Simultaneous determination of the major acidic metabolites of catecholamines and serotonin in urine by liquid chromatography with electrochemical detection after a one-step sample clean-up on Sephadex G-10; influence of vanilla and banana ingestion

A simple method is described for the simultaneous determination of vanilmandelic acid (VMA), 3,4-dihydroxyphenylacetic acid (DOPAC), 5-hydroxyindole-3-acetic acid (5-HIAA) and homovanillic acid (HVA) in urine. The compounds are isolated by a one-step sample clean-up on Sephadex G-10, separated by ion-pair reversed-phase liquid chromatography and detected electrochemically. A single analysis is completed within 65 min. Sample clean-up did not cause losses of the compounds of interest. The detection limits in urine were 0.4, 0.8, 1.0 and 1.6 mumol/l for VMA, DOPAC, 5-HIAA and HVA, respectively. 3,4-Dihydroxymandelic acid and vanillic acid (VA) were also detectable, but, under the chromatographic conditions used, they were not resolved from interfering components. VA and 5-HIAA could be analysed separately in the Sephadex G-10 eluate if more restrictive sampling conditions were used. Ingestion of bananas caused an increase of VMA, DOPAC, 5-HIAA and HVA in 24-h urine. After ingestion of vanilla an increased excretion of VA was observed, while the excretion of VMA, DOPAC and HVA was unaffected.     

Measurement of urinary vanilmandelic acid and homovanillic acid by high-performance liquid chromatography with electrochemical detection following extraction by ion-exchange and ion-moderated partition

An improved protocol has been developed to isolate homovanillic acid (HVA) and vanilmandelic acid (VMA) from urine with strong anion-exchange resin. The sample is diluted with acetate buffer and passed through a disposable column. HVA, uric acid, and many hydrophobic organic acids are removed with 1.0 M acetic acid--ethanol. Then VMA is eluted with 0.5 M phosphoric acid. Two isocratic mobile phases allow rapid high-performance liquid chromatographic measurement of VMA (5 min) and HVA (8 mins) on a 5-micron ODS column. Selective conditions were developed with dual-electrode coulometric detection to permit specific measurement of VMA, HVA, and internal standards, with less than 5% between-run variation.     

Analytical validation and clinical application of urinary vanillylmandelic acid and homovanillic acid by LC–MS/MS for diagnosis of neuroblastoma

Vanillylmandelic acid (VMA) and homovanillic acid (HVA) are clinical biomarkers for diagnosis of neuroblastoma (NB), which commonly occurs in the childhood. Development and application of a robust LC–MS/MS method for fast determination of these biomarkers for optimal laboratory testing of NB is essential in clinical laboratories. In present study, we developed and validated a simple liquid chromatography tandem mass spectrometry (LC–MS/MS) method for quick clinical testing of VMA and HVA for diagnosis of NB. The method was validated according to the current CLSI C62‐A and FDA guidelines. The age‐adjusted pediatric reference intervals and diagnostic performance were evaluated in both 24 h urine and random urine. Injection‐to‐injection time was 3.5 min. Inter‐ and intra‐assay coefficients of variation (CVs) were ≤3.88%. The lower limit of quantification and the limit of detection were 0.50 and 0.25 μmol/L for both VMA and HVA. Recoveries of VMA and HVA were in the ranges of 85–109% and 86–100% with CVs ≤5.76%. This method was free from significant matrix effect, carryover and interference. The establishment of age‐adjusted pediatric reference intervals by this LC–MS/MS method was favorable for the improvement in diagnostic performance, which was crucial for correct interpretation of test results from children in both 24 h and random urine.     

Determination of serum catecholamine metabolites in neuroblastoma by high performance liquid chromatography with electrochemical detection

A method for determining serum catecholamine metabolites such as vanillylmandelic acid (VMA), 3-methoxy-4-hydroxyphenyl glycol (MHPG) and homovanillic acid (HVA) in neuroblastoma by using high performance liquid chromatography and electrochemical detector is described. The separation of catecholamine metabolites was performed on a reverse phase column with an eluting system containing citric acid-potassium hydrogen phosphate buffer and methanol as the organic modifier. The experimental results showed that VMA and HVA levels in the serum of neuroblastoma patients were 15-30 times higher than that of the normal control group. The same phenomenon also occurred in patients with stage II neuroblastoma. Serum VMA, MHPG and HVA levels reduced to normal in patients suffering from neuroblastoma after surgery. Serum catecholamine metabolites analysed by using HPLC/ECD is more simple, sensitive and reliable than that by usual urine assay and might be used for the diagnosis of neuroblastoma even in early stage.     

