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HPLC Methods (hplc + methods)
Selected AbstractsRapid characterization of fatty alcohol ethoxylates by non-aqueous capillary electrophoresisELECTROPHORESIS, Issue 14 2008Mónica Arias Abstract Fatty alcohol ethoxylates (FAE) (a mixture of nonionic surfactants) have been characterized using NACE with UV detection. Phenyl polyurethane derivatives of these compounds were previously obtained by reaction with phenyl isocyanate. The derivatization reaction only requires microwave irradiation for 30,s (600,W). Phenyl polyurethanes were separated and characterized using a BGE containing a mixture of ammonium nitrate (15,mM), acetic acid (1.5%) and 9:1 v/v methanol/ACN. After optimization of the instrumental conditions for the separation, phenyl polyurethane compounds (formed from the corresponding FAE) with ethylene oxide numbers (EON) of 6 (certified standard and industrial samples), 7 and 10 (both as industrial samples), and 5.5 (microemulsion phase) were successfully separated and characterized. The properties of these FAE nonionic surfactants are very important in the petroleum industry, which requires characterization of the quality of the purchased materials as well as the final products in the microemulsion-oil-water stream process. This analytical objective has been achieved by the proposed NACE methods, allowing FAE to be distinguished from 5.5 to 10 EON with errors below 4%, and shows advantages against to HPLC methods. [source] Simultaneous detection of S -adenosylmethionine and S -adenosylhomocysteine in mouse and rat tissues by capillary electrophoresisELECTROPHORESIS, Issue 7-8 2003Eric O. Uthus Abstract A capillary electrophoresis method for the determination of S -adenosylmethionine (SAM) and S -adenosylhomocysteine (SAH) in rat liver and kidney and mouse liver is described. The method can also be used to determine SAM in whole blood. The method provides rapid (approximately 16 min sample to sample) resolution of both compounds in perchloric extracts of tissues. Separation was performed by using an uncoated 50 ,m ID capillary with 60 cm total length (50 cm to the detector window). Samples were separated at 22.5 kV and the separation running buffer was 200 mM glycine pH 1.8 (with HCl). The method compares favorably to HPLC methods (r,2 = 0.994 for SAM, r,2 = 0.998 for SAH) and has a mass detection limit of about 10 fmol for both SAM and SAH at a signal-to-noise ratio of 3. The method is linear over ranges of 1,100 ,M SAM and 1,250 ,M SAH. This method can be used to determine tissue concentrations of SAM and SAH, two metabolites that can provide insight into many biological processes. [source] The effects of ice storage on inosine monophosphate, inosine, hypoxanthine, and biogenic amine formation in European catfish (Silurus glanis) filletsINTERNATIONAL JOURNAL OF FOOD SCIENCE & TECHNOLOGY, Issue 10 2009Fatih Özogul Summary European catfish fillets in ice were evaluated by measuring nucleotide components and biogenic amine contents and these then compared with sensory and microbiological assessment during the 21 days of iced storage. Analyses were carried out using two different rapid HPLC methods for nucleotid degradation products and biogenic amine contents in European catfish fillets. Sensory evaluation showed that storage life of European catfish found to be 14,18 days. Initial inosine monophosphate (IMP) level was 12.6 ,mol g1 and then decreased during the rest of storage period. Inosine (INO) level increased rapidly until 7 days of storage. Hypoxanthine (Hx) level increased almost linearly with storage time. The most accumulated biogenic amines were putrescine, cadaverine, spermidine, spermine, and serotonin in all the European catfish fillets during the storage, although the formation of biogenic amines levels was fluctuated. Histamine was only detectable at 4 and 7 days of storage as low as 1 mg 100 g1 fish. Total viable count in European catfish increased rapidly with storage time and reached ,109 cfu g1 when the fillets were not acceptable for consumption. [source] Development of a Solid-Phase Extraction,Enzyme-Linked Immunosorbent Assay for the Determination of 17,-19-Nortestosterone