The Absolute Quantification of Endogenous Levels of Brain Neuropeptides In Vivo Using LC–MS/MS

Abstract

Neuropeptides seem to play an important role when the CNS is challenged. In order to obtain better insights into the central peptidergic effects, it is essential to monitor their concentration in the brain. Quantification of neuropeptides in dialysates is challenging due to their low extracellular concentrations (low pM range), their low microdialysis efficiencies, the need for acceptable temporal resolution, the small sample volumes, the complexity of the matrix and the tendency of peptides to stick to glass and polymeric materials. The quantification of neuropeptides in dialysates therefore necessitates the use of very sensitive nano-LC-MS/MS methods. A number of LC-MS/MS and microdialysis parameters need to be optimized to achieve maximal sensitivity. The optimized and validated methods can be used to investigate the in vivo neuropeptide release during pathological conditions, in this way initiating new and immense challenges for the development of new drugs.

Improved sensitivity of the nano ultra-high performance liquid chromatography-tandem mass spectrometric analysis of low-concentrated neuropeptides by reducing aspecific adsorption and optimizing the injection solvent

“…Indeed, developing bioanalytical methods for peptides is challenging, since their behaviour under various sample preparation and chromatographic conditions is still not well understood [11,12]. Additional challenges, discussed in detail by Van Eeckhaut et al [13], include the low extracellular concentration of peptides in the brain (ranging from sub-pico to femtomolar levels), the low microdialysis efficiency, the need for acceptable temporal resolution, the small sample volumes, the complexity of the matrix and the tendency of peptides to stick to several surfaces. Owing to the high sensitivity and selectivity obtained, miniaturized ultra-high performance liquid chromatography-tandem mass spectrometry (nano UHPLC-MS/MS) has become the method of choice for the quantification of neuropeptides in microdialysis samples [13][14][15][16][17][18].…”

“…Additional challenges, discussed in detail by Van Eeckhaut et al [13], include the low extracellular concentration of peptides in the brain (ranging from sub-pico to femtomolar levels), the low microdialysis efficiency, the need for acceptable temporal resolution, the small sample volumes, the complexity of the matrix and the tendency of peptides to stick to several surfaces. Owing to the high sensitivity and selectivity obtained, miniaturized ultra-high performance liquid chromatography-tandem mass spectrometry (nano UHPLC-MS/MS) has become the method of choice for the quantification of neuropeptides in microdialysis samples [13][14][15][16][17][18]. As the peptides of the neuromedin family differ considerably in size, ranging from 6 (neuromedin N) to 36 (neuromedin S) amino acid residues, as well as in physicochemical properties [4], a tailored method optimization strategy is required.…”

Reassessment of the antioxidative mixture for the challenging electrochemical determination of dopamine, noradrenaline and serotonin in microdialysis samples

“…To quantify changes in the extracellular release of the monoamines in a specific brain area in response to a pharmacological stimulus, the in vivo microdialysis sampling technique is typically applied [9,10], combined with ion-pair (ultra-) high performance liquid chromatography ((U)HPLC) coupled to electrochemical detection (ECD) [9,[11][12][13][14][15][16]. Thanks to the latest advances in miniaturized and high throughput techniques, researchers can start exploring more challenging, small brain areas which may contain particularly low extracellular monoamine levels (pM range).…”

An Ultrasensitive Nano UHPLC–Esi–MS/MS Method for The Quantification of Three Neuromedin-Like Peptides in Microdialysates

“…Compared with the classical neurotransmitters, neuropeptides are released in much lower amounts (ranging from sub-pico to femtomolar levels) because of their higher potency [1,13]. In addition, the microdialysis recovery of peptides is smaller due to their tendency to stick to the material of the microdialysis setup, such as the probe membrane and the tubing [13,14]. The low neuropeptide content in the collection tube can decrease even further due to instability in the dialysate matrix, a complex mixture of small endogenous compounds and salts [14].…”

“…To monitor brain neuromedin levels and dynamics as a function of time, microdialysis is used as in vivo sampling tool [12,13]. Compared with the classical neurotransmitters, neuropeptides are released in much lower amounts (ranging from sub-pico to femtomolar levels) because of their higher potency [1,13].…”