High-performance liquid chromatographic analysis and pharmacokinetics of terazosin in healthy volunteers

A high-performance liquid chromatographic (HPLC) analysis of terazosin in 1 ml of human plasma was developed using prazosin as an internal standard. The plasma sample was extracted with dichloromethane and ethylether and a 100-microl aliquot was injected onto the reversed-phase column. The mobile phase, 0.02 M sodium phosphate buffer:acetonitrile:tetrahydrofuran = 720:220:60 (v/v/v), was run at a flow rate of 0.8 ml/min and the column effluent was monitored using a florescence detector set at 370 and 250 nm for the emission and excitation wave numbers, respectively. The retention times for terazosin and prazosin were approximately 6.4 and 9.8 min, respectively, and the coefficients of variation of terazosin were generally low, below 6.4%. The present HPLC method was successful for the pharmacokinetic study of terazosin in healthy volunteers. Following oral administration of terazosin, 2 mg, to 20 healthy male volunteers, the area under the plasma concentration-time curve from time zero to time infinity was 421 +/- 71.8 ng h/ml and terminal half-life was 9.83 +/- 1.29 h.     

Development of a high-performance liquid chromatographic method for the quantification of chlorpyrifos, pyridostigmine bromide, N,N-diethyl-m-toluamide and their metabolites in rat plasma and urine

A method was developed for the separation and quantification of the insecticide chlorpyrifos (O,O-diethyl-O[3,5,6-trichloro-2-pyridinyl] phosphorothioate), its metabolites chlorpyrifos-oxon (O,O-diethyl-O[3,5,6-trichloro-2-pyridinyl] phosphate) and TCP (3,5,6-trichloro-2-pyridinol), the anti-nerve agent drug pyridostigmine bromide (PB; 3-dimethylaminocarbonyloxy-N-methyl pyridinium bromide), its metabolite N-methyl-3-hydroxypyridinium bromide, the insect repellent DEET (N,N-diethyl-m-toluamide), and its metabolites m-toluamide and m-toluic acid in rat plasma and urine. The method is based on using solid-phase extraction and high-performance liquid chromatography (HPLC) with reversed-phase C18 column, and gradient UV detection ranging between 210 and 280 nm. The compounds were separated using a gradient of 1-85% acetonitrile in water (pH 3.20) at a flow-rate ranging between 1 and 1.7 ml/min over a period of 15 min. The retention times ranged from 5.4 to 13.2 min. The limits of detection ranged between 20 and 150 ng/ml, while the limits of quantitation were between 150 and 200 ng/ml. Average percentage recovery of five spiked plasma samples was 80.2+/-7.9, 74.9+/-8.5, 81.7+/-6.9, 73.1+/-7.8, 74.3+/-8.3, 80.8+/-6.6, 81.6+/-7.3 and 81.4+/-6.5, and from urine 79.4+/-6.9, 77.8+/-8.4, 83.3+/-6.6, 72.8+/-9.0, 76.3+/-7.7, 83.4+/-7.9, 81.6+/-7.9 and 81.8+/-6.8 for chlorpyrifos, chlorpyrifos-oxon, TCP, pyridostigmine bromide, N-methyl-3-hydroxypyridinium bromide, DEET, m-toluamide and m-toluic acid, respectively. The relationship between peak areas and concentration was linear over a range between 200 and 2000 ng/ml.     

Determination of 2-hydroxyflutamide in human plasma by high-performance liquid chromatography and its application to pharmacokinetic studies

Flutamide is a potent antiandrogen used for the treatment of prostatic cancer. Flutamide undergoes extensive first-pass metabolism to the pharmacologically active metabolite 2-hydroxyflutamide. A simple, sensitive, precise, accurate and specific HPLC method, using carbamazepine as the internal standard, for the determination of 2-hydroxyflutamide in human plasma was developed and validated. After addition of the internal standard, the analytes were isolated from human plasma by liquid-liquid extraction. The method was linear in the 25 to 1,000 ng/ml concentration range (r>0.999). Recovery for 2-hydroxyflutamide was greater than 91.4% and for internal standard was 93.6%. The limit of quantitation was 25 ng/ml. Inter-batch precision, expressed as the relative standard deviation (RSD), ranged from 4.3 to 7.9%, and accuracy was better than 93.9%. Analysis of 2-hydroxyflutamide concentrations in plasma samples from 16 healthy volunteers following oral administration of 250 mg of flutamide provided the following pharmacokinetic data (mean+/-SD): Cmax, 776 +/- 400 ng/ml; AUC(0-infinity), 5,368 +/- 2,689 ng h/ml; AUC(0-t) 5,005 +/- 2,605 ng h/ml; Tmax 2.6 +/- 1.6 h; elimination half-life, 5.2 +/- 2.0 h.     

