Characterization of antioxidants present in hawthorn fruits

Hawthorn fruit extract has been shown to have many health benefits including being cardiovascular protective, hypotensive and hypocholesterolemic. The present study was carried out to characterize further the antioxidants of hawthorn fruit and their effect on the oxidation of human low density lipoprotein (LDL) and alpha-tocopherol. The dry hawthorn fruit was extracted successively with ether, ethyl acetate, butanol and water. The ethyl acetate fraction was only effective in inhibition of Cu(+2)-mediated LDL oxidation. The column chromatographic separation led to isolation of eight pure compounds; namely, ursolic acid, hyperoside, isoquercitrin, epicatechin, chlorogenic acid, quercetin, rutin and protocatechuic acid. All of these phenolic compounds, except ursolic acid, were protective to human LDL from Cu(+2)-mediated LDL oxidation. They were also effective in preventing the peroxy free radical-induced oxidation of alpha-tocopherol in human LDL. The inhibitory effect of these compounds on oxidation of LDL and alpha-tocopherol was dose-dependent at concentrations ranging from 5 to 40 μM. In addition, supplementation of 2% hawthorn fruit powder significantly elevated serum alpha-tocopherol by 18-20% in rats fed a 30% polyunsaturated canola oil diet, as compared with the control. The present results suggest that part of the mechanism for cardiovascular protective effects of hawthorn fruit might also involve the direct protection to human LDL from oxidation or indirect protection via maintaining the concentration of alpha-tocopherol in human LDL.     

Simultaneous determination of felbamate and three metabolites in rat and dog plasmas by high-performance liquid chromatography

An isocratic liquid chromatographic method employing one extraction step and a 150 mm × 4.6 mm I.D. Spherisorb ODS2, 3-μm HPLC column using UV-absorbance detection at 210 nm has been developed for the quantitation of felbamate and three felbamate metabolites in 0.100-ml aliquots of rat and dog plasmas. The linear quantitation range in rat plasma is 0.195–200 μg/ml for felbamate; 1.563–200 μg/ml for the p-hydroxy metabolite; 0.391–200 μg/ml for the 2-hydroxy metabolite; and 0.098–200 μg/ml for the monocarbamate metabolite. The linear quantitation range in dog plasma is 0.195–200 μg/ml for felbamate; 0.781–200 μg/ml for the p-hydroxy metabolite; 0.195–200 μg/ml for the 2-hydroxy metabolite; and 0.098–200 μg/ml for the monocarbamate metabolite.     

Intestinal absorption of hawthorn flavonoids--in vitro, in situ and in vivo correlations

Our previous studies identified hyperoside (HP), isoquercitrin (IQ) and epicatechin (EC) to be the major active flavonoid components of the hawthorn phenolic extract from hawthorn fruits demonstrating inhibitory effect on in vitro Cu(+2)-mediated low density lipoproteins oxidation. Among these three hawthorn flavonoids, EC was the only one detectable in plasma after the oral administration of hawthorn phenolic extract to rats. The present study aims to investigate the intestinal absorption mechanisms of these three hawthorn flavonoids by in vitro Caco-2 monolayer model, rat in situ intestinal perfusion model and in vivo pharmacokinetics studies in rats. In addition, in order to investigate the effect of the co-occurring components in hawthorn phenolic extract on the intestinal absorption of these three major hawthorn flavonoids, intestinal absorption transport profiles of HP, IQ and EC in forms of individual pure compound, mixture of pure compounds and hawthorn phenolic extract were studied and compared. The observations from in vitro Caco-2 monolayer model and in situ intestinal perfusion model indicated that all three studied hawthorn flavonoids have quite limited permeabilities. EC and IQ demonstrated more extensive metabolism in the rat in situ intestinal perfusion model and in vivo study than in Caco-2 monolayer model. Moreover, results from the Caco-2 monolayer model, rat in situ intestinal perfusion model as well as the in vivo pharmacokinetics studies in rats consistently showed that the co-occurring components in hawthorn phenolic extract might not have significant effect on the intestinal absorption of the three major hawthorn flavonoids studied.     

