Detection and pharmacokinetics of alginate oligosaccharides in mouse plasma and urine after oral administration by a liquid chromatography/tandem mass spectrometry (LC-MS/MS) method

A sensitive and simple liquid chromatography/tandem mass spectrometry (LC-MS/MS) method was developed for the detection of alginate oligosaccharides (AOs) in mouse plasma and urine after oral administration. In an AO mixture, dimer, trimer, and tetramer were detected by LC-MS/MS equipped with an anion-exchange column with extremely high sensitivity. By this method, we detected certain levels of AOs in samples prepared from mouse plasma and urine after a single oral administration of the AO mixture. Based on a calibration curve made with an AO trimer peak area as a standard, the maximum plasma and urine concentrations of AOs were estimated to be 24.5 microg/ml at 5 min and 425.5 microg/ml at 30 min, respectively. These results suggest that the LC-MS/MS method is well suited to pharmacokinetic analysis of AOs in an in vivo system, and that some of orally administered AOs, at least from dimer to tetramer, are absorbed by digestive organs promptly, and that unaltered, these oligomers were excreted into an urine after a single oral administration to a mouse.     

In vivo rat metabolism and pharmacokinetic studies of ginsenoside Rg3

Metabolism of an anti-tumor active component of Panax ginseng, ginsenoside (20R)-Rg(3), was studied for better understanding its pharmacokinetics in rat. LC-MS was used to determine Rg(3) and its metabolites in rat plasma, urine and feces samples. An average half-life of 18.5 min was obtained after the ginsenoside was intravenously dosed at 5 mg/kg. However, Rg(3) was not detected in rat plasma collected after oral administration at 100 mg/kg. Only 0.97-1.15% Rg(3) of the dosed amount was determined in feces. Hydrolysis and oxygenated metabolites were detected and identified in feces collected after oral administration by using LC-MS and MS-MS.     

Rapid and sensitive liquid chromatography-tandem mass spectrometry: assay development, validation and application to a human pharmacokinetic study

Rasagiline is a highly potent, selective and irreversible second-generation monoamine oxidase inhibitor with selectivity for type B of the enzyme (MAO-B). The present studies aimed at developing and validating a rapid and sensitive liquid chromatography-tandem mass spectrometric (LC-MS/MS) method for determination of rasagiline in human plasma and urine. LC-MS/MS analysis was carried out on a Finnigan LC-TSQ Quantum mass spectrometer using positive ion electrospray ionization (ESI(+)) and selected reaction monitoring (SRM). The assay for rasagiline was linear over the range of 0.01-40 ng/mL in plasma and 0.025-40 ng/mL in urine. It took 5.5 min to analyze a sample. The average recoveries in plasma and urine samples were both >85%. The RSD of precision and bias of accuracy were less than 15% and 10%, respectively, of their nominal values based on the intra- and inter-day analysis. The developed method was proved to be suitable for use in clinical pharmacokinetic study after single oral administration of 0.5, 1 and 2 mg rasagiline mesylate tablets in healthy Chinese volunteers.     

Liquid chromatography-electrospray ionization ion trap mass spectrometry for analysis of mesocarb and its metabolites in human urine

A method is described for the determination of metabolites of mesocarb in human urine by combining gradient liquid chromatography and electrospray ionization (ESI)-ion trap mass spectrometry. Seven metabolites (two isomers of hydroxymesocarb, p-hydroxymesocarb, two isomers of dihydroxymesocarb and two isomers of trihydroxymesocarb) and parent drug were detected in human urine after the administration of a single oral dose 10 mg of mesocarb (Sydnocarb, two tablets of 5 mg). Various extraction techniques (free fraction, enzyme hydrolyses and acid hydrolyses) and their comparison were carried out for investigation of the metabolism of mesocarb. After extraction procedure the residue was dissolved in methanol and injected into the column HPLC (Zorbax SB-C18 (Narrow-Bore 2.1 x 150 mm i.d., 5 microm particles)) with mobile phase (0.2 ml/min) of methanol/0.2 mM ammonium acetate. Conformation of the results and identification of all metabolites are performed by LC-MS and LC-MS/MS. The major metabolites of mesocarb in urine of the human were p-hydroxylated derivative of the phenylcarbamoyl group of the parent drug (p-hydrohymesocarb) and dihydroxylated derivative of mesocarb (two isomers of dihydroxymesocarb). This analytical method for dihydrohymesocarb was very sensitive for discriminating the ingestion of mesocarb longer than the parent drug or other metabolites in human urine. The dihydroxymesocarb was detected in urine until 168-192 h after administration of the drug.     

