Simultaneous Determination Of Tryptophan Research Articles

Simultaneous Determination of Tryptophan and 5-hydroxytryptophan in Dietary Supplements using Capillary Zone Electrophoresis and Capacitively Coupled Contactless Conductivity Detection

Simultaneous Determination of Tryptophan and 5-hydroxytryptophan in Dietary Supplements using Capillary Zone Electrophoresis and Capacitively Coupled Contactless Conductivity Detection

Simultaneous determination of tryptophan, 5-hydroxytryptophan, tryptamine, serotonin, and 5-HIAA in small volumes of mouse serum using UHPLC-ED

In this paper we report a simple and efficient method for the concurrent analysis of tryptophan, 5-HTP, tryptamine, serotonin, and 5-HIAA in mouse serum using UHPLC-ED after protein precipitation and dilution. These compounds are neuroactive and are of interest in studies of mood and behavior; They are also biomarkers for the presence of neuroendocrine tumors and are used in the diagnosis of these cancers. After a brief series of validation experiments, this method was applied to serum from mouse behaviour experiments.•A convenient UHPLC method with electrochemical detection for concomitant analysis of the serotonin pathway in serum, including, for the first time, tryptamine.•The method met all performance criteria established for use in our lab and was applied in rodent experiments.

Simultaneous determination of tryptophan, kynurenine, kynurenic acid and two monoamines in rat plasma by HPLC-ECD/DAD

A high-performance liquid chromatography method with a diode array and an electrochemical detection (HPLC-ECD/DAD) was developed to determine the levels of tryptophan (TRP), kynurenine (KYN), kynurenic acid (KYA), 5-hydroxytryptamine (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) in rat plasma. The prepared samples were separated on a BDS column (4.6 mm × 250 mm, 5 mm) with column oven temperature of 25 °C. The mobile phase consisted of 5% acetonitrile and a buffer solution, which contained 25 mmol/L sodium acetate and 0.01 mmol/L EDTA, adjusting pH to 4.5 with acetic acid, and it was pumped at a flow-rate of 1.0 mL/min. KYN and KYA were measured by a variable wavelength detector at wavelengths 360 nm and 333 nm respectively, TRP and vanillic acid (as IS) both were measured at 280 nm. Determination of 5-HT and 5-HIAA was accomplished at the electrochemical working potential of 700 mV. Total run time was 14 min. Several parameters of the developed method were validated including linearity, accuracy precision, and stability. The results showed the established method had good LOD and separation for all of the five compounds and IS in the biological matrix. The method is simple, fast, economical and accurate. The analytical method and the results could provide a reference for the clinical and scientific research of depression.

Simultaneous determination of tryptophan and its 31 catabolites in mouse tissues by polarity switching UHPLC-SRM-MS

Tryptophan (TRP) and its catabolites have attracted a lot of attention because of their clinical significance to human health. Recently, microbiome-gut-brain axis was found to have links to many diseases based on the imbalance of TRP catabolism. By using ultra-high performance liquid chromatography coupled to electrospray ionization triple quadrupole mass spectrometry, we present a rapid, robust and comprehensive method to determine 31 TRP catabolites covering three major pathways - kynurenic, serotonergic and bacterial degradation - within 5 min. Polarity switching was employed to analyze catabolites in both ionization modes simultaneously for greatly improved analytical throughput. The intra-day and inter-day precision were 0.5–15.8% and 1.5–16.7%, respectively. Accuracy was between 75.8 and 126.9%. The developed method was applied to study the tissue level of TRP catabolites in the liver, ileum, ileal contents, brain and plasma samples from 8 mice, and clear differences in the distribution of TRP catabolites were observed in different tissues. Ratios of key catabolites to TRP were used to evaluate the activities of specific enzyme and pathway in respective tissues. This method has potential in high throughput analysis of TRP catabolites in biological matrices, which can facilitate understanding the influence of TRP catabolites on microbiome-gut-brain axis and on human health.