Stereoselective high-performance liquid chromatographic determination of ketamine and its active metabolite, norketamine, in human plasma

A stereoselective high-performance liquid chromatographic method for the determination of the enantiomers of ketamine and its active metabolite, norketamine, in human plasma is described. The compounds were extracted from plasma by liquid-liquid extraction three times in a combination of cyclohexane with 2.5 M NaOH, 1 mM HCl and 1 M carbonate buffer. Stereoselective separation was achieved on a Chiralcel OD column with a mobile phase of n-hexane-2-propanol (98:2, v/v). The detection wavelength was 215 nm. The lower limits of the determination of the method were 5 ng/ml for ketamine and 10 ng/ml for norketamine. The intra- and inter-day coefficients of variation ranged from 2.9 to 9.8% and from 3.4 to 10.7% for all compounds, respectively. The method was sensitive and sufficiently reproducible for stereoselective monitoring of ketamine and norketamine in human plasma during pharmacokinetic studies after the administration of ketamine for analgesia.     

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

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

Determination of calcium dobesilate in human plasma using ion-pairing extraction and high-performance liquid chromatography

A rapid, simple reversed-phase high-performance liquid chromatographic method with ultraviolet absorbance detection has been developed for the determination of calcium dobesilate in human plasma. Sample processing is based on an ion-pairing extraction with tetra-n-butylammonium hydroxide as cationic pairing ion and dichloromethane. Separation of the investigated calcium dobesilate and 2,4-dihydroxybenzoic acid as internal standard was achieved on a Discovery RP-Amide C16 analytical column with 50 mM, pH 2.5, potassium dihydrogenphosphate buffer-acetonitrile (75:25, v/v) mobile phase. The wavelength was set at 305 nm. The limit of quantitation is 100 ng/ml and the calibration curve is linear up to 50 microg/ml. Within-day and between-day precision expressed as the relative standard deviation is about 10% and the accuracy of the determination did not deviate from 100% by more than +/-10%. The developed method was found to be suitable for application in human bioequivalence studies.     

Simultaneous determination of the histamine H1-receptor antagonist ebastine and its two metabolites, carebastine and hydroxyebastine, in human plasma using high-performance liquid chromatography.

Ebastine (CAS 90729-43-4) is an antiallergic agent which selectively and potently blocks histamine H1-receptors in vivo. A simple and sensitive high-performance liquid chromatography (HPLC) method is described for the simultaneous determination of ebastine and its two oxidized metabolites, carebastine (CAS 90729-42-3) and hydroxyebastine (M-OH), in human plasma. After a pretreatment of plasma sample by solid-phase extraction, ebastine and its metabolites were analyzed on an HPLC system with ultraviolet detection at 254 nm. Chromatography was performed on a cyano column (250x4.0 mm I.D.) at 40 degrees C with the mobile phase of acetonitrile-methanol-0.012 M ammonium acetate buffer (20:30:48, v/v/v) at a flow rate of 1.2 ml/min. Accurate determinations were possible over the concentration range of 3-1000 ng/ml for the three compounds using 1 ml plasma samples. The intra- and inter-day assay accuracy of this method were within 100+/-15% of nominal values and the precision did not exceed 12.4% of relative standard deviation. The lower limits of quantitation were 3 ng/ml for ebastine and its metabolites in human plasma. This method was satisfactorily applied to the determination of ebastine and its two oxidized metabolites in human plasma after oral administration of ebastine.     

Validation of a high-performance liquid chromatography method for tramadol and o-desmethyltramadol in human plasma using solid-phase extraction

An HPLC system using solid-phase extraction and HPLC with UV detection has been validated in order to determine tramadol and o-desmethyltramadol (M1) concentrations in human plasma. The method developed was selective and linear for concentrations ranging from 50 to 3,500 ng/ml (tramadol) and 50 to 500 ng/ml (M1) with mean recoveries of 94.36 +/- 12.53% and 93.52 +/- 7.88%, respectively. Limit of quantitation (LOQ) was 50 ng/ml. For tramadol, the intra-day accuracy ranged from 95.48 to 114.64% and the inter-day accuracy, 97.21 to 103.24%. Good precision (0.51 and 18.32% for intra- and inter-day, respectively) was obtained at LOQ. The system has been applied to determine tramadol concentrations in human plasma samples for a pharmacokinetic study.     

