Dihydroxybergamottin caproate as a potent and stable CYP3A4 inhibitor

We investigated the inhibitory activity of the furanocoumarin derivatives from grapefruit juice to the drug metabolizing enzyme, cytochrome P450 (CYP) 3A4. Although two known furanocoumarin dimers GF-I-1 (1) and GF-I-4 (2) showed potent CYP3A4 inhibition with IC50 value of 0.07 microM, a semi-synthetic dihydroxybergamottin caproate (11), which was more stable and more simple than the dimers, exhibited comparable activity against CYP3A4.     

Inhibition selectivity of grapefruit juice components on human cytochromes P450

Five compounds including furanocoumarin monomers (bergamottin, 6', 7'-dihydroxybergamottin (DHB)), furanocoumarin dimers (4-??6-hydroxy-71-?(1-hydroxy-1-methyl)ethyl-4-methyl-6-(7-oxo-7H- furo?3,2-g1benzopyran-4-yl)-4-hexenyl]oxy]-3,7-dimethyl- 2-octenyl]oxy]-7H-furo[3,2-g]?1benzopyran-7-one (GF-I-1) and 4-??6-hydroxy-7??4-methyl-1-(1-methylethenyl)-6-(7-oxo-7H-furo?3, 2-g1benzopyran-4-yl)-4-hexenyl?xy-3, 7-dimethyl-2-octenyl?xy-7H-furo?3,2-g1benzopyran-7-one (GF-I-4)), and a sesquiterpene nootkatone have been isolated from grapefruit juice and screened for their inhibitory effects toward human cytochrome P450 (P450) forms using selective substrate probes. Addition of ethyl acetate extract of grapefruit juice into an incubation mixture resulted in decreased activities of CYP3A4, CYP1A2, CYP2C9, and CYP2D6. All four furanocoumarins clearly inhibited CYP3A4-catalyzed nifedipine oxidation in concentration- and time-dependent manners, suggesting that these compounds are mechanism-based inhibitors of CYP3A4. Of the furanocoumarins investigated, furanocoumarin dimers, GF-I-1 and GF-I-4, were the most potent inhibitors of CYP3A4. Inhibitor concentration required for half-maximal rate of inactivation (K(I)) values for bergamottin, DHB, GF-I-1, and GF-I-4 were calculated, respectively, as 40.00, 5. 56, 0.31, and 0.13 microM, whereas similar values were observed on their inactivation rate constant at infinite concentration of inhibitor (k(inact), 0.05-0.08 min(-1)). Apparent selectivity toward CYP3A4 does occur with the furanocoumarin dimers. In contrast, bergamottin showed rather stronger inhibitory effect on CYP1A2, CYP2C9, CYP2C19, and CYP2D6 than on CYP3A4. DHB inhibited CYP3A4 and CYP1A2 activities at nearly equivalent potencies. Among P450 forms investigated, CYP2E1 was the least sensitive to the inhibitory effect of furanocoumarin components. A sesquiterpene nootkatone has no significant effect on P450 activities investigated except for CYP2A6 and CYP2C19 (K(i) = 0.8 and 0.5 microM, respectively).     

Blood prostaglandin A (PGA) levels in normal human subjects

Blood prostaglandin A levels were measured in ten healthy subjects under different conditions of collection and storage.Plasma levels ranged from 1.20 to 1.81 ng/ml (M ± SE = 1.50 ± 0.10) in 5 females to 1.23 to 1.68 ng/ml (1.45 ± 0.09) in 5 males, when centrifuged and frozen immediately after collection. Storate at 4°C for varying times up to 24 hours and at 22°C (room temperature) up to 4 hours did not affect mean plasma concentrations significantly, but increased the range obtained to 1.00 to 2.47 ng/ml for both male and female groups.Serum concentrations differed in males and females and were lower than corresponding mean plasma values for males and higher for females. Mean serum concentrations were 1.77 ± 0.08 ng/ml in females and 1.18 ± 0.05 ng/ml in males and did not change significantly up to 24 hours of storage at 4°C.These results suggest that prostaglandin A assayed in both plasma and serum under the conditions described is stable and should allow for greater flexibility in sampling under different experimental conditions.     

