Oryzalin,a dinitroaniline herbicide,binds to plant tubulin and inhibits microtubule polymerization in vitro

The effects of oryzalin, a dinitroaniline herbicide, on chromosome behavior and on cellular microtubules (MTs) were examined by light microscopy and immunogold staining, respectively, in endosperm cells from Haemanthus katherinae Bak. Brief treatments with 1.0·10^-8 M oryzalin reduced markedly the migration rate of anaphase chromosomes and 1.0·10^-7 M oryzalin stopped migration abruptly. Oryzalin (1.0·10^-7 M) depolymerized MTs and prevented the polymerization of new MTs at all stages of the mitotic cycle. The chromosome condensation cycle was unaffected by oryzalin. Endothelial cells from the heart of Xenopus leavis showed no chromosomal or microtubular rearrangements after oryzalin treatment. The inhibition by oryzalin of the polymerization of tubulin isolated from cultured cells of Rosa sp. cv. Paul's scarlet was examined in vitro by turbidimetry, electron microscopy and polymer sedimentation analysis. Oryzalin inhibited the rapid phase of taxol-induced polymerization of rose MTs at 24°C with an apparent inhibition constant (K _i ) of 2.59·10⁶ M. Shorter and fewer MTs were formed with increasing oryzalin concentrations, and maximum inhibition of taxol-induced polymerization occurred at approx. 1:1 molar ratios of oryzalin and tubulin. Oryzalin partially depolymerized taxol-stabilized rose MTs. Ligand-binding experiments with [¹⁴C]oryzalin demonstrated the formation of a tubulin-oryzalin complex that was time- and pH-dependent. The tubulin-oryzalin interaction (24°C, pH 7.1) had an apparent affinity constant (K _app) of 1.19·10⁵ M^-1. Oryzalin did not inhibit taxol-induced polymerization of bovinebrain MTs and no appreciable binding of oryzalin to brain tubulin or other proteins was detected. The results demonstrate pharmacological differences between plant and animal tubulins and indicate that the most sensitive mode of action of the dinitroaniline herbicides is the direct poisoning of MT dynamics in cells of higher plants.Abbreviations MT microtubule - SIB sucrose isolation buffer - TO tubulin-oryzalin complex     

Gas chromatographic determination of 2- and 3-dechloroethylifosfamide in plasma and urine

The metabolic oxidation of one of the chloroethyl groups of the antitumour drug ifosfamide leads to the formation of the inactive metabolites 2- and 3-dechloroethylifosfamide together with the neurotoxic metabolite chloroacetaldehyde. A very sensitive capillary gas chromatographic method, requiring only 50 μl of plasma or urine, has been developed to measure the amounts of the drug and the two inactive metabolites in a single run. Calibration curves were linear (r > 0.999) in the concentration ranges from 50 ng/ml to 100 μg/ml in plasma and from 100 ng/ml to 1 mg/ml in urine.     

Determination of benperidol and its reduced metabolite in human plasma by high-performance liquid chromatography and electrochemical detection

An isocratic high-performance liquid chromatographic method with electrochemical detection for the quantification of benperidol and its suggested reduced metabolite TVX Q 5402 in human plasma is described. The method included a two-step solid-phase extraction on reversed-phase and cation-exchange material, followed by separation on a cyanopropyl silica gel column (5 μm; 250 mm × 4.6 mm I.D.). The eluent was 0.15 M acetate buffer (pH 4.7) containing 25% acetonitrile (w/w). Spiperone served as internal standard. The inclusion of the cation-exchange step provided sample purity higher than those achieved with other methods. After extraction of 1 ml of plasma, concentrations as low as 0.5 ng/ml were detectable for both benperidol and the metabolite. In plasma samples collected from a schizophrenic patient treated with a single oral dose of 6 mg of benperidol, plasma levels of benperidol and of the metabolite could be measured from 20 min to at least 12 h after administration.     

