Sensitive determination of deuterated and non-deuterated tryptophan, tryptamine and serotonin by combined capillary gas chromatography and negative ion chemical ionization mass spectrometry

Sensitive methods for the determination of deuterated and non-deuterated tryptophan, tryptamine and serotonin by combined capillary gas chromatography and negative ion chemical ionization mass spectrometry were developed. [3,3-2H2]-L-Tryptophan, which was used as a tracer, was synthesized for studies of their in vivo metabolism. Tryptophan was converted into its trifluoroacetylmethyl derivative after prepurification with an AG 50W-X2 cation-exchange column. Tryptamine and serotonin were extracted with 20% butanol in diethyl ether and derivatized with trifluoroacetic anhydride. These derivatives were separated and determined by selected ion monitoring. In these determinations, [2',3,3,4',5',6',7'-2H7]-D,L-tryptophan, [alpha,alpha,beta,beta-2H4]tryptamine and [alpha,alpha,beta,beta-2H4]serotonin were used as internal standards.     

4-Hydroxylindole-acetic acid — The microbiological transformation product of indole-3-acetic acid

Conclusions The isolation of an unknown intermediary in the microbiological transformation of indole-3-acetic acid to 4-hydroxyindole-3-acetic acid may help us to determine the structure of 4-hydroxyindole-3-acetic acid.Translated from Izvestiya Akademii Nauk SSSR, Seriya Khimicheskaya, No. 5, pp. 1155–1158, May, 1975.The authors express their gratitude to G. K. Skryabin and M. N. Kolosov for assistance in carrying out the present work.     

Highly sensitive method for the determination of 1-methyl-1,2,3,4-tetrahydro-beta-carboline using combined capillary gas chromatography and negative-ion chemical ionization mass spectrometry

A sensitive method was developed for the determination of deuterated and non-deuterated 1-methyl-1,2,3,4-tetrahydro-beta-carboline by combined capillary gas chromatography and negative-ion chemical ionization mass spectrometry. 1-Methyl-1,2,3,4-tetrahydro-beta-carboline was converted into a trifluoroacetyl derivative after pretreatment with fluorescamine and extraction with ethyl acetate. The derivative was separated by capillary gas chromatography and determined by selected-ion monitoring. In the determination, [3,3,4,4-2H4]-1-methyl-1,2,3,4-tetrahydro-beta-carboline was used as an internal standard. The method developed in this work was used for the determination of deuterated and non-deuterated 1-methyl-1,2,3,4-tetrahydro-beta-carboline in human urine samples collected before and after administration of [3,3-2H2]-L-tryptophan.     

Determination of Indole-3-acetic Acid and Abscisic Acid by Double-Direction Dispersive Liquid–Liquid Microextraction Coupled with High-Performance Liquid Chromatography

A method for the separation and purification of indole-3-acetic acid and abscisic acid based on double-direction dispersive liquid–liquid microextraction coupled with high-performance liquid chromatography was developed. The reverse-direction dispersive liquid–liquid microextraction procedure was very efficient in the purification of the hormones. The forward-direction dispersive liquid–liquid microextraction procedure was very efficient in the enrichment of the analytes. Under optimum conditions, a linear response was observed over the ranges of 0.2 µg/g to 200.0 µg/g for indole-3-acetic acid and 0.1 µg/g to 200.0 µg/g for abscisic acid, with correlation coefficients of 0.9967 and 0.9983. The limits of detection for indole-3-acetic acid and abscisic acid were 0.02 µg/g and 0.01 µg/g. The recoveries by standard addition were 91.4% and 88.6% for indole-3-acetic acid and abscisic acid with corresponding relative standard deviations of 4.5% and 6.1% (n = 5). The developed method was used for the preconcentration and determination of indole-3-acetic acid and abscisic acid in Arabidopsis thaliana, and satisfactory results were obtained.     

