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PubMed1993

Immunoaffinity isolation of the sulfate conjugate of 4'-hydroxypropranolol from plasma.

Selective extraction of sulfate conjugates of basic drugs from biological matrices has been difficult because of their highly polar nature. Immunoaffinity isolation may be the best solution to this analytical problem. This was tested for a model compound, the metabolite 4'-hydroxypropranolol sulfate (HOPS), which was effectively extracted from plasma by a column containing antibodies to the parent drug propranolol. The specificity was very high, giving little interference from the biological material in subsequent high-performance liquid chromatographic analysis with fluorometric detection. The method for HOPS was highly reproducible and provided a sensitivity of 1 ng/ml plasma. The technique was applied to measurements of HOPS in plasma after therapeutic doses of propranolol as well as to the individual enantiomers after chiral derivatization.

Adult↗
PubMed1994

Quantitative analysis of phosphorothioate oligonucleotides in biological fluids using direct injection fast anion-exchange chromatography and capillary gel electrophoresis.

The analysis of antisense phosphorothioate DNA (SODN) in human plasma via direct injection using anion-exchange high-performance liquid chromatography (AE-HPLC) is presented. The method relies on the ability to selectively extract phosphorothioate DNA from undigested serum, plasma and urine on anion-exchange resins. The automated HPLC method can analyze a sample every 5 min with a limit of detection of 50 ng/ml (ppb). The DNA was collected, desalted and analyzed by capillary gel electrophoresis. Due to the high resolving power of this technique, a qualitative assessment of enzymatic degradation of the antisense oligonucleotide can be made.

Chromatography, Ion Exchange↗
PubMed1984

Assay of methotrexate and 7-hydroxymethotrexate by gradient-elution high-performance liquid chromatography and its application in a high-dose pharmacokinetic study.

A paired-ion high-performance liquid chromatographic method is described for the simultaneous determination of methotrexate (MTX) and its major metabolise, 7-hydroxymethotrexate (7-OH-MTX), in plasma and urine. In addition, this technique permits the separation of other known metabolites of MTX, such as DAMPA and MTX-polyglutamates. After selective extraction on an anion-exchange resin column, both compounds and the internal standard, aminopterin, were separated on a reversed-phase octadecylsilane column with UV-detection at 313 nm. The detection limits for plasma and urine samples were approximately 40 ng/ml (8.8 x 10(-8) M) for MTX and 100 ng/ml (2.1 x 10(-7) M) for 7-OH-MTX. This method was applied in pharmacokinetic studies following 24-h infusion of high-dose MTX in four patients during two successive treatments. After the end of the infusion, the mean apparent half-life for the metabolite was 19.1 h, while that for MTX was 8.8 h. A stepwise increase in the plasma concentrations of both MTX and its metabolite was observed during the second MTX infusion. This increase was reflected in the cumulative urinary excretion of both drug and its metabolite.

Journal Article↗
PubMed1989

Determination of isoniazid in pharmaceutical preparations by reaction with radiochloramine-B.

A method has been developed for the determination of isoniazid (INH) at microgram level in pharmaceutical preparations based on the oxidation of isoniazid by radiochloramine-B. Interference of vitamin C has been overcome by precipitation as lead ascorbate and that of rifampicin and p-aminosalicylic acid by selective extraction. Amounts as low as 25 micrograms of INH can be determined.

Aminosalicylic Acid↗
PubMed1991

Photobinding of 8-methoxypsoralen, 4,6,4'-trimethylangelicin and chlorpromazine to Wistar rat epidermal biomacromolecules in vivo.