Separation and determination of homovanillic acid and vanillylmandelic acid by capillary electrophoresis with electrochemical detection

A method of separation and determination of homovanillic acid (HVA) and vanillylmandelic acid (VMA) was developed based on capillary zone electrophoresis/amperometric detection with high sensitivity, good resolution and selectivity. In order to achieve complete separation and good response, several factors including pH, buffer concentration, separation voltage, detection potential and the length of separation capillary, were studied in detail. The method has been used to determine both HVA and VMA in human urine. Uric acid (UA) in human urine did not interference with their determination. The limit of detection of the method was 1.3×10⁻⁶ mol/l (1.4 fmol) for HVA and 7.9×10⁻⁷ mol/l (0.87 fmol) for VMA at a signal-to-noise ratio of 3.     

Direct determination of vanillylmandelic acid in human urine by reversed-phase HPLC

A reversed-phase HPLC method for the determination of epinephrine, norepinephrine, dopamine, and vanillylmandelic acid (VMA) has been developed. The concentration of VMA in the urine of hypertensive patients was measured by direct injection after centrifugation. The method is useful for the diagnosis of pheochromocytoma.     

Determination of selected neurotransmitter metabolites using monolithic column chromatography coupled with chemiluminescence detection

The oxidation of selected clinically important neurotransmitter metabolites with acidic potassium permanganate in the presence of polyphosphates evokes chemiluminescence of sufficient intensity to enable the sensitive determination of these species. Limits of detection for 5-hydroxyindole-3-acetic acid (5-HIAA), vanilmandelic acid (VMA; α,4-dihydroxy-3-methoxybenzeneacetic acid), 4-hydroxy-3-methoxyphenylglycol (MHPG), homovanillic acid (HVA, 4-hydroxy-3-methoxyphenylacetic acid) and 3,4-dihydroxyphenylacetic acid (DOPAC) were between 5 × 10⁻⁹ and 4 × 10⁻⁸ M, using flow-injection analysis methodology. In addition, we demonstrate the rapid determination of homovanillic acid and 5-hydroxyindole-3-acetic acid in human urine - without the need for extraction procedures - using monolithic column chromatography with chemiluminescence detection.     

Alcohol biomarker analysis: simultaneous determination of 5-hydroxytryptophol glucuronide and 5-hydroxyindoleacetic acid by direct injection of urine using ultra-performance liquid chromatography-tandem mass spectrometry

A direct ultra-performance liquid chromatography-tandem mass spectrometry method (UPLC-MS/MS) for simultaneous measurement of urinary 5-hydroxytryptophol glucuronide (GTOL) and 5-hydroxyindoleacetic acid (5-HIAA) was developed. The GTOL/5-HIAA ratio is used as an alcohol biomarker with clinical and forensic applications. The method involved dilution of the urine sample with deuterated analogues (internal standards), reversed-phase chromatography with gradient elution, electrospray ionisation and monitoring of two product ions per analyte in selected reaction monitoring mode. The measuring ranges were 6.7-10 000 nmol/l for GTOL and 0.07-100 micromol/l for 5-HIAA. The intra- and inter-assay imprecision, expressed as the coefficient of variation, was below 7%. Influence from ion suppression was noted for both compounds but was compensated for by the use of co-eluting internal standards. The accuracy in analytical recovery of added substance to urine samples was 96 and 98%, respectively, for GTOL and 5-HIAA. Method comparison with GC-MS for GTOL in 25 authentic patient samples confirmed the accuracy of the method with a median ratio between methods (GC-MS to UPLC-MS/MS) of 1.14 (r(2) = 0.975). The difference is explained by the fact that the GC-MS method also measures unconjugated 5-hydroxytryptophol naturally present in urine. The comparison with data for 5-HIAA obtained by an HPLC method demonstrated a median ratio of 1.05 between the methods. The UPLC-MS/MS method was capable of measuring endogenous GTOL and 5-HIAA levels in urine, which agreed with the literature data. In conclusion, a fully validated and robust direct method for the routine measurement of urinary GTOL and 5-HIAA was developed.     

