Simultaneous determination of twelve biogenic amines in human urine as potential biomarkers of inflammatory bowel diseases by capillary electrophoresis - tandem mass spectrometry.

Biogenic amines (BA) are a broad group of biologically active substances, the presence of which in the human body can provide important diagnostic information for many various pathologies, including chronic inflammation. In this work, a capillary electrophoresis (CE) hyphenated with tandem mass spectrometry (MS/MS) method was developed for the simultaneous determination of twelve BA (histamine, serotonin, dopamine, norepinephrine, epinephrine, putrescine, cadaverine, spermine, spermidine, tyramine, tryptamine, phenylethylamine) in human urine as potential biomarkers of inflammatory bowel diseases (IBD). The electrophoretic separations were carried out in an uncoated fused silica capillary (I.D. 50 µm) using 50 mM formic acid (pH 2.0) as a background electrolyte. A reliable identification of the analytes was based on the combination of time resolution in CE and mass resolution in triple quadrupole MS/MS. The total analysis time of the proposed CEMS/MS method was less than 10 min with the limits of detection in the range of 4.47-144 ng/mL. The intra- and inter-day accuracy ranged in the intervals 89.75-109.4% and 89.99-110.2%, respectively, with the RSD values for the intra- and inter-day precision lower than 14 and 13 %, respectively. The recovery values for the samples spiked at three concentration levels ranged from 81.73-105.6% with a precision not exceeding 9.9 %. The favorable performance parameters of the CEMS/MS method highlighted its usefulness for routine clinical applications. In this work, the CEMS/MS method was applied, for the first time, to the analytical profiling of the BA in clinical human samples. The obtained results showed a statistically significant decrease of serotonin and norepinephrine, and an increase of histamine and spermidine, in the studied group of IBD patients when compared with the control group. These findings could be utilized in studying and clarifying the mechanisms of IBD or relevant therapy.

Targeted analysis reveals alteration in pathway in 5p minus individuals.

Cri du Chat or 5p minus (5p-) syndrome is characterized by a deletion located on the chromosome 5 short (-p) arm and has an incidence rate of 1 in 50,000 individuals worldwide. This disease manifests in disturbances across a range of systems biochemicals. Therefore, a targeted metabolomics analysis was evaluated in patients with 5p- syndrome to help unravel the biochemical changes that occur in this disease. Urine samples were collected from people of both sexes aged 1-38 years old and analyzed by ultra-performance liquid chromatography coupled to mass spectrometry. Student' statistical test, metabolomic pathway analysis and metabolite set enrichment analysis were applied to the data. Alterations of some amino acids and amine biogenics levels were found in Cri du Chat Syndrome individuals. The alteration of most of these metabolites is associated with energy recuperation and glycolysis. In general, we found the catabolism of some metabolic pathways to be affected in 5p- patients.

Integration of three-phase microelectroextraction sample preparation into capillary electrophoresis.

A major strength of capillary electrophoresis (CE) is its ability to inject small sample volumes. However, there is a great mismatch between injection volume (typically <100 nL) and sample volumes (typically 20-1500 µL). Electromigration-based sample preparation methods are based on similar principles as CE. The combination of these methods with capillary electrophoresis could tackle obstacles in the analysis of dilute samples. This study demonstrates coupling of three-phase microelectroextraction (3PEE) to CE for sample preparation and preconcentration of large volume samples while requiring minimal adaptation of CE equipment. In this set-up, electroextraction takes place from an aqueous phase, through an organic filter phase, into an aqueous droplet that is hanging at the capillary inlet. The first visual proof-of-concept for this set-up showed successful extraction using the cationic dye crystal violet (CV). The potential of 3PEE for bioanalysis was demonstrated by successful extraction of the biogenic amines serotonin (5-HT), tyrosine (Tyr) and tryptophan (Trp). Under optimized conditions limits of detection (LOD) were 15 nM and 33 nM for 5-HT and Tyr respectively (with Trp as an internal standard). These LODs are comparable to other similar preconcentration methods that have been reported in conjunction with CE. Good linearity (R2 > 0.9967) was observed for both model analytes. RSDs for peak areas in technical replicates, interday and intraday variability were all satisfactory, i.e., below 14%. 5-HT, Tyr and Trp spiked to human urine were successfully extracted and separated. These results underline the great potential of 3PEE as an integrated enrichment technique from biological samples and subsequent sensitive metabolomics analysis.

