Liquid chromatographic-tandem mass spectrometric assay for the simultaneous quantification of Camptosar and its metabolite SN-38 in mouse plasma and tissues

A simple, rapid and sensitive LC-MS/MS bioanalytical method has been developed to simultaneously quantify Camptosar (CPT-11) and its active metabolite, SN-38, in mouse plasma and tissues. A single step protein precipitation with acetonitrile in 96-well plates was used for sample preparation. Camptothecin (CPT) was used as the internal standard. Fast separation of SN-38, CPT-11 and CPT was carried out isocratically on a C18, 2 mm x 50 mm, 5 microm HPLC column with a mobile phase containing acetonitrile and 20 mM ammonium acetate (pH 3.5) and a 2.5 min chromatographic run time. The API 4000 MS/MS system was operated in positive ionization multiple reaction monitoring mode, and the transitions for SN-38, CPT-11 and CPT were 393.4 --> 349.3, 587.6 --> 167.2 and 349.3 --> 305.3, respectively. The SN-38 and CPT-11 concentrations in samples were calculated from a standard curve of peak area ratios of the analyte to that of the internal standard using a 1/chi2 weighted linear regression. The quantitation limit of 0.5 ng/mL was achieved by using a low sample volume (100 microL) of plasma or tissue homogenates. The assay was linear over the concentration range of 0.5-500 ng/mL with acceptable precision and accuracy. The method was used for the quantification of CPT-11 and SN-38 in plasma and tissues to support a preclinical pharmacokinetics and tissue distribution study of CPT-11 in mice.     

High-performance liquid chromatographic assay for glucuronidation activity of 7-ethyl-10-hydroxycamptothecin (SN-38), the active metabolite of irinotecan (CPT-11), in human liver microsomes

A simple and sensitive assay for glucuronidation activity of 7-ethyl-10-hydroxycamptothecin (SN-38), the active metabolite of irinotecan (CPT-11), in human liver microsomes by high-performance liquid chromatography (HPLC) with fluorescence detection is reported. The method was validated for the determination of SN-38 glucuronide (SN-38G) with respect to specificity, linearity, recovery, stability, precision, accuracy, and limits of detection and quantitation. There was no interference from matrix and non-enzymatic reactions. The calibration curve for SN-38G was linear from 5 to 500 nM. Average recoveries ranged from 98 to 100% in spiked human liver microsome samples, and the SN-38G was stable at 4 degrees C for at least 72 h. The newly developed method was found to be more sensitive and selective than previous methods using thin layer chromatography and HPLC. The limit of quantitation for SN-38G was 5 nM (2.5 pmol/assay). The intra- and inter-day precision and accuracy were less than 7 and 4%, respectively. The intra- and inter-day precision of enzyme assay for UDP-glucuronosyltransferase (UGT) activity toward SN-38 in human liver microsomes was less than 4%. With this improved sensitivity, the kinetics of SN-38 glucuronidation in human liver microsomes could be determined more precisely. Therefore, this method is applicable to in vitro study on the side effects and drug interactions of CPT-11 using small amounts of biological sample.     

Simultaneous determination of the camptothecin analogue CPT-11 and its active metabolite SN-38 by high-performance liquid chromatography: application to plasma pharmacokinetic studies in cancer patients.

