Measurement of 4,5-dioxovaleric acid by high-performance liquid chromatography and fluorescence detection

In this work we describe a sensitive method for the detection of 4,5-dioxovaleric acid (DOVA). 4,5-Dioxovaleric acid is derivatized with 2,3-diaminonaphthalene to form 3-(benzoquinoxalinyl-2)propionic acid (BZQ), a product with favorable UV absorbance and fluorescence properties. The high-performance liquid chromatographic method with UV absorbance and fluorescence detection is simple and its detection limit is approximately 100 fmol. This method was used to detect 4,5-dioxovaleric acid formation during metal-catalyzed 5-aminolevulinic acid (ALA) oxidation. Iron and ferritin were active in the formation of 4,5-dioxovaleric acid in the presence of 5-aminolevulinic acid. In addition, HPLC–MS–MS assay was used to characterize BZQ. The determination of 4,5-dioxovaleric acid is of great interest for the study of the mechanism of the metal-catalyzed damage of biomolecules by 5-aminolevulinic acid. This reaction may play a role in carcinogenesis after lead intoxication. The high frequency of liver cancer in acute intermittent porphyria patients may also be due to this reaction.     

Quantification of urinary 5-aminolevulinic acid by gas chromatography-mass spectrometry

In this report, we describe a method for the specific quantification of urinary 5-aminolevulinic acid. It is based on gas chromatographic mass spectrometric measurement of the trimethylsilyl ester of the ethylacetoacetate pyrrole derivative of 5-aminolevulinic acid. Selected ion monitoring (SIM) was used for quantification using 3-hydroxymyristic acid as an internal standard. The detection limit of this method was 0.1 nmol/ml. This method was applied to the determination of urinary 5-aminolevulinic acid from a tyrosinemia type I patient and normal subjects, and 21.4 mmol/mol creatinine and 0.54+/- 0.49 mmol/mol creatinine (n = 7), respectively, were detected. Less than 0.2 ml urine was sufficient for the determination of 5-aminolevulinic acid in healthy subjects.     

Determination of lipoic acid in human plasma by high-performance liquid chromatography with electrochemical detection

A selective and sensitive method for the determination of lipoic acid in human plasma samples has been developed. After enzymatic hydrolysis of the sample, the liberated lipoic acid was extracted by a solid-phase cartridge and measured by HPLC using electrochemical detection. The detection limit was 1 ng/ml lipoic acid in plasma. The calibration curve was non-linear in the range 0.01–50 μg/ml but could be described by a power function. The average extraction recoveries were 82.5 and 85.1% at the 25 and 2500 ng/ml levels, respectively. Coefficients of variation for both within-day and day-to-day analysis were between 2.1 and 9.4%. The assay method is sensitive, reproducible and suitable for disposition studies of lipoic acid in humans.     

Determination of uric acid in human saliva by high-performance liquid chromatography with amperometric electrochemical detection

The aim of the present study is to establish a highly sensitive method for the determination of uric acid (UA) in human saliva. The monitoring of UA levels in less invasive biological samples such as saliva is suggested for the diagnosis and therapy of gout, hyperuricemia, and the Lesch-Nyhan syndrome, and for detecting such conditions as alcohol dependence, obesity, diabetes, high cholesterol, high blood pressure, kidney disease, and heart disease. Reversed-phase high-performance liquid chromatography with electrochemical detection (HPLC-ED) was employed for the determination of UA obtained by solid-phase extraction from saliva. To quantify UA, we compared the ED efficiencies of an amperometric ED (Ampero-ED) with a single electrode and a coulometric ED (Coulo-ED) with a multiple electrode array. The results showed that the detection limits (S/N=3) were 3 nM for Ampero-ED and 6 nM for Coulo-ED, and the linearity of the calibration curves of 60-6000 nM had correlation coefficients exceeding 0.999. In addition, the total analytical time was 10 min. In the sample preparation of UA in saliva, an Oasis MAX solid-phase cartridge was used. The recoveries of UA spiked at 0.6 and 3 microM in saliva were above 95% with a relative standard deviation (RSD) of less than 15%. Therefore, the present method may be used in the routine and diagnostic determination of UA in human saliva.     

Determination of 5-aminolevulinic acid in biological samples by high-performance liquid chromatography

5-Aminolevulinic acid (ALA), the common precursor of all naturally occurring tetrapyrroles, forms a stable condensation product with 2-amino-3-hydroxynaphthalene which can be identified by its fluorescence. Separation of the compound by reversed-phase high-performance liquid chromatography on RPC-18 columns allows its detection down to the picomolar range and can be successfully applied for ALA analysis in small biological samples. The reaction product of ALA with 2-amino-3-hydroxynaphthalene has been synthesized and characterized.     