Application of SPME supported by ionic liquids for the determination of biogenic amines by MEKC in clinical practice.

The analysis of biogenic amines (BAs) and their metabolites is helpful for the diagnosis of central nervous system disorders and other neuroendocrine and cancer disturbances. In the study, a developed micellar electrokinetic chromatography method, coupled with diode array detection (MEKC-DAD), was validated to monitor levels of adrenaline (A), noradrenaline (NA), dopamine (DA), L-Tryptophan (L-Tryp) and L-Tyrosine (L-Tyr) in real human urine samples. These neurotransmitters were isolated from urine samples using solid-phase microextraction (SPME) and methanol containing 1-ethyl-3-methylimidazolium tetrafluoroborate ionic liquid as the desorption phase. The method was linear for DA, A and L-Tyr in the range of 0.5-20 µg/mL and for NA and L-Tryp in the range of 0.25-20 µg/mL. The good linearity for BAs was confirmed by the correlation coefficient (R2) from 0.9989 for A to 0.9997 for NA and L-Tryp, respectively. The validation assays for accuracy, precision, limit of detection, limit of quantification, absolute recovery, and stability of the analytes were consistent with the requirements recommended by the FDA and ICH guidelines. Next, the validated SPME-MEKC method was successfully used for the quantification of A, NA, DA, L-Tryp and L-Tyr in real human urine samples collected from pediatric patients suffering from neuroblastoma, ganglioneuroblastoma, Wilms' tumor, rhabdoid tumor and lipoblastomatosis, as well as from healthy volunteers. Finally, the levels of BAs in cancer patients were evaluated as to whether they can be used as biomarkers of various health disturbances.

A validated UPLC-MS/MS assay for the quantification of amino acids and biogenic amines in rat urine.

A UPLC-MS/MS assay, employing a reversed-phase separation, has been applied to the analysis of a number of common amino acids and biogenic amines in rat urine. Analytes were derivatised, using 6­aminoquinolyl­N­hydroxysuccinimidyl carbamate (AccQTag Ultra™). Derivatisation with this reagent, by increasing the hydrophobicity of the analytes, enables better retention by improving reversed-phase chromatographic properties and also improves ionisation efficiency to enhance MS-detection. The method allows for the determination of 38 amino compounds in 7.5 min, including baseline resolution of critical isomers. The assay has been validated for the absolute quantification of 29 amino compounds in rat urine, over a concentration range of 0.6-200 µM, for the purpose of exploratory metabolite phenotyping. Acceptable linearity (R2 > 0.995) and intra- and inter-day accuracy (<20.7%) and precision (<20.1%) for these analytes was achieved. The limits of detection ranged from 1.2-12 fmol on column with 20 µL of sample. The remaining nine amines examined were not accurately quantified by this method but can be monitored for relative/fold change in biological samples. The use of the method is exemplified by the monitoring of changes in healthy male Sprague-Dawley rat urinary amino acid concentrations over a 7-day period.

Urinary Metabolites Associated with Blood Pressure on a Low- or High-Sodium Diet.

