Simultaneous determination of plasma creatinine,uric acid,kynurenine and tryptophan by high‐performance liquid chromatography: method validation and in application to the assessment of renal function

A high‐performance liquid chromatography with ultraviolet detection method has been developed for the simultaneous determination of a set of reliable markers of renal function, including creatinine, uric acid, kynurenine and tryptophan in plasma. Separation was achieved by an Agilent HC‐C₁₈ (2) analytical column. Gradient elution and programmed wavelength detection allowed the method to be used to analyze these compounds by just one injection. The total run time was 25 min with all peaks of interest being eluted within 13 min. Good linear responses were found with correlation coefficient >0.999 for all analytes within the concentration range of the relevant levels. The recovery was: creatinine, 101 ± 1%; uric acid, 94.9 ± 3.7%; kynurenine, 100 ± 2%; and tryptophan, 92.6 ± 2.9%. Coefficients of variation within‐run and between‐run of all analytes were ≤2.4%. The limit of detection of the method was: creatinine, 0.1 µmol/L; uric acid, 0.05 µmol/L; kynurenine, 0.02 µmol/L; and tryptophan, 1 µmol/L. The developed method could be employed as a useful tool for the detection of chronic kidney disease, even at an early stage. Copyright © 2014 John Wiley & Sons, Ltd.     

PROFLAVINE-SENSITIZED PHOTOOXIDATION OF TRYPTOPHAN AND RELATED PEPTIDES

Abstract— The photodynamic action of proflavine on the amino acids cystine, rnethionine, tyrosine, histidine, tryptophan and related peptides was tested in anhydrous formic and acetic acids. Only rnethionine and tryptophan were found to be photooxidized: the former, whether free or bound in a polypeptide chain, is quantitatively converted to methioninesulphoxide; the latter, when free, is fragmented to several compounds, which may be divided into two main classes: melanines and kynurenine derivatives. Whereas masking the carboxyl group of tryptophan has no effect on the photooxidation products, N-substituted derivatives of tryptophan are converted in high yields to the corre-sponding kynurenine compounds. The possible applications of the method to biologically active polypeptides are discussed.     

Determination of Kynurenine and Tryptophan in Human Plasma by Stacking-Micellar Electrokinetic Chromatography

Kynurenine and tryptophan are important biomarkers for disorders in nervous and immune systems, while the low content of kynurenine in human plasma makes the determination by routine approaches difficult. In this work, an on-line preconcentration method for capillary electrophoresis was developed for the quantification of kynurenine and tryptophan. A concomitant sweeping phenomenon was observed due to the combination of micellar electrokinetic chromatography and stacking. The poor sensitivity in capillary electrophoresis was improved so that the determination of low concentrations of kynurenine became possible. Parameters that may affect the efficiency of the on-line concentration were systematically investigated and optimized. A large volume sample injection was employed for sampling at 100 mbar for 112 sec. A voltage of ?20 kV was applied, and the ionic sample matrix was removed with stacking while analytes were kept in the capillary. The voltage was switched to +18 kV and micellar electrokinetic chromatography was performed for separation and determination of kynurenine and tryptophan. Based on peak heights, the concentration factors for kynurenine and tryptophan were 33 and 31, respectively. The analytical performance of the established method was studied; good linearity of kynurenine was observed in the range of 0.3–30 µM (R² = 0.9988), and 0.3–60 µM (R² = 0.9963) was obtained for tryptophan. LODs of 0.15 µM for kynurenine and 0.30 µM for tryptophan were obtained. The optimum method was successfully tested in human plasma; kynurenine and tryptophan were well identified, which suggests that this method has potential for the analysis of biological samples.     

Simultaneous determination of tryptophan and kynurenine in serum by HPLC with UV and fluorescence detection

Tryptophan metabolism is disturbed in mental depression, and the induction of indoleamine 2,3-dioxygenase increases kynurenine production. In order to determine this disturbance in patients with chronic hepatitis C and receiving interferon-based immunotherapy, a new and specific HPLC protocol was elaborated. For tryptophan, the assay was linear from 6.25 to 100 micromol L(-1), and the limits of detection (LOD) and quantitation (LOQ) for the method were 0.7 and 8.0 micromol L(-1). For kynurenine, the linearity of calibration was from 0.0625 to 6.25 micromol L(-1), with LOD and LOQ of 2 and 3 nmol L(-1). Reproducibility and repeatability were satisfactory. The method allowed study of human blood serum.     

