Quantitative analysis of diclazuril in animal plasma by liquid chromatography/electrospray ionization mass spectrometry

A novel, sensitive and specific method for the quantitative determination of diclazuril in animal plasma using liquid chromatography combined with electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) with negative ion detection is presented. Extraction of the samples was performed with a rapid deproteinization step using acetonitrile. Chromatography of diclazuril and the internal standard was achieved on a Nucleosil ODS 5-microm column, using a gradient elution with 0.01 M ammonium acetate and acetonitrile. To obtain the highest sensitivity and specificity possible, the mass spectrometer was operated in the multiple reaction monitoring (MRM) mode. The method was validated according to the requirements defined by the European Community. Calibration curves using plasma fortified between 1-100 ng/mL and 100-2000 ng/mL showed good linear correlation (r >or= 0.9991, goodness-of-fit coefficient 相似文献   

Simultaneous determination of cyproterone acetate and ethinylestradiol in tablets by derivative spectrophotometry

Derivative spectrophotometry offers a useful approach for the analysis of drugs in multi-component mixtures. In this study a third-derivative spectrophotometric method was used for simultaneous determination of cyproterone acetate and ethinylestradiol using the zero-crossing technique. The measurements were carried out at wavelengths of 316 and 226 nm for cyproterone acetate and ethinylestradiol respectively. The method was found to be linear (r2>0.999) in the range of 0.5-6 mg/100 ml for cyproterone acetate in the presence of 35 microg/100 ml ethinylestsradiol at 316 nm. The same linear correlation (r2>0.999) was obtained in the range of 10-80 microg/100 ml of ethinylestradiol in the presence of 2 mg/100 ml of cyproterone acetate at 226 nm. The limit of determination was 0.5 mg/100 ml and 10 microg/100 ml for cyproterone acetate and ethinylestradiol respectively. The method was successfully applied for simultaneous determination of cyproterone acetate and ethinylestradiol in pharmaceutical preparations without any interferences from excipients.     

An isocratic HPLC method for the quantitation of eicosanoids in human platelets

We describe here a modified protocol for the simultaneous quantification of specific eicosanoids formed during stimulation of human platelets in vitro with adenosine diphosphate. The eicosanoids thromboxane B(2) (TXB(2)), arachidonic acid (AA), 12-R-hydroxyeicosatetraenoic acid (12-R-HETE), 12-S-hydroxyheptadecatrienoic acid (12-S-HHTrE) and the internal standard prostaglandin B(1) (PGB(1)) were extracted from human platelets by liquid-liquid extraction using ethyl acetate. This was followed by derivatization and fluorescent detection prior to analysis by reversed phase liquid chromatography. The high-performance liquid chromatographic method consisted of ODS reversed-phase column (3 microm) and a mobile phase of acetonitrile-water (85:15). TXB(2) and AA plasma calibration curves were linear between 6.25 and 125 ng mL(-1) (r(2) > 0.997), whereas for 12-R-HETE and 12-S-HHTrE the curves were linear between 5.0 and 40 ng mL(-1) (r(2) > 0.998). All calibration curve standards had <15% CV (coefficient of variation) and between-run precision, and the percentage relative deviation for replicate (n = 6) quality controls was less than 5.5%. The method was adapted to allow the screening of drugs that may affect either one or both of the lipoxygenase and cyclo-oxygenase pathways.     

Liquid chromatographic high-throughput analysis of ketamine and its metabolites in human plasma using a monolithic silica column and solid phase extraction

