Determination of niacin metabolites 1-methyl-5-carboxylamide-2-pyridone and n-1-methylnicotinamide in urine by high-performance liquid chromatography

A test suitable for detecting latent niacin deficiency was developed. It measures the 24-h urinary output of the two major metabolites of niacin, 1-methyl-5-carboxylamide-2-pyridone and N-1-methylnicotinamide. The two metabolites were isolated from urine using separate ion-exchange extractions. They and their two internal standards were quantitated simultaneously by high-performance liquid chromatography using a reversed-phase ion pairing separation. Detection was by absorbance at 254 nm.     

Simultaneous measurement of nicotinamide and its catabolites,nicotinamide N-oxide,N1-methyl-2-pyridone-5-carboxamide,and N1-methyl-4-pyridone-3-carboxamide,in mice urine

Nicotinamide N-oxide is a major nicotinamide catabolite in mice but not in humans and rats. A high-performance liquid chromatographic method for the simultaneous measurement of nicotinamide, nicotinamide N-oxide, N¹-methyl-2-pyridone-5-carboxamide, and N¹-methyl-4-pyridone-3-carboxamide in mice urine was developed by modifying the mobile phase of a reported method for measurement of nicotinamide N-oxide.     

Effects of 1-methylnicotinamide and its metabolite N-methyl-2-pyridone-5-carboxamide on streptozotocin-induced toxicity in murine insulinoma MIN6 cell line

1-methylnicotinamide (MNA) is a primary metabolite of nicotinamide. In recent years several activities of MNA have been described, such as anti-inflammatory activity in skin diseases, induction of prostacyclin synthesis via COX-2, aortal endothelium protection in diabetes and hypertriglyceridaemia and increased survival rate of diabetic rats. 1-methylnicotinamide was also suggested to protect pancreatic cells from streptozotocin in vivo. Streptozotocin toxicity is known to be mediated by poly-ADP-ribose polymerase. Nicotinamide and its derivatives have been shown to ameliorate poly-ADP-ribose polymerase-dependent nucleotide pool reduction. We aimed to verify if 1-methylnicotinamide and its metabolite, N-methyl-2-pyridone-5-carboxamide, can protect insulinoma cells from streptozotocin-induced toxicity. We found that N-methyl-2-pyridone-5-carboxamide, but not 1-methylnicotinamide, restores the pool of ATP and NAD+ in streptozotocin-treated cells, but neither compound improved the cell viability. We conclude that inhibition of poly-ADP-ribose polymerase-dependent nucleotide pool reduction may not be sufficient to protect cells from streptozotocin toxicity.     

High-performance liquid chromatographic method with UV photodiode-array, fluorescence and mass spectrometric detection for simultaneous determination of galantamine and its phase I metabolites in biological samples

Galantamine, an alkaloid isolated from the bulbs and flowers of Caucasian snowdrop (Galanthus woronowii, Amaryllidaceae) and related species, is employed in human medicine for the treatment of various neuromuscular and neurodegenerative diseases. After the administration, the products of oxidative biotransformation (O-desmethyl-galantamine, N-desmethyl-galantamine, galantamine-N-oxide) and chiral conversion (epigalantamine) are formed in various concentrations from parent compound. For the identification and determination of galantamine and its phase I metabolites in blood plasma and tissues, a new bioanalytical method based on a reversed-phase high-performance liquid chromatography with UV photodiode-array, fluorescence and mass spectrometric detection was developed, validated and applied to pharmacokinetic and biotransformation studies. Sample preparation included a homogenization of the rat tissues (liver, brain, hypophysis) in a phosphate buffer 0.05 mol/L pH 7.4. Plasma samples and tissue homogenates were purified using a mixed-mode solid-phase extraction (Waters Oasis MCX cartridges). Galantamine, its above-mentioned metabolites and the internal standard codeine were separated on a Discovery HS F5 column (Supelco, 150 mmx4.6 mm I.D., 5 microm) at flow rate of 1 mL/min using a linear gradient elution. UV photodiode-array and mass spectrometric detection were employed for the identification of individual galantamine metabolites in various biomatrices, the fluorescence detection (lambdaexcit=280 nm/lambdaemiss=310 nm) was chosen for the quantification of galantamine and its metabolites. The developed method was applicable in liver tissue in the range from 0.50 to 63.47 nmol/g of galantamine, from 0.32 to 41.42 nmol/g of O-desmethyl-galantamine, from 0.54 to 69.40 nmol/g of N-desmethyl-galantamine and from 0.70 to 89.03 nmol/g of epigalantamine. Limit of detection was found to be 0.04 nmol/g for galantamine, 0.19 nmol/g for O-desmethyl-galantamine, and 0.07 nmol/g for N-desmethyl-galantamine and epigalantamine.     