Factors affecting the ultramicro-determination of tryptophan,kynurenine and their derivatives

The factors affecting the ultramicrodetermination of tryptophan and kynurenine were investigated by paper electrophoresis, paper chromatography and spectrophotometry. The electrolyte used in electrophoresis influences the mobility, the separation and the ultraviolet spectra of tryptophan and kynurenine. The pre-treatment of filter paper for chromatography did not influence the R_F of tryptophan and kynurenine, but interfered with their quantitative determination. The effect of ultraviolet irradiation of d-, l-, and dl-tryptophan as well as d-, l-, and dl-kynurenine was studied.     

A study of kynurenine fragmentation using electrospray tandem mass spectrometry

A combination of accurate mass measurement and tandem mass spectrometry (both product ion and precursor ion scans) have been used to characterize the major fragment ions observed in the ESI mass spectrum of kynurenine. Kynurenine is a metabolite of tryptophan found in the human lens and is thought to play a role in protecting the retina from UV-induced damage. Three major fragmentation pathways were evident, following initial elimination either of ammonia, H2O and CO or the imine form of glycine. The latter is proposed to occur via the formation of an ion-molecule complex. In the case of loss of H2O and CO from deaminated kynurenine, there is evidence for an acylium ion intermediate, which is not observed for the loss of H2O and CO directly from protonated kynurenine. Product ion scans of deuterated kynurenine enabled the elucidation of structural rearrangements that were not evident in the spectra of the native compound. Since UV filter compounds can often only be isolated in small quantities from the lens, this study forms the basis for the characterization of novel UV filter compounds using mass spectrometry. The approach presented here may also be useful for the characterization of related classes of small molecules.     

Development of simultaneous analysis of tryptophan metabolites in serum and gastric juice – an investigation towards establishing a biomarker test for gastric cancer diagnosis

To evaluate changes in tryptophan metabolism and discover diagnostic biomarkers for gastric cancer, a quantitative method was developed for tryptophan and its seven metabolites (indole‐3‐lactic acid, anthranilic acid, serotonin, nicotinic acid, kynurenic acid, kynurenine and 3‐indoxyl sulfate) in both human serum and gastric juice using liquid chromatography–tandem mass spectrometry (LC–MS/MS). Serum and gastric juice were prepared with a simple protein precipitation using aqueous 0.1% formic acid and acetonitrile. As a result, it was found that the kynurenine pathway of tryptophan metabolism was activated in gastric cancer and that the metabolic ratio of kynurenine/tryptophan, which reflects the enzyme activity of indoleamine‐2,3‐dioxygenase, was associated with the observed metabolic changes. Finally, the investigation of tryptophan metabolites, especially kynurenic acid, in serum and gastric juice might serve as biomarkers for gastric cancer. The findings in this study provide critical information of tryptophan metabolism which can be applied to a serum‐based diagnostic test for gastric cancer.     

Determination of tryptophan and its metabolites in human plasma and serum by high-performance liquid chromatography with automated sample clean-up system

An automated high-performance liquid chromatographic method that incorporates direct injection of biological samples followed by chromatographic sample clean-up in a precolumn is described for the determination of tryptophan and its metabolites in human plasma and serum. The system gave reproducible data with a coefficient of variation of less than 3% with a sample size of 100 microliters of human plasma. The major tryptophan metabolites found in 100 microliters of human plasma were kynurenine, indolelactic acid, indoleacetic acid, indolepropionic acid, serotonin and 5-hydroxyindoleacetic acid. The level of tryptophan and kynurenine in individuals was constant in comparison with other metabolites. Analysis of samples from normal controls, diabetics, gravida and their foetuses showed a tendency for tryptophan metabolites to be low in maternal plasma.     

Determination of neopterin, kynurenine, tryptophan and creatinine in human serum by high throuput HPLC

A new HPLC method for simultaneous determination of neopterin, creatinine, kynurenine and tryptophan in human serum was developed and validated. Monolithic stationary phase's technology (two monolithic columns RP-18e were connected with guard monolithic cartridge 4.6 mm × 50 mm + 3.0 mm × 100 mm and 4.6 × 10 mm) and special auto sampler for micro titration plates (samples are storage in dark cooled place protected against evaporation) were combined with easy sample preparation step. As mobile phase 15 mmol/L phosphate buffer at pH 4.50 was used. Neopterin and tryptophan were detected using fluorescent detection and kynurenine and creatinine were detected by diode-array detection. This method may be suitable for large sequences of samples in clinical research and routine practice.     