Determination of retigabine and its acetyl metabolite in biological matrices by on-line solid-phase extraction (column switching) liquid chromatography with tandem mass spectrometry

A HPLC assay with tandem mass spectrometric detection in the positive-ion atmospheric pressure chemical ionisation (APCI) mode for the sensitive determination of retigabine [(I), D-23129] and its acetyl metabolite [(II), ADW 21-360] in plasma was developed, utilising the structural analogue (D-10328), (III), as internal standard. Automated on-line solid-phase extraction of diluted plasma samples, based on 200-microl plasma aliquots, at pH 6.5, allowed a reliable quantification of retigabine and the acetyl metabolite down to 1 ng/ml. Injection of 500 microl of diluted plasma onto a C2 stationary phase-based column switching system in combination with a 75 mm x 4 mm reversed-phase analytical column at a flow-rate of 0.5 ml/min provided cycle times of 4 min per sample. The standard curves were linear from 1 to 1000 ng/ml using weighted linear regression analysis (1/x2). The method is accurate (mean accuracy < or = +/- 10%), precise (RSD < +/- 15%) and sensitive, providing lower limits of quantification in plasma of 1 ng/ml for retigabine (I), and 2.5 ng/ml for the metabolite (II) with limits of detection of 0.5 ng/ml for both analytes. Up to 200 unknowns may be analysed each 24 h per analyst.     

Liquid chromatographic determination of ketoconazole, a potent inhibitor of CYP3A4-mediated metabolism

A high-performance liquid chromatographic assay with UV detection has been developed for the determination of ketoconazole in human plasma. Quantitative extraction was achieved by a single solvent extraction involving a mixture of acetonitrile-n-butyl chloride (1:4, v/v). Ketoconazole and the internal standard (clotrimazole) were separated on a column packed with Inertsil ODS-80A material and a mobile phase composed of water-acetonitrile-tetrahydrofuran-ammonium hydroxide-triethylamine (45:50.2:2.5:0.1:0.1, v/v). The column effluent was monitored at a wavelength of 206 nm with a detector range set at 0.5. The calibration graph was linear in the range of 20-2000 ng/ml, with a lower limit of quantitation of 20.0 ng/ml. The extraction recoveries for ketoconazole and clotrimazole in human plasma were 93+/-9.7% and 83+/-10.0%, respectively. The developed method has been successfully applied to a clinical study to examine the pharmacokinetics of ketoconazole in a cancer patient.     

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 muBondapak 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%.     

Simultaneous determination of tramadol and its major active metabolite O-demethyltramadol by high-performance liquid chromatography with electrochemical detection

A novel, highly sensitive method was developed for simultaneous determination of tramadol and its main active metabolite O-demethyltramadol (ODMT) in rat plasma. The method involves a single-step extraction procedure and a specific determination by high-performance liquid chromatography with electrochemical detection, using an ethoxy analogue of tramadol (L-233) as internal standard. The dual-electrode detector was operated in the oxidation-screening mode. Absolute recoveries of tramadol and ODMT were about 80%. Calibration curves were linear over a concentration range of 10-1000 ng/ml for ODMT and 10-10000 ng/ml for tramadol with intra- and inter-day coefficients of variation not exceeding 10% and 15%, respectively. The limit of quantification for tramadol and ODMT was lower than 15 ng/ml and 10 ng/ml using 100 microl of plasma, respectively. The described method allows an adequate characterization of the plasma vs. time profiles for both compounds.     

Determination of a new asiatic acid derivative, AS 2-006A in rat plasma and urine, and human plasma by high-performance liquid chromatography

A high-performance liquid chromatographic method was developed for the determination of a new wound healing agent, AS 2-006A (ethoxymethyl 2-oxo-3, 23-O-isopropylideneasiatate), in rat plasma and urine, and human plasma. The sample preparation was simple: 2 volumes of acetonitrile were added to the biological samples to deproteinize it. A 50-microl aliquot of the supernatant was injected onto the reversed-phase column. The mobile phase employed was acetonitrile : H2O (9:1, v/v) and run at a flow rate of 1.1 ml/min. The column effluent was monitored by a UV detector set at 205 nm. The retention time for AS 2-006A was approximately 29.5 min. The detection limits for AS 2-006A in rat and human plasma were both 1 microg/ml, and in rat urine was 2 microg/ml. The coefficients of variation of the assay (within-day and between-day) were generally low (below 10.8%) for rat plasma and urine, and human plasma. No interferences from endogenous substances were found.     

