Plasma and cerebral spinal fluid tranexamic acid quantitation in cardiopulmonary bypass patients

A method for the determination of tranexamic acid (TXA) in human plasma and cerebral spinal fluid (CSF) was developed. Analyses were performed by ultra performance liquid chromatography with tandem mass spectrometry detection (UPLC–MS/MS) using ?-aminocaproic acid (ACA) as an internal standard. TXA and ACA were extracted from a 50 μL sample of plasma or CSF using a methanol protein crash protocol, and chromatographic separation was performed on an ACQUITY? TQD mass spectrometer using a UPLC C18 BEH 1.7 μm column with a water and methanol gradient containing 0.1% formic acid. The detection and quantitation was performed by positive ion electrospray ionization using the multiple reaction monitoring (MRM) mode. The method was linear over the concentration range of 0.1–10.0 μg/mL, with lower limit of quantitation of 0.1 μg/mL for TXA. The intra- and inter-assay precision was less than 12% and 13% respectively at the plasma and CSF TXA concentrations tested. The present method provides a relatively simple and sensitive assay with short turn-around-time. The method has been successfully applied to assess the plasma and CSF concentrations of tranexamic acid achieved with only one dosing regimen of tranexamic acid in patients undergoing cardiopulmonary bypass surgery (CPB).     

Simple high-performance liquid chromatographic method for the detection of phenylpropionylglycine in urine as a diagnostic tool in inherited medium-chain acyl-coenzyme A dehydrogenase deficiency

Deficiency of medium-chain acyl-CoA dehydrogenase is a frequent and treatable metabolic defect, which can be diagnosed by detection of phenylpropionylglycine in urine after an oral load of phenylpropionic acid. We studied the determination of phenylpropionylglycine in urine by isocratic ion-exclusion chromatography on a cation-exchange column using water–sulphuric acid (pH values between 2 and 4) as mobile phase. Phenylpropionylglycine, phenylpropionic acid and hippuric acid exhibited high retention factors with only a slight decline at increasing solvent pH. This resulted in a good separation from interfering substances after direct injection of urine. We hypothesize that π–π interactions between the aromatic carbonic acids and the ion-exchange resin are responsible for the strong retention on the stationary phase. We conclude that, even in asymptomatic patients, determination of phenylpropionylglycine in urine after a phenylpropionic acid load by ion-exclusion chromatography is a rapid and reliable diagnostic tool for the detection of medium-chain acyl-CoA dehydrogenase deficiency.     

Determination of carboxylic acids in wines by liquid ion-exclusion chromatography

Chromatographic behavior of aromatic and aliphatic acids was studied using an ion-exclusive column. Conditions were determined, under which the separation of the acids by single-column ion-exclusion chromatography with UV-detection was the most selective and efficient. The method developed by us was employed for determining the content of carboxylic acids in wines.     

Determination of Carboxylic Acids in Wines by Liquid Ion-Exclusion Chromatography

The chromatographic behavior of aromatic and aliphatic acids was studied using an ion-exclusive column. The conditions were determined under which the separation of the acids by single-column ion-exclusion chromatography with UV detection was the most selective and efficient. The method developed was employed for determining the content of carboxylic acids in wines.     

The origin of indoleacetic acid and indolepropionic acid in rat and human cerebrospinal fluid

Abstract: Using a new high performance liquid chromatographic method we have measured tryptophan, 5-hydroxyindoleacetic acid (5HIAA), indoleacetic acid (IAA), and indolepropionic acid (IPA) in rat and human CSF. Experiments on rats indicate that IPA in CSF is not derived from the CNS but from bacterial metabolism in the intestine. However, IAA in CSF is derived from CNS tryptamine metabolism. Some tryptamine that is formed peripherally diffuses across the blood-brain barrier and augments the tryptamine formed within the CNS. We have concluded from our data that (i) measurements on CSF are a useful way of studying trace amine metabolism in human CNS, but it is essential to establish the anatomical and metabolic origin of any metabolite found in the CSF; and (ii) tryptamine metabolism is more important in man than in the rat.     

