Rapid and sensitive liquid chromatography-tandem mass spectrometric method for the quantitation of metformin in human plasma

A rapid and sensitive liquid chromatography-tandem mass spectrometric (LC-MS-MS) method for the determination of metformin in human plasma using phenformin as internal standard has been developed and validated. Sample preparation of plasma involved acidification with acetic acid, deproteination with acetonitrile and washing with dichloromethane. Samples were then analyzed by HPLC on a short Nucleosil C18 column (5 microm, 50 mm x 4.6 mm i.d.) using a mobile phase consisting of acetonitrile:methanol:10mM ammonium acetate pH 7.0 (20:20:60, v/v/v) delivered at 0.65 ml/min. Detection was performed using an Applied Biosystems Sciex API 4000 mass spectrometer set at unit resolution in the multiple reaction monitoring (MRM) mode. Atmospheric pressure chemical ionization (APCI) was used for ion production. The assay was linear over the range 1-2000 ng/ml with intra- and inter-day precision of <8.6% and accuracy in the range 91-110%. The limit of detection was 250 pg/ml in plasma. The method was successfully applied to a clinical pharmacokinetic study of an extended-release tablet of metformin hydrochloride (500 mg) administered as a single oral dose.     

Determination of tiropramide in human plasma by liquid chromatography-tandem mass spectrometry

A rapid, sensitive and selective liquid chromatography-tandem mass spectrometric (LC/MS/MS) method for the determination of tiropramide in human plasma was developed. Tiropramide and internal standard, cisapride were extracted from human plasma by liquid-liquid extraction and analyzed on a Luna C8 column with the mobile phase of acetonitrile-ammonium formate (10mM, pH 4.5) (50:50, v/v). The analytes was detected using an electrospray ionization tandem mass spectrometry in the multiple-reaction-monitoring mode. The standard curve was linear (r=0.998) over the concentration range of 2.0-200 ng/ml. The intra- and inter-assay coefficients of variation ranged from 2.8 to 7.8 and 6.7 to 8.9%, respectively. The recoveries of tiropramide ranged from 50.2 to 53.1%, with that of cisapride (internal standard) being 60.9+/-5.3%. The lower limit of quantification for tiropramide was 2.0 ng/ml using 100 microl plasma sample. This method was applied to the pharmacokinetic study of tiropramide in human.     

Determination of vinorelbine in rabbit plasma by high-performance liquid chromatography with coulometric detection

A high-performance liquid chromatographic method was developed for the determination in plasma (400-μl sample) of a vinca alkaloid, vinorelbine. The analysis was performed by using an octadecylsilane column and heptanesulfonic acid as ion-pairing agent. This method used a new internal standard, teniposide, that permitted a good compromise between sensitivity and retention times (10.6 and 15.5 min for teniposide and vinorelbine, respectively). After a liquid-liquid extraction with diethyl ether, the extracts were injected into a reversed-phase system. The extraction efficiency was approximately 80% for both vinorelbine and the internal standard. The mobile phase was phosphate buffer (pH 3)-acetonitrile-methanol (50:30:20, v/v/v). Using coulometric detection, the limit of detection in plasma (400 μl) was 1 ng.ml. The intra-assay coefficients of variation were 10.95, 3.80 and 5.71% for 5, 500 and 1000 ng/ml, respectively, and the inter-assay coefficients of variation were 20.14, 14.27 and 10.67% for 5, 500 and 1000 ng/ml, respectively. A linear response was observed for the plasma calibration graph in the ranges 2.5–50 and 50–1000 ng/ml. This method was used to follow the time course of the concentration of vinorelbine in rabbit plasma after a single intravenous dose of vinorelbine (30 mg/m²) and seems to be suitable for studying the pharmacokinetics of vinorelbine in rabbit.     