Simultaneous determination of zidovudine and its monophosphate in mouse plasma and peripheral red blood cells by high-performance liquid chromatography

Novel prodrugs for the intracellular delivery of zidovudine monophosphate (AZTMP) have recently been designed. To investigate the bioconversion and pharmacokinetic profiles of these compounds, an analytical method for the simultaneous determination of zidovudine (AZT) and AZTMP in mouse plasma and peripheral red blood cells was developed. Mouse whole blood samples were treated with TBAHS, EDTA and NaH₂PO₄, and separated into plasma and red blood cell portions. Samples were processed by solid-phase extraction using Bond Elut C₁₈ cartridges. Chromatography was performed using an Hypersil ODS column and a mobile phase of 2.9% (v/v) acetonitrile and 97.1% (v/v) phosphate buffer, pH 7.50, with UV detection at 267 nm. The average extraction recoveries of AZTMP and AZT in plasma were approximately 85% and 97% over their linear ranges of 0.05–5 μg/ml and 0.125–25 μg/ml, respectively. Extraction recoveries of AZTMP and AZT from peripheral red blood cells averaged 56 and 69% over their linear ranges of 0.125–5 μg/ml and 0.125–25 μg/ml, respectively. The accuracy of the assay was 90–100%. The intra- and inter-day variations of the assay were less than 14%. The analytical method was found to be applicable, reliable and suitable for pharmacokinetic studies.     

Simple and rapid determination of irinotecan and its metabolite SN-38 in plasma by high-performance liquid-chromatography: application to clinical pharmacokinetic studies

Irinotecan (CPT-11) is an anticancer agent widely employed in the treatment of colorectal carcinoma. A simple, rapid and sensitive high-performance liquid chromatographic method for the simultaneous determination of CPT-11 and its metabolite SN-38 in plasma, and their preliminary clinical pharmacokinetics are described. Both deproteinisation of plasma specimens (100 μl) and addition of the internal standard, camptothecin (CPT), are achieved by incorporating to samples 100 μl of a solution of CPT (1 μg/ml) in acetonitrile–1 mM orthophosphoric acid (90:10); 200 μl of this acidified acetonitrile solution, drug-free, is also added to accomplish complete deproteinisation: this procedure reduces sample preparation time to a minimum. After deproteinisation, samples are treated with potassium dihydrogenphosphate (0.1 M) and injected into a Nucleosil C₁₈ (5 μm, 250×4.0 mm) column. Mobile phase consists of potassium dihydrogenphosphate (0.1 M)–acetonitrile (67:33), at a flow-rate of 1 ml/min. CPT-11, SN-38 and CPT are detected by fluorescence with excitation wavelength set at 228 nm and emission wavelengths of CPT-11, SN-38 and CPT fixed, respectively, at 450, 543 and 433 nm. The limits of quantitation for CPT-11 and SN-38 are 1.0 and 0.5 ng/ml, respectively. This method shows good precision: the within day relative standard deviation (RSD) for CPT-11 (1–10 000 ng/ml) is 5.17% (range 2.15–8.27%) and for SN-38 (0.5–400 ng/ml) is 4.33% (1.32–7.78%); the between-day RSDs for CPT-11 and SN-38, in the previously described ranges, are 6.82% (5.03–10.8%) and 4.94% (2.09–9.30%), respectively. Using this assay, plasma pharmacokinetics of CPT-11, SN-38 and its glucuronidated form, SN-38G, have been determined in one patient receiving 200 mg/m² of CPT-11 as a 90 min intravenous infusion. The peak plasma concentration of CPT-11 at the end of the infusion is 3800 ng/ml. Plasma decay is biphasic with a terminal half-life of 11.6 h. The volume of distribution at steady state (V_ss) is 203 l/m², and the total body clearance (Cl) is 14.8 l/h·m². The maximum concentrations of SN-38 and SN-38G reach 28.9 and 151 ng/ml, respectively.     

Applied environmental stresses to enhance the levels of polyphenolics in leaves of hawthorn plants

In this investigation, two species of Crataegus (hawthorn) were chosen because their polyphenolic constituents have recently received greater attention for the treatment of patients with severe heart disease. One-year-old plants of hawthorn ( Crataegus laevigata and C. monogyna ) were subjected to water-deficit (continuous water deprivation), cold (4°C), flooding (immersion of roots of plants in water) or herbivory (leaf removal) stress treatments (each of 10 days duration) in order to assess their effects on levels of polyphenolics, namely (-)-epicatechin, catechin, chlorogenic acid, vitexin, vitexin-2'- O -rhamnoside, acetylvitexin-2'- O -rhamnoside, hyperoside, quercetin, and rutin in the leaves. The working hypothesis followed is that one or more of these stress treatment will elicit increases in the levels of these polyphenolics. Cold stress causes increases in levels of vitexin-2'- O -rhamnoside, acetylvitexin-2'- O -rhamnoside, hyperoside, and quercetin in both Crataegus species. Water-deficit stress increased the productivity of chlorogenic acid, catechin, and (-)-epicatechin in both hawthorn species. Flooding and herbivory caused no net increases, and in some cases, decreases in levels of polyphenolics. These studies indicate that either water-deficit stress or cold stress treatments, or a combination of the two, can be used to enhance the levels of desired polyphenolics in the leaves of these two hawthorn species in a photobioreactor system. These results may have significance for hawthorn in adapting to water-deficit or cold stress and are important considerations for the use of hawthorn in the treatment of heart disease in humans.     

