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In recent years, the simultaneous monitoring of the monoamine neurotransmitters dopamine, noradrenaline and serotonin in vivo is advancing due to innovations in miniaturized and fast chromatographic techniques. However, the determination of the most hydrophilic compound, noradrenaline, in microdialysis samples by (ultra-)high performance liquid chromatography ((U)HPLC) with electrochemical detection (ECD) is impeded by a broad solvent front, caused by the addition of antioxidative agents. Hence, an elaborate reassessment of currently used antioxidative mixtures is necessary for further analytical improvements. The proposed mixture, containing 100mM acetic acid, 0.27mM Na2EDTA and 12.5μM ascorbic acid (pH 3.2), is less complex than previously described mixtures and shows minimal ECD interference. It stabilizes the three monoamines in standard solutions and in microdialysis samples, considering both autosampler stability at 4°C for 48h and long term stability at -20°C for a duration of six months. An in vivo microdialysis experiment demonstrates the possibility to monitor changes in extracellular levels of the three monoamines simultaneously in the rat hippocampus with UHPLC-ECD using the optimized antioxidative mixture., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Testing of new therapeutic strategies for Parkinson's disease (PD) is currently hampered by the lack of relevant and reproducible animal models. Here, we developed a robust rat model for PD by injection of adeno-associated viral vectors (rAAV2/7) encoding α-synuclein into the substantia nigra, resulting in reproducible nigrostriatal pathology and behavioral deficits in a 4-week time period. Progressive dopaminergic dysfunction was corroborated by histopathologic and biochemical analysis, motor behavior testing and in vivo microdialysis. L-DOPA treatment was found to reverse the behavioral phenotype. Non-invasive positron emission tomography imaging and magnetic resonance spectroscopy allowed longitudinal monitoring of neurodegeneration. In addition, insoluble α-synuclein aggregates were formed in this model. This α-synuclein rat model shows improved face and predictive validity, and therefore offers the possibility to reliably test novel therapeutics. Furthermore, it will be of great value for further research into the molecular pathogenesis of PD and the importance of α-synuclein aggregation in the disease process., (Copyright © 2015 Elsevier Inc. All rights reserved.)

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., (Copyright © 2014 Elsevier B.V. All rights reserved.)

In the drug-discovery setting, the development of new peptide and protein-based biopharmaceuticals attracts increased attention from the pharmaceutical industry and consequently demands the development of high-throughput LC-MS methods. Regulatory guidelines require bioanalytical methods to be validated not only in terms of linearity, sensitivity, accuracy, precision, selectivity and stability, but also in terms of carryover. Carryover results from the aspecific adsorption of analyte(s) to parts of the analytical system and thus introduces bias in both identification and quantification assays. Moreover, nonspecific binding occurs at the surface of materials used during sample preparation, such as pipette tips, sample tubes and LC-vials. Hence, linearity, sensitivity and repeatability of the analyses are negatively affected. Due to the great diversity in physicochemical properties of biomolecules, there is no general approach available to minimize adsorption phenomena. Therefore, we aim to present different strategies which can be generically applied to reduce nonspecific binding of peptides and proteins. In the first part of this review, a systematic approach is proposed to guide the reader through the different solvents which can be used to dissolve the analyte of interest. Indeed, proper solubilization is one of the most important factors for a successful analysis. In addition, alternative approaches are described to improve analyte recovery from the sample vial. The second part focuses on strategies to efficiently reduce adsorption at components of the autosampler, column and mass spectrometer. Thereby carryover is reduced while maintaining a sufficiently wide dynamic range of the assay., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The use of ion-pair ultra-high performance liquid chromatography (UHPLC) coupled with electrochemical detection (ECD) is of great interest for the fast and sensitive determination of the monoamine neurotransmitters dopamine, noradrenaline and serotonin in microdialysis samples. However, when applying high flow rates in ion-pair UHPLC, other peaks than the initial compound peaks appear on the chromatogram. This peak-splitting phenomenon is caused by disturbed ion-pair retention mechanisms. The influence of several chromatographic parameters is investigated. Peak-splitting is delayed to higher flow rates when increasing the concentration of ion-pair reagent or buffering agent in the mobile phase, when decreasing the percentage of organic modifier in the mobile phase, when applying a stationary phase with a smaller amount of packing material or when increasing the separation temperature. One or a combination of these conditions can be applied to analyze the monoamine neurotransmitters using ion-pair UHPLC-ECD at high flow rates., (Copyright © 2013 Elsevier B.V. All rights reserved.)