Anastrozole quantification in human plasma by high-performance liquid chromatography coupled to photospray tandem mass spectrometry applied to pharmacokinetic studies

A rapid, sensitive and specific method for quantifying the aromatase inhibitor (anastrozole) in human plasma using dexchlorpheniramine as the internal standard (I.S.) is described herein. The analyte and the I.S. were extracted from 200 microl of human plasma by liquid-liquid extraction using a mixture of diethyl ether:dichloromethane (70:30, v/v) solution. Extracts were removed and dried in the organic phase then reconstituted with 200 microl of acetonitrile:water (50:50; v/v). The extracts were analyzed by high performance liquid chromatography coupled with photospray tandem mass spectrometry (HPLC-MS-MS). Chromatography was performed isocratically on a Genesis, C18 4 microm analytical column (100 mm x 2.1mm i.d.). The method had a chromatographic run time of 2.5 min and a linear calibration curve ranging from 0.05-10 ng ml(-1). The limit of quantification (LOQ) was 0.05 ng ml(-1). This HPLC-MS-MS procedure was used to assess pharmacokinetic studies.     

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.     

High-performance liquid chromatographic determination of oxodipine enantiomers, a new 1,4-dihydropyridine, applied to stereoselectivity studies in man and dog

A specific and reproducible HPLC method using a Chiral-AGP column and UV detection was developed for the evaluation of the pharmacokinetic profile of oxodipine enantiomers in dog and man. Each enantiomer was determined in plasma in the concentration range 1–400 ng/ml using the internal standard calibration method with linear regression analysis. After extraction of oxodipine and the internal standard at alkaline pH with diethyl ether—n-hexane (50:50, v/v), this method permitted the determination of each enantiomer at levels down to 10 ng/ml in dog plasma and 25 ng/ml in human plasma with sufficient accuracy (relative error <11%, n = 6) and precision (coefficient of variation <16%, n = 6). The extracted plasma volume was 500 μl and after evaporation of the organic phase, the dry residue was dissolved in 100 μl of water—2-propanol; an aliquot of 80 μl was injected into the HPLC system.     

Simple high-performance liquid chromatographic method for the determination of ranitidine in human plasma

A simple high-performance liquid chromatographic method was developed for the determination of ranitidine in human plasma. Prior to analysis, ranitidine and the internal standard (metoprolol) were extracted from alkalinized plasma samples using dichloromethane. The mobile phase was 0.05 M potassium dihydrogenphosphate–acetonitrile (88:12, v/v) adjusted to pH 6.5. Analysis was run at a flow-rate of 1.3 ml/min and at a detection wavelength of 229 nm. The method is sensitive with a detection limit of 1 ng/ml at a signal-to-noise ratio of 3:1, while the quantification limit was set at 15 ng/ml. The calibration curve was linear over a concentration range of 15–2000 ng/ml. Mean recovery value of the extraction procedure was about 90%, while the within-day and between-day coefficients of variation and percent error values of the assay method were all less than 15%.     

Simultaneous determination of metformin and rosiglitazone in human plasma by liquid chromatography/tandem mass spectrometry with electrospray ionization: application to a pharmacokinetic study

A selective and sensitive high performance liquid chromatography-electrospray ionization-tandem mass spectrometry (ESI-MS/MS) method for simultaneous determination of metformin and rosiglitazone in human plasma using phenformin as internal standard (IS) has been first developed and validated. Plasma samples were precipitated by acetonitrile and the analytes were separated on a prepacked Phenomenex Luna 5u CN 100A (150 mm x 2.0 mm I.D.) column using a mobile phase comprised of methanol:30 mM ammonium acetate pH 5.0 (80:20, v/v) delivered at 0.2 ml/min. Detection was performed on a Finnigan TSQ triple-quadrupole tandem mass spectrometer in positive ion selected reaction monitoring (SRM) mode using electrospray ionization. The ion transitions monitored were m/z 130.27-->71.11 for metformin, m/z 358.14-->135.07 for rosiglitazone and m/z 206.20-->105.19 for the IS. The standard curves were linear (r(2)>0.99) over the concentration range of 5-3000 ng/ml for metformin and 1.5-500 ng/ml for rosiglitazone with acceptable accuracy and precision, respectively. The within- and between-batch precisions were less than 15% of the relative standard deviation. The limit of detection (LOD) of both metformin and rosiglitazone was 1 ng/ml. The method described is precise and sensitive and has been successfully applied to the study of pharmacokinetics of compound metformin and rosiglitazone capsules in 12 healthy Chinese volunteers.     

