Quantification of epimeric budesonide and fluticasone propionate in human plasma by liquid chromatography–atmospheric pressure chemical ionization tandem mass spectrometry

A highly sensitive and selective liquid chromatography–atmospheric pressure chemical ionization tandem mass spectrometry assay was developed and validated for simultaneous determination of epimeric budesonide (BUD) and fluticasone propionate (FP) in plasma. The drugs were isolated from human plasma using C₁₈ solid-phase extraction cartridges, and epimeric BUD was acetylated with a mixture of 12.5% acetic anhydride and 12.5% triethylamine in acetonitrile to form the 21-acetyl derivatives following the solid-phase extraction. Deuterium-labelled BUD acetate with an isotopic purity >99% was synthesized and used as the internal standard. The assay was linear over the ranges 0.05–10.0 ng/ml for epimeric BUD, and 0.02–4.0 ng/ml for FP. The inter- and intra-day relative standard deviations were <14.3% in the assay concentration range.     

Simultaneous quantification of beclomethasone dipropionate and its metabolite, beclomethasone 17-monopropionate in rat and human plasma and different rat tissues by liquid chromatography-positive electrospray ionization tandem mass spectrometry

A sensitive, rapid and selective liquid chromatography-positive electrospray ionization tandem mass spectrometry (LC-(ESI+)-MS-MS) method has been developed and validated for the simultaneous quantification of beclomethasone dipropionate (BDP) and its active metabolite, beclomethasone 17-monopropionate (17-BMP) in rat plasma and different tissues using fluticasone propionate (FP) as the internal standard. The method was validated over a linear range from 0.05 to 5 ng/ml for both analytes. A solid-phase extraction procedure was used for plasma samples and a liquid-liquid extraction procedure for tissues samples (lung, liver and kidney). The between-day and within-day coefficients of variation for all compounds were 相似文献   

Simultaneous determination of erythromycin propionate and base in human plasma by high-performance liquid chromatography-electrospray mass spectrometry

An analytical method for simultaneous determination of erythromycin propionate and its active metabolite, erythromycin base, in human plasma by high-performance liquid chromatography-electrospray mass spectrometry (HPLC-ESI-MS) was developed and validated. Roxithromycin was selected as the internal standard. The samples were directly injected after simple deproteinized procedure only. The separation was achieved on a Johnson Spherigel analytical column packed with 5 microm C18 silica, employing acetonitrile -0.1% formic acid aqueous solution (50:50) as mobile phase. The quantification of target compounds was obtained by using a selected ion monitoring (SIM) at m/z 790.7 for erythromycin propionate, m/z 734.7 for erythromycin base and m/z 837.8 for roxithromycin. The correlation coefficients of the calibration curves were better than 0.997 (n=6), in the ranges from 2 ng/ml to 1 microg/ml, and from 1 to 10 microg/ml for erythromycin propionate and base. The method can provide the necessary sensitivity, precision and accuracy to allow the simultaneous determination of both compounds in a patient's plasma following a single administration of erythromycin stinoprate capsule (500 mg erythromycin base equivalent).     

Micro-quantitative determination of ciprostene in plasma by gas chromatography—mass spectrometry coupled with an antibody extraction

A simple and highly sensitive method has been developed for the determination in plasma of ciprostene, 9β-methyl-6α-carbaprostaglandin I₂, using gas chromatography—mass spectrometry following solid-phase extraction on an immobilized antibody column. The anti-ciprostene antibody obtained from rabbit serum was coupled to an agarose support matrix, and the immobilized antibody thus prepared was used as an extraction phase for sample clean-up. The extracted drug was treated with pentafluorobenzyl bromide followed by bis(trimethylsilyl)trifluoroacetamide. The derivative was quantitatively analysed by negative-ion chemical ionization gas chromatography—mass spectrometry. The lower limit of quantitation was 50 pg/ml when 1 ml of human plasma was used. The plasma concentration of ciprostene in a dog treated with ciprostene at 2.5 μg/kg was determined successfully by this method.     

