Analysis of the second generation antidepressant venlafaxine and its main active metabolite O-desmethylvenlafaxine in human plasma by HPLC with spectrofluorimetric detection

A high-performance liquid chromatographic method has been developed for the determination of the recent serotonin and norepinephrine reuptake inhibitor (SNRI) venlafaxine and its main active metabolite, O-desmethylvenlafaxine, in human plasma. Separation was obtained by using a reversed-phase column (C8, 150 x 4.6 mm I.D., 5 microm) and a mobile phase composed of 75% aqueous phosphate buffer containing triethylamine at pH 6.8 and 25% acetonitrile. Fluorescence detection was used, exciting at lambda=238 nm and monitoring the emission at lambda=300 nm. Citalopram was used as the internal standard. A careful pre-treatment of plasma samples was developed, using solid-phase extraction with C1 cartridges (100 mg, 1 mL). The limit of quantification (LOQ) was 1.0 ng mL(-1) and the limit of detection (LOD) was 0.3 ng mL(-1) for both analytes. The method was applied with success to plasma samples taken from patients undergoing treatment with venlafaxine. Precision data, as well as accuracy results, were satisfactory and no interference from other drugs was found. Hence, the method is suitable for therapeutic drug monitoring of venlafaxine and its main metabolite in depressed patients' plasma.     

HPLC analysis of the novel antidepressant duloxetine in human plasma after an original solid-phase extraction procedure

Duloxetine is the most recent serotonin and norepinephrine reuptake inhibitor (SNRI) drug introduced for the therapy of depression. Thus, it is evident that there is a need for having on hand new reliable analytical methods for the determination of duloxetine plasma levels in depressed patients. The present paper deals with the development of a rapid and sensitive high-performance liquid chromatographic method for duloxetine analysis in human plasma. The assays were carried out using a C8 reversed-phase column and a mobile phase composed of 60% aqueous phosphate buffer containing triethylamine at pH 3.0 and 40% acetonitrile. The UV detector was set at 230 nm and loxapine was used as the internal standard. An original pre-treatment of plasma samples was developed, based on solid-phase extraction (SPE) with mixed-mode reversed phase-strong cation exchange cartridges (30 mg, 1 mL). The extraction yields values were higher than 90%. Linearity was found in the 2-200 ng mL(-1) duloxetine concentration range; the limit of quantitation was 2.0 ng mL(-1) and the limit of detection was 0.7 ng mL(-1). The method was applied to plasma samples from depressed patients undergoing therapy with duloxetine. Precision data and accuracy results were satisfactory and no interference from other drugs was found. Thus, the method seems to be suitable for the therapeutic drug monitoring of duloxetine in depressed patients' plasma.     

Simultaneous determination of clobazam and its major metabolite in human plasma by a rapid HPLC method

A rapid and specific HPLC method has been developed and validated for simultaneous determination of clobazam, the anticonvulsant agent, and its major metabolite in human plasma. The sample preparation was a liquid-liquid extraction with tuloene yielding almost near 100% recoveries of two compounds. Chromatographic separation was achieved with a Chromolith Performance RP-18e 100 mm x 4.6mm column, using a mixture of a phosphate buffer (pH 3.5; 10mM)-acetonitrile (70:30, v/v), in isocratic mode at 2 ml/min at a detection wave-length of 228 nm. The calibration curves were linear (r(2)>0.998) in the concentration range of 5-450 ng ml(-1). The lower limit of quantification was 5 ng ml(-1) for two compounds studied. The within- and between-day precisions in the measurement of QC samples at four tested concentrations were in the range of 0.89-9.1% and 2.1-10.1% R.S.D., respectively. The developed procedure was applied to assess the pharmacokinetics of clobazam and its major metabolite following administration of a single 10mg oral dose of clobazam to healthy volunteers.     