Determination of betamethasone and betamethasone 17-monopropionate in human plasma by liquid chromatography-positive/negative electrospray ionization tandem mass spectrometry

This study presents a high-performance liquid chromatography-positive/negative electrospray ionization tandem mass spectrometric (LC-ESI(+/-)-MS-MS) method for the determination of betamethasone (BOH) and betamethasone 17-monopropionate (B17P) in human plasma using beclomethasone dipropionate as the internal standard (I.S.). Both compounds were extracted from human plasma with ether-cyclohexane (4:1, v/v) and were separated by HPLC on a Hanbon Lichrospher C(18) column with a mobile phase of methanol-water (85:15, v/v) at a flow rate of 0.7ml/min. Calibration curves were linear over the range of 0.10-50ng/ml for BOH and 0.050-50ng/ml for B17P. The inter-run relative standard deviations were less than 14.4% for BOH and 12.3% for B17P. The intra-run relative standard deviations were less than 9.3% for BOH and 7.9% for B17P. The mean plasma extraction recovery for BOH and B17P were in the ranges of 82.7-85.9% and 83.6-85.3%, respectively. The method was successfully applied to study the pharmacokinetics of a new formulation of betamethasone phosphate/betamethasone dipropionate injection in healthy Chinese volunteers.     

An analytical method with a single extraction procedure and two separate high performance liquid chromatographic systems for the determination of artesunate, dihydroartemisinin and mefloquine in human plasma for application in clinical pharmacological studies of the drug combination

The combination of two sensitive, selective and reproducible reversed phase liquid chromatographic (RP-HPLC) methods was developed for the determination of artesunate (AS), its active metabolite dihydroartemisinin (DHA) and mefloquine (MQ) in human plasma. Solid phase extraction (SPE) of the plasma samples was carried out on Supelclean LC-18 extraction cartridges. Chromatographic separation of AS, DHA and the internal standard, artemisinin (QHS) was obtained on a Hypersil C4 column with mobile phase consisting of acetonitrile-0.05 M acetic acid adjusted to pH 5.2 with 1.0M NaOH (42:58, v/v) at the flow rate of 1.50 ml/min. The analytes were detected using an electrochemical detector operating in the reductive mode. Chromatography of MQ and the internal standard, chlorpromazine hydrochloride (CPM) was carried out on an Inertsil C8-3 column using methanol-acetonitrile-0.05 M potassium dihydrogen phosphate adjusted to pH 3.9 with 0.5% orthophosphoric acid (50:8:42, v/v/v) at a flow rate of 1.00 ml/min with ultraviolet detection at 284 nm. The mean recoveries of AS and DHA over a concentration range of 30-750 ng/0.5 ml plasma and MQ over a concentration of 75-1500 ng/0.5 ml plasma were above 80% and the accuracy ranged from 91.1 to 103.5%. The within-day coefficients of variation were 1.0-1.4% for AS, 0.4-3.4% for DHA and 0.7-1.5% for MQ. The day-to-day coefficients of variation were 1.3-7.6%, 1.8-7.8% and 2.0-3.4%, respectively. Both the lower limit of quantifications for AS and DHA were at 10 ng/0.5 ml and the lower limit of quantification for MQ was at 25 ng/0.5 ml. The limit of detections were 4 ng/0.5 ml for AS and DHA and 15 ng/0.5 ml for MQ. The method was found to be suitable for use in clinical pharmacological studies.     