Simultaneous HPLC analysis of triamcinolone acetonide and budesonide in microdialysate and rat plasma: application to a pharmacokinetic study

A specific and reliable HPLC-PDA method for the quantitative determination of triamcinolone acetonide, budesonide and fluticasone propionate (as internal standards) in small volumes of microdialysate and rat plasma was developed. An efficient solid-phase extraction (SPE) procedure for plasma samples yielded extremely clean extracts with overall recovery of 104.3% and 95.7% for triamcinolone acetonide (TA) and fluticasone propionate, respectively. Plasma extracts obtained after SPE and microdialysis samples were directly injected on a C18 column to separation. The method has been validated with good linearity, sensitivity, specificity and high accuracy (RE -5.28% to 9.14%) and precision (CV 0.50% to 6.62%) on both matrices. In stability studies, TA and budesonide were stable during storage and assay procedures. The method was applied to a pharmacokinetic study in rodents using microdialysis to determine protein unbound TA concentrations in blood and muscle.     

Simultaneous determination of blood concentrations of methohexital and its hydroxy metabolite by gas chromatography and identification of 4′-hydroxymethohexital by combined gas—liquid chromatography—mass spectrometry

A simple, sensitive and selective method is described for the simultaneous determination of low concentrations (less than 50 ng/ml) of underivatized methohexital and its hydroxy metabolite in small (0.1 ml) samples of human and rat plasma or whole blood by gas chromatography with nitrogen-selective detection.Moreover, the main metabolite in rat and man was identified as 4′-hydroxymethohexital by comparison of chromatograms from gas—liquid chromatography (GLC) with data obtained from GLC—mass spectrometry and ¹H-nuclear magnetic resonance spectrometry of this metabolite, produced both by incubating methohexital with isolated rat liver microsomes and by isolating this metabolite from rat urine.     

Determination of dioxopiperazine metabolites of quinapril in biological fluids by gas chromatography—mass spectrometry

The dioxopiperazine metabolites of quinapril in plasma and urine were extracted with hexane—dichloroethane (1:1) under acidic conditions. Following derivatization with pentafluorobenzyl bromide and purification of the desired reaction products using a column packed with silica gel, the metabolites were analysed separately by capillary column gas chromatography—electron-impact mass spectrometry with selected-ion monitoring. The limits of quantitation for the metabolites were 0.2 ng/ml in plasma and 1 ng/ml in urine. The limits of detection were 0.1 ng/ml in plasma and 0.5 ng/ml in urine, at a signal-to-noise ratio of > 3 and > 5, respectively. The proposed method is applicable to pharmacokinetic studies.     

Twice weekly fluticasone propionate added to emollient maintenance treatment to reduce risk of relapse in atopic dermatitis: randomised,double blind,parallel group study

Objective To explore the efficacy and safety of fluticasone propionate, cream and ointment, applied twice weekly in addition to maintenance treatment with emollients, in reducing the risk of relapse of chronic recurrent atopic dermatitis.Design Randomised, double blind, parallel group study of 20 weeks'' duration.Setting Dermatology outpatient clinics (6 countries, 39 centres).Participants Adult (aged 12-65) patients with moderate to severe atopic dermatitis who were experiencing a flare.Methods Participants applied fluticasone propionate (0.05% cream or 0.005% ointment; once or twice daily) regularly for four weeks to stabilise their condition. The patients whose disease was brought under control then continued into a 16 week maintenance phase, applying emollient on a daily basis with a bath oil as needed and either the same formulation of fluticasone propionate or its placebo base (emollient alone) twice weekly to the areas that were usually affected.Main outcome measure Time to relapse of atopic dermatitis during maintenance phase.Results 376 patients entered the stabilisation phase, and 295 continued into the maintenance phase. After 16 weeks in the maintenance phase, the disease remained under control in 133 patients (87 using fluticasone propionate twice weekly, 46 using emollient alone), 135 (40 fluticasone propionate, 95 emollient) had experienced a relapse, and 27 had discontinued. Median time to relapse was six weeks for emollient alone compared with more than 16 weeks for additional fluticasone propionate. Patients who applied fluticasone propionate cream twice weekly were 5.8 times less likely (95% confidence interval 3.1 to 10.8, P < 0.001) and patients using fluticasone propionate ointment 1.9 times less likely (1.2 to 3.2, P=0.010) to have a relapse than patients applying emollient alone. The groups showed no differences in adverse events.Conclusion After atopic dermatitis had been stabilised the addition of fluticasone propionate twice weekly to maintenance treatment with emollients significantly reduced the risk of relapse.     