Dietary salt intake has significant effects on arterial blood pressure and the development of hypertension. Mechanisms underlying salt-dependent changes in blood pressure remain poorly understood, and it is difficult to assess blood pressure salt-sensitivity clinically. Methods: We examined urinary levels of metabolites in 103 participants of the Dietary Approaches to Stop Hypertension (DASH)-Sodium trial after nearly 30 days on a defined diet containing high sodium (targeting 150 mmol sodium intake per day) or low sodium (50 mmol per day). Targeted chromatography/mass spectrometry analysis was performed in 24 h urine samples for 47 amino metabolites and 10 metabolites related to the tricarboxylic acid cycle. The effect of an identified metabolite on blood pressure was examined in Dahl salt-sensitive rats. Results: Urinary metabolite levels improved the prediction of classification of blood pressure salt-sensitivity based on race, age and sex. Random forest and generalized linear mixed model analyses identified significant (false discovery rate <0.05) associations of 24 h excretions of ß-aminoisobutyric acid, cystine, citrulline, homocysteine and lysine with systolic blood pressure and cystine with diastolic blood pressure. The differences in homocysteine levels between low- and high-sodium intakes were significantly associated with the differences in diastolic blood pressure. These associations were significant with or without considering demographic factors. Treatment with ß-aminoisobutyric acid significantly attenuated high-salt-induced hypertension in Dahl salt-sensitive rats. Conclusion: These findings support the presence of new mechanisms of blood pressure regulation involving metabolic intermediaries, which could be developed as markers or therapeutic targets for salt-sensitive hypertension.

Targeted metabolomic analysis of 33 amino acids and biogenic amines in human urine by ion-pairing HPLC-MS/MS: Biomarkers for tacrolimus nephrotoxicity after renal transplantation.

Calcineurin inhibitor nephrotoxicity, especially for the widely used tacrolimus, has become a major concern in post-transplant immunosuppression. Multiparametric amino acid metabolomics is useful for biomarker identification of tacrolimus nephrotoxicity, for which specific quantitative methods are highlighted as a premise. This article presents a targeted metabolomic assay to quantify 33 amino acids and biogenic amines in human urine by high-performance liquid chromatography coupled with tandem mass spectrometry. Chromatographic separation was carried out on an Agilent Zorbax SB-C18 column (3.0 × 150 mm, 5 µm) with addition of an ion-pairing agent in the mobile phase, and MS/MS detection was achieved in both the positive and negative multiple reaction monitoring modes. Good correlation coefficients (r2 > 0.98) were obtained for most analytes. Intra- and inter-day precision, stability, carryover and incurred sample reanalysis met with the acceptance criteria of the guidance of the US Food and Drug Administration. Analysis on urine from healthy volunteers and renal transplantation patients with tacrolimus nephrotoxicity confirmed symmetric dimethylarginine and serine as biomarkers for kidney injury, with AUC values of 0.95 and 0.81 in receiver operating characteristic analysis, respectively. Additionally, symmetric dimethylarginine exhibited a tight correlation with serum creatinine, and was therefore indicative of renal function. The targeted metabolomic assay was time and cost prohibitive for amino acid analysis in human urine, facilitating the biomarker identification of tacrolimus nephrotoxicity.

Practical Application of Biogenic Amine Profiles for the Diagnosis of Patients with Ischemic Stroke.

BACKGROUND: Ischemic stroke (IS) is still one of the major issues in medicine. Still, early diagnosis and misdiagnosis remain the main barriers for proper patient treatment and follow-up. Exploring new potential diagnostic biomarkers for IS is relevant to decrease patient morbidity and the occurrence of poststroke diseases. Biomedical analysis could bring new light to the background of IS and-in such a way-propose new bioanalytical tools for the early diagnosis, prognostication, and monitoring of IS. MATERIALS AND METHODS: This research aimed to present a discussion on the employment of biogenic amines (BAs), as well as their precursory amino acids and main metabolites, as a new panel of biomarkers for IS. Preliminary patient data were presented and the patients were described with respect to their clinical history and examination records, as well as scientific data gained from the liquid extraction-capillary electrophoresis determination of BAs in the patients' urine samples. RESULTS: The results showed the potential of BA screening using the developed sample preparation and analysis methods in urine during IS, and this will be further studied on a more numerous group of patients with IS to reveal the usefulness of BAs as a new panel of biomarkers for early IS diagnosis and prognostication. CONCLUSIONS: To our best knowledge, this methodology for the first time has been used for the simultaneous analysis of multiple small molecular biomarkers. In addition, the factors that might influence the determination of BAs in real samples were pointed out.

Determination of urinary biogenic amines' biomarker profile in neuroblastoma and pheochromocytoma patients by MEKC method with preceding dispersive liquid-liquid microextraction.