Simultaneous determination of tryptophan and 8 metabolites in human plasma by liquid chromatography/tandem mass spectrometry.

Tryptophan (Trp) is an essential amino-acid and the precursor of many biologically active substances such as kynurenine (KYN) and serotonin (5HT). Its metabolism is involved in different physiopathological states, such as cardiovascular diseases, cancer, immunomodulation or depression. Hence, the quantification of Trp catabolites, from both KYN and 5HT pathways, might be usefulfor the discovery of novel diagnostic and follow-up biomarkers. We have developed a simple method for quantification of Trp and 8 of its metabolites,involved in both KYN and 5HT pathways, using liquid chromatography coupled to tandem mass spectrometry. We also validated the methodin human plasma samples, according to NF EN ISO 15189 criteria. Our method shows acceptable intra- and inter-day coefficients of variation (CV) (<12% and <16% respectively). The linearity entirelycovers the human plasma range. Stabilities of whole blood and of residues weredetermined, as well as the use of 2 different types of collectiontube, enabling us to adapt our process. Matrix effects and reference values showed good agreement compared to the literature. We propose here a method allowing the simultaneous quantification of a panel of Trp catabolites, never used before to our knowledge. This method, witha quickchromatographic runtime (15min) and simple sample preparation, has beenvalidated according to NF EN ISO 15189 criteria. The method enables the detailed analysis of these metabolic pathways, which are thought to be involved in a number of pathological conditions.

Simultaneous Determination of Tryptophan, Kynurenine and Niacin in Serum of Periparturient Dairy Cows by High-Performance Liquid Chromatography with Diode Array Detection

Simultaneous Determination of Tryptophan, Kynurenine and Niacin in Serum of Periparturient Dairy Cows by High-Performance Liquid Chromatography with Diode Array Detection Tryptophan is an essential amino acid and substrate for important biochemical pathways, e.g. generating the neurotransmitter serotonin or building kynurenine and nicotinamide via the kynurenine pathway which can be induced by the enzyme tryptophan 2,3-dioxygenase (TDO) via glucocorticoid hormones or by indoleamine 2,3-dioxygenase (IDO) after immune activation. As the kynurenine to tryptophan ratio is regarded as indicator for an activated immune system, we developed an analytical method for the simultaneous determination of kynurenine, tryptophan and nicotinamide in serum via high-performance liquid chromatography (HPLC) und assessed their suitability as inflammatory markers in dairy cows. Validation parameters have shown that the HPLC method meets the requirements for routine analysis of tryptophan and its metabolites kynurenine and nicotinamide in serum with high linearity within the respective working ranges and limits of quantifications of 0.41 µmol L-1 for nicotinamide, 0.43 µmol L-1 for kynurenine and 3.40 µmol L-1 for tryptophan. The intra-day and inter-day variations were 2.3 and 3.6% (nicotinamide), 3.1 and 6.3% (kynurenine) and 1.9 and 5.2% (tryptophan), respectively. Tryptophan degradation via the kynurenine pathway was investigated in 10 periparturient dairy cows out of two dietary groups during the transition period. The course of tryptophan concentrations in cow serum around calving was similar to those found in pregnant women with a decrease until birth, followed by increased and normalized postpartum tryptophan concentrations. However, rising kynurenine concentrations and a simultaneous incline of the kynurenine to tryptophan ratio during gestation could not be confirmed in our study, apart from a peak of the kynurenine to tryptophan ratio at parturition which might be due to stress hormone-induced activation of hepatic TDO and extrahepatic IDO. Additional measurements of interferon-γ and glucocorticoids as putative inductors of TDO and IDO would be useful to verify the suitability of the kynurenine to tryptophan ratio as an immune marker in the bovine.

