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The authors in the current work suggested the potential repurposing of omarigliptin (OMR) for neurodegenerative diseases based on three new findings that support the preliminary finding of crossing BBB after a single dose study in the literature. The first finding is the positive results of the docking study with the crystal structures of A 2A adenosine (A2AAR) and acetylcholine esterase (AChE) receptors. A2AAR is a member of non-dopaminergic GPCR superfamily receptor proteins and has essential role in regulation of glutamate and dopamine release in Parkinson's disease while AChE plays a major role in Alzheimer's disease as the primary enzyme responsible for the hydrolytic metabolism of the neurotransmitter acetylcholine into choline and acetate. Docking showed that OMR perfectly fits into A2AAR binding pocket forming a distinctive hydrogen bond with Threonine 256. Besides other non-polar interactions inside the pocket suggesting the future of the marketed anti-diabetic drug (that cross BBB) as a potential antiparkinsonian agent while OMR showed perfect fit inside AChE receptor binding site smoothly because of its optimum length and the two fluorine atoms that enables quite lean fitting. Moreover, a computational comparative study of OMR docking, other 12 DPP-4 inhibitors and 11 SGLT-2 inhibitors was carried out. Secondly, glucagon-like peptide-1 (GLP-1) concentration in rats' brain tissue was determined by the authors using sandwich GLP-1 ELISA kit bio-analysis to ensure the effect of OMR after the multiple doses' study. Brain GLP-1 concentration was elevated by 1.9-fold following oral multiple doses of OMR (5 mg/kg/day, p.o. for 28 days) as compared to the control group. The third finding is the enhanced BBB crossing of OMR after 28 days of multiple doses that had been studied using LC -MS/MS method with enhanced liquid-liquid extraction. A modified LC-MS/MS method was established for bioassay of OMR in rats' plasma (10-3100 ng/mL) and rats' brain tissue (15-2900 ng/mL) using liquid-liquid extraction. Alogliptin (ALP) was chosen as an internal standard (IS) due to its LogP value of 1.1, which is very close to the LogP of OMR. Extraction of OMR from samples of both rats' plasma and rats' brain tissue was effectively achieved with ethyl acetate as the extracting solvent after adding 1N sodium carbonate to enhance the drug migration, while choosing acetonitrile to be the diluent solvent for the IS to effectively decrease any emulsion between the layers in the stated method of extraction. Validation results were all pleasing including good stability studies with bias of value below 20%. Concentration of OMR in rats' plasma were determined after 2 h of the latest dose from 28 days multiple doses, p.o, 5 mg/kg/day. It was found to be 1295.66 ± 684.63 ng/mL estimated from the bio-analysis regression equation. OMR passed through the BBB following oral administration and exhibited concentration of 543.56 ± 344.15 ng/g in brain tissue, taking in consideration the dilution factor of 10. The brain/plasma concentration ratio of 0.42 (543.56/1295.66) was used to illustrate the penetration power through the BBB after the multiple doses for 28 days. Results showed that OMR passed through the BBB more effectively in the multiple dose study as compared to the previously published single dose study by the authors. Thus, the present study suggests potential repositioning of OMR as antiparkinsonian agent that will be of interest for researchers interested in neurodegenerative diseases.

Enhancing drug extraction from human plasma is a challenging approach that critically affects pharmacokinetic and any further clinical studies based on the drug C min and C max values. It also has a serious impact on the sensitivity and the lower limit of quantification (LLOQ) value of the bio-analytical methods. An advanced liquid chromatography tandem mass spectrometry (LC-MS/MS) bio-analytical method of omarigliptin (25-1000 nM) was established in human plasma using one-step liquid-liquid extraction. Alogliptin was used as an internal standard (IS) to attain good recovery and reproducibility while reducing the effects of the matrix. Enhanced plasma extraction of omarigliptin was successfully achieved with tertiary butyl methyl ether-diethyl ether (TBME-DEE) mixture as the extracting solvent, while using acetonitrile as the diluent solvent for the IS to effectively decrease the formed emulsion. Multiple Reaction Monitoring (MRM) of the transition pairs of m / z 399.2 to 153.0 for omarigliptin and m / z 340.2 to 116.0 for alogliptin was employed in positive Electro Spray Ionization (ESI) mode. Human plasma samples were collected after 1.5 h (t max ) of Marizev ® (12.5 mg) tablets administration to healthy human volunteers showing average concentration of 292.18 nM. Validation results were all satisfactory including successful stability studies with bias below 12%. The proposed study will be valuable for ethnicity comparison studies that will be commenced on omarigliptin in Egypt by the authors in prospective study, following the FDA recommends, to evaluate possible sub-group dissimilarities that include pharmacokinetic parameters.

