Radioimmunoassay and gas chromatography/mass spectrometry for a novel antiglaucoma medication of a prostaglandin derivative,S-1033, in plasma

A radioimmunoassay (RIA) and a gas chromatographic/mass spectrometric (GC/MS) method for a new antiglaucoma medicament, the prostaglandin derivative sodium (5Z,9α,11α,13E)-9,11-dihydroxyprosta-5,13-dienoate (S-1033), in human and rabbit plasma were investigated. For a competitive RIA, antisera from rabbit and radioiodine-labeled S-1033 were prepared by immunizing a conjugate of S-1033 with bovine serum albumin and by the Bolton and Hunter method, respectively. Pretreatment by C₁₈ solid-phase extraction (SPE) for rabbit plasma sample and further purification by high-performance liquid chromatography (HPLC) for human plasma samples followed by the RIA (SPE/RIA and HPLC/RIA, respectively) were developed. The assay recoveries of SPE/RIA and HPLC/RIA were both excellent and the limits of quantitation were 320 and 10 pg ml⁻¹, respectively. GC/MS for plasma samples after solid-phase extraction and thin-layer chromatographic purification was also developed using deuterium-labeled S-1033 as internal standard. The limit of quantitation was 100 pg ml⁻¹ in human or rabbit plasma. Rabbit plasma samples after administration of this drug were measured by SPE/RIA and GC/MS and the assay results from both methods agreed well. The SPE/RIA, HPLC/RIA and GC/MS assay methods were suitable for measuring samples from preclinical studies, clincial studies and cross-validation, respectively.     

Quantitative analysis of a novel HIV fusion inhibitor (sifuvirtide) in HIV infected human plasma using high-performance liquid chromatography–electrospray ionization tandem mass spectrometry

A sensitive method for measuring sifuvirtide, a novel HIV fusion inhibitor peptide drug in HIV-1⁺ human plasma by liquid chromatography–tandem mass spectrometry (LC–MS/MS) was developed. The plasma samples were treated by solvent/detergent (S/D) method to inactivate viral activity before analysis. After protein precipitation sifuvirtide was determined by LC–MS/MS. A structure analog was used as internal standard (IS). The mass spectrometer was operated in positive ion and multiple reaction monitoring mode with transitions m/z 946.3 → 159.0 for sifuvirtide and 951.7 → 159.2 for IS. The intra-day precision ranged from 2.74% to 7.57% with accuracy from 91.63% to 102.53%. The inter-day precision ranged from 2.65% to 3.58% and the accuracy from 95.53% to 105.28%. Stability studies showed that sifuvirtide was stable both during the assay procedure and long-term storage. The lower limit of quantitation (LLOQ) was 9.75 ng ml⁻¹. The method was used for analyzing samples from phase IIa clinical study of sifuvirtide in China.     

Determination of pirenzepine in human plasma using liquid chromatography with tandem mass spectrometric detection

A sensitive (LOQ = 1 ng ml⁻¹) and specific method based on liquid chromatography with tandem mass spectrometric (MS/MS) detection has been developed and validated for the analysis of pirenzepine (I) in plasma. Sample preparation involved liquid-liquid extraction of drug and internal standard (IS) from basified plasma. The organic extract was evaporated to dryness, and the residue was reconstituted in the mobile phase and then injected into the liquid chromatography/MS/MS system. Drug, IS, and endogenous impurities were separated using reverse-phase chromatography. A Sciex API III tandem mass spectrometer equipped with a heated nebulizer was operated in the positive ion mode. Multiple reaction monitoring using the parent→daughter ion combinations of m/z 352 → 113 and 629 → 422 was used to quantify I and IS, respectively. The method was validated in the concentration range of 1–100 ng ml⁻¹ plasma with adequate assay precision and accuracy, and was utilized to support human safety and tolerability study with I.     