CPT-11 (I; 7-ethyl-10-[4-(1-piperidino)-1- piperidino]carbonyloxycamptothecin) is a new anticancer agent currently under clinical development. A sensitive high-performance liquid chromatographic assay suitable for the simultaneous determination of I and its active metabolite SN-38 (II) in human plasma, and their preliminary clinical pharmacokinetics, are described. Plasma samples were processed using a solid-phase (C18) extraction step allowing mean recoveries of I, II and the internal standard camptothecin (III) of 84, 99 and 72%, respectively. The extracts were chromatographed on a C18 reversed-phase column with a mobile phase composed of acetonitrile, phosphate buffer and heptanesulphonic acid, with fluorescence detection. The calibration graphs were linear over a wide range of concentrations (1 ng/ml-10 micrograms/ml), and the lower limit of determination was 1 ng/ml for both I and II. The method showed good precision: the within-day relative standard deviation (R.S.D.) (5-1000 ng/ml) was 13.0% (range 4.9-19.4%) for I and 12.8% (6.7-19.1%) for II; the between-day R.S.D. (5-10,000 ng/ml was 7.9% (5.4-17.5%) for I and 9.7% (3.5-15.1%) for II. Using this assay, plasma pharmacokinetics of both I and II were simultaneously determined in three patients receiving 100 mg/m2 I as a 30-min intravenous infusion. The mean peak plasma concentration of I at the end of the intravenous infusion was 2400 +/- 285 ng/ml (mean +/- standard error of the mean). Plasma decay was triphasic with half-lives alpha, beta and gamma of 5.4 +/- 1.8 min, 2.5 +/- 0.5 h and 20.2 +/- 4.6 h, respectively. The volume of distribution at steady state was 105 +/- 15 l/m2, and the total body clearance was 12.5 +/- 1.9 l/h.m2. The maximum concentrations of the active metabolite II reached 36 +/- 11 ng/ml.     

An Accurate Assay for Simultaneous Determination of Irinotecan and Its Active Metabolite SN-38 in Rat Plasma by LC with Fluorescence Detection

An accurate LC method was developed and validated for simultaneous determination of irinotecan (CPT-11) and its active metabolite SN-38 in rat plasma. Plasma samples were pretreated with 0.4 g mL^?1 sodium dodecyl sulfate to inactive the carboxylesterase and avoid the conversion of CPT-11 to SN-38. Chromatographic separation was achieved on a Diamaonsil C₁₈ column using acetonitrile–50 mM phosphate buffered solution (30:70, v/v) at pH 4.0 as the mobile phase with the flow rate of 1 mL min^?1. The linear quantitation ranges for CPT-11 and SN-38 were 5.05–3,030 and 3.15–315 ng mL^?1 with r ² > 0.99, respectively. The lower limit of quantification (LLOQ) was 2.33 ng mL^?1 for CPT-11 and 0.26 ng mL^?1 for SN-38 with intra- and inter-day relative standard deviation of <12% and the accuracy values of >90%. The method was proved to be accurate and sensitive enough and was successfully applied to a pharmacokinetic study of CPT-11 in rats.     

Development and validation of reversed phase liquid chromatographic method utilizing ultraviolet detection for quantification of irinotecan (CPT-11) and its active metabolite, SN-38, in rat plasma and bile samples: application to pharmacokinetic studies

A new, simple, sensitive and specific reversed-phase high performance liquid chromatographic (HPLC) method using ultraviolet detection was developed and validated for the analysis of CPT-11 (lambda(max)=254 nm, 365 nm) and its major active metabolite, SN-38 (lambda(max)=380 nm) in rat plasma and bile. The sample pre-treatment from plasma involved a single protein precipitation step with cold acetonitrile. In case of bile, liquid-liquid extraction with dichloromethane: tert-butyl methyl ether (3:7) was carried out. Topotecan, a structurally related camptothecin, was used as an internal standard. An aliquot of 50 microL was injected onto a C-18 column. The chromatographic separation was achieved by gradient elution consisting of acetonitrile and water (pH 3.0 adjusted with 20% o-phosphoric acid) at a flow rate of 1.0 ml/min. Total run time for each sample was 30 min. All the analytes viz. topotecan, CPT-11, SN-38 were well separated with retention times of 11.4, 13.4 and 15.5 min, respectively. Method was found to be selective, linear (R(2) approximately 0.999), accurate (recovery+/-15%) and precise (<5% C.V.) in the selected concentration ranges for both the analytes. The quantification limit for CPT-11 was 40 ngml(-1) and for SN-38 was 25 ngml(-1). The percent extraction efficiency was approximately 97% for CPT-11 and SN-38 from plasma while extraction recovery of CPT-11 and SN-38 from bile was approximately 70% and approximately 60%, respectively. The method was successfully used to determine plasma and biliary excretion time profiles of CPT-11 and SN-38, following oral and intravenous CPT-11 administration in rats. In the present study, irinotecan showed an absolute bioavailability of 30% as calculated from the pharmacokinetic data.     