Sensitive, rapid and easy analysis of three catecholamine metabolites in human urine and serum by liquid chromatography tandem mass spectrometry

A sensitive and easy analytical method for catecholamine metabolites including 4-hydroxy-3-methoxyphenylglycol sulfate (HMPG sulfate), vanillylmandelic acid (VMA) and homovanillic acid (HVA) determination was developed based on liquid chromatography-tandem mass spectrometry in a negative multiple reaction monitoring mode. The analytes were rapidly separated on a reversed-phase Waters Xbridge C18 column (150 × 2.1 mm i.d.) with the mobile phase of 15% (v/v) acetonitrile containing 2 mM ammonium formate and 85% (v/v) formic acid solution (0.05%, v/v). Mass spectrometric conditions, such as characteristic fragmentations and quantification ion transitions, both with chromatographic conditions including separation column type and mobile phase composition, were systematically investigated to get optimal sensitivity and specificity. The limits of detection were in the range of 0.03-0.7 ng/mL for the targets. Recovery rates of spiked urine samples with three different concentration levels (low, middle and high) were above 86% with precisions less than 5.7%. For serum analysis, acetonitrile chosen both as protein precipitation reagent and extraction solvent facilitates to reduce matrix effects. Recovery rates of spiked serum sample were in the range of 90.6% to 111.1% for three targets. The intra-day and inter-day precisions were satisfactory less than 8.7%. This proposed method was successfully applied to determine HMPG sulfate, HVA and VMA present in human urine and serum.     

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.     

A reliable and simple method for the assay of neuroendocrine tumor markers in human urine by solid-phase microextraction–gas chromatography-triple quadrupole mass spectrometry

Homovanillic acid (HVA), vanylmandelic acid (VMA), and 5-hydroxyindoleacetic acid (5-HIAA) are the metabolites of some catecholamines such as epinephrine, nor-epinephrine, dopamine and serotonin and their quantification is used in the diagnosis and management of patients with neurocrine tumors. A novel approach in the assay of these biomarkers in human urine samples by solid phase microextraction (SPME) combined with gas chromatography-triple quadrupole mass spectrometry (GC–QqQ-MS) is presented. A preliminary derivatization with ethyl chloroformate/ethanol was used and the corresponding derivatives were then extracted by SPME in immersion mode. The performance of five SPME fibers and three chloroformates were evaluated in univariate mode and the best results were obtained using the polyacrylate fiber and ethyl chloroformate. The variables affecting the efficiency of SPME analysis were optimized by the multivariate approach of “Experimental design” and, in particular, a central composite design (CCD) was applied. The optimum working conditions in terms of response values were achieved by performing analysis at room temperature with addition of NaCl (9.5%) and with an extraction time of 25.8 min. Identification and quantification of analytes were carried out by using a gas chromatography-triple quadrupole mass spectrometry (GC–QqQ MS) system in multiple reaction monitoring (MRM) acquisition. An evaluation of all analytical parameters shows that the proposed method provides satisfactory results. Very good linearities were, in fact, achieved in the tested calibration ranges with correlation coefficient values >0.99 for all the analytes and accuracies and RSDs calculated for between-run and tested at concentrations of 1, 10, and 80 mg L⁻¹ were ranging from 91.3% to 106.6%, and from 0.5 to 8.9%, respectively. Moreover, the LOD values obtained can be considered very satisfactory (1.3, 0.046 and 24.3 μg L⁻¹ for HVA, VMA and 5-HIAA, respectively). The developed protocol represents, therefore, a simple, rapid and selective tool for assaying these acidic biomarkers in urine samples for neuroendocrine cancer diagnosis.     

Unconjugated methoxylated catecholamine metabolites in human saliva. Quantitation methodology and comparison with plasma levels

A newly developed method for the simultaneous extraction and quantitation of the unconjugated levels of the catecholamine metabolites vanilmandelic acid (VMA), 3-methoxy-4-hydroxyphenylethylene glycol (MHPG) and homovanillic acid (HVA) in plasma by high performance liquid chromatography with electrochemical detection was modified and applied to studies of human saliva. The assay had a mean coefficient of variation under 3% for each of the metabolites. Levels of plasma VMA, MHPG and HVA were measured in 28 normal subjects and compared to their saliva levels, obtained before and after stimulation by mastication. Significant correlations were found between plasma and saliva MHPG and HVA, but there was no correlation between plasma and saliva VMA. Salivary MHPG and HVA can be reproducibly assayed and may be useful tools for indications of changes in central and peripheral catecholamine metabolism.     

