Determination of dopamine and its metabolites in small volumes of rat brain dialysates using small-bore liquid chromatography with electrochemical detection

A miniaturized liquid chromatographic system with electrochemical detection (LC-ED) was developed and applied to the analysis of dopamine and its metabolites in dialysate samples collected from the rat brain in vivo. An existing LC-ED system was down-scaled using a 1 mm I.D. small-bore column operated at a reduced flow-rate and with an injection volume of 1 microliter. With the small-bore system the limit of detection for dopamine of ca. 0.06 pg was almost 50 times less than with the conventional system, which represents a two-fold improvement in concentration sensitivity. Miniaturization was accomplished with negligible loss in resolution by using a conventional commercial amperometric detector with minor modifications. The results indicate that a number of useful advantages could be realized by the combination of this small-bore LC-ED system and the in vivo brain dialysis method.     

Pre-column fluorescence derivatization high-performance liquid chromatography of opioid peptides in rat brain and its use for enzymatic peptide characterization

A high-performance liquid chromatographic method involving fluorescence derivatization followed by separation on a reversed-phase polymer (octadecylated polyvinylalcohol copolymer gel) column is described for the determination of opioid peptides in rat brain tissues. The peptides extracted from brain tissues were converted into fluorescent derivatives by reaction with hydroxylamine, cobalt(II) ion and borate. The derivatives were separated on an Asahipak ODP-50 column by gradient elution of acetonitrile in the mobile phase containing borate buffer (pH 9.5). The detection limits (S/N = 3) for the peptides were 0.33-1.21 pmol per 100 microliters injected. The method actually permit the determination of leucine enkephalin, methionine enkephalin, methionine enkephalin-Arg-Phe and methionine enkephalin-Arg-Gly-Leu in the tissues. The method is also applied to the characterization of the peptides in the tissues by means of enzymatic degradations with carboxypeptidase A and trypsin.     

Analysis of proenkephalin A, proopiomelanocortin and protachykinin neuropeptides in human lumbar cerebrospinal fluid by reversed-phase high-performance liquid chromatography, radioimmunoassay and enzymolysis.

A comprehensive high-performance liquid chromatographic, radioimmunoassay, and enzymatic degradation scheme has been developed to analyze several intact neuropeptides and the corresponding peptides created by in vivo enzymolysis of precursors to study neuropeptides in human lumbar cerebrospinal fluid (CSF) and to test the hypothesis that defects in the metabolism (synthesis, degradation) of neuropeptide precursors, neuropeptides, and metabolites play a role in low back pain. CSF samples were obtained from three different patient groups: controls (C), whose low back pain was relieved without lidocaine; pharmacological responders (PR), whose pain was relieved by lidocaine and who were candidates for surgery; and pharmacological non-responders (PNR), whose pain was not relieved by lidocaine and a mid-thoracic anesthetic, and who were not candidates for surgery. The metabolic activity involved during synthesis and degradation of the peptides was assessed by measuring intact, native neuropeptide immunoreactivity in pre-incubated and post-incubated CSF samples, where samples were incubated at 37 degrees C for 1 h. Pre-incubation radioimmunoassay measurements reflected the content of intact peptides present in lumbar CSF at the time of sampling, and post-incubation measurements assayed the amount of peptide that had remained embedded within its precursors [cryptic methionine enkephalin (ME)] and that had been released by the action of CSF peptidases. Significant differences were found in post-incubation samples for the amount of proenkephalin A [ME, leucine enkephalin (LE)] and tachykinin [substance P (SP)] peptides. For example, significant differences were observed for ME-like immunoreactivity (C versus cryptic), SP-like immunoreactivity (PNR versus PR), and LE-like immunoreactivity (PR versus C). No significant differences were observed among the peptides within the pre-incubation samples.(ABSTRACT TRUNCATED AT 250 WORDS)     

Assay for enkephalin-degrading peptidases in rat brain tissues by high-performance liquid chromatography with on-line post-column fluorescence detection