High-performance liquid chromatography of the neuroactive steroids alphaxalone and pregnanolone in plasma using dansyl hydrazine as fluorescent label: application to a pharmacokinetic-pharmacodynamic study in rats

This report describes a rapid and sensitive analytical method for the quantification of the neuroactive steroids alphaxalone and pregnanolone in rat plasma using derivatization with dansyl hydrazine as fluorescent label. The method involves protein precipitation, alkaline derivatization and extraction of the compounds and internal standard pregnenolone with dichloromethane, followed by isocratic reversed-phase high-performance liquid chromatography on a 3-μm Microsphere C₁₈ column with fluorescence detection at wavelengths 332 nm and 516 nm for excitation and emission, respectively. The mobile phase consists of a mixture of 25 mM acetate buffer (pH 3.7)–acetonitrile (45:55, v/v for alphaxalone and 40:60, v/v for pregnanolone) with a flow-rate of 1 ml/min. The total run time was 35 min. In the concentration range of 0.010–10 μg ml⁻¹, the intra- and inter-assay coefficients of variation were less than 17% for both methods. In 50 μl plasma samples the corresponding limits of detection were 10 ng ml⁻¹ (signal-to-noise ratio=3). The utility of the analytical method was established by analyzing plasma samples from rats, which had received an intravenous administration of 5 mg kg⁻¹ alphaxalone or pregnanolone. Values for clearance, volume of distribution at steady state and terminal half life were 71.9 ml min⁻¹ kg⁻¹, 814 mg kg⁻¹ and 13.5 min for alphaxalone and 69.2 ml min⁻¹ kg⁻¹, 1638 ml kg⁻¹ and 27.8 min for pregnanolone, respectively. Due to its simplicity and sensitivity this method can be used on a routine basis for pharmacokinetic analysis of neuroactive steroids.     

Pharmacokinetics of H-Cicaprost in healthy volunteers

Cicaprost (5-{(E)-(1S,5S,6S,7R)-7-hydroxy-6-[(3S,4S)-3-hydroxy-4-methylnona-1,6-diinyl]-bicyclo[3.3.0]octan-3-yliden}-3-oxapentanoic acid, ZK, 96 480) is a novel PGI₂-derivative, which is chemically stable and not subject to metabolic degradation in rats and cynomolgus monkeys. The pharmacokinetics of Cicaprost were studied in six healthy volunteers (age: 54–74 y) after i.v. infusion (2.1 μ over 60 min) and p.o. dosage (7.6 μg) of the tritiated compound.All treatments were well-tolerated by the test subjects. At the end of the infusion plasma levels of 100 pg/ml were reached, declining biphasically with half-lives of 3–4 min and 64 ± 21 in. Total clearance was 3.8 ± 0.5 ml/min/kg. The oral dosage resulted in peak plasma levels of 251 ± 90 pg/ml occurring at 23 ± 5 min post dose. The terminal half-life in the plasma was 115 ± 30 in. Gastro-intestinal absorption and absolute bioavailability of Cicaprost was complete. After both routes of administration approx. 60 % of dose was excreted with the urine within 24 h, whereas fecal ³H-excretion lasted for several days and accounted for approx. 35 %. Radiochromatography revelaed that Cicaprost was metabolically stable in the plasma and urine. In the feces several degradation products were observed apart from approx. 30 % of the dose fraction being excreted unchanged by that route.The present results demonstrate that Cicaprost is an orally completely bioavailable, metabolically stable PGI₂-mimetic which may be an ideal candidate for oral therapy because of its pharmacokinetic characteristics.     

