Quantification of BNP7787 (dimesna) and its metabolite mesna in human plasma and urine by high-performance liquid chromatography with electrochemical detection

A sensitive and accurate assay was developed and validated to determine BNP7787 (dimesna), a new protector against cisplatin-induced toxicities, and its metabolite mesna in plasma and urine of patients. Both analytes were measured as mesna in deproteinized plasma or in urine diluted with mobile phase using high-performance liquid chromatography with an electrochemical detector provided with a wall-jet gold electrode. The assays for BNP7787 and mesna in deproteinized plasma were linear over the range of 1.6–500 μM and 0.63–320 μM, respectively. In plasma, the mean recovery of BNP7787 over the whole concentration range was 100.6% and of mesna 94.6%. The lower limits of quantitation (LLQs) of BNP7787 and mesna in deproteinized plasma were 1.6 μM and 0.63 μM, respectively. For both compounds the within- and between-day accuracy and precision of the assay was better than 12%. The assays for BNP7787 and mesna in urine were linear over the range of 0.8–1200 μM and 0.63–250 μM, respectively. In urine, the mean recovery of BNP7787 over the whole concentration range was 94.1% and of mesna 93.1%. The LLQ of BNP7787 in urine was 0.8 μM and of mesna 1.6 μM. The within- and between-day accuracy and precision of the assay for BNP7787 and mesna was lower than 15%. The stability of mesna in urine increased with an increasing concentration of mesna, lower temperature and addition of EDTA (1 g/l) and hydrochloric acid (0.2 M). BNP7787 in urine was stable for at least 24 h at temperatures in the range of −20°C up to 37°C and independent of the concentration. The developed assays are currently applied for samples of patients with solid tumors participating in a phase I trial of BNP7787 in combination with cisplatin.     

Capillary electrophoresis analysis of nitrite and nitrate in sub-microliter quantities of airway surface liquid

We developed a simple capillary electrophoresis (CE) method to measure nitrite and nitrate concentrations in sub-microliter samples of rat airway surface liquid (ASL), a thin (10–30 μm) layer of liquid covering the epithelial cells lining the airways of the lung. The composition of ASL has been poorly defined, in large part because of the small sample volume (1–3 μl per cm² of epithelium) and difficulty of harvesting ASL. We have used capillary tubes for ASL sample collection, with microanalysis by CE using a 50 mM phosphate buffer (pH 3), with 0.5 mM spermine as a dynamic flow modifier, and direct UV detection at 214 nm. The limit of detections (LODs), under conditions used, for ASL analysis were 10 μM for nitrate and 30 μM for nitrite (S/N=3). Nitrate and nitrite were also measured in rat plasma. The concentration of nitrate was 102±12 μM in rat ASL and 70±1.0 μM in rat plasma, whereas nitrite was 83±28 μM in rat ASL and below the LOD in rat plasma. After instilling lipopolysaccharide intratracheally to induce increased NO production, the nitrate concentration in ASL increased to 387±16 μM, and to 377±88 μM in plasma. The concentration of nitrite increased to 103±7.0 μM for ASL and 138±17 μM for plasma.     

Automated solid-phase extraction method for the determination of atovaquone in capillary blood applied onto sampling paper by rapid high-performance liquid chromatography

A bioanalytical method for the determination of atovaquone in 100 μl blood-spots by solid-phase extraction and high-performance liquid chromatography has been developed and validated. Atovaquone was extracted from the sampling paper in 0.2 M phosphoric acid and a structurally similar internal standard was added with acetonitrile before being loaded onto a C8 end-capped solid-phase extraction column. Atovaquone and internal standard were analysed by high-performance liquid chromatography on a C₁₈ J’Sphere ODS-M80 (150×4.0 mm) column with mobile phase acetonitrile–phosphate buffer, 0.01 M, pH 7.0 (65:35, v/v) and UV detection at 277 nm. The intra-assay precision was 2.7% at 12.00 μM and 13.5% at 1.00 μM. The inter-assay precision was 3.3% at 12.00 μM and 15.6% at 1.00 μM. The lower limit of quantification was 1.00 μM. The limit of detection was 0.50 μM.     