The activities of enkephalin-degrading peptidases such as enkephalinases A and B in rat brain tissues were simultaneously assayed by a high-performance liquid chromatographic method with fluorimetric detection with an automatic reaction system. Tyrosine and tyrosine-containing peptides produced enzymatically from the substrate, methionine-enkephaline, were separated by gradient elution on a reversed-phase column (TSK gel ODS-120T), and then converted into fluorescent derivatives for detection by reaction with hydroxylamine, cobalt(II) and borate reagents. The method permits the simple and sensitive detection of N-terminal tyrosine-containing fragments of the enkephalin peptide. The limits of detection are 5-20 pmol per assay tube for the N-terminal tyrosine-containing fragments. The enzyme activities in the regionally separated tissues were 54-191 pmol/min.mg protein for enkephalinase A and 79-153 pmol/min.mg protein for enkephalinase B, which were calculated from the formation of Tyr-Gly-Gly and Tyr-Gly, respectively, during the enzyme reaction.     

Quantification of rat brain neurotransmitters and metabolites using liquid chromatography/electrospray tandem mass spectrometry and comparison with liquid chromatography/electrochemical detection

Analytical methodology based on solid-phase extraction, polar reversed-phase liquid chromatography, and electrospray tandem mass spectrometry (LC/MS/MS) with isotope dilution was developed and validated for quantifying the neurotransmitters, dopamine and serotonin, and their major metabolites in brain tissue. Limits of detection (0.1-20 pg/mg tissue) were sufficient for analysis of multiple neurotransmitters in rat brain regions, including parietal cortex, hypothalamus, pituitary, substantia nigra, and striatum. Method performance was compared with contemporaneous measurements using a well-established procedure based on ion-pairing reversed-phase liquid chromatography and amperometric detection. The principal advantages of the LC/MS/MS method include a more robust sample purification procedure, an optimized chromatographic separation, and the qualitative and quantitative assurance that comes from coeluting isotopically labeled internal standards; however, sensitivity did not consistently improve upon that provided by amperometric detection. This methodology may be particularly useful for applications in which simultaneous determinations are required for drugs and their affected neurotransmitters in specific brain regions.     

Determination of reduced and total ubiquinones in biological materials by liquid chromatography with electrochemical detection

A convenient and reliable liquid chromatographic (LC) method with electrochemical detection (ED) was developed for the determination of reduced (ubiquinol) and total ubiquinones in biological materials. After extraction of samples with n-hexane, ubiquinol was separated on a reversed-phase column and assayed directly by ED. In order to determine the total amount of a ubiquinone in biological samples, the unbiquinone was converted into the corresponding reduced form by treatment with sodium borohydride. No significant interfering peak (plastoquinol-9, ubichromenol-9, etc.) was observed in the elution areas of ubiquinol-7 to -11. This LC-ED method was about 70 times more sensitive than the previous LC-UV method and was able to detect 150 pg of ubiquinol-10. The method was applied satisfactorily to the determination of the contents of ubiquinol homologues in biological materials. The content of ubiquinols is a major component of the total ubiquinones in human plasma and urine and rat plasma and liver, but a minor component in rat heart and kidney.     

Simultaneous detection of phosphatidylcholines and glycerolipids using matrix‐enhanced surface‐assisted laser desorption/ionization‐mass spectrometry with sputter‐deposited platinum film

