Highly Sensitive and Simple Method for Determination of Free 3-Methoxy-4-Hydroxyphenylglycol in Plasma by High-Performance Liquid Chromatography Using a Sep-Pak Alumina B Cartridge

Abstract

A novel sample clean-up procedure for the determination of free 3-methoxy-4-hydroxyphenylglycol (MHPG) in plasma in described. MHPG was purified with Sep-Pak alumina B cartridge followed by ethyl acetate extraction from the cartridge. High-performance liquid chromatography with amperometric detection was used for separation and detection of MHPG and the internal standard 3-ethoxy-4-hydroxyphenylglycol (EHPG). This method provided good, clean chromatograms with base-line separation of the appropriate peaks. This technique is sensitive, reliable and less time-consuming than other methods. Thus, only 0.5 ml of plasma is needed and the within-analysis and between-analysis coefficients of variation were 5.2% and 13% respectively. The plasma MHPG values in normal control were in good agreement with those using more complex methods.     

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.     

A Uricase Method for the Peak Identification of Uric Acid Appeared in a Liquid Chromatogram Amperometrically Monitored

Abstract

A uricase method for the peak identification of uric acid appeared in a liquid chromatogram monitored by aid of an electrochemical detector has been developed. Uricase (EC 1.7.3.3, from Candida utilis) catalyzes the conversion of uric acid to allantoin. We have found that uric acid can be oxidized under the chromatographic conditions employed in this study, whereas allantoin cannot be oxidized. The complete disappearance of a uric acid peak in a chromatogram of a biological sample after the uricase treatment indicates that the uric acid peak does not contain any other electroactive components. We observed the complete disappearance of the uric acid peaks in the chromatograms of human serum and gastric body.     

Electrochemical Detection of Retinoids Using Normal Phase HPLC

Abstract

Non-aqueous electrochemical (EC) detection of 13-cis-retinoic acid, all-trans retinoic, acitretin and vitamin A palmitate in non-aqueous solvents are reported.

Non-aqueous (EC) detection allows for normal-phase chromato-graphy of these compounds prior to detection. The normal phase system used a mobile phase of HEX/THF/AcOH for the separation of all four compounds. The stationary phase was either silica or PVA-sil. The lipophilic salts, t-butylammoniumtetrafluoroborate or t-butyl-ammoniumhexafluorophosphate necessary for EC detection were added post-column.

The limit of detection (LOD) for EC detection of these compounds is approximately 1 ng on column compared with an LOD by UV absorption of 2 ng on column.

The linear detection for these compounds with the EC detector was about two orders of magnitude.     

Enzymatic Estimation and Quantitative High-Pressure Liquid Chromatography of Fructose,Glucose and Sucrose in Powders from Rose Petals

Abstract

There was no significant difference between mean mass fractions of fructose, glucose and sucrose measured enzymatically and by liquid chromatography. So the chromatographic method can be used on powders from rose petals as the peaks of the chromatograms are really caused by the sugars.

A method is described for clean-up of the rose extract before injecting it into the high-pressure liquid chromatograph.     

Analysis of 3-Methoxy-4-Hydroxyphenylglycol in Human Cerebrospinal Fluid by Gas Chromatography Mass Spectrometry and Electrochemical Detection Liquid Chromatography

Abstract

Gas chromatography mass spectrometry [GCMS] and liquid chromatography with electrochemical detection [LCEC] have been compared in simultaneous determination of 3-methoxy-4-hydroxy-phenylglycol [MHPG] in human cerebrospinal fluid [CSF]. Both free and sulphate conjugated MHPG have been measured in these samples. By Spearman Rank Correlation non-parametric analysis both instrumental techniques related significantly for all quantitated samples.     