Simultaneous determination of tryptophan and its metabolites in plasma by high performance liquid chromatography with on-column derivatization

A high performance liquid chromatography-ultraviolet/fluorescence detection (HPLC-UV/FLD) with on-column derivatization was established to simultaneously determine tryptophan (Trp), kynurenine (Kyn), 5-hydroxyindole acetic acid (5-Hiaa) and kynurenic acid (Kyna). A Hypersil C-18 column (250 mm x 4.0 mm, 5 microm) was used for the analysis at 30 degrees C. The separation was carried out with the mobile phase consisting of 250 mmol/L zinc acetate (pH 5.5) and acetonitrile (95: 5, v/v) at a flow rate of 0.8 mL/min using 3-nitrotyrosine as internal standard (IS). The excitation (Ex) and emission (Em) wavelengths were set at 278 nm (lambda(ex))/343 nm (lambda(em)) for 5-Hiaa and 244 nm (lambda(ex))/400 nm (lambda(em)) for Kyna, while the wavelengths of ultraviolet detection were set at 360 nm for Kyn and IS, 302 nm for Trp. The recoveries were in the range of 91.62% to 114.17%. The linearities were from 2.50 micromol/L to 320.00 micromol/L for Trp, 0.32 micromol/L to 15.36 micromol/L for Kyn, 3.27 nmol/L to 104.60 nmol/L for 5-Hiaa, and 14.00 nmol/L to 464.80 nmol/L for Kyna. The detection limits were 0.078 micromol/L, 0.056 micromol/L, 0.690 nmol/L and 1.290 nmol/L for Trp, Kyn, 5-Hiaa, and Kyna, respectively. Thirty plasma samples of normal pregnant women and 28 plasma samples of healthy controls were tested, and the results exhibited that the concentrations of Trp, Kyn and Kyna in the plasma of the normal pregnant women were significantly different from those of the control group (all P < 0.01). The method is simple and sensitive with good reproducibility, and it is suitable for clinical measurements.

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

Objective To establish an accurate method for simultaneous determination of plasma Kyn and Trp by HPLC-UV detection.Methods Kyn and Trp were separated on Agilent Hypersil ODS column using 3-nitrotyrosine as internal standard.The mobile phase consisted of 15 mmol/L sodium acetateacetic acid (pH 5.5):acetonitrile 94∶ 6(v/v) at a rate of 0.8 ml/min.The chromatographic separation was performed at 25 ℃.The eluate was monitored with programmed wavelength setting at 360 nm from 0 to 4 min for Kyn and at 302 nm from 4 to 5 min for Trp.The method was applied to determination of plasma Kyn and Trp in 8 chronic glomerulonephritis,10 idiopathic thrombocytopenic purpura,15 chronic hepatitis B virus patients and 15 healthy controls from September to December in 2010.The differences were compared using ANOVA and SNK methods.Results The retention time of Kyn and Trp were 2.9 min and 4.4 min,respectively.For Kyn,the assay was linear from 0.44 μmol/L to 18.30 μmol/L.For Trp,the linearity was from 3.67 μmol/L to 470.00 μmol/L.The detection limits were 0.014 μmol/L for Kyn and 0.122 μmol/L for Trp,respectively.The within-day CVs were ＜ 3％ and the between-day CVs were ＜ 4％.The mean recoveries yield were in the range of 92.29 to 104.40.The plasma concentrations of Kyn were ( 1.59 ± 0.28),(2.73 ± 0.56),(2.69 ± 0.44) and ( 1.54 ± 0.48 ) μmol/L,the plasma concentrations of Trp were (59.8 ± 10.0),(46.1 ± 11.7),(58.5 ±8.0) and (41.4±13.1) μmol/L,the Kyn/Trp were (0.027 4±0.007 5),(0.061 6 ±0.016 5),(0.046 7 ±0.009 1) and (0.038 3 ±0.007 5)in controls,chronic glomerulonephritis patients,idiopathic thrombocytopenic purpura patients and chronic hepatitis B virus patients,respectively.There were significance difference of Kyn,Trp and Kyn/Trp amony the four groups (F=23.734,8.463,20.921,all P＜0.01).Conclusion The method is simple,fast,and suitable for applicability to clinical measurement. Key words: Tryptophan; Kynurenine; Chromatography,high pressure liquid