Levels in Antifatigue Functional FoodsJOURNAL OF FOOD SCIENCE, Issue 8 2009Yan Zhang ABSTRACT:, 17,-19-nortestosterone (17,-NT) has been illegally used in antifatigue functional foods to promote muscle growth and improve endurance. A rapid and sensitive solid-phase extraction,enzyme-linked immunosorbent assay (SPE-ELISA) method was developed and successfully applied to analyze the levels of 17,-NT in antifatigue functional foods. A polyclonal antibody against 17,-NT was produced from rabbits immunized with the 17,-NT-BSA conjugate, and a competitive direct enzyme-linked immunosorbent assay was developed for the rapid detection of 17,-NT. The concentration causing 50% inhibition (IC50) and the limit of detection (LOD) were found to be 0.08 and 0.0055 ng/mL, respectively; this was better than methods previously reported that had a LOD of 2.4 ng/mL. C18 cartridges were investigated for use in removing the effects of matrix in foods, and the sample purification protocol was optimized. Using the developed SPE-ELISA method, recoveries of functional food samples were obtained in the range of 71% to 91.5%. Moreover, 2 kinds of antifatigue functional foods were analyzed using the established ELISA and HPLC methods. The correlation coefficient of the results obtained using the 2 methods was greater than 0.98. Thus, the preliminary evaluation of the SPE-ELISA method proved that it is a specific, sensitive, and precise tool that can be used for the practical detection of 17,-NT in various antifatigue functional food samples. [source] HPLC methods for the purification of [11C]-labelled radiopharmaceuticals: reversal of the retention order of products and precursorsJOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS, Issue 5 2009Szabolcs Lehel Abstract Preparative HPLC methods have been developed for a number of [11C]-methylated PET tracers, which enable elution of the labelled compounds prior to their precursors, thus reducing the overall synthesis time and avoiding contamination of the final product with precusor. This reversal of retention order has been achieved for [11C]DASB, [11C]raclopride, [11C]FLB 457, [11C]carfentanil, and 2-fluoro-[N -methyl- 11C]apomorphine, enabling collection of the purified radiopharmaceuticals from the HPLC system after 5,7,min. Furthermore, by using ethanol as the organic modifier, residual solvent analysis prior to human injection could be avoided and three of the radiopharmaceuticals could be injected directly following simple dilution and sterile filtration. Copyright © 2009 John Wiley & Sons, Ltd. [source] Radiosynthesis and in vivo study of [18F]1-(2-fluoroethyl)-4-[(4-cyanophenoxy)methyl]piperidine: a promising new sigma-1 receptor ligandJOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS, Issue 8 2005Jun Zhao Abstract The novel sigma-1 receptor PET radiotracer [18F]1-(2-fluoroethyl)-4-[(4-cyanophenoxy)methyl]piperidine ([18F]WLS1.002, [18F]-2) was synthesized (n=6) by heating the corresponding N -ethylmesylate precursor in an anhydrous acetonitrile solution containing [18F]fluoride, Kryptofix K222 and potassium carbonate for 15 min. Purification was accomplished by reverse-phase HPLC methods, providing [18F]-2 in 59±8% radiochemical yield (EOB), with specific activity of 2.89±0.80 Ci/µmol (EOS) and radiochemical purity of 98.3±2.1%. Rat biodistribution studies revealed relatively high uptake in many organs known to contain sigma-1 receptors, including the lungs, kidney, heart, spleen, and brain. Good clearance from normal tissues was observed over time. Blocking studies (60 min) demonstrated high (>80%) specific binding of [18F]-2 in the brain, with reduction also noted in other organs known to express these sites. Copyright © 2005 John Wiley & Sons, Ltd. [source] Solid phase peptide synthesis on epoxy-bearing methacrylate monolithsJOURNAL OF PEPTIDE SCIENCE, Issue 12 2004E. Vlakh Abstract Monoliths based on a copolymer of glycidyl methacrylate (GMA) and ethylene dimethacrylate (EDMA) can be used directly as sorbents for affinity chromatography after solid phase peptide synthesis. The quality of the synthesized products, the amount of grown peptides on a support and the reproducibility of the process must be considered. A