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.     

Rapid quantification of indinavir in human plasma by high-performance liquid chromatography with ultraviolet detection

A rapid, sensitive and specific high-performance liquid chromatography (HPLC) procedure for the quantification of indinavir, a potent human immunodeficiency virus (HIV) protease inhibitor, in human plasma is described. Following C18 solid-phase extraction, indinavir was chromatographed on a reversed-phase C8 column using a simple binary mobile phase of phosphate buffer-acetonitrile (60:40, v/v). UV detection at 210 nm led to an adequate sensitivity without interference from endogenous matrix components. The limit of quantification was 25 ng/ml with a 0.1 ml plasma sample. The standard curve was linear across the range from 25 to 2500 ng/ml with an average recovery of 91.4%. The mean relative standard deviations for concentrations within the standard curve ranged between 1.4 and 9.7%. Quality control standards gave satisfactory intra- and inter-assay precision (R.S.D. from 3.5 to 15.8%) and accuracy within 15% of the nominal concentration. Sample handling experiments, including HIV heat inactivation, demonstrated analyte stability under expected handling processes. The assay is suitable for the analysis of samples from adult and pediatric patients infected with HIV.     

Simplified method to quantify furosemide in urine by high-performance liquid chromatography and ultraviolet detection.

Simplified reversed-phase high-performance liquid chromatographic method with ultraviolet detection at 280 nm without extraction procedure is described to quantify furosemide in rabbit and human urine. An internal standard was not used. The lower limit of quantitation was 0.750 microg/ml using 50 microl urine samples (100 microl of total injection volume), and linear response was tested from 0.750 to 250 microg/ml in both humans and rabbits. Within and between-day accuracy and precision were always below 10% at all analyzed concentrations. Validation data showed that this method is linear, sensitive, selective, specific, accurate and reproducible.     

Determination of neostigmine in human plasma and cerebrospinal fluid by high-performance liquid chromatography with ultraviolet detection

A high-performance liquid chromatographic assay coupled with ultraviolet detection has been developed for the determination of neostigmine in human plasma and cerebrospinal fluid. A novel solid-phase extraction procedure was first used for this analyte and allowed good recovery (89+/-4.5%) together with ease and speed of execution. The method was sensitive, reproducible (C.V.<4.5%) and accurate (100+/-6.6%) over the range 2.6-167.0 ng/ml neostigmine concentrations in plasma or cerebrospinal fluid, and was applied successfully to study the pharmacokinetics of neostigmine in patients suffering from chronic postoperative abdominal pain.     

Determination of the cephalosporin antibiotic cephradine in human plasma by high-performance liquid chromatography with ultraviolet detection

A simple and sensitive high-performance liquid chromatographic method, for the determination of cephradine in human plasma samples has been developed and validated. Cephradine and cephaloridine (internal standard) were extracted from human plasma by perchloric acid protein precipitation followed by centrifugation. Aliquots of the extracts were analysed by reversed-phase high-performance liquid chromatography (HPLC) utilising a polymeric reversed-phase PLRP-S column, followed by ultraviolet detection at 260 nm. The method has a working dynamic range from 0.2 to 30.0 microg/ml from 200 microl human plasma. The precision of the method at 0.2 microg/ml was 4.9% (intra-assay) and negligible (inter-assay) as calculated by one-way analysis of variance and the accuracy of the method at 0.2 microg/ml was -4.1% in terms of percentage bias. This method has been successfully applied to clinical studies including an oral bioequivalence study comparing the pharmacokinetics of 500 mg tablets of Kefdrin with 500 mg tablets of Velosef in healthy human volunteers.     

Determination of a new oral cephalosporin, S-1090, in human plasma and urine by direct injection high-performance liquid chromatography with ultraviolet detection and column switching.

Direct injection high-performance liquid chromatographic (HPLC) methods with column switching and UV detection were developed for the rapid and accurate determination of S-1090 in human plasma and urine. An internal-surface reversed-phase pre-column and a C18 analytical column were used for the plasma assay. Two pre-columns packed with cyano and phenyl materials and a C18 analytical column were used for the urine assay. The calibration curves for plasma and urine assays were linear in the ranges 0.09-9 microg/ml and 0.5-100 microg/ml of S-1090, respectively. The relative standard deviations for plasma and urine assays were less than 6% with low relative errors. The established HPLC methods were demonstrated to be useful for clinical pharmacokinetic studies after oral administration of S-1090.     

