Enzymatic Determination of Urinary Total 7α-Hydroxy Bile Acids

Abstract

An enzymatic determination of urinary 7^α-hydroxy bile acids is described. The principle of the method is as follows: after hydrolysis with β-glucuronidase and solvolysis, the ethyl acetate extract is washed with alkalin solution and water, then the alkali and water washes of the ethyl acetate extract are combined and the solution is acidified to pH 1 and sodium chloride added. Shake solution with ethyl acetate to re-extract the acidic fraction and the ethyl acetate layer is evaporated. Add enzyme color solution of 7_α-hydroxysteroid dehydrogenase (7_α-HSD, from E. coli) to a tube of extract residue and then after incubation, measure the absorbance of the solution.

Excretion values of urinary total 7_α-hydroxy bile acids was measured with patients of acute hepatitis and normal subjects and excretion pattern of 7_α-hydroxy bile acids by the use of chromatographic fractionation was also shown.     

Extraction Rapide de la Cafeine Urinaire Sur Cartouche C18 Et Dosage Par Chromatographie Gazeuse

Abstract

A rapid method for urinary caffeine extraction on C₁₈ cartridge before GC determination is proposed in view of doping controls in sport. The choice of the standard and of the analytical procedure has been directed by the necessity of rapid determination.

Caffeine extraction on C₁₈ reverse-phase is achieved with a good efficiency (recovery : 98 %, CV = 3 %), so that use of an internal standard was not found necessary. Amobarbital was chosen as internal standard in GC because of several advantages : retention time, thermal stability, interesting properties in mass fragmentometry.     

Synthesis of 3,4-Substituted Phenylmethylene-(2-carboxyphenyl)amines and Their Antitumor Activity

Previously unknown azomethines 3a-z and 4a-j were prepared in methanol by condensation of anthranilic acid 1 with vanillin, vanillal, and esters of these compounds 2.__________Translated from Khimiya Prirodnykh Soedinenii, No. 2, pp. 164–169, March–April, 2005.     

Improved High Performance Liquid Chromatographic Method for the Quantification of 3-Methylhistidine in Serum and Urine

Abstract

We optimized an high-performance liquid chromatographic (HPLC) method for the determination of 3-methylhistidine (3-MeHis) in biological fluids. After pre-column derivatization with OPA, analytical separation was achieved on a reversed-phase C18 column by a simple gradient between sodium propionate buffer and acetonitrile. the method is accurate, reproducible and sensitive, and allows the determination of urinary 3-MeHis levels in about 55 min. Other additional 16 amino acids may be easily quantified while the 3-MeHis peak is well resolved from an unknown urinary compound potentially interfering.     

Electrode Hybride Pour la Determination de Phosphates et de Polyphosphates. Application aux Problemes Lies a l'Environnement

Abstract

The determination of phosphates and polyphosphates has been effected using an hybrid electrode, which consists of a glucose oxydase enzymic membrane and a Solanum tuberosum tissue slice. This membrane, rich in acid phosphatase, catalyses the glucose-6-phosphate hydrolysis. This reaction is quantitatively inhibited by phosphate. Several factors involved in the electrode response, like substrate concentration, pH, ionic strength and type of buffer are discussed in detail. At a 4.10^?4 M glucose-6-phosphate concentration, the linear ranges of phosphates and polyphosphates are, respectively, 6.10^?5 M to 1.6.10^?3 M and 3.10^?5 M to 1.10^?3 M. The urinary phosphate contents determinated by this biosensor are in good agrement with those obtained by usual spectrophotometric techniques.     

Quantitative Determination of Iron and Aluminium in Some Alloys and Silicate Rocks After a Cation Exchange Separation on Zirconium(IV) Phospho and Silico Arsenates

Abstract

A rapid and quantitative method has been developed for the analysis of some iron and aluminium based alloys and silicate rocks using zirconium(IV) based arsenophosphate and arsenosilicate cation exchangers. The method is simple, reproducible and precise with a standard deviation <3%, for the direct determination of iron and aluminium in rocks and alloys. The low standard deviation values suggest that the method should be useful for the standardization purposes.     

Application de la Polarographie Sinusoidale au Dosage Sanguin de L'indomethacine

Abstract

Alternative current polarography has been applied to indomethacin determination in serum, after dichloromethan extraction at pH 3.0. Solvent is evaporated and the residue submited to hydrolysis by NaOH. The indole derivative obtained is nitrosated by nitrous acid at pH 3,5. The indole-nitrosamine formed is measured by polarography at pH 1,3 and compared with an added quantity of the pure nitrosated product. The proposed method is selective and sensitive; it allows measurement of concentrations in serum as low as o,30 μg ml^?1 (0.83. 10^?9 moles ml^?1).     