Voltammetric determination of 5-hydroxydole-3-acetic acid in human gastric juice

A method for the determination of the major serotonin metabolite — 5-hydroxyindole-3-acetic acid (5-HIAA) in human gastric juice by cyclic voltammetry was described. The measurement conditions were investigated. The potential window was chosen from +0.1 to +0.9 V, the supporting electrolyte was 0.025 M PBS solution (pH 2.0). The method allowed determination in the concentration range from 2.0 ×10⁻⁷ to 2.0 ×10 ⁻⁵ M and a detection limit of 80 nM. When samples of gastric juice were analyzed with the method, we obtained the mean content of 5-HIAA in the gastric juice. Meanwhile, interference from other ions and substances were examined. The experimental results indicate that the method for the determination of gastric juice samples is successful.     

Simultaneous determination of serotonin and 5-hydroxyindole-3-acetic acid in human urine by automated precolumn derivatization and semi-microbore column liquid chromatography with fluorescence detection.

An improved high-performance liquid-chromatographic (HPLC) assay for the simultaneous determination of serotonin (5-HT) and 5-hydroxyindole-3-acetic acid (5-HIAA) in human urine is reported. Following the automated precolumn derivatization of urinary 5-HT and 5-HIAA with benzylamine, the derivatives are separated by isocratic elution on a reversed-phase C18 semi-microbore column, and are fluorometrically detected at an excitation wavelength of 345 nm and an emission wavelength of 480 nm. The detection limits of 5-HT and 5-HIAA are 7 and 5 nmol/l in urine (0.7 and 0.5 fmol/20-microl injection). The proposed automatic method permits a highly selective and sensitive determination of 5-HT and 5-HIAA in human urine without any sample purification.     

Mass fragmentographic determination of 5-hydroxytryptamine and 5-hydroxyindole-3-acetic acid in brain tissue using deuterated internal standards.

A mass fragmentographic method for the determination of 5-hydroxytryptamine (5-HT) and 5-hydroxyindole-3-acetic acid (5-HIAA) in the same extract of rat brain tissue is decribed. Deuterium-labelled analogues were used as internal standards. 5-HT and 5-HIAA were separated by solvent extraction and pentafluoropropionyl derivatives were prepared for the mass fragmentographic analysis. Multiple ion analysis confirmed the identity of 5-HT and 5-HIAA in the rat brain. At the mass numbers used routinely for the determination of 5-HT and 5-HIAA in the rat brain. At the mass numbers used routinely for the determination of 5-HT and 5-HIAA, the experimental error was below 3% (calculated from mean values of 0.05 and 0.24 nmole, respectively). The recovery of the authentic compounds added to brain extracts was more than 95%. The levels of 5-HT and 5-HIAA in the rat brain were 2.95 +/- 0.16 and 0.64 +/- 0.18 nmole/g, respectively. More than 100 samples could be analyzed within 3 days. The presence of 5-hydroxytryptophol in rat brain was also investigated, but none could be detected either as a conjugate or as the free alcohol.     

Characterization of monohydroxylated derivatives of the anticancer agent flavone-8-acetic acid by liquid chromatography with on-line UV and mass spectrometry

The experimental anticancer agent flavone-8-acetic acid (FAA) is metabolized into several monohydroxylated derivatives using mouse microsomes. Because these metabolites could be involved in the biological effects of FAA, the aim of this study was to characterize all its possible monohydroxylated derivatives. To do so, we have developed a methodology using reversed-phase high-performance liquid chromatography (RP-HPLC) coupled with ultraviolet (UV) detection and mass spectrometry (MS) to analyze and identify FAA derivatives hydroxylated at the 2', 3', 4', 3, 5, 6, or 7 position. In RP-HPLC, 4'-, 3'-, 2'-, 6-, and 7-OH-FAA eluted before FAA, whereas 3- and 5-OH-FAA eluted after FAA. UV spectra showed a bathochromic shift of band I for all derivatives and of band II for 5- and 6-OH-FAA. In addition, the position of the OH group could be determined by the presence of certain product ions in MS. Ions at m/z 133 and 151 were specific for 2'-, 3'-, 4'-, and 3-OH-FAA, whereas the ion at m/z 177 was specific for 3-OH-FAA only. The ions m/z 133, 151 and 167 were specific for 2'-OH-FAA. Ions at m/z 149 were specific for the presence of the OH group on cycle A only (i.e., 5-, 6- or 7-OH-FAA). The presence of both product ions m/z 149 and 179 were specific for 7-OH-FAA. Finally, ions at m/z 149 and several product ions of even m/z values were specific for 5-OH-FAA. In conclusion, the methodology described can be used to identify all possible monohydroxylated FAA derivatives.     