Determination of pilocarpine, isopilocarpine, pilocarpic acid and isopilocarpic acid in human plasma and urine by high-performance liquid chromatography with tandem mass spectrometric detection

A method is described for the determination of pilocarpine and its degradation products isopilocarpine, pilocarpic acid and isopilocarpic acid in human plasma and urine. The method is based on a simple sample preparation step – ultrafiltration for plasma and dilution for urine samples – followed by a reversed-phase liquid chromatographic separation of the analytes and detection by means of tandem mass spectrometry. Parameters affecting the performance of these steps are discussed. The high sensitivity and selectivity of the method allow low ng/ml concentrations to be determined for all compounds in plasma and undiluted urine, which enables the investigation of the metabolic fate and elimination of pilocarpine after oral administration to humans.     

Analysis of methadone and metabolites in biological fluids with gas chromatography—mass spectrometry

The analysis of methadone and its metabolites in biological fluids by gas chromatography—mass spectrometry is described with deuterated methadone and metabolites as internal standards. The method allowed the determination of 20 ng methadone in 0.5 ml of plasma or saliva. Mean saliva to plasma ratio of methadone for two patients was determined to be 0.51 ± 0.13. Methadone and 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) in urine were measured by selected ion monitoring. Gas chromatography—mass spectrometry was found to have advantages over conventional gas chromatographic methods in terms of ratio analysis. 1,5-Dimethyl-3,3-diphenyl-2-pyrrolidone previously reported as a metabolite was shown to result primarily from the decomposition of EDDP free base.     

Development and validation of a high-performance liquid chromatography–tandem mass spectrometry assay for the determination of sanfetrinem in human plasma

A rapid, selective and accurate high-performance liquid chromatography–tandem mass spectrometry assay for the quantification of sanfetrinem in human plasma has been developed and validated. The performance of manual and automated sample preparation was assessed; 50 μl of plasma sample was deproteinized with acetonitrile, followed by dilution with water and injection onto the LC system. Chromatographic separation was achieved on a Phenomenex Luna C₁₈(2), 50×2.0 (5 μm) column with a mobile phase consisting of water–acetonitrile with 0.1% formic acid followed by detection with a Perkin-Elmer API3000 mass spectrometer in multiple reaction monitoring mode. The lower limit of quantification was improved by five times compared to the UV method previously reported. A range of concentration from 10 ng/ml to 5 μg/ml was covered. The method was applied to the quantification of sanfetrinem in human plasma samples from healthy volunteers participating in a clinical study.     

Bioanalysis of erythromycin 2'-ethylsuccinate in plasma using phase-system switching continuous-flow fast atom bombardment liquid chromatography—mass spectrometry

A specific method for the determination of erythromycin 2'-ethylsuccinate (EM-ES) in plasma is described. The method involves a liquid—liquid extraction procedure followed by the analysis of extracts using phase-system switching (PSS) continuous-flow fast atom bombardment (CF-FAB) liquid chromatography—mass spectrometry (LC—MS). In PSS EM-ES is enriched after analytical separation on a short trapping column, from which it is desorbed to the LC—MS interface. In this way, favourable mobile phases can be used for the LC separation and for the MS detection. Using the PSS approach a flow-rate reduction from 1.0 ml/min in the LC system to 15 μl/min going into the mass spectrometer was achieved without splitting. The determination limit for EM-ES was 0.1 μg/ml.     

Analysis of aplidine (dehydrodidemnin B), a new marine-derived depsipeptide, in rat biological fluids by liquid chromatography–tandem mass spectrometry

