Determination of isosorbide-5-mononitrate in human plasma by high-resolution gas chromatography

A method for the determination of isosorbide-5-mononitrate (5-ISMN) in human plasma by capillary gas chromatography with electron-capture detection was developed and applied to clinical samples. 9-Fluorenone was used as an internal standard, ethyl acetate was employed for liquid-liquid extraction. The advantage of the extraction procedure is the possibility of a direct injection of the plasma extract, without solvent removal/reconstitution of the sample. The precision and accuracy of the method were satisfactory in the concentration range 10-1600 ng/ml. The lower limit of quantification was 10 ng/ml.     

Sex- and species-related differences in the biotransformation of isosorbide dinitrate by various tissues of the rabbit and rat

The biotransformation of isosorbide dinitrate (ISDN) by various tissues of the rabbit and rat was examined. Incubation of 2 X 10(-7) M ISDN at 37 degrees C with tissue homogenates of liver, lung, kidney, intestine, skeletal muscle, aorta, and erythrocytes from the rabbit and rat resulted in a significant disappearance of ISDN after a 30-min incubation (also, 5-min incubation for liver). The disappearance of ISDN in each tissue homogenate was accompanied by an equimolar production of the mononitrate metabolites, isosorbide-2-mononitrate (2-ISMN) and isosorbide-5-mononitrate (5-ISMN), with the exception of liver homogenates where the loss of ISDN could not be accounted for by mononitrate formation. The relative rate of ISDN disappearance in various tissue homogenates was for the male rabbit, liver greater than lung approximately intestine greater than kidney greater than erythrocytes approximately skeletal muscle approximately aorta; for the female rabbit, liver greater than kidney approximately lung approximately intestine greater than erythrocytes approximately skeletal muscle approximately aorta; and for the male rat, liver greater than intestine greater than erythrocytes greater than skeletal muscle greater than lung approximately kidney. A sex difference in the percent disappearance of ISDN was observed in homogenates of lung and intestine from male and female rabbits. In addition, a sex difference in the ratio of metabolite (2-ISMN/5-ISMN) formed by denitration of ISDN was seen in homogenates of lung, skeletal muscle, and erythrocyte lysate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Patterns of isosorbide dinitrate and glyceryl trinitrate metabolites formed by selected segments of the rabbit gastrointestinal tract

Homogenates of selected segments of the rabbit gastrointestinal tract (GIT) were studied for their ability to biotransform isosorbide dinitrate (ISDN) and glyceryl trinitrate (GTN) to their mono- and di-nitrate metabolites, respectively. In addition, preferential formation of certain metabolites was investigated by examination of the patterns of metabolites formed by the various homogenates. After a 30-min incubation of ISDN with GIT homogenates (pH 7.4, 37 degrees C), the percent disappearance of ISDN and the ratio of isosorbide-2-mononitrate (2-ISMN) to isosorbide-5-mononitrate (5-ISMN) were as follows: stomach, 32%, 0.8; duodenum, 65%, 0.1; jejunum, 59%, 0.2; ileum, 38% , 1.2; cecum, 33%, 2.7; and colon, 32%, 3.4. After a 5-min incubation of GTN with GIT homogenates, the percent disappearance of GTN and the ratio of glyceryl-1,3-dinitrate (1,3-GDN) to glyceryl-1,2-dinitrate (1,2-GDN) were as follows: duodenum, 54%, 0.65; ileum, 73%, 0.68; and colon, 61%, 0.17. Incubation of 2 x 10(-7) M ISDN with mucosal and muscularis homogenates of duodenum, jejunum, and ileum resulted in significant losses of ISDN with an equimolar formation of the mononitrate metabolites. Most of the metabolic activity for ISDN resided in the mucosal layer of each section. The ratio of 2-ISMN to 5-ISMN varied in each section (stomach to colon) and cross section (mucosal versus muscularis) of the GIT. We conclude that the metabolism of ISDN and GTN by the GIT may contribute to the high clearance of these organic nitrates, and the low oral bioavailability of ISDN. Also, multiple mechanisms appear to be involved in the biotransformation of ISDN and GTN in the rabbit GIT.     