Simultaneous determination of JTT-501 and its main metabolite in human plasma by liquid chromatography-ionspray mass spectrometry

An LC-MS-MS analytical method was developed for the determination of a new antidiabetic agent, JTT-501 and its main metabolite (JTP-20604) in human plasma. The compounds were isolated from plasma by protein precipitation before analysis by HPLC with atmospheric pressure positive ionisation MS-MS detection. An isotopically labelled analog of JTT-501 was used as the internal standard. Linearity was demonstrated over the calibration range of about 5-10000 ng/ml for both compounds. The assay was validated with respect to accuracy, precision and analyte stability. This method was used for the determination of plasma concentrations for the two compounds in a clinical tolerability study. A cross-validation exercise between two different mass spectrometers, used for the determination of clinical samples, is also reported.     

High-performance liquid chromatographic assay with fluorescence detection for the routine monitoring of the antidepressant mirtazapine and its demethyl metabolite in human plasma

A validated HPLC method for the simultaneous quantitative analysis of the antidepressant mirtazapine and its demethyl metabolite in human plasma is described. The active constituents including internal standard were extracted from 1 ml of plasma with hexane and separated on a μBondapak Phenyl column with fluorescence detection. The lower limit of quantification was 0.5 ng/ml, without significant interferences with endogenous or exogenous components. Inter- and intra-assay accuracy determined at quality control levels of 2, 10 and 80 ng/ml were, respectively, 104.6–113.7% and 105.1–117.7% for mirtazapine, and 91.7–99.3% and 89.9–103.7% for demethylmirtazapine. In all cases the precision was below 6.8%.     

Rapid determination of gefitinib and its main metabolite, O-desmethyl gefitinib in human plasma using liquid chromatography-tandem mass spectrometry

A novel, rapid and specific liquid chromatography-tandem mass spectrometric (LC-MS/MS) method was developed and validated for the simultaneous quantification of gefitinib and its predominant metabolite, O-desmethyl gefitinib in human plasma. Chromatographic separation of analytes was achieved on an Alltima C18 analytical HPLC column (150 mm × 2.1 mm, 5 μm) using an isocratic elution mode with a mobile phase comprised acetonitrile and 0.1% formic acid in water (30:70, v/v). The flow rate was 300 μL/min. The chromatographic run time was 3 min. The column effluents were detected by API 4000 triple quadrupole mass spectrometer using electrospray ionization (ESI) in positive mode. Linearity was demonstrated in the range of 5-1000 ng/mL for gefitinib and 5-500 ng/mL for O-desmethyl gefitinib. The intra- and inter-day precisions for gefitinib and O-desmethyl gefitinib were ≤10.8% and the accuracies ranged from 89.7 to 104.7% for gefitinib and 100.4 to 106.0% for O-desmethyl gefitinib. This method was used as a bioanalytical tool in a phase I clinical trial to investigate the possible effect of hydroxychloroquine on the pharmacokinetics of gefitinib. The results of this study enabled clinicians to ascertain the safety of the combination therapy of hydroxychloroquine and gefitinib in patients with advanced (Stage IIIB-IV) non-small cell lung cancer (NSCLC).     

High-performance liquid chromatographic determination of indalpine,a new non-tricyclic antidepressant,in human plasma : Identification and simultaneous measurement of its major plasma metabolite

Indalpine or 4-[2-(3-indolyl)ethyl]piperidine, a selective inhibitor of 5-hydroxytryptamine uptake in central monoamine neurons, has proved to be an effective agent in the treatment of chronically ill depressed patients. We have developed a rapid high-performance liquid chromatographic method for the simultaneous determination of indalpine and its major metabolite in human plasma. Isolation by high-performance liquid chromatography and identification of this metabolite by mass spectrometry are also described.     

Quantitative determination of pravastatin and R-416, its main metabolite in human plasma, by liquid chromatography-tandem mass spectrometry