HPLC Determination of Monoamines in Rat Brain after Enzymatic Treatment with Ascorbate Oxidase and Sulfatase

Abstract

The simultaneous analysis of norepinephrine, dopamine and serotonin along with their respective metabolites, MHPG, DOPAC and 5HIAA is accomplished in rat brain by using a recently developed HPLC technique. Sulfated MHPG is decongegated with sulfatase for detection purposes and endogenous ascorbate is diminished with ascorbate oxidase to reduce the front.     

Simultaneous determination of 3,4-dihydroxymandelic acid, 4-hydroxy-3-methoxymandelic acid, 3,4-dihydroxyphenylglycol, 4-hydroxy-3-methoxyphenylglycol, and their precursors, in human urine by HPLC with electrochemical detection

Summary A method has been developed for the quantification of urinary 3,4-dihydroxymandelic acid (DOMA), 4-hydroxy-3-methoxymandelic acid (VMA), 3,4-dihydroxyphenylglycol (DHPG), and 4-hydroxy-3-methoxyphenyglycol (MHPG). Separation and determination of these compounds in biological samples was previously thought to be very difficult. In this work the separation has been achieved by reversed-phase high-performance liquid chromatography with step-wise gradient elution with three mobile phases. The conditions for coulometric detection have been optimized for effective determination of these compounds. In analysis of a sample of human urine, after a simple deproteinization proceudre, DOMA, VMA, DHPG, and MHPG were separated from interferences and quantified successfully; the average levels of these compounds in six different samples were 33.87±1.03, 1202±41.3, 31.3±1.92, and 80.6±2.15 μg (24 h)⁻¹, respectively. Their precursors E, MN, DOPA, DA, NE, DOPAC, HVA, 3MT, and NMN, and the indolamine 5HT and its metabolite 5HIAA (a list of abbreviations is given at the end of the paper) can also be determined simultaneously in the same chromatographic run. The overlapping peak of DHPG was resolved by deconvolution.     

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.     

Automatic Headspace Sampling for Determination of Ethylene Dibromide Residues in Cereals

Abstract

The possibility was investigated to apply a commercially available automatic head-space sampler in trace analysis of ethylene dibromide (EDB, 1,2-dibromoethane), in cereals.

Samples of rice and wheat flour were thermostatted in closed vials at 70[ddot]C for 30min. The top gas was then automatically introduced into a gas chromatograph equipped with an electron capture detector. Quantitation was performed using external standards (untreated samples spiked with solutions of EDB in N,N′-dimethylacetamide).

The relative standard deviation of the method was 3.4% for rice and 4.5% for wheat flour, at a residue level of 0.008 mg/kg. The detection limit was 0.001 mg/kg (the official EC residue tolerance is 0.01 mg/kg).

Preliminary experiments with other fumigants were carried out as well.

The headspace technique in question has the following advantages over other methods for determining EDB residues in cereals: 1. No sample pre-treatment like extraction, steam distillation, purge and trap etc.; 2. Automated sample handling; 3. “Clean” chromatograms.     

Reverse-Phase Liquid Chromatographic Analysis of the Acid-Catalyzed Rearrangement of Nadh,Separation of New Products and an Analysis of the First Two Reactions of the Rearrangement

Abstract

The previously unresolved products of acid-catalyzed rearrangement of NADH have been separated into seven peaks by liquid chromatography on microparticle ODS. The peak with the second highest retention volume was identified as O²,6-B-cyclotetrahydronicotinamide adenine dinucleotide. Based on the order of appearance and disappearance of the peaks, and on their production in the presence of glyceraldehyde-3-phosphate dehy-drogenase (EC 1.2.1.12), four of the peaks have been assigned to specific products in the reaction sequence originally proposed by Oppenheimer and Kaplan [Biochemistry, 13, 4675, 1974]. The three remaining peaks (in addition to AMP) are previously unidentified products of the acid-catalyzed rearrangement of 6-B-cyclotetrahydronicotinamide adenine dinucleotide. The first step in the rearrangement of NADH to 6-B-cyclotetrahydronicotinamide adenine dinucleotide was shown to be dependent on the pH and ionic strength of the buffer, but neither the first product nor any other product incorporated the buffer into its structure.     

