To support pharmacokinetic and drug metabolism studies, LC-MS/MS plays more and more an essential role for the quantitation of drugs and their metabolites in biological matrices. With the new challenges encountered in drug discovery and drug development, new strategies are put in place to achieve high-throughput analysis, using serial and parallel approaches. To speed-up method development and validation, generic approaches with the direct injection of biological fluids is highly desirable. Column-switching, using various packing materials for the extraction columns, is widely applied. Improvement of mass spectrometers performance, and in particular triple quadrupoles, also strongly influences sample preparation strategies, which remain a key element in the bioanalytical process.
Recently, linear ion traps (LITs) have been combined with quadrupole (Q), time-of-flight (TOF) and Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry (MS). LITs can be used either as ion accumulation devices or as commercially available, stand-alone mass spectrometers with MSn capabilities. The combination of triple quadrupole MS with LIT technology in the form of an instrument of configuration QqLIT, using axial ejection, is particularly interesting, because this instrument retains the classical triple quadrupole scan functions such as selected reaction monitoring (SRM), product ion (PI), neutral loss (NL) and precursor ion (PC) while also providing access to sensitive ion trap experiments. For small molecules, quantitative and qualitative analysis can be performed using the same instrument. In addition, for peptide analysis, the enhanced multiply charged (EMC) scan allows an increase in selectivity, while the time-delayed fragmentation (TDF) scan provides additional structural information. Various methods of operating the hybrid instrument are described for the case of the commercial Q TRAP (AB/MDS Sciex) and applications to drug metabolism analysis, quantitative confirmatory analysis, peptides analysis and automated nanoelectrospray (ESI-chip-MS) analysis are discussed.
The present work investigates various method development aspects for the quantitative analysis of pharmaceutical compounds in human plasma using matrix-assisted laser desorption/ionization and multiple reaction monitoring (MALDI-MRM). Talinolol was selected as a model analyte. Liquid-liquid extraction (LLE) and protein precipitation were evaluated regarding sensitivity and throughput for the MALDI-MRM technique and its applicability without and with chromatographic separation. Compared to classical electrospray liquid chromatography/mass spectrometry (LC/ESI-MS) method development, with MALDI-MRM the tuning of the analyte in single MS mode is more challenging due to interfering matrix background ions. An approach is proposed using background subtraction. With LLE and using a 200 microL human plasma aliquot acceptable precision and accuracy could be obtained in the range of 1 to 1000 ng/mL without any LC separation. Approximately 3 s were required for one analysis. A full calibration curve and its quality control samples (20 samples) can be analyzed within 1 min. Combining LC with the MALDI analysis allowed improving the linearity down to 50 pg/mL, while reducing the throughput potential only by two-fold. Matrix effects are still a significant issue with MALDI but can be monitored in a similar way to that used for LC/ESI-MS analysis.
In nonclinical drug development targeting the central nervous system (CNS), the quantitative determination of extracellular brain concentrations of neurotransmitters is a key challenge. In some CNS disorders, the monitoring of the modified profile of neurotransmitter release such as that of histamine may explain the mechanism of action of the drug candidate. Microdialysis is a commonly used method for sampling extracellular levels of neurotransmitters/drug candidates in small laboratory animals. Detection and quantification of extracellular levels of neurotransmitters remain an analytical and technical challenge owing to the low concentrations of neurotransmitters collected, the small microdialysis sample size, and the high amount of inorganic salts. A precolumn derivatization strategy prior to hydrophilic interaction liquid chromatography (HILIC)-tandem mass spectrometry analysis is proposed to quantify histamine release after administration of a CNS research compound. Derivatization using propionic anhydride dissolved in organic solvent combined with the HILIC approach effectively eliminated three time-consuming steps, organic layer transfer, dry down, and reconstitution, all of which are required by traditional reversed-phase liquid chromatography. The formation of propionylated amides, performed under mild conditions, required no further sample cleanup. After a dual microdialysis probe implantation into the prefrontal cortex (neurotransmitters) and in the inferior vena cava of rat (drug candidate), microdialysate fractions were collected every 15 min for 8 h and stored frozen at -20 °C until analysis. The method was validated using 10 μL microdialysate, achieving low limits of quantitation of 83.4 and 84.5 pg.mL(-1) for histamine and 1-methylhistamine, respectively. These limits were suitable to assess kinetic release of neurotransmitters and are compatible with those obtained by microdialysis sampling. This method provided the required selectivity, sensitivity, accuracy, and precision to assess release kinetics of histamine and 1-methylhistamine in several hundred rat brain microdialysates after intravenous infusion of CNS drug candidates.
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