Simultaneous determination of felbamate and four metabolites in rat cerebrospinal fluid by high-performance liquid chromatography

An isocratic liquid chromatographic method for direct sample injection has been developed for the quantitation of felbamate and four metabolites in rat cerebrospinal fluid. The method uses 0.050- or 0.025-ml aliquots of cerebrospinal fluid diluted with equal volumes of internal standard. Chromatography is performed on a 150 mm × 4.6 mm I.D. Spherisorb ODS2, 3-μm HPLC column eluted with a phosphate buffer—acetonitrile—methanol (820:120:60, v/v/v) mobile phase and ultraviolet absorbance detection at 210 nm. The linear quantitation ranges are: felbamate and the 2-hydroxy metabolite 0.195–200 μg/ml, the propionic acid metabolite 0.195–50.0 μg/ml, the p-hydroxy metabolite 0.781 to 50.0 μg/ml, and the monocarbamate metabolite 0.098–50.0 μg/ml.     

Chemical Characterization and Multidirectional Biological Effects of Different Solvent Extracts of Arum elongatum: in Vitro and in Silico Approaches

Arum elongatum (Araceae) is widely used traditionally for the treatment of abdominal pain, arterial hypertension, diabetes mellitus, rheumatism and hemorrhoids. This study investigated the antioxidant properties, individual phenolic compounds, total phenolic and total flavonoid contents (HPLC/MS analysis), reducing power and metal chelating effects of four extracts obtained from A. elongatum (ethyl acetate (EA), methanol (MeOH), methanol/water (MeOH/water) and infusion). The inhibitory activity of the extracts were also determined against acetylcholinesterase, butyrylcholinesterase, tyrosinase, amylase and glucosidase enzymes. The MeOH/water extracts contained the highest amount of phenolic contents (28.85 mg GAE/g) while the highest total flavonoid content was obtained with MeOH extract (36.77 mg RE/g). MeOH/water demonstrated highest antioxidant activity against DPPH⋅ radical at 38.90 mg Trolox equivalent per gram. The infusion extract was the most active against ABTS⁺⋅ (133.08 mg TE/g). MeOH/water extract showed the highest reducing abilities with the CUPRAC value of 102.22 mg TE/g and the FRAP value of 68.50 mg TE/g. A strong metal chelating effect was observed with MeOH/water extract (35.72 mg EDTAE/g). The PBD values of the extracts ranged from 1.01 to 2.17 mmol TE/g. EA extract displayed the highest inhibitory activity against AChE (2.32 mg GALAE/g), BChE (3.80 mg GALAE/g), α-amylase (0.56 mmol ACAE/g) and α-glucosidase (9.16 mmol ACAE/g) enzymes. Infusion extract was the most active against tyrosinase enzyme with a value of 83.33 mg KAE/g. A total of 28 compounds were identified from the different extracts. The compounds present in the highest concentration were chlorogenic acids, 4-hydroxybenzoic acid, caffeic acid, p-coumaric acid, ferulic acid, isoquercitrin, delphindin 3,5-diglucoside, kaempferol-3-glucoside and hyperoside. The biological activities of A. elongatum extracts could be due to the presence of compounds such as gallic acid, chlorogenic acids, ellagic acid, epicatechin, catechin, kaempferol, 4-hydroxybenzoic acid, caffeic acid, p-coumaric acid, ferulic acid, quercetin, isoquercitrin, and hyperoside. Extracts of A. elongatum showed promising biological activities which warrants further investigations in an endeavor to develop biopharmaceuticals.     