Er-Zhi-Wan (EZW) is a traditional Chinese medicine with many clinical applications and used as a health product in East Asia. Five active ingredients (salidroside, specnuezhenide, nuezhenoside, luteolin, and oleanolic acid) were screened out from EZW to develop an in vitro rapid evaluation method for the classification of in vivo drug absorption behavior by biopharmaceutics classification system (BCS). Ultra-performance liquid chromatography was used for quantitative analysis. Solubility and permeability were assayed by equilibrium solubility and multiple models: everted rat intestinal sac model, cultured Caco-2 cells, octanol-water partition coefficient (LogP) method. The BCS properties of drugs were predicted using software applications, and the correlations of measured and predicted values of factors affecting oral drug absorption were calculated. The results were verified by measuring the absolute bioavailability of the active ingredients. Salidroside, specnuezhenide, and nuezhenoside were classified as BCS class III drugs, and luteolin was classified as a BCS class III/I drug because of the difference in LogP and intestinal permeability. Oleanolic acid was classified as a BCS class II/IV drug in acidic media and BCS class I/III drug in other media. Overall, EZW may be classified as a BCS class III drug, and permeability was identified as the primary factor limiting absorption. The results provide a novel method for the evaluation of the in vivo absorption of oral traditional Chinese medicines., (© 2019 John Wiley & Sons, Ltd.)

Aims: To characterize the population pharmacokinetics (PK) and pharmacodynamics (PD) of the once-weekly dipeptidyl peptidase-4 (DPP-4) inhibitor omarigliptin in healthy subjects and patients with type 2 diabetes mellitus, and use these models to support the dosing recommendation for patient labelling including patients with renal impairment., Methods: PK and PD were assessed from a total of 9827 omarigliptin concentrations collected from 1387 healthy subjects and patients participating in Phase 1, 2 and 3 studies examining single- or multiple-dose weekly administration of omarigliptin at doses ranging from 0.25 to 400 mg. Population PK and PD analyses were performed using nonlinear mixed effect modelling., Results: A semi-mechanistic 2-compartment model with linear unbound clearance and concentration-dependent binding of omarigliptin to the DPP-4 enzyme in both the central and peripheral compartments adequately described omarigliptin PK. Key covariates on omarigliptin PK included reduced unbound clearance with renal impairment. A direct effect sigmoid maximum inhibitory efficacy model adequately described the relationship between omarigliptin plasma concentrations and DPP-4 inhibition. These models supported the current Japan label instructions that the approved omarigliptin 25-mg once-weekly dose be halved in patients with severe renal impairment and in those with end-stage renal disease. Also, if patients missed a dose, the next dose of omarigliptin should be taken as soon as remembered up to and including the day before the next scheduled dose. No other clinically important covariates were identified., Conclusion: The models in the present analysis adequately described PK and PD characteristics of omarigliptin and supported the dosing and administration section of the omarigliptin label., (© 2019 The British Pharmacological Society.)

Xuanmai Ganjie Granules (XMGJ), a widely used Chinese herbal formula in the clinic, is used for treatment of sore throats and coughs. Despite the chemical constituents having been clarifying by our previous studies, both of the metabolism and pharmacokinetic studies of XMGJ are unclear. This study aimed to explore the disposition process of XMGJ in vivo. A sensitive and selective ultra-high performance liquid chromatography with quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS) method was developed to analyze the absorbed components and metabolites in rat plasma and urine after oral administration of XMGJ. A total of 42 absorbed components, including 16 prototype compounds and 26 metabolites, were identified or tentatively characterized in rat plasma and urine after oral administration of XMGJ. Moreover, the pharmacokinetic studies of five compounds of XMGJ were investigated using ultra-high liquid chromatography with tandem mass spectrometry method. The results indicated that liquiritin, harpagoside, glycyrrhetic acid, liquiritigenin, formononetin and their metabolites might be the major components involved in the pharmacokinetic and metabolism process of XMGJ. This research showed a comprehensive investigation of XMGJ in vivo, which could provide a meaningful basis for further material basis and pharmacological as well as toxicological research., (© 2018 John Wiley & Sons, Ltd.)