Determination of timiperone in rat plasma by high-performance liquid chromatography with electrochemical detection

We report a sensitive new method for the determination of timiperone in rat plasma by using high-performance liquid chromatography with electrochemical detection. The method involves extraction of plasma samples with heptane-isoamyl alcohol at pH>8, followed by back-extraction into dilute acetic acid. Separation was accomplished by reversed-phase high-performance liquid chromatography on an ODS column with the mobile phase consisting of 0.1 M phosphate buffer (pH 3.5)-acetonitrile-methanol (65:20:15, v/v). Recovery was greater than 80%. Calibration curve was linear over the concentration range 0.5–50.0 ng/ml. The limit of quantitation of timiperone was 0.5 ng/ml plasma.     

Determination of succinylcholine in human plasma by high-performance liquid chromatography with electrochemical detection

An alternative HPLC method for the quantification of the depolarizing neuromuscular blocking agent succinylcholine in human plasma is described. Drug spiked plasma and patient plasma samples were extracted using a C₁ solid-phase cartridge. Succinylcholine was separated on a Cyano column and quantitated using electrochemical detection at a potential of 450 mV and 750 mV. Mobile phase consisted of a mixture of phosphoric acid–acetonitrile–methanol (45:35:25) adjusted to an apparent pH of 5. Standard curves for the quantitation were linear in the range of 250–8000 ng/ml. Between-day and within-day relative standard deviations were 5.1% and 1.7%, respectively. Mean drug recovery and accuracy was 68% and 104%, respectively.     

Determination of catecholamines in human plasma by high-performance liquid chromatography with electrochemical detection

In the present study, assays were improved for the determination of catecholamines in human plasma. High-performance liquid chromatography with electrochemical detection was employed for quantitative analysis. The influence of various parameters on chromatographic performance, such as the composition and the pH of the mobile phase, and the detection potential, was investigated. An accurate solid-phase extraction procedure, after catecholamine complexation with diphenylborate, was developed. The efficiency yield for all catecholamines was in the range 92–98%. Relative standard deviation values for repeatability and for intermediate precision were less than 2% and 3%, respectively, for all three analytes.     

Determination of urinary 5-hydroxytryptophol by high-performance liquid chromatography with electrochemical detection

A high-performance liquid chromatographic method for the routine determination of elevated urinary levels of the serotonin metabolite 5-hydroxytryptophol (5-HTOL) is described. Urine samples were treated with β-glucuronidase, and 5-HTOL was isolated by solid-phase extraction on a small Sephadex G-10 column prior to injection onto an isocratically eluted C₁₈ reversed-phase column. Detection of 5-HTOL was performed electrochemically at +0.60 V vs. Ag/AgCl. The limit of detection was ca. 0.05 μM, and the intra-assay coefficients of variation were below 6% with urine samples containing 0.2 and 2.1 μM 5-HTOL and a standard solution of 2.0 μM (n = 5). The recovery of 5-HTOL after the sample clean-up procedure was close to 100%. A good correlation (r² = 0.97; n = 12) was obtained between the present method and a sensitive and specific gas chromatographic—mass spectrometric method. The total (free plus conjugated) 5-HTOL levels in urine were normally below 0.2 μM, but after an acute dose of alcohol they increased to 0.5–15 μM.     

Determination of captopril in human blood by high-performance liquid chromatography with electrochemical detection

A new method for quantitation of captopril in human blood is described. Captopril was derivatized with N-(4-dimethylaminophenyl)maleimide into the electrochemically active adduct. The derivative was separated and determined by high-performance liquid chromatography with an electrochemical detector on a reversed-phase column. The proposed method was satisfactory for determination of captopril in whole blood with respect to accuracy and precision. The detection limit of captopril thereby obtained was 10 ng/ml. The blood levels of captopril in patients orally given an officinal dose were measured by the present method.     