On-line microdialysis coupled with microbore liquid chromatography for the determination of unbound chloramphenicol and its glucuronide in rat blood

On-line microdialysis coupled with microbore liquid chromatography was used to investigate the pharmacokinetics of chloramphenicol and its glucuronide in rat blood. A microdialysis probe was inserted into a jugular vein of male Sprague–Dawley rats. Chloramphenicol succinate (20 mg/kg, intravenously) was then administered via a femoral vein. Dialysates were automatically injected onto a LC system, via an on-line injector. Samples were eluted with a mobile phase containing acetonitrile-10 mM monochloroacetic acid (30:70, v/v, pH 3.0). The UV detector wavelength was set at 278 nm. The limit of quantitation for chloramphenicol was 10 ng/ml. The in vitro recoveries of chloramphenicol and chloramphenicol glucuronide at 500 ng/ml were 32.2±0.3% and 11.4±0.7%, respectively (n=6). Intra- and inter-assay accuracy and precision of the analyses were ≤10% in the range of 0.01 to 5.0 μg/ml.     

Development of a simplified, sensitive high-performance liquid chromatographic method using fluorescence detection to determine the concentration of UCN-01 in human plasma

UCN-01 is a naturally derived anticancer agent isolated in the culture broth of actinomyces streptomyces. We have developed a sensitive high-performance liquid chromatographic method for the determination of UCN-01 in human plasma. UCN-01 was isolated from human plasma after intravenous administration, by using 100% ice-cold acetonitrile liquid–liquid phase extraction. Liquid chromatographic separation was achieved by isocratic elution on a phenyl analytical column. The mobile phase consisted of acetonitrile–0.5 M ammonium acetate (45:55) with 0.2% triethylamine added as a modifier. The UCN-01 peak was identified from other peaks using fluorescence excitation energy and emission energy wavelengths of 310 and 410 nm, respectively. Retention time for UCN-01 was 4.2±0.5 min. The UCN-01 peak was baseline resolved, with nearest peak at 2.6 min distance. No interfering peaks were observed at the retention time of UCN-01. Peak area amounts from extracted samples were proportional over the dynamic concentration range used: 0.2 to 30 μg/ml. Mean recoveries of UCN-01 at concentrations of 0.5 and 25 μg/ml were 89 and 90.2%, respectively. Relative standard deviations for UCN-01 calibration standards ranged from 1.89 to 2.31%, with relative errors ranging from 0.3 to 11.6%. Assay precision for UCN-01 based on quality control samples of 0.50 μg/ml was ±4.86% with an accuracy of ±5.7%. For drug extracted from plasma the lowest limit of detection was 0.1 μg/ml, with the lowest limit of quantitation being 0.2 μg/ml. This method is suitable for routine analysis of UCN-01 in human plasma at concentration from 0.2 to 30 μg/ml.     

High-performance liquid chromatographic assay for cefepime in serum

A simple, rapid, specific and sensitive high-performance liquid chromatographic method was developed for the determination of cefepime 1-[[6R, 7R)-7-[2-(2-amino-4-thiazolyl)glyoxylamido]-2-carboxy-8-oxo-5-thia-1-azabicyclo-[4.2.0] oct-2-en-3-yl]methyl]-1-methylpyrrolidinium hydroxide, inner salt, 7²-(Z)-(O-methyloxime) in human serum. Separation was achieved on a reversed-phase Ultrasphere XL-ODS column (75×4.6 mm I.D.). The mobile phase was 7% acetonitrile in 20 mM ammonium acetate (pH 4). Cefepime eluted in the range of 1.8–2.2 min. Detection was by UV absorbance at 254 nm. The lower limit of quantitation of cefepime in plasma was 0.5 μg/ml. The average absolute recovery was 106.2±2.1%. The linear range was from 0.1 to 50 μg/ml, with a correlation coefficient greater than 0.999. The within-day C.V.s for human samples were 4.9 and 2.3% for 1 and 50 μg/ml, respectively. The between-day C.V.s for human serum samples were 14.5, 7.4 and 6.7 for 1, 25 and 50 μg/ml, respectively. Cefepime was found to be unstable in serum at room temperature. For delayed assay, samples must be stored at −80°C.     

Liquid chromatographic determination of sodium mercaptoundecahydrododecaborate in rat urine and plasma after precolumn derivatization

A high-performance liquid chromatographic (HPLC) method was developed for the determination of disodium mercaptoundecahydrododecaborate (BSH) in biological fluids. Monobromobimane was used as a precolumn derivatizing agent. A stable derivative was obtained. The derivative was separated on a C₁₈ column using reversed-phase ion-pairing chromatography and detected by a spectrophotometric detector at 373 nm. The detection limit was 200 ng/ml (0.1 ppm boron). Calibration curves were prepared for rat urine and plasma samples. The calibration curves were linear in the range of 1 μg/ml to 100 μg/ml for urine samples and 0.2 μg/ml to 50 μg/ml for plasma samples.     