A sensitive method for the quantification of fluticasone propionate in human plasma by high-performance liquid chromatography/atmospheric pressure chemical ionisation mass spectrometry

A highly sensitive and selective method has been developed for the quantification of fluticasone propionate (FP) in human plasma. The drug was isolated from human plasma using C₁₈ solid-phase extraction cartridges. The analysis was based on high-performance liquid chromatography/atmospheric pressure chemical ionisation mass spectrometry (HPLC/APCI/MS), using the 22R epimer of budesonide (BUD) acetate, synthesised using acetic anhydride, as internal standard. The mass spectrometer was operated in APCI mode with selected ions at tune masses of 473.2 and 501.2 m/z, corresponding to the MH⁺ of acetylated (22R)BUD and FP, respectively. The mobile phase used was a mixture of 50% ethanol in water with a flow rate of 0.45 ml min⁻¹. The system was optimised by tuning the capillary and tube lens with a concentrated solution of FP. The recovery of FP from human plasma was 86.3%. Linearity of response was obtained over the concentration range 0.2–4.0 ng ml⁻¹. The intra-assay and inter-assay variability were 6.3 and 2.9%, respectively. The lower limit of quantification was 0.2 ng ml⁻¹ when a solid-phase extraction preceded the HPLC/APCI/MS.     

Simultaneous determination of 3-chlorotyrosine and 3-nitrotyrosine in human plasma by direct analysis in real time–tandem mass spectrometry

A novel method for the simultaneous determination of 3-nitrotyrosine (NT) and 3-chlorotyrosine (CT) in human plasma has been developed based on direct analysis in real time–tandem mass spectrometry (DART–MS/MS). Analysis was performed in the positive ionization mode using multiple reaction monitoring (MRM) of the ion transitions at m/z 216.2/170.1 for CT, m/z 227.2/181.1 for NT and m/z 230.2/184.2 for the internal standard, d³-NT. The assay was linear in the ranges 0.5–100 μg/mL for CT and 4–100 μg/mL for NT with corresponding limits of detection of 0.2 and 2 μg/mL. Intra- and inter-day precisions and accuracies were respectively <15% and ±15%. Matrix effects were also evaluated. The method is potentially useful for high throughput analysis although sensitivity needs to be improved before it can be applied in clinical research.KEY WORDS: 3-Nitrotyrosine, 3-Chlorotyrosine, Determintion, DART–MS/MS, Human plasma     

Determination of olprinone in human plasma utilizing liquid chromatography tandem mass spectrometry

A sensitive and rapid method was developed for quantification of olprinone in human plasma utilizing liquid chromatography tandem mass spectrometry (LC–MS/MS). An aliquot of 1 mL plasma sample was extracted with ethyl acetate–dichloromethane. Separation of olprinone and the milrinone (internal standard, IS) from the interferences was achieved on a C₁₈ column followed by MS/MS detection. The analytes were monitored in the positive ionization mode. Multiple reaction monitoring using the transition of m/z 251 → m/z 155 and m/z 212 → m/z 140 was performed to quantify olprinone and IS, respectively. The method had a total chromatographic run time of 3 min and linear calibration curves over the concentration range of 0.5–60 ng/mL. The lower limit of quantification (LLOQ) was 0.5 ng/mL. The intra- and inter-day precisions were less than 16.3% for low QC level, and 7.1% for other QC levels, respectively. The intra- and inter-day relative errors were ranged between −12.2% and 3.7% for three QC concentration levels. The validated method was successfully applied to the quantification of olprinone concentration in human plasma after intravenous (i.v.) administration of olprinone at a constant rate of infusion of 2 μg/(kg min) for 5 min in order to evaluate the pharmacokinetics.     

Implementation of a Radioreceptor Assay for Dexmedetomidine

Abstract: We have implemented a radioreceptor assay for dexmedetomidine, a novel α₂-adrenoceptor agonist. Receptor-bearing membranes were prepared from rat cerebral cortex and ³H-clonidine, 4 nM, was used as the labeled ligand. Dexmedetomidine displaced ³H-clonidine in a linear fashion over a concentration of 2 × 10^-10 to 2 × 10^-8 M. The detection limit of dexmedetomidine (i.e. 10% of radiolabeled ligand displaced) in this assay was 50 pg.ml^-1 which is comparable to that seen with the reference method which utilizes gas chromotography with mass spectrometer (GC/MS) in series (Vuorilehto et al. 1989). Endogenous catecholamines, which can displace the radiolabeled ligand from its binding site, could easily be eliminated with a one-step extraction procedure. A comparison was made with the reference method (GC/MS) in 47 human plasma samples; the correlation coefficient (r²) was 0.61 (P<0.001). The radioreceptor assay was also successfully applied for determining dexmedetomidine concentration in rabbit samples. These data indicate that the radioreceptor assay can be utilized for characterizing the pharmacokinetics of novel α₂ agonists which are now being introduced into the clinical practice of anaesthesia.     