Bioconversion of isosorbide dinitrate into isosorbide mononitrate by the protozoan Tetrahymena thermophila: relationship to glutathione transferase levels

Summary Growing cells of Tetrahymena thermophila (T. t.) metabolized, after 72 h, 80% of isosorbide dinitrate (ISDN) to isosorbide 5-mononitrate (5-ISMN) and isosorbide 2-mononitrate (2-ISMN) in a ratio 5-ISMN/2-ISMN=2.6 as evaluated by HPLC. The level of glutathione S-transferases (GSH-ST) determined by following the reactions with of 1-chloro-2,4-dinitrobenzene (CDNB), o-dinitrobenzene (o-DNB) or ISDN, showed the inductive effect of ISDN (0.5 mg/ml) on the level of this enzyme. The enzymatic activity, evaluated at 72 h, showed a twofold increase compared with the control. The GSH-ST activity correlated well with the rate of ISDN bioconversion.     

Quantification of isosorbide 5-mononitrate in human plasma by liquid chromatography-tandem mass spectrometry using atmospheric pressure photoionization

Isosorbide 5-mononitrate (5-ISMN) is an organic nitrate widely used for its vasodilating properties in the treatment of angina pectoris. In the present study, an efficient, sensitive, robust method was developed for the determination and quantification of isosorbide 5-mononitrate, in human plasma, by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS), using photospray ionization. Isosorbide 5-mononitrate was extracted from 0.5 mL human plasma by liquid-liquid extraction (LLE). The method had a chromatographic run of 2.0 min using a C(8) analytical column (100 mm x 2.1 mm i.d.) and the linear calibration curve over the range was linear from 20 to 2000 ng mL(-1) (r(2)>0.995). The between-run precision, based on the relative standard deviation replicate quality controls, was 7.9% (60 ng mL(-1)), 5.2% (300 ng mL(-1)) and 7.0% (1800 ng mL(-1)). The between-run accuracy was 94.9%, 94.1% and 88.8% for the above-mentioned concentrations, respectively. The method herein described was employed in a bioequivalence study of two tablet formulations of isosorbide 5-mononitrate 40 mg.     

Improved extraction procedure for the assay of isosorbide-5-mononitrate in serum

A procedure is described for the extraction of isosorbide-5-mononitrate from serum with diethyl ether-n-butyl acetate (15:1, v/v). After extraction, the diethyl ether is evaporated at room temperature and the nitrate remains dissolved in the relatively non-volatile n-butyl acetate phase. Glassware must be used throughout as the recovery of isosorbide-5-mononitrate is not reproducible when plastic tubes or plastic pipettes are used. The extraction recovery of isosorbide-5-mononitrate from serum is 67 ± 7%, measured relative to external standards.     

Bioconversion of isosorbide dinitrate by various microorganisms

Summary A total of 19 microorganisms, selected from genera of bacteria, fungi and yeasts, were screened for their ability to hydrolyse isosorbide dinitrate (ISDN) to mononitrates. Cunninghamella echinulata and Cunninghamella elegans showed rates of bioconversion of ISDN of 74% and 88% respectively, measured after 73 h. However, the two strains exhibited opposite stereoselectivity, as reflected in the ratios of isosorbide 5-mononitrate (5-ISMN) to isosorbide 2-mononitrate (2-ISMN). These were 2.57 and 0.75 for C. echinulata and C. elegans, respectively.     

High performance liquid chromatography-electrospray ionization mass spectrometric determination of isosorbide 5-mononitrate in human plasma

A novel, selective and sensitive high performance liquid chromatography-mass spectrometric (HPLC-MS) method has been developed for the determination of isosorbide 5-mononitrate (5-ISMN) in human plasma. With acetaminophen as internal standard, sample pretreatment involved one-step extraction with diethyl ether of 0.5 mL plasma. Analysis was performed on an ACQUITY UPLC BEH C(18) column (100 mm x 2.1mm, 1.7 microm) with mobile phase consisting of acetonitrile-water (20:80, v/v). The detection was carried out by means of electrospray ionization mass spectrometry in negative ion mode with selected ion recording (SIR). Standard curves were linear (r(2)> or =0.99) over the concentration range of 1.04-1040 ng/mL. The lower limit of quantification (LLOQ) was 1.04 ng/mL. The intra- and inter-day precisions (RSDs) were less than 8.6% and 13.4%, respectively, and the accuracy (RE) was within +/-0.45%. The method herein described was fully validated and successfully applied to the pharmacokinetic study of 5-ISMN in compound extended-release tablets in 18 healthy male volunteers after oral administration.     