Radical scavenging and cytochrome P450 3A4 inhibitory activity of bergaptol and geranylcoumarin from grapefruit

Grapefruit juice has been shown to increase the oral bioavailability of several clinically important drugs by inhibiting first pass metabolism. Several compounds in grapefruit juice have shown different biological activities. Unique among them are furocoumarins with potent inhibitory activity against cytochrome P450 enzymes. In the present study, two bioactive compounds were isolated from grapefruit juice and grapefruit peel oil. The purity of the isolated compounds has been analyzed by HPLC. Structures of the compounds were elucidated by extensive NMR and mass spectral studies and identified as bergaptol and geranylcoumarin. The isolated compounds were tested for their radical scavenging activity using 2,2'-azobis (3-ethylbenz-thiazoline-6-sulfonic acid) (ABTS) and 2,2-diphenyl-1-picrylhydrazil (DPPH) methods at different concentrations. Bergaptol showed very good radical scavenging activity at all the tested concentrations. Furthermore, these compounds were evaluated for their inhibitory activity against CYP3A4 enzyme. Bergaptol and geranylcoumarin were found to be potent inhibitors of debenzylation activity of CYP3A4 enzyme with an IC(50) value of 24.92 and 42.93 microM, respectively.     

The role of plasma arachidonic acid metabolites in the pathogenesis and the prognosis of Henoch-Schönlein Purpura

Henoch-Schönlein Purpura (HSP) involve small vessel inflammation. Arachidonate biochemical pathways play an important role in the pathogenesis of vascular inflammation. The aim of this study was to investigate the change in the ratio of plasma arachidonic acid metabolites in the patients with HSP and evaluate the association between clinical activity and prostanoid activity in the acute phase of HSP. Plasma prostaglandin E₂ (PGE₂)-like activities were found to be 7.2 ± 0.8 ng/ml in control group (n=12) while it was 5.3 ± 0.6 ng/ml in the patients with HSP (n=12). Plasma leukotriene C₄ (LTC₄)-like activities were found to be 16.0 ± 1.1 ng/ml in control while it was 30.9 ± 4.3 ng/ml in the patients. The differences of LTC₄-like activities and the ratios between the HSP patients and the controls were significant (p < 0.01, p < 0.001 respectively), but no significant difference was found in PGE₂-like activities. Plasma LTC₄-like activity and ratio were also significantly increased in the patients with high clinical score (p < 0.05, p < 0.02 respectively). These results suggested that not only cyclooxygenase products but also LTs may play an important role in vascular inflammation. Therefore ratio must be taken into consideration in the pathogenesis and the prognosis of HSP.     

The analysis of arachidonic acid metabolites in normal, uninvolved and lesional psoriatic skin

Collection of exudate from suction bullae is a commonly used method for sampling human skin for mediator analysis. It is satisfactory on skin of normal structure but is unreliable on lesional psoriatic skin in which there are major structural changes and excessive scaling. Collection of exudates from abraded sites was found to be a suitable alternative method for psoriatic skin. Arachidonic acid and 12-HETE, but not PGE₂, were significantly higher in exudate from abraded lesional psoriatic skin (494 ± 88, 45.9 ± 4.2 and 9.6 ± 1.8 ng/ml respectively, mean ± sem, n = 5) compared to uninvolved skin (154 + 38, 18.5 + 5.1 and 7.7 ± 1.9 ng/ml) or skin of normal volunteers (119 ± 37, 14.5 ± 6.7 and 4.5 ± 1.6 ng/ml, n = 7) which were similar. The coefficient of variation for exudate collection and mediator analysis was usually less that 55%. The analysis of lipoxygenase and cyclooxygenase products was simplified by the use of chlorobutane to exctract preferentially arachidonic acid and HETEs from neutral aqueous solutions.     