A rapid and sensitive liquid chromatographic (LC) assay was developed for the simultaneous determination of ketamine (KE) and its two main metabolites, namely, norketamine (NK) and dehydronorketamine (DHNK) in human plasma. Each compound together with an internal standard (Labetalol) was extracted from the plasma matrix using solid phase extraction (SPE). The applicability of monolithic LC phases in the field of quantitative bioanalysis has been evaluated. The existing method with UV detection set at 220 nm was successfully transferred from a conventional reversed phase column to a 10 cm × 4.6 mm i.d. monolithic silica column. By simply increasing the mobile phase flow-rate, run times were about six-fold reduced and consumption of mobile phase were about two-fold decreased, while the chromatographic resolution of the analytes remain unaffected. The method was validated over the range 25-2000 ng/mL for KE, 25-1500 ng/mL for NK, and 15-750 ng/mL for DHNK. The method proved to be precise (within-run precision ranges from 2.2 to 7.2% and between-run precision ranges from 3.7 to 8.2%) and accurate (within-run accuracies ranged from 1.3 to 7.2% and between-run accuracies ranged from 1.5 to 8.7%). The mean absolute recoveries were 95.3, 96.9, and 103.9% for KE, NK and DHNK, respectively. The limit of quantitation (LOQ) and limit of detection (LOD) for KE and NK in human plasma were 25 and 12.5 ng/mL, respectively, and for DHNK were 15 and 7.5 ng/mL (S/N = 3). The assay should be suitable for use in routine determination of KE and its metabolites in human plasma.     

Development and validation of an improved method for the quantitation of sertraline in human plasma using LC-MS-MS and its application to bioequivalence studies

A rapid and sensitive LC-MS-MS method for the quantitation of sertraline in human plasma was developed and validated. Sertraline and the internal standard, telmisartan, were cleaned up by protein precipitation from 100 μL of plasma sample, and analyzed on a TC-C18 column (5 μm, 150 × 4.6 mm i.d.) using 70% acetonitrile and 30% 10 mM ammonium acetate (0.1% formic acid) as mobile phase. The method was demonstrated to be linear from 0.1 ng/mL to 50 ng/mL with the lower limit of quantitation of 0.1 ng/mL. Intra- and inter-day precision were below 4.40% and 3.55%. Recoveries of sertraline at low, medium, and high levels were 88.0 ± 2.3%, 88.2 ± 1.9%, and 90.0 ± 2.0%, respectively. The method was successfully applied to a bioequivalence study of sertraline after a single oral administration of 50 mg sertraline hydrochloride tablets.     

Development and validation of liquid chromatography–mass spectrometry method for the determination of telmisartan in human plasma

A sensitive liquid chromatographic–electrospray ionization mass spectrometric method was developed and validated for fast determination of telmisartan in human plasma. Plasma of 0.1 mL was deprotienated with methanol, centrifugation, evaporation to dryness and dissolving in mobile phase, samples were separated using a Hypersil-Keystone C18 reversed-phase column (150 mm × 2.1 mm i.d., 5 μm), together with a mobile phase containing of acetonitrile–10 mM ammonium acetate (42:58, v/v), 0.2% acetic acid and was isocratically eluted at a flow rate of 0.2 mL/min. Telmisartan and its internal standard, valsartan, were measured by electrospray ion source in positive selective ion monitoring mode. The method demonstrated linearity from 1 to 2000 ng/mL (r = 0.9988). The limit of quantification for telmisartan in plasma was 1 ng/mL with good accuracy and precision. The mean sample extract recovery of the method were higher than 82 and 78% for telmisartan and internal standard (IS), respectively. The within-run and between-run precision ranged from 3.4 to 8.9% and 5.9 to 11.2% (relative standard deviation, R.S.D.), respectively.     

On-line desalting and determination of morphine, morphine-3-glucuronide and morphine-6-glucuronide in microdialysis and plasma samples using column switching and liquid chromatography/tandem mass spectrometry

A sensitive and reproducible method for the determination of morphine and the metabolites morphine-3-glucuronide (M3G) and morphine-6-glucuronide (M6G) was developed. The method was validated for perfusion fluid used in microdialysis as well as for sheep and human plasma. A C18 guard column was used to desalt the samples before analytical separation on a ZIC HILIC (hydrophilic interaction chromatography) column and detection with tandem mass spectrometry (MS/MS). The mobile phases were 0.05% trifluoroacetic acid (TFA) for desalting and acetonitrile/5 mM ammonium acetate (70:30) for separation. Microdialysis samples (5 microL) were directly injected onto the system. The lower limits of quantification (LLOQ) for morphine, M3G and M6G were 0.50, 0.22 and 0.55 ng/mL, respectively, and the method was linear from LLOQ to 200 ng/mL. For plasma, a volume of 100 microL was precipitated with acetonitrile containing internal standards (deuterated morphine and metabolites). The supernatant was evaporated and reconstituted in 0.05% TFA before the desalting process. The LLOQs for sheep plasma were 2.0 and 3.1 ng/mL and the ranges were 2.0-2000 and 3.1-3100 ng/mL for morphine and M3G, respectively. For human plasma, the LLOQs were 0.78, 1.49 and 0.53 ng/mL and the ranges were 0.78-500, 1.49-1000 and 0.53-500 ng/mL for morphine, M3G and M6G, respectively.     