Photoinduced binding of drugs to endogenous biomacromolecules may cause both toxic and therapeutic effects. For example, photobinding of certain phenothiazines to biomolecules possibly underlies their phototoxic and photoallergic potential, whereas photobinding of furocoumarins to epidermal DNA is held responsible for their advantageous effects in the photochemotherapy of psoriasis. Usually, the in vitro photobinding of drugs is investigated. However, under in vivo conditions, the metabolism and distribution of the drug and the light absorption by endogenous compounds will significantly affect the photobinding of drugs to biomolecules. Therefore, in the present study, the photobinding of 8-methoxypsoralen (8-MOP), 4,6,4'-trimethylangelicin (TMA) (two therapeutically used furocoumarins) and chlorpromazine (CPZ) (a member of the phenothiazines) was investigated in vivo. The compounds were applied topically on the shaven skin of Wistar rats; one group was exposed to UVA and the other was kept in a dimly lit environment. Immediately, and at certain time intervals after UVA exposure, members of the two groups were sacrificed. By separating epidermal lipids, DNA/RNA and proteins by a selective extraction method, irreversible binding of 8-MOP, TMA or CPZ to each of these biomacromolecules was determined. In contrast with in vitro experiments, photobinding of CPZ to epidermal DNA/RNA was not found in vivo; apparently the bioavailability in the nucleus is very low. Compared with TMA, 8-MOP was observed to bind more extensively to epidermal DNA/RNA (again in contrast with findings from in vitro experiments) and proteins, but less extensively to lipids. The rates of removal of photobound 8-MOP and TMA were comparable. Photobound CPZ was more slowly removed from epidermal proteins and lipids than the furocoumarins. The observed in vivo photobinding is discussed with respect to the UVA-induced (side) effects of these drugs.

Animals↗
PubMed1996

Activation of protein phosphatase-1 isoforms and glycogen synthase kinase-3 beta in muscle from mdx mice.

Three Protein Phosphatase-1 (PP1) isoforms (PP1 alpha, PP1 gamma-1 and PP1 delta) are found in skeletal muscle. These are bound to regulatory subunits, such as inhibitor 2 (I2) in the cytosol and G in the glycogen and microsomal fractions. In vitro, the PP1-12 complex is activated by Glycogen Synthase Kinase-3 (GSK-3 or FA). We investigated the activities and protein levels of the three PP1 isoforms and of GSK-3 in muscle of mdx dystrophic mice. PP1 was assayed as phosphorylase phosphatase, in the presence of 5 nM okadaic acid (which inhibits PP2A). Peptide antibodies were produced and used to investigate PP1 alpha, PP1 gamma-1 and PP1 delta. GSK-3 was assayed using a previously described peptide. This was synthesized in a pre-phosphorylated from, which avoids the additional use of Casein Kinase II. Higher PP1 activity was assayed in the cytosol from mdx rather than from control muscles. Immunoprecipitation indicated that only PP1 alpha and PP1 gamma-1 were more active. This was most likely due to enzyme activation, since the immunodetected proteins were unchanged. On the other hand, the immunodetected PP1 delta was lower in the glycogen and microsomal fractions from mdx muscle. GSK-3 was more active in the mdx extract Selective immunoprecipitation of GSK-3 alpha and GSK-3 beta indicated that both isoforms were activated. In the case of GSK-3 beta, the immunodetected protein was also increased. The changes described herein may be related to the pathological events occurring in the mdx muscle. These include increased protein degradation and turnover, and fibre regeneration. In fact, the decreased PP1 delta may be due to protein degradation and the increased GSK-3 may be the consequence of increased protein turnover or regeneration. The apparent correlation between the increased PP1 alpha and PP1 gamma-1 activities and the increased GSK-3 may agree with the hypothesis that GSK-3 activates the newly synthesized PP1.

Amino Acid Sequence↗
PubMed2005

The determination of homocysteine-thiolactone in human plasma.

The thioester homocysteine-thiolactone, a reactive metabolite of homocysteine, has been implicated in human cardiovascular disease. However, data on the levels of homocysteine-thiolactone in humans are limited, mostly due to a lack of facile and reliable assays. Here we describe a sensitive assay for the determination of plasma homocysteine-thiolactone and demonstrate its utility with a cohort of 60 healthy human subjects. Plasma homocysteine-thiolactone is first separated from macromolecules by ultrafiltration and then selectively extracted with chloroform/methanol. Further purification of plasma homocysteine-thiolactone is achieved by high-performance liquid chromatography on a cation exchange microbore column. The detection and quantification is by monitoring fluorescence after postcolumn derivatization with o-phthaldialdehyde. The limit of detection is 0.36 nM. Using this assay, homocysteine-thiolactone concentrations in plasma from normal healthy human subjects (n=60) were found to vary from zero to 34.8 nM, with an average of 2.82+/-6.13 nM. In 29 of the 60 human plasma samples analyzed, homocysteine-thiolactone levels were below the detection limit. Homocysteine-thiolactone represented from 0 to 0.28%, on average 0.023+/-0.05%, of plasma total homocysteine.