A sensitive and rapid liquid chromatography tandem mass spectrometry method for quantitative determination of 7-ethyl-10-hydroxycamptothecin (SN-38) in human plasma containing liposome-based SN-38 (LE-SN38)

7-Ethyl-10-hydroxycamptothecin (SN-38) is an active metabolite of Irinotecan (CPT-11), an anticancer pro-drug. To support clinical pharmacokinetic studies for liposome based formulation of SN-38 (LE-SN38) in cancer patients, a rapid, simple and sensitive liquid chromatography tandem mass spectrometry (LC-MS/MS) method has been developed and validated for the quantification of total SN-38 in human plasma. Sample preparation was carried out by one-step protein precipitation using cold acetonitrile with 0.5% acetic acid (v/v). Camptothecin was used as an internal standard (IS). Chromatographic separation of SN-38 and IS was achieved using a Synergi Hydro-RP column (C(18), 50 x 2 mm, 4 micro m), with a gradient elution of acetonitrile and 0.1% acetic acid. After ionization in electrospray source (positive ions), the acquisition was performed in the multiple reactions monitoring mode. Quantitation was accomplished using the precursor-->product ion combinations of m/z 393.1-->349.2 for SN-38 and 349.1-->305.1 for IS. The quantification limit of 0.05 ng/mL was achieved by using much lower volume (0.2 mL) of plasma and in the presence of LE-SN38. The method was validated over the concentration range of 0.05-400 ng/mL. Accuracy was within +/-12% of nominal at all concentration levels. Inter-day and intra-day precisions expressed as percentage coefficient of variation (%CVs) for quality control (QC) samples were less than 14 and 5%, respectively.     

Determination of the Camptothecin Derivatives CPT-11 and SN-38 in Urine and Saliva by Micellar Electrokinectic Chromatography

The anti-cancer synthetic drug irinotecan (CPT-11) and its active metabolite SN-38 have been determined by micellar electrokinetic capillary chromatography (MEKC). The detection of the analytes was made at 368 nm and their separation took less than 7 min using a borate buffer (pH 8.8 at 25 mmol L^?1) solution containing sodium dodecyl sulfate (45 mmol L^?1) and acetonitrile (13.5% v/v). On-line analyte concentration (normal stacking mode) and the use of a highly sensitive cell (Z shaped cell) improved detection limits (at the 10^?8 mol L^?1 level). Recovery in fortified human saliva was 108 ± 5%, in agreement with the result achieved with the reference HPLC method. For the analysis of urine from rats submitted to a single dose of CPT-11 and SN-38, camptothecin was used as internal standard enabling recoveries close to 100% when compared to the results achieved using HPLC.     

A solid phase extraction method for the screening and determination of pyrethroid metabolites and organochlorine pesticides in human urine.

A screening method has been developed for the determination of 23 organochlorine pesticides (OCPs) and 3-pyrethroid metabolities [cis- and trans-3-(2,2-dichlorovinyl)-2,2-dimethyl-(1-cyclopropane) carboxylic acid, cis-3-(2,2-dibromovinyl)-2,2-dimethyl-(1-cyclopropane) carboxylic acid and 3-phenoxybenzoic acid] from human urine. OCPs were directly detected in urine samples while pyrethroid metabolites required acid-induced hydrolysis to convert their conjugates into free acids; all compounds were then cleaned-up/preconcentrated using solid phase extraction. Determination and quantitation was achieved by gas chromatography with a mass spectrometer detector operating in selected ion monitoring mode. Limits of detection varied between 0.1 and 0.3 ng/mL with linear ranges from 0.3 to 700 ng/mL; the precision of the method was high (4.3-7.2%). Recoveries of all analytes from urine samples fortified at levels of 30 ng/mL for each OCP and 15 ng/mL for each pyrethroid metabolite ranged from 88 to 101% (captan gave the lowest recovery). The results obtained from the analysis of real urine samples show the suitability of the proposed method for monitoring people exposed to organochlorine and pyrethroid pesticides.     