Development and validation of a GC-EI-MS method with reduced adsorption loss for the quantification of olanzapine in human plasma

A simple and sensitive GC-EI-MS method using solvent extraction and evaporation was developed for the determination of olanzapine concentrations in plasma samples. Because olanzapine and promazine, which was used as the internal standard (IS), are nitrogenous bases, they can adsorb to the weakly acidic silanol groups on the surfaces of glass centrifuge tubes during solvent extraction and evaporation. Silylation of the glass tubes, addition of triethylamine (TEA), and use of a sample solution with a basic pH could prevent adsorption loss. The extraction method involved mixing plasma (500 μL) in a silylated glass tube with a promazine solution (2 μg/mL, 25 μL) in methanol containing 1% TEA. After addition of aqueous sodium carbonate (0.5 mol/L, pH 11.1, 1 mL) and extraction into 3 mL of dichloromethane/n-hexane (1:1, v/v) containing 1% TEA, the organic phase was evaporated to dryness in a silylated glass tube. The residue was dissolved in ethyl acetate containing 1% TEA (50 μL). For GC-EI-MS analysis, the calibration curves of olanzapine in human plasma were linear from 0.5 to 100 ng/mL. Intra- and interday precisions in plasma were both less than 7.36% (coefficient of variation), and the accuracy was between 94.6 and 110% for solutions with concentrations greater than 0.5 ng/mL. The limit of quantification was 0.5 ng/mL in plasma. The assay was applied to therapeutic drug monitoring in samples from three schizophrenic patients.     

Liquid chromatography-mass spectrometric method for determination of the non-imidazole H3-receptor antagonist UPR1056 in rat plasma

The non-imidazole H3 receptor antagonist UPR1056 was dosed in plasma samples from rats individually administered with a single i.p. dose of 1.25 mg/kg by means of a newly validated HPLC-MS method. UPR1056 was extracted from rat plasma by protein precipitation with acetonitrile and was separated by linear gradient elution, employing water and methanol both additioned with 0.05% trifluoroacetic acid as mobile phases. UPR1056 was detected in MS using an electrospray ion source operating in positive ion mode. Acquisition was performed in single ion monitoring mode at m/z=349.3. The method was validated over the concentration range of 17.43-1743 ng/mL (50-5000 pmol/mL). Within- and between-run precision for the low, mid and high quality controls (QC) levels were 6.75% or less and accuracy ranged from 95.8 to 107.6%. The lower limit of quantification was 17.43 ng/mL. The analysis of the time course of UPR1056 concentrations over the 24-h period revealed a C(max) of 1147 ng/mL after 2 h from peripheral administration of the non-imidazole H(3)-receptor antagonist, with a prolonged elimination half-life of over 9 h.     

A rapid and sensitive high-performance liquid chromatography-electrospray ionization-triple quadrupole mass spectrometry method for the quantitation of oxycodone in human plasma

A sensitive method for the determination of oxycodone concentrations in plasma by high-performance liquid chromatography (HPLC)-electrospray ionization-triple quadrupole mass spectrometry is described. The method is rugged, reliable, selective, and rapid with a run time of 2 min. One milliliter of plasma is made basic and extracted with 2-mL duplicate portions of 2% isoamyl alcohol in n-butyl chloride. The combined extracts are then evaporated to dryness, reconstituted in 100 microL of the mobile phase (15% methanol-85% water containing 0.1% acetic acid), and injected onto the HPLC. The limit of quantitation is 1 ng/mL, and the estimated limit of detection is 33 pg/mL (signal-to-noise = 3). Standard curves are linear over the range of 1 to 100 ng/mL with all correlation coefficient values greater than 0.9989. The method is used to determine the concentration of oxycodone in human plasma following the intravenous infusion of doses ranging from 5 to 15 mg in which the analysis of over 3000 plasma samples is required.     

