Quantification Of Neurotransmitters Research Articles

In vivo study on the neurotransmitters and their metabolites change in depressive disorder rat plasma by ultra high performance liquid chromatography coupled to tandem mass spectrometry.

A sensitive and versatile, ultra-high performance, liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method coupled to pre-column derivatization for the simultaneous determination of 5-hydroxytryptamine (5-HT), 5-hydroxyindoleacetic acid (5-HIAA), dopamine (DA), norepinephrine (NE), homovanillic acid (HVA), γ-aminobutyric acid (GABA) and glutamic acid (Glu) was developed and validated in rat plasma. The analytes were dansylated under strong alkaline conditions after protein precipitation extraction, which were analyzed on a BEH C18 column using a gradient elution. The lower limit of quantification (LLOQ) values for 5-HT, 5-HIAA, DA, NE, HVA, GABA and Glu were 1.00, 1.00, 0.991, 0.992, 1.02, 1000, and 5030 pmol/mL, respectively. Good linearity was obtained (r > 0.99) and the intra- and inter-day precisions of the method (relative standard deviation, RSD%) were lower than 12%. The method was novel, sensitive and specific which can provide an alternative method for the quantification of neurotransmitters and their metabolites in plasma samples.

Derivatization for the simultaneous LC/MS quantification of multiple neurotransmitters in extracellular fluid from rat brain microdialysis

Quantification of amino acid based neurotransmitters in extracellular fluids, such as those in the neuron synapse, presents a challenge to the analytical chemistry because of the absence of UV- or fluorescence-detectable functional groups and the low sensitivity in mass spectrometric detection. This report describes a novel use of the succinimide reagent, N-α-Boc-l-tryptophan hydroxysuccinimide ester (Boc-TRP), for the pre-column derivatization to simultaneously quantify multiple neurotransmitters in the rat brain microdialysis samples. The Boc-TRP derivatization was rapid and quantitative in phosphate the buffer (pH 7.4) at room temperature. The derivatized neurotransmitters were suitable for rapid LC/MS quantification with less than 3-min chromatographic separation. The Boc-group in the derivatized product generated unique fragmentation patterns in the triple quadrupole mass spectrometric analysis under Multiple Reaction Monitoring mode and significantly increased the specificity and sensitivity. The derivatization and rapid LC/MS quantification method developed in this study showed a linear dynamic range from single digit nM to 1000nM with coefficient greater than 0.990. At the LOQ, the accuracy ranged from 95 to 108% and the precision (CV%) was less than 20%. Since there was no concentration and reconstitution in the sample workup process, this derivatization approach simplified the neurotransmitter quantification of the brain microdialysis samples.

Pattern Recognition of Dopamine, Epinephrine, Norepinephrine, Serotonin, Acethylcholine, GABA, and Glutamate in Biological Samples Using Stochastic Sensing

Pattern recognition is essential in chemical analysis of biological fluids. Reliable and sensitive methods for neurotransmitters analysis are needed. Therefore, we developed for pattern recognition of neurotransmitters dopamine, epinephrine, norepinephrine, serotonin, acethylcholine, GABA, and glutamate a method based on stochastic sensing. Microsensors based on diamond paste modified with 5,10,15,20-tetraphenyl-21H,23H-porphyrine, hemin and protoporphyrin IX in stochastic were designed and characterized. The proposed method can perform pattern recognition of the seven neurotransmitters on biological fluids at a lower determination level than chromatographic methods. The pattern recognition showed high reliability (RSD<1% for more than 6 months) for the simultaneous determination of the seven neurotransmitters in whole blood, cerebrospinal liquid, urine, zebra fish and snails. Accordingly with the statistic evaluation of the results at 99.00% confidence level, the proposed sensors can be used for pattern recognition and quantification of neurotransmitters with high reliability. The best stochastic microsensor was the one based on diamond paste modified with protoporphyrin IX.Acknowledgements The present work was supported by the European Commission through the DENAMIC project (Contract-No. 282957) and by UEFISCDI, Romania Contract-no. 186/23.10.2012.