High-performance liquid chromatographic determination of pyridostigmine bromide, nicotine, and their metabolites in rat plasma and urine

This study reports on the development of a rapid and simple method for the determination of the antinerve agent drug pyridostigmine bromide (3-dimethylaminocarbonyloxy-N-methyl pyridinium bromide) (PB), its metabolite N-methyl-3-hydroxypyridinium bromide, nicotine (S-1-methyl-5-(3-pyridyl)-2-pyrrolidine), and its metabolites nornicotine (2-(3-pyridyl)pyrrolidine) and cotinine (S-1-methyl-5-(3-pyridyl)-2-pyrrolidone) in rat plasma and urine. The compounds are extracted and eluted by methanol and acetonitrile using C18 Sep-Pak cartridges and separated using high-performance liquid chromatography by a gradient of methanol, acetonitrile, and water (pH 3.2) at a flow rate of 0.8 mL/min in a period of 14 min. UV detection was at 260 nm for nicotine and its metabolites and at 280 nm for PB and its metabolite. The limits of detection ranged between 20 and 70 ng/mL, and the limits of quantitation were 50-100 ng/mL. The average percent recovery of five spiked plasma samples were 85.7 +/- 7.3%, 80.4 +/- 5.8%, 78.9 +/- 5.4%, 76.7 +/- 6.4%, and 79.7 +/- 5.7% and for urine were 85.9 +/- 5.9%, 75.5 +/- 6.9%, 82.6 +/- 7.9%, 73.6 +/- 5.9%, and 77.7 +/- 6.3% for nicotine, nornicotine, cotinine, PB, and N-methyl-3-hydroxypyridinium bromide, respectively. The calibration curves for standard solutions of the compounds of peak areas and concentration are linear for a range between 100 and 1,000 ng/mL. This method is applied in order to analyze the previously mentioned chemicals and metabolites following their oral administration in rats.     

A solid phase extraction reversed-phase HPLC method for the simultaneous determination of methoprene, permethrin and selected metabolites in rat plasma and urine.

A method was validated and applied for the analysis of the insect growth regulator methoprene [Isopropyl (2E,4E)-11-methoxy-3,7,11-trimethyldodeca-2,4-dienoate], its metabolite methoprene 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 using solid-phase extraction and reversed-phase high performance liquid chromatography. The analytes were separated using gradient of 55-100% acetonitrile in water (pH 4.0) at a flow rate ranging between 0.6 and 1.0 mL/min over a period of 20 min, and UV detection at 210 and 254 nm. The retention times ranged from 7.3 to 18.4 min. The limits of detection ranged between 50 and 100 ng/ml, while limits of quantitation were 100-150 ng/mL. Average percentage recovery of five spiked plasma samples was 83.6 +/- 3.9, 80.1 +/- 5.4, 82.1 +/- 4.4, 83.7 +/- 3.9 and 83.1 +/- 4.7, and from urine 79.3 +/- 4.3, 82.0 +/- 5.4, 80.7 +/- 4.2, 78.9 +/- 5.7 and 83.9 +/- 4.5 for methoprene, methoprene acid, permethrin, m-phenoxybenzyl alcohol and m-phenoxybenzoic acid, respectively. The method was linear and reproducible over the range of 100-1000 ng/mL. This method was applied to analyze the above chemicals and metabolites following their combined administration in rats.     