Measurement of kynurenic acid in mammalian brain extracts and cerebrospinal fluid by high-performance liquid chromatography with fluorometric and coulometric electrode array detection

Kynurenic acid is a broad-spectrum excitatory amino acid (EAA) receptor antagonist which is present in the mammalian central nervous system. We describe a method for the measurement of kynurenic acid using isocratic reverse-phase high-performance liquid chromatography (HPLC) with fluorometric detection enhanced by Zn2+ as a postcolumn reagent. The method requires no prior sample preparation procedures other than extraction with 0.1 M HClO4. The reliability of the primary fluorometric method was verified by comparing measurements of tissue concentrations of kynurenic acid in human cerebral cortex and putamen using three different methods of separation with fluorometric detection, as well as four methods utilizing HPLC with coulometric electrode array system (CEAS) detection. All seven methods produced comparable results. The concentration of kynurenic acid in human cerebral cortex was 2.07 +/- 0.61 pmol/mg protein, and in human putamen, 3.38 +/- 0.81 pmol/mg protein. Kynurenic acid was also found to be present in human cerebrospinal fluid (CSF) at a concentration of 5.09 +/- 1.04 nM. The regional distribution of kynurenic acid in the rat brain was examined. Kynurenic acid concentrations were highest in brainstem (149.6 fmol/mg protein) and olfactory bulb (103.9 fmol/mg protein) and lowest in thalamus (26.0 fmol/mg protein). There were no significant postmortem changes in kynurenic acid concentrations in cerebral cortex, hippocampus, and striatum at intervals ranging from 0 to 24 h. Perfusion of the cerebral vasculature with normal saline prior to sacrifice did not significantly alter kynurenic acid content in rat hippocampus, cerebral cortex, or striatum. The analytical methods described are the most sensitive (10-30 fmol injection-1) and specific (utilizing both excitation and emissions properties and electrochemical reaction potentials, respectively) methods for determining kynurenic acid in brain tissue extracts and CSF. These methods should prove useful in examining whether kynurenic acid modulates EAA-mediated neurotransmission under physiologic conditions, as well as in determining the role of kynurenic acid in excitotoxic neuronal death.     

Analysis of Poly-beta-Hydroxybutyrate in Rhizobium japonicum Bacteroids by Ion-Exclusion High-Pressure Liquid Chromatography and UV Detection

Ion-exclusion high-pressure liquid chromatography (HPLC) was used to measure poly-beta-hydroxybutyrate (PHB) in Rhizobium japonicum bacteroids. The products in the acid digest of PHB-containing material were fractionated by HPLC on Aminex HPX-87H ion-exclusion resin for organic acid analysis. Crotonic acid formed from PHB during acid digestion was detected by its intense absorbance at 210 nm. The Aminex-HPLC method provides a rapid and simple chromatographic technique for routine analysis of organic acids. Results of PHB analysis by Aminex-HPLC were confirmed by gas chromatography and spectrophotometric analysis.     

Simultaneous determination of 3-methoxy-4-hydroxyphenylglycol, 5-hydroxyindoleacetic acid, and homovanillic acid in cerebrospinal fluid with high-performance liquid chromatography using electrochemical detection

An improved high-performance liquid chromatographic method with electrochemical detection (HPLC-EC) for the simultaneous determination of 3-methoxy-4-hydroxyphenylglycol (MHPG), 5-hydroxyindoleacetic acid (5-HIAA), and homovanillic acid (HVA) in cerebrospinal fluid (CSF) of humans and nonhuman primates is described. Quantitation is based on the use of an internal standard, 5-fluoro-HVA. Sample preparation consists of mixing an aliquot of CSF with a solution of the internal standard followed by ultrafiltration. The precision of the method is high, with within-run and between-run coefficients of variation of 2-6% and less than 10%, respectively, in the concentration ranges of the metabolites encountered in human lumbar CSF. Accuracy was tested by comparing the present HPLC method with specific gas chromatographic-mass spectrometric (GS-MS) assays for MHPG and HVA and a GC-MS-validated HPLC assay for 5-HIAA: the correlations obtained were 0.968 for MHPG, 0.989 for 5-HIAA, and 0.999 for HVA, with no systematic bias between the methods. The use of ascorbate as a preserving agent for monoamine metabolites in CSF was not found to be necessary when proper care was exercised in sample handling and storage. The analysis of samples with up to 2% ascorbic acid was possible as well, but MHPG had to be assayed separately using an extraction procedure and an alternative internal standard, 3-ethoxy-4-hydroxyphenylglycol.     

Analysis of Poly-β-Hydroxybutyrate in Rhizobium japonicum Bacteroids by Ion-Exclusion High-Pressure Liquid Chromatography and UV Detection

Ion-exclusion high-pressure liquid chromatography (HPLC) was used to measure poly-β-hydroxybutyrate (PHB) in Rhizobium japonicum bacteroids. The products in the acid digest of PHB-containing material were fractionated by HPLC on Aminex HPX-87H ion-exclusion resin for organic acid analysis. Crotonic acid formed from PHB during acid digestion was detected by its intense absorbance at 210 nm. The Aminex-HPLC method provides a rapid and simple chromatographic technique for routine analysis of organic acids. Results of PHB analysis by Aminex-HPLC were confirmed by gas chromatography and spectrophotometric analysis.     