Simultaneous immunochemical detection of stanozolol and the main human metabolite, 3'-hydroxy-stanozolol, in urine and serum samples

Two enzyme-linked immunosorbent assays (ELISAs) have been established for the analysis of stanozolol (St) and 3′-hydroxy-stanozolol (3′OH-St), the main metabolite found in humans. The immunizing hapten N2′-(5-valeric acid)-androst-2-eno[3,2-c]-pyrazol-17a-methyl-17b-ol (hapten 8) has been designed with the aid of molecular modeling and theoretical tools to allow immunochemical detection of both compounds. Using an ELISA based on a homologous antisera/coating antigen combination, St can be selectively quantified without significant interference of the St metabolites or other steroids potentially present in the biological samples. On the other hand, St immunoreactivity equivalents due to the additional presence of 3′OH-St can also be quantified using an ELISA based on a heterologous antisera/coating antigen combination, in which the metabolite can be detected with 51% cross-reactivity. Thus, As147/5BSA detects 3′OH-St and St in buffer with IC₅₀ values of 1.46 and 0.68 μg L⁻¹, respectively. In contrast, As147/8BSA is highly specific for St with an IC₅₀ of 0.16 μg L⁻¹ and a limit of dection of just 0.022 μg L⁻¹. Performance of both assays in urine and serum samples has been evaluated and demonstrate that inappropriate use of stanozolol by athletes or young people can be detected in these matrices after simple cleanup methods, with IC₅₀ values below the minimum performance required levels established by the World Antidoping Agency.     

Simultaneous analysis of insect repellent DEET, sunscreen oxybenzone and five relevant metabolites by reversed-phase HPLC with UV detection: application to an in vivo study in a piglet model

N,N-Diethyl-m-toluamide (DEET) and oxybenzone are two essential active ingredients in insect repellent and sunscreen preparations. We developed and validated a simple, sensitive, and selective HPLC assay to simultaneously measure DEET, oxybenzone and five primary metabolites of DEET and oxybenzone in biological samples including plasma, urine and skin strips. The compounds were separated on a reversed-phase C18 column using three-stage gradient steps with methanol and water. DEET and two relevant metabolites were detected at 254 nm, while oxybenzone and three relevant metabolites were detected at 289 nm. The limit of detection was 0.6 ng for DEET and 0.5 ng for oxybenzone, respectively. The developed method was further applied to analyze various biological samples from an in vivo animal study that evaluated concurrent use of commercially available insect repellent and sunscreen preparations.     

Novel tryptamine-related substances, 5-sulphatoxydiacetyltryptamine, 5-hydroxydiacetyltryptamine,and reduced melatonin in human urine and the determination of those compounds, 6-sulphatoxymelatonin,and melatonin with fluorometric HPLC

During our studies to establish a method for identifying tryptamine-related substances in human urine, we detected three large peaks of unknown origin in an HPLC chromatogram. Fluorometric HPLC and HPLC-TOF-MS/MS analyses led to the identification of these substances as 6-sulphatoxymelatonin, 5-sulphatoxydiacetyltryptamine, and reduced melatonin. This is the first report of the latter two compounds in human urine. Here, we report the results of two fluorometric HPLC assays of these three substances, as well as melatonin, 6-hydroxymelatonin, and 5-hydroxydiacetyltryptamine, using synthesized standards and discuss the possibility that 5-hydroxydiacetyltryptamine (the parent substance of 5-sulphatoxydiacetyltryptamine) and reduced melatonin have radical scavenging activity.     

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.     

High-performance liquid chromatographic assay with ultraviolet detection for the determination of etoperidone and two active metabolites, 5-(1-hydroxyethyl)etoperidone and 1-(3-chlorophenyl)piperazine, in plasma

A selective and sensitive high-performance liquid chromatographic assay with ultraviolet detection for the determination of the antidepressant drug etoperidone and two active metabolites in plasma is described. The drug, metabolites and internal standard are isolated from plasma using a two-step liquid—liquid extraction procedure. The resulting sample is chromatographed on a C₁₈ column (10 cm × 2.1 mm I.D.) with ultraviolet detection at 254 nm. Standard curves are linear for each compound over the concentration range 2–1000 ng/ml. The accuracy and precision of the assay, expressed as the percentage deviation of measured values from the true value and the relative standard deviation (inter-run), are ≤ 10% at all concentrations except the minimum quantification limit. Using an automated injector and computerized data acquisition, eighty samples can be routinely processed in one day. The assay has been successfully used for the analysis of plasma samples from pharmacokinetic studies in mice, rats, dogs and humans.     