Liquid chromatography-tandem mass spectrometry analysis of anisodamine and its phase I and II metabolites in rat urine

A sensitive and specific method for the analysis of anisodamine and its metabolites in rat urine by liquid chromatography-electrospray ionization tandem mass spectrometry (LC-MS/MS) was developed. Various extraction techniques (free fraction, acid hydrolyses and enzyme hydrolyses) and their comparison were carried out for investigation of the metabolism of anisodamine. After extraction procedure the pretreated samples were injected on a reversed-phase C18 column with mobile phase (0.2 ml/min) of methanol/0.01% triethylamine solution (adjusted to pH 3.5 with formic acid) (60:40, v/v) and detected by MS/MS. Identification and structural elucidation of the metabolites were performed by comparing their changes in molecular masses (DeltaM), retention-times and full scan MS(n) spectra with those of the parent drug. At least 11 metabolites (N-demethyl-6beta-hydroxytropine, 6beta-hydroxytropine, tropic acid, N-demethylanisodamine, hydroxyanisodamine, anisodamine N-oxide, hydroxyanisodamine N-oxide, glucuronide conjugated N-demethylanisodamine, sulfate conjugated and glucuronide conjugated anisodamine, sulfate conjugated hydroxyanisodamine) and the parent drug were found in rat urine after the administration of a single oral dose 25mg/kg of anisodamine. Hydroxyanisodamine, anisodamine N-oxide and the parent drug were detected in rat urine for up 95 h after ingestion of anisodamine.     

Determination of the active metabolite of prulifloxacin in human plasma by liquid chromatography-tandem mass spectrometry

A liquid chromatographic-tandem mass spectrometric method (LC-MS/MS) for the determination of ulifloxacin, the active metabolite of prulifloxacin, in human plasma is described. After sample preparation by protein precipitation with methanol, ulifloxacin and ofloxacin (internal standard) were chromatographically separated on a C(18) column using a mobile phase consisting of methanol, water and formic acid (70:30:0.2, v/v/v) at a flow rate of 0.5 ml/min and then were detected using MS/MS by monitoring their precursor-to-product ion transitions, m/z 350-->m/z 248 for ulifloxacin and m/z 362-->m/z 261 for ofloxacin, in selected reaction monitoring (SRM) mode. Positive electrospray ionization was used for the ionization process. The linear range was 0.025-5.0 microg/ml for ulifloxacin with a lower limit of quantitation of 0.025 microg/ml. Within- and between-run precision was less than 6.6 and 7.8%, respectively, and accuracy was within 2.0%. The recovery ranged from 92.1 to 98.2% at the concentrations of 0.025, 0.50 and 5.0 microg/ml. Compared with the reported LC method, the present LC-MS/MS method can directly determine the ulifloxacin in human plasma without any need of derivatization. The present method has been successfully used for the pharmacokinetic studies of a prulifloxacin formulation product after oral administration to healthy volunteers.     

Validated LC-MS/MS methods for the determination of risperidone and the enantiomers of 9-hydroxyrisperidone in human plasma and urine