Automated determination of reboxetine by high-performance liquid chromatography with column-switching and ultraviolet detection

A fully automated method including column-switching and isocratic high-performance liquid chromatography (HPLC) was developed for quantitative analysis of the new antidepressant reboxetine, a noradrenaline reuptake inhibitor. After serum injection into the HPLC system and on-line sample clean-up on a silica C8 (10x4.0 mm I.D.) clean-up column with an eluent consisting of 2.5% acetonitrile in deionized water, the chromatographic separation was performed on an analytical column (Lichrospher CN; 250x4.6 mm I.D.) with an eluent of acetonitrile-aqueous potassium phosphate buffer (0.008 M, pH 6.4) (50:50). The UV detector was set at 273 or 226 nm. The limit of quantification was about 15 ng/ml at 273 nm and about 4 ng/ml at 226 nm. The day-to-day relative standard deviation ranged between 2.7 and 6.7% with recovery rates > or = 90%. Linear regression analyses revealed correlation coefficients > 0.998. The method can be applied to therapeutic drug monitoring of reboxetine as well as pharmacokinetic studies.     

Direct determination of tramadol glucuronides in human urine by high-performance liquid chromatography with fluorescence detection.

A sensitive and selective reversed-phase high-performance liquid chromatography method has been developed for the direct determination of three glucuronides of the centrally acting analgesic tramadol (1). Separation of these glucuronides into their diastereomers was achieved by HPLC using ion pair chromatography with nonanesulfonic acid sodium salt and LiChrospher 100 RP 18 as stationary phase. Quantification of O-demethyltramadol glucuronide and N,O-didemethyltramadol glucuronide in human urine was performed by fluorescence detection. The urine samples were purified by a two-step solid-phase extraction. The glucuronides were found to be highly enriched in the 1S,2S-diastereomers. The results of a study with three healthy volunteers are presented.     

Determination of terbutaline enantiomers in human urine by coupled achiral-chiral high-performance liquid chromatography with fluorescence detection

A coupled achiral-chiral high-performance liquid chromatographic system with fluorescence detection at excitation/emission wavelengths of 276/306 nm has been developed for the determination of the enantiomers of terbutaline, (S)-(+)-terbutaline and (R)-(-)-terbutaline in urine. Urine samples were prepared by solid-phase extraction with Sep-pak silica, followed by HPLC. The terbutaline was preseparated from the interfering components in urine on Phenomenex silica column and the terbutaline enantiomers and betaxolol were resolved and determined on a Sumichiral OA-4900 chiral stationary phase. The two columns were connected by a switching valve equipped with silica precolumn. The precolumn was used to concentrate the terbutaline in the eluent from the achiral column before back flushing onto the chiral phase. For each enantiomer the assay was linear between 1 and 250 ng/ml (R2=0.9999) and the detection limit was 0.3 ng/ml. The intra-day variation was between 4.6 and 11.6% in relation to the measured concentration and the inter-day variation was 4.3-11.0%. It has been applied to the determination of (S)-(+)-terbutaline and (R)-(-)-terbutaline in urine from a healthy volunteer dosed with racemic terbutaline sulfate.     

Determination of 8-oxoguanine in human plasma and urine by high-performance liquid chromatography with electrochemical detection.

A highly sensitive and selective method for determining 8-oxoguanine in plasma and urine was developed by high-performance liquid chromatography with electrochemical detection. The compound was separated by gradient elution on a C18 reversed-phase column with a mobile phase of acetonitrile and 0.1 M sodium acetate, pH 5.2. 8-Hydroxy-2'-deoxyguanosine was used as internal standard. 8-Oxoguanine was detected electrochemically by setting the potential to +300 mV vs. Pd reference. The sensitivity of the assay was 22 ng/ml with a signal-to-noise ratio of 7:1. The within-day relative standard deviations for 8-oxoguanine quality control samples with concentrations of 3340, 1340 and 84 ng/ml were 3.6, 4.3 and 5.7% for plasma, and 4.1, 4.6 and 6.2% for urine, respectively. The day-to-day relative standard deviations for the same samples were 3.8, 6.8 and 7.1% for plasma, and 3.9, 7.0 and 7.9% for urine, respectively. The method is designed to study the pharmacokinetics and metabolic fate of O6-benzylguanine in a phase I clinical trial. Previously, O6-benzyl-8-oxoguanine was identified as the primary metabolite of O6-benzylguanine in humans. We now demonstrate that 8-oxoguanine is a further metabolite of O6-benzylguanine.     