Rapid screening procedures for identification of succinic acid producers

Succinic acid, an intermediate of tricarboxylic acid cycle, is produced and accumulated by anaerobic microorganisms. The long-standing interest in the production of this organic acid is because it is a key compound in producing more than 30 commercially important products. The detection of succinic acid is generally carried out by gas chromatography (GC), enzymatic assays, ion-exclusion chromatography (IEC) or by high performance liquid chromatography (HPLC). However, these methods are time consuming, require sophisticated instrumentation and are expensive. In the present investigation we are reporting two rapid, cost effective screening methods for the detection of this important organic acid. These methods can be utilized to screen a large number of microbes producing succinic acid in a very short span of time.     

Separation and determination of liver uric acid and allantoin

We previously described the only satisfactory procedure yet achieved for separating uric acid and allantoin from rat liver. The procedure was based on trichloroacetic acid (TCA) extraction, acid hydrolysis, treatment with Hg-acetate, and cation- and anion-exchange chromatography. After separation, allantoin was quantified by a colorimetric method, and uric acid enzymatically using uricase. Since this procedure is too time-consuming, we propose an improved version which avoids the need for anion-exchange chromatography and the complex assay of catabolic compounds. The new method consists of a very fast and simple HPLC separation and direct determination of uric acid and allantoin at 220 nm. The method can be used for fresh tissue or after treatment of the tissue with labeled precursor.     

Effects of acute tryptophan depletion on plasma and cerebrospinal fluid tryptophan and 5-hydroxyindoleacetic acid in normal volunteers

Brain serotonin synthesis and metabolism (turnover), as indicated by CSF concentrations of 5-hydroxyindoleacetic acid (5-HIAA), may depend on plasma concentrations of the essential amino acid L-tryptophan (TRP). We investigated the biochemical effects of acute plasma TRP depletion (ATD) in normal volunteers undergoing a 36-h CSF collection via lumbar drain. Six subjects who were in good health were put on a low-TRP diet (160 mg/day) 24 h before lumbar puncture; this diet was continued for the first 22 h of the CSF collection. At hour 22, subjects ingested a TRP-deficient 15-amino acid drink shown previously to deplete plasma TRP. Total plasma TRP, free plasma TRP, and CSF TRP subsequently decreased 86.3, 86.5, and 92.3%, respectively. CSF 5-HIAA decreased by 32.8%. Plasma total and free TRP concentrations were both decreased at approximately 2 h following ingestion of the TRP-free amino acid drink and were lowest approximately 6 h after ATD; CSF TRP and 5-HIAA were decreased at 2.5 h and approximately 4 h after ATD, respectively. CSF TRP was lowest 8.0 h later. CSF 5-HIAA continued to decrease 14 h after the TRP-deficient amino acid drink was given.     

Application of liquid chromatography-mass spectrometry to measure the concentrations and study the synthesis of short chain fatty acids following stable isotope infusions

A new method involving zinc sulphate deproteinization was developed to study short chain fatty acids (SCFA) production in the colon and subsequent occurrence of SCFA in blood. SCFA were baseline separated in a 30 min cycle using ion-exclusion chromatography and detected by mass spectrometry. Concentrations could be measured down to 10 microM and isotopomeric distributions could be assessed, enabling the conduction of tracer studies to study changes in SCFA synthesis. The applicability of the method was tested in an extensively characterized pig model yielding portal SCFA concentrations ranging from 70 microM (butyric acid) to 150 microM (propionic acid) to 440 microM (acetic acid) prior to butyrate tracer infusion, reaching butyric acid isotopic steady state within 2 h.     

Characterization by high performance liquid chromatography of angiotensin peptides in the plasma and cerebrospinal fluid of the dog

A high performance liquid chromatography (HPLC) method is described for the separation of angiotensin (Ang) peptides and their subsequent quantification by radioimmunoassay in plasma and cerebrospinal fluid (CSF). The use of the ion-pair solvent heptafluorobutyric acid in gradient HPLC achieves baseline resolution of Ang I, Ang II, and the C-terminal fragments des-[Asp1]-Ang I, des-[Asp1]-Ang II, des-[Asp1,Arg2]-Ang II and des-[Asp1,Arg2,Val3]-Ang II in approximately 25 min. Recovery of synthetic Ang standards after phenylsilica extraction and HPLC separation was greater than 70% for each peptide in both plasma and CSF. Ang I and Ang II were shown to be the major immunoreactive Ang components in plasma, and Ang II, des-[Asp1,Arg2]-Ang II and des-[Asp1,Arg2,Val3]-Ang II in CSF.     