Use of a naphthylethylcarbamoylated-β-cyclodextrin chiral stationary phase for the separation of drug enantiomers and related compounds by sub- and supercritical fluid chromatography

Enantiomeric separation of a variety of drugs and related compounds was achieved on an (S)-naphthylethylcarbamoylated-β-cyclodextrin (S-NEC-CD) chiral stationary phase (CSP) using sub- and supercritical fluid chromatography (SFC). Compounds previously resolved on native or derivatized cyclodextrin CSPs in liquid chromatography (LC) using reversed phase or polar organic mobile phase modes could be resolved in SFC using a simple carbon dioxide/methanol eluent. Resolution of cromakalim, which is not possible on the S-NEC-CD column in LC, was readily accomplished in SFC. The importance of modifier, temperature, and pressure was assessed in relation to retention, selectivity, and resolution. The nature of the modifier and the modifier concentration were found to be crucial parameters. © 1996 Wiley-Liss, Inc. Contribution of the National Institute of Standards and Technology. Not subject to copyright.     

Simple and sensitive high performance liquid chromatographic method for the simultaneous quantitation of the lactone and carboxylate forms of topotecan in human plasma

A selective and highly sensitive isocratic high performance liquid chromatographic (HPLC) method is described for simultaneous determination of lactone and carboxylate species of topotecan, in plasma. The method utilizes a protein precipitation step with cold methanol (-20 degrees C) for sample preparation followed by separation on a Novapack C(18) column using ammonium acetate buffer, acetonitrile and triethylamine (84:16:1.5, v/v) containing tetrabutyl ammonium hydrogen sulfate (TBAHS) (2 mM) with a pH of 5 as the mobile phase. The eluted peaks were detected by a fluorescence detector was set at an excitation wavelength of 380 nm and an emission wavelength of 527 nm. The method was validated in the range of lactone and carboxylate forms of topotecan concentrations from 0.05 to 75 ng/ml. Intra- and inter-day precision expressed by the relative standard deviation was less than 8.50% and inaccuracy did not exceed 10% for lactone and carboxylate forms of topotecan. The limit of quantitation was 0.05 ng/ml using 0.50 ml plasma. Stability studies in plasma and plasma extract indicated that topotecan is stable for at least 2 weeks at -70 degrees C.     

Aqueous two-phase extraction of 2,3-butanediol from fermentation broths using an ethanol/ammonium sulfate system

Separation of 2,3-butanediol from the fermentation broth is a difficult task that has become a bottleneck in industrial production. Aqueous two-phase systems composed of hydrophilic solvents and inorganic salts could be used to extract 2,3-butanediol from fermentation broth. The ethanol/ammonium sulfate system was investigated in detail, including phase diagram, effect of phase composition on partition, removal of cells and biomacromolecules from the broths and recycling of ammonium sulfate. The highest partition coefficient (7.10) and recovery of 2,3-butanediol (91.7%) were obtained by a system composed of 32% (w/w) ethanol and 16% (w/w) ammonium sulfate. The maximum selective coefficient of 2,3-butanediol to glucose was 30.74 in the experimental range. In addition, cells and proteins could be simultaneously removed from the fermentation broth. The removal ratio of cells and proteins reached 99.7% and 91.2%, respectively. The recovery of ammonium sulfate in the bottom phase reached 97.14% when two volumes of methanol were added to the salt-rich phase.     

Purification of (S)-3-cyano-5-methylhexanoic acid from bioconversion broth using an acetone/ammonium sulfate aqueous two-phase system

(S)-3-Cyano-5-methylhexanoic acid ((S)-CMHA) is the key chiral intermediate of pregabalin. In this paper, an aqueous two-phase system (ATPS) was developed to extract (S)-CMHA from nitrilase-catalyzed bioconversion broth. Inorganic salts and hydrophilic solvents were screened to form ATPS, among which an acetone/ammonium sulfate ATPS was investigated in detail, including phase diagram, effect of phase composition and stability of (S)-CMHA. The maximum product recovery of 99.15% was obtained by an optimized ATPS system composed of 15% (w/w) ammonium sulfate and 35% (w/w) acetone with the removal of 99% cells and 86.27% proteins. The total (S)-CMHA yield reached 92.11% after back-extraction. The recycling use of ammonium sulfate was investigated, and 93.10% of salt in the salt-rich phase was recovered with the addition of methanol. The results demonstrated the efficiency of the two-step extraction process for separation of (S)-CMHA.     