Two liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) methods are described, one for the quantitative determination of risperidone and the enantiomers of its active metabolite 9-hydroxyrisperidone (paliperidone) in human plasma and the other for the determination of the enantiomers of 9-hydroxyrisperidone in human urine. The plasma method is based on solid-phase extraction of 200 microl of sample on a mixed-mode sorbent, followed by separation on a cellulose-based LC column with a 13.5-min mobile phase gradient of hexane, isopropanol and ethanol. After post-column addition of 10 mM ammonium acetate in ethanol/water, detection takes place by ion-spray tandem mass spectrometry in the positive ion mode. Method validation results show that the method is sufficiently selective towards the enantiomers of 7-hydroxyrisperidone and capable of quantifying the analytes with good precision and accuracy in the concentration range of 0.2-100 ng/ml. An accelerated (run time of 4.3 min) and equally valid method for the enantiomers of 9-hydroxyrisperidone alone in plasma is obtained by increasing the mobile phase flow-rate from 1.0 to 2.0 ml/min and slightly adapting the gradient conditions. The urine method is based on the same solid-phase extraction and chromatographic approach as the accelerated plasma method. Using 100 microl of sample, (+)- and (-)-9-hydroxyrisperidone can be quantified in the concentration range 1-2000 ng/ml. The accelerated method for plasma and the method for urine can be used only when paliperidone is administered instead of risperidone, as there is insufficient separation of the 9-hydroxy enantiomers from the 7-hydroxy enantiomers, the latter ones being present only after risperidone administration.     

LC-MS/MS characterization of the urinary excretion profile of the mycotoxin deoxynivalenol in human and rat

The understanding of mycotoxins transfer to biological fluids is challenged by the difficulties in performing and replicating in vivo experiments as well as the lack of suitable methods of analysis to detect simultaneously a range of chemically different metabolites at trace levels. LC-MS/MS has been used herein to study the urinary excretion profile of the mycotoxin deoxynivalenol in human and Wistar rat. Deoxynivalenol and deoxynivalenol glucuronide were found in both human and rat urines, whereas de-epoxydeoxynivalenol and its glucuronide conjugate were only detected in rat urine. The presence of two deoxynivalenol glucuronide isomers in Wistar rat urine has been shown for the first time. Structure confirmation of the detected metabolites was provided by the analysis of fragmentation patterns. A solid phase extraction clean up procedure allowing recoveries in the range 72-102% for deoxynivalenol, de-epoxydeoxynivalenol, and their glucuronide conjugates was optimized. A multiple reaction monitoring method for the simultaneous determination of all investigated metabolites was elaborated allowing the direct detection of deoxynivalenol metabolites without the hydrolysis step. Deoxynivalenol urinary levels in the range 0.003-0.008 μg/ml were detected in healthy human subjects, whereas deoxynivalenol and de-epoxynivalenol levels between 1.9-4.9 μg/ml and 1.6-5.9 μg/ml, respectively were found in administered rat urine. These findings emphasize the relevance of the highly selective and sensitive LC-MS/MS technique for the direct detection and characterization of deoxynivalenol metabolites in complex biological matrices.     

An efficient method for the purification and quantification of a galactose-specific lectin from vegetative tissues of Dolichos lablab

The differences among individual bile acids (BAs) in eliciting different physiological and pathological responses are largely unknown because of the lack of valid and simple analytical methods for the quantification of individual BAs and their taurine and glycine conjugates. Therefore, a simple and sensitive LC-MS/MS method for the simultaneous quantification of 6 major BAs, their glycine, and taurine conjugates in mouse liver, bile, plasma, and urine was developed and validated. One-step sample preparation using solid-phase extraction (for bile and urine) or protein precipitation (for plasma and liver) was used to extract BAs. This method is valid and sensitive with a limit of quantification ranging from 10 to 40 ng/ml for the various analytes, has a large dynamic range (2500), and a short run time (20 min). Detailed BA profiles were obtained from mouse liver, plasma, bile, and urine using this method. Muricholic acid (MCA) and cholic acid (CA) taurine conjugates constituted more than 90% of BAs in liver and bile. BA concentrations in liver were about 300-fold higher than in plasma, and about 180-fold higher in bile than in liver. In summary, a reliable and simple LC-MS/MS method to quantify major BAs and their metabolites was developed and applied to quantify BAs in mouse tissues and fluids.     

