Improved method for the rapid determination of isosorbide dinitrate in human plasma and its application in pharmacokinetic studies

A rapid, accurate and highly sensitive method was developed for the determination of isosorbide dinitrate in human plasma. Concentrations in the lower nanogram and subnanogram range are determined by a one-step extraction of 2 ml plasma, containing 4 ng/ml nitroglycerine as internal standard, with 5.5 ml n-pentane. The extract is subjected to gas—liquid chromatography—electron capture detection analysis. The lower limit of quantitation is 200 pg/ml, but concentrations as low as 50 pg/ml are still detectable. The method allows the quantitative determination of isosorbide dinitrate plasma levels in man following a 5 mg sublingual administration up to four hours after application.     

Determination of 3-hydroxy-guanfacine in biological fluids by electron-capture gas—liquid chromatography

An electron-capture gas—liquid chromatographic method was developed for measuring 3-hydroxy-guanfacine, the main metabolite of guanfacine in human plasma and urine. After extraction, the metabolite was derivatized by condensing the amidino group with hexafluoroacetylacetone and by methylating the NH and OH groups with methyl iodide. The obtained derivative possessed good bioanalytical gas chromatographic properties, using a capillary column. The O-glucuronide was measured after enzymatic hydrolysis. Unchanged guanfacine could be determined in urine together with its 3-hydroxy metabolite by this method.     

The mutagenicity testing of tertiary-butyl alcohol, tertiary-butyl acetate™ and methyl tertiary-butyl ether in Salmonella typhimurium

Tertiary-Butyl alcohol (TBA), tertiary-butyl acetate™ (TBAc™) and methyl tertiary-butyl ether (MTBE) are chemicals to which the general public may be exposed either directly or as a result of their metabolism. There is little evidence that they are genotoxic; however, an earlier publication reported that significant results were obtained in Salmonella typhimurium TA102 mutagenicity tests with both TBA and MTBE. We now present results of testing these chemicals and TBAc™ against S. typhimurium strains in two laboratories. The emphasis was placed on testing with S. typhimurium TA102 and the use of both dimethyl sulphoxide and water as vehicles. Dose levels up to 5000 μg/plate were used and incubations were conducted in both the presence and absence of liver S9 prepared from male rats treated with either Arochlor 1254 or phenobarbital-β-naphthoflavone. The experiments were replicated, but in none of them was a significant mutagenic response observed, thus the current evidence indicates the TBA, TBAc™ and MTBE are not mutagenic in bacteria.     

Practical gas—liquid chromatographic method for the determination of amino acids in human serum

Application of the gas—liquid chromatographic method previously reported by us was made to the analysis of the 22 amino acids including asparagine and glutamine in serum. The method permitted that aqueous serum samples obtained after deproteinization with perchloric acid were directly subjected to derivatization without any further clean-up procedure such as ion-exchange chromatography. The N-ethyloxycarbonyl methyl esters, which were prepared in the same manner as the N-isobutyloxycarbonyl methyl esters, were introduced for the determination of leucine, isoleucine, arginine and tyrosine. Both derivatives were prepared by two-step procedures involving alkyloxycarbonylation in aqueous media and esterification with diazomethane, and simultaneously analyzed by using the dual set of columns with the same thermal conditions. The advantages of this method are that the sample pretreatment and derivatization are very simple and rapid, and that both asparagine and glutamine along with other amino acids in serum can be determined.     

Capillary gas chromatographic analysis of serum bile acids as the n-butyl ester–trimethylsilyl ether derivatives

Gas chromatographic separations of n-butyl ester-trimethylsilyl ether derivatives of several common bile acids were compared with those of the corresponding methyl ester-trimethylsilyl ether derivatives on a CP-Sil-5 CB capillary column. Both types of derivatives were similarly resolved from each other. However, the n-butyl ester-trimethylsilyl ether derivatives of the bile acids showed longer retention times than the corresponding methyl ester-trimethylsilyl ethers and unlike the methyl ester-trimethylsilyl ether derivatives, were completely resolved from and eluted later than the trimethylsilyl ethers of common plasma sterols including sitosterol. A simplified method of plasma work-up for quantitation of bile acids and application of the above method in quantification of plasma bile acids in humans is described.     