Room-temperature phosphorimetric determination of biogenic indole compounds adsorbed on a thin-layer chromatographic plate

A simple and sensitive method is described for the determination of biogenic indole compounds adsorbed on cellulose or alumina plates for thin-layer chromatography by room-temperature phosphorimetry. The optimum conditions were investigated for 5-hydroxyindole-3-acetic acid and indole-3-acetic acid. The compounds are spotted on the plates, which are then sprayed successively with sodium citrate or sodium acetate, and sodiu iodide solutions. The plates are dried completely under a stream of dry nitrogen, and immediately dipped in molten paraffin. The phosphorescence is stable for at least 3 h even in moist air. The limits of detection for nine biogenic indole compounds tested are between 2 and 300 pmol per sample spot.     

Labelling indole-3-acetic acid with tritium

A simple method of obtaining indole-3-acetic acid/ growth stimulating plant hormone/ labelled with tritium has been described. The preparate has specific radioactivity of 455 MBq/mmol, and 50% yield as compared to the initial amount of used substance.     

Simultaneous Electrochemical Determination of Indole-3-acetic Acid and Salicylic Acid in Pea Roots using a Multiwalled Carbon Nanotube Modified Electrode

Indole-3-acetic acid and salicylic acid are essential phytohormones with profound effects on growth and development as well as stress responses of plants. Accumulating evidence has suggested that these compounds mediate the same biological process by collaboration or antagonistic actions. Simultaneous determination of indole-3-acetic acid and salicylic acid may significantly improve the understanding on mechanisms of their interaction. In this study, the simultaneous determination of these analytes was electrochemically performed using a multiwalled carbon nanotubes-chitosan modified glassy carbon electrode. Based on differential pulse voltammetry, indole-3-acetic acid and salicylic acid were determined from 0.67 to 48.82 micromolar with detection limits of 0.1 micromolar. In addition, no interferences were observed from other molecules. This method was employed for the simultaneous determination of the analytes in pea root extracts. The approach may be extended for additional study of the interaction of indole-3-acetic acid and salicylic acid in plants.     

Rapid and sensitive detection of auxins and flavonoids in plant samples by high-performance liquid chromatography coupled with tandem mass spectrometry

Simultaneous determination of indole-3-acetic acid and methyl indole-3-acetic acid ester in small amounts of plant tissue is essential for elucidating their mutual transformation mechanism and the in vivo function of methyl indole-3-acetic acid ester. Rapid quantification of flavonoids in the same sample is important for clarifying their roles in the transport of auxins and other phytohormones. Herein, we describe a simple method for the simultaneous determination of indole-3-acetic acid and its methyl ester in the roots of the Arabidopsis thaliana seedlings and a protocol for the rapid extraction and quantification of quercetin and kaempferol in these seedlings. High-performance liquid chromatography coupled with electrospray ionization time-of-flight tandem mass spectrometry was used for the detection of all the compounds. Negative data for indole-3-acetic acid and positive data for methyl indole-3-acetic acid ester were collected in two successive files with a single injection of the extracted sample. Under optimized conditions, the limit of detection for the four compounds was 2 ng/mL for indole-3-acetic acid, 0.5 ng/mL for methyl indole-3-acetic acid ester, 5 ng/mL for quercetin, and 1 ng/mL for kaempferol, respectively. Because of the high sensitivity of the assay, only 2-10 mg of the plant material was required to obtain quantitative results.     