Pattern recognition of neurotransmitters using multimode sensing

Pattern recognition is essential in chemical analysis of biological fluids. Reliable and sensitive methods for neurotransmitters analysis are needed. Therefore, we developed for pattern recognition of neurotransmitters: dopamine, epinephrine, norepinephrine a method based on multimode sensing. Multimode sensing was performed using microsensors based on diamond paste modified with 5,10,15,20-tetraphenyl-21H,23H-porphyrine, hemin and protoporphyrin IX in stochastic and differential pulse voltammetry modes. Optimized working conditions: phosphate buffer solution of pH 3.01 and KCl 0.1mol/L (as electrolyte support), were determined using cyclic voltammetry and used in all measurements. The lowest limits of quantification were: 10(-10)mol/L for dopamine and epinephrine, and 10(-11)mol/L for norepinephrine. The multimode microsensors were selective over ascorbic and uric acids and the method facilitated reliable assay of neurotransmitters in urine samples, and therefore, the pattern recognition showed high reliability (RSD<1% for more than 6 months) for the simultaneous determination of dopamine, epinephrine and norepinephrine from urine and whole blood samples. The proposed method can perform pattern recognition of the three neurotransmitters on biological fluids at a lower determination level than chromatographic methods. The sampling of the biological fluids referees only to the buffering (1:1, v/v) with a phosphate buffer pH 3.01, while for chromatographic methods the sampling is laborious. Accordingly with the statistic evaluation of the results at 99.00% confidence level, both modes can be used for pattern recognition and quantification of neurotransmitters with high reliability. The best multimode microsensor was the one based on diamond paste modified with protoporphyrin IX.

Quantification of neurotransmitters in mouse brain tissue by using liquid chromatography coupled electrospray tandem mass spectrometry.

A simple and rapid liquid chromatography tandem mass spectrometry method has been developed for the determination of BH4, DA, 5-HT, NE, EP, Glu, and GABA in mouse brain using epsilon-acetamidocaproic acid and isotopically labeled neurotransmitters as internal standards. Proteins in the samples were precipitated by adding acetonitrile, and then the supernatants were separated by a Sepax Polar-Imidazole (2.1 mm × 100 mm, i.d., 3 μm) column by adding a mixture of 10 mM ammonium formate in acetonitrile/water (75 : 25, v/v, 300 μl/min) for BH4 and DA. To assay 5-HT, NE, EP, Glu, and GABA; a Luna 3 μ C18 (3.0 mm × 150 mm, i.d., 3 μm) column was used by adding a mixture of 1% formic acid in acetonitrile/water (20 : 80, v/v, 350 μl/min). The total chromatographic run time was 5.5 min. The method was validated for the analysis of samples. The calibration curve was linear between 10 and 2000 ng/g for BH4 (r2 = 0.995) , 10 and 5000 ng/g for DA (r2 = 0.997) , 20 and 10000 ng/g for 5-HT (r2 = 0.994) , NE (r2 = 0.993) , and EP (r2 = 0.993) , and 0.2 and 200 μg/g for Glu (r2 = 0.996) and GABA (r2 = 0.999) in the mouse brain tissues. As stated above, LC-MS/MS results were obtained and established to be a useful tool for the quantitative analysis of BH4, DA, 5-HT, NE, EP, Glu, and GABA in the experimental rodent brain.

Comprehensive analysis of neurotransmitters from regenerating planarian extract using an ultrahigh‐performance liquid chromatography/mass spectrometry/selected reaction monitoring method

Absolute quantification of neurotransmitters (NTs) from biological systems is imperative to track how changes in concentration of active neurochemicals may affect biological behavior. A sensitive method for the absolute quantification of multiple NTs in a single method is highly needed. A stable-isotope dilution ultrahigh-performance liquid chromatography/mass spectrometry/selected reaction monitoring (UHPLC/MS/SRM) assay has been developed for a sensitive and quantitative assessment of NTs in planaria. We used this method for the simultaneous quantification of 16 NTs. All analytes showed a linear relationship between concentrations (0.78-50 ng/mL), regression coefficients higher than 0.97, accuracy (91-109%) and low coefficients of variation (CVs). The inter-day CVs for the lowest quality controls (1.56 ng/mL) were in the range between 2-11%. The levels of most of the NTs were similar in both sexual and asexual planarians except for glutamic acid, which was about two-fold higher in asexual compared to sexual planarians. We identified high levels of serotonin and failed to detect tryptamine suggesting that the pathway essential for the conversion of tryptophan into tryptamine is absent in planarians. Interestingly, we also found high levels of dopamine and L-DOPA in regenerating planarians suggesting their possible role in regeneration. For the first time, we developed novel methodology based on UHPLC/MS/SRM and quantified 16 NTs with high sensitivity and specificity from sexual and asexual strains of planarian Schmidtea mediterranea. This method will also have great application in quantifying various NTs with great precision in different model systems.