Degradation of long-chain n-alkanes by the yeast Candida maltosa

Summary The yeast Candida maltosa precultivated on liquid n-alkanes utilized different solid n-alkanes (especially C₂₀–C₂₅) in the presence of pristane as an organic phase with rates comparable to, or somewhat larger than, those of liquid n-alkanes. Analysis of cellular fatty acids indicated an assimilation of solid n-alkanes via monoterminal oxidation. The resulting fatty acids with substrate chain length were chain-shortened by C₂ units down to an optimal range of chain length from C₁₆ to C₁₈ and incorporated into cellular, lipids directly or after desaturation. The intermediates of chain-shortening with numbers of carbon atoms higher than C₁₈, as well as the unusually long-chain fatty acids of substrate chain length, were detected in trace amounts only. Even-carbon-numbered and odd-numbered fatty acids predominated in experiments with evenchain and odd-chain n-alkanes, respectively. Studies with cerulenin indicated that de novo synthesis of fatty acids was negligible. Oxidation of solid n-alkanes by the yeast C. maltosa yielded fatty acid patterns similar to those of cells grown on liquid n-alkanes.     

Effect of organic solvents on oxygen mass transfer in multiphase systems: Application to bioreactors in environmental protection

The absorption of oxygen in aqueous–organic solvent emulsions was studied in a laboratory-scale bubble reactor at a constant gas flow rate. The organic and the gas phases were dispersed in the continuous aqueous phase. Volumetric mass transfer coefficients (k_La) of oxygen between air and water were measured experimentally using a dynamic method. It was assumed that the gas phase contacts preferentially the water phase. It was found that addition of silicone oils hinders oxygen mass transfer compared to air–water systems whereas the addition of decane, hexadecane and perfluorocarbon PFC40 has no significant influence. By and large, the results show that, for experimental conditions (organic liquid hold-up ≤10% and solubility ratio ≤10), the k_La values of oxygen determined in binary air–water systems can be used for multiphase (gas–liquid–liquid) reactor design with applications in environmental protection (water and air treatment processes).     

Determination of the aza alkyl lysophospholipid 3-methoxy-2-N,N-methyloctadecylaminopropyloxyphosphorylcholine in rat plasma by liquid chromatography—particle beam—mass spectrometry

A sensitive and specific method for the determination of the aza alkyl lysophospholipid (AALP) 3-methoxy-2-N,N-methyloctadecylaminopropyloxyphosphorylcholine (I) in rat plasma is described. The target molecule was analyzed by high-performance liquid chromatography (HPLC)—mass spectrometry (MS) after one single liquid—liquid extraction with chloroform—methanol (2:1, v/v). 1,2-Didecanoyl-sn-glycero-3-phosphocholine was used as internal standard. HPLC was carried out using a polymeric reversed-phase column; the coupling to the mass spectrometer was a particle beam (PB) interface, and the ionization method was electron impact (EI). This simple and rugged method permits the measurement of I in rat plasma in the range of 25 ng/ml–5 μg/ml with good accuracy and precision and is used in pharmacokinetic studies.     

Determination of exemestane, a new aromatase inhibitor, in plasma by high-performance liquid chromatography with ultraviolet detection

A sensitive and selective high-performance liquid chromatographic method for the determination of 6-methylen-androsta-1,4-diene-3,17-dione (exemestane) and its 17-dihydro metabolite in human plasma has been developed. The analytes and internal standard (Norgestrel) were extracted from plasma samples with a methylene chloride—iso—octane mixture; the organic phase was dried and the residue was reconstituted with an acetonitrile—water mixture, then analyzed by reversed-phase liquid chromatography. Quantification was achieved by ultraviolet detection of the eluate. The linearity, precision and accuracy of the method were evaluated. No interference from the constituents of human blank plasma was observed. The lower limit of quantification was 10 ng/ml plasma. The suitability of the method for in vivo samples was checked by analysis of plasma samples drawn from healthy male volunteers who had received a 200-mg single oral dose of the test compound.     