Determination of megestrol acetate and cyproterone acetate in serum of patients with advanced breast cancer by high-performance liquid chromatography

A reversed-phase high-performance liquid chromatographic method with ultraviolet detection of megestrol acetate and cyproterone acetate in human sera is described. The proposed assay is linear up to 1400 ng/ml (r = 0.999) and has a detection limit of 5 ng/ml. Recoveries of both compounds in spiked sera were ca. 95%; inter-assay coefficients of variation were 4.0 and 3.1% and intra-assay values were 1.3 and 1.4%, respectively. For validation of the method we also developed a gas chromatographic-mass spectrometric method for both steroids. The results obtained by the two methods showed good correlation: for megestrol acetate r = 0.98, n = 31, p less than 0.0001, and for cyproterone acetate r = 0.94, n = 0, p less than 0.0001. Large inter-individual differences in the serum concentrations of both substances were found in groups of patients with metastatic breast cancer receiving the same oral load of either steroid.     

Determination of oxymatrine in human plasma by LC-MS and study on its pharmacokinetics

A sensitive and selective liquid chromatographic-mass spectrometric method is built to determine oxymatrine in human plasma. After a liquid-liquid extraction for samples, samples are analyzed on a C18 column interfaced with a mass spectrometer. Positive electrospray ionization is employed as the ionization source. The mobile phase is methanol-water containing 10 mmol/L ammonium acetate (60:40) at the flow rate of 0.8 mL/min. The method is linear in the concentration range of 10-1000 ng/mL. The lower limit of quantitation is 10 ng/mL. The intra- and inter-day relative standard deviation across three validation runs over the entire concentration range is less than 14.27%. The accuracy determined at three concentrations (20, 100, and 500 ng/mL for oxymatrine) is within +/- 10.0% in terms of relative error. The method herein described is successfully applied to the evaluation of pharmacokinetic profiles of oxymatrine tablets pills in 18 healthy volunteers. The results show AUC, Tmax, Cmax, and T1/2 between the testing formulation and reference formulation have no significant difference (P > 0.05). Relative bioavailability is 104.2 +/- 13.8%.     

Simultaneous analysis of twenty-one glucocorticoids in equine plasma by liquid chromatography/tandem mass spectrometry

A method for the simultaneous separation, identification, quantification and confirmation of the presence of 21 glucocorticoids (GCC) in equine plasma by liquid chromatography coupled with triple stage quadrupole tandem mass spectrometry (LC/TSQ-MS/MS) is described. Plasma sample augmented with the 21 GCC was extracted with methyl tert-butyl ether (MTBE) and analyzed by positive electrospray ionization. Desoxymetasone or dichlorisone acetate was used as the internal standard (IS). Quantification was performed by IS calibration. For each drug, one major product ion was chosen and used for screening for that drug. Analyte confirmation was performed by using the three most intense product ions formed from the precursor ion and the corresponding mass ratios. The recovery of the 21 GCC when spiked into blank plasma at 5 ng/mL was 45-200% with coefficient of variation (CV) from 0.3-18%. The limit of detection (LOD) and that of quantification (LOQ) for most of the analytes were 50-100 pg/mL and 1 ng/mL, respectively, whereas that of confirmation (LOC) was 100-300 pg/mL depending on the analyte. Intra- and inter-day precisions expressed as CV for quantification of 1 and 10 ng/mL was 1.0-17%, and 0.51-19%, respectively, and the accuracy was from 84-110%. The linear concentration range for quantification was 0.1-100 ng/mL (r(2) > 0.997). Estimated measurement uncertainty was from 11-37%. This study was undertaken to develop a method for simultaneous screening, identification, quantification and confirmation of these agents in post-race equine plasma samples. The method has been successfully applied to screening of a large number of plasma samples obtained from racehorses in competition and in pharmacokinetic studies of dexamethasone in the horse and concurrent changes in endogenous GCC, hydrocortisone and cortisone. The method is simple, sensitive, selective and reliably reproducible.     