HPLC Separation of Tautomeric Compounds of 4-Aminoisoxazolyl- 1,2-Naphthoquinone. III

Abstract

This paper reports the development of a simple, precise and specific HPLC procedure for the determination of two new methylated isoxazolylnaphthoquinones with an amine group in the 4-position of the isoxazole ring, which are examples of exocyclic tautomers.

The analytical procedure involved the use of the internal standardization method which was applied for the qualitative and quantitative determination of both compounds using a C-18 Lichrosorb column in an isocratic mode and sulfadiazine as internal standard.     

Application of an LC–MS/MS Method for the Simultaneous Quantification of Homovanillic Acid and Vanillylmandelic Acid for the Diagnosis and Follow-Up of Neuroblastoma in 357 Patients

Homovanillic acid (HVA) and vanillylmandelic acid (VMA) are end-stage metabolites of catecholamine and are clinical biomarkers for the diagnosis of neuroblastoma. For the first time in Korea, we implemented and validated a liquid chromatography tandem mass spectrometry (LC–MS/MS) assay to measure urinary concentrations of HVA and VMA according to Clinical and Laboratory Standards Institute guidelines. Our LC–MS/MS assay with minimal sample preparation was validated for linearity, lower limit of detection (LOD), lower limit of quantification (LLOQ), precision, accuracy, extraction recovery, carryover, matrix effect, and method comparison. A total of 1209 measurements was performed to measure HVA and VMA in spot urine between October 2019 and September 2020. The relationship between the two urinary markers, HVA and VMA, was analyzed and exhibited high agreement (89.1% agreement, kappa’s k = 0.6) and a strong correlation (Pearson’s r = 0.73). To our knowledge, this is the first study to utilize LC–MS/MS for simultaneous quantitation of spot urinary HVA and VMA and analyze the clinical application of both markers on a large scale for neuroblastoma patients.     

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.     

Spectrofluorimetric determination of hydrogen peroxide scavenging activity

Homovanillic acid (HVA) is widely used for the detection and imaging of oxidative enzymes—peroxidase, glucose oxidase and xanthine oxidase, but antioxidant activity has not been determined so far with the use of HVA. We have developed a simple, sensitive and in-field spectrofluorimetric method for the determination of hydrogen peroxide (H₂O₂) scavenging activity. The assay is based on the oxidation of HVA to its fluorescent biphenyl dimer in the presence of H₂O₂ and peroxidase. The presence of substances with H₂O₂ scavenging activity prevents the oxidation of HVA by removing H₂O₂. The decrease in fluorescence intensity is proportional to the antioxidative (H₂O₂ scavenging) activity. The method was evaluated using Trolox (6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid), BHA (3-t-butyl-4-hydroxyanisole) and ferulic, vanillic, caffeic, chlorogenic, protocatechuic and oxalic acids. Additionally, tea and herb infusions known for their antioxidant properties were evaluated.     

Capillary Electrophoresis as A Diagnostic Tool: Determination of Biological Constituents Present in Urine of Normal and Pathological Individuals

Abstract

A quantitative ultraviolet detection method for the determination of urinary metabolites is described using capillary zone electrophoresis. The determination of these metabolites is simple, fast. reproducible and utilizes very small amounts of sample. This method is linear between 1.0 × 10^?4 and 1.0 × 10^?2 M for creatinine and between 1.0 × 10^?1 and 1.0 M for urea. The ultraviolet method shows detection limits for creatinine in the picogram (femtomol) range, and for urea in the nanogram (picomol) range.     

Ethanol as a Solvent in the Determination of Arsenic in Marine Extracts by Atomic Absorption Spectrophotometry

Abstract

Arsenic is determined in aqueous solutions produced from marine raw materials by atomic absorption spectrophotometry. Conversion of the aqueous extract to an ethanol-extract increases the sensitivity of determination by a factor of 4 to 5. The method is applied to some selected samples produced from marine raw materials.     

Ultraviolet Spectrophotometric Determination of Selenium by 1-Pyrrolidinecarbodithioate Method

Abstract

An ultraviolet spectrophotometric method for the determination of selenium has been developed using ammonium 1-pyrrolidinecarbo-dithioate. The selenium(IV)-1-pyrrolidinecarbodithioate complex is extracted with chloroform and the absorbance of the extract measured at 303 mμ. Conformity to Beer's law was found for 0.5 to 9 ppm of selenium.     