Application of Quartz Crystal Nanobalance for Simultaneous Determination of Vanillylmandelic and Homovanillic Acids by a Net Analyte Signal-Based Method

Homovanillic acid (HVA) and vanillylmandelic acid (VMA) were selectively determined by quartz crystal nanobalance sensor in conjunction with net analyte signal (NAS)-based method called HLA/GO. An orthogonal design was applied for the formation of calibration and prediction sets including HVA, VMA, and some common and structurally similar urine compounds. The selection of the optimal time range involved the calculation of the NAS regression plot in any considered time window for each test sample. The searching of a region with maximum linearity of NAS regression plot (minimum error indicator) and minimum of predicted error sum of squares value was carried out by applying a moving window strategy. Based on the obtained results, the differences on the adsorption profiles in the time range between 1 and 300 s were used to determine mixtures of compounds by HLA/GO method. Several figures of merit like selectivity, sensitivity, analytical sensitivity, and limit of detection were calculated for both compounds. The results showed that the method was successfully applied for the determination of VMA and HVA.     

Determination of urinary 5-hydroxy-3-indoleacetic acid by an automated HPLC method

A HPLC method for the quantitative determination of 5-hydroxy-3-indoleacetic acid (5-HIAA) in urine is described. The method is based on ion-pair chromatography, reversed phase (RP) column material and specific fluorimetric detection at 300 nm and 355 nm. Sample preparation and gradient elution were avoided by using a column-switching technique. The sensitivity of the assay was excellent for clinical routine analysis, with a detection limit of 0.2 mg/L 5-HIAA. No endogenous or exogenous interference problems arose. Intra- and interassay precision was good, with observed coefficients of variation of 1.5 to 2.6% and 2.1%, respectively. Recoveries were 93 to 98%. The system described can be used for clinical diagnosis and therapy follow-up of carcinoid tumors. It has been running for over a year without disturbances and with a minimum of technical attendance.     

Self-quenching in the electrochemiluminescence of tris(2,2'-bipyridyl) ruthenium(ii) using metabolites of catecholamines as co-reactants

Electrochemiluminescence (ECL) of Ru(bpy)(3)(2+) using metabolites of catecholamines: homovanillic acid (HVA) and vanillylmandelic acid (VMA) as co-reactants were investigated in aqueous solution for the first time. When HVA and VMA were co-existent in the buffer solution containing Ru(bpy)(3)(2+), ECL peaks were observed at a potential corresponding to the oxidation of Ru(bpy)(3)(2+), and the ECL intensity was increased noticeably when the concentrations of HVA and VMA were at lower levels. The linear calibration range was from 8.0 × 10(-5) to 1.0 × 10(-9) M for HVA and VMA. The detection limit (S/N = 3) of HVA and VMA was 4.0 × 10(-10) M. The formation of the excited state Ru(bpy)(3)(2+*) was confirmed to result from the reaction between Ru(bpy)(3)(3+) and the intermediates of HVA or VMA radicals. Moreover, it was found that the ECL intensity was quenched significantly when the concentrations of HVA and VMA were relatively higher. The mechanism of self-quenching processes involved in the Ru(bpy)(3)(2+)-HVA and -VMA ECL systems are proposed in this study.     

Measurement of plasma vanillylmandelic acid by liquid chromatography with electrochemical detection

A liquid chromatographic method with electrochemical detection is described for measuring plasma 3-methoxy-4-hydroxymandelic acid (VMA). Plasma is deproteinized by gel filtration and VMA is extracted into ethyl acetate, which is evaporated. VMA is oxidized to vanillin, which is purified by toluene extraction and quantified by high-performance liquid chromatography. The recovery of VMA through the entire procedure is 52 +/- 10% (mean +/- S.D., n = 19). The plasma VMA concentration in healthy young volunteers varies between 4.39 and 14.6 ng/ml, a range that is in excellent agreement with data obtained with mass fragmentography.     

3-methoxy-4-hydroxyphenylglycol, 5-hydroxyindoleacetic acid, and homovanillic acid in human cerebrospinal fluid. Storage and measurement by reversed-phase high-performance liquid chromatography and coulometric detection using 3-methoxy-4-hydroxyphenyllactic acid as an internal standard