High-performance liquid chromatographic analysis of amoxicillin in human and chinchilla plasma, middle ear fluid and whole blood

We extended the application of a sensitive high-performance liquid chromatography assay of amoxicillin developed in this laboratory for human plasma and middle ear fluid (MEF) to other sample matrices including chinchilla plasma or MEF and human and chinchilla whole blood with minor modification and validated the limit of quantitation at 0.25 μg/ml with a 50-μl sample size for human and chinchilla plasmas or MEFs. Amoxicillin and cefadroxil, the internal standard, were extracted from 50 μl of the samples with Bond Elut C₁₈ cartridges. The extract was analyzed on a Keystone MOS Hypersil-1 (C₈) column with UV detection at 210 nm. The mobile phase was 6% acetonitrile in 5 mM phosphate buffer, pH 6.5 and 5 mM tetrabutylammonium. The within-day coefficients of variation were 2.7–9.9 (n=4) and 1.7–7.2% (n=3) for chinchilla plasma and MEF samples, respectively; 2.8–8.1% (n=3) and 2.9–4.7% (n=3) for human and chinchilla whole blood, respectively. An alternative mobile phase composition for chinchilla plasma and MEF samples reduced the analysis time significantly.     

In vitro cleavage of paracetamol glucuronide by human liver and kidney β-glucuronidase: determination of paracetamol by capillary electrophoresis

A capillary electrophoresis (CE) method was developed using paracetamol glucuronide as a novel probe for human β-glucuronidase activity. Using UV detection without prior sample clean-up procedures, fast and reliable quantitation of the released paracetamol was possible. The method showed good precision, accuracy and sensitivity with a limit of detection of 0.25 μM (38 ng/ml) and a limit of quantitation of 1 μM (151 ng/ml). The suitability of the method has been shown for enzyme kinetic studies using different liver and kidney homogenates, respectively. Our data clearly demonstrate that paracetamol glucuronide is cleaved by human β-glucuronidase thereby releasing paracetamol. The CE method presented is not only a valuable tool for measuring human β-glucuronidase activity, but also allows investigation of the contribution of deglucuronidation of paracetamol glucuronide to the disposition of paracetamol.     

Sensitive assay of trimethylamine N-oxide in liver microsomes by headspace gas chromatography with flame thermionic detection

To compare the trimethylamine N-oxygenase activity of liver microsomes from house musk shrew (Suncus murinus) and rat, a sensitive method for the quantitation of trimethylamine (TMA) N-oxide was developed using gas chromatography with flame thermionic detection. The limit of quantification was 0.5 μM and the calibration curve was linear at least up to 5 μM in incubations containing liver microsomal preparations from Suncus. The intra-day RSD values ranged from 10.4 to 12.8 at 0.5 μM and from 3.5 to 6.7 at 5 μM. The inter-day RSD values were 11.6 and 6.5 at 0.5 and 5 μM, respectively. This method provides a sensitive assay for TMA N-oxygenase activity in liver microsomes. Using this method we found that Suncus was capable of N-oxidizing trimethylamine at a very slow rate.     

Simultaneous determination of urinary free cortisol and 6β-hydroxycortisol by liquid chromatography–atmospheric pressure chemical ionization tandem mass spectrometry and its application for estimating hepatic CYP3A induction

A reversed-phase high-performance liquid chromatography coupled to atmospheric pressure chemical ionization tandem mass spectrometry (HPLC–APCI-MS–MS) assay was developed to simultaneously determine monkey urinary free cortisol (C) and 6β-hydroxycortisol (6β-OHC) in 8 min. Urine sample (0.5 ml) containing fludrocortisone acetate (F-C) as the internal standard was extracted with ethyl acetate for 5 min with an extraction efficiency of 90% and 75% for C and 6β-OHC, respectively. A Perkin-Elmer Sciex API 3000 triple quadruple instrument was used for mass spectrometric detection and the column eluent was directed to a heated nebulizer probe. The assay was linear over the range 0.25–10 μM for each analyte. The intra- and inter-day relative standard deviation (RSD) over the entire concentration range for both analytes was less than 10%. Accuracy determined at three concentrations (0.8, 2.0 and 8.0 μM) ranged between 95.5 and 108%. The method described herein is suitable for the rapid and efficient measurement of 6β-OHC/C ratio in Rhesus monkey urine following administration of known hepatic CYP3A inducers and can be used to estimate potential CYP3A induction by drug candidates in the process of early drug development.     