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.     

Determination of Arene Oxides by a Gas Chromatography-Mass Spectrometry System: Thermal Reactions of 9, 10-Epoxy-9, 10-Dihydrophenanthrene

Abstract

An analytical technique involving the use of a gas chromatograph coupled to a mass spectrometer-computer system, has been developed to detect arene oxides using 9, 10-epoxy-9, 10-dihydro-phenanthrene as a model substrate. The gas chromatograph was equipped with a hydrogen flame ionization detector and a high-resolution glass capillary column coated with SE-52 (methyl phenyl silicone). Two simultaneous thermal reactions (deoxygenation and rearrangement) of 9, 10-epoxy-9, 10-dihydrophenanthrene in the gas chromatograph were observed. The method developed was compared with a conventional method utilizing a packed glass column. Under the latter conditions neither thermal reactions nor 9, 10-epoxy-9, 10-dihydrophenanthrene were detected. The identification of the reactant and products was achieved by comparison of retention times and mass spectral fragmentation patterns obtained from authentic samples.     

Gas Liquid Chromatographic Determination of Acetaminophen in Blood Plasma

Abstract

An improved procedure for the GLC determination of acetaminophen (N-acetyl-p-aminophenol) in the presence of N-butyryl-p-aminophenol (internal standard) is described. The method is based on the extraction of acetaminophen from plasma with ethyl acetate containing a known amount of N-butyryl-p-aminophenol. Following a clean-up step with a basic buffer solution and neutralization with acid, both compounds are reextracted into ethyl acetate. The ethyl acetate is evaporated to dryness and the residue dissolved in 5 μl of pyridine and 15 μl of acetic anhydride at 42°C. One to 2 μl samples are injected directly into the gas chromatograph. This extraction process does not give rise to troublesome interfering peaks in the chromatogram. In addition, it prevents late-eluting peaks which inhibit efficient processing of samples. The recovery of acetaminophen is approximately 54%, and the limit of quantitation 0.5 μg/ml of plasma. Data are presented to illustrate the practicality of the method for bioavailability evaluation from acetaminophen plasma levels after oral administration of 325 mg of an acetaminophen dosage form.     

High-Performance Liquid Chromatography of Domoic Acid,a Marine Neurotoxin,with Application to Shellfish and Plankton

Abstract

A recent outbreak of poisoning resulting from the consumption of cultured blue mussels (Mytilus edulis L.) from a localized area in Eastern Canada has been attributed to the presence of domoic acid (1), a relatively rare neurotoxic amino acid, previously found only in some algae of the family Rhodomelaceae. Studies on aqueous extracts of shellfish tissue indicated that the toxin and several of its isomers could be separated (and isolated in sufficient amounts for subsequent structural identification) by reversed-phase high-performance liquid chromatography (HPLC) with ultraviolet (UV) diode array detection (DAD). Aqueous acetonitrile containing 0.1% v/v trifluoroacetic acid was used as mobile phase. As the retention time and characteristic UV absorption spectrum of 1 (λ_max = 242 nm) permit unequivocal identification, the HPLC-DAD procedure was refined with a microbore column to provide a rapid (5 min), sensitive (0.3 ng detection limit) and reproducible assay method for the determination of 1 in shellfish tissue. Extraction was accomplished by boiling homogenized shellfish tissue for 5 min with distilled water. Extracts were taken through an octadecylsilica solid phase extraction clean-up prior to HPLC. This method has been applied to a variety of shellfish and phytoplankton samples.

BRIEF

Reversed-phase HPLC with ultraviolet diode array detection was used to analyze shellfish tissue and phytoplankton extracts for domoic acid. A rapid (5 min) and sensitive (0.3 ng detection limit) assay is presented.     