determination of the quantity of the introducing ,-Ala (and, consequently, the total amount of synthesized peptide) was carried out. Three peptides complementary to recombinant tissue plasminogen activator (t-PA) have been synthesized using Fmoc-chemistry on GMA-EDMA disks. The peptidyl ligands were analysed by amino acid analysis, ES-MS and HPLC methods. The affinity binding parameters were obtained from frontal elution data. The results were compared with those established for GMA-EDMA affinity sorbents formed by the immobilization of the same but separately synthesized and purified ligands. The immobilization on GMA-EDMA disks was realized using a one-step reaction between the amino groups of the synthetic ligand and the original epoxy groups of monolithic material. The affinity constants found for two kinds of sorbent did not vary significantly. Finally, the directly obtained affinity sorbents were tested for t-PA separation from a cellular supernatant. Copyright © 2004 European Peptide Society and John Wiley & Sons, Ltd. [source] Nitrogen effects on total flavonoids, chlorogenic acid, and antioxidant activity of the medicinal plant Chrysanthemum morifoliumJOURNAL OF PLANT NUTRITION AND SOIL SCIENCE, Issue 2 2010Dahui Liu Abstract Chrysanthemum morifolium (Ramat.) has a long history of cultivation and use as a traditional medicine and tea plant in China. A greenhouse experiment with potted soil,quarz mixture studied the effects of nitrogen supply (0, 56, 112, 167, 224, 334, 501, 556, and 668 mg N,kg,1) on concentrations and ratios of total flavonoids and chlorogenic acid in the flowers of C. morifolium using spectrophotometric and HPLC methods. The antioxidant activity of the flowers was determined as the radical scavenging activities of hydroxyl, superoxide anion, and 2,2-diphenyl-1-picrylhydrazyl hydrate (DPPH) free radicals. A high N supply decreased the concentrations of total flavonoids by 18%,35% and that of chlorogenic acid by 8%,60% compared to a low N-supply rate. At the same time, increasing N supply significantly decreased the antioxidant activity of the flowers. The antioxidant activity of C. morifolium flowers was significantly positively correlated with the concentrations of total flavonoids and chlorogenic acid. We conclude that an N supply in excess of 300 mg (kg soil),1 will negatively affect the antioxidant activity and thereby reduce the quality of C. morifolium flowers. [source] High-performance liquid chromatography and capillary electrophoresis for the analysis of maize proteinsJOURNAL OF SEPARATION SCIENCE, JSS, Issue 2 2006José M. Rodriguez-Nogales Abstract Methods for the analysis of maize proteins using HPLC and CE are reviewed. Most of the references cited in this review concern HPLC methods. Size-exclusion HPLC and especially RP-HPLC methods have been developed for characterization of normal and genetically modified maize, cultivar differentiation, and prediction of quality. Few CE methods for the analysis of maize proteins were found in the existing literature. Most of these methods focus on optimization of the separation of maize proteins using CZE and SDS-capillary gel electrophoresis. [source] A comparative study of several HPLC methods for determining free amino acid profiles in honeyJOURNAL OF SEPARATION SCIENCE, JSS, Issue 9-10 2005José Luis Bernal Abstract A study of the viability of three derivatizing reagents for obtaining amino acid profiles in honey through high performance liquid chromatography (HPLC) is presented. A method using diode array detection based on a reaction with diethyl ethoxymethylene malonate (DEMM) and two other methods using fluorescence detection based on derivatization with fluorenylmethyl chloroformate (FMOC-Cl) and 6-aminoquinolyl- N -hydroxysuccinimidyl carbamate (AQC) have been developed. The three methods yield detection limits close to the ppb level, but vary in relation to other analytical characteristics. The use of methyl chloroformate derivatives allows the profile to be obtained with the greatest sensitivity within a short time frame. On applying such methods to honey samples of diverse botanical origin, we observe that the proline values obtained are always lower than those