Procedure for the sample preparation and handling for the determination of amino acids, monoamines and metabolites from microdissected brain regions of the rat

A method is described for the analysis of amino acids, monoamines and metabolites by high-performance liquid chromatography with electrochemical detection (HPLC–ED) from individual brain areas. The chromatographic separations were achieved using microbore columns. For amino acids we used a 100×1 mm I.D. C₈, 5 μm column. A binary mobile phases was used: mobile phase A consisted of 0.1 M sodium acetate buffer (pH 6.8)–methanol–dimethylacetamide (69:24:7, v/v) and mobile phase B consisted of sodium acetate buffer (pH 6.8)–methanol–dimethylacetamide (15:45:40, v/v). The flow-rate was maintained at 150 μl/min. For monoamines and metabolites we used a 150×1 mm I.D. C₁₈ 5 μm reversed-phase column. The mobile phase consisted of 25 mM monobasic sodium phosphate, 50 mM sodium citrate, 27 μM disodium EDTA, 10 mM diethylamine, 2.2 mM octane sulfonic acid and 10 mM sodium chloride with 3% methanol and 2.2% dimethylacetamide. The potential was +700 mV versus Ag/AgCl reference electrode for both the amino acids and the biogenic amines and metabolites. Ten rat brain regions, including various cortical areas, the cerebellum, hippocampus, substantia nigra, red nucleus and locus coeruleus were microdissected or micropunched from frozen 300-μm tissue slices. Tissue samples were homogenized in 50 or 100 μl of 0.05 M perchloric acid. The precise handling and processing of the tissue samples and tissue homogenates are described in detail, since care must be exercised in processing such small volumes while preventing sample degradation. An aliquot of the sample was derivatized to form the tert.-butylthiol derivatives of the amino acids and γ-aminobutyric acid. A second aliquot of the same sample was used for monamine and metabolite analyses. The results indicate that the procedure is ideal for processing and analyzing small tissue samples.     

Determination of 5-fluorouracil in microvolumes of human plasma by solvent extraction and high-performance liquid chromatography

In the present study, a new reversed-phase HPLC method has been developed and validated for the quantitative determination of 5-fluorouracil (5-FU) in human plasma using only 100-μl samples. The sample extraction and clean-up procedure involved a simple liquid–liquid extraction after addition of 5-chlorouracil (5-CU), used as internal standard, with 5 ml ethyl acetate. Chromatographic separations were performed on an Inertsil ODS-3 column (250×4.6 mm ID; 5 μM particle size), eluted with a mobile phase composed of acidified water (pH 2.0). The column effluent was monitored by UV absorption measurement at a wavelength of 266 nm. The calibration curves were constructed over a range of 0.20–50.0 μM and were fitted by weighted (1/x) linear regression analysis using the ratio of peak heights of 5-FU and 5-CU versus concentrations of the nominal standards. Extraction recoveries over the total range averaged 92 and 93% for 5-FU and 5-CU, respectively. The lower limit of quantitation was established at 0.20 μM (26 ng/ml), with within-run and between-run precisions of 4.2 and 7.0%, respectively, and an average accuracy of 109.3%. The within-run and between-run precisions at four tested concentrations analyzed in quintuplicate over a time period of four days were <1.4 and <4.4%, respectively. The accuracy at the tested concentrations ranged from 98.4 to 102.3%. Compared to previously described validated analytical methods for 5-FU, our present assay provides equivalent to superior sensitivity using only microvolumes of sample.     

High-performance liquid chromatographic procedures for the quantitative determination of paclitaxel (Taxol) in human urine

A reversed-phase high-performance liquid chromatographic (RP-HPLC) method has been developed and validated for the quantitative determination of paclitaxel in human urine. A comparison is made between solid-phase extraction (SPE) and liquid-liquid extraction (LLE) as sample pretreatment. The HPLC system consists of an APEX octyl analytical column and acetonitrile-methanol-0.2 μM ammonium acetate buffer pH 5 (4:1:5, v/v) as the mobile phase. Detection is performed by UV absorbance measurement at 227 nm. The SPE procedure involves extraction on Cyano Bond Elut columns. n-Butylchloride is the organic extraction fluid used for the LLE. The recoveries of paclitaxel in human urine are 79 and 75% for SPE and LLE, respectively. The accuracy for the LLE and SPE sample pretreatment procedures is 100.4 and 104.9%, respectively, at a 5 μg/ml drug concentration. The lower limit of quantitation is 0.01 μg/ml for SPE and 0.25 μg/ml for LLE. Stability data of paclitaxel in human urine are also presented.     