Matrix‐assisted laser desorption/ionisation (MALDI) imaging mass spectrometry (IMS) allows for the simultaneous detection and imaging of several molecules in brain tissue. However, the detection of glycerolipids such as diacylglycerol (DAG) and triacylglycerol (TAG) in brain tissues is hindered in MALDI‐IMS because of the ion suppression effect from excessive ion yields of phosphatidylcholine (PC). In this study, we describe an approach that employs a homogeneously deposited metal nanoparticle layer (or film) for the detection of glycerolipids in rat brain tissue sections using IMS. Surface‐assisted laser desorption/ionisation IMS with sputter‐deposited Pt film (Pt‐SALDI‐IMS) for lipid analysis was performed as a solvent‐free and organic matrix‐free method. Pt‐SALDI produced a homogenous layer of nanoparticles over the surface of the rat brain tissue section. Highly selective detection of lipids was possible by MALDI‐IMS and Pt‐SALDI‐IMS; MALDI‐IMS detected the dominant ion peak of PC in the tissue section, and there were no ion peaks representing glycerolipids such as DAG and TAG. In contrast, Pt‐SALDI‐IMS allowed the detection of these glycerolipids, but not PC. Therefore, using a hybrid method combining MALDI and Pt‐SALDI (i.e., matrix‐enhanced [ME]‐Pt‐SALDI‐IMS), we achieved the simultaneous detection of PC, PE and DAG in rat brain tissue sections, and the sensitivity for the detection of these molecules was better than that of MALDI‐IMS or Pt‐SALDI alone. The present simple ME‐Pt‐SALDI approach for the simultaneous detection of PC and DAG using two matrices (sputter‐deposited Pt film and DHB matrix) would be useful in imaging analyses of biological tissue sections. Copyright © 2015 John Wiley & Sons, Ltd.     

Conformation Changes of Enkephalin in Coordination with Pb2+ Investigated by Gas Phase Hydrogen/Deuterium Exchange Mass Spectrometry Combined with Theoretical Calculations

In this study, the effects of lead ions(Pb²⁺) on the conformations of leucine encephalin(LE) and methionine encephalin(ME) in gas phase were studied using hydrogen/deuterium exchange mass spectrometry(HDX-MS) and quantum chemistry theoretical calculations at the molecular level. The HDX-MS result revealed that the complexes with the monovalent compounds [LE+Pb–H]⁺ and [ME+Pb–H]⁺had a 1:1 stoichiometric ratio, and different HDX reactivates were observed in a follow of [ME+H]⁺>[LE+H]⁺>[LE+Pb–H]⁺> [ME+Pb–H]⁺. Combining the collision-induced dissociation energies of the complexes and their HDX results, it was found that the more stable the complex, the harder it was for HDX. In addition, the favo-rable conformations of the complexes were obtained by theoretical calculations, revealing that the similar coordination type with diffe-rent bond lengths was obtained. Then, the proton affinity(PA) values of the optimized complexes were calculated to interpret the HDX observations, indicating that the higher the PA values, the more difficult it was for HDX. Overall, the experiments and theoretical calculations revealed that Pb²⁺ could induce conformational changes of LE and ME, and generate ME into a more rigid conformation than LE. The results will prompt further fundamental investigations on the conformational properties of LE/ME in coordination with Pb²⁺.     

A simple and sensitive assay for eflornithine quantification in rat brain using pre‐column derivatization and UPLC‐MS/MS detection

Eflornithine (α‐difluoromethylornithine) has been used to treat second‐stage (or meningoencephalitic‐stage) human African trypanosomiasis and currently is under clinical development for cancer prevention. In this study, a new ultraperformance liquid chromatography–tandem mass spectrometry (UPLC‐MS/MS)‐based assay was developed and validated for the quantification of eflornithine in rat brain. To improve chromatographic retention and MS detection, eflornithine was derivatized with 6‐aminoquinolyl‐N‐hydroxysuccinimidyl carbamate for 5 min at room temperature prior to injection. Derivatized eflornithine was separated on a reverse‐phase C₁₈ UPLC column with a 6‐min gradient; elution occurred at approximately 1.5 min. Prior to derivatization, eflornithine was reproducibly extracted from rat brain homogenate by methanol protein precipitation (~70% recovery). Derivatized eflornithine was stable in the autosampler (6 °C) for at least 24 h. This new assay had acceptable intra‐ and interday accuracy and precision over a wide dynamic range (5000‐fold) and excellent sensitivity with a lower limit of quantification of 0.1 µm (18 ng/mL) using only 10 μL of rat brain homogenate. The validated eflornithine assay was applied successfully to determine eflornithine distribution in different regions of rat brain in an in situ rat brain perfusion study. Copyright © 2014 John Wiley & Sons, Ltd.     