Determination of ranolazine in human plasma by liquid chromatographic-tandem mass spectrometric assay

A highly sensitive liquid chromatographic-tandem mass spectrometric method (LC-MS-MS) is developed to quantitate ranolazine in human plasma. The analyte and internal standard tramadol are extracted from plasma by liquid-liquid extraction using diethyl ether-dichloromethane (60:40 v/v), and separated on a Zorbax extend C(18) column using methanol-10mM ammonium acetate (60:40 v/v, pH 4.0) at a flow of 1.0 mL/min. Detection is carried out by multiple reaction monitoring on a QtrapTM LC-MS-MS system with an electrospray ionization interface. The assay is linear over the range 10-5000 ng/mL with a limit of quantitation of 10 ng/mL and a lower limit of detection (S/N > 3) of 1 ng/mL. Intra- and inter-day precision are < 3.1% and < 2.8%, respectively, and the accuracy is in the range 96.7-101.6%. The validated method is successfully used to analyze the drug in samples of human plasma for pharmacokinetic studies.     

High-performance liquid chromatography-electrospray ionization-mass spectrometric determination of emedastine difumarate in human plasma and its pharmacokinetics

A selective and sensitive method employing high-performance liquid chromatography (HPLC)-electrospray ionization (ESI)-mass spectrometry is developed and validated for the determination of emedastine difumarate in human plasma. With naphazoline hydrochloride as the internal standard, emedastine difumarate is extracted from plasma with ethyl acetate. The organic layer is evaporated, and the residue is redissolved in the mobile phase. An aliquot of 10 microL is chromatographically analyzed on a prepacked Phenomenex Luna 5u CN 100A (150 x 2.0-mm i.d.) column, using a mobile phase comprised of methanol-water (20 mM CH(3)COONH(4), pH 4.0) (80:20, v/v). Standard curves are linear (r(2) = 0.9990) over the concentration range of 0.05-30 ng/mL and had good accuracy and precision. The within- and between-batch precisions did not exceed 15% for the relative standard deviation. The lower limit of detection is 0.01 ng/mL. The validated HPLC-ESI-MS method is successfully used to study emedastine difumarate pharmacokinetics in 12 healthy volunteers.     

Determination of methadone and its metabolites by high performance liquid chromatography following solid-phase extraction in rat plasma.

This paper describes a rapid, quantitative liquid chromatographic analysis and extraction of methadone and its two major metabolites from rat plasma, using difenoxin as the internal standard. Using a C18 column, resolution of all sample components and the internal standard is achieved with a mobile phase of 25:75 acetonitrile-0.08% diethylamine in 1000 mL water, pH 2.3, at a flow rate of 1.5 mL/min. The injection volume is 100 microL. Standards are linear over the range 25-100 ng, with a lower limit of detection for methadone of 0.25 ng. Within- and between-run coefficients of variation (CV) are 1.24% and 2.94%, respectively. Extraction of methadone and its metabolites from rat plasma uses a solid-phase extraction technique that is highly efficient. Extraction efficiencies of 90.3%, 99.6%, 85.9% and 93.8% were achieved for methadone, its primary and secondary metabolites, and difenoxin, respectively.     

A reversed-phase high-performance liquid chromatographic method for the determination of zafirlukast in pharmaceutical formulations and human plasma

Zafirlukast (ZAF) is a leukotriene receptor antagonist used in the treatment of chronic asthma. In this study, a simple and sensitive reversed-phase, high-performance liquid chromatographic method was developed for the determination of ZAF in pharmaceutical formulations and human plasma. Piribedil was used as an internal standard. Analysis was carried out on a Nucleosil C18 100 A (150 mm x 4.6 mm id, 5 Vm) column with acetonitrile-pH 3.0 acetate buffer (70 + 30, v/v) as the mobile phase at a flow rate of 0.8 mL/min. The peak was detected by an ultraviolet detector set at a wavelength of 240 nm. The retention times were about 3.9 min for piribedil and 5.8 min for ZAF. The developed method was applied to the determination of ZAF in its pharmaceutical formulation and spiked human plasma. For quantification of ZAF in spiked plasma, proteins were precipitated with ethanol before chromatographic analysis. The calibration range was linear from 49.69-437.50 ng/mL in spiked plasma. The absolute recovery from spiked plasma was 98.73 +/- 0.42% at a concentration of 254.78 ng/mL of ZAF. No endogenous substances from plasma were found to interfere.     