Determination of histamine, methylhistamines and histamine—o-phthaldialdehyde complexes by two high-performance liquid chromatographic procedures : Application to biological samples

Two high-performance liquid chromatographic procedures were proposed to measure histamine. The first, with UV detection and a strong acid cation exchanger (Partisil 10, SCX Whatman), made it possible to isolate histamine and some methylated derivatives. The second, with a C₁₈ sorbent (μBondapak, Waters, 10 μm particle size) eluted with ion-pairing phases, made it possible to isolate the histamine—o-phthaldialdehyde complexes. This last procedure allied with a chromatographic purification step gave lower or identical amounts of histamine than those described in human urine (16 ± 7 μg per 24 h), canine whole blood (1.5 ± 1 ng/ml) and human gastric juice (2.3 ± 1.4 ng/ml). The two procedures gave the concentration of a histamine-like compound isolated from the antral mucosa.     

Analysis of wines, grape juices and cranberry juices forAlternaria toxins

Sixty six samples of red and white wine from Ontario (VQA), British Columbia (VQA), Québec (“vins artisanaux”), imported wines (from Italy, South America and USA) and Canadian and US grape and cranberry juices were analysed for theAlternaria mycotoxins alternariol (AOH) and alternariol monomethyl ether (AME). After cleanup on aminopropyl SPE columns, AOH and AME were initially determined by reversed phase LC with UV detection. Positive sample extracts were re-analysed by LC-tandem negative ion electrospray mass spectrometry (MS/MS) in multiple reaction mode. Overall mean method recoveries measured by LC-UV were 93% for AOH and 81% for AME. Limits of detection in wine (and juice) by LC-UV for AOH were 0.8 (0.4) ng/ml and for AME were 0.5 (0.4) ng/ml; they were below 0.01 ng/ml by LC-MS/MS. As determined by LC-MS/MS, AOH was found in 13/17 Canadian red wines at levels of 0.03 to 5.02 ng/ml and in 7/7 imported red wines at 0.27–19.4 ng/ml, usually accompanied by lower concentrations of AME. Red grape juices (5 positive/10 samples) contained only sub ng/ml levels of AOH or AME except for one sample (39 ng AME/ml). White wines (3/23 samples), white grape juices (0/4 samples) and cranberry juices (1/5 samples) contained little AOH/AME (≤1.5 ng/ml). Presented at the World Mycotoxin Forum, Noordwijk, The Netherlands, November 10–11, 2005     

Effect of ibuprofen on menstrual blood prostaglandin levels in dysmenorrheic women

In a randomized crossover study 15 dysmenorrheic women were treated during two consecutive menstrual periods, once with the potent prostaglandin-synthesis inhibitor: ibuprofen and once with an identical looking placebo. Each patient was medicated for 12 hours during the first day of her menstrual flow and was subsequently fitted with a cervical cup for the collection of menstrual blood during three hours. In these samples the concentrations of prostaglandin (PG)F and PGE were measured by radioimmunoassay.The patients receiving placebo had high PGF levels 135 ± 27 ng/ml (Mean ± S.E.) which were significantly reduced by Ibuprofen to 24 ± 5 ng/ml (P<0.001). The PGE concentrations decreased from 5 ± 1 ng/ml to 2 ± 1 ng/ml (P<0.05). Ibuprofen also reduced the menstrual pain significantly (P<0.001). These results substantiate the earlier conclusion that a causal relationship exists between effective treatment with PG-synthesis inhibitors and decrease in menstrual blood PG levels, intrauterine pressure and dysmenorrheic pain.     

