Automatic determination of urinary 4-hydroxy-3-methoxymandelic (vanillylmandelic) acid by liquid chromatography with electrochemical detection

An automated liquid chromatographic method for the determination of urinary concentrations of 4-hydroxy-3-methoxymandelic acid (VMA) is described. Urine samples are purified by solid-phase extraction on an anion-exchange cartridge and automated on-line chromatographic elution is carried out using a Varian AASP (advanced automated sample processor) system. The column effluent is monitored with an electrochemical detector using a glassy carbon working electrode. The method allows the determination of VMA in 0.05 ml of normal urine with a relative standard deviation of less than 3%. The analysis time can be shortened by use of back-flushing technique, and the correlation with a classical (but non-automated) VMA analysis method is excellent.     

Improved synthesis of 3-methoxy-4-hydroxymandelic acid by glyoxalic acid method

The most important industrial process for the synthesis of vanillin is performed in two steps involving an condensation reaction of glyoxalic acid with guaiacol followed by an oxidative decarboxylation of the intermediary 3-methoxy-4-hydroxymandelic acid (MHPA) formed, thereby producing not only vanillin, but also byproducts, which have to be eliminated. In the present study, we focused our efforts on the first step of vanillin synthesis, namely, the condensation reaction governing the yield of vanillin. The factors influencing the stability of glyoxalic acid were preliminarily evaluated to provide significant referential value for suppressing the dismutation of glyoxalic acid in the condensation reaction, and the results indicate that the stability of glyoxalic acid largely depends on the pH, temperature, and holding time of the feed solution. Then the process of the condensation reaction was optimized under the factors affecting the stability of glyoxalic acid, as well as the molar ratio of guaiacol to glyoxalic acid. Under the optimized conditions, the maximum yield of the condensation reaction can reach to 88%.     

Sensitive determination of 3-methoxy-4-hydroxyphenylglycol (MHPG) in human plasma by HPLC with electrochemical detection

Summary This study deals with the development of a new HPLC method for the determination of 3-methoxy-4-hydroxyphenylglycol (MHPG), the main noradrenaline metabolite in human plasma. A Varian reversed-phase column (C₈; 250 mm×4.6 mm i.d.; 5 μm particles) was used as the stationary phase and an aqueous solution of citric acid, 1-octanesulfonic acid, EDTA, and methanol was used as the mobile phase. Coulometric electrochemical detection (ED) was used to obtain the highest sensitivity. Isolation of MHPG from plasma was accomplished by means of a new solid-phase extraction procedure after a protein precipitation step. The extraction yield of MHPG from plasma was very high (>97%). Linearity was observed in the 0.5–25 ng mL⁻¹ concentration range; the limit of detection was 0.2 ng mL⁻¹ and the limit of quantitation was 0.5 ng mL⁻¹. Repeatability (RSD,%) for plasma samples was found to be <3.2% and intermediate precision was <4.3%. The method was applied to the determination of MHPG in the plasma of healthy subjects under experimentally-induced psychological stress.     

Direct determination of 4-hydroxy-3-methoxyphenylacetic (homovanillic) acid in urine by high-performance liquid chromatography with amperometric detection

The improvement of high-performance liquid chromatographic analysis with electrochemical detection for urinary homovanillic acid is described. The method permits the chromatographic resolution of authentic homovanillic acid from coeluting interfering compounds in human and nonhuman primate, and rat urine. The electrochemically derived results are compared with post-column derivatized fluorescence results, and quality-control checks necessary to maintain assay precision in automated analysis are described.     

Gunshot residue determination by high-performance liquid chromatography with electrochemical detection

A procedure for the detection of gunshot residue via the organic constituent diphenylamine is described. The method incorporates high-performance liquid chromatography with electrochemical detection.     

The determination of oxalic acid in urine by high performance liquid chromatography with electrochemical detection

A system for the determination of oxalic acid in human urine using ion exchange-ion pair, high performance liquid chromatography with electrochemical detection (HPLCEC) is described. Urine is acidified with HCl, and excess CaCl2 is added to precipitate oxalate ion. The precipitate is isolated, redissolved in dilute sulfuric acid, and separated on a strong cation exchange column using an acetic acid-solium acetate-tetrabutylammonium tetrafluoroborate mobile phase adjusted to pH 2.8. Using an electrochemical detector at 1.25 volts vs. the saturated calomel electrode (SCE), oxalic acid exhibits a linear dynamic range from 1 to 1000 mg/liter with a detection limit of 0.1 mg/liter. Quantitative data are obtained by the method of standard addition in the clinically significant range from 5 to 40 mg/liter. Percentage recovery for spiked urine samples was 97.8% with a relative standard deviation of 2.5%.     

Attomole catechins determination by capillary liquid chromatography with electrochemical detection.

Attomole quantities of catechins were determined by a capillary liquid chromatography system with electrochemical detection (CLC-ECD) and the system is applied to the determination of catechins in human plasma. The eight catechins: catechin (C), epicatechin (EC), gallocatechin (GC), epigallocatechin (EGC), catechin gallate (Cg), epicatechin gallate (ECg), gallocatechin gallate (GCg), and epigallocatechin gallate (EGCg), were separated within 10 min using a capillary column (0.2 mm i.d.) and a mobile phase of phosphoric acid (85%)-methanol-water (0.5:27.5:72.5, v/v/v), and were detected at +0.85 V vs. Ag/AgCl. Peak heights were found to be linearly related to the amount of catechins injected, from 200 amol to 500 fmol (r > 0.998). The detection limits of the catechins were 61 amol for EGC, 75 amol for EC, 54 amol for GC, 61 amol for C, 67 amol for GCg, 75 amol for EGCg, 75 amol for ECg and 89 amol for Cg (S/N = 3). Because the present method is highly sensitive and allows facile pretreatment for plasma sample, the time courses of concentrations of catechins (GCg, EC, EGCg, ECg, and Cg) and their conjugates in human plasma obtained from a 10 microl plasma sample after ingestion of green tea could be determined.     

