Simultaneous high-performance liquid chromatographic determination of norepinephrine, serotonin, acetylcholine and their metabolites in the cerebrospinal fluid of anaesthetized normotensive rats.

A high-performance liquid chromatographic method with electrochemical detection was developed for the simultaneous determination of the levels of norepinephrine (NE), serotonin (5-HT), acetylcholine (ACh) and their metabolites in the cerebrospinal fluid (CSF) of anaesthetized rats. The response curve for each compound was linear for the concentration way of interest. The within- and between-day coefficients of variation (C.V.) for NE, 5-HT and their metabolites were less than 7.85% and 15.67%, respectively, and those for ACh and choline were less than 3.08% and 6.27%, respectively. This simultaneous determination should be useful for elucidating the noradrenergic, serotonergic and cholinergic nerve activity in the central nervous system.     

Simultaneous liquid chromatographic determination of seventeen of the major monoamine neurotransmitters, precursors and metabolites. II. Assessment of human brain and cerebrospinal fluid concentrations

The optimized chromatographic method procedure presented in Part I was employed for the assessment of human brain and cerebrospinal fluid neurotransmitters levels. The optimized sample preparation and chromatographic conditions permitted a rapid (less than 25 min), sensitive and semi-automated high-performance liquid chromatographic analysis which measures all major monoamine neurotransmitters, precursors and metabolites in human brain and cerebrospinal fluid. The brain specimen was deproteinized with perchloric acid (containing Na2EDTA and sodium sulphite), the internal standard and heparin were added and the samples were sonicated, centrifuged, filtered and injected directly into the chromatographic system. Cerebrospinal fluid was handled in a similar manner except that sonication was excluded. The regional distribution of monoamine neurotransmitter concentrations in human brain and cerebrospinal fluid is presented.     

Nontarget metabolomics profiling of neuromyelitis optica spectrum disorder

To investigate the characteristic of neuromyelitis optica spectrum disorder (NMOSD) in plasma and cerebrospinal fluid, UHPLC–MS was used to identify metabolites by metabolite extraction and on‐machine detection. Multivariate analysis methods were used to complete differential metabolite screening and metabolic pathway analysis. The content of eight substances, such as tryptophan and l ‐glutamic acid in the plasma of NMOSD patients, was higher than that of the healthy control group. Moreover, no differential metabolite was found in the plasma of patients with AQP‐4 antibody‐positive and antibody‐negative NMOSD. The content of five substances including 3‐hydroxybutyric acid in cerebrospinal fluid of patients with NMOSD was reduced. We demonstrated that the distribution of metabolites in plasma between NMOSD patients and healthy counterparts was significantly different. However, there is no significant difference in plasma metabolites between AQP‐4 antibody positive and negative NMOSD. There were some differences in metabolites between the cerebrospinal fluid of NMOSD patients and that of healthy controls. A variety of amino‐acid abnormalities, sphingomyelin dysfunction, energy metabolism and mitochondrial dysfunction are involved in the pathogenesis of NMOSD.     

Simple and rapid micro-analytical procedures for the estimation of milacemide and its metabolite glycinamide in rat plasma and cerebrospinal fluid by high-performance liquid chromatography

A high-performance liquid chromatographic technique is described for the determination of milacemide and its primary metabolite glycinamide in rat plasma and cerebrospinal fluid. Milacemide and glycinamide are derivatized with fluorescamine to form a chromophore and a fluorophore and subsequent analysis using ultraviolet and fluorescence detectors, respectively. The extraction procedures are simple with a limit of detection 2 and 0.5 micrograms/ml for milacemide in plasma and cerebrospinal fluid, respectively, and 0.5 micrograms/ml for glycinamide in plasma or cerebrospinal fluid. The within-batch coefficients of variation for both analytes were less than 3%. Since only a small amount of sample is required, these techniques are well suited for the study of milacemide pharmacokinetics in the rat.     

Simultaneous Determination of Fourteen Catecholamines and Their Metabolites by High Performance Liquid Chromatography with Electrochemical Detection

Abstract

A simple method has been developed for simultaneous determination of 14 catecholamines and their metabolites in cerebrospinal fluid and brain tissue by reversed-phase, ion-pair high-performance liquid chromatography with electrochemical detection. The time required for complete separation and analysis of all compounds was less than 35 min. Quantitation was based on the use of an internal standard isoproterenol. The mobile phase consisted of a 91:9 (v/v) mixture of 0.1 M formic acid and acetonitrile containing sodium-1-octane sulfonic acid. Using this method, analysis of neurotransmitters in brain tissue can be accomplished without a clean-up procedure.     