Presence of intestinal, liver and heart/adipocyte fatty-acid-binding protein types in the liver of a chimaera fish

Five fatty-acid-binding proteins from the liver of the elephant fish (Callorhynchus callorhynchus), a chimaera fish that belongs—together with the elasmobranchs—to the ancient chondrichthyes class were isolated and characterized. The purification procedures for these proteins involved gel filtration, anion-exchange chromatography, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a last step. They were submitted to “in gel” tryptic or cyanogen bromide digestion and the resulting peptides were separated by high performance liquid chromatography and then sequenced by Edman degradation. According to their partial amino acid sequences, one of them presents the highest identity with fatty-acid-binding proteins from human and catfish liver, another three with those from mammalian heart or adipose tissue and the fifth with the mammalian intestinal fatty-acid-binding protein. The presence of various members of this protein family, as now found in elephant fish and previously in catfish (Rhamdia sapo) liver, does not occur in mammalian liver which expresses only one a characteristic fatty-acid-binding protein.     

Determination of a new immunosuppressant, mycophenolate mofetil, and its active metabolite, mycophenolic acid, in rat and human body fluids by high-performance liquid chromatography

Mycophenolate mofetil, a new immunosuppressant, is a morpholinoethyl ester of mycophenolic acid. A new selective, sensitive and simple high-performance liquid chromatographic method was developed for the determination of mycophenolic acid and mycophenolate mofetil in biological samples. The preparation of samples was based on liquid—liquid extraction. The compounds were separated on a CN column using acetonitrile—0.01 M phosphate buffer (1:4, v/v) as the mobile phase. UV detection was used at wavelengths 215 and 304 nm. The detection limit was 5 ng per injection volume. This method enabled pharmacokinetic and pharmacodynamic studies in humans and rats.     

Comparative determination of plasma cholesterol and triacylglycerol levels by automated gas—liquid chromatographic and autoanalyzer methods

Plasma samples obtained during a prevalence study of hyperlipemia in a free living urban population were analyzed for total cholesterol and triacylglycerol content by automated high-temperature gas—liquid chromatographic (GLC) and automated colorimetric (Auto-Analyzer, AA11) methods. The analyses were done over a three-year period. The methods gave excellent overall correlation for both total cholesterol (r = 0.9811) and total triacylglycerols (r = 0.9739). Detailed comparisons of the results obtained by the two methods with natural samples over the entire concentration range, indicated that the GLC method gave cholesterol values 5–10 mg% lower and triacylglycerol values 10–20 mg% lower than the corresponding AA11 determinations. The differences between the two methods are attributed to an overestimation of the cholesterol and triacylglycerol levels by the AA11 method due to presence of variable amounts of interfering chromogens in the plasma extracts. The between-method relative error ranged from 3 to 5% for cholesterol and from 5 to 10% for triacylglycerols. The within-day standard deviation of GLC averaged 2.3 mg% for cholesterol and 3.5 mg% for triacylglycerols. The between-day standard deviation of the GLC method averaged about 6 mg% for both cholesterol and triacylglycerols. The within-day, within GLC, relative error averaged 1.12% for cholesterol and 2.66% for tri-acylglycerols. The apparent high precision and high accuracy of the GLC method recommend it as an alternative to the indirect methods of plasma cholesterol and triacylglycerol analysis, especially where a smaller throughput of samples is not a limitation and where both total amount and composition of the lipids is of interest.     

The D/H ratio and the evolution of water in the terrestrial planets

The presence of liquid water at the surface of the Earth has played a major role in the biological evolution of the Earth. None of the other terrestrial planets — Mercury, Venus and Mars — has liquid water at its surface. However, it has been suggested, since the early seventies, from both geological and atmospheric arguments that, although Venus and Mars are presently devoid of liquid water, their surfaces could have been partially or completely covered by water at some time of their evolution. There are many possible diagnostics of the long-term evolution of the planets, either from the present characteristics of their surfaces or from their present atmospheric compositions. Among them, the present value of the D/H ratio is of particular interest, although its significance in terms of long term evolution has been challenged by some authors. Recent progress has been made in this field. We now have evidence for higher D/H ratios on Mars and Venus than on Earth, with an enrichment factor of the order of 5 on Mars, and about 100 on Venus. Any scenario for the evolution of these planets must take this into account. The most recent models on the evolution of Mars and Venus are reviewed in light of these new measurements.Presented at the Session Water in the Solar System and Its Role in Exobiology during the 26th General Assembly of the European Geophysical Society, 22–26 April 1991 in Wiesbaden, Germany.     