A quantitative method was developed and validated for rapid and sensitive analysis of pravastatin and R-416, the main metabolite of pravastatin, in human plasma. The analytes were extracted from plasma samples by a solid phase extraction method using a Bond Elut C(8). The method involved the use of liquid chromatography coupled with atmospheric pressure chemical ionization (APCI) and selected reaction monitoring (SRM) mass spectrometry. A pravastatin analog, R-122798, was used as the internal standard (I.S.). Separation of pravastatin, R-416 and the I.S. was accomplished using a reverse-phase column (C(18)). The components eluted were ionized by the APCI source (negative ion) and subsequently detected by a highly selective triple quadrupole mass spectrometer in the SRM mode. Linear standard curves were obtained from 0.1 ng/mL (lower limit of quantification, LLOQ) to 100 ng/mL. The intra-assay precisions (coefficient of variation) for the samples at the LLOQ were 1.8% for pravastatin and 1.6% for R-416. The intra-assay accuracy values were 95.8-107.6% for pravastatin, and 92.6-109.0% for R-416, respectively. Precision and accuracy of quality control (QC) samples were determined at concentrations of 0.5, 10 and 80 ng/mL for all analytes. The intra- and inter-assay precision calculated from QC samples were within 10% for pravastatin and within 11% for R-416. The overall recoveries for pravastatin and R-416 were 75.7-82.1% and 68.6-74.3%, respectively. Pravastatin and R-416 were stable in human plasma for 3 weeks at -20 degrees C in a freezer, up to 6h at room temperature, and up to 48 h at 6 degrees C. This assay method was successfully used to evaluate the pravastatin and R-416 levels in healthy volunteers following oral administration of Mevalotin.     

Simultaneous determination of OSI-774 and its major metabolite OSI-420 in human plasma by using HPLC with UV detection

A new method was developed and validated for quantitating OSI-774 and its metabolite OSI-420 in human plasma. Sample preparation involved initial extraction with methyl t-butyl ether followed by back extraction with HCl and re-extraction with methyl t-butyl ether. This extraction process resulted in significant improvement in the specificity, reproducibility and sensitivity. The analytes were separated on a Water Symmetry C18 analytical column and the mobile phase consisted of acetonitrile-0.05 M potassium phosphate buffer (42:58, v/v) (pH 4.8), and monitored at a wavelength 345 nm. Values of between- and within-day precision and accuracy for both OSI-774 and OSI-420 were <20%. This method was successfully applied to study steady-state pharmacokinetics of OSI-774 and OSI-420 in a phase II clinical trial.     

Simultaneous determination of intact cisplatin and its metabolite monohydrated cisplatin in human plasma

Cisplatin is a cytotoxic platinum compound, used in the treatment of several solid tumors. Cisplatin and to a greater extent its hydrolysis product monohydrated cisplatin are responsible for side-effects like nephrotoxicity. A sensitive, accurate and precise method was developed to simultaneously determine cisplatin and monohydrated cisplatin in plasma. The compounds were separated by high-performance liquid chromatography and quantified by off-line furnace atomic absorption spectrophotometry. The linear ranges for cisplatin and monohydrated cisplatin in deproteinized plasma were 60-600 and 87.5-700 nM, respectively. From plasma, the mean recovery of cisplatin was 83.2% and that of monohydrated cisplatin 79.1%. The lower limits of quantification of cisplatin and monohydrated cisplatin in deproteinized plasma were 60 and 87.5 nM, respectively. Over the whole calibration range, the within- and between-day accuracy of intact cisplatin ranged from 100.7 to 111.4 and 94.8-102.0%, respectively. The within- and between-day accuracy of monohydrated cisplatin ranged from 107.1 to 113.3 and 101.4-104.9%, respectively. The within-day and between-day precision of cisplatin ranged from 3.4 to 11.5 and 7.3-10.3%, respectively. For monohydrated cisplatin, the within-day and between-day precision ranged from 3.7 to 6.2 and 5.6-7.9%, respectively. Currently, the developed assay has been implemented in pharmacokinetic studies of patients treated with cisplatin alone or in combination with other drugs.     

Development and validation of a rapid HPLC method for simultaneous determination of tramadol, and its two main metabolites in human plasma