Validation and implementation of a liquid chromatography/tandem mass spectrometry assay for quantitation of the total and unbound RO4929097, a γ-secretase inhibitor targeting Notch signaling, in human plasma

A reversed-phased liquid chromatography with tandem mass spectrometry (LC-MS/MS) method was developed and validated for quantitation of the total and unbound RO4929097, a γ-secretase inhibitor targeting Notch signaling, in human plasma. Sample preparation involved a liquid-liquid extraction with ethyl acetate. Chromatographic separation was achieved on a Waters X-Terra? MS C(18) column with an isocratic mobile phase consisting of methanol/0.45% formic acid in water (60:40, v/v) running at a flow rate of 0.2 ml/min for 6 min. The lower limits of quantitation (LLOQs) were 5 ng/ml for the total RO4929097 in plasma and 0.5 ng/ml for the unbound drug in phosphate buffer solution (PBS). Calibration curves were linear over RO4929097 concentration range of 5-2000 ng/ml in plasma for the total drug and 0.5-200 ng/ml in PBS for the unbound drug. The intra-day and inter-day accuracy and precision were within the generally accepted criteria for bioanalytical method (<15%). The method has been successfully employed to characterize the total and unbound plasma pharmacokinetics of RO4929097 after its oral administration in cancer patients.     

Development and validation of a LC-MS/MS method for the determination of viaminate in human plasma

A sensitive and specific method for determination of viaminate in human plasma by using high-performance liquid chromatography coupled with electrospray tandem mass spectrometry (LC-MS/MS) was developed in this study. The plasma samples were simply deproteinated, extracted, evaporated, and then reconstituted in 200 microl of methanol prior to analysis. Chromatographic separation was carried out on a Shimadzu VP-ODS column (250 mm x 2.0 mm, 5 microm) with a mobile phase of methanol-water (95:5, v/v) at a flow rate of 0.2 ml/min. Quantification was performed in the negative-ion electrospray ionization mode by selected ion monitoring of the product ions at m/z 164 for viaminate and m/z 109 for testosterone propionate which was used as the internal standard. The corresponding parent ions were m/z 446 and m/z 345. A linear calibration curve was observed within the concentration range of 0.10-200 ng/ml. The lowest limit of quantitation (LLOQ) was 0.1 ng/ml. The extraction-efficiency at three concentrations was 100.7, 93.6, and 99.7%. Practical utility of this new LC-MS/MS method was confirmed in pilot pharmacokinetic studies in humans following oral administration.     

Development of a novel LC-MS/MS method for the determination of letosteine in human plasma and its application on pharmacokinetic studies

Letosteine has been found to be effective in treating patients with chronic bronchopneumopathies in clinical practice. To provide robust support for its pharmacokinetic and clinical studies, a rapid and sensitive method based on liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) was developed and validated for the analysis of letosteine in plasma samples. After protein precipitation, the plasma samples were separated on a reversed-phase C(18) column in less than 1.5 min. The LC-MS/MS system was performed in the positive ion multiple-reaction-monitoring (MRM) mode to produce intensive product ions of m/z 280.1→160.0 for letosteine and m/z 248.1→121.1 for the internal standard, tinidazole. The method was found to have excellent linearity (r ≥ 0.9974), precision (RSD ≤ 5.83%), extraction recovery (71.8-73.0%) and stability (RE, -8.45% to 9.03%) over a concentration range of 0.1140-152.0 μgL(-1). Compared to the previous published radioactive method, LC-MS/MS method showed many advantages including shorter analysis time, simpler preparation procedure, increased sensitivity as well as lower safety risks. In addition, this method was successfully applied to study the pharmacokinetics of letosteine following a single and multiple dose oral administration in Chinese healthy volunteers.     

Determination of amiodarone and desethylamiodarone in horse plasma and urine by high-performance liquid chromatography combined with UV detection and electrospray ionization mass spectrometry