Determination of tryptophan and kynurenine in human plasma by liquid chromatography-electrochemical detection with multi-wall carbon nanotube-modified glassy carbon electrode

A novel method was developed for the simultaneous determination of kynurenine and tryptophan by high‐performance liquid chromatography with electrochemical detection at multi‐wall carbon nanotube (MWCNT)‐modified glassy carbon electrode. The separation and detection conditions were optimized. The typical HPLC experiments were conducted by using a reversed‐phase ODS column with a mobile phase consisting of stock acetate buffer (pH 5)–methanol (4:1, v/v) using an isocratic elution at the flow rate of 1.0 mL/min. The obtained LODs for kynurenine and tryptophane were 0.5 and 0.4 µmol/L, respectively. The analytical method for human plasma samples was validated and confirmed by LC‐UV and LC‐MS. The recoveries were in the range of 84.8–110%, and the precision was lower than 5.9%. Copyright © 2010 John Wiley & Sons, Ltd.     

High-voltage electrophoretic separation of tryptophan metabolites of the kynurenine pathway

An analytical separation method for tryptophan and its seven metabolites of the kynurenine pathway by high-voltage paper electrophoresis is presented. Anthranilic acid, 3-hydroxyanthranilic acid, kynurenic acid, kynurenine, nicotinic acid, quinolinic acid, xanthurenic acid and unmetabolized tryptophan are measured in urine. Using radioactive labelling and scintillation counting as a quantification method, the relative standard deviation varied from 3.5% to 14.4%, corresponding to kynurenine and nicotinic acid, respectively. The recovery of labelled tryptophan added to urine was 95%. An advantage of the electrophoretic method is the minor tailing of spots and, hence, a good resolution of the components. For the monovalent anions of the kynurenine pathway metabolites, a linear correlation (r = 0.9996) was found between the experimental relative electrophoretic mobility and the quantity M-2/3, where M is the molecular mass of the anion.     

Simultaneous analysis of kynurenine and tryptophan in human plasma by capillary electrophoresis with UV detection

Plasma kynurenine (Kyn)/tryptophan ratio has been proposed as a useful marker for the monitoring activation of the cellular immune system. Here, we describe an easy capillary electrophoresis method with UV detection for the separation and detection of Kyn and tryptophan in human plasma using methltryptophan as internal standard. The plasma samples were simply treated with acentonitrile for the elimination of proteins, the supernatant was evaporated, and the dried sample was resuspended with water and directly injected on the capillary without sample derivatization procedures. The use of a run buffer composed by 100 mmol/L Bis-Tris propane at pH 2.15 allowed to baseline resolve the analytes within 9 min. Precision tests indicated a good repeatability of our method both for times (CV< 0.53%) and areas (CV< 2.8%). Moreover, a good reproducibility of intra-assay and interassay tests was obtained (CV < 3.9% and CV < 7.6%, respectively). The obtained limit of detections for Kyn and tryptophane, evaluated at 226 nm, were 0.15 and 0.40 μmol/L, respectively. The method suitability was tested by measuring analyte levels both in healthy volunteers, acute myocardial infarction and chronic kidney disease patients.     

Chromatographic analysis of tryptophan metabolites

The kynurenine pathway generates multiple tryptophan metabolites called collectively kynurenines and leads to formation of the enzyme cofactor nicotinamide adenine dinucleotide. The first step in this pathway is tryptophan degradation, initiated by the rate‐limiting enzymes indoleamine 2,3‐dioxygenase, or tryptophan 2,3‐dioxygenase, depending on the tissue. The balanced kynurenine metabolism, which has been a subject of multiple studies in last decades, plays an important role in several physiological and pathological conditions such as infections, autoimmunity, neurological disorders, cancer, cataracts, as well as pregnancy. Understanding the regulation of tryptophan depletion provide novel diagnostic and treatment opportunities, however it requires reliable methods for quantification of kynurenines in biological samples with complex composition (body fluids, tissues, or cells). Trace concentrations, interference of sample components, and instability of some tryptophan metabolites need to be addressed using analytical methods. The novel separation approaches and optimized extraction protocols help to overcome difficulties in analyzing kynurenines within the complex tissue material. Recent developments in chromatography coupled with mass spectrometry provide new opportunity for quantification of tryptophan and its degradation products in various biological samples. In this review, we present current accomplishments in the chromatographic methodologies proposed for detection of tryptophan metabolites and provide a guide for choosing the optimal approach.     

Screening for defects in tryptophan metabolism

We introduced a two-step procedure for the detection of defects in metabolism of tryptophan: (1) HPTLC (described previously) is suitable when starting the investigation, (2) two HPLC methods with isocratic elution and spectrophotometric detection are used at the next step, when pathological findings are to be confirmed and the individual metabolites quantified. The first method enables the assessment of tryptophan, 5-hydroxyindolylacetic acid, indolylacetic acid, indolylacryloylglycine, indolylacrylic acid and its possible precursors, namely indolyllactic and indolylpropionic acids. The second procedure is intended for the monitoring of anthranilic, 3-hydroxyanthranilic, kynurenic and xanthurenic acids, kynurenine, 3-hydroxykynurenine and indoxyl-sulfate. The same pre-treated sample is used for all methods.     

