A comparative study of UniSpray and electrospray sources for the ionization of neuropeptides in liquid chromatography tandem mass spectrometry.

Despite the extensive use of electrospray ionization (ESI) for the quantification of neuropeptides by liquid chromatography-tandem mass spectrometry (LC-MS/MS), poor ionization and transmission efficiency are described for this ionization interface. A new atmospheric pressure ionization source, named UniSpray, was recently developed and commercialized. In this study, the LC-MS performance of this new ionization interface is evaluated and compared with ESI for the quantification of seven neuropeptides. Besides comparison of signal intensities and charge state distributions, also signal-to-noise (S/N) ratios and accuracy and precision were assessed. Additionally, matrix effects of human precipitated plasma and rat microdialysate were evaluated as well as the effect of three supercharging agents on the ionization of the seven neuropeptides. UniSpray ionization resulted in signal intensities four to eight times higher at the optimal capillary/impactor voltage for all seven neuropeptides. S/N values at the other hand only increased by not more than a twofold when the UniSpray source was used. Moreover, UniSpray ionization resulted in a shift towards lower charge states for some neuropeptides. Evaluation of the matrix effects by a post-column infusion set-up resulted in different infusion profiles between ESI and UniSpray. The charge state distributions of the neuropeptides obtained with UniSpray are highly comparable with ESI. Finally, the effect of the supercharging agents on the ionization of the neuropeptides tends to be peptide-dependent with both ionization sources.

Assessing mixtures of supercharging agents to increase the abundance of a specific charge state of Neuromedin U.

With increasing evidence of the important role of peptides in pathophysiological processes, a trend towards the development of highly sensitive bioanalytical methods is ongoing. Inherent to the electrospray ionization process of peptides and proteins is the production of multiple charge states which may hamper proper and sensitive method development. Supercharging agents allow modifying the maximal charge state and the corresponding distribution of charges, thereby potentially increasing the number of ions reaching the detector in selected reaction monitoring mode. In this study, the use of mixtures of charge state modifying additives, i.e. m-nitrobenzylalcohol (mNBA), sulfolane and dimethyl sulfoxide (DMSO), to specifically increase the abundance of one charge state of interest has been investigated. Screening experiments were performed to define an experimental domain, which was then further investigated via a response surface design to predict the optimal combination and concentration of superchargers. Using a combination of mNBA and DMSO (0.008% and 0.5% m/v respectively), we were able to increase the abundance of the +4 charge state of the investigated peptide neuromedin U from 64% to 87%. Unfortunately, charge state coalescence did not result in repeatable sensitivity improvements in this case study. However, it remains an attractive approach during method development of peptide bioanalytical methods, as coalescence to a particular intermediate charge state is difficult to obtain by using only one supercharging agent.

Biodegradable Amphipathic Peptide Hydrogels as Extended-Release System for Opioid Peptides.

Chronic pain is currently treated with opioids that offer unsatisfactory long-term analgesia and produce serious side effects. There is a clear need for alternative therapies. Herein, peptide-based hydrogels are used as extended-release drug delivery carriers. Two different formulations were developed: the drug is coformulated within the hydrogel; the drug is an integral part of the hydrogelator. Both strategies afford a prolonged and significant antinociception up to 72 h after subcutaneous administration in mice.

Synthesis and in Vitro Evaluation of Stabilized and Selective Neuromedin U-1 Receptor Agonists.

Neuromedin U (NMU) is a multifunctional neuropeptide which is characterized by a high conservation through all species. Herein, we describe the synthesis of a novel set of NMU-analogs based on the truncated NMU-8. Through combination of previously reported modifications, an elaborate structure-activity relationship study was performed aiming for the development of peptides with an increased selectivity toward NMU receptor 1 (NMUR1). Compound 7 possessed the highest NMUR1 selectivity (IC50 = 0.54 nM, selectivity ratio = 5313) together with an increased potency (EC50 = 3.7 nM), an 18% increase of the maximal effect at NMUR1, and a higher resistance against enzymatic degradation as compared to the native NMU-8. The development of a potent NMUR1 agonist with extended half-life could represent an attractive tool to further unveil the role of NMUR1 in NMU signaling.

Development of potent and proteolytically stable human neuromedin U receptor agonists.