Calibration↗
PubMed2006

Long-term reduction of Trypanosoma cruzi infection in sylvatic mammals following deforestation and sustained vector surveillance in northwestern Argentina.

Long-term variations in the dynamics and intensity of sylvatic transmission of Trypanosoma cruzi were investigated around eight rural villages in the semiarid Argentine Chaco in 2002-2004 and compared to data collected locally in 1984-1991. Of 501 wild mammals from 13 identified species examined by xenodiagnosis, only 3 (7.9%) of 38 Didelphis albiventris opossums and 1 (1.1%) of 91 Conepatus chinga skunks were infected by T. cruzi. The period prevalence in opossums was four-fold lower in 2002-2004 than in 1984-1991 (32-36%). The infection prevalence of skunks also decreased five-fold from 4.1-5.6% in 1984-1991 to 1.1% in 2002-2004. Infection in opossums increased with age and from summer to spring in both study periods. The force of infection per 100 opossum-months after weaning declined more than six-fold from 8.2 in 1988-1991 to 1.2 in 2002-2004. Opossums were mainly infected by T. cruzi lineage I and secondarily by lineage IId in 1984-1991, and only by T. cruzi I in 2002-2004; skunks were infected by T. cruzi IId in 1984-1991 and by IIc in 2002-2004. The striking decline of T. cruzi infection in opossums and skunks occurred in parallel to community-wide insecticide spraying followed by selective sprays leading to very low densities of infected Triatoma infestans in domestic and peridomestic habitats since 1992; to massive deforestation around one of the villages or selective extraction of older trees, and apparent reductions in opossum abundance jointly with increases in foxes and skunks. These factors may underlie the dramatic decrease of T. cruzi infection in wild reservoir hosts.

Animal Diseases↗
PubMed2005

Sorption and desorption of radioselenium on calcareous soil and its solid components studied by batch and column experiments.

The effect of different calcareous soil solid components on the sorption and desorption of radioselenium was investigated by using a selective extraction method. The distribution coefficients, the breakthrough curves and the displacement curves were experimentally determined by batch and column experiments. The experiments were carried out at pH 7.8 +/- 0.2, T=20 +/- 1 degrees C and in the presence of 0.001 M CaCl2. It is found that all sorption isotherms are linear, the sorption-desorption hysteresis for untreated and treated soils to remove organic matter, CaCO3 and organic matter plus CaCO3 is obvious; and the retention of radioselenium can probably be attributed to clay and oxides. The contributions of organic matter and CaCO3 to the sorption of radioselenium are significant, and the interaction effects among organic matter, CaCO3 and other components play an important role in the sorption of radioselenium in calcareous soil. The content of interlaminary space of compacted soil plays an important role in the sorption of radioselenium in compacted soil. The presence of CaCO3 retards the transport of radioselenium in soils, CaCO3 is an important trap of radioselenium. It is very difficult for the effluent concentration to reach the influent concentration, although radioselenium is very quickly detected in the effluent solution. The breakthrough curves (BTCs) can be simulated by a one-dimensional convection-dispersion transport model.

Adsorption↗
PubMed2004

Calcium phosphate sol-gel-derived thin films on porous-surfaced implants for enhanced osteoconductivity. Part II: Short-term in vivo studies.

Osseointegration rates of porous-surfaced Ti6Al4V implants with control (unmodified sintered coatings) were compared to porous-surfaced implants modified through the addition of either an Inorganic or Organic Route-formed-Ca-P film. Implants were placed in distal femoral rabbit condyle sites and, following a 9-day healing period, implant fixation strength was evaluated using a pull-out test. Three groups of ten rabbits each were evaluated. Inorganic Route Ca-P-coated implants were compared with control implants in Group I. Organic Route Ca-P-coated implants with control implants in Group II, and Inorganic- with Organic Route-Ca-P-coated implants in Group III. Maximum pull-out force and interface stiffness were compared while selected extracted implants were examined by SEM to characterise failure surfaces. Both types of Ca-P coatings significantly enhanced the early rate of bone ingrowth and fixation as evidenced by higher pull-out force and interface stiffness compared with controls. However, there was no significant difference between Ca-P-coated implants prepared using the two different methods. The enhanced osteoconductivity observed with the Organic Route-formed films despite the absence of any obvious new surface topographic features introduced with the films suggests that the increased rate of bone ingrowth was due primarily to altered surface chemistry rather than changes in topography, at least for these sintered porous-surfaced implants.