Rapid high-performance liquid chromatographic method for the determination of ketamine and its metabolite dehydronorketamine in equine serum

A simple, rapid and sensitive high-performance liquid chromatographic procedure has been developed for the determination of ketamine and dehydronorketamine in equine serum. Sample preparation consisted of mixing equal volumes of serum and acetonitrile—phosphoric acid (85%)—water (20:2:78, v/v/v), followed by ultrafiltration through a 10 000 molecular mass cut-off filter. Separation of these two analytes in the ultrafiltrate was accomplished on a reversed-phase phenyl column eluted with methanol—acetonitrile—phosphate buffer solution. Ketamine and dehydronorketamine were detected by a variable photometric UV-Vis detector set at 215 nm, and confirmed by a photodiode array detector operated in the 200–320 nm range. The limit of detection for ketamine was 5–15 ng/ml in equine serum. Additionally, the dehydronorketamine peak identity was tentatively confirmed by thermospray liquid chromatography—mass spectrometry.     

Simple high-performance liquid chromatographic method for the determination of PGT/1A, a new immunostimulating drug, in biological fluids

This paper describes a simple high-performance liquid chromatographic method for the determination of PGT/1A (3- -pyroglutamyl- -thiazolidine-4-carboxylic acid), a new immunostimulating drug, in plasma and urine. The column was packed with LiChrospher-NH₂ (5 μm), the mobile phase was 0.02 M monobasic potassium phosphate (pH 3.2 with concentrated phosphoric acid)—acetonitrile (25:75, v/v), the flow-rate was 1.2 ml/min, the detection wavelength was 210 nm and the apparatus was a Varian Model 5000. Plasma (1 ml) was added to 1.2 ml of acetonitrile and the supernatant injected; the urine was diluted 1:5. The retention time of PGT/1A was 9.4 min in plasma and 9.9 min in urine. The method was validated for recovery, accuracy and reproducibility. The results after intravenous injection in twelve volunteers are also given.     

Partition coefficients (n-octanol/water) of N-butyl-p-aminobenzoate and other local anesthetics measured by reversed-phase high-performance liquid chromatography

For the determination of the logarithmic partition coefficients between n-octanol and water (log P_o/w) of local anesthetics, the pH of the aqueous phase needs to be adjusted to high values to ensure that the local anesthetics are in the unionized form. Using the shake-flask or the stir-flask method, this high pH may catalyze hydrolysis, leading to increasing amounts of impurities in time. These impurities exclude non-selective quantification methods like UV spectrometry and require repetitive quantitative analysis of both liquid phases resulting in a tedious and time-consuming method. A rapid reversed-phase HPLC method was developed to measure log P_o/w of the local anesthetics N-butyl-p-aminobenzoate, methyl-p-aminobenzoate, benzocaine, procaine, mepivacaine, prilocaine, lidocaine, bupivacaine, etidocaine, tetracaine and oxubuprocaine.     

High-performance liquid chromatographic determination of naproxen, ibuprofen and diclofenac in plasma and synovial fluid in man

High-performance liquid chromatographic assay procedures have been developed for naproxen, ibuprofen and diclofenac in human plasma and synovial fluid samples. A single liquid—liquid extraction procedure was used to isolate each compound from acidified biological matrix prior to the quantitative analysis. A Spherisorb ODS column (12.5 cm × 4.6 mm I.D.) was used for all the chromatography. Naproxen was eluted with a mobile phase of methanol—Sörensen's buffer at pH 7 (37:63, v/v). Ibuprofen and diclofenac were eluted using mobile phases of methanol—water at pH 3.3 (65:35, v/v and 63:37, v/v, respectively). Diphenylacetic acid was used as the internal standard for the assay of naproxen and flurbiprofen was used in the analysis of ibuprofen and diclofenac. Inter- and intra-day coefficients of variation were less than 7%. The assays were used in clinical studies of the three drugs in osteo- and rheumatoid arthritis patients.     