Validated LC-MS/MS assay for the quantitative determination of nalbuphine in human plasma and its application to a pharmacokinetic study

A solid‐phase extraction–liquid chromatographic–tandem mass spectrometry method for the determination of nalbuphine concentrations in human plasma has been developed. Samples (1 mL) were extracted using a Strata™‐X solid phase extraction cartridges. Chromatographic separation of nalbuphine and naloxone (internal standard) was achieved on a Phenomenex Kinetex PFP (2.6 μm, 100 A, 100 × 2.1 mm) column using a mobile phase consisting of 0.1% formic acid, 15 mM ammonium acetate in deionized water and acetonitrile (60:40, v/v). The flow rate was 0.3 mL/min and the total run time was 2 min. Detection of the analytes was achieved using positive ion electrospray ionization via multiple reactions monitoring mode. The mass transitions were m/z 358 → 340 for nalbuphine and m/z 328 → 310 for naloxone. The assay was linear over the concentration range 0.50–500.00 ng/mL, with correlation coefficients ≥0.995. The lower limit of quantitation was set at 0.5 ng/mL plasma based on an average signal‐to‐noise ratio of 44.79. The intra‐ and inter‐day precision was less than 8.07% in terms of relative standard deviation and accuracy ranged from 94.97 to 106.29% at all quality control levels. The method was applied successfully to determine nalbuphine concentrations in human plasma samples obtained from subjects receiving intravenous administration of nalbuphine. The method is rapid, sensitive, selective and directly applicable to human pharmacokinetic studies involving nalbuphine. Copyright © 2011 John Wiley & Sons, Ltd.     

Liquid chromatography/electrospray ionization tandem mass spectrometry for the quantification of mitiglinide in human plasma: validation and its application to pharmacokinetic studies

A sensitive and specific method was developed and validated for the determination of mitiglinide in human plasma using liquid chromatographic separation with electrospray ionization tandem mass spectrometric detection. Acidified plasma samples were extracted with ethyl acetate. The chromatographic separation was performed on an Agilent Zorbax Eclipse Plus C(18) column with a mobile phase of methanol-10 mm ammonium acetate solution at a flow rate of 0.3 mL/min. Analytes were detected with an Agilent 6410 Triple qudrupole mass spectrometer equipped with an electrospray ionization source in positive multiple reaction monitoring mode: m/z 316.2 (precursor ion) to 298.2 (product ion) for mitiglinide and m/z 318.2 (precursor ion) to 120.2 (product ion) for the internal standard. This method was validated over a linear range of 0.5-4000 ng/mL for mitiglinide in human plasma. The lower limit of quantification (LLOQ) was 0.5 ng/mL, while a relative standard deviation (RSD) was less than 3.9%. The intra- and inter-run precision (as RSD, %) obtained from three validation runs were all less than 15%. The validated method was successfully used to analyze human plasma samples for application in pharmacokinetic studies.     

Determination of roxatidine in human plasma by liquid chromatography/electrospray mass spectrometry and application to a clinical pharmacokinetic study

A rapid and sensitive liquid chromatography/mass spectrometry (LC/MS) method was developed and validated for the determination of roxatidine in human plasma. Roxatidine was extracted by single liquid-liquid extraction with tert-butyl methyl ether, and the chromatographic separation was performed on a C8 column. The total analytical run time was relatively short (5 min), and the limit of assay quantification was 2 ng/mL using 0.1 mL of human plasma. Roxatidine and the internal standard, propranolol, were monitored in selected ion monitoring (SIM) mode at m/z 307.3 and 260.3, respectively. The standard curve was linear over a concentration range from 2-500 ng/mL, and the correlation coefficients were >0.999. The mean intra- and inter-day assay accuracy ranged from 103.4-108.8% and 102.3-110.0%, respectively, and the mean intra- and inter-day precision was between 3.3-8.8% and 5.3-6.2%, respectively. The developed assay method was successfully applied to a pharmacokinetic study in human volunteers after oral administration of roxatidine acetate hydrochloride at a dose of 75 mg.