Simultaneous determination of isosorbide dinitrate and its mononitrate metabolites in human plasma by capillary gas chromatography with electron-capture detection

A method for the simultaneous determination of isosorbide dinitrate (ISDN) and its mononitrate metabolites (2- and 5-ISMN) in human plasma by capillary gas chromatography with electron-capture detection was developed. Two internal standards were used: isomannide dinitrate (IMDN) for the determination of ISDN and isomannide mononitrate (IMMN) for the determinations of 2- and 5-ISMN. After addition of the internal standards, the compounds were isolated from plasma by solid-liquid extraction. They were determined by gas chromatography using an electron-capture detector. The reproducibility and accuracy of the method were found suitable in the range of concentrations 2.5–83 ng/ml for ISDN, 2.6–208 ng/ml for 2-ISMN and 2.3–1010 ng/ml for 5-ISMN. The limit of quantitation (LOQ) was about 2.5 ng/ml for each compound. The method was applied to clinical samples.     

Analysis of conjugated bile acids by packed-column supercritical fluid chromatography

A rapid method has been developed for the simultaneous separation of the polar glycine- and taurine-conjugated bile acids by packed-column supercritical fluid chromatography. Samples were analysed on a cyanopropyl-bonded silica column with ultraviolet detection at 210 nm and carbon dioxide modified with methanol as the mobile phase. The influence of the stationary phase, modifier concentration, temperature, column pressure and modifier identity on retention was also studied. This new chromatographic method is applicable to the assay of conjugated bile acids in duodenal bile samples from patients with hepatobiliary diseases.     

Comparison of polysaccharide-based and protein-based chiral liquid chromatography columns for enantioseparation of drugs

Two different columns—Lux Cellulose-1 and Chiralpak CBH—were evaluated for their chiral recognition abilities for eight drugs comprising three β-blockers, one antacid, and four cathinones in polar-organic elution mode and reversed-phase elution mode, respectively. The factors that affected the enantioseparation were tested and optimized to develop a suitable chiral separation method whose LC conditions are compatible with MS detection. In polar-organic elution mode with the Lux Cellulose-1 column, methanol and acetonitrile were tested as the main components of the mobile phase. In addition, the effects of adding isopropanol as organic modifier, acidic additives (formic acid), and basic additives (diethylamine) were evaluated. In reversed-phase elution mode with the Chiralpak CBH column, the effect of type and concentration of organic modifier (isopropanol, acetonitrile, and methanol), the mobile phase pH (6.4 and 5.0), and buffer concentration (1mM-20mM ammonium acetate) were evaluated. The best enantioseparation was achieved with the Chiralpak CBH column with a mobile phase composed of 5mM ammonium acetate aqueous (pH = 6.4)/methanol (95/5, v/v) at a flow rate of 0.1 mL/min and a temperature of 30°C. Under these conditions, six of eight chiral drugs were baseline separated.     

Purification of single-strand DNA binding protein from an Escherichia coli lysate using counter-current chromatography,partition and precipitation

Single-strand DNA binding protein (SSB) from Escherichia coli lysate was purified by counter-current chromatography (CCC) using the ammonium sulfate precipitation method in a coiled column. About 5 ml of E. coli lysate was separated by CCC using a polymer phase system composed of 16% (w/w) polyethylene glycol (PEG) 1000 and 17% (w/w) ammonium sulfate aqueous polymer two-phase solvent system. The precipitation of proteins in the lysate took place in the CCC column, and the SSB protein was eluted in the fraction 51-56. Many other impurities were either eluted immediately after the solvent front or precipitated in the column. The identities of the proteins in the fractions and in the precipitate were confirmed by SDS-polyacrylamide gel electrophoresis with Coomassie Brilliant Blue staining.     