Quantitative determination of neurotransmitters, metabolites and derivates in microdialysates by UHPLC–tandem mass spectrometry

The main objective of the present work is to study the time-course of rat brain neurotransmitters in vivo after an oral challenge with a nutritional ingredient or an external stimulus, such as a chemical agent. An ultrahigh performance liquid chromatography–tandem mass spectrometry method for the identification and quantification of neurotransmitters, metabolites and derivates in microdialysates from rat brain was previously developed. Betaine, glutamine, glutamic acid, gamma-aminobutyric acid, phosphocholine, glycerophosphocholine, cytidine 5'-diphosphocholine, choline, acetylcholine, dopamine, norepinephrine, serotonin, tyrosine, epinephrine, tryptophan and 5-hydroxyindoleacetic acid were selected as analytes. The method involves direct injection of samples of microdialysates from rat brain onto the chromatographic equipment and quantification with a triple quadrupole mass spectrometer detector using an electrospray ionization interface in positive mode. The limits of detection ranged from 0.1 to 50ngmL−1 and the limits of quantification from 0.3 to 200ngmL−1. The inter- and intra-day variability were lower than 15%. Recovery rates ranged from 85% to 115%.

Detection and Quantification of Neurotransmitters in Dialysates

Sensitive analytical methods are needed for the separation and quantification of neurotransmitters obtained in microdialysate studies. This unit describes methods that permit quantification of nanomolar concentrations of monoamines and their metabolites (high-performance liquid chromatography [HPLC] electrochemical detection), acetylcholine (HPLC-coupled to an enzyme reactor), and amino acids (HPLC-fluorescence detection, capillary electrophoresis with laser-induced fluorescence detection).

Investigating candidate neuromodulatory systems underlying parasitic manipulation: concepts, limitations and prospects

Studies addressing the functional basis of parasitic manipulation suggest that alteration of the neuromodulatory system is a common feature of manipulated hosts. Screening of the neuromodulatory system has so far been carried out by performing ethopharmacological analysis, biochemical quantification of neurotransmitters and neuromodulators, and/or immunocytochemistry. Here, we review the advantages and limitations of such approaches through the analysis of case studies. We further address whether the analysis of candidate neuromodulatory systems fits the current view of manipulation as being multidimensional. The benefits in combining ethopharmacology with more recent molecular tools to investigate candidate neuromodulatory pathways is also emphasized. We conclude by discussing the value of a multidisciplinary study of parasitic manipulation, combining evolutionary (parasite transmission), behavioural (syndrome of manipulation) and neuroimmunological approaches.

The involvement of the spinal release of glutamate and nitric oxide in peripheral noxious stimulation-induced pain-related behaviors—Study in mouse spinal microdialysis

The quantification of neurotransmitters in the dorsal horn of the spinal cord under pain conditions is important to investigate the mechanism of pain transmission. Microdialysis is widely used for the quantification of the release of endogenous substances in various tissues; however, most of the experiments have been conducted in rats. In the present study, we measured the spinal release of glutamate and NO2/NO3 under pain conditions induced by formalin or capsaicin using mouse spinal microdialysis. We found an association between formalin- or capsaicin-induced nociceptive behaviors and the release of glutamate and NO2/NO3 in the spinal cord.