Tramadol, an analgesic agent, and its two main metabolites O-desmethyltramadol (M1) and N-desmethyltramadol (M2) were determined simultaneously in human plasma by a rapid and specific HPLC method. The sample preparation was a simple extraction with ethyl acetate. Chromatographic separation was achieved with a Chromolith Performance RP-18e 50 mm x 4.6 mm column, using a mixture of methanol:water (13:87, v/v) adjusted to pH 2.5 by phosphoric acid, in an isocratic mode at flow rate of 2 ml/min. Fluorescence detection (lambda(ex)=200 nm/lambda(em)=301 nm) was used. The calibration curves were linear (r(2)>0.997) in the concentration range of 2.5-500 ng/ml, 1.25-500 ng/ml and 5-500 ng/ml for tramadol, M1 and M2, respectively. The lower limit of quantification was 2.5 ng/ml for tramadol, 1.25 ng/ml for M1 and 5 ng/ml for M2. The within- and between-day precisions in the measurement of QC samples at four tested concentrations were in the range of 2.5-9.7%, 2.5-9.9% and 5.9-11.3% for tramadol, M1 and M2, respectively. The developed procedure was applied to assess the pharmacokinetics of tramadol and its two main metabolites following administration of 100mg single oral dose of tramadol to healthy volunteers.     

Simultaneous determination of gemcitabine and its metabolite in human plasma by high-performance liquid chromatography

Gemcitabine (dFdC) is a pyrimidine antimetabolite with broad spectrum activity against tumors. In this paper, a normal-phase high-performance liquid chromatographic method was developed for the determination of the parent drug (dFdC) and its metabolite (dFdU) in human plasma. The described sample preparation procedure for determination of dFdC and dFdU is rapid, sensitive, reproducible and simple. The linear regression equations obtained by least square regression method, were area under the curve=0.0371 concentration (ng ml(-1))+192.53 and 1.05.10(-4) concentration (ng ml(-1))-1.2693 for dFdC and dFdU, respectively. The assay for dFdC and dFdU described in the present report has been applied to plasma samples from a bladder cancer patient.     

Reversed-phase liquid chromatographic determination of idarubicin and its 13-hydroxy metabolite in human plasma

A method is given for the determination of idarubicin and its main metabolite, idarubicinol, in plasma from cancer patients. Idarubicin and idarubicinol are extracted from 2-ml samples of buffered plasma (pH 8.1) using chloroform-1-heptanol (9:1). After reextraction into phosphoric acid (0.1 M), separation is performed by reversed-phase liquid chromatography on a LiChrosorb RP-2 column (5 μm) with a mobile phase of acetonitrile-water, acidified with phosphoric acid. The absolute recovery in the range 5–100 ng/ml was greater than 83% with a precision better than 8% (relative standard deviation), using photometric detection at 484 nm. Proper handling of whole blood samples containing idarubidin is essential to avoid metabolic conversion into idarubicinol. Prolonged storage of the drug and its main metabolite under alkaline conditions should be avoided to prevent chemical degradation.     

Development and validation of an LC-MS/MS method for the quantitative determination of aripiprazole and its main metabolite, OPC-14857, in human plasma

An accurate, sensitive, reproducible, and selective liquid chromatography/tandem mass spectrometry (LC-MS/MS) method for determination of aripiprazole and its main metabolite, OPC-14857, in human plasma was developed and validated. Chromatographic separation was achieved isocratically on a C18 reversed-phase column within 7.5 min. The calibration curve, ranging from 0.1 to 100 ng/ml, was fitted to a 1/y2-weighted linear regression model. The assay showed no significant interference. Lower limit of quantitation (LLOQ) for both analytes was 0.1 ng/ml using 0.4 ml of plasma. Intra- and inter-assay precision and accuracy values for aripiprazole and OPC-14857 were within regulatory limits.     

Direct and fast capillary zone electrophoretic method for the determination of Gleevec and its main metabolite in human urine

A capillary zone electrophoretic (CZE) method was investigated for the determination of Gleevec and its main metabolite (N-demethylated piperazine derivative) in human urine using a fused-silica capillary (75 microm I.D.x60 cm total length, 10 cm effective length). The separation was performed with an hydrodynamic injection time of 10 s (0.5 p.s.i.) a voltage of -25 kV, a capillary temperature of 25 degrees C and a 100 mM phosphoric acid adjusted to pH 2 with the addition of triethanolamine. Under these conditions, the analysis takes about 5 min. A linear response over the 0.4-30.0 mg l(-1) concentration range was investigated for two compounds. A dilution of the sample was the only step necessary before the electrophoresis analysis. Detection limits of 0.1 mg l(-1) for Gleevec and its metabolite (S/N=3) were obtained. The developed method is easy, rapid and sensitive and has been applied to determine Gleevec and its main metabolite in clinical urine samples.     