A capillary gas-liquid chromatographic method for the assay of the neuroleptic drug zotepine in human serum or plasma

A capillary gas-liquid chromatographic method suitable for the assay of the atypical neuroleptic drug zotepine in human serum or plasma was developed. A liquid-liquid extraction with three subsequent extraction steps was applied for sample preparation. The minimum detectable concentration was 1.0 ng ml⁻¹. The within-day relative standard deviation (RSD) (n = 6) was 5.3% at 5 ng ml⁻¹, 3.6% at 10 ng ml⁻¹ and 6.1% at 100 ng ml⁻¹. The day-to-day RSD (n = 6) was 9.3% at 10 ng ml⁻¹ and 5.1% at 100 ng ml⁻¹. Steady-state serum levels of four schizophrenic patients were measured.     

Clinical analysis of sampatrilat,a combined renal endopeptidase and angiotensin-converting enzyme inhibitor: I: assay in plasma of human volunteers by atmospheric-pressure ionisation mass-spectrometry following derivatisation with BF3–methanol

Sampatrilat is a dual inhibitor of angiotensin converting enzyme (ACE) and neutral endopeptidase (NEP) under development for the treatment of hypertension and congestive heart failure. In order to support the early clinical development (with oral administration and an expected low bioavailability), a sensitive and selective assay was required. A method for plasma was developed and validated employing HPLC–APCI–MS–MS. The plasma samples were extracted on solid-phase extraction cartridges, derivatised with BF₃–methanol, diluted, extracted again and then subjected to HPLC–APCI–MS–MS. Derivatisation was necessary because the two carboxyl group in the molecule prevented efficient ionisation in the heated nebuliser source. The calibration range was from 0.5 to 20 ng ml⁻¹ and the lower limit of quantification was 0.5 ng ml⁻¹. Imprecision and inaccuracy were determined on three separate occasions at three concentrations (0.5, 5 and 20 ng ml⁻¹) and shown to be lower than 10% in every case.     

Analysis and pharmacokinetics of olanzapine (LY170053) and two metabolites in rat plasma using reversed-phase HPLC with electrochemical detection

A sensitive HPLC assay for measurement of the antipsychotic drug, olanzapine, in plasma has been developed. The assay has a limit of quantitation of 1 ng ml⁻¹ in plasma and utilizes solid-phase extraction and electrochemical detection. The method provides a linear response for olanzapine over a concentration range of 1–100 ng ml⁻¹ with coefficients of determination greater than 0.9912. The inter-assay precision was 15.9% at the limit of detection and ranged from 7.33% to 8.47% over the range of 5–100 ng ml⁻¹. The intra-assay precision was in the range 0.97%–26.0%. The inter-assay accuracy ranged from 98.9 to 118% and the intra-assay accuracy ranged from 92.5% to 125% of the theoretical value. In addition, the assay was extended to measure the plasma levels of two metabolites of olanzapine, namely the N-desmethyl- and the 2-hydroxymethyl analogs. The utility of the assay was demonstrated following the administration of a single oral dose of ¹⁴C-olanzapine to rats where, at several time-points after dosing, the plasma was assayed for total radioactivity, levels of olanzapine, and the two metabolites. Olanzapine and two of its metabolites accounted for less than 50% of the total plasma radiocarbon; olanzapine accounting for approximately 39% at the C_max, N-desmethyl for 5% and 2-hydroxymethyl for 8% respectively. The plasma elimination half-times for olanzapine and the two metabolites were approximately the same, ranging from 3.3 to 4.4 h.     