Enzymatic Determination of Urinary Total 17β-Hydroxysteroids

Abstract

An enzymatic determination of urinary total 17β-hydroxysteroids is described. The principle of the method is as follows: after hydrolysis with β-glucuronidase and solvolysis, the ethyl acetate layer, which is washed with alkaline solution and water, is transferred to two test tubes, then each of them is evaporated. To one test tube is added 3β,17β-hydroxysteroid dehydrogenase (EC 1.1.1.51, from P. testosteroni), NAD⁺, 2-ρ-iodophenyl-3-ρ-nitrophenyl-5-phenyltetrazolium chloride (INT), and diaphorase (EC 1.6.99.2, from C. kluyveri) for determination of total 3β,17β-hydroxysteroids. To another test tube is added 3β-hydroxysteroid oxidase (EC 1.1.3.6, from B. sterolicum) for determination of total 3β-hydroxysteroids. The value of 17β-hydroxysteroids is calculated by subtracting the value for 3β-hydroxysteroids from the value for the 3β,17β-hydroxysteroids.     

Synthesis of <Emphasis Type="Italic">m</Emphasis>-carborane-C-carboxylic acid hexahydrobenzo[<Emphasis Type="Italic">a</Emphasis>]acridine esters

The possibility was demonstrated of selective preparation of m-carborane-C-carboxylic acid hexahydrobenzo[a]acridine esters both by the reaction of 1,3-diketones with azomethines obtained by condensation of m-carborane-C-carboxylic acid vanillin and vanillal esters with 1- or 2-naphthylamine and by the cascade heterocyclization of 1- or 2-naphthylamine, m-carborane-C-carboxylic acid vanillin and vanillal esters, and CH-acids.     

A Novel Protein Assay Method Using Tetraphenylporphin tetrasulfonate (TPPS4)

Abstract

A sensitive protein assay method which involves the reaction of TPPS₄ with protein is described. When protein is added to TPPS₄ solution, an absorption band with the maximum at 488 nm appears and the absorbance is proportional to the concentration of protein. Just Like the Soret absorption of the porphyrin, the new band is very narrow and there is no overlap at all between them, which means the free dyes would not give any background for the detection of the protein-TPPS₄ complexes. A new spectrophotometric method for determination of protein has been constructed and applied to the determination of human plasma protein and urinary protein; The assay using microtiter plates has also been studied.     

Spectrophotometric Determination of Chlorpromazine and Levomepromazine Hydrochlorides In Injectables and Drops By Reaction With Ferric Ion

Abstract

The principal aim of this research was the standardization of a spectrophotometric method using the reaction with ferric ion for quantitative determination of these substances in pharmaceutical preparations. By reaction with ferric ion chlorpromazine gave a pink compound with maximum absorption at 525 nm. the violet product obtained by the reaction with ferric ion and levomepromazine had a maximum at 565 nm. Beer's law was obeyed in a wide range of concentrations for both compounds. the method was applied to simulated and commercially available samples. the efficiency of the method was confirmed by recovery tests.     

Simultaneous Determination of Estrogen and Progesterone Receptors Using Human Uterus as a Standard

Abstract

Numerous reports have shown a high degree of correlation between the presence of estrogen and progesterone receptors in cancerous breast tumors and response to endocrine therapy. There are several methods in use for measurement of these receptors, but all are time consuming and require large amounts of tumor tissue. Our laboratory needed a faster and simpler method to determine receptor concentration. We describe here an assay which utilizes human uterus extract with known receptor concentration as a standard to determine receptor concentration of tumor tissues. This method is simple, fast and will allow determination of estrogen and progesterone receptor in up to ten patient samples at one time.     

HPLC Analysis of Methoxamine in Rabbit Plasma and Pharmaceutical Formulations

Abstract

A rapid, selective and simple high performance liquid chromatographic (HPLC) assay for methoxamine HC1 has been developed. The analytical procedure involved the use of internal standardization method (1-norephedrine). It has been applied for the qualitative and quantitative determination of methoxamine HC1 in rabbit plasma and in pharmaceutical formulations using an adsorption column in an isocratic mode, with resulting relative standard deviations of 1.7% and 3.3%, respectively. The applicability of the assay procedure to pharmacokinetic studies was demonstrated. Detection limits were as low as 15ng for a 30 ul injection and the determination time was less than 6 minutes.