Capillary electrophoretic chiral separation of hydroxychloroquine and its metabolites in the microsomal fraction of liver homogenates

A rapid, selective, and low-cost chiral capillary electrophoretic method was developed for the simultaneous analysis of hydroxychloroquine (HCQ) and its three chiral metabolites: desethylchloroquine (DCQ), desethylhydroxychloroquine (DHCQ), and bisdesethylchloroquine (BDCQ) in the microsomal fraction of liver homogenates. After liquid-liquid extraction using toluene as extracting solvent, the drug and metabolites were resolved on a fused-silica capillary (50 microm ID, 50 cm total length, and 42 cm effective length), using 100 mmol/L of Tris/phosphate buffer, pH 9.0 containing 1% w/v sulfated-beta-CD and 30 mg/mL hydroxypropyl-beta-CD. Detection was carried out at 220 nm. The extraction procedure was efficient in removing endogenous interferents, and low values (相似文献   

Simultaneous determination of YM-64227, a phosphodiesterase type 4 inhibitor, and its five metabolites in dog plasma by high-performance liquid chromatography with fluorescence detection

We developed and validated a reversed-phase high-performance liquid chromatographic method with fluorescence detection for the simultaneous determination of YM-64227 [4-cyclohexyl-1-ethyl-7-methylpyrido(2,3-d)pyrimidin-2-(1H)-one], a novel and selective phosphodiesterase type 4 inhibitor, and its fi ve hydroxylated metabolites in dog plasma. The plasma samples were extracted with tert-butyl methyl ether under alkali conditions. The analytes were well separated on a phenyl ethyl column (5 microm, 250 x 4.6 mm i.d.), opreating at 40 degrees C and using an acetonitrile-acetic acid gradient at a fl ow rate of 1.0 mL/min. The fluorescence signal was monitored at an excitation and emission wavelength of 330 and 400 nm, respectively. No interfering peak was observed at the retention time of YM-64227, its metabolites or the internal standard. The validated quantitation range of the method was 0.4-200 ng/mL for all analytes using 0.5 mL of the plasma sample. The recovery of analytes in the extraction process was more than 65.5%. The intra- and inter-assay precision was less than 5.1 and 12.6%, respectively, and the intra- and inter-assay accuracy ranged from -8.1 to 11.8% and -8.0 to 9.9%, respectively. Using this assay, the plasma concentration of YM-64227 and metabolites can be determined after the oral administration of YM-64227 to beagle dogs.     

Liquid chromatographic high-throughput analysis of the new ultra-short acting hypnotic 'HIE-124' and its metabolite in mice serum using a monolithic silica column

For the first time, a fast, high-performance liquid chromatography (HPLC) method was developed and validated for the simultaneous determination of the new ultra-short hypnotic HIE-124 and its metabolite in mice serum. Each compound, together with carbamazepine (internal standard) was extracted from the serum matrix using liquid-liquid extraction (LLE). Chromatographic resolution of the analytes was performed on a Chromolith Speed Rod monolithic silica column (100 mm × 4.6 mm i.d.) under isocratic conditions using a mobile phase of 65:35 (v/v), 20 mM phosphate buffer (pH 7.0 adjusted with phosphoric acid)-acetonitrile. The elution of the analytes were monitored at 240 nm and conducted at ambient temperature. Because of high column efficiency the mobile phase was pumped at a flow rate of 2.5 mL min(-1). The total run time of the assay was 2 min. The method was validated over the range of 60-2000 ng mL(-1) for HIE-124 and 200-1600 ng mL(-1) for the metabolite (r(2) = 0.99). The limit of detection (LOD) for HIE-124 and its metabolite were 20 ng mL(-1) and 65 ng mL(-1), respectively. The proposed method was validated in compliance with ICH guidelines, in terms of accuracy, precision, limits of detection and quantitation and other aspects of analytical validation. The developed method could be used for the trace analyses of HIE-124 and its metabolite in serum and was finally used for the pharmacokinetic study investigation of HIE-124 in mice serum.     

Column switching and high-performance liquid chromatography in the analysis of amitriptyline, nortriptyline and hydroxylated metabolites in human plasma or serum.