毛细管电泳对脑外伤脑脊髓液中兴奋性氨基酸的测定

用毛细管电泳分离测定了脑外伤脑脊髓液中的活性氨基酸，考察了进样方式、 缓冲液浓度、有机添加剂、氨基酸的衍生条件对脑脊髓液中微量活性氨基酸测定的 影响，最终在采用外加苯甲酸作为内标的优化电泳条件下，对活性氨基酸的检测限 低于1.7 * 10~(-7)mol/L，测定结果的相对标准偏差小于2%。     

Enzymic determination of cerebrospinal fluid lipid fractions

Colorimetric peroxidase-coupled procedures for the determination of several cerebrospinal fluid (CSF) lipid classes are described. These methods were modified to increase the effectiveness of each cerebrospinal fluid lipid assay by using the sample as the primary diluent for a highly concentrated reagent in an inverse concentration technique. Direct enzymic assays for the determination of CSF cholesterol (free and total), choline phospholipids, and triglycerides were adapted from existing assays to require less than 0.5 ml of sample per assay. This made determinations of the several lipid analytes possible even when samples were from pediatric specimens. In a study model, 51 pediatric CSF samples were analyzed for these lipid constituents. Mean values and standard deviations were determined. Within and between-run studies were performed by sampling from a pool of cerebrospinal fluid specimens. Within-run coefficients of variation for the several proposed procedures were less than 3% while the between-run findings for all of the procedures were less than 5%.     

Combined thin-layer chromatography-photography-densitometry for the quantification of ifosfamide and its principal metabolites in urine, cerebrospinal fluid and plasma.

A method has been devised for the determination of the anticancer drug ifosfamide and its principal metabolites in urine, plasma and cerebrospinal fluid (CSF). The urine and CSF samples are absorbed onto Amberlite XAD-2 eluting the compounds of interest with methanol. Plasma is deproteinated using cold acetonitrile and centrifuged to yield a clear supernatant. The eluate and supernatant are analyzed by thin-layer chromatography, with spot visualization using 4-(4-nitrobenzyl)pyridine. The plates are photographed for subsequent densitometeric analysis. The intra-assay coefficient of variation for each compound in both urine and plasma was less than 10% and the lower limit of detection was 1 microgram/ml. The method provides a means of determining the full spectrum of metabolic products of ifosfamide in patients and will allow detailed investigation of variability in metabolism and pharmacokinetics of this drug.     

High-performance liquid chromatographic determination of diclofenac and its monohydroxylated metabolites in biological fluids

Sensitive and selective high-performance liquid chromatographic assays for diclofenac and its monohydroxylated metabolites in biological fluids are described. Using ultraviolet detection at 282 nm, diclofenac is assayed in plasma at concentrations down to 10 ng/ml; total (free + conjugated) diclofenac and its monohydroxylated metabolites (the sum of 3'- + 4'-hydroxydiclofenac and 5-hydroxydiclofenac) are assayed in urine after chemical hydrolysis at concentrations down to 200 ng/ml. The applicability of the described assays is shown.     

Simultaneous determination of aceclofenac and three of its metabolites in human plasma by high-performance liquid chromatography

Aceclofenac [[2-(2',6'-dichlorophenyl)amino]phenylacetoxyacetic acid] is a phenylacetic acid derivative with potent analgesic and anti-inflammatory properties and an improved gastro-intestinal tolerance. In the present study, a liquid-liquid extraction-based reversed-phase HPLC method with UV detection was validated and applied for the analysis of aceclofenac and three of its metabolites (4'-hydroxy-aceclofenac, diclofenac, 4'-hydroxy-diclofenac) in human plasma. The analytes were separated using an acetonitrile-phosphate buffer gradient at a flow rate of 1 mL/min, and UV detection at 282 nm. The retention times for aceclofenac, diclofenac, 4'-hydroxy-aceclofenac, 4'-hydroxy-diclofenac and ketoprofen (internal standard) were 69.1, 60.9, 46.9, 28.4 and 21.2 min, respectively. The validated quantitation range of the method was 10-10000 ng/mL for aceclofenac, 4'-hydroxy-aceclofenac and diclofenac, and 25-10000 ng/mL for 4'-hydroxy-diclofenac. The developed procedure was applied to assess the pharmacokinetics of aceclofenac and its metabolites following administration of a single 100 mg oral dose of aceclofenac to three healthy male volunteers.     

High-performance liquid chromatographic determination of naproxen, ibuprofen and diclofenac in plasma and synovial fluid in man.

High-performance liquid chromatographic assay procedures have been developed for naproxen, ibuprofen and diclofenac in human plasma and synovial fluid samples. A single liquid-liquid extraction procedure was used to isolate each compound from acidified biological matrix prior to the quantitative analysis. A Spherisorb ODS column (12.5 cm x 4.6 mm I.D.) was used for all the chromatography. Naproxen was eluted with a mobile phase of methanol-S?rensen's buffer at pH 7 (37:63, v/v). Ibuprofen and diclofenac were eluted using mobile phases of methanol-water at pH 3.3 (65:35, v/v and 63:37, v/v, respectively). Diphenylacetic acid was used as the internal standard for the assay of naproxen and flurbiprofen was used in the analysis of ibuprofen and diclofenac. Inter- and intra-day coefficients of variation were less than 7%. The assays were used in clinical studies of the three drugs in osteo- and rheumatoid arthritis patients.     