found using the official spectrophotometric method, thereby underlining the advisability of using HPLC methods to reduce uncertainty in these results. [source] Supercritical carbon dioxide extraction of sea buckthorn (Hippophae rhamnoides L.) pomaceJOURNAL OF THE SCIENCE OF FOOD AND AGRICULTURE, Issue 13 2007Dániel Cossuta Abstract BACKGROUND: The goal of this work was to utilize the sea buckthorn pomace, which is the by-product of a sea buckthorn juice process. Pilot plant supercritical fluid extraction (SFE) experiments were performed in a 5 × 10,3 m3 volume high-pressure vessel. The effects of pressure and temperature on extraction yield and recoveries of biologically active components were studied using a 32 full factorial design. The pressure and temperature were varied over the ranges of 30,46 MPa and 313,353 K, respectively. The extract samples were analysed by TLC-densitometry, UV/VIS spectrofotometry and HPLC methods. RESULTS: The obtained yields changed between 142,164 g kg,1, according to the solvent power of the supercritical fluid. The recoveries of the different minor components were (g minor components kg,1 dried raw material): 2.50,4.25 sitosterol, 0.20,1.60 ursolic acid, 0.04,0.18 carotenoid, 0.35,0.42 total tocopherol. CONCLUSION: By evaluation the designed experiments 46 MPa and 333 K were chosen as the optimum conditions. Copyright © 2007 Society of Chemical Industry [source] Simultaneous quantification of the main organic acids and carbohydrates involved in tomato flavour using capillary zone electrophoresisJOURNAL OF THE SCIENCE OF FOOD AND AGRICULTURE, Issue 10 2002Salvador Roselló Abstract A capillary zone electrophoresis (CZE) procedure for the simultaneous determination of the major organic acids (oxalate, malate and citrate) and carbohydrates (fructose, glucose and sucrose) in Lycopersicon fruits is reported. Comparison of this method with routine HPLC methods indicates that the CZE method offers several attractive features (speed, resolution, sensitivity and cost) which significantly improve the determination of these compounds. Detection limits were better than 1.6,µg,ml,1 for organic acids and from 13 to 24,µg,ml,1 for carbohydrates; repeatabilities were better than 2.1% for migration times and between 1.4 and 7.3% for peak areas. The proposed protocol is very useful to characterise large series of tomato samples not only in breeding programmes but also in systematic and routine analysis in the tomato industry. © 2002 Society of Chemical Industry [source] Determination of marker constituents from Cissus quadrangularis Linn. and their quantitation by HPTLC and HPLCPHYTOCHEMICAL ANALYSIS, Issue 2 2001Manisha Mehta Abstract Four marker constituents, namely, onocer-7-ene-3,,21,-diol, ,-amyrin, ,-amyrone and 3,3,,4,4,-tetrahydroxybiphenyl of an Ayurvedic crude drug Cissus quadrangularis Linn. are defined for standardisation purposes. 3,3,,4,4,-Tetrahydroxybiphenyl has been isolated for the first time from this drug. The contents of the marker constituents were quantitatively determined by HPTLC and HPLC methods in samples collected from five different geographic zones of India. Copyright © 2001 John Wiley & Sons, Ltd. [source] Inhibition of cytochrome P450 enzymes by rhein in rat liver microsomesPHYTOTHERAPY RESEARCH, Issue 2 2009Jing-cheng Tang Abstract Rhein, an active ingredient extensively found in plants such as Aloe, Cassitora L., rhubarb and so on, has been used for a long time in China. Pharmacological tests revealed that rhein not only had a strong antibacterial action, but also may be useful in cancer chemotherapy as a biochemical modulator. Its therapeutic action and toxicity is still the subject of considerable research. With microsome incubation assays in vitro and HPLC methods, the inhibition of rat liver CYP1A2, CYP2C9, CYP2D6, CYP2E1 and CYP3A enzymes by rhein were studied kinetically. The results showed the most inhibition of CYP2E1 by rhein (Ki = 10 µm, mixed); CYP3A and CYP2C9 were also inhibited by rhein, Ki = 30 µm (mixed) and Ki = 38 µm (mixed), respectively; rhein revealed some inhibition of CYP1A2 (Ki = 62 µm, uncompetitive) and CYP2D6 (Ki = 74 µm, mixed). Drug,drug interactions, especially cytochrome P450 (CYP)-mediated interactions, cause an enhancement or attenuation in the efficacy of co-administered drugs. Inhibition of the five major CYP enzymes observed for rhein suggested that changes in pharmacokinetics of co-administered drugs were likely to occur. Therefore, caution should be paid to the possible drug interaction of medicinal plants containing rhein and CYP substrates. Copyright © 2008 John Wiley & Sons, Ltd. [source] Pharmacokinetics of luteolin and tetra-acetyl-luteolin assayed by HPLC in rats after oral administrationBIOMEDICAL CHROMATOGRAPHY, Issue 8 2010Xiujie Chen Abstract Accurate and reproducible HPLC methods were developed and validated for the determination of concentrations of luteolin (LT) and tetra-acetyl-luteolin (TALT) in rat plasma. HPLC analyses were performed on an Agilent TC-C18 column protected by a guard Agilent Zorbax Eclipse Plus. The mobile phase for LT was a binary mixture of acetonitrile,water (40:60, v/v) containing 0.5% phosphoric acid at a flow rate of 1.0,mL/min, and that for TALT was a binary mixture of methanol,water (70,:,30, v/v) containing 0.5% glacial acetic acid at the same flow rate. The UV detection wavelength for both analytes was set at 350,nm. The calibration curve was linear over the range of 40,1800,ng/mL, the lower limit of quantitation was 40,ng/mL and the lower limit of detection was 20,ng/mL for both LT and TALT. The intra- and inter-day precision (RSD) values for all samples were within 7.9%. The concentration,time curves of LT and TALT after oral administration (30,mg/kg) were both fitted to a two-compartment model. The pharmacokinetic characteristics of TALT were better than that of LT in the maximum plasma concentration (Cmax) and the area under the concentration,time curve (AUC). Copyright © 2010 John Wiley & Sons, Ltd. [source] Monitoring of fluoroquinolone residual levels in chicken eggs by microbiological assay and confirmation by liquid chromatographyBIOMEDICAL CHROMATOGRAPHY, Issue 1 2008Hee-Jung Cho Abstract The primary objective of this study was to develop a simple, rapid, and efficient method for the simultaneous determination of four fluoroquinolone residues, ciprofloxacin (CFX), danofloxacin (DFX), enrofloxacin (EFX) and norfloxacin (NFX), in chicken eggs. The samples were first monitored by microbiological assay using Escherichia coli as the reference organism, and were then quantified using HPLC with a fluorescence detector. Egg samples were extracted by the liquid-phase extraction process, and the analytes were analyzed via an ODS column using a mixture of acetonitrile and 0.4% phosphoric acid,0.4% triethylamine (15: 85, v/v) as a mobile phase (pH = 2) without purification. The calibration curves were linear (r2 , 0.999) over a concentration range of 0.1,1.0 µg/mL. The majority of the mean recoveries at four different fortification levels, 0.1, 0.2, 0.5 and 1.0 ppm, ranged from 73.7 ± 7.2% to 87.1 ± 12.7%, and the repeatability (as the relative standard deviation) from three repetitive determinations of recovery was between 1.03 and 18.83%. The calculated limit of quantitation (LOQ) was 9 ppb for CFX, EFX and NFX and 0.6 ppb for DFX. Both the bioassay and HPLC methods were applied to 120 total egg samples collected from the six major cities in the Republic of Korea. The bioassay, showed that two samples were positive (i.e contained inhibiting substances). On the other hand, the results of HPLC only identified and quantified the residues of enrofloxacin (from 0.43 to 1.02 ppm) in three samples out of 120. We concluded that the bioassay can be used as a routine screening method for the presence of fluoroquinolones in chicken eggs, which can be confirmed and quantified using LC. Copyright © 2007 John Wiley & Sons, Ltd. [source] Determination of oleanolic acid, ursolic acid and amygdalin in the flower of Eriobotrya japonica Lindl. by HPLCBIOMEDICAL CHROMATOGRAPHY, Issue 7 2007Chunhua Zhou Abstract Simple and accurate HPLC methods were developed for the determination of oleanolic acid (OA), ursolic acid (UA) and amygdalin in loquat (Eriobotrya japonica Lindl.) flower, which is commonly used for the treatment of various diseases as a traditional Chinese medicine. HPLC assay was performed