A rapid method for the quantification of amiodarone and desethylamiodarone in animal plasma using high-performance liquid chromatography combined with UV detection (HPLC-UV) is presented. The sample preparation includes a simple deproteinisation step with acetonitrile. In addition, a sensitive method for the quantification of amiodarone and desethylamiodarone in horse plasma and urine using high-performance liquid chromatography combined with electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) is described. The sample preparation includes a solid-phase extraction (SPE) with a SCX column. Tamoxifen is used as an internal standard for both chromatographic methods. Chromatographic separation is achieved on an ODS Hypersil column using isocratic elution with 0.01% diethylamine and acetonitrile as mobile phase for the HPLC-UV method and with 0.1% formic acid and acetonitrile as mobile phase for the LC-MS/MS method. For the HPLC-UV method, good linearity was observed in the range 0-5 microg ml(-1), and in the range 0-1 microg ml(-1) for the LC-MS/MS method. The limit of quantification (LOQ) was set at 50 and 5 ng ml(-1) for the HPLC-UV method and the LC-MS/MS method, respectively. For the UV method, the limit of detection (LOD) was 15 and 10 ng ml(-1) for amiodarone and desethylamiodarone, respectively. The LODs of the LC-MS/MS method in plasma were much lower, i.e. 0.10 and 0.04 ng ml(-1) for amiodarone and desethylamiodarone, respectively. The LODs obtained for the urine samples were 0.16 and 0.09 ng ml(-1) for amiodarone and desethylamiodarone, respectively. The methods were shown to be of use in horses. The rapid HPLC-UV method was used for therapeutic drug monitoring after amiodarone treatment, while the LC-MS/MS method showed its applicability for single dose pharmacokinetic studies.     

Development of an LC-MS/MS method for the simultaneous quantification of aripiprazole and dehydroaripiprazole in human plasma

A selective, sensitive, and accurate liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the simultaneous determination of aripiprazole and its active metabolite dehydroaripiprazole in human plasma has been developed using papaverine as internal standard (IS). LC-MS/MS analysis was carried out on a Finnigan LC-TSQ Quantum mass spectrometer using positive ion electrospray ionization (ESI⁺) and selected reaction monitoring (SRM). The assays for aripiprazole and dehydroaripiprazole were linear over the ranges of 0.1 to 600 ng/ml and 0.01 to 60 ng/ml, respectively. The average recoveries in plasma samples both were better than 85%. The intra- and interrun precision and accuracy values were found to be within the assay variability criteria limits according to the US Food and Drug Administration guidelines. The developed method was proved to be suitable for use in a clinical pharmacokinetic study after a single oral administration of a 5-mg aripiprazole tablet in healthy Chinese volunteers.     

Sensitive semi-microcolumn high-performance liquid chromatographic method for the determination of DU-6681, the active parent drug of a new oral carbapenem antibiotic,DZ-2640, in human plasma and urine using a column-switching system as sample clean-up procedure

DZ-2640 is a new oral carbapenem antibiotic having a dihydro-pyrroloimidazole ring as a side chain and a pivaloyloxymethyl (POM) ester prodrug of DU-6681, the active parent compound. A simple and sensitive column-switching semi-microcolumn high-performance liquid chromatographic method for the determination of DU-6681 in human plasma and urine has been developed. Human plasma was diluted with an equal volume of 1 M MOPS buffer (pH 7.0) and the mixture was filtered through an Ultrafree C3GV. The resulting filtrate was injected without further cleanup onto the HPLC system. Human urine was diluted with an equal volume of 1 M MOPS buffer (pH 7.0) and the mixture was directly injected onto the HPLC system. The analyte was detected by monitoring the column effluent with UV light at a wavelength of 300 nm, which resulted in the limit of quantitation of 0.008 μg/ml of plasma and 0.32 μg/ml of urine. Calibration curves were linear in the range of 0.008 to 5.85 μg/ml in plasma and 0.32 to 104.4 μg/ml in urine. The present methods showed greatly increased sensitivity for DU-6681 compared to conventional HPLC methods and also showed satisfactory recovery, selectivity, precision, and accuracy. Stability studies showed that 1 M MOPS buffer (pH 7.0) acted as a stabilizer. In plasma and urine diluted with equal volume of the buffer, DU-6681 showed good stability at −80°C for up to 4 weeks with no significant loss of the drug.     