Simultaneous determination of tryptophan, kynurenine, kynurenic and xanthurenic acids in honey by liquid chromatography with diode array, fluorescence and tandem mass spectrometry detection

A liquid chromatography method using diode array-fluorescence detection and atmospheric pressure chemical ionization mass spectrometry (LC-DAD-FLD and LC–APCI-MS/MS) was developed to quantify the levels of tryptophan (TRP), kynurenine (KYN), kynurenic (KYNA) and xanthurenic (XA) acids in honey. This procedure involved isolating the compounds of interest via solid-phase extraction (SPE) with mixed-mode polymeric cartridges. Chromatographic separation of the analytes was performed in isocratic mode on a Synergi 4μ Hydro-RP 80Å (150 × 4.60 mm i.d.) analytical column at 30 °C. The mobile phase of 20 mM ammonium formate (pH 4) and methanol was passed at a flow rate of 0.5 mL/min. In replicate sets of spiked honey samples, the average analyte recoveries ranged from 60 to 98% for TRP, 55 to 120% for KYN, 65 to 106.5 for KYNA and 56 to 114% for XA. Detection limits ranged from 4 to 36 μg/kg for LC-DAD-FLD to 0.2 and 1.0 μg/kg for LC–APCI-MS/MS. A strong matrix effect was found when MS/MS was employed, necessitating calibration using the standard addition method on matrix-matched standards for each honey type. The method was used to quantify each of the compounds of interest in 17 honey samples of distinct botanical origins.

Simultaneous determination of tryptophan and its key metabolites by high performance liquid chromatography with programmed wavelength ultraviolet detection

A high performance liquid chromatographic (HPLC) method with programmed wavelength ultraviolet (UV) detection was established to simultaneously determine tryptophan (Trp) and its key metabolites kynurenine (Kyn) and 5-hydroxytryptamine (5-HT). A BDS-Hypersil-C8 column (150 mm x 4.6 mm, 5 microm) was used for the analysis at 25 degrees C. The separation was carried out with the mobile phase consisting of 10 mmol/L sodium acetate-acetic acid (pH 4. 5) and acetonitrile (94: 6, v/v) using theophylline as internal standard (IS) at a flow rate of 0.6 mL/min. The eluates were monitored by the programmed wavelength UV detection at 360 nm for Kyn and IS, 220 nm for 5-HT and 302 nm for Trp. The mean recoveries were in the range of 87% to 113%. The linearities were from 3.97 to 400 micromol/L for Trp, 0.421 to 20.2 micromol/L for Kyn and 4.36 to 980.5 nmol/L for 5-HT. The detection limits were 0.134 micromol/L for Trp, 0.016 micromol/L for Kyn and 2.03 nmol/L for 5-HT. Fifteen plasma samples of patients with depression and fifteen plasma samples of healthy controls were tested, and the results demonstrated that the metabolism of Trp in the plasma from the depression group was significantly different from that of the control group.

Simultaneous determination of tryptophan, kynurenine and 5-hydroxytryptamine by HPLC: Application in uremic patients undergoing hemodialysis

Objectives To develop a reliable HPLC method for the simultaneous determination of plasma tryptophan, kynurenine and 5-hydroxytryptamine to analyze tryptophan metabolism. Design and methods Separation was carried out on a C8 column with the mobile phase composed of acetate buffer (pH 4.5) and acetonitrile using theophylline as internal standard. The eluates were monitored by ultraviolet detection with programmed wavelength. Results Analysis was achieved in less than 8.0 min. The limits of quantification were 3.97 μmol/L, 4.36 nmol/L and 0.421 μmol/L for tryptophan, 5-hydroxytryptamine and kynurenine, respectively. Reproducibility and recovery were satisfactory. Twenty healthy adults and 20 uremic patients undergoing hemodialysis were analyzed using the present method. Tryptophan metabolism was found to be disturbed in uremic patients and was improved obviously after hemodialysis. Conclusions The developed HPLC method is simple, reliable and suitable for monitoring tryptophan metabolism in uremic patients undergoing hemodialysis.