Simultaneous determination of cyclandelate and its metabolite in human plasma by capillary column gas-liquid chromatography

A method was developed for the simultaneous determination of cyclandelate and mandelic acid concentrations in plasma, involving extraction from plasma followed by trimethylsilylation and chromatography of the derivatives on a glass capillary column with hydrogen flame-ionization detection. Calibration graphs were linear down to at least 20 μg/ml for each substance. The precision was excellent with a pooled relative standard deviation of 6.3% and 6.4% for cyclandelate and mandelic acid serum samples, respectively. Concentrations below 500 ng/ml of each substance could be detected in human plasma. The method was developed for use in bioavailability and metabolism studies.     

Determination of sulphasalazine and its main metabolite sulphapyridine and 5-aminosalicylic acid in human plasma by liquid chromatography/tandem mass spectrometry and its application to a pharmacokinetic study

A simple and sensitive liquid chromatography/positive-ion electrospray ionization mass spectrometry (LC-ESI-MS/MS) method has been developed for the simultaneous determination of sulphasalazine (SASP) and its main metabolite sulphapyridine (SP) and 5-aminosalicylic acid (5-ASA) with 100 μL of human plasma using dimenhydrinate as the internal standard (I.S.). The API-3000 LC-MS/MS was operated under the multiple reaction-monitoring mode (MRM) using the electrospray ionization technique. Protein precipitation process was used to extract SASP, SP, 5-ASA and I.S. from human plasma. The total run time was 9.0 min and the elution of SASP, SP and 5-ASA was at 4.8 min, 2.5 min and 2.0 min, respectively. The separation was achieved with a mobile phase consisting of 0.2% formic acid, 2 mM ammonium acetate in water (mobile phase A) and 0.2% formic acid, 2 mM ammonium acetate in methanol (mobile phase B) by using gradient elution on a XBP Phenyl column (100 mm × 2.1 mm, 5 μm). The developed method was validated in human plasma with a lower limit of quantitation of 10 ng/mL for SASP, SP and 5-ASA, respectively. A linear response function was established for the range of concentrations 10-10,000 ng/mL (r>0.99) for SASP and 10-1000 ng/mL (r>0.99) for SP and 5-ASA. The intra and inter-day precision values for SASP, SP and 5-ASA met the acceptance as per FDA guidelines. SASP, SP and 5-ASA were stable during stability studies, i.e., long term, auto-sampler and freeze/thaw cycles. The method was successfully applied for the evaluation of pharmacokinetics of SASP, SP and 5-ASA after single oral doses of 250 mg SASP to 10 healthy volunteers.     

Analytical method development and validation of mianserin hydrochloride and its metabolite in human plasma by LC-MS

Mianserin is a tetracyclic antidepressant drug and administered as racemate of R (-) and S (+) mianserin hydrochloride in a dose of 30-90 mg/day in divided doses. Liquid chromatography-mass spectroscopy (LC-MS) is a tool, which is widely used for determination of drug and their metabolites in biological fluids because of its high sensitivity and precision. Here we describe a liquid chromatography mass spectroscopy method for simultaneous determination of mianserin and its metabolite, N-desmethylmianserin, from human plasma using a liquid-liquid extraction with hexane:isoamylalcohol (98:2) and back extraction with 0.005 M formic acid solution. This method is specific and linear over the concentration range of 1.00-60.00 ng/ml for mianserin and 0.50-14.00 ng/ml for N-desmethylmianserin in human plasma. The lowest limits of quantification (LLQ) is 1.00 ng/ml for mianserin and 0.50 ng/ml for N-desmethylmianserin. Intraday and interday precision (%C.V.) is <10% for both mianserin and N-desmethylmianserin. The accuracy ranges from 94.44 to 112.33% for mianserin and 91.85-100.13% for N-desmethylmianserin. The stability studies showed that mianserin and N-desmethylmianserin in human plasma are stable during short-term period for sample preparation and analysis. The method was used to assay mianserin and its metabolite, N-desmethylmianserin, in human plasma samples obtained from subjects who had been given an oral tablet of 30 mg of mianserin.