Methanogenesis from Methanol and Methylamines and Acetogenesis from Hydrogen and Carbon Dioxide in the Sediments of a Eutrophic Lake

¹⁴C-tracer techniques were used to examine the metabolism of methanol and methylamines and acetogenesis from hydrogen and carbon dioxide in sediments from the profundal and littoral zones of eutrophic Wintergreen Lake, Michigan. Methanogens were primarily responsible for the metabolism of methanol, monomethylamine, and trimethylamine and maintained the pool size of these substrates below 10 μM in both sediment types. Methanol and methylamines were the precursors for less than 5 and 1%, respectively, of the total methane produced. Methanol and methylamines continued to be metabolized to methane when the sulfate concentration in the sediment was increased to 20 mM. Less than 2% of the total acetate production was derived from carbon dioxide reduction. Hydrogen consumption by hydrogen-oxidizing acetogens was 5% or less of the total hydrogen uptake by acetogens and methanogens. These results, in conjunction with previous studies, emphasize that acetate and hydrogen are the major methane precursors and that methanogens are the predominant hydrogen consumers in the sediments of this eutrophic lake.     

Simultaneous quantification of dopamine, 5-hydroxytryptaine and four metabolically related compounds by means of reversed-phase high-performance liquid chromatography with electrochemical detection

A method for simultaneously quantifying dopamine, 5-hydroxytryptamine (5-HT) and four metabolically related compounds has been developed, permitting more efficient neurochemical examination of these often interrelated biogenic amine systems. The method uses high-performance liquid chromatographic separation of these compounds on a C₁₈ reversed-phase column with a buffered mobile phase containing methanol as an organic modifier and heptanesulfonate as an ion-pair reagent. Using 5-hydroxy-N-methyltryptamine as an internal standard and electrochemical detection, chromatography time is less than 12 min. Sample preparation simply involves the addition of internal standard, homogenization in the mobile phase, centrifugation and injection of the supernatant into the chromatograph. The method is sensitive to a tissue content of these compounds of less than 1 ng. The utility of this method for neuropharmacological—neurochemical studies is illustrated with studies using inhibitors of monoamine oxidase (pargyline) and aromatic amino acid decarboxylase (RO 4-4602).     

New retention mechanism of mononucleotides with buffer concentrations in ion-suppressing RP-HPLC

HPLC separation of ionic samples tends to be more complicated and difficult to understand than that of non-ionic compounds. On the other hand, band spacing is much more easily manipulated for ionic than for neutral samples. Ion-suppressing RP-HPLC method was used with organic modifier and aqueous buffer solution. In this work, five mononucleotides of cytidine-5-monophosphate (5′-CMP) disodium salt, uridine-5-monophosphate disodium salt (5′-UMP), guanosine-5-monophosphate disodium salt (5′-GMP), inosine-5-monophosphate disodium salt (5′-IMP), and adenosine-5-monophosphate disodium salt (5′-AMP) were examined. Acetic acid and sodium phosphate were used as buffers, and methanol as an organic modifier. A new relationship between the retention factor and the buffer concentration at a fixed modifier content (5% of methanol) could be expressed by fol|lowing:k=(k ₋₁+k ₀ (K _B/K _S)C _B ^a)/(1+(K _B/K _S)C _B ^a), whereC _B was the concentration of buffer. Using this relationship, the calculated values closely matched the experimental data. The derived relationship showed that as the buffer concentration increased, the retention factor approached a certain value, and this was buffer dependent.     

利用次溴酸盐氧化结合盐酸羟胺还原法测定大气气溶胶样品铵态氮同位素

硝酸铵、硫酸铵和硫酸氢铵等铵盐作为PM_2.5中主要的二次无机气溶胶污染物,在灰霾形成过程中发挥了重要作用.关于其来源及转化过程的研究备受关注.本文在前人研究基础上,基于稳定同位素分析技术改进了样品进样量,增加了调节浸提液pH的步骤,给出了一种能够快速、准确测量大气样品中铵态氮同位素的化学转化方法.即向含有0.25 μg·mL^-1铵态氮的4 mL大气气溶胶滤膜样品浸提液中加入碱性次溴酸盐溶液,使铵盐(NH₄⁺)氧化为亚硝酸盐(NO₂^-);然后通过调节pH值,在 pH<0.3 的条件下,使用盐酸羟胺将亚硝酸盐(NO₂^-)还原为氧化亚氮气体(N₂O),最终通过Precon-GasBench-IRMS系统的连用实现大气气溶胶样品中的铵态氮同位素的测定.该方法所需进样含量低,避免了剧毒易制爆试剂的使用,重复测试精确度可达到0.2‰(n=10).通过调节大气气溶胶滤膜样品浸提液的pH可提高还原效率(约100%).测量精确度和准确度满足了大气气溶胶中颗粒态铵的测定,有助于进一步开展大气铵盐的来源、化学转化过程、传输沉降等研究.