Determination of drug-protein interactions by microdialysis coupled with liquid chromatography and electrochemical detection based on a nickel hexacyanoferrate modified electrode

Microdialysis sampling coupled with liquid chromatography-electrochemical detection (LC-ED) was developed and applied to determine the interaction of thiopurine (TP) with bovine serum albumin (BSA). A nickel hexacyanoferrate (NiHCNFe) modified electrode was fabricated and used as the working electrode in LC-ED for the determination of TP. Cyclic voltammetric experiments demonstrate that this chemically modified electrode (CME) can effectively catalyze the electrooxidation of TP. The mechanism of the catalytic oxidation of TP at the CME involves an 'EC' process. In the LC-ED, the CME also shows good stability and reproducibility for the determination of TP. The limit of detection is 5.0 x 10(-7) mol l(-1) for 6-TP with a signal-to-noise ratio of 3. The utility of microdialysis as a quantitative sampling technique for in vitro studies of drug-protein interactions was studied. The microdialysis experiments were performed in phosphate buffer solution (pH 7.4) containing different molar ratios of the drug and protein at 37 degrees C. The collected microdialysis sample with unbound TP was analyzed at the NiHCNFe CME in the LC-ED. The relative recovery of TP determined in vitro is 18.3% at a perfusion rate of 1.0 microl min(-1) and the RSD is about 2.1%. The association constant and the number of binding sites on a BSA molecule calculated with the Scatchard equation are 3.72 x 10(-3) (1 mol(-1)) and 1.51, respectively. This method provides a fast, sensitive and simple technique for the study of drug-protein interactions.     

Improved method for the determination of 5,6-dihydroxytryptamine and 5,7-dihydroxytryptamine in tissue using high-performance liquid chromatography with electrochemical detection.

A liquid chromatographic method with electrochemical detection is described for the determination of the 5-hydroxytryptamine (5-HT) neurotoxins 5,6-dihydroxytryptamine (5,6-DHT) and 5,7-dihydroxytryptamine (5,7-DHT) in rat brain tissue. This method has also been used for the determination of 5-hydroxyindoleacetic acid, homovanillic acid and 5-HT in other tissue samples. The method is based on extraction of the indoles from brain samples with perchloric acid followed by reversed-phase liquid chromatography with electrochemical detection. The detection limit is 1 ng per 100 mg of tissue. This paper describes a quick and reliable method of assaying the 5-HT neurotoxins 5,6-DHT and 5,7-DHT in brain tissue, which is improved compared to currently available assays.     

Direct determination of 4-hydroxy-3-methoxyphenylacetic (homovanillic) acid in urine by high-performance liquid chromatography with amperometric detection

The improvement of high-performance liquid chromatographic analysis with electrochemical detection for urinary homovanillic acid is described. The method permits the chromatographic resolution of authentic homovanillic acid from coeluting interfering compounds in human and nonhuman primate, and rat urine. The electrochemically derived results are compared with post-column derivatized fluorescence results, and quality-control checks necessary to maintain assay precision in automated analysis are described.     

Electrospray mass spectrometry for the analysis of opioid peptides and for the quantification of endogenous methionine enkephalin and β-endorphin