Post-column enzyme reactors for chemiluminometric detection of glucose, 1,5-anhydroglucitol and 3-hydroxybutyrate in an anion-exchange chromatographic system

A liquid chromatographic system consisting of a co-immobilized 3-hydroxybutyrate dehydrogenase-NADH oxidase reactor and an immobilized pyranose oxidase reactor in series and a chemiluminometer was developed for the simultaneous determination of glucose, 1,5-anhydroglucitol and 3-hydroxybutyrate in plasma. The enzymes were immobilized on toresylated poly(vinyl alcohol) beads. Separation was achieved on a TSK gel SAX column (40×4 mm I.D.) with an eluent of 50 mM NaOH containing 30 mM sodium butyrate. The hydrogen peroxide produced was detected by measuring the chemiluminescence emitted on admixing with luminol and potassium hexacyanoferrate(III). The calibration curves were linear from 0.8 to 500 μM (7 ng−4 μg) for glucose, from 0.8 to 400 μM (7 ng−3 μg) for 1,5-anhydroglucitol and from 1 to 700 μM (5 ng−4 μg in a 50-μl injection) for 3-hydroxybutyrate. The sample throughput was four per hour. The reactors were stable for at least ten days.     

Determination of urinary 5-hydroxytryptophol by high-performance liquid chromatography with electrochemical detection

A high-performance liquid chromatographic method for the routine determination of elevated urinary levels of the serotonin metabolite 5-hydroxytryptophol (5-HTOL) is described. Urine samples were treated with β-glucuronidase, and 5-HTOL was isolated by solid-phase extraction on a small Sephadex G-10 column prior to injection onto an isocratically eluted C₁₈ reversed-phase column. Detection of 5-HTOL was performed electrochemically at +0.60 V vs. Ag/AgCl. The limit of detection was ca. 0.05 μM, and the intra-assay coefficients of variation were below 6% with urine samples containing 0.2 and 2.1 μM 5-HTOL and a standard solution of 2.0 μM (n = 5). The recovery of 5-HTOL after the sample clean-up procedure was close to 100%. A good correlation (r² = 0.97; n = 12) was obtained between the present method and a sensitive and specific gas chromatographic—mass spectrometric method. The total (free plus conjugated) 5-HTOL levels in urine were normally below 0.2 μM, but after an acute dose of alcohol they increased to 0.5–15 μM.     

Interdoublet Sliding in Bovine Spermatozoa: Its Relationship to Flagellar Motility and the Action of Inhibitory Agents

Interdoublet sliding rates were assessed in bull sperm, utilizing a freeze–thaw procedure to allow axonemal disintegration. The sliding rate at 23°C increased with increasing MgATP concentrations up to 1 mMATP, to plateau at 8 μm/sec. The analyzed interdoublet shear in both live and demembranated (Triton X-100-extracted) bull sperm reactivated with 1 mMATP established maximal microtubule sliding rates at 6 μm/sec during flagellar beating. Therefore,in vitrosliding rates were sufficient to account for the beat in intact flagella. The effect of inhibitors of flagellar motility onin vitrosliding rates was evaluated. While 8 μMvanadate minimally reduced the sliding rate (to ≈ 4 μm/sec), only 0.5 μMvanadate was sufficient to terminate reactivated bull sperm motility. Nickel ion (0.66 mM) terminated all spontaneous motility, while only reducing microtubule sliding rates to ≈ 5.0 μm/sec. Exposing intact bull sperm to theophylline (1 mM), and incubating the subsequently demembranated sperm in cAMP (3 μM), improved flagellar motility, but had little impact on microtubule sliding rates as determined by axonemal disintegration. Furthermore, deactivating live sperm with 2 mMKCN and 4 mM2-deoxy- -glucose renders the subsequently reactivated sperm immotile (as long as exogenous cAMP is absent). Yet, this treatment only reduced the sliding rate by 38%. Paradoxically, 4 mMMgADP reduced the sliding rates most dramatically (86%), whereas demembranated sperm models retain a strong, coordinated beating pattern in the presence of MgADP. These results demonstrate that there is no direct relationship between interdoublet sliding rates and the capacity for coordinated flagellar beating.     