Comparison of [14C]oryzalin Uptake in Root Segments of a Sensitive and a Resistant Species

Uptake of the dinitroaniline herbicide oryzalin (3,5-dinitro-N⁴,N⁴-dipropylsulfamlamide) and its effect on root growth werestudied using 5 mm corn (Zea mays L.) and pea (Pisum sativum)root apices. Pea root growth was much less susceptible to oryzalinthan corn root growth. Uptake studies showed that pea root apicesalso accumulated much less [¹⁴C]oryzalin and had a lower bindingaffinity for this herbicide. [¹⁴C]oryzalin was not metabolizedin root apices from either species. Thus, the differential susceptibilityto oryzalin in the case of corn versus pea can be explained,at least in part, by differences in oryzalin uptake and accumulationby roots. Oryzalin, dinitroaniline herbicides, Zea mays, Pisum sativum     

Routine determination of flumequine in kidney tissue of pig using automated liquid chromatography

A high-performance liquid chromatographic assay is described as a routine analytical method for the determination of flumequine (FLU) and its hydroxylated metabolite (OH-FLU) in pig kidney tissue. Kidney samples (2 g) containing FLU and OH-FLU were extracted by liquid-liquid extraction with ethyl acetate (10 ml). Analytical separations were performed by reversed-phase HPLC with fluorometric detection at 252 nm excitation and 356 nm emission under gradient conditions. The mobile phase was acetonitrile-2.7·10⁻³ M oxalic acid in water (pH 2.5). The assay is specific and reproducible within the flumequine range of 0.050–2.5 μg/g and recovery at 0.050 μg/g was 94.8%.     

Purification of “colony-forming” cells from immature rat testis

Follicle stimulating hormone (FSH) enhances colony formation (as a result of reaggregation) by dissociated 10-day-old rat testis cells in primary culture. The purpose of this study was to examine various cytological characteristics of the FSH-responsive cells and develop techniques for their purification. The ability of testis cells to form colonies in response to FSH (5 μg/ml) was tested at various ages and was found to be maximal at 15 days of age. No colony formation occurred at ages greater than 20 days. Using colony formation as an assay for the FSH-responsive cells, techniques were developed for their purification. Colony cells were purified on a continuous bovine serum albumin (BSA) step gradient (1 ml/step). In fractions purified in this manner and subsequently cultured 24 h with FSH (5 μg/ml) 99.4% of attached cells were colony cells. Light and electron microscopy indicated that the colony cells (1) were one cell type; (2) were not germinal cells; (3) ultrastructurally resembled in situ Sertoli cell of the immature rat testis; and (4) contained a nucleolus with satellite karyosomes, structures which are characteristic of rat Sertoli cells. The mitotic index of the purified cells was 0.014% following 24 h in 10⁻⁵ M colchicine. Based on these data, it was concluded that the FSH responsive cells in culture are Sertoli cells.     

γ-Tocopherol biokinetics and transformation in humans

Background: The uptake and biotransformation of γ-tocopherol (γ-T) in humans is largely unknown. Using a stable isotope method we investigated these aspects of γ-T biology in healthy volunteers and their response to γ-T supplementation.

Methods: A single bolus of 100 mg of deuterium labeled γ-T acetate (d²-γ-TAC, 94% isotopic purity) was administered with a standard meal to 21 healthy subjects. Blood and urine (first morning void) were collected at baseline and a range of time points between 6 and 240 h post-supplemetation. The concentrations of d² and d⁰-γ-T in plasma and its major metabolite 2,7,8-trimethyl-2-(b-carboxyethyl)-6-hydroxychroman (-γ-CEHC) in plasma and urine were measured by GC-MS. In two subjects, the total urine volume was collected for 72 h post-supplementation. The effects of γ-T supplementation on α-T concentrations in plasma and α-T and γ-T metabolite formation were also assessed by HPLC or GC-MS analysis.