Electrospray ionization mass spectrometry was used to characterize several different neuropeptides, whose molecular weights ranged from 555 to 3463 Da, and to quantify endogenous methionine enkephalin (ME) and β -endorphin (β E) extracted from a human pituitary gland. Methionine enkephalin and leucine enkephalin both yield only an [M + H] + ion with electrospray mass spectrometry; the other peptides produce a series of multiply charged even-electron molecular ions of the general nature [M + nH]ⁿ+ in proportion to the number of basic amino acid units present, with no evidence of fragmentation. The electrospray mass spectra are characterized by low background noise. The quantiftcation of ME is based on a comliarison of the ion current due to the [M + H] + ion of native and of a deuterated ME ([²H₅ s?⁴Phe]?ME) internal standard. The calibration curve is linear in the range of ca. 1-35 pmol synthetic ME. The amounts of ME determined in three separate human pituitary extracts were 9.1, 8.2, and 4.7 pmol/mg protein. The corresponding amount of ME in a canine pituitary was 39.8 pmol/mg protein. To quantify β E, the ion current due to the [M + 5H]⁵ + ion was monitored and compared to an external calibration curve obtained by analyzing solutions of synthetic β E in the range 5 μmol-50 pmol. The analysis of a human pituitary yielded 660 fmol β E/mg protein.     

Simultaneous determination of biogenic amines and morphine in discrete rat brain regions by high-performance liquid chromatography with electrochemical detection

A simple and sensitive method has been developed for the simultaneous determination of norepinephrine, epinephrine, dopamine, 5-hydroxytryptamine, 5-hydroxyindoleacetic acid, and morphine in discrete rat brain regions by reversed-phase high-performance liquid chromatography with electrochemical detection. Perchloric acid extracts of the tissue were directly injected into the chromatographic system. Each of these compounds gave a linear response over the range of 20-160 ng/ml cerebellar homogenate (0.4-3.2 ng on column). Recoveries of these compounds, added to the homogenates, were complete when compared with standards dissolved in perchloric acid. The average between-run coefficients of variation for all these compounds were lower than 7.4% over the range of 20-160 ng/ml, and the within-run coefficients of variation at 20 ng/ml were lower than 8.7%. The present method has been applied to a study of the effects of intraperitoneal administration of morphine on biogenic amines in several discrete rat brain regions.     

Determination of vitamin B6 vitamers and pyridoxic acid in biological samples.

For the determination of vitamin B6 vitamers (pyridoxal phosphate, pyridoxamine phosphate, pyridoxal, pyridoxine, pyridoxamine) and 4-pyridoxic acid in biological samples such as plasma, cerebrospinal fluid and rat brain regions, a sensitive micromethod using high-performance liquid chromatography (HPLC) with fluorescence detection in combination with post-column derivatization is described. Metaphosphoric acid tissue extracts with deoxypyridoxine as an internal standard were injected into the HPLC system with a binary gradient elution at a flow-rate of 1.2 ml/min. The excitation wavelength of the fluorescence detector was set at 328 nm and the emission wavelength at 393 nm with a 15-nm slit width for the photocell. This method allows the assay of vitamin B6 vitamers within 30 min in one chromatographic run. The present method has been applied extensively for the measurement of vitamin B6 vitamer levels in discrete brain regions of small animals, cells in culture and biopsy samples.     

Experimental design optimization of the capillary electrophoresis separation of leucine enkephalin and its immune complex

To optimize the capillary electrophoretic separation conditions for leucine enkephalin (LE) and the immune complex of the LE and anti-LE reaction, an analysis using a three-level, three-factorial Box-Behnken design was performed. Three separation parameters, buffer pH (X(1)), buffer concentration (X(2)), and applied voltage (X(3)), were chosen to observe the effect on separation responses. The responses were theoretical plate number, migration time of the LE peak, and resolution between the peaks. The optimum conditions and process validation were determined using statistical regression analysis and surface plot diagrams. The capillary electrophoresis optimum separation conditions were established to be 75 mM phosphate buffer at pH 7.00 with an applied separation voltage of 15 kV. By using the analysis technique, the prediction of responses was satisfactory and process verification yielded values within the +/-5% range of the predicted efficiency.     