The Combination of Pyrolytic Esterification and Ion Pair Extraction for the Analysis of Organic Acids

Abstract

Pyrolytic esterification and ion pair extraction were combined to furnish a rapid convenient method for analysis of organic acids. The extraction was not 100% efficient but the response of the gas chromatograph was linearly aroportional to the concentration of acid. However, it was necessary to have a large excess of the quaternary ammonium salt present.     

Comparison of Aqueous,Micellar and Microemulsion Carriers in Flow Injection Analysis. The Base Hydrolysis of Acetylsalicylic Acid

Abstract

The potential applicability of surfactant solutions as carrier streams in flow injection analysis is examined. The reaction rates determined for the base catalyzed hydrolysis of acetylsalicylic acid in aqueous, micellar and microemulsion solutions show a rate enhancement of 40% for the static microemulsion system when compared to the aqueous solution. However, when the identical microemulsion solution is employed as a carrier stream in flow injection analysis with ultraviolet detection, this enhancement in rate is not observed. To our knowledge, all previous work employing microemulsions in FIA has been concerned only with detection enhancement, here we present direct comparisons between aqueous and microemulsion carriers concerning rates of reaction, peak dispersion and analytical figures of merit. The loss in relative sensitivity can be traced to the increased dispersion in the microemulsion system (D = 14.36) when compared to the aqueous carrier (D = 12.52). Additionally, an increased skewness was observed in the peaks obtained with a microemulsion carrier, yielding further information about the physical dispersion process occuring in the sample plug.     

Liquid Chromatographic-Electro-Chemical Technique for Determination of Ethylenethiourea Residues

Abstract

A high performance liquid chromatographic-electrochemical (HPLC-EC) technique was developed to selectively determine ethylenethiourea (ETU) at residue levels without derivatization. ETU was eluted from a C-8 column with water, a phosphoric acid electrolyte solution was added to the column eluate, and then ETU was detected with an electrochemical detector containing a Au/Hg working electrode. The HPLC-EC system produced a sharp chromatographic peak for ETU that was detected by the Au/Hg electrode at an applied potential of +0.36 V. With detector sensitivity adjusted so that 10 ng ETU produced a 50% full scale deflection peak (1% baseline noise), the detector's response was linear from 2 to 400 ng ETU. No peaks were observed in potato and spinach controls, and only small apparent ETU peaks of 7 and 3 ppb, respectively, were found in apple and grape controls. Detector response was equivalent to 90% of actual ETU added (0.1 ppm) to purified spinach extracts. Crop coextractives from apples, grapes and potatoes did not affect detector response to ETU at the 0.1 ppm fortification level.     

Activity of SiDbCl in the Electrooxidation of Ascorbic Acid,Dopamine, and Uric Acid

Selective electroanalytical responses for ascorbic acid, dopamine and uric acid at a carbon modified electrode based on 3‐n‐propyl‐1‐azonia‐4‐azabicyclo[2.2.2]octane silsesquioxane chloride (SiDbCl) is reported. The overlapped peaks observed at an unmodified electrode are resolved into three well defined voltammetric peaks allowing the simultaneous determination of the three species. Detection limits of 37, 0.3 and 0.1 μmo L⁻¹ of ascorbic acid, dopamine and uric acid, respectively, were calculated from calibration curves based on differential pulse voltammetric experiments performed in Britton ‐ Robinson buffer solution at pH 7.04.     

Urinary Uric Acid Determination by Reversed-Phase High Pressure Liquid Chromatography

Abstract

Reversed-phase high pressure liquid chromatography with UV detection was proven to be a powerful method for the separation and quantitation of urinary uric acid. We have compared three different treatments for urine samples previous chromatographic injection: alkaline methanol extraction, ethylacetate extraction and centrifugation. It was also studied storage conditions for urine samples.

Our findings show that the method has high specificity and reproducibility for urinary uric acid. Samples are stables and require only centrifugation previous injection to the chromatograph.