Study of the kynurenine pathway of tryptophan metabolism by capillary electrophoresis and mass spectrometry

A procedure has been proposed for the electrophoretic determination of main tryptophan metabolites demonstrating neurotoxic properties, i.e., kynurenine, 3-hydroxykynurenine, and kynurenic acid, with UV detection at 227 nm. The limit of detection at the signal-to-noise ratio equal to 3 makes 0.3 μg/mL. Individuals of fruit flies have been studied as biological objects. The chances for the autooxidation 3-hydroxykynurenine in biological objects are proved by mass spectrometry.     

Determination of choline and acetylcholine levels in rat brain regions by liquid chromatography with electrochemical detection

Regional choline (Ch) and acetylcholine (ACh) in rat brain were clearly determined by high-performance liquid chromatography with electrochemical detection. The method is based on that of Potter et al.: the hydrogen peroxide that is enzymatically produced from both compounds is measured and a successful improvement of the method, particularly for purification, is described. Recoveries were 96.1 +/- 1.4% for Ch and 95.6 +/- 2.2% for ACh and amounts as low as 10 pmol could be determined. Prior to measuring the compounds, a newly developed magnetic field microwave instrument (10 kW) was utilized for the rapid inactivation of brain enzymes. The levels of Ch and ACh in brain regions were compared with those reported elsewhere.     

Application of a novel micro-injector in the determination of indole derivatives in the rat pineal gland by capillary electrophoresis with electrochemical detection

A novel micro-injector has been fabricated for capillary electrophoresis (CE). It was successfully used for the determination of some indole derivatives for example melatonin (MT), serotonin (5-HT), tryptophan (Trp), and 5-hydroxy-tryptophane (5-HTrp) in the rat pineal gland by capillary electrophoresis with electrochemical detection (CE-EC). CE was performed in 0.20 mol L(-1) phosphate buffer (pH 8.0). The compounds investigated can be well separated and detected within 15 min. The working electrode used was a 300-microm diameter carbon electrode positioned opposite the outlet of the capillary. The relationship between peak current and analyte concentration was highly linear in the range from 0.10 to 500 micromol L(-1); detection limits (S/N = 3) were 0.03-0.13 micromol L(-1). The proposed method has been successfully used to analyze real biological samples.     

Fluorescence of tryptophan-containing peptides on paper or silica gel after treatment with formaldehyde, formaldehyde-ozone or formaldehyde-hydrochloric acid.

Sensitive and specific procedures for the chromatographic detection of tryptophan and tryptophan-containing peptides are described. Formaldehyde gas induces strong and characteristic fluorescence from tryptophan and peptides with NH2-terminal tryptophan residues on silica gel. On filter-paper, the detection of small amounts of these compounds requires the additional use of an oxidant, such as ozone. Treatment with formaldehyde-hydrochloric acid was used as a method for inducing fluorescence from tryptophan-containing peptides regardless of the position of the tryptophan residue in the peptide molecule. This reaction is useful for the chromatographic demonstration of small amounts of such peptides on both paper and silica gel. The spectral properties of the fluorophores of such tryptophan-containing peptides are distinctive and serve to distinguish them from all other known biogenic compounds that are capable of giving fluorescence with formaldehyde.     

Determination of tryptophan in raw materials, rat brain and human plasma by RP-HPLC technique

This paper describes tryptophan (TRP) estimation in raw human plasma and rat brain by reversed-phase high-performance liquid chromatography (RP-HPLC). Estimation was carried out on a Purospher STAR C18 column using water-acetonitrile (90:10 v/v, at pH 2.7) mixture at a rate of 1.5 mL/min as mobile phase. Eluents were monitored at 273 nm by an ultraviolet detector. The method was linear (R(2) > 0.999), precise (intra-day and inter-day precision <2%) in the range of 0.25-20 μg/mL. The detection and quantification limits were 0.0144 μg/mL and 0.0437 μg/mL, respectively. In human plasma, Day 1 and Day 2 precision were 0.054-2.29% and 1.66-3.7%; whereas precisions in rat brain were 1.23-2.3% and 0.677-4.2%, respectively. The method was applied to study TRP level in human smokers and in arthritic rat brain. An efficient RP-HPLC method was developed for TRP determination that worked for clinical and research purposes.