Determination of vigabatrin in human plasma and urine by high-performance liquid chromatography with fluorescence detection

A sensitive and specific HPLC method has been developed for the assay of vigabatrin in human plasma and urine. The assay involves derivatization with 4-chloro-7-nitrobenzofurazan, solid-phase extraction on a silica column and isocratic reversed-phase chromatography with fluorescence detection. Aspartam was used as an internal standard. The assay was linear over the concentration range of 0.2–20.0 μg/ml for plasma and 1.0–15.0 μg/ml for urine with a lower limit of detection of 0.1 μg/ml using 0.1 ml of starting volume of the sample. Both the within-day and day-to-day reproducibilities and accuracies were less than 5.46% and 1.6%, respectively. After a single oral dose of 500 mg of vigabatrin, the plasma concentration and the cumulative urinary excretion of the drug were determined.     

Differential processing of substance P and neurokinin A by plasma dipeptidyl(amino)peptidase IV, aminopeptidase M and angiotensin converting enzyme

In addition to plasma metabolism of substance P (SP) by angiotensin converting enzyme (ACE; EC 3.4.15.1) (<1.0 nmol/min/ml), the majority of SP hydrolysis by rat and human plasma was due to dipeptidyl(amino)peptidase IV (DAP IV; EC 3.4.14.5) (3.15–5.91 nmol/min/ml), which sequentially converted SP to SP(3–11) and SP(5–11). In turn, the SP(5–11) metabolite was rapidly hydrolyzed by rat and human plasma aminopeptidase M (AmM; EC 3.4.11.2) (24.2–25.5 nmol/min/ml). The K_m values of SP for DAP IV and of SP(5–11) for AmM ranged from 32.7 to 123 μM. In contrast, neurokinin A (NKA) was resistant to both ACE and DAP IV but was subject to N-terminal hydrolysis by AmM (3.76–10.8 nmol/min/ml; K_m=90.7 μM. These data demonstrate differential processing of SP and NKA by specific peptidases in rat and human plasma.     

High-performance liquid chromatographic methods for the determination of a new carbapenem antibiotic, L-749,345, in human plasma and urine

A column-switching, reversed-phase high-performance liquid chromatographic (HPLC) method for the determination of a new carbapenem antibiotic assay using ultraviolet detection has been developed for a new carbapenem antibiotic L-749,345 in human plasma and urine. A plasma sample is centrifuged and then injected onto an extraction column using 25 mM phosphate buffer, pH 6.5. After 3 min, using a column-switching valve, the analyte is back-flushed with 10.5% methanol–phosphate buffer for 3 min onto a Hypersil 5 μm C₁₈ BDS 100×4.6 mm analytical column and then detected by absorbance at 300 nm. The sample preparation and HPLC conditions for the urine assay are similar, except for a longer analytical column 150×4.6 mm. The plasma assay is specific and linear from 0.125 to 50 μg/ml; the urine assay is linear from 1.25 to 100 μg/ml.     

Determination of Guan-Fu Base A, a new anti-arrhythmic, in human plasma by gas chromatography and electron–capture detection

A gas chromatographic–electron capture detection (GC–ECD) method has been developed for determining Guan-Fu Base A (GFA), an experimental anti-arrhythmic, in human plasma. The method was based on one-step liquid–liquid extraction with toluene and chemical derivatization with pentafluoropropionic anhydride followed by GC–ECD. The derivatives of GFA and metoprolol (Met, internal standard) were confirmed by gas chromatography–mass spectrometry (GC–MS) to be dipentafluoropropionyl-GFA and dipentafluoropropionyl-Met. The method was linear over the concentration ranges of 0.1–20.0 and 1.0–30.0 μg/ml with the detection limit of 0.05 μg/ml at S/N=5. The intra- and inter-assay precisions were less than 6 and 10%, and accuracy 99.70±3.30 and 97.60±5.99%, respectively. The absolute recoveries were 81.88, 77.35, 80.79 and 83.85% for GFA at concentrations of 0.5, 1.0, 5.0 and 14.0 μg/ml and 88.24% for Met at 3.0 μg/ml, respectively.     