To simultaneously measure 3-methoxy-4-hydroxyphenylglycol (MHPG), 5-hydroxyindoleacetic acid (5HIAA), and homovanillic acid (HVA) in human cerebrospinal fluid (CSF), we used an acetonitrile protein precipitation, reversed-phase high-performance liquid chromatography with coulometric detection, and 3-methoxy-4-hydroxyphenyllactic acid (MHPLA) as an internal standard for all three metabolites. MHPG, 5HIAA, HVA, and MHPLA were stable for one month when stored in CSF at -70 degrees C. Three determinations were made in triplicate for each of seven subjects over a 30-day storage period and the coefficients of variation within subject for these determinations ranged from 0.075 to 0.165 for MHPG, 0.045 to 0.148 for 5HIAA and 0.053 to 0.181 for HVA. Means and standard deviations of CSF concentrations were 10.7 +/- 3.0 ng/ml for MHPG, 22.4 +/- 9.9 ng/ml for 5HIAA, and 39.9 +/- 21.4 ng/ml for HVA. This method provides simple sample preparation, sensitivity, and cost advantages, as well as simultaneous extraction and quantitation of MHPG, 5HIAA, and HVA using an internal standard.     

Separation of vanillylmandelic (VMA) and homovanillic (HVA) acids from urine for assay by a two resin column system

A procedure is described for separating vanillylmandelic acid (VMA) and homovanillic acid (HVA) from urine so that they can be assayed by chemical means or gas chromatography. The process comprises passing a threefold diluted urine over cation exchange column (AG 50W-X12) to remove catecol amines and amino acids. The VMA and HVA is then adsorbed from the diluted urine to an anion exchange column (AG 2-X8). After washing with water, the HVA and VMA are eluted with an NaCl-K₂CO₃ solution. The VMA can be determined by oxidation to vanillin directly. By extraction with n-pentanone-2, after acidification and evaporation to dryness, the residue may be redissolved for processing or treated with a methylating reagent for gas chromatography. For the latter purpose, 4-methoxy benzophenone is recommended as the internal standard.     

Determination of three Tumor Biomarkers (Homovanillic Acid,Vanillylmandelic Acid,and 5‐Hydroxyindole‐3‐Acetic Acid) Using Flow Injection Analysis with Amperometric Detection

Flow injection analysis with amperometric detection (FIA‐AD) at screen‐printed carbon electrodes (SPCEs) in optimum medium of Britton‐Robinson buffer (0.04 mol ? L^?1, pH 2.0) was used for the determination of three tumor biomarkers (homovanillic acid (HVA), vanillylmandelic acid (VMA), and 5‐hydroxyindole‐3‐acetic acid (5‐HIAA)). Dependences of the peak current on the concentration of biomarkers were linear in the whole tested concentration range from 0.05 to 100 μmol ? L^?1, with limits of detection (LODs) of 0.065 μmol ? L^?1 for HVA, 0.053 μmol ? L^?1 for VMA, and 0.033 μmol ? L^?1 for 5‐HIAA (calculated from peak heights), and 0.024 μmol ? L^?1 for HVA, 0.020 μmol ? L^?1 for VMA, and 0.012 μmol ? L^?1 for 5‐HIAA (calculated from peak areas), respectively.     

Development of a 6-hydroxychroman-based derivatization reagent: application to the analysis of 5-hydroxytryptamine and catecholamines by using high-performance liquid chromatography with electrochemical detection

We developed a novel derivatization reagent, (2R)-2,5-dioxopyrrolidin-1-yl-2,5,7,8-tetramethyl-6-(tetrahydro-2H-pyran-2-yloxy)chroman-2-carboxylate (NPCA), for electrochemical (EC) detection in HPLC. NPCA was synthesized from (R)-(+)-6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (alpha-CA), which exhibits intense EC response. NPCA successfully yielded alpha-CA derivatives of primary amines by a two-step derivatization procedure. Following pre-column derivatization with NPCA, a simultaneous determination of alpha-CA derivatives of neuroactive monoamines [dopamine (DA), epinephrine, and 5-hydroxytryptamine (5-HT)], their monoamine oxidase metabolites (3,4-dihydroxyphenylacetic acid, homovanillic acid, and 5-hydroxyindole-3-acetic acid) and their catechol-O-methyltransferase metabolites [3-methoxytyramine (3-MT) and normetanephrine (NMN)] was completely achieved using our HPLC-EC method. Using an HPLC equipped with coulometric electrode-array detection system, the resultant alpha-CA derivatives of NMN, 5-HT, DA and 3-MT showed intense EC responses, that were approximately 1.3, 1.4, 1.1 and 1.4-fold higher than the corresponding native forms, respectively. The detection limits were in the range of approximately 16-60 fmol on column (signal-to-noise ratio 3). The proposed HPLC method was applied to determine 5-HIAA, HVA, alpha-CA-5-HT and alpha-CA-DA in rat urine. As a consequence, these analytes were successfully determined with satisfactory precisions.