A LC-MS/MS method for the specific, sensitive, and simultaneous quantification of 5-aminolevulinic acid and porphobilinogen

Accurate determinations of 5-aminolevulinic acid (ALA) and porphobilinogen (PBG) in physiologic fluids are required for the diagnosis and therapeutic monitoring of acute porphyrias. Current colorimetric methods are insensitive and over-estimate ALA and PBG due to poor specificity, while LC-MS/MS methods increase sensitivity, but have limited matrices. An LC-MS/MS method was developed to simultaneously determine ALA and PBG concentrations in fluids or tissues which were solid phase extracted, butanol derivatized, and quantitated by selective reaction monitoring using (13)C(5), (15)N-ALA and 2,4-(13)C(2)-PBG internal standards. ALA was separated from interfering compounds on a reverse phase C8-column. For ALA and PBG, the matrix effects (87.3-105%) and process efficiencies (77.6-97.8% and 37.2-41.6%, respectively) were acceptable in plasma and urine matrices. The assay was highly sensitive for ALA and PBG (LLOQ=0.05 μM with 25 μL urine or 100 μL plasma), and required ～4 h from extraction to results. ALA and PBG accuracy ranged from 88.2 to 110% (n=10); intra- and inter-assay coefficients of variations were <10% for urine and plasma. In clinical applications, patients with mutation-confirmed acute porphyrias had normal to slightly increased urinary ALA and PBG levels when asymptomatic, and high levels during acute attacks, which decreased with hemin therapy. In AIP mice, baseline ALA and PBG levels in urine, plasma, and liver were increased after phenobarbital induction 28-/63-, 42-/266-, and 13-/316-fold, respectively. This LC-MS/MS method is rapid, specific, highly sensitive, accurate, and simultaneously measures ALA and PBG in urine, plasma, and tissues permitting porphyria clinical diagnoses, therapeutic monitoring, and research.     

High performance liquid chromatographic method for the determination of cetirizine and ambroxol in human plasma and urine--a boxcar approach

A column switching high performance liquid chromatographic method with estimable sensitivity and accuracy was developed for the determination of cetirizine and ambroxol in human plasma using nebivolol as the internal standard. Plasma samples were prepared by liquid-liquid extraction in methylene chloride and a mixture of diethylether (80:20, v/v). The extracted samples were injected into a multifunctional clean-up column Supelcosil LCABZ (50 mm × 4.6 mm, 5 μm particle size) using mobile phase 1 comprising acetonitrile-phosphate buffer (pH 3.5; 20 mM) (20:80, v/v). The eluate of cetirizine and ambroxol were separated to an analytical Kromasil C(8) micro bore column (50 mm × 0.3 mm, 5 μm particle size) via a column switching device. A Kromasil C(18) analytical column (250 mm × 2.1 mm, 5 μm particle size) was used as a separation column. Mobile phase 2 consisting acetonitrile-triethylamine (0.5%) in phosphate buffer (pH 3.5; 20mM) (55:45, v/v) was used for the compound elution. The eluents were detected at 230 nm with photodiode array detector. An aliquot of 150 μl of plasma sample was introduced into the pretreatment column via the auto sampler using mobile phase 1 at a flow rate of 0.5 ml/min, column switching valve being positioned at A. The pretreatment column retained cetirizine, ambroxol and nebivolol (IS) in the column leaving the residual proteins of plasma eluted in void volume and drained out. The switching valve was shifted to position B at 7.5 min. Cetirizine, ambroxol and IS were eluted from the pretreatment column between 7. 5 and 11.5 min and introduced to the concentration column. Finally, cetirizine, ambroxol and IS were introduced to the separation column by switching valve using mobile phase 2 at a flow rate of 0.4 ml/min. During the analysis the pretreatment column was washed for the next analysis and resume to the position A. The total run time was 25 min for a sample. The procedure was repeated for urine analysis also. The method was linear from 2 to 450 ng/ml and 7-300 ng/ml for cetirizine and ambroxol respectively in plasma and 1-500 ng/ml and 5-400 ng/ml, respectively for cetirizine and ambroxol in urine. Intra-day and inter-day precision of cetirizine and ambroxol was below 15% in terms of coefficient of variation and accuracy of cetirizine and ambroxol was ranged from 94 to 101.6% and 91.1 to 100.2%, respectively. The method demonstrated high sensitivity and selectivity and therefore, applied to evaluate pharmacokinetics of cetirizine and ambroxol in healthy human volunteer after a single oral administration. Urine samples obtained from healthy human volunteers and clinical subjects with renal impairment have also been analyzed by the method to compare the elimination pattern. The method was precise and accurate for the estimation of cetirizine and ambroxol both in blood and in urine.     