Aplidine (dehydrodidemnin B) is a new marine-derived depsipeptide with a powerful cytotoxic activity, which is under early clinical investigation in Europe and in the US. In order to investigate the pharmacokinetic properties of this novel drug, an HPLC–tandem mass spectrometry method was developed for the determination of aplidine in biological samples. Didemnin B, a hydroxy analogue, was used as internal standard. After protein precipitation with acetonitrile and extraction with chloroform, aplidine was chromatographed with a RP octadecylsilica column using a water–acetonitrile linear gradient in the presence of formic acid at the flow-rate of 500 μl/min. The method was linear over a 5–100 ng/ml range (LOD=0.5 ng/ml) in plasma and over a 1.25–125 ng/ml range (LOD=0.2 ng/ml) in urine with precision and accuracy below 14.0%. The intra- and inter-day precision and accuracy were below 12.5%. The extraction procedure recoveries for aplidine and didemnin B were 69% and 68%, respectively in plasma and 91% and 87%, respectively in urine. Differences in linearity, LOQ, LOD and recoveries between plasma and urine samples seem to be matrix-dependent. The applicability of the method was tested by measuring aplidine in rat plasma and urine after intravenous treatment.     

Simultaneous determination of flupirtine and its major active metabolite in human plasma by liquid chromatography–tandem mass spectrometry

A rapid, selective and sensitive HPLC–tandem mass spectrometry method was developed and validated for simultaneous determination of flupirtine and its active metabolite D-13223 in human plasma. The analytes and internal standard diphenhydramine were extracted from plasma samples by liquid–liquid extraction, and chromatographed on a C₁₈ column. The mobile phase consisted of acetonitrile–water–formic acid (60:40:1, v/v/v), at a flow rate of 0.5 ml/min. Detection was performed on a triple quadrupole tandem mass spectrometer by selected reaction monitoring (SRM) mode via atmospheric pressure chemical ionization (APCI). The method has a limit of quantitation of 10 ng/ml for flupirtine and 2 ng/ml for D-13223, using 0.5-ml plasma sample. The linear calibration curves were obtained in the concentration range of 10.0–1500.0 ng/ml for flupirtine and 2.0–300.0 ng/ml for D-13223. The intra- and inter-run precision (RSD), calculated from quality control (QC) samples was less than 7.2% for flupirtine and D-13223. The accuracy as determined from QC samples was less than 5% for the analytes. The overall extraction recoveries of flupirtine and D-13223 were determined to be about 66% and 78% on average, respectively. The method was applied for the evaluation of the pharmacokinetics of flupirtine and active metabolite D-13223 in volunteers following peroral administration.     

Determination of an arylether antiarrhythmic and its N-dealkyl metabolite in rat plasma and hepatic microsomal incubates using liquid chromatography–tandem mass spectrometry

A method was developed and validated for the quantification of (±)-trans-[2-morpholino-1-(1-naphthaleneethyloxy]cyclohexane monohydrochloride (RSD1070) and its N-dealkyl metabolite in rat plasma and hepatic microsomal incubates. Chromatographic separations were achieved using reversed-phase high-performance liquid chromatography coupled with positive ion electrospray ionization and detection by tandem mass spectrometry. The assay was linear from 2.5 to 100 ng/ml and this range was used for validation. Inter- and intra-assay variability (n=6), extraction recovery, and stability in plasma were assessed. The estimated limit of quantitation was in the range 2.5–3 ng/ml for both analytes in rat plasma. The analytical method was used in a pharmacokinetic study of RSD1070 in rats after a single i.v. bolus of 12 mg/kg.     

Determination of celecoxib in human plasma and rat microdialysis samples by liquid chromatography tandem mass spectrometry

Methods for the determination of celecoxib in human plasma and rat microdialysis samples using liquid chromatography tandem mass spectrometry are described. Celecoxib and an internal standard were extracted from plasma by solid-phase extraction with C₁₈ cartridges. Thereafter compounds were separated on a short narrow bore RP C₁₈ column (30×2 mm). Microdialysis samples did not require extraction and were injected directly using a narrow bore RP C₁₈ column (70×2 mm). The detection was by a PE Sciex API 3000 mass spectrometer equipped with a turbo ion spray interface. The compounds were detected in the negative ion mode using the mass transitions m/z 380→316 and m/z 366→302 for celecoxib and internal standard, respectively. The assay was validated for human plasma over a concentration range of 0.25–250 ng/ml using 0.2 ml of sample. The assay for microdialysis samples (50 μl) was validated over a concentration range of 0.5–20 ng/ml. The method was utilised to determine pharmacokinetics of celecoxib in human plasma and in rat spinal cord perfusate.     