Comparison of high-performance liquid chromatography with electrochemical detection and gas chromatography—mass fragmentography for the assay of salsolinol, dopamine and dopamine metabolites in food and beverage samples

High-performance liquid chromatography with electrochemical detection (HPLC—ED) and combined gas chromatograph—mass spectrometry in the single-ion monitoring mode (GC—MS-SIM) have been used for the determination of salsolinol, dopamine, 3,4-dihydroxyphenylacetic acid, 3,4-dihydorxyphenylethanol and norepinephrine in a selection of food and beverage samples. The unique specificity of the SIM mode allows a simple one-step extraction to be used even for complex sample matrices. We have been able to demonstrate the quantitative and qualitative advantages offered by GC—MS over HPLC—ED by direct comparison of the chromatographic data obtained. We demostrate that the specificity of SIM and the benefits offered by the incorporation of deuterated internal standards make GC—MS-SIM the method of choice for valid identification and precise quantitation of salsolinol, dopamine and dopamine metabolites in a complex sample matrix.     

High-performance liquid chromatographic determination of 3′-hydroxy-5′-(4-isobutyl-1-piperazinyl)benzoxazinorifamycin (KRM-1648) and its deacetyl metabolite in plasma, whole blood, urine and tissue samples in rats

A reversed-phase high-performance liquid chromatographic method was developed for the determination of 3′-hydroxy-5′-(4-isobutyl-1-piperazinyl)benzoxazinorifamycin (KRM-1648, I), a new rifamycin derivative, and its 25-deacetyl metabolite (KRM-1671, II) in plasma, whole blood, tissues and urine from rats. I and II were coextracted with an internal standard from each sample matrix by solid-phase extraction (Bond Elut). Plasma and urine were directly loaded onto Bond Elut, while whole blood and tissues were homogenized and extracted with methanol or dichloromethane—chloroform prior to Bond Elut extraction. The extracts were chromatographed on Shim-pack CLC-ODS(M) using acetonitrile—0.02 M citrate buffer containing 0.1 M sodium perchlorate (2:1, v/v), and peaks were detected at 643 nm. The validation data showed that the assays for I and II in plasma, whole blood, tissues and urine were selective, accurate and reproducible.     

Synthesis of (1R,2S)-1-(3'-chloro-4' -methoxyphenyl)-1,2-propanediol (trametol) and (1R,2S)-1-(3',5'-dichloro-4'-methoxyphenyl)-1,2-propanediol,chlorinated fungal metabolites in the natural environment

(1R,2S)-1-(3'-Chloro-4'-methoxyphenyl)-1,2propanediol (Trametol, 3), a metabolite of the fungus Trametes sp. IVP-F640 and Bjerkandera sp. BOS55, was synthesized by employing Sharpless asymmetric dihydroxylation as the key step. Similarly, the (1R,2S)-isomer of 1-(3',5'-dichloro-4'-methoxyphenyl)-1,2-propanediol (4), another metabolite of Bjerkandera sp. BOS55, was synthesized by asymmetric dihydroxylation.     

Clearer demonstration of calcium/calmodulin-dependent events in synaptosomes by use of the differential effects of two calmodulin antagonists, N-(aminohexyl)-5-chloro-1-naphthalenesulfonamide and N-(6-aminohexyl)-1-naphthalenesulfonamide

1. In order to demonstrate more clearly calcium/calmodulin-dependent events, the differential effects of two calmodulin antagonists, W-7 and W-5, on synapsin I phosphorylation and norepinephrine release associated with calcium influx, were investigated using 32Pi in synaptosomes derived from rat cerebral cortex. 2. The calcium ionophore (A23187)-stimulatory effect on synapsin I phosphorylation and norepinephrine release was markedly reduced by W-7 and slightly reduced by W-5; whereas neither the strong nor the weak calmodulin antagonist had an effect on A23187-stimulated synaptosomal uptake of calcium. 3. Preincubation with H-8 reduced both W-5- and W-7-inhibited A23187-stimulated synapsin I phosphorylation by the same amount but did not affect their inhibitory effect nor the ionophore-stimulated norepinephrine release, thereby suggesting that W-5 may serve as an appropriate control for non-calmodulin-mediated effect of both calmodulin antagonists.