Neuromedin U (NMU) is a highly conserved endogenous peptide that is involved in a wide range of physiological processes such as regulation of feeding behavior, the stress response and nociception. The major limitation to use NMU as a therapeutic is its short half-life. Here, we describe the development of a set of novel NMU-analogs based on NMU-8, by introducing unnatural amino acids into the native sequence. This approach shows that it is possible to generate molecules with increased potency and improved plasma stability without major changes of the peptidic nature or the introduction of large conjugates. When compared to the native NMU-8 peptide, compounds 16, 18 and 20 have potent agonist activity and affinity for both NMU receptors. Selectivity towards NMUR1 was observed when the Phe residue in position 4 was modified, whereas higher potencies at NMUR2 were found when substitutions of the Pro residue in position 6 were executed. To study the effect of the modifications on the proteolytic stability of the molecules, an in vitro stability assay in human plasma at 37 °C was performed. All analyzed analogs possessed an increased resistance against enzymatic degradation in human plasma resulting in half-lifes from 4 min for NMU-8, up to more than 23 h for compound 42.

LC-method development for the quantification of neuromedin-like peptides. Emphasis on column choice and mobile phase composition.

In this study, the separation of four neuromedin-like peptides is investigated on four different core-shell stationary phases. Moreover, the effect of the mobile phase composition, i.e. organic modifier (acetonitrile and methanol) and additive (trifluoroacetic acid, formic acid, acetic acid, ammonium formate and ammonium acetate) on the chromatographic performance is studied. An improvement in chromatographic performance is observed when using the ammonium salt instead of its corresponding acid as additive, except for the column containing a positively charged surface (C18+). In general, the RP-Amide column provided the highest separation power with different mobile phases. However, for the neuromedin-like peptides of interest, the C18+ column in combination with a mobile phase containing methanol as organic modifier and acetic acid as additive provided narrower and higher peaks. A three-factor, three-level design is applied to further optimize the method in terms of increased peak height and reduced solvent consumption, without loss in resolution. The optimized method was subsequently used to assess the in vitro microdialysis recovery of the peptides of interest. Recovery values between 4 and 8% were obtained using a perfusion flow rate of 2µL/min.

Challenges for the in vivo quantification of brain neuropeptides using microdialysis sampling and LC-MS.

In recent years, neuropeptides and their receptors have received an increased interest in neuropharmacological research. Although these molecules are considered relatively small compared with proteins, their in vivo quantification using microdialysis is more challenging than for small molecules. Low microdialysis recoveries, aspecific adsorption and the presence of various multiply charged precursor ions during ESI-MS/MS detection hampers the in vivo quantification of these low abundant biomolecules. Every step in the workflow, from sampling until analysis, has to be optimized to enable the sensitive analysis of these compounds in microdialysates.

Mixed α/ß-Peptides as a Class of Short Amphipathic Peptide Hydrogelators with Enhanced Proteolytic Stability.

Peptide hydrogels are a highly promising class of materials for biomedical application, albeit facing many challenges with regard to stability and tunability. Here, we report a new class of amphipathic peptide hydrogelators, namely mixed α/ß-peptide hydrogelators. These mixed α/ß-gelators possess good rheological properties (high storage moduli) and form transparent self-supporting gels with shear-thinning behavior. Infrared spectroscopy indicates the presence of ß-sheets as the underlying secondary structure. Interestingly, self-assembled nanofibers of the mixed α/ß-peptides display unique structural morphologies with alteration of the C-terminus (acid vs amide) playing a key role in the fiber formation and gelation properties of the resulting hydrogels. The incorporation of ß3-homoamino acid residues within the mixed α/ß-peptide gelators led to an increase in proteolytic stability of the peptides under nongelating conditions (in solution) as well as gelating conditions (as hydrogel). Under diluted conditions, degradation of mixed α/ß-peptides in the presence of elastase was slowed down 120-fold compared to that of an α-peptide, thereby demonstrating beneficial enzymatic resistance for hydrogel applications in vivo. In addition, increased half-life values were obtained for the mixed α/ß-peptides in human blood plasma, as compared to corresponding α-peptides. It was also found that the mixed α/ß-peptides were amenable to injection via needles used for subcutaneous administrations. The preformed peptide gels could be sheared upon injection and were found to quickly reform to a state close to that of the original hydrogel. The shown properties of enhanced proteolytic stability and injectability hold great promise for the use of these novel mixed α/ß-peptide hydrogels for applications in the areas of tissue engineering and drug delivery.

An improved microbore UHPLC method with electrochemical detection for the simultaneous determination of low monoamine levels in in vivo brain microdialysis samples.