Gas chromatography-mass spectrometry method for determining the methanol and acetic acid contents of pectin using headspace solid-phase microextraction and stable isotope dilution

A simple, fast, and direct procedure was developed for the simultaneous determination of the methanol and acetic acid present as esters in the plant cell wall polysaccharide pectin. After base-hydrolysis of esters and acidification of pectin samples, headspace solid-phase microextraction (SPME) was performed using a Carboxen-PDMS fiber assembly. Methanol and acetic acid were separated by gas chromatography with a Chrompak PoraPlot Q capillary column and detected using electron impact mass spectrometry with selected ion monitoring. Stable deuterated isotopomers (d3-methanol and d3-acetic acid) were used as internal standards and for constructing calibration curves, providing accurate and absolute quantification of analytes. The methanol and acetic acid contents in 1 mg quantities of fruit and vegetable pectins were readily quantified by this procedure.     

Indolic urinary melanogens: separation and identification by gas chromatography with selected-ion monitoring mass spectrometry of 5-hydroxy-6-methoxyindole-2-carboxylic and 5-methoxy-6-hydroxyindole-2- carboxylic acids.

Two isomeric urinary melanogens, 5-hydroxy-6-methoxyindole-2-carboxylic acid and 5-methoxy-6-hydroxyindole-2-carboxylic acid, have been separated by gas chromatography with selected-ion monitoring mass spectrometry. After chemical synthesis of one of these two isomers, 5-methoxy-6-hydroxyindole-2-carboxylic acid, and the establishment of the mass spectrum of its trimethylsilylated derivative, a 30-ml sample of a melanotic 24-h urine was adjusted to pH 1 and extracted twice with 10 ml of ethyl acetate. The extract was evaporated to dryness and the residue derivatized with methyl-8, followed by Tri-Sil/TBT. Silylated derivatives were analysed by gas chromatography with selected-ion monitoring mass spectrometry. The mass spectrum of the 5-methoxy-6-hydroxyindole-2-carboxylic acid allowed the determination of the retention times of both isomers.     

Analysis of Fluorescent Compounds in Urine by Liquid Chromatography

Abstract

High speed separation of fluorescent compounds was examined. The retention time of 21 compounds was measured in two reversed phase modes and an ion-exchange mode liquid chromatography. Furthermore, urine samples of new-born babies, cancer patients and normal subjects were analyzed by the above systems. Several peaks were positively identified from the retention time, however there were many unknown fluorescent compounds. Among them, two peaks were found on the chromatograms in the reversed phase modes. These compounds were very polar and could not be identified, however the ratio of these peak height was used for classification of urine samples. Furthermore, indole-3-acetic acid and 5-hydroxyindole-3-acetic acid in urine were selectively analyzed on an ion-exchange resin with isocratic eluent after filtration.     

Spectrophotometric determination of tryptophan by reaction with nitrous acid

The heterocyclic nucleus in tryptophan is oxidised by nitrous acid at elevated temperature to produce a phenolic intermediate which further reacts with nitrous acid to form a nitro compound that has a golden yellow colour in alkaline medium. The maximum absorbance is obtained at 400 nm with maximum molar absorptivity of 9.44 x 10(3) l.mole(-1). cm(-1). Derivatives of tryptophan such as indole-3-acetic acid, tryptamine and tryptophanamide, as well as indole, produce the same colour, but some others, e.g., 5-hydroxytryptamine and 5-hydroxyindole-3-acetic acid do not give the colour reaction. A plausible mechanism is proposed to explain this behaviour.     

Fluorimetric determination of indole derivatives by fluorophore generation in copper(II)-sulphuric acid solution

A spectrofluorimetric procedure for the determination of indole-3-acetic acid, indole-3-propanoic acid and indole-3-butyric acid by derivatization with copper sulphate—sulphuric acid solution has been developed. The optimum reaction conditions, the effect of interferents and the advantages associated with the use of first- and second-derivative synchronous spectrofluorimetry have been studied. The detection limits are 3, 12 and 6 ng/ml for indole-3-acetic, indole-3-propanoic and indole-3-butyric acid, respectively.     