High-performance liquid chromatographic determination, with ultraviolet detection, of S-adenosyl-L-methionine and S-adenosyl-L-homocysteine in rat tissues and simultaneously of normetanephrine and metanephrine for phenylethanolamine-N-methyltransferase or catechol-O-methyltransferase activities

A high-performance liquid chromatographic method, with ultraviolet detection, for the determination of S-adenosyl-L-methionine and S-adenosyl-L-homocysteine, and simultaneously of normetanephrine and metanephrine, is presented. The separation was carried out by reversed-phase ion-pair isocratic chromatography. This procedure was applied to the study of the content of S-adenosyl-L-methionine and S-adenosyl-L-homocysteine in rat brain and adrenal glands and to the measurement of phenylethanolamine-N-methyltransferase or catechol-O-methyltransferase activities in rat adrenal gland.     

High-performance liquid chromatographic determination of substance P-like arginine-containing peptide in rat brain by on-line post-column fluorescence derivatization with benzoin

A high-performance liquid chromatographic method with fluorescence detection is described for the determination of substance P, one of the neuropeptides, in the hypothalamus tissue of rat brain. The detection is based on on-line post-column fluorescence derivatization selective for arginine-containing peptides. The endogenous substance P-like arginine-containing peptide extracted from the tissue and [D-Phe11]-neurotensin as an internal standard were separated from various interfering substances on a reversed-phase column (TSKgel ODS-120T) by gradient elution with acetonitrile-phosphate buffer (pH 2.3). The peptides in the eluate were then automatically converted into fluorescent derivatives for detection by reaction with benzoin. Arginine-containing fragments produced by the enzyme reaction of substance P in the chromatographic fraction with trypsin were also detected, for the identification of the endogenous substance P-like arginine-containing peptide. The method was sensitive enough to permit the quantitative determination of the peptide at a concentration as low as 580 fmol/mg of protein in the brain homogenate. The concentration values of the substance P-like arginine-containing peptide in the tissue were 9.45 +/- 1.50 pmol/mg of protein (six determinations).     

Simultaneous determination of cefazolin in rat blood and brain by microdialysis and microbore liquid chromatography

A sensitive microbore liquid chromatographic method combined with the minimally invasive technique of microdialysis was devised for simultaneously and continuously monitoring the levels of unbound blood and brain cefazolin in rats. Microdialysis probes were inserted into the jugular vein and brain striatum for blood and brain sampling, respectively. Chromatographic conditions consisted of a mobile phase of methanol-acetonitrile-100 mM monosodium phosphoric acid (20:10:70, v/v, pH 4.5) pumped through a microbore reversed-phase column at a flow rate of 0.05 mL/min. The ultraviolet detection wavelength was set at 270 nm. An on-line design allowed direct and continuous analysis of protein-free samples in the dialysate. Microdialysis probes, being home-made, were screened for acceptable in vivo recovery. Chromatographic resolution and detection were validated for response linearity as well as intra-day and inter-day variabilities. This method was then applied to pharmacokinetic profiling of protein unbound cefazolin in both the blood and brain following intravenous administration (10 mg/kg, i.v., n = 6). Rapid appearance of cefazolin in the rat brain striatal dialysate following drug injection suggested good blood-brain barrier penetration. According to a non-compartmental pharmacokinetics model, the area under the concentration (AUC) vs time ratio of cefazolin in rat brain and blood was 6%.     

Electrochemical Analysis of Liposome-encapsulated Colistimethate Sodium

In this study, the neutral and cationic liposomal formulations of Colistimethate sodium (CMS), an antibiotic for multi-drug resistant gram-negative bacteria, were prepared and electrochemical quantification of CMS from these liposomes were achieved. This is the first study of the electrochemical detection of CMS released from liposomes. First, the electrochemical properties of CMS were analysed, then the encapsulation efficiency, and the release kinetic of CMS from liposomes were determined with Differential Pulse Voltammetry (DPV) measurements. In addition, Cyclic Voltammetry were applied to determine oxidation signal of CMS. A higher encapsulation efficiency was found in the cationic liposome compared to the neutral liposome. Moreover, CMS was controlled released from liposomes with zero-order drug release kinetics.