A liquid chromatographic method with fluorescence detection was developed and validated for the quantification of the antidepressant reboxetine (RBX), a selective noradrenalin reuptake inhibitor, in rat brain microdialysates. After modification of the method in terms of sample preparation and sensitivity, it was also validated for the quantification of RBX in rat plasma samples. To enable fluorescence detection, a pre-column derivatization step with 9-fluorenylmethyl chloroformate was included. Separations were performed on a reversed phase C₁₈ column using gradient elution. The retention time for RBX was found to be 8.8 min. The assay of RBX in brain microdialysis samples showed a linear relationship in the calibration curve from 2 to 200 ng/mL, with a correlation coefficient ≥0.999. The limit of detection (LOD) and the lower limit of quantification (LLOQ) were 0.6 and 2.0 ng/mL respectively. The intra-day and the inter-day precision (RSD %) ranged between 1.5% and 11.7% with an average recovery of 101.2±8.2% (mean±SD, n=40). For the analysis of plasma samples, the calibration curve was linear between 20 and 700 ng/mL with a correlation coefficient ≥0.999. LOD and LLOQ were 6 and 20 ng/mL respectively. The intra-day and the inter-day precision (RSD %) ranged between 1.7% and 11.5% with an average recovery of 98.5±7.3% (mean±SD, n=40). We demonstrated the applicability of the method to determine the concentration-time profiles of RBX in brain and plasma following systemic administration., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Liquid chromatography coupled to atmospheric pressure ionization tandem mass spectrometry is currently the method of choice for the quantitative determination of drugs in biological matrices. The advantages of this technique include high specificity, sensitivity and throughput. However, co-eluting matrix components, which are not observed in the chromatogram, can have a detrimental effect on the analysis, since they can cause ion suppression or enhancement of the analyte. The evaluation of matrix effects on the quantitative analysis of drugs in biological fluids is an important and sometimes overlooked aspect of assay validation. In this review, the influence of matrix effects on bioanalytical LC-MS/MS methods is discussed and illustrated with some examples. In addition, possible solutions to reduce or eliminate matrix effects are highlighted. A literature overview of validated LC-MS/MS methods published from January till June 2008 is also included. Although matrix effects are investigated in most papers, there is no consensus on how matrix effects should be evaluated during method validation. In addition, the definition of specificity should be changed for LC-MS/MS based methods.

Microdialysis (MD) is an in vivo sampling technique used to investigate biochemical events in the extracellular fluid of animal and human tissues. MD produces protein- and cell-free, aqueous samples which can be analyzed without further sample clean-up. Liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) is a sensitive and selective analysis technique which is suitable to quantify low concentrated target analytes in microdialysates. This paper reviews the LC-MS/MS methods which are described for the quantification of endogenous molecules, such as neurotransmitters and peptides, and of exogenous molecules, such as drugs, in microdialysates. Since miniaturization of the LC-MS/MS methods is the key to obtain maximal sensitivity of the analytical technique, this feature is discussed in the paper. In addition, critical issues related to the quantification of low concentrated molecules in microdialysates are described such as the presence of matrix effects, the low MD efficiency and the sticking of, for instance, neuropeptides.

Enhanced sensitivity is a well known benefit of miniaturised LC-electrospray (ESI)-MS/MS methods. The suitability of miniaturised LC-MS/MS for quantification of small molecules in dialysates was investigated using the anti-epileptic drug oxcarbazepine, its active metabolite, 10,11-dihydro-10-hydroxycarbamazepine, and the internal standard for microdialysis probe calibration, 2-methyl-5H-dibenz(b,f)azepine-5-carboxamide, as test compounds. ESI-MS detection is sensitive to matrix effects. Therefore, dialysate matrix effects were investigated by comparing the responses of standards made in water, Ringer's solution (salt solution used as perfusion fluid) and blank dialysate matrix. Due to the occurrence of ion suppression or enhancement, direct injection of dialysis samples onto the analytical column could not be applied for quantification of small molecules in dialysis samples. Column switching was necessary for desalting and preconcentration of the dialysates. However, this approach was not able to completely eliminate salt effects when the injection volume exceeded 1 microL. No differences in response between Ringer's solution and dialysate matrix were detected at capillary and nano-dimensions. Calibration standards should be prepared with Ringer's solution instead of water for quantitative analysis of microdialysates. A microbore, capillary and nano-LC-ESI-MS/MS method were compared in terms of method feasibility, linearity, sensitivity, accuracy and precision. Downscaling to capillary and nano-dimensions resulted in a gain in detection sensitivity of 5 and 50, respectively. Miniaturised LC-MS/MS was found to be fit for quantification of small molecules in dialysates with acceptable accuracy and method precision.

A microbore LC-MS/MS method is developed and validated for the quantification of the anti-epileptic drug oxcarbazepine (OXC) and its active metabolite 10,11-dihydro-10-hydroxycarbamazepine (MHD) in rat brain microdialysates, together with the internal standard for microdialysis probe calibration, 2-methyl-5H-dibenz(b,f)azepine-5-carboxamide (m-CBZ). The benefits of gradient versus isocratic separation are shown, next to the improved sensitivity resulting from the addition of 0.1% formic acid to the mobile phase. The coupling of microdialysis with ESI-MS requires sample desalting for which column switching was applied. Using weighed regression to calculate the calibration curves (1-1000 ng/mL), the assay was validated in terms of linearity, accuracy and precision, yielding a sensitive (limit of quantification is 1 ng/mL) and selective method for quantification of OXC, MHD and m-CBZ. By applying this method, we were able to determine the extracellular concentrations of OXC and MHD during at least 4h after intraperitoneal (i.p.) administration of 10 mg/kg OXC.

A capillary zone electrophoresis method has been developed for the detection of 0.1% of (R)-levochlorpheniramine maleate in samples of (S)-dexchlorpheniramine maleate. Using 1.5 mM carboxymethyl-beta-cyclodextrin in an acidic background electrolyte, resolution values of more than 10 were obtained. Under these conditions the R-enantiomer is migrating in front of the bulk S-enantiomer. The assay was validated for linearity (2-10 microg/ml; R2 = 0.9992), selectivity [(RS)-pheniramine maleate and (RS)-brompheniramine maleate], limit of detection (0.25 microg/ml), limit of quantification (0.75 microg/ml), analytical precision (intra- and inter-day variability), repeatability of the method (RSD = 5.0%) and accuracy. In samples of dexchlorpheniramine maleate from two different manufacturers, concentrations of, respectively, 0.15% and 1.95% (m/m) of levochlorpheniramine maleate were detected. The method was compared to the HPLC method described in the European Pharmacopoeia III monograph.