Simultaneous determination of the three major monoamine metabolites in cerebrospinal fluid by high-performance liquid chromatography with electrochemical detection

A simple method is described for the simultaneous determination of the three monoamine metabolites, 4-hydroxy-3-methoxyphenylacetic acid, 4-hydroxy-3-methoxyphenylethyleneglycol and 5-hydroxyindole-3-acetic acid, in cerebrospinal fluid by high-performance liquid chromatography with electrochemical detection. Quantitation is accomplished by the standard addition technique. Chromatographic peak heights are corrected for volume effects by comparison with the signal obtained for an added auxiliary reference substance. Sample preparation is kept to a minimum, involving precipitation of proteins by means of perchloric acid and subsequent neutralization. The reproducibility was estimated to be 10%. For one of the metabolites, 4-hydroxy-3-methoxyphenylethyleneglycol, a correlation between the results obtained by this method and a mass fragmentographic method was made, and a satisfactory correlation (r = 0.904, slope = 0.914, intercept 3.26 ng/ml) found. The sensitivity of the method is in the picogram range. The methodology has been applied to measure biogenic amine metabolites in both rabbit and human cerebrospinal fluid. The levels found are in agreement with previously reported values.     

HPLC method for determination of diclofenac in human plasma and its application to a pharmacokinetic study in Turkey

A simple high-performance liquid chromatography (HPLC) method has been developed for determination of diclofenac in human plasma. The method was validated on Ace C(18) column using UV detection. The mobile phase consisted of 20 mM phosphate buffer (pH 7) containing 0.1% trifluoroacetic acid-acetonitrile (65:35, v/v). Calibration curve was linear between the concentration range of 75-4000 ng/mL. Intra- and inter-day precision values for diclofenac in plasma were less than 3.6, and accuracy (relative error) was better than 5.3%. The limits of detection and quantification of diclofenac were 25 and 75 ng/mL, respectively. Also, this assay was applied to determine the pharmacokinetic parameters of diclofenac in healthy Turkish volunteers who had been given 50 mg diclofenac.     

Nuclear magnetic resonance spectroscopy in solving the analytical problems of medicine: Analysis of cerebrospinal fluid

The samples of cerebrospinal fluids from patients with ischemic stroke and motor neuron disease and from persons with no neurological diseases were studied by ¹H NMR spectroscopy. Conditions for cerebrospinal fluid analysis were optimized. Twenty-five low-molecular-weight resident metabolites were identified and quantitatively determined in the cerebrospinal fluid. It was found that the concentrations of lactate, citrate, glucose, creatine, alanine, acetate, pyruvate, acetoacetate, formate, 2-hydroxybutyrate, 3-hydroxybutyrate, tyrosine, phenylalanine, valine, 2-hydroxyisovalerate, glutamine, fructose, isoleucine, and mannose in patients with ischemic stroke were several times higher than those in patients from the control group. Differences between the compositions of cerebrospinal fluid in the control group and a group of patients with motor neuron disease were detected with respect to the concentrations of 3-hydroxybutyrate, histidine, acetate, and mannose. The statistical processing of cerebrospinal fluid analysis data with the use of the method of principal components makes it possible to distinguish among the groups of cerebrospinal fluid samples from patients with ischemic stroke and motor neuron disease and persons from the control group. The results and the proposed approach to obtaining data can form the basis of the development of diagnostic models.     

Measurement of total plasma and cerebrospinal fluid homocysteine by fluorescence following high-performance liquid chromatography and precolumn derivatization with o-phthaldialdehyde.

Precolumn derivatization of amino acids with o-phthaldialdehyde followed by high-performance liquid chromatographic separation and fluorescence detection is used in many clinical and experimental laboratories for the measurement of primary amino acids. This technique was adapted for the measurement of homocysteine in plasma and cerebrospinal fluid (CSF) following alkylation of the free sulphydryl group with iodoacetate. The minimum detection limits are less than 1 microM in plasma and 80 nM in CSF. Within-day and between-day coefficients of variation for plasma and for CSF are less than 10%. Values for normal plasma homocysteine range from 6.04 to 16.2 microM and for CSF from 0.28 to 0.66 microM.     

