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In this article, we describe the process of sample preparation and method development for the analysis of polar small molecules using benzoyl chloride (BzCI) derivatization and liquid chromatography--tandem mass spectrometry [...]

Change history: In this Article, an extraneous label appeared in Fig. 4b, and has been removed in the online version., Author(s): Ali Mohebi [sup.1] , Jeffrey R. Pettibone [sup.1] , Arif A. Hamid [sup.2] , Jenny-Marie T. Wong [sup.3] , Leah T. Vinson [sup.4] , Tommaso Patriarchi [sup.5] , Lin [...]

Method qualification is a key step in the development of routine analytical monitoring of pharmaceutical products. However, when relying on published monographs that describe longer method times based on older high‐performance liquid chromatography column and instrument technology, this can delay the overall analysis process for generated drug products. In this study, high‐throughput ultrahigh pressure liquid chromatography techniques were implemented to decrease the amount of time needed to complete a 24‐run sequence to identify linearity, recovery, and repeatability for both drug assay and impurity analysis in 16 min. Multiple experimental parameters were tested to identify a range of experimental settings that could be used for the sequence while still maintaining this fast analysis time. The full sequence was replicated on a different system and with different columns, further demonstrating its robustness. [ABSTRACT FROM AUTHOR]

The faster drugs of abuse reach the brain, the greater is the risk of addiction. Even small differences in the rate of drug delivery can influence outcome. Infusing cocaine intravenously over 5 vs. 90–100 s promotes sensitization to the psychomotor and incentive motivational effects of the drug and preferentially recruits mesocorticolimbic regions. It remains unclear whether these effects are due to differences in how fast and/or how much drug reaches the brain. Here, we predicted that varying the rate of intravenous cocaine infusion between 5 and 90 s produces different rates of rise of brain drug concentrations, while producing similar peak concentrations. Freely moving male Wistar rats received acute intravenous cocaine infusions (2.0 mg/kg/infusion) over 5, 45 and 90 s. We measured cocaine concentrations in the dorsal striatum using rapid‐sampling microdialysis (1 sample/min) and high‐performance liquid chromatography‐tandem mass spectrometry. We also measured extracellular concentrations of dopamine and other neurochemicals. Regardless of infusion rate, acute cocaine did not change concentrations of non‐dopaminergic neurochemicals. Infusion rate did not significantly influence peak concentrations of cocaine or dopamine, but concentrations increased faster following 5‐s infusions. We also assessed psychomotor activity as a function of cocaine infusion rate. Infusion rate did not significantly influence total locomotion, but locomotion increased earlier following 5‐s infusions. Thus, small differences in the rate of cocaine delivery influence both the rate of rise of drug and dopamine concentrations, and psychomotor activity. A faster rate of rise of drug and dopamine concentrations might be an important issue in making rapidly delivered cocaine more addictive. [ABSTRACT FROM AUTHOR]

Neuropeptides are an important class of neurochemicals; however, measuring their concentration in vivo by using microdialysis sampling is challenging due to their low concentration and the small samples generated. Capillary liquid chromatography with mass spectrometry (cLC-MS) can yield attomole limits of detection (LOD); however, low recovery and loss of sample to adsorptive surfaces can still hinder detection of neuropeptides. We have evaluated recovery during sampling and transfer to the cLC column for a selection of 10 neuropeptides. Adding acetonitrile to sample eliminated carryover and improved LOD by 1.4- to 60-fold. The amount of acetonitrile required was found to have an optimal value that correlated with peptide molecular weight and retention time on a reversed phase LC column. Treating AN69 dialysis membrane, which bears negative charge due to incorporated sulfonate groups, with polyethylenimine (PEI) improved recovery by 1.2- to 80-fold. The effect appeared to be due to reducing electrostatic interaction between peptides and the microdialysis probe because modification increased recovery only for peptides that carried net positive charge. The combined effects improved LOD of the entire method by 1.3- to 800-fold for the different peptides. We conclude that peptides with both charged and hydrophobic regions require combined strategies to prevent adsorption and yield the best possible detection. The method was demonstrated by determining orexin A, orexin B, and a novel isoform of rat β-endorphin in the arcuate nucleus. Dialysate concentrations were below 10 pM for these peptides. A standard addition study on dialysates revealed that while some peptides can be accurately quantified, some are affected by the matrix. [ABSTRACT FROM AUTHOR]

The article presents a study that determines the role of striatal dysfunction protein torsin A in the dystonic-like movements and loss of striatal cholinergic neurons.

Hypoglycemia initiates the counter-regulatory response (CRR), in which the sympathetic nervous system, glucagon and glucocorticoids restore glucose to appropriate concentrations. During starvation, low leptin levels restrain energy utilization, enhancing long-term survival. To ensure short-term survival during hypoglycemia in fasted animals, the CRR must overcome this energy-sparing program and nutrient depletion. Here we identify in mice a previously unrecognized role for leptin and a population of leptin-regulated neurons that modulate the CRR to meet these challenges. Hypoglycemia activates neurons of the parabrachial nucleus (PBN) that coexpress leptin receptor (LepRb) and cholecystokinin (CCK) (PBN LepRbCCK neurons), which project to the ventromedial hypothalamic nucleus. Leptin inhibits these cells, and Cckcre-mediated ablation of LepRb enhances the CRR. Inhibition of PBN LepRb cells blunts the CRR, whereas their activation mimics the CRR in a CCK-dependent manner. PBN LepRbCCK neurons are a crucial component of the CRR system and may be a therapeutic target in hypoglycemia. [ABSTRACT FROM AUTHOR]