Animals↗
PubMed2006

Resolution of complex monosaccharide mixtures from plant cell wall isolates by high pH anion exchange chromatography.

The use of high pH anion exchange chromatography combined with pulsed amperometric detection has been established as an effective and sensitive method for the separation, detection and quantification of monosaccharides from a wide range of sources. However, careful examination of the separation conditions required is necessary to ensure that a complete monosaccharide profile can be determined from structures such as the plant cell wall which is a complex network of both neutral and charged polysaccharides. This study has investigated the optimal conditions required for the analysis of such a challenging mixture, including both the stationary and mobile phase minimising co-elution and reducing method complexity. The preferred methods have been used to successfully identify and quantify the monosaccharide components of a selected extract from the plant cell wall of the primitive higher plant Equisetum arvense.

Anion Exchange Resins↗
PubMed2005

The speciation and bioavailability of mercury in sediments of Haihe River, China.

Twenty-one sediment samples in 11 sites along the Haihe River and Dagu Drainage River, Tianjin, China, were analyzed to investigate the pollution status and bioavailability of mercury (Hg). The results showed that the Haihe River was slightly polluted with Hg when flowing through Tianjin city. On the contrary, the sediments collected from Dagu Drainage River, an important drainage river in Tianjin, were found to have very high Hg concentrations and the highest concentration reached 8779.1 ng g(-1) (dry weight). The methylmercury (MeHg) concentrations accounted for 0.1-2.4% (average: 0.9%) of total mercury (HgT) and were strongly influenced by HgT (r=0.91, p=0.99, n=20) and total organic carbon (TOC; r=0.76, p=0.99, n=20) contents in sediments. Moreover, a five-step sequential selective extraction (SSE) procedure was used to study the bioavailability of Hg in sediments. The mercury in sediments existed mainly as element Hg and mercury sulfide, which accounted for 46.5% and 39.0% of HgT, respectively. The percentage of exchangeable Hg (defined as water soluble Hg plus 'human stomach acid' soluble Hg) was only 0.1-4.6%. The distribution of exchangeable Hg showed an obvious difference to that of HgT, indicating that the HgT concentrations were absolutely insufficient to evaluate the risk of Hg in sediments.

Biological Availability↗
PubMed2006

Cu and Zn adsorption onto non-residual and residual components in the natural surface coatings samples (NSCSs) in the Songhua River, China.

Natural surface coatings samples (NSCSs) from the surface of river shingles were employed to investigate the roles of non-residual and residual components of the NSCSs in controlling Cu and Zn adsorption via the selective extraction techniques and statistical analysis. The results indicate that the greatest contribution to metals adsorption on a molar basis was from Mn oxides in the non-residual fraction. Metals adsorption capacities of Mn oxides exceeded those of Fe oxides by one order of magnitude, fewer roles were found attributing to adsorption by organic materials (OM), and the estimated contribution of the residual fraction to metals adsorption was insignificant. These results implied that Mn oxides were the most important component in controlling heavy metals in aquatic environments. Experiments with Cu and Zn adsorption measured together showed that Cu severely interfered with Zn adsorption to the NSCSs and vice versa under the conditions of the two coexisted ions adsorption.

Adsorption↗
PubMed2004

Application of cytochrome b DNA sequences for the authentication of endangered snake species.

In order to enforce the conservation program and curbing the illegal trading and consumption of endangered snake species, the value of cytochrome b sequence in the authentication of snake species was evaluated. As an illustration, DNA was extracted, selected cytochrome b DNA sequences amplified and sequenced from six snakes commonly consumed in Hong Kong. Cataloging with sequences available in public, a cytochrome b database containing 90 species of snakes was constructed. In this database, sequence homology between snakes ranged from 70.68 to 95.11%. On the other hand, intraspecific variation of three tested snakes was 0-0.98%. Using the database, we were able to determine the identity of six meat samples confiscated by the Agriculture, Fisheries and Conservation Department, HKSAR.