Simultaneous determination of tryptophan and tyrosine in binary mixture by zero-crossing second derivative spectrophotometry.

Rapid and accurate binary mixture resolution of tryptophan and tyrosine was performed. Differential-derivative spectrophotometry with a zero-crossing measurement technique was used for the quantitative determination of tryptophan and tyrosine in laboratory-prepared mixtures. Neither sample pretreatment nor separation were required. Linear calibration graphs of differential second derivative values were (at 222.4 and 217.9 nm for tryptophan and tyrosine , respectively) versus concentration in the ranges 0.1–20.0 and 1.0– 50.0 µg ml_1, and the linearity was satisfactory (r = 0.9987 and r = 0.9997), for tryptophan and tyrosine , respectively) were obtained .The relative standard deviations were found to be less than 1.03%, indicating reasonable repeatibility of method.The results obtained by the proposed method has been statistically compared by means of Student t-test and by the variance ratio F-test where shows a good agreement .

Simultaneous determination of tryptophan and kynurenine in plasma samples of children patients with Kawasaki disease by high-performance liquid chromatography with programmed wavelength ultraviolet detection

A simple, fast, sensitive and specific high-performance liquid chromatography (HPLC) method is developed for simultaneous determination of kynurenine (Kyn) and tryptophan (Trp) with ultraviolet (UV) detection setting programmed wavelength. The separation was carried out on an Agilent Hypersil ODS column (125 mm × 4.0 mm, 5 μm) in less than 6 min and the eluate was monitored by the programmed wavelength detection setting at 360 nm from 0 min to 4 min for Kyn, and at 278 nm from 4 min to 6 min for Trp in a single run with UV detector. The linearities of the method were from 0.20 μmol/L to 21.2 μmol/L for Kyn and 2.25–678.0 μmol/L for Trp, and the detection limits were 0.028 μmol/L for Kyn and 0.053 μmol/L for Trp, respectively. Satisfactory precisions and recoveries were obtained by this method. The assay was employed to analyze plasma samples of children patients with Kawasaki disease (KD). The result showed great difference between Kawasaki disease and control group.

Simultaneous determination of tryptophan, uric acid and ascorbic acid at iron(III) doped zeolite modified carbon paste electrode

A new chemically modified electrode is constructed based on iron(III) doped zeolite modified carbon paste electrode (Fe 3+Y/ZCME). The electrode was evaluated as a sensor for sub-micromolar determination of tryptophan (Trp), uric acid (UA) and ascorbic acid (AA) in aqueous solutions. The measurements were carried out by application of the differential pulse voltammetry (DPV) method in phosphate buffer solution with pH 3.5. Iron(III) loaded in zeolite can increase anodic peak currents by adsorption of Trp, UA and AA on electrode surface The analytical performance was evaluated with respect to the carbon paste composition, pH of solution, accumulation time and accumulation potential. The prepared electrode shows voltammetric responses with high sensitivity and selectivity for Trp, UA and AA in optimal conditions, which makes it very suitable for simultaneous determination of these compounds. The linear calibration range for AA in the presence of 50 μM UA and 50 μM Trp was 0.6 μM to 100 μM, with a correlation coefficient of 0.9992, and a detection limit of 0.21 μM ( S/ N = 3). A linear relationship was found for UA in the range of 0.3–700 μM containing 10 μM AA and 50 μM Trp, with a correlation coefficient of 0.9990 and a detection limit of 0.08 μM. The linear calibration range for Trp in the presence of 10 μM AA and 50 μM UA was 0.2–150 μM, with a correlation coefficient of 0.9996, and a detection limit of 0.06 μM. The proposed method was successfully applied for determination Trp, UA and AA in biological systems and pharmaceutical 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.

Simultaneous determination of tryptophan and glutathione in individual rat hepatocytes by capillary zone electrophoresis with electrochemical detection at a carbon fiber bundle--Au/Hg dual electrode.