Sensitive determination of clarithromycin in human plasma by high-performance liquid chromatography with spectrophotometric detection

A rapid, selective and sensitive high-performance liquid chromatographic method with spectrophotometric detection was developed for the determination of clarithromycin in human plasma. Liquid-liquid extraction of clarithromycin and norverapamil (as internal standard) from plasma samples was performed with n-hexane/1-butanol (98:2, v/v) in alkaline condition followed by back-extraction into diluted acetic acid. Chromatography was carried out using a CN column (250 mm x 4.6 mm, 5 microm) under isocratic elution with acetonitrile-50 mM aqueous sodium dihydrogen phosphate (32:68, v/v), pH 4.5. Detection was made at 205 nm and analyses were run at a flow-rate of 1.0 ml/min at 40 degrees C. The analysis time was less than 11 min. The method was specific and sensitive with a quantification limit of 31.25 ng/ml and a detection limit of 10 ng/ml in plasma. The mean absolute recovery of clarithromycin from plasma was 95.9%, while the intra- and inter-day coefficient of variation and percent error values of the assay method were all less than 9.5%. Linearity was assessed in the range of 31.25-2000 ng/ml in plasma with a correlation coefficient of greater than 0.999. The method was used to analyze several hundred human plasma samples for bioavailability studies.     

Determination of haloperidol and its reduced metabolite in human plasma by liquid chromatography-mass spectrometry with electrospray ionization

A sensitive and accurate liquid chromatographic-electrospray mass spectrometric (LC-ES-MS) method for the determination of haloperidol (H) and reduced haloperidol (RH) in human plasma is presented, using chlorohaloperidol as the internal standard. A 2-ml volume of plasma subjected to basic (NaOH) extraction, acid (HCl) back-extraction, acid wash and basic (NaOH) re-extraction. The extraction solvent was hexane-isoamyl alcohol (99:1, v/v) for the whole procedure. A Nucleosil C₁₈ column (150×1 mm) was used for high-performacne liquid chromatography, together with 2 mM HCOONH₄-acetonitrile (55:45, v/v; pH 3.0) as the mobile phase. For each drug, four characteristic ions were monitored. Linearity was assessed in the ranges 0.1–50 and 0.25–50 ng/ml for H and RH, respectively. Recoveries were 58 and 70% and detection limits were 0.075 and 0.100 ng/ml for H and RH, respectively. Correlation coefficients were better than 0.999 for both compounds. R.S.D.s for repeatability and reproducibility at 0.25 ng/ml were 11.1 and 8.5% for H and 9.4 and 11.2% for RH, respectively. One of the main advantages of (LC-ES-MS) over other detection systems is the increase in selectivity obtained by monitoring three ions of confirmation for each of the drugs.     

Alternative method for determination of pyrimethamine in plasma by high-performance liquid chromatography

A rapid, selective, sensitive and reproducible reversed-phase high-performance liquid chromatography (HPLC) procedure for the quantitative determination of pyrimethamine (PYR) in plasma is described. The procedure involved the two-step extraction of PYR and the internal standard, quinine (QN) with acetonitrile and dichloromethane at basic pH. Chromatographic separation consisted of the mobile phase (methanol-water containing 0.005 M octanesulfonic acid, 50:50, v/v) running through the column (Techopak-10 C₁₈) at a flow-rate of 1.6 ml/min. Detection was at UV wavelength of 240 nm. The mean recoveries of PYR and QN at a concentration range of 50 and 500 ng/ml were 98.9 and 89%, and 94.7 and 96% for PYR and QN. The within-day coefficients of variation were 2.1–5.1% for PYR and 5.9% for QN. The day-to-day coefficients of variation were 2.1–4.1% for PYR and 5% for QN. The minimum detectable concentrations for PYR and QN in plasma were 3 and 10 ng/ml. The method was found to be suitable for use in clinical pharmacokinetic study.     

Liquid chromatographic method for the determination of ganciclovir and/or acyclovir in human plasma using pulsed amperometric detection

A rapid high-performance liquid chromatographic method for the quantitation of pseudoephedrine in human plasma is presented. The sample preparation involved liquid-liquid extraction of pseudoephedrine from alkalised plasma with hexane-isoamylalcohol (9:1, v/v) and back-extraction of the drug to 0.02 M hydrochloric acid. Liquid chromatography was performed on an octadecylsilica column (50 x 4 mm, 5 microm particles); the mobile phase consisted of acetonitrile-phosphate buffer containing 0.1% of triethylamine, pH 2.4 (5:95, v/v). The run time was 4 min. The spectrophotometric detector was operated at 195 nm. Codeine was used as the internal standard. The limit of quantitation was 5.8 ng/ml using 0.5 ml of plasma. Within-day and between-day precision expressed by relative standard deviation was less than 7% and inaccuracy did not exceed 8%. The assay was applied to the analysis of samples from a pharmacokinetic study.     