Animals↗
PubMed2004

Nitric oxide metabolism in mammalian cells: substrate and inhibitor profiles of a NADPH-cytochrome P450 oxidoreductase-coupled microsomal nitric oxide dioxygenase.

Human intestinal Caco-2 cells metabolize and detoxify NO via a dioxygen- and NADPH-dependent, cyanide- and CO-sensitive pathway that yields nitrate. Enzymes catalyzing NO dioxygenation fractionate with membranes and are enriched in microsomes. Microsomal NO metabolism shows apparent KM values for NO, O2, and NADPH of 0.3, 9, and 2 microM, respectively, values similar to those determined for intact or digitonin-permeabilized cells. Similar to cellular NO metabolism, microsomal NO metabolism is superoxide-independent and sensitive to heme-enzyme inhibitors including CO, cyanide, imidazoles, quercetin, and allicin-enriched garlic extract. Selective inhibitors of several cytochrome P450s and heme oxygenase fail to inhibit the activity, indicating limited roles for a subset of microsomal heme enzymes in NO metabolism. Diphenyleneiodonium and cytochrome c(III) inhibit NO metabolism, suggesting a role for the NADPH-cytochrome P450 oxidoreductase (CYPOR). Involvement of CYPOR is demonstrated by the specific inhibition of the NO metabolic activity by inhibitory anti-CYPOR IgG. In toto, the results suggest roles for a microsomal CYPOR-coupled and heme-dependent NO dioxygenase in NO metabolism, detoxification, and signal attenuation in mammalian cells.

Caco-2 Cells↗
PubMed2006

In vivo sampling with solid phase microextraction.

This review discusses the most recent developments and future challenges in the application of solid phase microextraction (SPME) for sampling of live biological samples. The emphasis is placed on applications of fiber SPME for analysis of volatile emissions and drugs in biological fluids. The method development section highlights the main parameters that need to be considered in the case of in vivo experiments: extraction techniques, selection of extraction phases, calibration procedures, determination of free concentrations, and automation.

Drug Evaluation, Preclinical↗
PubMed2004

96-Well liquid-liquid extraction liquid chromatography-tandem mass spectrometry method for the quantitative determination of ABT-578 in human blood samples.

We report here a quantitative method for the analysis of ABT-578 in human whole blood samples. Sample preparation was achieved by a semi-automated 96-well format liquid-liquid extraction (LLE) method. Aluminum/polypropylene heat seal foil was used to enclose each well of the 96-well plate for the liquid-liquid extraction. A liquid chromatography combined with tandem mass spectrometry (LC-MS/MS) method with pre-column regeneration was developed for the analysis of sample extracts. Selective reaction monitoring (SRM) of the mass transitions m/z 983-935 and m/z 931-883 was employed for the detection of ABT-578 and internal standard, respectively. The ammonium adduct ions [M + NH(4)](+) generated from electrospray ionization were monitored as the precursor ions. The assay was validated for a linear dynamic range of 0.20-200.75ng/ml. The correlation coefficient (r) was between 0.9959 and 0.9971. The intra-assay CV (%) was between 1.9 and 13.5% and the inter-assay CV (%) was between 4.7 and 11.3%. The inter-assay mean accuracy was between 86.4 and 102.5% of the theoretical concentrations.

Chromatography, Liquid↗
PubMed2004

Determination of the immunosuppressant mycophenolic acid in human serum by solid-phase microextraction coupled to liquid chromatography.

A solid phase microextraction (SPME)-HPLC-UV method for the determination of the immunosuppressant mycophenolic acid (MPA) in human serum samples was developed for the first time. The procedure, that employed a carbowax/templated resin (Carbowax/TPR-100) as fiber coating, required a very simple sample pretreatment, an isocratic elution, and provides an highly selective extraction. The linear range was 0.2-100 microg x ml(-1). Recovery was practically unchanged (63+/- 4%) passing from 0.2 to 100 microg x ml(-1) level. Within-day and between-days coefficient of variation ranged from 5.9 to 6.5% and from 8.8 to 9.2%, respectively. A detection limit of 0.05 microg x ml(-1) was estimated in spiked serum. The method was successfully applied to the determination of MPA in serum of a patient under mycophenolate mophetil ester (MMF) therapy, as demonstrated by the relevant concentration-time profiles.

Chromatography, High Pressure Liquid↗