The unbalanced secretion of biogenic amines (BAs) is considered to be a relevant biochemical biomarker in the screening for neuroendocrine tumors, such as: neuroblastoma and pheochromocytoma. However, there is still a need to improve the bioanalytical procedures for BA determination in biological samples due to their instability (photo- and thermosensitivity, easy oxidation) and low concentration in the body fluids. In this study, the primary analytical challenge was to optimize the method of extraction of seven compounds from among BAs and their precursors from urine samples. Several methods based on liquid-liquid extraction (LLE) or solid phase extraction (SPE) techniques were tested. By optimization of the extraction and data analysis using chemometric tool, the dispersive liquid-liquid microextraction (DLLME) has been chosen due to its low solvents consumption, high efficiency of isolation, preconcentration and suitable clean-up of biological matrix. Further, α-cyclodextrin-modified micellar electrokinetic chromatography (MEKC) with ultraviolet detection (UV) has been applied for quantification of the analyzed biologically active compounds with limits of detection (LOD) and limits of quantification (LOQ) at 0.15 and 0.5µgmL-1, respectively. Finally, the optimized and validated DLLME-MEKC-UV method has been employed for the analysis of real urine samples, obtained from 6 children with neuroendocrine tumors and 6 healthy children. It was stated that concentrations of BA could serve to differentiate between the patients and healthy children. This pilot study indicates that the elaborated fast and sensitive DLLME-MEKC-UV method for determination of panel of biomarkers could be successfully applied in everyday clinical practice to help to confirm the clinical diagnosis of neuroendocrine tumors in children.

Separation of Key Biogenic Amines by Capillary Electrophoresis and Determination of Possible Indicators of Sport Fatigue in Athlete's Urine.

This article aims to build up a simple, rapid and accurate capillary zone electrophoresis (CZE) method for the separation of biogenic amines (BAs). Here, 10 key BAs (phenethylamine, histamine, tryptamine, tyramine, 5-hydroxytryptamine, octopamine, dopamine, norepinephrine, epinephrine and carnosine) owning significant functions were chosen for method development. The baseline separation and identification of 10 standards of the mixture by CZE were eventually achieved in 150.0 mmol/L phosphate buffer (Na2HPO4-NaH2PO4) containing 1.0 mmol/L borax at a pH of 6.1. The addition of borax was found effective for improving the isomeric separation of octopamine and dopamine. The proposed method allowed the limits of detections of BAs to be in the range of 0.2-1.2 µmol/L at UV detection (200 nm); the relative standard deviation of the migration time and the peak area were in the ranges 0.08-0.12 and 2.74-4.63% (n = 5), respectively. Formaldehyde (a possible antiseptic in urine) and five main matrices in urine were studied for the identification of BAs. Finally, profiling of BAs in actual urine from athletes was carried out. Currently, only phenethylamine, norepinephrine and carnosine were designated by spiking the standards. In addition, their variation in athletes' urine has been checked at different states of sport fatigue with the goal of obtaining possible indicators of sport fatigue.

Monitoring of biogenic amines and drugs of various therapeutic groups in urine samples with use of HPLC.

A high-performance liquid chromatography method for simultaneous separation and determination of biogenic amines [dopamine, epinephrine, serotonin and its six metabolites (normetanephrine, metanephrine, 3,4-dihydroxyphenylacetic acid, 4-hydroxy-3-methoxyphenylglycol, homovanilic acid and 5-hydroxyindoloacetic acid)] with drugs from different therapeutically groups [analgesics (paracetamol, metamizol), diuretics (furosemide) and antibiotics (cefazolin, fluconazole)] was developed. A chromatographic column with pre-column with octadecylsilane phase (C18e ) and two detectors - diode array serial connected and fluorescence - was used. Gradient elution of mixture of acetate buffer (pH 4.66) and methanol as a mobile phase was applied. The limit of detection (LOD) of 8-10 ng/mL and limit of quantitation (LOQ) of 24-30 ng/mL for biogenic amines, as well as the LOD of 50-100 ng/mL and the LOQ of 150-300 ng/mL for drugs, were determined. The applied sample preparation method allowed recoveries of 93% for the biogenic amines and 92% for the drugs to be achieved. The developed procedure has been applied to simultaneous determination of the examined compounds in urine samples and could be used in clinical analysis.