Assay of 2-naphthol in human urine by high-performance liquid chromatography

This paper describes a novel liquid chromatographic method for the quantitation of 2-naphthol in human urine. Urine samples were extracted after enzymatic hydrolysis of glucuronides and sulfates; 2-naphthol was then separated using reversed-phase high-performance liquid chromatography. The corresponding detection limits were 0.04 ng/ml for the standard sample in acetonitrile and 0.13 ng/ml for urine samples. The level of urinary 2-naphthol in 100 Korean shipyard workers was analyzed using this new method. The level ranged from 0.21 ng/ml (0.26 μmol/mol creatinine) to 34.19 ng/ml (59.11 μmol/mol creatinine), and the mean±standard deviation was 5.08 ng/ml (6.60 μmol/mol creatinine)±5.75 ng/ml (9.22 μmol/mol creatinine). The mean±standard deviation of urinary 2-naphthol level of smokers, 7.03 ng/ml (8.49 μmol/mol creatinine)±6.16 ng/ml (10.23 μmol/mol creatinine), was significantly higher than that of non-smokers, 2.49 ng/ml (4.10 μmol/mol creatinine)±3.92 ng/ml (7.03 μmol/mol creatinine).     

Sensitive, high-throughput gas chromatographic–mass spectrometric assay for fluoxetine and norfluoxetine in human plasma and its application to pharmacokinetic studies

A sensitive, robust gas chromatographic–mass spectrometric assay suitable for use in pharmacokinetic or bioequivalence studies is presented for the selective serotonin reuptake inhibitor, fluoxetine, and its major metabolite, norfluoxetine (N-desmethylfluoxetine). This method employs solid-phase extraction followed by acetylation with trifluoroacetic anhydride and analysis of the derivatives using selected ion monitoring. The lower limit of quantification was 1.0 ng/ml, and the assay was linear for both analytes from 1 to 100 ng/ml. Mean recoveries following solid-phase extraction at concentrations of 5.0, 20 and 100 ng/ml were 91% (fluoxetine) and 87% (norfluoxetine). Assay precision (as mean RSD) and accuracy (as mean relative error) for both analytes were tested at the same three nominal concentrations and were found to be within 10% in all cases. Analysis of fluoxetine concentrations in plasma samples from 18 volunteers following administration of a single 40 mg dose of fluoxetine provided the following pharmacokinetic data (mean±SD): C_max, 32.73±9.21 ng/ml; AUC_0–∞, 1627±1372 ng/ml h; T_max, 3.08 h (median); k_e, 0.022±0.007 h⁻¹; elimination half-life, 37.69±21.70 h.     

Effect of 2-deoxy- -glucose on acetylcholine and histamine levels in gastric juice of pylorus-ligated rats anesthetized with urethane

Acetylcholine (ACh) in gastric juice was detected and measured by pretreatment of acetylcholinesterase inhibitor, 1 mM eserine (1 ml/rat, p.o.), in pylorus-ligated rats, by liquid chromatography with electrochemical detection. In order to elucidate whether or not the ACh level in gastric juice reflects the activity of cholinergic neurons, the effect of 2-deoxy- -glucose (2-DG), a vagus stimulant, on the levels of ACh, histamine and gastric acid in gastric juice was investigated in pylorus-ligated rats anesthetized with urethane (1.25 g/kg, i.p.). Under the non-anesthetic condition, ACh, histamine and gastric acid levels were 100±25 pmol/h, 120±10 ng/h, and 240±32 μequiv./h, respectively. These levels were completely inhibited by urethane anesthesia. Under the anesthetized condition, 2-DG (50–200 mg/kg, i.v.) significantly increased ACh and histamine levels in gastric juice, as well as acid secretion. The 2-DG (200 mg/kg, i.v.)-induced increases in these levels were completely inhibited by vagotomy. These results suggest that ACh level measured in gastric juice reflects the activity of cholinergic transmission. Furthermore, these results also support the conclusion that vagus stimulation facilitates not only cholinergic transmission but also histaminergic transmission related to gastric acid secretion.     