Approaches to the biosynthesis of the cecropia C18-Juvenile Hormone

The juvenile hormone of the silkmoth Hyalophora cecropia—methyl 7-ethyl-3,11-dimethyl-cis-10,11-oxido-trans,trans-2,6-tridecadienoate—is a rare bis-homosesquiterpenoid whose biosynthesis has not been explained. In an attempt to do so, I have studied the incorporation of ¹⁴C- and ³H-labelled precursors into the carbon skeleton of C₁₈-Juvenile Hormone (JH) both in adult male insects and in short-term tissue cultures of corpora allata, the glands which are known to secrete the hormone. In these studies, also, novel combinations of microchemical derivatization procedures and radiochemical purification sequences, involving thin-layer, high-pressure liquid, and electron capture gas chromatography were used to assess the extent of isotope incorporation into sub-nanogram amounts of biosynthetic JH. Thus, in in vivo incubations I recovered label derived from acetate, glucose, methionine, and to a lesser extent from iso-leucine and valine. Bis-homofarnesol, farnesol, leucine, 3-methyl-2-pentenol, propionate, and mevalonate did not contribute significantly to the formation of the carbon skeleton. In parallel in vitro incubations I recovered label in the JH fraction when glucose, acetate, or methionine served as precursor.     

Determination of the new podophyllotoxin derivative NK 611 in plasma by high-performance liquid chromatography with ultraviolet detection

A high-performance liquid chromatographic method has been developed for the determination of the new podophyllotoxin derivative NK 611 in plasma samples. A solid—liquid extraction procedure with C₁₈ extraction columns was used for extraction of plasma samples containing NK 611. The adsorbed NK 611 was eluted from the extraction columns with methanol—acetonitrile (50:50, v/v). The elution liquid was injected into a reversed-phase system consisting of a Chrompack C₁₈ column. The mobile phase was acetonitrile—20 mM phosphate buffer, pH 7 (30:70, v/v). The UV detection mode allows sensitive determination of NK 611 in plasma within phase I trials. The limit of detection was 10 ng/ml, the limit of quantitation 35 ng/ml (for 1 ml of extracted plasma and 20-μl injection volume). The calibration curve is linear within the concentration range 100–1000 ng/ml. The recovery of NK 611 from spiked plasma samples was approximately 80%.     

Features of Crossability, Haploidy and Polyembryony in Hybrid Combinations between Cultivated Barley Hordeum vulgare L. (2n = 14) and Wheat-Rye Substitution Lines Triticum aestivum L., Cultivar Saratovskaya 29/Secale cereale L., Cultivar Onokhoiskaya

The role of individual chromosomes of rye in the manifestation of crossability and seedling development in hybrid combinations between cultivated barley Hordeum vulgare L., cultivar Nepolegayushchii (2n = 14) and five wheat-rye substitution lines Triticum aestivum L., cultivar Saratovskaya 29/Secale cereale L., cultivar Onokhoiskaya (2n = 40 wheat + 2 rye chromosomes). Crossability, which was measured by two parameters—frequency of set grains and frequency of grains with embryos—was shown to be significantly affected by each of the five rye chromosomes examined: 1R, 2R, 3R, 5R, and 6R; the development of barley haploids was affected by rye chromosomes 1R, 3R, and 5R. We were the first to demonstrate that polyembryony could be induced by mutual effects of barley cytoplasm and rye chromosome 1R. Possible mechanisms controlling the development of haploids and twins in hybrid combinations H. vulgare × T. aestivum/S. cereale are discussed. The conclusion is drawn that hybrid combinations between cultivated barley and wheat-rye substitution lines can serve as new models for studying incompatibility mechanisms in distant crosses and genetic control of parthenogenesis.__________Translated from Genetika, Vol. 41, No. 6, 2005, pp. 784–792.Original Russian Text Copyright © 2005 by Pershina, Belova, Devyatkina, Rakovtseva, Kravtsova, Shchapova.