[Determination of four biogenic amine metabolites in urine by high-performance liquid chromatography].

OBJECTIVE: To establish a method for simultaneously determining vanilmandelic acid (VMA), 5-hydroxyindoleacetic (5-HIAA), 3, 4-dihydroxyphenylacetic acid (DOPAC), and homovanillic acid (HVA) in urine by high-performance liquid chromatography (HPLC). METHODS: After being filtered with a 0.45 µm membrane syringe filter, the urinary samples were injected directly into the HPLC system using a C18 chromatographic column and a fluorescence detector. The excitation and emission wavelengths were chose as 280 nm and 315 nm, respectively, and the urinary samples were carried with a mobile phase of methanol-0.1 mol/L phosphate buffered solution (V/V = 20:80) at a flow rate of 1.0 ml/min and an injection volume of 20 µl. RESULTS: Using the method reported here, the correlation coefficients of VMA, 5-HIAA, DOPAC, and HVA were 0.9999, 0.9998, 0.9997, 0.9999, respectively, over linear ranges of 0-2.5, 0-2.0, 0-2.0, and 0-2.5 µg/ml, the minimum detectable concentrations were 0.006, 0.008, 0.012, and 0.0082 µg/ml, the average precisions were 4.2%, 3.7%, 4.9%, and 3.6%, and the recovery rates were 91%∼102%, 93%∼101%, 94%∼101%, and 89%∼ 102%. CONCLUSION: This determination method is simple, efficient, accurate, and sensitive for the simultaneous detection of VMA, 5-HIAA, DOPAC, and HVA in urine.

LC-MS/MS in endocrinology: what is the profit of the last 5 years?

Currently, chromatography (GC but more commonly HPLC) is the analytical method of choice for several hormones, either because the immunoassays suffer from extensive crossreactivity or because chromatography permits simultaneous measurements of hormones. However, sometimes the conventional detection systems with HPLC methods do not meet desired specificity. With the increase of robust and affordable LC-MS/MS systems, the next step forward in specificity was taken. LC-MS/MS is rapidly being incorporated in the endocrine laboratories. To be useful in the clinical diagnostic practice, it is of utmost importance that methods are both analytically and clinically vaidated, as until now, the majority of applications of LC-MS/MS in the clinical laboratories are 'home-made' methods, therefore special case must be taken. This review aims to focus on Clinical and Laboratory Standards Institute or comparable validated LC-MS/MS methods for targeted hormone analysis used for diagnostic purposes in human samples, published in the last 5 years.

Comprehensive profiling analysis of bioamines and their acidic metabolites in human urine by gas chromatography/mass spectrometry combined with selective derivatization.

A comprehensive analytical method was developed for the profiling of biogenic amines in human urine using GC/MS in SIM mode. Biogenic amines and their acidic metabolites were converted into their volatile O-trimethylsilyl/N-heptafluorobutyryl (OTMS/-NHFBA) derivatives for GC/MS analysis. Dual hexamethyldisilazane (HMDS)/-N-methyl-bis-heptafluorobutyramide (MBHFBA) derivatizations have been shown to be quite effective, with high derivatization yields and the absence of side products for primary biogenic amines. In this study, selective derivatization conditions by HMDS/MBHFBA were optimized in terms of the reagent amount, reaction temperature and reaction time period. The highest derivatization reaction yield was obtained at 40°C for 10min for OTMS derivatization and 80°C for 5min for N-HFBA derivatization. The use of MCX SPE cartridges with different SPE elution solvents was effective for the pre-concentration and selective cleanup of the biogenic amines and their acidic metabolites in human urine. The selection of appropriate ions in SIM mode provided reliable quantification and identification and a reduction in background effects. The established method was validated in terms of linearity, limits of detection (LOD), limits of quantification (LOQ), precision, and accuracy. The present method was linear (r(2)>0.996), reproducible (relative standard deviation range 1.1-6.9%), and accurate (range 87.9-111.9%), with LOQs of 0.17-17.84ng/mL. The biogenic amine profiling of human urine was successfully accomplished by GC/MS in SIM mode combined with selective HMDS/MBHFBA derivatization and MCX SPE cleanup.