Determination of a 'GW cocktail' of cytochrome P450 probe substrates and their metabolites in plasma and urine using automated solid phase extraction and fast gradient liquid chromatography tandem mass spectrometry

A mass spectrometry based method for the simultaneous determination of an in vivo Greenford-Ware or 'GW cocktail' of CYP450 probe substrates and their metabolites in both human plasma and urine is described. The probe substrates, caffeine, diclofenac, mephenytoin, debrisoquine, chlorzoxazone and midazolam, together with their respective metabolites and stable isotope labelled internal standards, are simultaneously extracted from the biological matrix using solid phase extraction in 96-well microtitre plate format, automated by means of a custom built Zymark robotic system. The extracts are analysed by fast gradient high performance liquid chromatography (HPLC) with detection by tandem mass spectrometry (MS/MS) using thermally and pneumatically assisted electrospray ionisation in both positive and negative ion modes and selected reaction monitoring. The methods are specific, accurate and precise with intra- and inter-assay precision (%CV) of less than 15% for all analytes.     

The separation and detection of alkaline oligoclonal IgG bands in cerebrospinal fluid using immobilised pH gradients

A method is described for the separation and detection of highly alkaline IgG bands in unconcentrated cerebrospinal fluid (CSF). These bands are frequently found in the cerebrospinal fluid of patients with inflammatory diseases of the central nervous system, particularly in the case of multiple sclerosis, and their detection is an important aid in clinical diagnosis. An isoelectric focusing technique using an immobilised pH gradient in polyacrylamide gel has been developed over the pH range 7-10, producing a linear and stable pH gradient with excellent resolution. After electrofocusing, the protein patterns were blotted onto polyvinylidene difluoride membranes and visualised using anti-human IgG followed by an enzyme-labelled second antibody. Blotting could be carried out by capillary diffusion for up to 16 h duration without any loss in resolution. Using this method, highly alkaline intrathecal IgG bands were found in the cerebrospinal fluid of all of the 14 multiple sclerosis patients. There were also 2 patients with alkaline IgG bands in their cerebrospinal fluid who were not diagnosed as multiple sclerosis. By contrast, no alkaline IgG bands with an isoelectric point (pI) greater than 8.6 were found in any of the serum samples studied (n = 50) from patients with various neurological disorders including multiple sclerosis.     

Determination of cefotaxime and desacetylcefotaxime in plasma and urine by high-performance liquid chromatography

A high-performance liquid chromatographic method is described for the analysis of the anti-bacterial agent cefotaxime and desacetylcefotaxime in physiological fluids. Plasma or serum samples were mixed with chloroform--acetone to remove proteins and most lipid material. The aqueous phase was then freeze-dried, reconstituted in mobile phase and chromatographed on a reversed-phase column using UV detection at 262 nm. Urine was analysed directly after centrifugation to remove particulate matter. The detection limit was 0.5--1.0 micrograms/ml for serum and 5 micrograms/ml for urine. The method has been applied to the analyses of cefotaxime and desacetylcefotaxime in plasma, serum, urine, cerebrospinal fluid, saliva, and pus from infected wound secretions. Two additional metabolites, which are lactones in which the beta-lactam ring has been opened, could be separated by this method.     

Predicting Drug Interaction of Clopidogrel on Microbial Metabolism of Diclofenac

Seven fungal cultures were studied for the metabolism of diclofenac in order to elucidate the nature of enzymes involved in biotransformation, as diclofenac is a specific substrate to cytochrome P450 (CYP) 2C9 isozyme in mammals. The metabolites were identified by high-performance liquid chromatography–diode array detection and liquid chromatography–tandem mass spectroscopy analysis. The study included clopidogrel, a selective inhibitor of CYP2C9 isozyme, to inhibit the metabolism of diclofenac. Two-stage fermentation protocol was used to study the diclofenac metabolism and its inhibition by clopidogrel. Among the cultures studied, four have shown positive indication for drug interaction, since clopidogrel inhibited the metabolism of diclofenac in a dose-dependent manner. The results indicate that microbial cultures possess enzyme systems similar to mammals and they can be used to predict drug interactions in mammalian systems.     

Metabolic Profiling and Quantitative Analysis of Cerebrospinal Fluid Using Gas Chromatography–Mass Spectrometry: Current Methods and Future Perspectives

Cerebrospinal fluid is a key biological fluid for the investigation of new potential biomarkers of central nervous system diseases. Gas chromatography coupled to mass-selective detectors can be used for this investigation at the stages of metabolic profiling and method development. Different sample preparation conditions, including extraction and derivatization, can be applied for the analysis of the most of low-molecular-weight compounds of the cerebrospinal fluid, including metabolites of tryptophan, arachidonic acid, glucose; amino, polyunsaturated fatty and other organic acids; neuroactive steroids; drugs; and toxic metabolites. The literature data analysis revealed the absence of fully validated methods for cerebrospinal fluid analysis, and it presents opportunities for scientists to develop and validate analytical protocols using modern sample preparation techniques, such as microextraction by packed sorbent, dispersive liquid–liquid microextraction, and other potentially applicable techniques.