Results: At baseline, mean plasma α-T concentration was approximately 15 times higher than γ-T (28.3 vs. 1.9 µmol/l). In contrast, plasma γ-CEHC concentration (0.191 µmol/l) was 12 fold greater than α-CEHC (0.016 µmol/l) while in urine it was 3.5 fold lower (0.82 and 2.87 µmol, respectively) suggesting that the clearance of α-CEHC from plasma was more than 40 times that of γ-CEHC. After d²-γ-TAC administration, the d² forms of γ-T and γ-CEHC in plasma and urine increased, but with marked inter-individual variability, while the d⁰ species were hardly affected. Mean total concentrations of γ-T and γ-CEHC in plasma and urine peaked, respectively, between 0–9, 6–12 and 9–24 h post-supplementation with increases over baseline levels of 6–14 fold. All these parameters returned to baseline by 72 h. Following challenge, the total urinary excretion of d²-γ-T equivalents was approximately 7 mg. Baseline levels of γ-T correlated positively with the post-supplementation rise of (d⁰ + d²) – γ – T and γ-CEHC levels in plasma, but correlated negatively with urinary levels of (d⁰ + d²)-γ-CEHC. Supplementation with 100 mg γ-TAC had minimal influence on plasma concentrations of α-T and α-T-related metabolite formation and excretion.

Conclusions: Ingestion of 100mg of γ-TAC transiently increases plasma concentrations of γ-T as it undergoes sustained catabolism to CEHC without markedly influencing the pre-existing plasma pool of γ-T nor the concentration and metabolism of α-T. These pathways appear tightly regulated, most probably to keep high steady-state blood ratios α-T to γ-T and γ-CEHC to α-CEHC.     

Rapid determination of chloroprocaine and its major metabolite, 2-chloroaminobenzoic acid, in plasma by high-performance liquid chromatography

A sensitive and specific high-performance liquid chromatographic method for determination of the 2-chloroprocaine, local anesthetic of ester type, and its major metabolite 2-chloroaminobenzoic acid, has been developed and validated. A single-step extraction procedure is employed followed by high-performance liquid chromatographic separation using reversed-phase column and analysis using variable length UV detection. Lidocaine was used as internal standard for 2-chloroprocaine measurement and p-aminobenzoic acid was used as internal standard for 2-chloroaminobenzoic acid analysis. The analysis of spiked plasma demonstrated good accuracy and precision of the method with limit of detection 0.1 μg/ml for 2-chloroprocaine and 0.5 μg/ml for 2-chloroaminobenzoic acid. The method has been used for pharmacokinetic studies in laboratory animals.     

Sulfated Polysaccharides Are Required for Collagen-Induced Vascular Tube Formation

We have previously shown that soluble type I collagen can induce vascular tube formation when it contacts the apical side of a confluent endothelial monolayer. In this study we have examined which soluble agent(s) are required for collagen-induced tube formation. Human neo-natal foreskin microvascular endothelial cells, maintained in basal medium, were preincubated with each test agent for 2 h prior to the addition of solubilised type I collagen (100 μg/ml). After 6 h, tube formation was quantitated using image analysis and expressed as the mean area of tube formation (mm²) per microscopic field of view. Collagen-induced tube formation did not occur in the presence of endothelial cell growth supplement, basic fibroblast growth factor, or normal pooled human serum. In contrast, the addition of heparin at 5 or 50 μg/ml caused extensive tube formation (0.22 ± 0.07 and 0.30 ± 0.12 mm², respectively) whereas at 500 μg/ml little tube formation occurred (0.03 ± 0.02 mm²). Protamine sulfate, an antagonist of heparin, inhibited collagen-induced tube formation in a dose-dependent manner. Pentosan polysulfate, dextran sulfate, heparan sulfate, and chondroitin sulfate mimicked the action of heparin. Partially sulfated heparin (de-N-sulfated heparin) stimulated less tube formation compared to heparin (0.15 ± 0.06 mm² at 50 μg/ml). The nonsulfated polysaccharides, xylan and dextran, had no effect on tube formation. In summary, sulfated polysaccharides are required for collagen-induced vascular tube formation in vitro. The sulfation of these molecules appears to be vital for collagen-induced tube formation.     