Simultaneous determination of three Panax notoginseng saponins at sub-nanograms by LC-MS/MS in dog plasma for pharmacokinetics of compound Danshen tablets

Compound Danshen tablets are composed of Panax notoginseng, Salvia miltiorrhiza and Borneol. The tablets are prescribed for treatment of cardiovascular diseases in China. The present study aimed at developing a specific and sensitive LC-MS/MS method to simultaneously determine three bioactive P. notoginseng saponins, i.e., notoginsenoside R1, ginsenoside Rg1 and Rb1, in dogs after a single oral administration of the compound tablets in order to obtain the clinically relevant saponin-related pharmacodynamics of the tablets in patients. The R1, Rg1 and Rb1 were extracted from dog plasma with acetone-methanol (80:20, v/v), separated by reversed phase liquid chromatography and determined by tandem mass spectrometry (LC-MS/MS) with positive electrospray ionization (ESI). The developed method reached lower limit of quantitation (LLOQ) at 0.10 ng/ml for the three saponins. The method was validated in terms of selectivity, matrix effects, linearity, precision and accuracy, and then was applied to a pharmacokinetic study of the three bioactive saponins simultaneously in dogs after a single oral administration of compound Danshen tablets at a clinical equivalent dose. The C(max) and AUC((0-∞)) for R1, Rg1 and Rb1 were 1.91, 3.34 and 28.6 ng/ml, and 7.5, 11.0, and 1712 (h ng/ml), respectively.     

Sensitive analytical method for the novel H1-receptor antagonist HSR-609 in human plasma and urine by high-performance liquid chromatography with pre-column fluorescent derivatization

A simple and sensitive method for quantitation of HSR-609 (I) in human plasma and urine was developed using HPLC with the fluorescence labelling reagent 4-(N,N-dimethylaminosulfonyl)-7-N-piperazino-2,1,3-benzoxadiazole (DBD-PZ). Compound I was extracted from human plasma and urine, and derivatized by reaction with DBD-PZ in the presence of Mukaiyama reagent A, an equimolar solution of 2,2′-dipyridyl disulfide (DPDS) and triphenylphosphine (TPP) in acetonitrile. The reaction mixture was cleaned up by liquid-liquid extraction following the derivatization. The conjugate was analyzed by ion-pair HPLC with fluorometric detection. The quantitation limits for I were 0.5 ng/ml in plasma and 5 ng/ml in urine. Using this method, plasma concentration and urinary excretion of I were studied after oral administration of I to human volunteers.     

A rapid and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the determination of hydroxysafflor yellow A in human plasma: application to a pharmacokinetic study

A sensitive and specific liquid chromatography-tandem mass spectrometry (LC-MS/MS) method has been developed and validated for the determination of hydroxysafflor yellow A (HSYA) in human plasma. HSYA was extracted from human plasma by using solid-phase extraction technique. Puerarin was used as the internal standard. A Shim-pack VP-ODS C(18) (150mm x 4.6mm, 5 microm) column and isocratic elution system composing of methanol and 5mM ammonium acetate (80:20, v/v) provided chromatographic separation of analytes followed by detection with mass spectrometry. The mass transition ion-pair was followed as m/z 611.19-->491.19 for HSYA and m/z 415.19-->295.10 for puerarin. The proposed method has been validated with a linear range of 1-1000 ng/ml for HSYA with a correlation coefficient >/=0.999. The lower limit of quantitation was 1 ng/ml. The intra-batch and inter-batch precision and accuracy were within 10%. The average extraction recovery was 81.7%. The total run time was 5.5 min. The validated method was successfully applied to the study on pharmacokinetics of HSYA in 12 healthy volunteers after a single oral administration of safflower oral solution containing 140 mg of HSYA.