Pyrimidine-fused compounds are of great interest for the discovery of potent bioactive agents. This study describes the synthesis of novel pyranopyrimidines 3a-f and pyranotriazolopyrimidines 4a-d derivatives via the cyclocondensation reaction of α-functionalized iminoether 2, which was obtained from 2-amino-3-cyanopyrane 1, with a series of primary aromatic amines and hydrazides, respectively. Structures of all synthesized compounds were established on the basis of spectroscopic methods including 1 H NMR, 13 C NMR and ES-HRMS. They were finally tested for their anticoagulant and anti-tyrosinase activities. Significant results have been obtained and the structure-activity relationship (SAR) was discussed with the help of molecular docking analysis., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Devil's claw is used for the treatment of inflammatory symptoms and degenerative disorders in horses since many years, but without the substantive pharmacokinetic data. The pharmacokinetic parameters of harpagoside, the main active constituent of Harpagophytum procumbens DC ex Meisn., were evaluated in equine plasma after administration of Harpagophytum extract FB 8858 in an open, single-dose, two-treatment, two-period, randomized cross-over design. Six horses received a single dose of Harpagophytum extract, corresponding to 5 mg/kg BM harpagoside, and after 7 days washout period, 10 mg/kg BM harpagoside via nasogastric tube. Plasma samples at certain time points (before and 0-24 hr after administration) were collected, cleaned up by solid-phase extraction, and harpagoside concentrations were determined by LC-MS/MS using apigenin-7-glucoside as internal standard. Plasma concentration-time data and relevant parameters were described by noncompartmental model through PKSolver software. Harpagoside could be detected up to 9 hr after administration. C max was found at 25.59 and 55.46 ng/ml, t 1/2 at 2.53 and 2.32 hr, respectively, and t max at 1 hr in both trials. AUC 0-inf was 70.46 and 117.85 ng hr ml -1 , respectively. A proportional relationship between dose, C max and AUC was observed. Distribution (V z /F) was 259.04 and 283.83 L/kg and clearance (CL/F) 70.96 and 84.86 L hr -1  kg -1 , respectively. Treatment of horses with Harpagophytum extract did not cause any clinically detectable side effects., (© 2018 The Authors. Journal of Veterinary Pharmacology and Therapeutics Published by John Wiley & Sons Ltd.)

Although salinomycin sodium (SS) has shown in-vitro potential to inhibit cancer stem cell growth and development, its low water solubility makes it a poor candidate as an oral chemotherapeutic agent. To improve the bioavailability of SS, SS was encapsulated here using D-α-tocopherol polyethylene glycol succinate (TPGS)-emulsified poly(lactic-co-glycolic acid) (PLGA) nanoparticles and compared with its parent SS in terms of absorption, pharmacokinetics, and efficacy in suppressing nasopharyngeal carcinomas stem cells. The pharmacokinetics of SS and salinomycin sodium-loaded D-α-tocopherol polyethylene glycol succinate-emulsified poly(lactic-co-glycolic acid) nanoparticles (SLN) prepared by nanoprecipitation were analyzed in-vivo by timed-interval blood sampling and oral administration of SS and SLN to rats. Sensitive liquid chromatography-mass spectrometry (LC-MS) was developed to quantify plasma drug concentrations. SS and SLN transport in Caco-2 cells was also investigated. The therapeutic efficacy of SS and SLN against cancer stem cells was determined by orally administering the drugs to mice bearing CNE1 and CNE2 nasopharyngeal carcinoma xenografts and then evaluating CD133 cell proportions and tumorsphere formation. The in-vivo trial with rats showed that the Cmax, AUC(0-t), and Tmax for orally administered SLN were all significantly higher than those for SS (P<0.05). These findings were corroborated by a Caco-2 cell Transwell assay showing that relative SLN absorption was greater than that of SS on the basis of their apparent permeability coefficients (Papp). Significantly, therapeutic SLN efficacy against nasopharyngeal carcinoma stem cells was superior to that of SS. TPGS-emulsified PLGA nanoparticles effectively increase SS solubility and bioavailability. SLN is, therefore, promising as an oral chemotherapeutic agent against cancer stem cells.