High-performance liquid chromatographic determination of a potent AII receptor antagonist (DMP 811) in plasma

An HPLC assay for DMP 811, 4-ethyl-2-propyl-1-[(2′-(1H-tetrazol-5-yl)biphenyl-4-yl)-methyl]imidazole-5-carboxylic acid (I) in rat and dog plasma has been developed. Compound I was isolated from plasma using a liquid—liquid back extraction procedure. The extraction recovery was greater than 81%. Separation of I from endogenous components in plasma was achieved on an E. Merck C₈ column using a mobile phase of 0.05 M ammonium acetate, brought to pH 3.75 with acetic acid, and acetonitrile (78:22, v/v). The eluent was monitored by fluorescence with excitation and emission set at 235 and 370 nm, respectively. The assay was linear from 2 to 2000 ng/ml. Inter- and intra-day coefficients of variation for the rat-plasma assay ranged from 0.9 to 5.2% (5–2000 ng/ml) and 2.7 to 16.5% (2–2000 ng/ml), respectively. The respective coefficients of variation for the dog-plasma assay were 1.9 to 5.6% and 1.2 to 14.0%. The percent differences from the accuracy results were 12% or less. Using 0.5 ml of plasma for extraction, the minimum quantifiable limit was 2 ng/ml. This method has been used to quantify plasma levels of I in rats or dogs following 3–10 mg/kg i.v. or p.o. doses.     

High-performance liquid chromatographic determination of iothalamic acid in human plasma and urine

A simple, rapid and sensitive method for the determination of iothalamic acid (IA) in both plasma and urine is reported. After extraction with ethyl acetate, IA was determined by strong anion-exchange high-performance liquid chromatography with ultraviolet detection at 254 nm. The lower limit of detection was 0.5 μg/ml. The average recovery was 73 and 57% from plasma and urine, respectively. Linearity was found over the investigated concentration range (up to 500 μg/ml for plasma and up to 10.0 mg/ml for urine). The reproducibility of the technique was good (coefficient of variation less than 6%) as was the precision and accuracy (coefficient of variation less than 2.5%). No interference from endogenous substances or any of the common drugs tested was found.     

Stereoselective determination of R-(+)- and S-(−)-remoxipride, a dopamine D2-receptor antagonist, in human plasma by chiral high-performance liquid chromatography

A stereoselective high-performance liquid chromatographic (HPLC) method is described for the selective and sensitive quantitation in human plasma of R-(+)- and S-(−)-enantiomers of remoxipride. Remoxipride was extracted from basified plasma into hexane-methyl-tert.-butyl ether (20:80, v/v), washed with sodium hydroxide (1.0 M), then back-extracted into phosphoric acid (0.1 M). A structural analog of remoxipride was used as an internal standard. The sample extracts were chromatographed using a silica-based derivatized cellulose chiral column, Chiralcel OD-R, and a reversed-phase eluent containing 30–32% acetonitrile in 0.1 M potassium hexafluorophosphate. Ultraviolet (UV) absorbance detection was performed at 214 nm. Using 0.5-ml plasma aliquots, the method was validated in the concentration range 0.02-2.0 μg/ml and was applied in the investigation of systemic inversion of remoxipride enantiomers in man.     

Determination of a chemoprotective agent, 2-(allylthio)pyrazine, in plasma, urine and tissue homogenates by high-performance liquid chromatography

A high-performance liquid chromatographic method was developed for the determination of a chemoprotective agent, 2-(allylthio)pyrazine (I), in human plasma and urine, and in rat blood and tissue homogenate using diazepam as an internal standard. The sample preparation was simple; 2.5 volumes of acetonitrile were added to the biological sample to deproteinize it. A 50–100 μl aliquot of the supernatant was injected onto a C₁₈ reversed-phase column. The mobile phase employed was acetonitrile–water (55:45, v/v), and it was run at a flow-rate of 1.5 ml/min. The column effluent was monitored using an ultraviolet detector at 330 nm. The retention times for I and the internal standard were 4.0 and 5.1 min, respectively. The detection limits of I in human plasma and urine, and in rat tissue homogenate (including blood) were 20, 20 and 50 ng/ml, respectively. The coefficients of variation of the assay (within-day and between-day) were generally low (below 6.1%) in a concentration range from 0.02 to 10 μg/ml for human plasma and urine, and for rat tissue homogenate. No interferences from endogenous substances were found.