Determination of rutin in human plasma by high-performance liquid chromatography utilizing solid-phase extraction and ultraviolet detection

The degradation kinetics of aplidine were investigated using reversed-phase high-performance liquid chromatography combined with UV detection. Aplidine consists of at least two isomers that undergo interconversion at a low rate. Influences of pH, temperature, buffer ions and ionic strength on the degradation kinetics were studied. The log kobs) -pH profile can be divided into three parts, a proton, a solvent and a hydroxyl-catalysed section. The stability-indicating properties of the used analysis technique as well as the identities of the main degradation products were checked using gradient liquid chromatography and mass spectrometric detection. The overall degradation rate constant as a function of the temperature under acidic and alkaline conditions obeys the Arrhenius equation. No catalytic influences were observed with phosphate and carbonate buffers and, in addition, the ionic strength showed no substantial effect on the stability, as expected. Results from gradient LC-MS indicated that hydrolysis of the ester groups present in the ring structure was the main degradation route. There is no difference in degradation rate constants for the individual isomers.     

Determination of celecoxib in human plasma by normal-phase high-performance liquid chromatography with column switching and ultraviolet absorbance detection

A method is described for the determination of celecoxib in human plasma. Samples were extracted using 3M Empore membrane extraction cartridges and separated under normal-phase HPLC conditions using a Nucleosil-NO2 (150x4.6 mm, 5 microm) column. Detection was accomplished using UV absorbance at 260 nm. The HPLC method included a column switching procedure, in which late eluting compounds were diverted to waste, to reduce run-time to 12 min. The assay was linear in the concentration range of 25-2000 ng/ml when 1-ml aliquots of plasma were extracted. Recoveries of celecoxib were greater than 91% over the calibration curve range. Intraday precision and accuracy for this assay were 5.7% C.V. or better and within 2.3% of nominal, respectively. The assay was used to analyze samples collected during human clinical studies.     

Determination of clozapine and its N-desmethyl metabolite by high-performance liquid chromatography with ultraviolet detection

A rapid high-performance liquid chromatographic method has been developed for the simultaneous determination of the atypical antipsychotic drug clozapine and its principal metabolite, N-desmethyl clozapine in human plasma. After liquid-liquid extraction the compounds were separated in a reversed-phase column and measured by ultraviolet absorption at 230 nm. For both compounds inter-day variations were <3.8%, and, based on a plasma sample volume of 2 ml, the limits of quantification were 25 ng/ml. Analytical interference from coadministered psychoactive drugs and their metabolites was also studied, and no interference was found from the most commonly used antidepressants and antipsychotic drugs. The assay is sufficiently sensitive and easy to use for the analysis of plasma samples in human clinical trials and therapeutic drug monitoring.     

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 micromol/mol creatinine) to 34.19 ng/ml (59.11 micromol/mol creatinine), and the mean+/-standard deviation was 5.08 ng/ml (6.60 micromol/mol creatinine)+/-5.75 ng/ml (9.22 micromol/mol creatinine). The mean+/-standard deviation of urinary 2-naphthol level of smokers, 7.03 ng/ml (8.49 micromol/mol creatinine)+/-6.16 ng/ml (10.23 micromol/mol creatinine), was significantly higher than that of non-smokers, 2.49 ng/ml (4.10 micromol/mol creatinine)+/-3.92 ng/ml (7.03 micromol/mol creatinine).     

Determination of ketorolac in human plasma by reversed-phase high-performance liquid chromatography using solid-phase extraction and ultraviolet detection

An improved high-performance liquid chromatographic method has been developed to measure human plasma concentrations of the analgesic nonsteroidal anti-inflammatory drug ketorolac for use in pharmacokinetic studies. Samples were prepared for analysis by solid-phase extraction using Bond-Elut PH columns, with nearly complete recovery of both ketorolac and the internal standard tolmetin. The two compounds were separated on a Radial-Pak C18 column using a mobile phase consisting of water-acetonitrile-1.0 mol/l dibutylamine phosphate (pH 2.5) (30:20:1) and detected at a UV wavelength of 313 nm. Using only 250 microl of plasma, the standard curve was linear from 0.05 to 10.0 microg/ml.     