The simultaneous determination of the monoamines dopamine (DA), noradrenaline (NA) and serotonin (5-HT) in in vivo microdialysis samples remains challenging because of the low extracellular neurotransmitter levels in different brain regions, specific sample characteristics, and the quest for high temporal resolution and a multi-target strategy in neuropharmacological research. A fast and sensitive microbore (1.0mm i.d. column) UHPLC method coupled to electrochemical detection (ECD) is developed by means of design of experiments with the emphasis on sufficient retention of NA within an acceptable total analysis time. Indeed, NA is the earliest eluting compound and often interferes with the broad solvent front originating from the sample matrix. The sensitive UHPLC-ECD assay (LLOQ of 100pM for NA and 150pM for DA and 5-HT) with an analysis time of 8min for standard solutions and 20min for in vivo microdialysis samples originating from rat hippocampus, prefrontal cortex and striatum, is validated applying accuracy profiles. The combination of in vivo microdialysis and microbore UHPLC-ECD has shown to be particularly suitable for future contributions to neuropharmacological research on the monoaminergic system.

Miniaturized ultra-high performance liquid chromatography coupled to electrochemical detection: Investigation of system performance for neurochemical analysis.

The interest in implementation of miniaturized ultra-high performance liquid chromatography (UHPLC) in neurochemical research is growing because of the need for faster, more selective and more sensitive neurotransmitter analyses. The instrument performance of a tailor designed microbore UHPLC system coupled to electrochemical detection (ECD) is investigated, focusing on the quantitative monoamine determination in in vivo microdialysis samples. The use of a microbore column (1.0mm I.D.) requires miniaturization of the entire instrument, though a balance between extra-column band broadening and injection volume must be considered. This is accomplished through the user defined Performance Optimizing Injection Sequence, whereby 5 µL sample is injected on the column with a measured extra-column variance of 4.5-9.0 µL(2) and only 7 µL sample uptake. Different sub-2 µm and superficially porous particle stationary phases are compared by means of the kinetic plot approach. Peak efficiencies of about 16000-35000 theoretical plates are obtained for the Acquity UPLC BEH C18 column within 13 min analysis time. Furthermore, the coupling to ECD is shown suitable for microbore UHPLC analysis thanks to the miniaturized flow cell design, sufficiently fast data acquisition and mathematical data filtering. Ultimately, injection of in vivo samples demonstrates the applicability of the system for microdialysis analysis.

An ultrasensitive nano UHPLC-ESI-MS/MS method for the quantification of three neuromedin-like peptides in microdialysates.

AIM: An ultrasensitive nano UHPLC-ESI-MS/MS method is developed to simultaneously monitor three low-concentration neuromedin-like peptides in microdialysates. RESULTS: Peptide preconcentration and sample desalting is performed online on a trap column. A shallow gradient slope at 300 nl/min on the analytical column maintained at 35°C, followed by two saw-tooth column wash cycles, results in the highest sensitivity and the lowest carryover. The validated method allows the accurate and precise quantification of 0.5 pM neurotensin and neuromedin N (2.5 amol on column), and of 3.0 pM neuromedin B (15.0 amol on column) in in vivo microdialysates without the use of internal standards. CONCLUSION: The assay is an important tool for elucidating the role of these neuromedin-like peptides in the pathophysiology of neurological disorders.

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.

Obtaining maximal sensitivity of nano UHPLC-MS/MS methods is primordial to quantify picomolar concentrations of neuropeptides in microdialysis samples. Since aspecific adsorption of peptides to Eppendorf tubes, pipette tips and UHPLC vials is detrimental for method sensitivity, a strategy is presented to reduce adsorption of these peptides during standard preparation. Within this respect, all procedural steps from dissolution of the lyophilized powder until the injection of the sample onto the system are investigated. Two peptides of the neuromedin family, i.e. neuromedin B and neuromedin N, and a neuromedin N-related neuropeptide, neurotensin, are evaluated. The first part of this study outlines a number of parameters which are known to affect peptide solubility. The main focus of the second part involves the optimization of the sample composition in the UHPLC vial by using design of experiments. Contradictory findings are observed concerning the influence of acetonitrile, salts and matrix components. They are found important for injection of the peptides into the system, but crucially need to be excluded from the dilution solvent. Furthermore, the type of surface material, temperature and the pipetting protocol considerably affect the adsorption phenomenon. Statistical analysis on the results of the central composite design reveals that the highest peptide responses are obtained with the injection solvent consisting of 13.1% V/V ACN and 4.4% V/V FA. This aspect of the optimization strategy can be identified as the main contributor to the gain in method sensitivity. Since the reduction of peptide adsorption and the optimization of the injection solvent resulted in a clear and quantifiable signal of the three peptides, optimization of both issues should be considered in the early stage of method development, in particular when the analysis of low-concentration peptide solutions is envisaged.