Application of solid-phase extraction in the method development for determination of SEPA,an acronym for soft enhancement of percutaneous absorption,in human,rat, and rabbit serum using GC–FID method

A new nonaqueous topical minoxidil formulation containing SEPA (2-n-nonyl-1,3-dioxolane) for enhancement of percutaneous absorption was under evaluation. SEPA does not have chromophore for either ultraviolet or fluorescence detection using liquid chromatography and has no functional groups for derivatization. Therefore, a direct gas-chromatographic method with flame-ionization detection (GC–FID) was developed. Owing to the limited detection response of the FID detection, it needs a selective and concentrated extract for GC–FID analysis to improve the assay sensitivity to meet the requirement for pharmacokinetic evaluation after topical application. In addition, SEPA is a very volatile compound. Any extraction procedures involving evaporation will result in a poor recovery. The application of solid-phase extraction (SPE) makes it possible to achieve a selective and a 10-fold concentrated extract with an absolute extraction recovery of approximately 90%, which greatly improved the assay sensitivity. This method involved the extraction of SEPA and the internal standard (2-n-heptyl-1,3-dioxolane) from serum (0.1–1 ml) with 100 μl of hexane-chloroform (1:1, v:v) using a 50 mg 1.0 ml⁻¹ phenyl SPE column (Varian, Harbor City, CA, USA), followed by direct GC–FID analysis on a fused-silica column chemically bonded with cross-linked methyl silicone gum phase (Hewlett Packard Ultra-1, 12 m×0.2 mm×0.33 μm, Avondale, PA, USA). The assay demonstrated a lower limit of quantitation of 2.5 ng ml⁻¹ and a linear range of 2.5 to 250 ng ml⁻¹ with intra- and inter-assay precision and accuracy of ≤10%.     

High-performance liquid chromatography-mass spectrometry-mass spectrometry analysis of morphine and morphine metabolites and its application to a pharmacokinetic study in male Sprague–Dawley rats

A high-performance liquid chromatography tandem mass spectrometry-mass spectrometry (LC-MS-MS) assay was developed for the analyses of morphine, morphine glucuronides and normorphine in plasma samples from rats. The analytes were extracted by using C₂ solid-phase extraction cartridges. The extraction recoveries were 100% for morphine, 84% for morphine-3-glucuronide, 64% for morphine-6-glucuronide and 88% for normorphine. Both intra- and inter-assay variabilities were below 11%. Using a plasma sample size of 100 μl, the limits of detection were 13 nmol l⁻¹ (3.8 ng ml⁻¹) for morphine, 12 nmol l⁻¹ (5.5 ng ml⁻¹) for morphine-3-glucuronide, 26 nmol l⁻¹ (12 ng ml⁻¹) for morphine-6-glucuronide and 18 nmol l⁻¹ (5.0 ng ml⁻¹) for normorphine, at a signal-to-noise ratio of 3. The present assay was applied to a pharmacokinetic study in rats after intraperitoneal administration of morphine.     

Rapid and sensitive assay for trantinterol,a novel β2-adrenoceptor agonist,in human plasma using liquid chromatography–tandem mass spectrometry

A rapid and sensitive assay for trantinterol, a novel β₂-adrenoceptor agonist, in human plasma has been developed. Samples containing the analyte and internal standard, clenbuterol, were analyzed by liquid chromatography–tandem mass spectrometry after liquid–liquid extraction with diethyl ether:dichloromethane (60:40, v/v). Separation was performed on a Venusil MP C₁₈ column (50 mm × 4.6 mm, 5 μm) using methanol:1% formic acid (50:50, v/v) as mobile phase and monitored by multiple reaction monitoring of the precursor-to-product ion transitions of trantinterol at m/z 311.2 → 238.1 and clenbuterol at m/z 277.2 → 203.1. The total run time was only 1.5 min and the method was linear over the concentration range 1–1000 pg/mL with a lower limit of quantitation of 1 pg/mL. Intra- and inter-day precisions (relative standard deviation) were below 7% and 12%, respectively, with accuracy (relative error) below 8%. The method was successfully applied to a pharmacokinetic study involving oral administration of a 50 μg trantinterol tablet to healthy volunteers.     

Simplified reversed-phase HPLC method with spectrophotometric detection for the assay of verapamil in rat plasma

A high-performance liquid chromatographic (HPLC) method was developed for the assay of verapamil in rat plasma. After deproteinization of the plasma sample with an acetonitrile-perchloric acid (8:2) mixture containing dextromethorphan, the internal standard, an aliquot of the supernatant was directly analyzed on a cyanopropylsilane column with methanol-acetonitrile-triethylamine acetate buffer (10:30:60) as the mobile phase and detection at 235 mm. At a flow rate of 1.5 ml min⁻¹, a complete analysis was completed in less than 6 min. The method was linear for verapamil concentrations in the range 0.5–10 μg ml⁻¹ (r=0.9999). Recoveries for the same drug concentrations from spiked rat plasma ranged from 85.6-93.0% (n=8). The mean RSD values for intraday and interday assay reproducibility (n=3) were, in both cases, less than 0.9%. The limit of detectability was about 0.1 μg ml⁻¹. The method was found useful to monitor the plasma levels of verapamil in rats that had received this drug by the nasal, oral and intravenous routes of administration.     