Development of a high-performance liquid chromatographic method to determine the concentration of karenitecin, a novel highly lipophilic camptothecin derivative, in human plasma and urine

Karenitecin is a novel, highly lipophilic camptothecin derivative with potent anticancer potential. We have developed a sensitive high-performance liquid chromatographic method for the determination of karenitecin concentration in human plasma and urine. Karenitecin was isolated from human plasma and urine using solid-phase extraction. Separation was achieved by gradient elution, using a water and acetonitrile mobile phase, on an ODS analytical column. Karenitecin was detected using fluorescence detection at excitation and emission wavelengths of 370 and 490 nm, respectively. Retention time for karenitecin was 16.2 +/- 0.5 min and 8.0 +/- 0.2 min for camptothecin, the internal standard. The karenitecin peak was baseline resolved, with the nearest peak at 3.1 min distance. Using normal volunteer plasma and urine from multiple individuals, as well as samples from the 50 patients analyzed to date, no interfering peaks were detected. Inter- and intra-day coefficients of variance were <4.4 and 7.1% for plasma and <4.9 and 11.6% for urine. Assay precision, based on an extracted karenitecin standard plasma sample of 2.5 ng/ml, was +4.46% with a mean accuracy of 92.4%. For extracted karenitecin standard urine samples of 2.5 ng/ml assay precision was +2.35% with a mean accuracy of 99.5%. The mean recovery of karenitecin, at plasma concentrations of 1.0 and 50 ng/ml, was 81.9 and 87.8% respectively. In urine, at concentrations of 1.5 and 50 ng/ml, the mean recoveries were 90.3 and 78.4% respectively. The lower limit of detection (LLD) for karenitecin was 0.5 ng/ml in plasma and 1.0 ng/ml in urine. The lower limit of quantification (LLQ) for karenitecin was 1 ng/ml and 1.5 ng/ml for plasma and urine, respectively. Stability studies indicate that when frozen at -70 degrees C, karenitecin is stable in human plasma for up to 3 months and in human urine for up to 1 month. This method is useful for the quantification of karenitecin in plasma and urine samples for clinical pharmacology studies in patients receiving this agent in clinical trials.     

Gas chromatographic-mass spectrometric analysis of perillyl alcohol and metabolites in plasma.

Perillyl alcohol (POH), a metabolite of d-limonene and a component of the lavender oil, is currently in Phase I clinical trials both as a chemopreventative and chemotherapeutic agent. In vivo, POH is metabolized to less active perillic acid (PA) and cis- and trans-dihydroperillic acids [DHPA, 4-(1'-methylethenyl)-cyclohexane-1-carboxylic acid]. Previous pharmacokinetic studies using a GC-MS method detected POH metabolites but not POH itself; thus these studies lacked information on the parent drug. The present report describes a sensitive GC-MS method for the quantitation of POH and metabolites using stable-isotopically labeled internal standards. The residue obtained from CH2Cl2 extraction of a plasma sample was silylated. The products were separated on a capillary column and analyzed by an ion-trap GC-MS using NH3 chemical ionization. POH-d3 was used as the internal standard for POH while 13C-PA-d2 was used as the internal standards for the metabolites. The quantitation limits for POH, PA, cis- and trans-DPA were <10 ng/ml using 1-2 ml plasma. The assay was validated in rat and human plasma. The assay was linear from 2 to 2000 ng/ml for POH, 10 to 1000 ng/ml for PA and trans-DHPA, and 20 to 1000 ng/ml for cis-DHPA monitored. The within-run and between-run coefficients of variation were all <8%. Preliminary pharmacokinetic data from a rat following i.v. administration of POH at 23 mg/kg and from a patient receiving POH at 500 mg/m2 p.o. was also provided. Intact POH, PA, cis- and trans-DHPA were all detected in plasma in both cases. Two new major metabolites were found in human and one in the rat plasma.     

High-performance liquid chromatographic assay of malagashanine in rat plasma and urine and its pharmacokinetic application

A reversed-phase HPLC method was developed for quantitative analysis of malagashanine in rat plasma and urine. Malagashanine and internal standard were extracted from alkalinized rat plasma. Urine analysis was performed by direct injection onto the HPLC system. Acetonitrile-aqueous 25 mM sodium acetate solution at pH 6.25 (45:55, v/v) was used as the mobile phase. The eluate was monitored by using UV detection at 250 nm. The assay was linear within the concentration range of 10-1000 ng/ml. Both intra- and inter-day accuracy and precision were within acceptable limits. The method was applied to study the pharmacokinetics of malagashanine in rats.     