On-line sample preconcentration technique based on a dynamic pH junction in CE-amperometric detection for the analysis of biogenic amines in urine.

It is difficult to detect biogenic amines (BAs) in biological fluids because of their very low concentrations. In this paper, we reported an on-line sample preconcentration method in CE-amperometric detection (CE-AD) based on a dynamic pH junction, and a concentration enhancement of approximately 100-fold was achieved compared with the classical CE-AD methods in the simultaneous analysis of six BAs in urine (dopamine, epinephrine, norepinephrine, tyramine, tryptamine, and serotonin). The technique is proposed based on the sharp pH change generated at the boundary between an acidic sample and the basic BGE zone. Under optimized conditions, all analytes were successfully focused and well separated within 20 min with high efficiency and sensitivity (LODs at S/N = 3 ranging from 5.34 to 68.3 nM). For the analysis of urine samples by this method, satisfactory recoveries were obtained without a complicated pretreatment step or derivatization process. Therefore, it is self-evident that this approach for the analysis of real biological samples has great potential in the future.

Sensitive profiling of biogenic amines in human urine by capillary electrophoresis with field amplified sample injection.

In order to monitor biogenic amines in human urine, a method based on field-amplified sample injection combined with capillary electrophoresis and direct UV absorption detection was developed. Dopamine, tyramine, tryptamine, serotonin and epinephrine were effectively separated and identified in human urine samples, and detection limits were 0.072, 0.010, 0.027, 0.010 and 0.120 µmol/L, respectively. Detection limits comparable to laser-induced fluorescence detection or solid phase extraction combined with capillary electrophoresis were achieved. Parameters affecting electrophoretic system detection sensitivity were investigated. Optimal separation conditions were obtained using as background electrolyte a pH 6.5 mixture of 2-(morpholino)ethanesulfonic acid 20 mmol/L and 30 mmol/L phosphate buffer, containing 0.05% hydroxypropylcellulose and 10% v/v methanol. Injections of the sample solution were performed by applying a voltage of 12 kV for 50 s. Recovery and accuracy ranged between 89.4 and 94.9%, and 89 and 112%, respectively. The method was successfully applied on actual urine samples (from a healthy volunteer): target bioamine content was consistent with endogenous levels reported in the literature. The proposed method is simple, fast and inexpensive and can be conveniently employed in work-related stress studies. The affordability and noninvasive sampling of the method allow epidemiological studies on large number of exposed persons to be performed.

Simultaneous determination of sixteen underivatized biogenic amines in human urine by HPLC-MS/MS.

The broad group of biogenic amines includes polyamines and catecholamines, whose presence in human tissues and biological fluids can give important diagnostic information and act as marker of many pathologies. In particular, polyamines are involved in cancer cell growth while catecholamines act as neurotransmitters and hormones. Their simultaneous determination in biological tissues and fluids is therefore an important task. A high-performance liquid chromatography tandem mass spectrometry method is presented here for the simultaneous determination in urine of 16 biogenic amines: adrenaline (epinephrine), agmatine, cadaverine, dopamine, histamine, 3-methoxytyramine, noradrenaline (norepinephrine), norephedrine, octopamine, 2-phenylethylamine, putrescine, serotonin, spermidine, spermine, tryptamine, and tyramine. The method does not require any derivatization step. To guarantee the maximum of sensitivity, the mass spectrometer works in selected reaction monitoring mode, monitoring for each analyte the two most intensive transitions. Method validation includes the evaluation of limits of detection (that range from 0.3 to 6.6 µg L(-1)), limits of quantitation (that range from 1.0 to 21.9 µg L(-1)), linearity range (three orders of magnitude), recovery, intra- and inter-day precision on both concentration, and retention time. Recovery (R) is shown not to depend on the analyte concentration: the average R percent ranges from 72.9 to 100.0 %. Particular attention is devoted to the matrix effect and the correlated phenomena of ion enhancement or suppression in mass spectrometry detection.

