it depended on the organ and tissue thickness. However, once extraction efficiency was determined and applied, the droplet-based approach provided satisfactory quantitation accuracy and precision for assay validations. Thus, once the extraction efficiency was calibrated for a given tissue type, tissue thickness and drug, the droplet-based approach provides a non-labour-intensive and high-throughput means to acquire spatially resolved quantitative analysis of multiple samples of the same type. Published in 2016. This article is a U.S. Government work and is in the public domain in the USA.