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.     

Simultaneous determination of selegiline-N-oxide, a new indicator for selegiline administration, and other metabolites in urine by high-performance liquid chromatography-electrospray ionization mass spectrometry

In order to discriminate selegiline (SG) use from methamphetamine (MA) use, the urinary metabolites of SG users have been investigated using high-performance liquid chromatography (HPLC)-electrospray ionization mass spectrometry (HPLC-ESI-MS). Selegiline-N-oxide (SGO), a specific metabolite of SG, was for the first time detected in the urine, in addition to other metabolites MA, amphetamine (AP) and desmethylselegiline (DM-SG). A combination of a Sep-pak C18 cartridge for the solid-phase extraction, a semi-micro SCX column (1.5 mm I.D.x 150 mm) for HPLC separation and ESI-MS for detection provided a simple and sensitive procedure for the simultaneous determination of these analytes. Acetonitrile-10 mM ammonium formate buffer adjusted to pH 3.0 (70:30, v/v) at a flow-rate of 0.1 ml/min was found to be the most effective mobile phase. Linear calibration curves were obtained over the concentration range from 0.5 to 100 ng/ml for all the analytes by monitoring each protonated molecular ion in the selected ion monitoring (SIM) mode. The detection limits ranged from 0.1 to 0.5 ng/ml. Upon applying the scan mode, 10-20 ng/ml were the detection limits. Quantitative investigation utilizing this revealed that SGO was about three times more abundant (47 ng/ml, 79 ng/ml) than DM-SG in two SG users' urine samples tested here. This newly-detected, specific metabolite SGO was found to be an effective indicator for SG administration.     

Chromatographic method for the determination of diazepam, pyridostigmine bromide, and their metabolites in rat plasma and urine

This study describes a chromatographic method for the determination of diazepam, an anxiolytic drug that is also used as an antidote against nerve agent seizures, its metabolites N-desmethyldiazepam, and temazepam, the anti-nerve agent drug pyridostigmine bromide (PB; 3-dimethylaminocarbonyloxy-N-methyl pyridinium bromide) and its metabolite N-methyl-3-hydroxypyridinium bromide in rat plasma and urine. The compounds were extracted using C18 Sep-Pak Vac 3cc (500 mg) cartridges and separated using isocratic mobile phase of methanol, acetonitrile and water (pH 3.2) (10:40:50) at a flow-rate of 0.5 ml/min in a period of 12 min, and UV detection ranging between 240 and 280 nm. The limits of detection for all analytes ranged between 20 and 50 ng/ml, while limits of quantitation were 100 ng/ml. Average percentage extraction recoveries of five spiked plasma samples were 79.1+/-7.7, 83.5+/-6.4, 83.9+/-5.9, 71.3+/-6.0 and 77.7+/-5.6, and from urine 79.4+/-7.9, 83.1+/-6.9, 73.6+/-7.7, 74.3+/-7.1 and 77.6+/-5.9 for diazepam, N-desmethyldiazepam, temazepam, pyridostigmine bromide, and N-methyl-3-hydroxypyridinium bromide, respectively. The relationship between peak areas and concentration was linear over the range between 100 and 1000 ng/ml. This method was applied to determine the above analytes following a single oral administration in rats as a tool to study the pharmacokinetic profile of each compound, alone and in combination.     

Quantification of perifosine, an alkylphosphocholine anti-tumour agent, in plasma by pneumatically assisted electrospray tandem mass spectrometry coupled with high-performance liquid chromatography

An HPLC assay with tandem mass spectrometric detection in the positive-ion Turbo-Ion-Spray (TISP) mode for the fast and sensitive determination of perifosine ((I), D-21266) in human plasma was developed, utilising the structural analogue, miltefosine ((II), D-18506), as internal standard. Automated solid-phase extraction of diluted plasma samples, based on 250-microl plasma aliquots, at pH 6.5, allowed a reliable quantification of perifosine down to 4 ng/ml. Injection of 200 microl of plasma extracts onto a 100x3 mm normal-phase analytical column at a flow-rate of 0.5 ml/min provided retention-times of 2.4 and 2.1 min for perifosine (I) and the internal standard (II), respectively. The standard curves were linear from 4 to 2000 ng/ml using weighted linear regression analysis (1/Y2). The inter-assay and intra-assay accuracies for the calibration standards were within +0.9% and -0.2%, exhibiting precisions (C.V.) of +/-6.5 and +/-7.3%, respectively. Up to 100 unknowns may be analysed each 24 h per analyst.