Determination of cibenzoline in plasma and urine by high-performance liquid chromatography

A rapid, sensitive and selective high-performance liquid chromatographic (HPLC) assay was developed for the determination of cibenzoline (Cipralan ^TM) in human plasma and urine. The assay involves the extraction of the compound into benzene from plasma or urine buffered to pH 11 and HPLC analysis of the residue dissolved in acetonitrile---phosphate buffer (0.015 mol/1, pH 6.0) (80:20). A 10-μ ion-exchange (sulfonate) column was used with acetonitrile—phosphate buffer (0.015 mol/1, pH 6.0) (80:20) as the mobile phase. UV detection at 214 nm was used for quantitation with the di-p-methyl analogue of cibenzoline as the internal standard.The recovery of cibenzoline in the assay ranged from 60 to 70% and was validated in human plasma and urine in the concentration range of 10–1000 ng/ml and 50–5000 ng/ml, respectively. A normal-phase HPLC assay was developed for the determination of the imidazole metabolite of cibenzoline. The assays were applied to the determination of plasma and urine concentrations of cibenzoline and trace amounts of its imidazole metabolite following oral administration of cibenzoline succinate to two human subjects.     

High-performance liquid chromatographic determination of tianeptine in plasma applied to pharmacokinetic studies

An improved analytical method for the quantitative measurement of tianeptine and its main metabolite MC₅ in human plasma was designed. Extraction involved ion-paired liquid–liquid extraction of the compounds from 1.0 ml of human plasma adjusted to pH 7.0. HPLC separation was performed using a Nucleosil C₁₈, 5 μm column (150×4.6 mm I.D.) and a mixture of acetonitrile and pH 3, 2.7 g l⁻¹ solution of sodium heptanesulfonate in distilled water (40:60, v/v) as mobile phase. UV detection was performed using a diode array detector in the 200–400 nm passband, and quantification of the analytes was made at 220 nm. For both tianeptine and MC₅ metabolite, the limit of quantitation was 5 μg l⁻¹ and the calibration curves were linear from 5 to 500 μg l⁻¹. Intra- and inter-assay precision and accuracy fulfilled the international requirements. The recovery of tianeptine and its metabolite from plasma was, respectively, 71.5 and 74.3% at 20 μg l⁻¹, 71.2 and 70.8% at 400 μg l⁻¹. The selectivity of the method was checked by verifying the absence of chromatographic interference from pure solutions of the most commonly associated therapeutic drugs. This method, validated according to the criteria established by the Journal of Chromatography B, was applied to the determination of tianeptine and MC₅-metabolite in human plasma in pharmacokinetic studies.     

Effects of salinity on solute clearance from the freshwater bivalve, Dreissena polymorpha Pallas

Clearance of polyethylene glycol (PEG), inulin, or dextran that had been injected into the hemolymph of the mussel, Dreissena polymorpha, was measured in animals acclimated to pondwater (PW) or 10% seawater (SW). In addition, we measured the clearance of PEG from mussels acutely transferred into 10% SW and following return to PW after acclimation to 10% SW. Clearance values calculated for PW-acclimated mussels ranged from 2.0 to 3.3 ml (g dry tissue ċ h)^-1 and declined to 0.28 ml (g dry tissue ċ h)^-1 in 10% SW-acclimated animals. Transferring mussels into 10% SW resulted in a reduction in PEG clearance from the blood, coincident with the reduction of osmotic gradient. When 10% SW-acclimated mussels were returned to PW the clearance of PEG increased to rates observed in PW-acclimated animals within 1 h. The PEG clearance remained constant during the re-acclimation to PW even though the osmotic gradient declined from about 100 to 30 mosmol kg^-1. Clearance of the solutes used in this study was likely to be a measurement of renal filtration rate. The clearance values appeared to be maximal when the animals were in PW. The limited capacity to increase clearance in the face of an osmotic challenge may be a critical factor in restricting D. polymorpha to freshwater or lower salinity environments with small ranges in salinity.     