Simultaneous determination of timolol maleate, rosuvastatin calcium and diclofenac sodium in pharmaceuticals and physiological fluids using HPLC-UV

A novel HPLC-UV method was developed for the simultaneous determination of timolol (TM), rosuvastatin (RST), and diclofenac sodium (DS) in pharmaceuticals, human plasma and aqueous humor using naproxen sodium as internal standard (IS). The target compounds were analyzed on Hypersil BDS C(18) column (250 mm × 4.6 mm, 5 μm), applying 0.2% triethylamine (TEA) and acetonitrile (ACN) (40:60, v/v), in isocratic mode as mobile phase, pH 2.75 adjusted with 85% phosphoric acid at a flow rate of 1 ml/min. The column oven temperature was kept at 45°C and the peak response was monitored at 284 nm after injecting a 50 μl sample into HPLC system. The direct liquid-liquid extraction procedure was applied to human plasma and bovine aqueous humor samples using mobile phase as an extraction solvent after deproteination with methanol. The different HPLC experimental parameters were optimized and the method was validated according to standard guidelines. The recoveries of the suggested method in human plasma were 98.72, 96.04, and 95.14%, for TM, RST, and DS, while in aqueous humor were 94.99, and 98.23%, for TM, and DS, respectively. The LOD values were found to be 0.800, 0.500, and 0.250 ng/ml, for TM, RST, and DS, respectively, while their respective LOQ values were 2.00, 1.50, and 1.00 ng/ml. The co-efficient of variation (CV) were in the range of 0.1492-1.1729% and 1.0516-4.0104%, for intra-day and inter-day studies, respectively. The method was found accurate in human plasma and bovine aqueous humor and will be applied for the quantification of these compounds in plasma, and aqueous humor samples using animal models and in pharmaceuticals.     

Ultra-performance liquid chromatography-tandem mass spectrometric method for the determination of strychnine and brucine in mice plasma

A selective, simple and efficient method-ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was developed for determination of two toxic alkaloids, namely strychnine and brucine in mice plasma. The UPLC separation was carried out using a 1.7 μm BEH C(18) column (50 mm × 2.1 mm) with a mobile phase consisting of methanol:0.1% formic acid (25:75, v/v), hence providing high efficiency, high resolution and excellent peak shape for the analytes and internal standard. The method was validated over the range of 2.48-496.4 ng/ml for strychnine and 2.64-528 ng/ml for brucine, respectively. Intra- and inter-day accuracy ranged from 95.0% to 107.9% for strychnine, 93.4% to 103.3% for brucine, and the precisions were within 13.8%. The extraction recoveries of both the two alkaloids exceed 81.9%. With a simple and minor sample preparation procedure and short run-time (<3 min), the proposed method was applicable for the pharmacokinetic and toxicological analysis of strychnine and brucine in vivo.     

Liquid chromatographic-tandem mass spectrometric method for the quantitation of huperzine A in dog plasma

A rapid and sensitive LC-MS-MS method for the determination of huperzine A in dog plasma using huperzine B as internal standard has been developed and validated. The analyte and internal standard were extracted from plasma using n-hexane-dichloromethane-2-propanol (300:150:15, v/v/v), chromatographed on a C(18) column (5 microm, 50 mm x 4.6 mm i.d.) with a mobile phase consisting of acetonitrile-methanol-10mM ammonium acetate (35:40:25, v/v/v), and detected using a tandem mass spectrometer with a TurboIonSpray ionization interface. The run time was only 2 min. The assay was linear over the concentration range 0.05-20 ng/ml and intra- and inter-day precision over this range were <5.3% with good accuracy. The limit of detection in plasma was 0.01 ng/ml. The method was successfully applied to define plasma concentration-time curves of huperzine A in dogs after the last dose of an intramuscular injection (10 microg/kg per day for 15 days) of a sustained-release formulation of huperzine A.     