An improved radiometric assay of plant peroxidases based in the decarboxylation of indole-3-[1-14C]acetic acid

A radiometric microassay has been developed to measure the indole-3-acetic acid oxidizing activity of plant peroxidases. This was based in a reappraisal of the pre-existing assay of the indole-3-acetic acid oxidase activity based in the decarboxylation of indole-3-[1-¹⁴C]acetic acid. The improvement consists in the measurement of the indole-3-acetic acid decarboxylation by the determination of the decrease of radioactivity due to the decarboxylation of indole-3-[1-¹⁴C]acetic acid carried out in open vessels, during the steady-state (peroxidative) phase of the oxidation rate. In contrast to the previously reported radiometric methods, the reliability (kinetic stoichiometry of the decarboxylation) of our microassay was studied, and it was supported by the fact that, for steady-state conditions, a kinetic correlation between the disappearance of labelled substrate and the appearance of the first decarboxylate product takes place. The sensitivity and reproducibility of the improved assay was tested with crude protein samples taken from cellular homogenates of lupin hypocotyls.     

Binding behavior of amino acid conjugates of indole-3-acetic acid to immobilized human serum albumin

The affinity of indole-3-acetic acid (IAA), indole-3-propionic acid, indole-3-butyric acid and 24 of their amino acid conjugates to immobilized human serum albumin, as expressed by the retention factor k (determined by HPLC), was dependent on (1) lipophilicity, (2) chirality and (3) functional groups in the amino acid moiety; in some cases conformation plays an additional role. Two lipophilicity-related parameters afforded quantitative correlations with k: retention on a C18 reversed-phase column (experimental approach) and the distance between the hydrophilic and hydrophobic poles of the molecules (in silico approach). Most compounds examined are possible metabolic precursors of IAA, an experimental tumor therapeutic.     

Determination of melatonin and monoamines in rat pineal using reversed-phase ion-interaction chromatography with fluorescence detection

A method is reported for the ion-interaction, reversed-phase separation of 24 compounds (chiefly monoamines) arising from the metabolism of tyrosine and tryptophan. These compounds were separated as two groups. The first group comprised 3,4-dihydroxyphenylethylene glycol, tyrosine, 3-methoxy-4-hydroxyphenyl glycol, 5-hydroxytryptophan, norepinephrine, 3,4-dihydroxyphenylacetic acid, epinephrine, 5-hydroxyindole-3-acetic acid, homovanillic acid, 5-hydroxytryptophol, dopamine, tryptophan. N-acetylserotonin, N-acetyltryptophan, 5-methoxytryptophan and serotonin. The mobile phase consisted of a 6.8:93.2 (v/v) mixture of acetonitrile and an aqueous solution containing 0.16 M ammonium phosphate, 0.06 M citric acid, 0.15 mM disodium EDTA, 10 mM dibutylamine and 6 mM sodium 1-octanesulphonate at pH 4.50. The second group of compounds comprised 6-hydroxymelatonin, 5-methoxyindole-3-acetic acid, indole-3-acetic acid, 5-methoxytryptamine, tryptamine, 5-methoxytryptophol, melatonin and tryptophol. The mobile phase consisted of a 16:84 (v/v) mixture of acetonitrile and an aqueous solution containing 0.05 M ammonium phosphate, 0.05 M citric acid, 0.15 mM disodium EDTA, 25 mM dibutylamine and 5 mM sodium 1-octanesulphonate at pH 5.30. Detection was by fluorescence measurement (lambda ex = 280 nm, lambda em = 340 nm). The proposed method exhibited linear calibration over the biochemically significant concentration range, with detection limits in the 10-200 pg range. Excellent precision for peak areas and retention times was observed, even over a period of 24 h. The applicability of amperometric detection (at 0.72V) is also demonstrated. The method is applied to the determination of monoamines in individual rat pineals. Low nanogram levels of tyrosine, norepinephrine, 5-hydroxyindole-3-acetic acid, tryptophan, serotonin and 6-hydroxymelatonin, and picogram levels of 5-hydroxytryptophan, 5-hydroxytryptophol, 5-methoxyindole-3-acetic acid, indole 3-acetic acid, 5-methoxytryptophol and melatonin were indicated in most of the samples.