Disruption of the Cytokeratin Cytoskeleton and Inhibition of Hepatocytic Autophagy by Okadaic Acid

To learn whether autophagy might be dependent on any of the major cytoskeletal elements, the effect of various cytoskeleton inhibitors on autophagy and cytoskeletal organization was studied in isolated rat hepatocytes. Autophagy, measured as the sequestration of endogenous lactate dehydrogenase, was completely inhibited in isolated rat hepatocytes by the protein phosphatase inhibitor okadaic acid (30 nM). Only small effects were seen with vinblastine (10 μM) or cytochalasin D (10 μM). Indirect immunofluorescence microscopy with antibody to a 55-kDa cytokeratin, corresponding to human cytokeratin 8 (CK8), revealed that whereas control cells contained a well-organized network of cytokeratin intermediate filaments, okadaic acid disrupted this network into small spherical aggregates. Treatment with cytochalasin D or vinblastine, which disrupt microfilaments and microtubules, respectively, had no detectable effect on the cytokeratin filament distribution. Neither the microtubule network (detected by indirect immunofluorescence with antibodies against α- and β-tubulin) nor the actin microfilament network (detected by rhodamine-palloidin) was disrupted by okadaic acid. Naringin (100 μM), a putative protein kinase-inhibitory flavonoid, offered complete protection against the autophagy-inhibitory and cytokeratin-disruptive effects of okadaic acid. Two other flavonoids, genistein (100 μM) and prunin (100 μM) as well as KN-62 (10 μM), a specific inhibitor of Ca²⁺/calmodulin-dependent kinase II), likewise displayed a good ability to protect against the effect of okadaic acid upon cytokeratin organization, while no such protection was seen with H-89 (20 μM), an inhibitor of the cyclic nucleotide-dependent protein kinases, or with H-7 (100 μM), which in addition inhibits protein kinase C. The results suggest that the cytokeratin cytoskeleton of hepatocytes is subject to rapid control by phosphorylation and dephosphorylation and that cytokeratin filaments may somehow be involved in the autophagic process.     

Determination of the halothane metabolites trifluoroacetic acid and bromide in plasma and urine by ion chromatography

Halothane (CF₃CHClBr), a widely used volatile anesthetic, undergoes extensive biotransformation in humans. Oxidative halothane metabolism yields the stable metabolites trifluoroacetic acid and bromide which can be detected in plasma and urine. To date, analytical methodologies have either required extensive sample preparation, or two separate analytical procedures to determine plasma and urine concentrations of these analytes. A rapid and sensitive method utilizing high-performance liquid chromatography-ion chromatography (HPLC-IC) with suppressed conductivity detection was developed for the simultaneous detection of both trifluoroacetic acid and bromide in plasma and urine. Sample preparation required only ultrafiltration. Standard curves were linear (r²≥0.99) from 10 to 250 μM trifluoroacetic acid and 2 to 5000 μM bromide in plasma and 10 to 250 μM trifluoroacetic acid and 2 to 50 μM bromide in urine. The assay was applied to quantification of trifluoroacetic acid and bromide in plasma and urine of a patient undergoing halothane anesthesia.     

On-line sample preparation of paraquat in human serum samples using high-performance liquid chromatography with column switching

A new ion-pair high-performance liquid chromatographic method with column-switching has been developed for the determination of paraquat in human serum samples. The diluted serum sample was injected onto a precolumn packed with LiChroprep RP-8 (25-40 μm) and polar serum components were washed out by 3% acetonitrile in 0.05 M phosphate buffer (pH 2.0) containing 5 mM sodium octanesulfonate. After valve switching to inject position, concentrated compounds were eluted in the back-flush mode and separated on an Inertsil ODS-2 column with 17% acetonitrile in 0.05 M phosphate buffer (pH 2.0) containing 10 mM sodium octanesulfonate. The total analysis time per sample was about 30 min and mean recovery was 98.5±2.8% with a linear range of 0.1–100 μg/ml. This method has been successfully applied to serum samples from incidents by paraquat poisoning.     

Determination of temozolomide in human plasma and urine by high-performance liquid chromatography after solid-phase extraction

As a part of a pilot clinical study, a high-performance reversed-phase liquid chromatography analysis was developed to quantify temozolomide in plasma and urine of patients undergoing a chemotherapy cycle with temozolomide. All samples were immediately stabilized with 1 M HCl (1 + 10 of biological sample), frozen and stored at −20°C prior to analysis. The clean-up procedure involved a solid-phase extraction (SPE) of clinical sample (100 μl) on a 100-mg C₁₈-endcapped cartridge. Matrix components were eliminated with 750 μl of 0.5% acetic acid (AcOH). Temozolomide was subsequently eluted with 1250 μl of methanol (MeOH). The resulting eluate was evaporated under nitrogen at RT and reconstituted in 200 μl of 0.5% AcOH and subjected to HPLC analysis on an ODS-column (MeOH-0.5% AcOH, 10:90) with UV detection at 330 nm. The calibration curves were linear over the concentration range 0.4–20 μg/ml and 2–150 μg/ml for plasma and urine, respectively. THe extraction recovery of temozolomide was 86–90% from plasma and 103–105% from urine over the range of concentrations considered. The stability of temozolomide was studied in vitro in buffered solutions at RT, and in plasma and urine at 37°C. An acidic pH (<5–6) shoul be maintained throughout the collection, the processing and the analysis of the sample to preserve the integrity of the drug. The method reported here was validated for use in a clinical study of temozolomide for the treatment of metastatic melanoma and high grade glioma.     