Analytical approach to determine biogenic amines in urine using microextraction in packed syringe and liquid chromatography coupled to electrochemical detection.

The goal of this work was to develop and validate an analytical method for the detection and quantification of the biogenic amines serotonin (5-HT), dopamine (DA) and norepinephrine (NE), using microextraction in packed syringe (MEPS) and liquid chromatography coupled to electrochemical detection (HPLC-ED) in urine. The method was validated according to internationally accepted guidelines from the Food and Drug Administration. Linearity was established between 50 and 1000 ng/mL for 5-HT and between 5 and 1000 ng/mL for DA and NE, with determination coefficients (R(2)) >0.99 for all compounds. The limits of quantification and detection were respectively 50 and 20 ng/mL for 5-HT, and 5 and 2 ng/mL for DA and NE. Within- and between-run precision ranged from 0.84 to 9.41%, while accuracy ranged from 0.79 to 12.76% for all compounds. The intermediate precision and accuracy were 1.50-8.36 and 0.54-13.51%, respectively. The method was found suitable for clinical routine analysis of the studied compounds, using a sample volume of 0.5 mL. This is the first study employing a commercially available MEPS column for the simultaneous detection and quantification of 5-HT, DA and NE in urine by coulometric detection.

Study of the photocatalytic transformation of synephrine: a biogenic amine relevant in anti-doping analysis.

The occurrence of some cases of positive results in anti-doping analysis of octopamine requires clarification as to whether its methylated derivative synephrine could be a metabolic precursor of octopamine itself. Synephrine is a natural phenylethylamine derivative present in some food supplements containing Citrus aurantium, permitted in sport regulations. A simulative laboratory study had been done using a photocatalytic process, to identify all possible main and secondary transformation products, in a clean matrix; these were then sought in biological samples obtained from three human volunteers and four rats treated with synephrine; the parent compound and its new potential metabolic products were investigated in human urine and rat plasma samples. The transformation of synephrine and octopamine and the formation of intermediate products were evaluated, adopting titanium dioxide as photocatalyst. Several products were formed and characterized using the HPLC-HRMS(n) technique. The main intermediates identified in these experimental conditions were compared with the major synephrine metabolites found in in vivo studies on rats and humans. Some more oxidized species, already formed in the photocatalytic process, were also found in urine and plasma samples of treated animals. These new findings could be of interest in further metabolism studies. The main photocatalytic pathway involving synephrine appears to be N-demethylation to give octopamine. On the contrary, we demonstrate the inconsistency of this reaction in both rat and human in vivo determinations, resulting in forensic importance.

Micellar electrokinetic chromatography-chemiluminescent detection of biogenic amines using N-(4-aminobutyl)-N-ethylisoluminol as derivatization reagent and trivalent copper chelate as chemiluminescence enhancer.

A facile, sensitive and universal method was established for analysis of biogenic amines using micellar electrokinetic chromatography coupled with chemiluminescent (CL) detection. It was found that diperiodatocuprate (III) (K(5)[Cu(HIO(6))(2)], DPC), a transition metal chelate at unstable high oxidation state, could effectively enhance the reaction between luminol-type compound and hydrogen peroxide, to produce very strong CL signal. In addition, triethylamine was found to be able to effectively improve the yield of the derivatization reaction between biogenic amines and a luminol-type derivatization reagent, N-(4-aminobutyl)-N-ethylisoluminol (ABEI). Based on these facts, three biogenic amines were pre-column derivatized with ABEI, and post-column detected using high sensitive luminol-hydrogen peroxide-DPC CL system. Since the background was quite low, and the signal was quite strong, a considerable improved sensitivity was obtained. The presented method had been successfully applied to simultaneously analyze glycine, proline and phenylalanine with the detection limits (S/N=3) of 0.030 µmol L(-1), 0.23 µmol L(-1) and 0.21 µmol L(-1), respectively. To evaluate its potential application value, glycine in saliva and urine samples was detected using this method, and satisfied results were obtained. This approach can be further extended to detection of many other compounds such as peptides and drugs by using luminol-type derivatization reagent.