Quantification of monoamine neurotransmitters and melatonin in sea lamprey brain tissues by high performance liquid chromatography–electrospray ionization tandem mass spectrometry

A rapid liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed and validated for simultaneous quantification of three monoamine neurotransmitters (NTs) and melatonin in sea lamprey brain tissues. Separation was performed on a reversed-phase column with mobile phases of 1mM perfluoroheptanoic acid (PFHA) water solution/acetonitrile and mass spectra were acquired in positive electrospray ionization multiple reaction monitoring (MRM) mode. Solid-phase extraction (SPE) was employed to purify and extract the target compounds from the tissue samples. The matrix effects as well as the influence of two extraction methods, protein precipitation (PPT) and SPE, on matrix effects were examined for the first time on the quantification of NTs from brain tissue extracts. The matrix effects with SPE (2.4 to −14.9%) were about 30% lower on average than those with the ACN PPT method (−29 to −38%). The recoveries of three types of SPE cartridges were tested and Bond-Elut C18 cartridge was selected to process the samples because of its good extraction efficiencies (71.3–95.3%) and low matrix effects (−6.6 to −14.9%) for all four analytes. This method exhibited excellent linearity for all analytes with regression coefficients higher than 0.99. The limits of detection were between 0.03ng/mL (melatonin) and 0.14ng/mL (norepinephrine). The precisions, expressed as coefficients of variation (CV), ranged from 4.8 to 14.1% for intra-day analyses and from 6.1 to 16.2% for inter-day analyses. Brain tissues from 360 sea lampreys were analyzed by the developed method and the concentrations for four analytes were found to be at the level of nanogram per gram of brain tissues. To our knowledge, this is the first report on the quantification of NTs and melatonin in the sea lamprey using the LC–MS/MS method.

Analysis of biogenic amines in a single Drosophila larva brain by capillary electrophoresis with fast-scan cyclic voltammetry detection.

Drosophila, the fruit fly, is a common model organism in biology; however, quantifying neurotransmitters in Drosophila is challenging because of the small size of the central nervous system (CNS). Here, we develop neurotransmitter quantification by capillary electrophoresis with fast-scan cyclic voltammetry (CE-FSCV) detection, which allows peak identification by both migration time and the cyclic voltammogram, in contrast to traditional amperometric detection which provides no chemical identification. Tissue content of biogenic amine neurotransmitters was determined in a single CNS dissected from a Drosophila larva. Low detection limits, 1 nM for dopamine and serotonin, 2.5 nM for tyramine, and 4 nM for octopamine, were achieved using field-amplified sample stacking by diluting the homogenized tissue with percholoric acid and acetonitrile. Two different strains of wild-type flies, Oregon R and Canton S, have similar dopamine and serotonin levels but different octopamine content. When flies are fed NSD-1015, which inhibits dopamine decarboxylase (Ddc) a synthesis enzyme in the dopamine and serotonin pathways, dopamine significantly decreases by 52%. A genetically altered driver line, Ddc-GAL4, had lower serotonin and dopamine content as did w(118) flies. When the Ddc-GAL4 line was used to produce flies overexpressing the serotonin synthesis enzyme tryptophan hydroxylase (Ddc-GAL4;UAS-Trh), the serotonin tissue content was greater than for Ddc-GAL4 but not significantly different than the wild-type. These results show that CE-FSCV is useful for monitoring the impact of genetic and pharmacological manipulations on the content of multiple neurotransmitters in a CNS from a Drosophila larva.

The Effect of the SDS Concentration on the Electrochemical Response of Adrenaline at Acid pH

The effect that micellar aggregates have on the electrochemical response of some compounds is indeed interesting, because adsorption and solubility of electrochemically active compounds may induce changes in the redox potential, in the charge transfer and on the diffusion coefficients of the species predominating in solution. This work studies the behavior of adrenaline in the presence of different SDS concentrations on carbon paste electrodes, CPE, with particular reference to its diffusion-controlled redox mechanism, which has an associated chemical reaction step. On consideration of its heterogeneous reaction constant, ko, the overall process is said to be quasi-reversible with one electron transferred. Therefore, it is rightly interpreted that the adrenaline's oxidation mechanism changes drastically in the presence of SDS, whereby the latter favors oxidation of the former, also becoming adsorption-controlled. These results indeed support the applicability of an analytical procedure for the neurotransmitter's quantification

Detection and Quantification of Neurotransmitters in Dialysates

Sensitive analytical methods are needed for the separation and quantification of neurotransmitters obtained in microdialysate studies. This unit describes methods that permit quantification of nanomolar concentrations of monoamines and their metabolites (high-pressure liquid chromatography electrochemical detection), acetylcholine (HPLC-coupled to an enzyme reactor), and amino acids (HPLC-fluorescence detection; capillary electrophoresis with laser-induced fluorescence detection).