Measurement of plasma uracil using gas chromatography–mass spectrometry in normal individuals and in patients receiving inhibitors of dihydropyrimidine dehydrogenase

A sensitive gas chromatographic–mass spectrometric method is described for reliably measuring endogenous uracil in 100 μl of human plasma. Validation of this assay over a wide concentration range, 0.025 μM to 250 μM (0.0028 μg/ml to 28 μg/ml), allowed for the determination of plasma uracil in patients treated with agents such as eniluracil, an inhibitor of the pyrimidine catabolic enzyme, dihydropyrimidine dehydrogenase. Calibration standards were prepared in human plasma using the stable isotope, [¹⁵N₂]uracil, to avoid interference from endogenous uracil and 10 μM 5-chlorouracil was added as the internal standard.     

Zooplankton-induced unicell-colony transformation in Scenedesmus acutus and its effect on growth of herbivore Daphnia

The effects of colonial ecomorphs of the green alga Scenedesmus acutus on growth of Daphnia cucullata and D. pulex were examined. In ecologically relevant densities (up to 200 animals l^-1) the relatively small D. cucullata did not induce colony formation in Scenedesmus acutus, whereas the larger congener D. pulex significantly promoted colony formation. Both clearance rate and population growth rate (r) were significantly lower in D. cucullata when fed colonial Scenedesmus then when fed unicellular food. However, for D. pulex no effects of food type were observed. These results show that large Daphnia may influence its food in such a way that smaller congeners and competitors are negatively affected.     

Simultaneous determination of the camptothecin analogue CPT-11 and its active metabolite SN-38 by high-performance liquid chromatography: application to plasma pharmacokinetic studies in cancer patients

CPT-11 {I; 7-ethyl-10-[4-(1-piperidino)-1-piperidino]carbonyloxycamptothecin} is a new anticancer agent currently under clinical development. A sensitive high-performance liquid chromatographic assay suitable for the simultaneous determination of I and its active metabolite SN-38 (II) in human plasma, and their preliminary clinical pharmacokinetics, are described. Plasma samples were processed using a solid-phase (C₁₈) extraction step allowing mean recoveries of I, II and the internal standard camptothecin (III) of 84, 99 and 72%, respectively. The extracts were chromatographed on a C₁₈ reversed-phase column with a mobile phase composed of acetonitrile, phosphate buffer and heptanesulphonic acid, with fluorescence detection. The calibration graphs were linear over a wide range of concentrations (1 ng/ml–10 μg/ml), and the lower limit of determination was 1 ng/ml for both I and II. The method showed good precision: the within-day relative standard deviation (R.S.D.) (5–1000 ng/ml) was 13.0% (range 4.9–19.4%) for I and 12.8% (6.7–19.1%) for II; the between-day R.S.D. (5–10 000 ng/ml was 7.9% (5.4–17.5%) for I and 9.7% (3.5–15.1%) for II. Using this assay, plasma pharmacokinetics of both I and II were simultaneously determined in three patients receiving 100 mg/m² I as a 30-min intravenous infusion. The mean peak plasma concentration of I at the end of the intravenous infusion was 2400 ± 285 ng/ml (mean ± standard error of the mean). Plasma decay was triphasic with half-lives α, β and γ of 5.4 ± 1.8 min, 2.5 ± 0.5 h and 20.2 ± 4.6 h, respectively. The volume of distribution at steady state was 105 ± 15 l/m², and the total body clearance was 12.5 ± 1.9 l/h · m². The maximum concentrations of the active metabolite II reached 36 ± 11 ng/ml.     

High-performance liquid chromatography analysis, preliminary pharmacokinetics, metabolism and renal excretion of methylprednisolone with its C6 and C20 hydroxy metabolites in multiple sclerosis patients receiving high-dose pulse therapy