Plasma transport and tissue distribution of β-carotene, vitamin A and retinol-binding protein in domestic cats

The objective of this study was to determine retinol, retinyl esters and retinol-binding protein (RBP) as well as carotenoids in plasma, urine, liver and kidneys of randomly selected domestic cats. Retinol (240±64 ng/ml, mean±S.D.) represented one-third of total retinyl esters (736±460 ng/ml) in plasma. Retinyl esters were stearate, palmitate and oleate representing 61±6, 36±13 and 5±3% of total retinyl esters, respectively. In half of the cats, retinyl esters (22±21 ng/ml) were found in the urine. Vitamin A in the livers (4317±1956 μg/g) was significantly higher than in the kidney cortex and medulla (14.16±8.92 and 7.59±4.52 μg/g, respectively, both P<0.001). RBP was detected in the plasma but not in the urine. Immunoreactive RBP was observed in hepatocytes and in the cells of the proximal tubules. β-Carotene was present in plasma but never in tissues. The results show that similar to canines differences in vitamin A metabolism in cats are related to the occurrence of retinyl esters in plasma. They differ, however, with regard to the tissue distribution of β-carotene and the excretion of vitamin A in the urine.     

Pentobarbital anesthesia: Lack of effect on venous prostaglandins in dogs

Venous prostaglandins A, E, and F were determined by radioimmunoassay in 10 dogs before and one hour after administration of sodium pentobarbital (35 mg/Kg, iv). In the conscious state, PGA was 0.34 ± 0.04 ng/ml (mean ± SE), PGE 0.20 ± 0.01 ng/ml, and PGF 0.25 ± 0.03 ng/ml. During pentobarbital anesthesia, these levels were unchanged (p >0.05). Thus, pentobarbital anesthesia had no effect on peripheral venous prostaglandin levels.     

Effect of 15-HETE on cerebral arterioles of newborn pigs

Effects of topical application of 15-HETE on pial arteriolar diameter and cortical perirachnoid cerebrospinal fluid (CSF) prostanoid concentrations were investigated in chloralose-anesthetized newborn pigs. Pial arteriolar diameters were measured using a closed cranial window, and CSF samples from under the window were collected for prostanoid analysis after applying artificial CSF without drug and CSF containing 15-HETE (1, 10, 100, 1000 ng/ml). 15-HETE caused significant dose-related constriction from 162 ± 17.0 μm (control diameter) to 136 ± 14.5 and 129 ± 18.7 μm (100 and 1000 ng/ml, respectively). The concentration of PGE₂ (but not of PGF_2α or 6-keto-PGF_1α increased in CSF at 100 and 1000 ng/ml of 15-HETE. Pial arteriolar responses to 15-HETE were determined before and after indomethacin treatment (5 mg/kg, i.v.). 15-HETE (100 ng/ml) constricted pial arterioles before indomethacin (diameter change, −15 ± 10%); after indomethacin, constriction was potentiated in response to the same dose (diameter change, −26 ± 7%). These data support the hypothesis thet, in newborn piglets, 15-HETE exerts a vasoconstrictor effect on pial arterioles, which appears to be attenuated by 15-HETE-induced stimulation of dilator prostanoids.     

Simultaneous determination of pyridostigmine bromide, N,N-diethyl-m-toluamide, permethrin, and their metabolites in rat plasma and urine by high-performance liquid chromatography