Determination of ginsenoside compound K in human plasma by liquid chromatography–tandem mass spectrometry of lithium adducts

Ginsenoside compound K (GCK), the main metabolite of protopanaxadiol constituents of Panax ginseng, easily produces alkali metal adduct ions during mass spectrometry particularly with lithium. Accordingly, we have developed a rapid and sensitive liquid chromatography–tandem mass spectrometric method for analysis of GCK in human plasma based on formation of a lithium adduct. The analyte and paclitaxel (internal standard) were extracted from 50 µL human plasma using methyl tert-butyl ether. Chromatographic separation was performed on a Phenomenex Gemini C18 column (50 mm×2.0 mm; 5 μm) using stepwise gradient elution with acetonitrile–water and 0.2 mmol/L lithium carbonate at a flow rate of 0.5 mL/min. Detection was performed in the positive ion mode using multiple reaction monitoring of the transitions at m/z 629→449 for the GCK-lithium adduct and m/z 860→292 for the adduct of paclitaxel. The assay was linear in the concentration range 1.00–1000 ng/mL (r²>0.9988) with intra- and inter-day precision of ±8.4% and accuracy in the range of −4.8% to 6.5%. Recovery, stability and matrix effects were all satisfactory. The method was successfully applied to a pharmacokinetic study involving administration of a single GCK 50 mg tablet to healthy Chinese volunteers.KEY WORDS: LC−MS/MS, Ginsenoside compound K, Lithium adduct ion, Pharmacokinetics     

A specific enzyme immunoassay (EIA) with selective extraction for quantitation of a topical anti-inflammatory agent,SCH 40120, in human plasma

SCH 40120 is a potent anti-inflammatory agent under development for the topical treatment of dermal inflammatory and allergic disorders such as atopic dermatitis, contact dermatitis and psoriasis. We have previously described a sensitive enzyme immunoassay (EIA) for SCH 40120 in unextracted human plasma to support clinical studies. However, severe cross-reaction with unknown metabolites was observed during validation using samples from rats dosed with ¹⁴C-SCH 40120. Therefore, a selective extraction procedure was developed to remove the unknown plasma metabolites of SCH 40120 prior to EIA quantitation. The modified EIA using extracted plasma was cross-validated with an LC method using plasma samples from dosed subjects (human and rat), thereby confirming the specificity of the assay. The EIA can reliably quantitate SCH 40120 in plasma samples from 100 pg ml⁻¹ to 10 ng ml⁻¹ with good linearity, accuracy and precision, and is suitable for pharmacokinetic studies in man.     

Simultaneous Determination of Atorvastatin and Aspirin in Human Plasma by LC–MS/MS: Its Pharmacokinetic Application

A simple, rapid, and sensitive liquid chromatography tandem mass spectro-metric (LC–MS/MS) assay method has been developed and fully validated for the simultaneous quantification of atorvastatin and aspirin in human plasma using a polarity switch. Proguanil and furosemide were used as the internal standards for the quantification of atorvastatin and aspirin, respectively. The analytes were extracted from human plasma by the liquid–liquid extraction technique using methyl tert-butyl ether. The reconstituted samples were chromatographed on a Zorbax XDB Phenyl column by using a mixture of 0.2% acetic acid buffer, methanol, and acetonitrile (20:16:64, v/v) as the mobile phase at a flow rate of 0.8 mL/min. Prior to detection, atorvastatin and aspirin were ionized using an ESI source in the multiple reaction monitoring (MRM) mode. The ions were monitored at the positive m/z 559.2→440.0 transition for atorvastatin and the negative m/z 179.0→136.6 transition for aspirin. The calibration curve obtained was linear (r² ≥ 0.99) over the concentration range of 0.20–151 ng/mL for atorvastatin and 15.0–3000 ng/mL for aspirin. The method validation was performed as per FDA guidelines and the results met the acceptance criteria. A run time of 3.0 min for each sample made it possible to analyze more than 300 human plasma samples per day. The proposed method was found to be applicable to clinical studies.     