A method for single-cell analysis was developed by combining capillary zone electrophoresis (CZE) with electrochemical detection (ECD) using a dual electrode consisting of two different types of electrode material (carbon fiber and Au/Hg). In this method, the parallel mode was used. Different potentials were applied to both electrodes of the dual electrode. Tryptophan and glutathione, which could not be simultaneously detected by normal CZE-ECD, could be simultaneously and selectively detected by CZE-ECD at the dual electrode in one run, respectively. The CZE-ECD system with the dual electrode was applied to determine them in individual rat hepatocytes.

Simultaneous determination of tryptophan, 5-hydroxytryptophan, 5-hydroxytryptamine, 5-hydroxyindoleacetic acid, 4-hydroxy-3-methoxyphenylacetic acid and 3-methoxy-4-hydroxyphenylglycol in human cerebrospinal fluid

Serotonin (5-hydroxytryptamine) metabolism may be influenced by its precursor tryptophan. A method utilizing reversed-phase high-performance liquid chromatography and electrochemical and ultraviolet detection with a mobile phase composed of acetate buffer and methanol has been developed for determination of tryptophan, its metabolites 5-hydroxytryptophan, serotonin, 5-hydroxyindoleacetic acid, as well as 4-hydroxy-3-methoxyphenylacetic acid (homovanillic acid) and 3-methoxy-4-hydroxyphenylglycol in human cerebrospinal fluid (CSF). The electrochemical potential is set at 0.6 V in order to reduce the background current. Since tryptophan is not electroactive at this potential, it is detected by ultraviolet absorbance. The present method is simple, rapid, specific and accurate as compared with a previously reported method. No sample pretreatment is necessary and it takes ca. 20 min to run a sample. The concentrations of the compounds measured in CSF are similar to those obtained by HPLC in previous reports, although there are still arguments about the true level of serotonin in CSF.

Rapid Simultaneous Determination of Tryptophan and Tyrosine in Synthetic Peptides by Derivative Spectroscopy

A method for the simultaneous quantitative determination of tryptophan and tyrosine in synthetic peptides by second-order derivative diode-array spectroscopy is reported. The method does not require hydrolysis of the peptides or a derivatization reaction; the sample is dissolved in 0.1 N NaOH and directly scanned between 262 and 264 nm to detect tyrosine and between 304 and 306 nm to detect tryptophan. From these results the peptide content of the synthetic sample can be easily calculated in a very simple and fast way. The results obtained by the described method with several peptides are compared with those obtained by classical high-performance liquid chromatographic analysis of amino acids after peptide hydrolysis. A very good correlation was found both for the tryptophan/tyrosine molar ratio and the peptide content.

Simultaneous determination of tryptophan and its metabolites in mouse brain by high-performance liquid chromatography with fluorometric detection

A new method for the determination of tryptophan and its metabolites in a single mouse brain using high-performance liquid chromatography (HPLC) with fluorometric detection is described. Tryptophan, serotonin, 5-hydroxyindoleacetic acid, indoleacetic acid, and tryptophol were clearly separated by a C8 reverse-phase column. Tissue preparation is performed only to centrifuge homogenates of brain prior to the injection to HPLC. The sensitivity is in the range from 10 to 15 pg.

Simultaneous determination of tryptophan and its 5-hydroxy metabolites in human cerebrospinal fluid by reversed phase liquid chromatography with electrochemical detection

A sensitive ion-pair reversed phase liquid chromatographic method for determining tryptophan, 5-hydroxytryptophan, 5-hydroxytryptamine (serotonin) and 5-hydroxyindole acetic acid has been developed. To separate these indoles in cerebrospinal fluid both octadecyl- and phenyl-bonded columns were tested. The effect of an ion-pair reagent (n-heptyl sulphonate) and pH of the mobile phase on the separation was examined. With the method described the concentrations of the four indoles can be determined within 45 min without sample pretreatment. The sensitivity obtained with electrochemical detection is 0.45 mumol/l for tryptophan and 2-4 nmol/l for 5-hydroxyindoles in human cerebrospinal fluid samples.