A rapid and simple method was developed for the separation and quantification of the anti nerve agent drug pyridostignmine bromide (PB; 3-dimethylaminocarbonyloxy-N-methyl pyridinium bromide) its metabolite N-methyl-3-hydroxypyridinium bromide, the insect repellent DEET (N,N-diethyl-m-toluamide), its metabolites m-toluamide and m-toluic acid, the insecticide permethrin (3-(2,2-dichloro-ethenyl)-2,2-dimethylcyclopropanecarboxylic acid(3-phenoxyphenyl)methylester), and two of its metabolites m-phenoxybenzyl alcohol, and m-phenoxybenzoic acid in rat plasma and urine. The method is based on using C₁₈ Sep-Pak^® cartridges for solid-phase extraction (SPE) and high-performance liquid chromatography (HPLC) with reversed-phase C₁₈ column, and gradient UV detection ranging between 208 and 230 nm. The compounds were separated using gradient of 1 to 99% acetonitrile in water (pH 3.20) at a flow-rate ranging between 0.5 and 1.7 ml/min in a period of 17 min. The retention times ranged from 5.7 to 14.5 min. The limits of detection were ranged between 20 and 100 ng/ml, while limits of quantitation were 150–200 ng/ml. Average percentage recovery of five spiked plasma samples were 51.4±10.6, 71.1±11.0, 82.3±6.7, 60.4±11.8, 63.6±10.1, 69.3±8.5, 68.3±12.0, 82.6±8.1, and from urine 55.9±9.8, 60.3±7.4, 77.9±9.1, 61.7±13.5, 68.6±8.9, 62.0±9.5, 72.9±9.1, and 72.1±8.0, for pyridostigmine bromide, DEET, permethrin, N-methyl-3-hydroxypyridinium bromide, m-toluamide, m-toluic acid, m-phenoxybenzyl alcohol and m-phenoxybenzoic acid, respectively. The relationship between peak areas and concentration was linear over the range between 100 and 5000 ng/ml. This method was applied to analyze the above chemicals and metabolites following their administration in rats.     

3-methoxy-4-hydroxyphenylglycol, 5-hydroxyindoleacetic acid, and homovanillic acid in human cerebrospinal fluid : Storage and measurement by reversed-phase high-performance liquid chromatography and coulometric detection using 3-methoxy-4-hydroxyphenyllactic acid as an internal standard

To simultaneously measure 3-methoxy-4-hydroxyphenylglycol (MHPG), 5-hydroxyindoleacetic acid (5HIAA), and homovanillic acid (HVA) in human cerebrospinal fluid (CSF), we used an acetonitrile protein precipitation, reversed-phase high-perforamance liquid chromatography with coulometric detection, and 3-methoxy-4-hydroxyphenyllactic acid (MHPLA) as an internal standard for all three metabolites. MHPG, 5HIAA, HVA, and MHPLA were stable for one month when stored in CSF at −70°C. Three determinations were made in triplicate for each of seven subjects over a 30-day storage period and the coefficients of variation within subject for these determinations ranged from 0.075 to 0.165 for MHPG, 0.045 to 0.148 for 5HIAA and 0.053 to 0.181 for HVA. Means and standard deviations fo CSF concentrations were 10.7 ± 3.0 ng/ml for MHPG, 22.4 ± 9.9 ng/ml for 5HIAA, and 39.9 ± 21.4 ng/ml for HVA. This method provides simple sample preparation, sensitivity, and cost advantages, as well as simultaneous extraction and quantitation of MHPG, 5HIAA, and HVA using an internal standard.     

The effect of grapefruit juice and seville orange juice on the pharmacokinetics of dextromethorphan: the role of gut CYP3A and P-glycoprotein

The objective of this study was to determine the effects of grapefruit juice and seville orange juice on dextromethorphan (DM) pharmacokinetics. Eleven healthy volunteers were studied over a 3-week period consisting of 5 study days each separated by a three-day washout. All subjects refrained from drinking caffeine containing beverages (coffee, soda, etc.) 8 h before orally taking DM (30 mg) with 200 ml water, 200 ml grapefruit juice, 200 ml water, 200 ml seville orange juice, and 200 ml water on Study Days 1 to 5. Aliquots of urine samples were assayed and analysed for DM, and the DM metabolites dextrorphan, 3-methoxymorphinan and 3-hydroxymorphinan using a validated HPLC method employing a phenyl column and a fluorescence detection. Results suggests that DM could provide some useful information on P-glycoprotein or related membrane efflux protein activity in the human gastro-intestinal tract. Bioavailability (F) of DM increased significantly with grapefruit and seville orange juice, but only returned to half the baseline value after three days of washout. This confirms that grapefruit and seville orange juice are long-lasting and perhaps irreversible inhibitors of gut CYP3A/P-glycoprotein. Grapefruit and seville orange juice appeared to have the same overall effect on DM pharmacokinetics. In addition, this paper presents a novel method of phenotyping for CYP2D6, CYP3A and P-glycoprotein using DM as a probe.     