Overview of brain microdialysis.

The technique of microdialysis enables sampling and collecting of small-molecular-weight substances from the interstitial space. It is a widely used method in neuroscience and is one of the few techniques available that permits quantification of neurotransmitters, peptides, and hormones in the behaving animal. More recently, it has been used in tissue preparations for quantification of neurotransmitter release. This unit provides a brief review of the history of microdialysis and its general application in the neurosciences. The authors review the theoretical principles underlying the microdialysis process, methods available for estimating extracellular concentration from dialysis samples (i.e., relative recovery), the various factors that affect the estimate of in vivo relative recovery, and the importance of determining in vivo relative recovery to data interpretation. Several areas of special note, including impact of tissue trauma on the interpretation of microdialysis results, are discussed. Step-by-step instructions for the planning and execution of conventional and quantitative microdialysis experiments are provided.

Biogenic amines and pterins in cerebrospinal fluid: some pitfalls with interpretation

The quantification of neurotransmitters or their metabolites in cerebrospinal fluid (CSF) can provide valuable clues for the detection of (often treatable) genetic disorders or their biosynthesis or salvage pathways. These analyses are technically demanding since the concentrations to be assessed are in the nanomolar range. To date, only a few laboratories worldwide offer these analyses. Since some metabolites are unstable at room temperature and sensitive to light or oxygen, special preparation of sampling tubes is helpful and the samples should be immediately frozen at the bedside. Furthermore, due to a craniocaudal gradient, a sampling protocol with numbered tubes should be adhered to. In addition to the technical difficulties, interpretation of the biochemical findings in lumbar CSF is much more complex than the interpretation of results in urine or blood. This article will focus on some pitfalls associated with the interpretation of analyses of dopamine, serotonin and pterin metabolites, and examines clinical data from pediatric patients to further illustrate those pitfalls.

Evaluation of reversed phase columns designed for polar compounds and porous graphitic carbon in “trapping” and separating neurotransmitters

Quantification of neurotransmitters as biologically active analytes in neurological samples is of high interest for studying their effect on multiple targets. This work is part of a strategy involving two-dimensional liquid chromatography (2D LC) system with mass spectrometry (MS) detection. The concept of the on-line LC system is the coupling of reversed phase liquid chromatography (RPLC, the second separation dimension) to ion-exchange chromatography (IEC, the first dimension). Our objective in this study is to find the appropriate second dimension column, ensuring that samples of neurotransmitters are refocused and separated on it. Silica-based columns designed specifically to retain polar compounds were tested in LC conditions and compared with results obtained with a porous graphitic carbon (PGC, Hypercarb) column. These polar embedded, polar endcapped, and high-density alkyl chain columns successfully separated analytes in question using mobile phase systems with high percentage of water, or even pure water. Only Hypercarb column provided efficient retention of the most polar neurotransmitters and could be used for trapping and preconcentrating the compounds without rapid breakthrough.

Nanomolar derivatizations with 5-carboxyfluorescein succinimidyl ester for fluorescence detection in capillary electrophoresis

The ability of 5-carboxyfluorescein succinimidyl ester (CFSE) to derivatize amino acids at very low concentrations has been investigated. CFSE can derivatize nanomolar concentrations of amino acids, which represents a 1000-fold improvement compared to fluorescein isothiocyanate (FITC). Although the reactive probe is not fluorogenic, few hydrolysis products result from derivatization, leading to relatively clean electropherograms compared to FITC derivatization. Derivatizations of γ-aminobutyric acid suggest that CFSE may be a practical amine-reactive fluorescent probe for the quantification of neurotransmitters in microdialysates.

Detection and Quantification of Neurotransmitters in Dialysates

AbstractThis unit describes methods for the separation and quantification of neurotransmitters, peptides, and second messengers in microdialysis samples by high‐pressure liquid chromatography (HPLC) coupled to either electrochemical detection (EC) or fluorescence detection. The protocols describe commonly used procedures for the detection of catecholamines and indoleamines (HPLC‐EC detection; detection of acetylcholine (HPLC‐EC detection with enzyme reactor), and detection of amino acids (HPLC‐fluorescence detection). Each of these protocols affords detection limits in the low nanomolar range.