High-performance liquid chromatographic determination of vinorelbine in human plasma and blood: application to a pharmacokinetic study

A sensitive and specific high-performance liquid chromatographic method with fluorescence detection (excitation wavelength: 280 nm; emission wavelength: 360 nm) was developed and validated for the determination of vinorelbine in plasma and blood samples. The sample pretreatment procedure involved two liquid–liquid extraction steps. Vinblastine served as the internal standard. The system uses a Spherisorb cyano analytical column (250×4.6 mm I.D.) packed with 5 μm diameter particles as the stationary phase and a mobile phase of acetonitrile–80 mM ammonium acetate (50:50, v/v) adjusted to pH 2.5 with hydrochloric acid. The assay showed linearity from 1 to 100 ng/ml in plasma and from 2.5 to 100 ng/ml in blood. The limits of quantitation were 1 ng/ml and 2.5 ng/ml, respectively. Precision expressed as RSD was in the range 3.9 to 20% (limit of quantitation). Accuracy ranged from 92 to 120%. Extraction recoveries from plasma and blood averaged 101 and 75%, respectively. This method was used to follow the time course of the concentration of vinorelbine in human plasma and blood samples after a 10-min infusion period of 20 mg/m² of this drug in patients with metastatic cancer.     

Rapid determination of loratadine in small volume plasma samples by high-performance liquid chromatography with fluorescence detection

A simple and rapid high-performance liquid chromatographic method with fluorescence detection was developed for the determination of loratadine in small volume plasma samples. Liquid-liquid extraction of loratadine and diazepam (as internal standard) from plasma samples was performed with n-butyl alcohol/n-hexane (2:98, v/v) in alkaline condition followed by back-extraction into diluted perchloric acid. Chromatography was carried out using a C8 column (250 x 4.6 mm, 5 microm) under isocratic elution with acetonitrile-20 mM sodium dihydrogen phosphate-triethylamine (43:57:0.02, v/v), pH 2.4. Analyses were run at a flow-rate of 1.0 ml/min at room temperature. The method was specific and sensitive with a quantitation limit of 0.62 ng/ml and a detection limit of 0.2 ng/ml at a signal-to-noise ratio of 3:1. The mean absolute recovery of loratadine from plasma was 84%, while the intra-and inter-day coefficient of variation and percent error values of the assay method were all less than 9.7%. Linearity was assessed in the range of 0.62-20 ng/ml in plasma with a correlation coefficient of greater than 0.999. The method has been used to analyze several hundred human plasma samples for bioavailability studies.     

Liquid chromatographic method for the determination of plasma itraconazole and its hydroxy metabolite in pharmacokinetic/bioavailability studies

A sensitive and selective high-performance liquid chromatographic method was developed for the determination of itraconazole and its active metabolite, hydroxyitraconazole, in human plasma. Prior to analysis, both compounds together with the internal standard were extracted from alkalinized plasma samples using a 3:2 (v/v) mixture of 2,2,4-trimethylpentane and dichloromethane. The mobile phase comprised 0.02 M potassium dihydrogen phosphate-acetonitrile (1:1, v/v) adjusted to pH 3.0. Analysis was run at flow-rate of 0.9 ml/min with excitation and emission wavelengths set at 260 and 365 nm, respectively. Itraconazole was found to adsorb on glass or plastic tubes, but could be circumvented by prior treating the tubes using 10% dichlorodimethylsilane in toluene. Moreover, rinsing the injector port with acetonitrile helped to overcome any carry-over effect. This problem was not encountered with hydroxyitraconazole. The method was sensitive with limit of quantification of 3 ng/ml for itraconazole and 6 ng/ml for hydroxyitraconazole. The calibration curve was linear over a concentration range of 2.8-720 ng/ml for itraconazole and 5.6-720 ng/ml for the hydroxy metabolite. Mean recovery value of the extraction procedure for both compounds was about 85%, while the within-day and between-day coefficient of variation and percent error values of the assay method were all less than 15%. Hence, the method is suitable for use in pharmacokinetic and bioavailability studies of itraconazole.