Solid-phase extraction and high-performance liquid chromatographic determination of tamoxifen and its major metabolites in plasma

Tamoxifen (TAM) is a triphenylethylene anti-oestrogen, commonly used in the treatment of breast cancer. Patients receiving tamoxifen therapy may experience both de novo and acquired resistance. As one of the mechanisms for this may be extensive peripheral bio-transformation of tamoxifen, there has been considerable interest in the pharmacokinetics and metabolism of tamoxifen. A reversed-phase high-performance liquid chromatography separation has been developed to determine the levels of tamoxifen and its major metabolites in human plasma. The method is highly sensitive (2 ng/ml) and selective for tamoxifen, cis-tamoxifen (CIS), 4-hydroxytamoxifen (4-OH) and desmethyltamoxifen (DMT). A μBondapak C₁₈ 10 μm column (30 cm × 3.9 mm I.D.) was used, with a mobile phase of methanol-1% triethylamine at pH 8 (89:11, v/v). Sample preparation was carried out using a C₂ (500 mg sorbent, 3 ml reservoirs) solid phase extraction method, and extraction efficiencies were approximately 60% for TAM and its metabolites. Accuracy and precision, as determined by spiking plasma samples with a mixture of tamoxifen and its metabolites, ranged from 85–110% (± 5–10%) at 1 μg/ml, 101–118% (± 8–20%) at 0.1 μg/ml and 111–168% (± 43–63%) at 0.01 μg/ml. Results from 59 patients show mean values of 54 ng/ml for 4-OH; 190 ng/ml for DMT; 93 ng/ml for TAM and 30 ng/ml for CIS (detected in three patients only). This methodology can be applied routinely to the determination of TAM and its metabolites in plasma from patients undergoing therapy.     

A high-performance liquid chromatography assay with ultraviolet detection for olanzapine in human plasma and urine

Olanzapine is a commonly used atypical antipsychotic medication for which therapeutic drug monitoring has been proposed as clinically useful. A sensitive method was developed for the determination of olanzapine concentrations in plasma and urine by high-performance liquid chromatography with low-wavelength ultraviolet absorption detection (214 nm). A single-step liquid–liquid extraction procedure using heptane-iso-amyl alcohol (97.5:2.5 v/v) was employed to recover olanzapine and the internal standard (a 2-ethylated olanzapine derivative) from the biological matrices which were adjusted to pH 10 with 1 M carbonate buffer. Detector response was linear from 1–5000 ng (r²>0.98). The limit of detection of the assay (signal:noise=3:1) and the lower limit of quantitation were 0.75 ng and 1 ng/ml of olanzapine, respectively. Interday variation for olanzapine 50 ng/ml in plasma and urine was 5.2% and 7.1% (n=5), respectively, and 9.5 and 12.3% at 1 ng/ml (n=5). Intraday variation for olanzapine 50 ng/ml in plasma and urine was 8.1% and 9.6% (n=15), respectively, and 14.2 and 17.1% at 1 ng/ml (n=15). The recoveries of olanzapine (50 ng/ml) and the internal standard were 83±6 and 92±6% in plasma, respectively, and 79±7 and 89±7% in urine, respectively. Accuracy was 96% and 93% at 50 and 1 ng/ml, respectively. The applicability of the assay was demonstrated by determining plasma concentrations of olanzapine in a healthy male volunteer for 48 h following a single oral dose of 5 mg olanzapine. This method is suitable for studying olanzapine disposition in single or multiple-dose pharmacokinetic studies.