Separation of the catecholamine metabolites 4-hydroxy-3-methoxy- and 3-hydroxy-4-methoxymandelic acid by reversed-phase high performance liquid chromatography

The catecholamine metabolites 4-hydroxy-3-methoxy- and 3-hydroxy-4-methoxymandelic acid can be completely separated by reversed-phase high performance liquid chromatography with chemically bonded octadecylsilane as stationary phase and a citrate/ammonium phosphate buffer (pH 4.5) containing 8% methanol as mobile phase. The two isomers can be electrochemically detected and produce different hydrodynamic voltammograms.     

Determination of micromolar bromate concentrations by adsorptive-catalytic stripping votammetry of the molybdenum-3-methoxy-4-hydroxymandelic acid complex

Adsorptive-catalytic stripping voltammetry is commonly used for monitoring trace metals. This study reverses the scheme by developing a sensitive procedure for measuring the catalyst. The catalytic action of bromate upon the cathodic response of the adsorbed molybdenum-3-methoxy-4-hydroxymandelic acid (VMA) complex has been exploited for measuring bromate down to the micromolar concentration level. Experimental variables affecting the response, including the metal and ligand concentrations or the accumulation time and potential, were characterized and optimized to yield a highly linear response (up to at least 1x10(-4) mol l(-1)) and good precision (RSD of 3.5%; n=10; 2x10(-6) mol l(-1) bromate). Analogous adsorptive-catalytic stripping protocols may be utilized for the voltammetric detection of other catalysts.     

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.     

Improved high-performance liquid chromatographic method for the routine determination of unconjugated 3-methoxy-4-hydroxyphenylethyleneglycol in human plasma using solid-phase extraction and electrochemical detection

An improved semi-automated high-performance liquid chromatographic method is described for the routine determination of unconjugated 3-methoxy-4-hydroxyphenylethyleneglycol in plasma. The 3-ethoxy analogue of the compound is used as an internal standard. The method is based on purification of 0.5-ml plasma samples with phenyl-type reversed-phase extraction columns, reversed-phase separation with an acetate-citrate-methanol mobile phase with an octadecyl-bonded column, and dual-electrode coulometric detection with oxidation at +0.44 V and reduction at -0.25 V. The precision and accuracy of the assay are satisfactory: the lower limit of reliable detection corresponds to a plasma concentration of 1.5 nM. The validity of the determination is demonstrated by an 18% mean increase in plasma levels of 3-methoxy-4-hydroxyphenylethyleneglycol during physical exercise (duration 16 min, n = 13) and a 50% mean reduction in plasma levels induced by a single dose of the monoamine oxidase inhibitor, moclobemide (n = 8). The method is suitable for routine use in pharmacological and physiological experiments.     

Separation and determination of aminohalogenbenzophenones by high-performance liquid chromatography with electrochemical detection.

A high-performance liquid chromatographic method with electrochemical detection has been developed for the determination of three aminohalogenbenzophenones: 2-amino-2',5-dichlorobenzophenone, 2-amino-5-chlorobenzophenone and 2-amino-5-bromo-2'-fluorobenzophenone, metabolites of benzodiazepinooxazoles and other psychotropic drugs. A mobile phase of methanol-water (65:35), containing 5 mM KH2PO4 appeared to be the optimal when a 4-microns, 60-A Nova-Pak C18 column and a flow-rate of 0.75 ml/min (130 bar) were used. The temperature was optimized at 30 degrees C. The amperometric detector, equipped with glassy carbon electrode, was operated at 1.3 V versus Ag/AgCl in the DC mode. The method was applied to the determination of these compounds at two concentration levels: ppm and ppb (ng/cm3) using 2-amino-5-chlorobenzophenone as internal standard. The limit of determination was 750 pg/ml of biological fluid for each compound, and recoveries greater than 97% were obtained for spiked samples of urine and serum, using C18 Sep-Pak cartridges in the sample clean-up procedure.     

Simple high-performance liquid chromatographic method for the concurrent determination of the amine metabolites vanillylmandelic acid, 3-methoxy-4-hydroxyphenylglycol, 5-hydroxyindoleacetic acid, dihydroxyphenylacetic acid and homovanillic acid in urine using electrochemical detection

A simple method for the concurrent analysis of the noradrenaline metabolites vanillylmandelic acid and 3-methoxy-4-hydroxyphenylglycol, the dopamine metabolites dihydroxyphenylacetic acid and homovanillic acid, and the serotonin metabolite 5-hydroxyindoleacetic acid in human urine is described. Following organic extraction of the metabolites from acidified urine, they are separated by single-step gradient elution high-performance liquid chromatography on a reversed-phase column. Detection and quantification are achieved with an electrochemical detector using a carbon-paste electrode; samples can be injected at 40-min intervals. Optimisation of analytical parameters is described, and examples of the application of the method in the fields of clinical chemistry and clinical neuroscience are given. This provides a convenient method for the concurrent study of the metabolism of three major biogenic amines, and is readily adaptable for studies on cerebrospinal fluid and brain tissue.