on a reversed-phase C18 column and all three compounds were detected at 210 nm with a flow rate of 1.0 mL/min. The mobile phase consisted of methanol (A) and 0.03 mol/L phosphate buffer (pH 2.8) (B) with a ratio of 88:12 (A:B, v/v) for simultaneous detection of OA and UA, and 25:75 (A:B, v/v) for detection of amygdalin. The established methods showed good precision and accuracy with overall intra-day and inter-day variation of 0.99,3.55 and 1.05,4.05%, respectively, and overall recoveries of 97.37,99.32% for the three compounds. Application of these methods to determine the OA, UA and amygdalin contents in loquat flower showed that cultivar had a minor effect on the contents of all three compounds, with average amounts of 0.38,0.51 mg OA/g dry weight (DW), 2.15,2.68 mg UA/g DW and 1.23,1.56 mg amygdalin/g DW among five loquat cultivars tested. However, developmental stages and flower tissues showed significant effect on the contents of all three bioactive components. Copyright © 2007 John Wiley & Sons, Ltd. [source] Separation and quantification of N -acetyl- l -cysteine and N -acetyl-cysteine-amide by HPLC with fluorescence detectionBIOMEDICAL CHROMATOGRAPHY, Issue 5 2006Wei Wu Abstract N- acetyl- l -cysteine (NAC) is a well-known antioxidant that is capable of facilitating glutathione (GSH) biosynthesis and replenishing intracellular GSH under oxidatively challenging circumstances. N- acetyl-cysteine-amide (NACA), the amide form of NAC, is a newly designed and synthesized thiol-containing compound which is believed to be more lipophilic and permeable through cell membranes than NAC. The metabolic and antioxidant effects of these compounds in vitro and in vivo are under investigation. However, an analytical method that can separate and quantify both compounds simultaneously is not yet available, to the best of our knowledge. Because of their structural similarities, the two compounds are difficult to separate using earlier HPLC methods which were designed for NAC quantification. Therefore, the goal of this work was to develop an HPLC method with fluorescence detection for simultaneous quantification of NAC and NACA in biological blood and tissue samples. A gradient HPLC program with fluorescence detection (,ex = 330 nm, ,em = 376 nm) using N -(1-pyrenyl)maleimide (NPM) as the derivatizing agent was developed. The calibration curves were linear over a concentration range of 25,5000 nm (r2 > 0.997). The coefficients of variation for within-run precision and between-run precision ranged from 0.67 to 5.23% and for accuracy ranged from 0.98 to 10.54%; the percentage relative recovery ranged from 94.5 to 102.8%. This new method provides satisfactory separation of NAC and NACA, along with other biological thiols, in 20 min with a 5 nm limit of detection (LOD) per 5 µL injection volume. Copyright © 2005 John Wiley & Sons, Ltd. [source] Column selection and method development for the separation of nucleoside phosphotriester diastereoisomers, new potential anti-viral drugs.BIOMEDICAL CHROMATOGRAPHY, Issue 6 2005Application to cellular extract analysis Abstract Analytical HPLC methods using derivatized cellulose and amylose chiral stationary phases used in normal and reversed-phase modes were developed for the diastereoisomeric separation of mononucleotide prodrugs (pronucleotides) of 3,-azido-2,,3,-dideoxythymidine (AZT). The resolutions were performed with two silica-based celluloses using normal and reversed-phase methodologies: Tris-3,5-dimethylphenylcarbamate (Chiralcel OD-H and Chiracel OD-RH) and Tris-methylbenzoate (Chiralcel OJ and OJ-R). Two amyloses phases, Tris-3,5-dimethylphenylcarbamate (Chiralpak AD) and Tris-(S)-1-phenylethylcarbamate (Chiralpak AS), were used in normal-phase mode. Additionally, we developed separation using two stationary phases with immobilized cyclodextrins in reversed-phase and polar-organic modes. The mobile phase and the chiral stationary phase were varied to achieve the best resolution. Different types and concentration of aliphatic alcohols, acetonitrile or water in the mobile phase were also tested for the different separation modes. An optimal baseline separation (Rs > 1.5) was readily obtained with all silica-based