Direct measurement of homovanillic, vanillylmandelic and 5-hydroxyindoleacetic acids in urine by capillary electrophoresis

Separation conditions in CE, with a neutral coated capillary and reversed polarity, have been optimised to make direct measurement of vanillylmandelic acid, homovanillic acid and 5-hydroxyindoleacetic acid possible in urine samples without pre-treatment. The method developed has been validated, presenting adequate parameters for linearity, accuracy and precision. Detection limits range from 0.03 to 2.5 microM. Finally the method has been applied to urine samples taken from patients, both adults and children, in hospital. Some of them were also measured by immunoassay and HPLC-electrochemical detection and results have been compared.     

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.     

Separation of copper(II) and aluminium(III) from fresh waters by solid phase extraction on a complexing resin column

The solid‐phase extraction (SPE) of copper(II) and aluminium(III) from fresh waters on an ion‐exchange complexing resin containing iminodiacetic groups (Chelex 100) has been examined. Quantitative recovery of the metal ions was related to the breakthrough profile that, for some samples, could not be evaluated directly. A method is suggested for evaluation, instead, of the sorption curves, on the basis of passing different volumes of sample through the column. This enables evaluation of important properties, for instance the central point of the breakthrough curve, V_f. The column used was a small one, containing 0.10 g dry Chelex 100. The metal ion was eluted with a small volume of acid solution, 10 mL of 0.5 mol L^–1 HNO₃; this resulted in good preconcentration factors. For copper(II) it was found that fresh waters of similar composition could have different V_f in the same column. This was ascribed to different reaction coefficients (α_M(I)) of copper(II) in the considered samples, which affects V_f. By use of the proposed SPE procedure it is possible to evaluate the reaction coefficient of copper(II). The values of α_M(I) for two different drinking waters at pH 7.7 were found to be 3.70×10¹² and less than 4.40×10¹¹. Similar results were obtained for aluminium(III).     

Simple and fast chromatographic method for the simultaneous determination of vanillylmandelic acid and homovanillic acid in human urine.

A high-performance liquid chromatographic method for the determination of vanillylmandelic acid and homovanillic acid is described. The method is fast despite the great polarity differences between the two acids. Moreover the sample pretreatment is quick and it does not need complex or expensive equipment. The only requirement is the disposition of two pumps (or at least two eluent reservoirs) operated alternatively by means of a switching valve placed before the injection device. This makes the method available for most routine laboratories.     

Separation of palladium(II) and platinum(II) chlorides by means of a guanidine resin

The preparation and properties of a new, weakly basic ion-exchanger containing suilonylguanidine as the active groups are described. Palladium and platinum chlorides can be quantitatively separated by this resin. Under certain conditions platinum does not react with the resin, whereas palladium is adsorbed and can then be eluted with 3 M hydrochloric acid.     

Separation and preconcentration of iron(II) and iron(III) from natural water on a melamine-formaldehyde resin

A combined method for the preconcentration and selective spectrophotometric determination of both valencies of iron, i.e., Fe(II) and Fe(III), down to 0.4 mug l(-1) has been developed. Iron(III) from synthetic and natural water samples has been concentrated on a melamine-formaldehyde resin at pH 5; iron(II) was not retained under identical conditions. The oxidized iron was concentrated on a second resin column. The iron in both columns was eluted with 1 M HCl solution and separately analyzed by the 1,10-phenanthroline-citrate spectrophotometric method. The effect of pH, adsorption and elution rates, and interferences on the developed procedure were investigated. Metal ions that can be retained by the resin at moderate concentrations, e.g., Al(3+), do not cause interference in more dilute solutions encountered in natural water samples. At least 160-fold volume enrichment can be easily obtained using an adsorption flowrate of 50 ml min(-1). A hydrothermal water sample was analyzed by the recommended procedure and by a literature method, and the results were statistically compared by t- and F-tests.     

Quantitation of mercapturic acids from acrylamide and glycidamide in human urine using a column switching tool with two trap columns and electrospray tandem mass spectrometry

A sensitive and specific electrospray tandem mass spectrometry method using a column switching unit with two trap columns was established to quantify the mercapturates (MAs) of acrylamide (AA) and glycidamide (GA) in human urine. A specially endcapped material was applied for trapping the hydrophilic MAs and a pre-trap column was used to remove lipophilic compounds from the directly injected urine to protect the trap column. The limits of quantitation for AA-MA and GA-MA in urine were 0.5 microg/L and 1 microg/L, respectively. Urine was spiked with deuterated internal standards and injected directly into LC-MS/MS. Urine of smokers (n=13) revealed the highest concentrations of AA-MA and GA-MA in the range of 61-706 microg/L and 5-54 microg/L, respectively. Lower levels for AA-MA (14-102 microg/L) and GA-MA (1-11 microg/L) were detected in non-smokers (n=13).     

Routine analysis of catecholamines and metabolites in urine by a liquid chromatographic column switching system

A reversed-phase liquid chromatographic technique including a column switching system has been adapted for the routine measurement of catecholamines and their metabolites (14 compounds) in urine. From 1 ml of urine all the compounds and the internal standards were obtained according to combined extraction procedures involving organic solvent, anionic and weakly cationic resins. Finally four extracts (catecholamines, methoxamines, acidic and neutral derivatives) had to be chromatographed throughout a wholly automated apparatus. For each run, the column switching system determined the analytical columns to be used to obtain the separation of the compounds from interferences due to other co-extracted endogenous substances, while the analysis times remained between 20 and 40 min. Such a system allowed the rapid clean-up of columns (in direct- and back-flush mode) carried out between two consecutive injections. By coupling on-line fluorimetric and electrochemical detections the specificity of the technique could be checked, since the ratio of the responses of both detectors was an index of the purity of the peaks. Finally the advanced automation of the equipment allowed weekly the evaluation of catecholamines and the whole range of their known metabolites in 36 urine samples.     

Separation of helium and hydrocarbon mixtures by a two-membrane column

The permeabilities to pure gases can be directly used in the calculation of mixed gas permeation through a rubbery membrane (silicone rubber), but the permeabilities of cellulose acetate membranes to mixed gases are considerably affected by the presence of hydrocarbons. The efficiency of separation increases with decreasing pressure ratio and reaches a maximum when the pressure ratio becomes zero. The enrichment of the less permeable gas can be easily achieved in a stripper. Combining two strippers made out of two different membranes, highly concentrated products (99%) are continuously obtained at both ends of the column. As a result, the overall separation factors are greatly increased.     

Separation of transition metals on a poly-iminodiacetic acid grafted polymeric resin column with post-column reaction detection utilising a paired emitter-detector diode system

The selectivity, retention and separation of transition metals on a short (2 mm x 50 mm) column packed with a poly-iminodiacetic acid functionalised polymer 10 microm resin (Dionex ProPac IMAC-10) are presented. This stationary phase, typically used for the separation of proteins, is composed of long chain poly-iminodiacetic acid groups grafted to a hydrophilic layer surrounding a 10 microm polymeric bead. Through the use of a combination of a multi-step pH and picolinic acid gradient, the separation of magnesium, iron, cobalt, cadmium, zinc, lead and copper was possible, followed by post-column reaction with 4-(2-pyridylazo) resorcinol (PAR) and absorbance detection at 510 nm using a novel and inexpensive optical detector, comprised of two light emitting diodes with one acting as a light source and the other as a detector. Column efficiency for selective transition metals was in excess of N=10,000, with the baseline separation of seven metal cations in <3 min possible under optimised conditions. Detection limits of between 5 and 81 microg/L were possible based upon a 50 microL injection volume.