Determination of cyclovirobuxine D in human plasma by liquid chromatography tandem mass spectrometry and application in a pharmacokinetic study

A sensitive and reliable method based on liquid chromatography tandem mass spectrometry (LC–MS/MS) for the quantitation of cyclovirobuxine D in human plasma has been developed and validated. Sample preparation by solid phase extraction was followed by separation on a CN column with a mobile phase of methanol–water (95:5, v/v) containing 0.2% formic acid. Mass spectrometric detection in the positive ion mode was carried out by selected reaction monitoring (SRM) of the transitions at m/z 403.0→372.0 for cyclovirobuxine D and m/z 325.0→234.0 for citalopram (internal standard). The method was linear in the range 10–200 ng/L with LLOQ of 10 ng/L, recovery >85%, and no significant matrix effects. Intra- and inter-day precisions were all <9% with accuracies of 94.0–104.8%. The method was successfully applied to a pharmacokinetic study involving a single oral administration of a 2 mg cyclovirobuxine D tablet to twenty-two healthy Chinese volunteers.     

LC–MS/MS assay for olanzapine in human plasma and its application to a bioequivalence study

This paper describes a selective and sensitive assay for the determination of olanzapine (OLZ) in human plasma based on liquid chromatography–tandem mass spectrometry (LC–MS/MS). The analyte and quetiapine as internal standard (IS) were extracted from 200 μL plasma via solid phase extraction on Waters Oasis HLB cartridges. Chromatographic separation was achieved on an ACE 5C18-300 column (100 mm×4.6 mm, 5 μm) under isocratic conditions in a run time of 3.5 min. Mass spectrometric detection involved electrospray ionization in the positive ion mode followed by multiple reaction monitoring (MRM) of the transitions at m/z 313/256 for OLZ and m/z 384/253 for the IS. The assay was linear in the range 0.10–40.0 ng/mL with a lower limit of quantitation and limit of detection of 0.10 and 0.012 ng/mL, respectively. Intra- and inter-day precision (as coefficient of variation) and relative recovery were <5.0% and >90%, respectively. The method was successfully applied to a bioequivalence study of 5 and 10 mg OLZ disintegrating tablets in 40 healthy Indian males with reproducibility by incurred sample reanalysis in the range ?7.43 to 8.07%.     

Characterization of benzimidazole and other oxidative and conjugative metabolites of brimonidine in vitro and in rats in vivo using on-line H/D exchange LC-MS/MS and stable-isotope tracer techniques

The characterization of brimonidine metabolites presents some challenges since brimonidine and its metabolites generate few structurally informative fragment ions in the LC-MS/MS spectra. The objective of the current study is to use on-line hydrogen/deuterium (H/D) exchange LC-MS/MS and stable-isotope tracer techniques to further characterize unknown brimonidine metabolites in vitro and in vivo. Brimonidine and D₄-brimonidine were co-incubated in rat and human microsomes and rabbit aldehyde oxidase in vitro. In addition, the urine was collected from rats co-administered orally with brimonidine and D₄-brimonidine. The hepatic microsomal and urinary metabolites were then characterized by H/D LC-MS/MS system. In addition to previously characterized 2-oxobrimonidine, 3-oxobrimonidine and 2,3-dioxobrimonidine, the results show that oxidation occurs at quinoxaline ring producing oxo-hydroxybrimonidine and hydroxyquinoxaline metabolites. The hydroxyquinoxaline metabolite was only observed in microsomal incubations with hydroxylation at the 7- or 8- position. The dehydro-hydroxybrimonidine metabolites were characterized as 2-oxo or 3-oxo -4′,?5′-dehydrobrimonidine. A novel metabolite ((4-bromo-1H-benzoimidazol-5-yl)-imidazolidin-2-ylidene-amine) of benzimidazole derivative of brimonidine in rats in vivo was identified and confirmed with reference standard. In conclusion, on-line H/D exchange LC-MS/MS and stable-isotope tracer techniques are useful for the characterization of brimonidine metabolites.