A gradient eluent HPLC analysis in human plasma and urine was developed and validated for methylprednisolone (MP), its prodrug methylprednisolone-21-hemisuccinate (MPS) with the metabolites 6β-hydroxy-6α-methylprednisolone (MPA), 20-hydroxymethylprednisolone (MPC), 6β-hydroxy-20α-hydroxymethylprednisolone (MPB), 6β-hydroxy-20β-hydroxymethylprednisolone (MPE), 20-carboxymethylprednisolone (MPD), methylprednisolone-glucuronide (MPF) and 21-carboxymethylprednisolone (MPX). The column was Cp Spherisorb C8 5 μm, 250 mm×4.6 mm I.D. (Chrompack, Bergen op Zoom, The Netherlands) with a guard column 75 mm×2.1 mm, packed with pellicular reversed-phase. The eluent was a mixture of acetonitrile and 0.067 M KH₂PO₄ buffer, pH 4.5. At t=0, the eluent consisted of 2% acetonitrile and 98% buffer (v/v). Over the following 35 min the eluent changed linearly until it attained a composition of 50% acetonitrile and 50% buffer (v/v). At 37 min (t=37) the eluent was changed over 5 min to the initial composition, followed by equilibration over 3 min. The flow-rate was 1.5 ml/min and UV detection was achieved at 248 nm. Preliminary pharmacokinetic data were obtained from one patient who showed illustrative plasma concentration–time curves and renal excretion-time profiles after a short-lasting infusion (0.5 h) of 1 g of methylprednisolone hemisuccinate. The half-life of prodrug methylprednisolone-21-hemisuccinate (MPS) was 0.3 h, that of metabolite MPX (21-carboxy MP) was 0.4 h and that of the parent drug methylprednisolone (MP) was 1.4 h. The half-lives of the metabolites are almost similar (4 h). The main compounds in the urine are methylprednisolone hemisuccinate (prodrug, 15.0%), methylprednisolone (parent drug, 14.6%), metabolite MPD (20-carboxy, 11.7%), and metabolite MPB (13.2%). The renal clearance values of metabolites MPB, MPC and MPD are approximately 500 ml/min, that of MP is 100 ml/min.     

High-performance liquid chromatographic analysis of sulfinpyrazone and its metabolites in biological fluids

A rapid, sensitive, and specific high-performance liquid chromatographic method is described for the quantitative analysis of sulfinpyrazone and its sulfone and p-hydroxy metabolites in plasma and urine. The method uses two different procedures for sample preparation: (1) a rapid and convenient procedure using a single extraction with 1-chlorobutane and subsequent back-extraction into sodium hydroxide solution for the analysis of sulfinpyrazone and its sulfone metabolite, and (2) a more time consuming procedure using triple extraction with ethylene dichloride, a buffer wash, and back extraction into the base for the additional analysis of the p-hydroxy metabolite. The lower limit of sensitivity for sulfinpyrazone is 50 ng/ml. Concentrations of sulfinpyrazone between 0.05 to 0.1 and 50 μg/ml were measured with an average coefficient of variation of 3.9%, ranging from 1.5 to 6.1%.     

High-performance liquid chromatographic determination of trimebutine and its major metabolite, N-monodesmethyl trimebutine, in rat and human plasma

A rapid, selective and very sensitive ion-pairing reversed-phase HPLC method was developed for the simultaneous determination of trimebutine (TMB) and its major metabolite, N-monodesmethyltrimebutine (NDTMB), in rat and human plasma. Heptanesulfonate was employed as the ion-pairing agent and verapamil was used as the internal standard. The method involved the extraction with a n-hexane–isopropylalcohol (IPA) mixture (99:1, v/v) followed by back-extraction into 0.1 M hydrochloric acid and evaporation to dryness. HPLC analysis was carried out using a 4-μm particle size, C₁₈-bonded silica column and water–sodium acetate–heptanesulfonate–acetonitrile as the mobile phase and UV detection at 267 nm. The chromatograms showed good resolution and sensitivity and no interference of plasma. The mean recoveries for human plasma were 95.4±3.1% for TMB and 89.4±4.1% for NDTMB. The detection limits of TMB and its metabolite, NDTMB, in human plasma were 1 and 5 ng/ml, respectively. The calibration curves were linear over the concentration range 10–5000 ng/ml for TMB and 25–25000 ng/ml for NDTMB with correlation coefficients greater than 0.999 and with within-day or between-day coefficients of variation not exceeding 9.4%. This assay procedure was applied to the study of metabolite pharmacokinetics of TMB in rat and the human.