5-Hydroxyindoleacetic acid and homovanillic acid concentrations in cerebrospinal fluid in patients with Alzheimer's disease, depression and mild cognitive impairment

In this study we investigated the cerebrospinal fluid (CSF) concentrations of 5-hydroxyindoleacetic acid (5-HIAA) and homovanillic acid (HVA) in Alzheimer (AD) patients (n=75), patients with mild cognitive impairment (MCI, n=9) and patients with depression (n=7). CSF HVA was significantly elevated in AD with depression (Geriatric Depression Scale, 15 point version GDS>5) in comparison to AD without depression (p<0.05, ANOVA) and CSF HVA showed a significant positive correlation with the GDS score of AD-patients (p=0.03, Spearman Rho: 0.38, Spearman Rank Correlation). In the group of AD patients CSF 5-HIAA was positively correlated with cerebrospinal fluid beta-amyloid 1-42 (Abeta42), p<0.05, Spearman Rho: 0.3, Spearman Rank Correlation, but not with CSF tau. Additionally, there was a significant positive correlation between cerebrospinal fluid 5-HIAA and HVA in the group of AD patients (p<0.0001, Rho: 0.47, Spearman Rank correlation). Neither 5-HIAA nor HVA in CSF could differentiate between mild cognitive impairment, depression and AD. The results of this study support the hypothesis that the serotonergic system plays a role in the course of AD. They further suggest an important role of dopamine metabolism in depression within AD patients.     

A simple and rapid electrophoretic method for the separation of glucuronic acid and iduronic acid

A new electrophoretic method using Titan III cellulose acetate plates has been developed for the separation and quantitation of glucuronic acid and iduronic acid. This method is quite simple, and glucuronic acid and iduronic acid can be separated within 50 min. This method was applied to the analyses of uronic acids in chondroitin sulfates A and C, and dermatan sulfate.     

Measurement of acid monoamine metabolites in human and animal cerebrospinal fluid

Techniques for measuring 5-hydroxyindolacetic acid (5-HIAA) and homovanillic acid (HVA) have been modified to permit the use of smaller samples for measuring these acid monoamine metabolites in human and animal CSF. Levels of both HVA and 5-HIAA were approximately three times as high in human ventricular CSF as in human lumbar CSF. Lumbar CSF levels of 5-HIAA in neurologic patients were significantly higher than those in psychiatric patients. Values were obtained for HVA in dog cisternal CSF and for 5-HIAA in dog, rabbit, and cat cisternal CSF.     

Improvement in HPLC separation of acetic acid and levulinic acid in the profiling of biomass hydrolysate

5-Hydroxymethylfurfural (HMF) and furfural could be separated by the Aminex HPX-87H column chromatography, however, the separation and quantification of acetic acid and levulinic acid in biomass hydrolysate have been difficult with this method. In present study, the HPLC separation of acetic acid and levulinic acid on Aminex HPX-87H column has been investigated by varying column temperature, flow rate, and sulfuric acid content in the mobile phase.The column temperature was found critical in resolving acetic acid and levulinic acid. The resolution for two acids increased dramatically from 0.42 to 1.86 when the column temperature was lowered from 60 to 30 °C. So did the capacity factors for levulinic acid that was increased from 1.20 to 1.44 as the column temperature dropped. The optimum column temperature for the separation was found at 45 °C. Variation in flow rate and sulfuric acid concentration improved not as much as the column temperature did.     

Liquid chromatographic assay of dityrosine in human cerebrospinal fluid

A micro-scale method for separation and measurement of dityrosine in human cerebrospinal fluid (CSF) is described utilizing liquid-liquid extraction and ion-paired, reversed-phase high-performance liquid chromatography with fluorimetric detection. A mobile phase containing 1-heptanesulfonic acid linearly increased in methanol from 0 to 100% over 30 min allows the resolution of dityrosine from other fluorescent compounds with excitation at 285 nm and emission at 410 nm. As little as 0.15 ml CSF sample can be utilized with a detection limit of 60 pg dityrosine on the column. This method facilitates the use of CSF dityrosine as a measure of free radical mediated protein damage in the central nervous system.     

Determination of isomeric acid dopamine metabolites in human cerebrospinal fluid by mass fragmentography

A method for the quantitative determination of the isomers 4-hydroxy-3-methoxyphenylacetic acid (HVA) and 3-hydroxy-4-methoxyphenylacetic acid (iso-HVA) in cerebrospinal fluid (CSF) by mass fragmentography has been developed. The heptafluorobutyryl methyl ester derivations of the two compounds could not be separated by gas chromatography. The relative intensity of the base peak (m/e 333) and the molecular ion (m/e 392) in the mass spectra were, however, quite different and allowed a separate determination. Contradictory to a previous report, it was found that the concentration of iso-HVA was less than 2% of the HVA level in the investigated 12 CSF samples from patients with different diseases.