Determination of plasma homovanillic acid by liquid chromatography with electrochemical detection

We describe a simple method for extracting homovanillic acid (HVA) from plasma. An aliquot of 0.5 ml of the internal standard solution (3-hydroxy-4-methoxycinnamic acid in 0.2 mol/l phosphoric acid) and 0.5 ml of the sample are applied to a 1-ml Bond Elut C₁₈ column prewashed with methanol and 0.2 mol/l phosphoric acid. The sample is drawn through the column at low speed. The column is washed with water and eluted with dichloromethane. The eluate is evaporated under vacuum at ambient temperature and the residue reconstituted with 250 μl of the mobile phase. A 10-μl aliquot of the resulting solution is injected onto a 150 mm × 4.6 mm I.D. column packed with 5-μm octadecylsilyl silica particles (Beckman). Peaks are detected coulometrically in the screening-oxidation mode with E₁ = +0.25 V and E₂ = +0.38 V. In the resulting chromatogram, HVA and the internal standard give sharp peaks and are well separated from solvent and other endogenous electroactive acids. The extraction recovery is 90–95% which allows the determination of 0.5 μg/l analyte.     

Determination of 5-methyltetrahydrofolic acid in plasma and spinal fluid by high-performance liquid chromatography,using on-column concentration and electrochemical detection

Chromatographic conditions for the determination of 5-methyltetrahydrofolic acid in plasma and spinal fluid are described, involving simple pretreatment of the sample. Electrochemical detection was used. The linear range of the method is more than 10³. Recovery from plasma and spinal fluid is 100%, and the detection limit of the method is 2·10^?9M, sufficient for the detection of endogenous plasma and spinal fluid levels. The detection conditions are discussed. Endogenous concentrations of the compound in plasma and spinal fluid were determined and correlated with a folate bioassay. Plasma concentrations have been shown after the administration of leucovorin which is used in anticancer therapy.     

Simultaneous determination of 3-methoxy-4-hydroxyphenylglycol, 5-hydroxyindoleacetic acid, and homovanillic acid in cerebrospinal fluid with high-performance liquid chromatography using electrochemical detection

An improved high-performance liquid chromatographic method with electrochemical detection (HPLC-EC) for the simultaneous determination of 3-methoxy-4-hydroxyphenylglycol (MHPG), 5-hydroxyindoleacetic acid (5-HIAA), and homovanillic acid (HVA) in cerebrospinal fluid (CSF) of humans and nonhuman primates is described. Quantitation is based on the use of an internal standard, 5-fluoro-HVA. Sample preparation consists of mixing an aliquot of CSF with a solution of the internal standard followed by ultrafiltration. The precision of the method is high, with within-run and between-run coefficients of variation of 2-6% and less than 10%, respectively, in the concentration ranges of the metabolites encountered in human lumbar CSF. Accuracy was tested by comparing the present HPLC method with specific gas chromatographic-mass spectrometric (GS-MS) assays for MHPG and HVA and a GC-MS-validated HPLC assay for 5-HIAA: the correlations obtained were 0.968 for MHPG, 0.989 for 5-HIAA, and 0.999 for HVA, with no systematic bias between the methods. The use of ascorbate as a preserving agent for monoamine metabolites in CSF was not found to be necessary when proper care was exercised in sample handling and storage. The analysis of samples with up to 2% ascorbic acid was possible as well, but MHPG had to be assayed separately using an extraction procedure and an alternative internal standard, 3-ethoxy-4-hydroxyphenylglycol.     

Determination of dopexamine hydrochloride in human blood by high-performance liquid chromatography with electrochemical detection

A method is described for the determination of dopexamine hydrochloride at concentrations of 5 to 1000 ng/ml in human blood using electrochemical detection. The method uses a Hypersil ODS column and a mobile phase containing heptane sulphonate, orthophosphoric acid, diisopropylamine and disodium EDTA. Blood samples are stabilised immediately after collection by the use of dipotassium EDTA and a high concentration of sodium metabisulphite. The sample preparation procedure consists of a simple de-proteinisation with perchloric acid. The method is accurate, with inter-assay accuracies ranging from 100 to 104%, and is free of interference by blood from different individuals. Known and potential metabolites of dopexamine hydrochloride and a wide range of drugs do not interfere with the method. The method is precise with inter-assay coefficients on variation of 10.6% at 5 ng/ml and of less than 4.2% at higher concentrations. Stabilised blood samples may be stored for over six months at −25°C prior to analysis.     