High-performance liquid chromatographic method for the determination of the major quinine metabolite, 3-hydroxyquinine, in plasma and urine

The determination of 3-hydroxyquinine in urine and plasma samples is described. Extraction was performed using a mixture of toluene–butanol (75:25, v/v), followed by back-extraction into the mobile phase, which consisted of 0.1 M phosphate buffer, acetonitrile, tetrahydrofuran and triethylamine. A reversed-phase liquid chromatography system with fluorescence detection and a CT-sil C₁₈ column were used. The within-assay coefficient of variation of the method was 2% at the higher concentration values in plasma, 2.95 μM, 4% at 227 nM and 9% at the lower limit of quantitation, 4.5 nM. In urine, the coefficient of variation was 11% at the lower concentration, 227 nM and was 3% at 56.8 μM. The between-assay coefficient of variation was 4% at the low concentration (5.1 nM) in plasma, 2% at 276.8 nM and 3% at 1.97 μM. In urine, the between assay coefficient of variation was 4% at 204.6 nM, 3% at 5.12 μM and 2% at 56.8 μM.     

Simultaneous quantitation of etoposide and its catechol metabolite in human plasma using high-performance liquid chromatography with electrochemical detection

Etoposide, a highly active and widely used antineoplastic agent, is O-demethylated to its active catechol metabolite. A high-performance liquid chromatographic assay method for the simultaneous quantitation of etoposide and etoposide catechol in human plasma was established. Etoposide and etoposide catechol were extracted from plasma using chloroform and methanol followed by phase separation, evaporation of the organic phase, and reconstitution of the residue. Chromatography was accomplished using a reversed-phase phenyl analytical column (390 mm×3.9 mm I.D.) with a mobile phase of 76.6% 25 mM citric acid–50 mM sodium phosphate (pH 2.4)–23.4% acetonitrile pumped isocratically at 1 ml/min with electrochemical detection. The limit of detection for etoposide was 1.2 nM and for etoposide catechol was 0.2 nM. The precision (CV) for etoposide ranged from 0.7 to 3% and for the catechol metabolite from 1 to 6%; accuracy of predicted values ranged from 97 to 106% and 94 to 103%, respectively. The assay was linear from 0.1 to 10 μM for etoposide and from 0.005 to 0.5 μM for etoposide catechol in plasma. Recovery of etoposide and etoposide catechol ranged from 93 to 95% and 90 to 98%, respectively. Stability of etoposide and etoposide catechol in human plasma containing ascorbic acid stored at −70°C for one year was demonstrated. This assay procedure is suitable for evaluation of etoposide and etoposide catechol pharmacokinetics in plasma following etoposide administration.     

High-performance liquid chromatographic determination of the antifungal drug fluconazole in plasma and saliva of human immunodeficiency virus-infected patients

A high-performance liquid chromatographic (HPLC) assay has been developed for the determination of the antifungal drug fluconazole in saliva and plasma of patients infected with the human immunodeficiency virus (HIV). Samples can be heated at 60°C for 30 min to inactivate the virus without loss of the analyte. The sample pretreatment involves a liquid-liquid extraction with chloroform-1-propanol (4:1, v/v). The chromatographic analysis is performed on a Lichrosorb RP-18 (5 μm) column by isocratic elution with a mobile phase of 0.01 M acetate buffer (pH 5.0)-methanol (70:30, v/v) and ultraviolet (UV) detection at 261 nm. The lower limit of is 100 ng/ml in plasma (using 500-μl samples) and 1 μg/ml in saliva (using 250-μl samples) and the method is linear up to 100 μg/ml in plasma and saliva. At a concentration of 5 μg/ml the within-day and between-day precision in plasma are 7.1 and 5.7%, respectively. In saliva the within-day and between-day precision is 10.8% (at 5 μg/ml). The methodology is now being used in pharmacokinetic studies in HIV-infected patients in our hospital.