Automated protein precipitation by filtration in the 96-well format

The use of automated protein precipitation by filtration in the 96-well format as a rapid sample preparation technique for high throughput bioanalysis using liquid chromatography tandem mass spectrometry is reported. A robotic sample processor is used to aspirate sequentially a plasma sample and acetonitrile separated by air gaps. These are then mixed by being dispensed into individual channels of a 96-well filter block. The resulting supernatant is separated from the precipitated plasma proteins by the application of gentle vacuum using a custom manifold. The filtered supernatants are collected into a deep well microtitre plate, evaporated to dryness using a heated 96-well dry down station and reconstituted in water prior to analysis. The efficiency of the extraction procedure is measured by the Lowry method for determining protein concentration. This method was used to optimise both the volume and the order of reagent addition, and to compare several prototype 96-well filter blocks. Using the optimised procedure a specific, precise and accurate method was developed for the β-agonist salbutamol in rabbit plasma with a calibration range of 1 to 100 ng/ml from 100 μl of sample.     

Minimizing matrix effects in the development of a method for the determination of salmeterol in human plasma by LC/MS/MS at low pg/mL concentration levels

Salmeterol is an inhaled bronchodilator drug used for treatment of asthma. Its concentrations in plasma are very low or undetectable by previously developed methods. The present paper describes a method for analysis of salmeterol in human plasma with 2.5 pg/mL lower limit of quantitation. Despite the basic character of the drug the method uses mixed mode anion-exchange solid phase extraction for sample preparation combined with a column switching approach to minimize matrix effects. Samples are separated and detected by LC/MS/MS. The method is easy to use, only requires 0.5 mL of plasma and was validated for use in bioanalytical applications. The method does not suffer from interference from co-administered fluticasone propionate.     

Determination of norepinephrine in small volume plasma samples by stable-isotope dilution gas chromatography–tandem mass spectrometry with negative ion chemical ionization

A stable-isotope based gas chromatography–tandem mass spectrometry–negative ion chemical ionization method was developed for the determination of norepinephrine (NE) levels in small volumes (25–100 μl) of plasma. NE was stabilized in plasma by the addition of semicarbazide and spiked with deuterium-labeled norepinephrine internal standard. The analytes were isolated from the plasma by solid-phase extraction using phenylboronic acid columns and derivatized using pentafluoropropionic anhydride. The derivatized analytes were chromatographed on a capillary column and detected by tandem mass spectrometry with negative ion chemical ionization. Unparalleled sensitivity and selectivity were obtained using this detection scheme, allowing the unambiguous analysis of trace levels of NE in small-volume plasma samples. Linear standard curves were obtained for NE over a mass range from 1 to 200 pg per sample. The method had a limit of quantitation of 10 pg NE/ml plasma when using a 100-μl sample aliquot (1 pg/sample). Accuracy for the analysis of plasma samples spiked with 10 to 200 pg NE/ml typically ranged from 100±10%, with RSD values of less than 10%. The methodology was applied to determine the effect of clonidine on plasma NE levels in conscious spontaneously hypertensive rats. Administration of clonidine (30 μg/kg) produced an 80% reduction in plasma NE accompanied by a 30% reduction in heart and mean arterial pressure that persisted >90 min after drug administration. The ability to take multiple samples from individual rats allowed the time course for the effect of clonidine to be mapped out using only one group of animals.     

Determination of 9α,11β-prostaglandin F2 in human urine and plasma by gas chromatography—mass spectrometry

Sensitive and specific assay methods for 9α,11β-prostaglandin F₂ (9α,11β-PGF₂) by gas chromatography—mass spectrometry with electron impact ionization are described. The mass spectrometric assay for 9α,11β-PGF₂ was based on the use of the methyl ester—dimethylisopropylsilyl ether derivative, and pentadeuterated PGF_2α as a convenient internal standard. The calibration graph for 9α,11β-PGF₂ was linear from 5 pg to 100 ng for both the standard and spiked biological samples. The limit of detection was 50 pg/ml for urine and 25 pg/ml for plasma (signal-to-noise RATIO = 2.3). The method was applied to the determination of 9α,11β-PGF₂ in urine and plasma samples from patients with bronchial asthma.