Stereoselective determination of cisapride, a prokinetic agent, in human plasma by chiral high-performance liquid chromatography with ultraviolet detection: application to pharmacokinetic study

We have developed a simple, sensitive, specific and reproducible stereoselective high-performance liquid chromatography technique for analytical separation of cisapride enantiomers and measurement of cisapride enantiomers in human plasma. A chiral analytical column (ChiralCel OJ) was used with a mobile phase consisting of ethanol-hexane-diethylamine (35:64.5:0.5, v/v/v). This assay method was linear over a range of concentrations (5-125 ng/ml) of each enantiomer. The limit of quantification was 5 ng/ml in human plasma for both cisapride enantiomers, while the limit of detection was 1 ng/ml. Intra- and inter-day C.V.s did not exceed 15% for all concentrations except at 12.5 ng/ml for EII (+)-cisapride, which was approximately 20 and 19%, respectively. The clinical utility of the method was demonstrated in a pharmacokinetic study of normal volunteers who received a 20 mg single oral dose of racemic cisapride. The preliminary pharmacokinetic data obtained using the method we describe here provide evidence for the first time that cisapride exhibits stereoselective disposition.     

Simultaneous determination of fentanyl and midazolam using high-performance liquid chromatography with ultraviolet detection

When measuring fentanyl and midazolam simultaneously in the same plasma sample with standard high-performance liquid chromatography-ultraviolet (HPLC-UV) detection, overlap of the fentanyl peak by the midazolam peak occurs, which makes fentanyl determination impossible. We tested the hypothesis that by acidifying the methanol mobile phase with 0.02% perchloric acid, 70%, it would be possible to separate both peaks. The UV detector was set at 200 nm. Calibration curves for fentanyl (range 0-2000 pg/ml) and midazolam (range 0-400 ng/ml) were linear (r>0.99). The detection limits were 200 pg/ml (fentanyl) and 10 ng/ml (midazolam). Precision and accuracy for intra- and inter-assay variability as well as in-line validation with quality control samples (QCS) were acceptable (<15 and 20%, respectively), except for fentanyl QCS of 200 pg/ml (17.8% precision). Although less sensitive than gas chromatography-mass spectrometry (GC-MS), reliable measurements of fentanyl, simultaneously with midazolam, can be performed with this HPLC-UV system.     

Determination of the catechol-O-methyltransferase inhibitor tolcapone and three of its metabolites in animal and human plasma and urine by reversed-phase high-performance liquid chromatography with ultraviolet detection.

Reversed-phase HPLC procedures were developed for the determination of tolcapone (Ro 40-7592) and its metabolites Ro 40-7591, Ro 61-1448, and Ro 47-1669 in plasma and in urine samples. One of the procedures for plasma involved the determination of tolcapone and its metabolite Ro 40-7591 and the other, the determination of the two other metabolites. The urine assay enabled the simultaneous determination of tolcapone and all metabolites in one run. Sample preparation in plasma involved protein precipitation with acetonitrile. Urine was simply diluted. The compounds of interest were monitored in the UV at 270 nm. The limits of quantification were 0.05 microg/ml for each compound (plasma assay) and 0.2 microg/ml for the urine assay. The mean inter-assay precisions (C.V.) were < or = 6% (plasma assay) and < or = 8% (urine assay). The procedures were successfully applied to the sample analysis of animal pharmacokinetic (rat, dog, mouse, rabbit and cynomolgus monkey) and clinical pharmacology studies.     

Determination of famotidine in human plasma and urine by high-performance liquid chromatography.

An improved, rapid and specific high-performance liquid chromatographic assay was developed for the determination of famotidine in human plasma and urine. Plasma samples were alkalinized and the analyte and internal standard (cimetidine) extracted with water-saturated ethyl acetate. The extracts were reconstituted in mobile phase, and injected onto a C18 reversed-phase column; UV detection was set at 267 nm. Urine samples were diluted with nine volumes of a mobile phase-internal standard mixture prior to injection. The lower limits of quantification in plasma and urine were 75 ng/ml and 1.0 microg/ml, respectively; intra- and inter-day coefficients of variation were < or =10.5%. This method is currently being used to support renal function studies assessing the use of intravenously administered famotidine to characterize cationic tubular secretion in man.