A column-switching system for the direct injection of plasma or serum samples, followed by isocratic high-performance liquid chromatography and ultraviolet detection, is described for the simultaneous quantitation of the tricyclic antidepressant amitriptyline, its demethylated metabolite nortriptyline and the E- and Z-isomers of 10-hydroxyamitriptyline and 10-hydroxynortriptyline. The method included adsorption of amitriptyline and metabolites on a reversed-phase C8 clean-up column (10 microns; 20 mm x 4.6 mm I.D.), washing of unwanted material to waste and, after on-line column-switching, separation on a cyanopropyl analytical column (5 microns; 250 mm x 4.6 mm I.D.). The compounds of interest were separated and eluted using acetonitrile-methanol-0.01 M phosphate buffer (pH 6.8) (578:188:235, v/v) within less than 20 min. Various drugs frequently co-administered with amitriptyline or other antidepressants did not interfere with the determinations. In plasma samples spiked with 25-300 ng/ml, the recoveries were between 84 and 112% and the inter-assay coefficients of variation were 3-11%. After a minor modification, as little as 5 ng/ml could be quantitated. There were linear correlations (r greater than 0.99) between drug concentrations of 5-500 ng/ml and the detector signal. The method allows routine measurements of amitriptyline, nortriptyline and hydroxylated metabolites in blood plasma or serum of patients treated with amitriptyline or nortriptyline, and enables the results to be reported within 1 h.     

An improved HPLC method for rapid quantitation of diazepam and its major metabolites in human plasma

A rapid and specific HPLC method was developed and validated for simultaneous determination of diazepam and its main active metabolites, desmethyldiazepam, oxazepam and temazepam in human plasma. Plasma samples were extracted using toluene. HPLC system included a Chromolith Performance RP-18e 100 mm x 4.6mm column, using 10mM phosphate buffer (pH 2.5)-methanol-acetonitrile (63:10:27, v/v) as mobile phase running at 2 mL min(-1). UV detector (lambda=230 nm) was used. The calibration curves were linear in the concentration range of 2-800 ng mL(-1) for diazepam and 2-200 ng mL(-1) for the three metabolites (r(2)>0.99). The lower limit of quantification was 2 ng mL(-1) for all analytes. Within and between-day precisions in the measurement of QC samples were in the range of 1.8-18.0% for all analytes. The developed procedure was used to assess the pharmacokinetics of diazepam and its main metabolites following single dose administration of 10mg diazepam orally to healthy subjects.     

Liquid chromatography high resolution mass spectrometry for the determination of baclofen and its metabolites in plasma: Application to therapeutic drug monitoring

Baclofen is used to manage alcohol dependence. This study describes a simple method using liquid chromatography coupled to high‐resolution mass spectrometry (LC‐HR‐MS) developed in plasma samples. This method was optimized to allow quantification of baclofen and determination of metabolic ratio of its metabolites, an oxidative deaminated metabolite of baclofen (M1) and its glucuronide form (M2). The LC‐HR‐MS method on Exactive® apparatus is a newly developed method with all the advantages of high resolution in full‐scan mode for the quantification of baclofen and detection of its metabolites in plasma. The present assay provides a protein precipitation method starting with 100 μL plasma giving a wide polynomial dynamic range (R ² > 0.999) between 10 and 2000 ng/mL and a lower limit of quantitation of 3 ng/mL for baclofen. Intra‐ and inter‐day precisions were <8.1% and accuracies were between 91.2 and 103.3% for baclofen. No matrix effect was observed. The assay was successfully applied to 36 patients following baclofen administration. Plasma concentrations of baclofen were determined between 12.2 and 1399.9 ng/mL and metabolic ratios were estimated between 0.4 and 81.8% for M1 metabolite and on the order of 0.3% for M2 in two samples.     