Determination of acetylsalicylic acid and salicylic acid in skin and plasma by high-performance liquid chromatography

This study describes a HPLC method to determine the concentrations of acetylsalicylic acid (ASA) and salicylic acid (SA) in human stratum corneum and in plasma. The stratum corneum layers for ASA/SA analysis were removed from three patients with postherpetic hyperalgesia treated with topical and oral aspirin. Blood samples were also collected from the same patients. Tape strippings were placed in acetonitrile and sonicated for 15 min. After centrifuging, aliquots of the supernatant were injected into the chromatograph. ASA and SA from plasma samples were extracted on Isolute C₈ columns. Due to interfering peaks in the tape samples, HPLC conditions were slightly different for tape and plasma samples. ASA and SA were separated on a LiChrospher 100 RP-18 column at 1 ml/min using a water–phosphate buffer (pH 2.5)–acetonitrile mobile phase (35:40:25, v/v/v). A linear response to quantities of ASA from 0.1 to 100 μg/cm² and of SA from 0.1 to 5 μg/cm² in tape and to quantities of ASA 0.1 to 2 μg/ml and 1 to 50 μg/ml was obtained and the recovery from tape and plasma samples was over 98%. The method is sensitive (0.1 μg/cm²) and specific enough to allow the determination of the drugs in the skin not only after topical but also after oral administration. A good sensitivity was also obtained in plasma (0.1 μg/ml) allowing study of the kinetics of ASA and SA in plasma after oral administration. Concentrations of ASA after topical administration were 100–200 times higher than after oral administration. Plasma levels of ASA and SA after oral administration were similar to those previously found. No ASA or SA were detected in plasma after topical ASA administration.     

Determination of formaldehyde in blood plasma by high-performance liquid chromatography with fluorescence detection

An HPLC method was developed for the determination of formaldehyde in human blood plasma. The method was based on the determination of the fluorescent product of the chemical reaction between formaldehyde and ampicillin. A 0.2-ml aliquot of blood plasma was reacted directly with ampicillin under acidic and heating conditions. The reaction product was extracted from the matrix with diethyl ether and analyzed by reversed-phase HPLC with fluorescence detection. Recoveries of spiked formaldehyde at the low ppm (μg/ml) level were between 93% and 102% with relative standard deviations less than 8%. The limits of detection and quantitation of formaldehyde in blood plasma samples were 0.46 μg/ml and 0.87 μg/ml, respectively.     

Analysis of perillic acid in plasma by reversed-phase high-performance liquid chromatography with ultraviolet detection

A simple and sensitive isocratic high-performance liquid chromatographic (HPLC) method with UV detection for the quantitation of perillic acid, a major circulating metabolite of perillyl alcohol and d-limonene, in plasma is described. Sample preparation involved protein precipitation and subsequent transfer and dilution with 10 mM NaHCO₃. The mobile phase consisted of acetonitrile (36%) and 0.05 M ammonium acetate buffer pH 5.0 (64%). Separations were achieved on a C₁₈ column and the effluent monitored for UV absorption at the analytes' respective UV_max. Separation was excellent with no interference from endogenous plasma constituents. This method was found suitable for quantifying drug concentrations in the range of 0.25 to 200.0 μg/ml using a 0.05-ml plasma sample, and was used to study the plasma pharmacokinetics of perillic acid in mice.     

Determination of morphine in cerebrospinal fluid and plasma by high-performance liquid chromatography with electrochemical detection

Two methods for the extraction of morphine from cerebrospinal fluid or plasma with quantitation by high-performance liquid chromatography with electrochemical detection were compared for accuracy, precision and ease of preparation. One procedure was a standard extraction procedure and the other utilized a commercially available liquid—liquid extraction column. Both methods produced linear calibration curves over the concentration range of 1–200 ng/ml with coefficients of correlation of 0.999. Since the electrochemical detector is capable of detecting 20 pg of morphine, biological samples as small as 0.1 to 0.4 ml can be quantified with an average relative precision of 4.1 ± 3.9% over the concentration range 1–200 ng/ml. The potential clinical importance of the assay is demonstrated using a time course distribution study of morphine in the cerebrospinal fluid and plasma of a Rhesus monkey.     

Purification and characterization of 5-aminolevulinic acid dehydratase from Methanosarcina barken

Abstract 5-Aminolevulinic acid dehydratase from the archaebacterium Methanosarcina barken resembles the mammalian and yeast enzymes in its activation by Zn²⁺, whereas its activation by K⁺ resembles the characteristic of bacterial enzymes. This enzyme is activated with Ni²⁺ which is a component of F₄₃₀, a cofactor present mainly in methanogens. The M _r of 280000 for the native enzyme and 30 000 ± 2000 for the individual subunit suggest that the enzyme is composed of eight apparently indentical subunits similar to mammalian and yeast enzymes. The enzyme has two pH optima, at 8.5 and 9.4. Higher levels of 5-aminolevulinic acid dehydratase in acetate-grown cells suggest the possibility that regulation and control of this enzyme could be different on various growth substrates.