celluloses and amyloses using a normal-phase methodology. The different columns gave complementary results in term of resolution. Limits of detection and quantification were 0.12,0.20 and 0.40,0.67 µm, respectively. This analytical method was applied in a preliminary study for the pronucleotide 2 quantification in cellular extract. Copyright © 2005 John Wiley & Sons, Ltd. [source] Rapid determination of enzyme activities of recombinant human cytochromes P450, human liver microsomes and hepatocytesBIOPHARMACEUTICS AND DRUG DISPOSITION, Issue 9 2003Anima Ghosal Abstract Cytochrome P450 (CYP) substrates that yield fluorescent metabolites were used for rapid screening of drug metabolism activities of 13 recombinant human cytochromes P450, human liver microsomes and human hepatocytes. Reproducible results were obtained using a fluorescent plate reader (CytoFluor) more expediently than those generated using conventional HPLC methods. Typically, results for 96 samples were obtained with the plate reader in less than 10 min as opposed to 15,35 min/sample required by conventional HPLC. The fluorescent substrates used to measure CYP activities were as follows: 3-cyano-7-ethoxycoumarin (CEC) for CYP1A1, CYP1A2, CYP2C9 and CYP2C19; 7-ethoxyresorufin (7-ER) for CYP1A1, CYP1A2 and CYP1B1; 3-[2-(N,N -diethyl- N -methylammonium)ethyl]-7-methoxy-4-methylcoumarin (AMMC) for CYP2D6; dibenzylfluorescein (DBF) for CYP3A4, CYP3A5 and CYP2C8; 7-methoxy-4-trifluoromethylcoumarin (7-MFC) for CYP2E1, CYP2B6 and CYP2C18; and coumarin for CYP2A6. The chemical inhibition and correlation data indicated that the following substrates can be used as specific functional probes for individual cytochrome P450 present in human liver microsomes: coumarin for CYP2A6 (r=0.82), AMMC for CYP2D6 (r=0.83) and DBF for CYP3A4 (r=0.92). The fluorescent plate reader was found to be useful for the rapid assessment of CYP activities (positive control) in both intact cells and subcellular fractions. Copyright © 2003 John Wiley & Sons, Ltd. [source] Effect Of Uranyl Nitrate-Induced Renal Failure On Morphine Disposition And Antinociceptive Response In RatsCLINICAL AND EXPERIMENTAL PHARMACOLOGY AND PHYSIOLOGY, Issue 1-2 2000Jacoba T Van Crugten SUMMARY 1. The aims of the present study were to administer morphine (14.0 ,mol/kg, s.c.) to male Hooded Wistar rats and to determine the effect of uranyl nitrate-induced renal failure on: (i) the antinociceptive effect of morphine; (ii) the pharmacokinetics of morphine and morphine-3-glucuronide (M3G); and (iii) the relationship between antinociceptive effect and the pharmacokinetics of morphine in plasma and brain. 2. Renal failure was induced by a single s.c. injection of uranyl nitrate and kinetic/dynamic studies were performed 10 days after its administration, when creatinine clearance was 17% of the control group. Antinociceptive effect was measured by the tail-flick method at various times up to 2 h post-drug administration. Concentrations of morphine and M3G in plasma and brain and concentrations of creatinine in urine and serum were determined by specific HPLC methods. 3. After morphine administration, the area under the antinociceptive effect,time curve was decreased by 44% in renal failure rats. There were no differences between control and renal failure rats in: (i) plasma morphine concentration,time curves; (ii) brain morphine concentration,time curves; and (iii) plasma M3G concentration,time curves. Morphine-6-glucuronide was not detected in any plasma or brain sample from rats administered morphine and no M3G was detected in brain. 4. For both control and renal failure rats, the relationships between antinociceptive effect and plasma morphine concentration were characterized by counterclockwise hysteresis loops, probably reflecting a delay for the relatively polar morphine to cross the blood,brain barrier. The relationship between antinociceptive effect and brain morphine concentration in control rats revealed no evidence of acute tolerance and was described by a sigmoidal function. In contrast, the relationship in renal failure rats was characterized by clockwise hysteresis, which is consistent with acute tolerance development. [source] | |||||||||||||