On-line extraction coupled with liquid chromatography tandem mass spectrometry for quantitation of pravastatin and its metabolite in human serum

A high-throughput bioanalytical method for simultaneous quantitation of pravastatin and its metabolite (M1) in human serum was developed and validated using on-line extraction following liquid chromatography tandem mass spectrometry (LC-MS/MS). The on-line extraction was accomplished by the direct injection of a 50 microL serum sample, mixed 4:1 with an aqueous internal standard solution, into one of the extraction columns with aqueous 1 mm formic acid at flow rate of 3 mL/min. The separation and analysis were achieved by back-eluting the analytes from the extraction column and the analytical column to the mass spectrometer with an isocratic mobile phase consisting of 62% aqueous 1 mm formic acid and 38% acetonitrile at a flow rate of 0.8 mL/min. The second extraction column was being equilibrated while the first column was being used for analysis, and vice versa. The standard curve range was 0.500-100 ng/mL for pravastatin and M1. The lower limit of quantitation, 0.500 ng/mL for all the analytes, was achieved when 50 microL of human serum was used. The intra- and inter-day precisions were within 7.4%, and the accuracy was between 95 and 103%. The on-line extraction was finished in 0.5 min and total analysis time was 2.5 min per sample.     

New sensitive liquid chromatography method coupled with tandem mass spectrometric detection for the clinical analysis of vinorelbine and its metabolites in blood, plasma, urine and faeces

A new sensitive and specific liquid chromatographic method coupled with tandem mass spectrometric detection was set up and validated for the simultaneous quantitation of vinorelbine, its main metabolite, 4-O-deacetylvinorelbine and two other minor metabolites, 20'-hydroxyvinorelbine and vinorelbine 6'-oxide. All these compounds, including vinblastine (used as internal standard) were deproteinised from blood, plasma and faeces (only diluted in urine), analysed on a cyano column and detected on a Micromass Quattro II system in the positive ion mode after ionisation, using an electrospray ion source. Under tandem mass spectrometry conditions, the specific product ions led one to accurately quantify vinorelbine and its metabolites in all biological fluids. In whole blood, linearity was assessed up to 200 ng/ml for vinorelbine and up to 50 ng/ml for the metabolites. The limit of quantitation was validated at 250 pg/ml for both vinorelbine and 4-O-deacetylvinorelbine. In the other biological media, the linearity was assessed within a same range and the limit of quantitation was adjusted according to the expected concentrations of each compound. This method was initially developed in order to identify the metabolite structures and to elucidate the metabolic pathway of vinorelbine. Thanks to its high sensitivity, this method has enabled the quantitation of vinorelbine and all its metabolites in whole blood over 168 h (i.e., 4-5 elimination half lives) whilst the previous liquid chromatographic methods allowed their measurement for a maximum of 48-72 h. Therefore, using this method has improved the reliability of the pharmacokinetic data analysis of vinorelbine.     

Assay of cortisol and its metabolite in the rat urine by HPLC-MS method

A technique has been developed for the assay of cortisol and its metabolite (6-β-hydroxycortisol) in the rat urine. Analysis was performed using high-performance liquid chromatography with mass-selective detector. The limits of the quantification of cortisol and its metabolite are 1.0 and 2.5 ng/mL, respectively.     

Measurement of mephenytoin (3-methyl-5-ethyl-5-phenylhydantoin) and its demethylated metabolite by selective ion monitoring

Mephenytoin (3-methyl-5-ethyl-5-phenylhydantoin) and its demethylated metabolite Nirvanol (5-ethyl-5-phenylhydantoin) were measured by a selective ion monitoring technique. This method was used in the analysis of both compounds in plasma from a patient receiving 50 mg and 400 mg of mephenytoin in single oral doses. Both compounds were extracted from plasma and ethylated prior to analysis by electron-impact mass spectrometry. Alkylation, using ethyl iodide in 2-butanone, occurred in the N-1 and N-3 positions of the hydantoin ring when concentrated KOH was added to the reaction mixture. The lower limits of quantitation for mephenytoin and Nirvanol were 10 ng/ml and 50 ng/ml, respectively, and were found to be reproducible within 8% upon repeated quantification.