The aim of this study was to establish and validate a rapid and sensitive LC-MS/MS method for the simultaneous determination of specnuezhenide and its bioactive metabolite salidroside in rat plasma. Protein precipitation was carried out and the analytes were separated on a Waters Acquity UPLC HSS T3 column (2.1 × 100 mm, 1.8 μm). A mobile phase consisting of acetonitrile and 0.1% formic acid aqueous solution was used for elution under gradient conditions at a flow rate of 0.4 mL/min. Quantification was performed in the negative multiple reaction monitoring mode with precursor-to-product transitions at m/z 685.2 → 453.1 for specnuezhenide, m/z 229.3 → 119.0 for salidroside and 493.2 → 147.1 for the internal standard. The method showed good linearity, accuracy, precision and stability in the range 0.5-500.0 ng/mL for specnuezhenide and salidroside. The values of the matrix effect were within the range of 100.02-111.87% for both analytes, while the mean extraction recovery was within the range 64.19-78.26%. The intra- and inter-day precisions (RSD) were <13.49% and the accuracy (RR) ranged from 93.59 to 102.24%. This study was successfully utilized for the pharmacokinetic study of specnuezhenide in rats after oral and intravenous administration. The oral bioavailability of specnuezhenide was 1.93%., (© 2018 John Wiley & Sons, Ltd.)

Omarigliptin is a novel long-acting dipeptidyl peptidase-4 inhibitor used for the treatment of type 2 diabetes. In this work, a sensitive and selective ultra-high pressure liquid chromatography tandem mass spectrometry method was developed and validated for the determination of omarigliptin in rat plasma. Sample preparation was performed by protein precipitation with acetonitrile. Chromatographic separation of analytes was achieved on an RRHD Eclipse Plus C18 column (2.1 × 50 mm, 1.8 μm), using gradient mobile phase (0.1% formic acid-acetonitrile) at a flow rate of 0.4 mL/min. Detection was performed in multiple reaction monitoring mode, with target fragment ions m/z 399.1 → 152.9 for omarigliptin and m/z 237.1 → 194 for the internal standard. The total run time was 4 min. Retention time of omarigliptin and internal standard was 1.25 and 2.12 min, respectively. Relative standard deviation (%) of the intra- and inter-day precision was below 10.0%, and accuracy was between 97.9% and 105.3%. Calibration curve was established over the range 2-5000 ng/mL with good linearity. The lower limit of quantification and limit of detection of omarigliptin were 2 and 0.25 ng/mL, respectively. Mean recoveries were in the range 87.3-95.1% for omarigliptin. No matrix effect was observed in this method. This novel method has been successfully applied to a pharmacokinetic study of omarigliptin in rats. The absolute bioavailability of omarigliptin was identified as high as 87.31%., (Copyright © 2017 John Wiley & Sons, Ltd.)

Purpose: The objective of this clinical trial was to assess the efficacy and safety of omarigliptin monotherapy in young adult patients with type 2 diabetes mellitus (T2DM). Unexpected efficacy results in this trial led to a series of investigations that identified the use of prohibited medication by a substantial number of trial patients., Methods: Patients with T2DM who were ≥18 to <45 years of age and either drug-naive or not on an antihyperglycemic agent for ≥12 weeks with inadequate glycemic control were randomized in a double-blind manner to receive omarigliptin 25 mg once weekly (n = 102) or placebo once weekly (n = 101) for 24 weeks. The objectives of the trial were to assess the effect of treatment with omarigliptin on glycemic parameters, including levels of glycosylated hemoglobin (HbA 1c ), 2-hour postmeal glucose, and fasting plasma glucose, and to assess the safety and tolerability of omarigliptin. Additional investigations into trial conduct included the measurement of drug levels for omarigliptin and metformin in blood samples collected for future biomedical research, available for approximately one half of the patients., Findings: The mean age of trial participants was 39.2 years, approximately 60% were male, mean body mass index was 32.5 kg/m2, and mean duration of diabetes was 3.1 years. The mean baseline HbA 1c value was 7.9% in the omarigliptin group and 8.1% in the placebo group. After 24 weeks, the least squares mean change (95% CI) in HbA 1c value from baseline was -0.33% (-0.60 to -0.06) in the omarigliptin group and -0.45% (-0.72 to -0.18) in the placebo group, with a between-group difference of 0.12% (-0.26 to 0.49; P = 0.535). Similarly, no between-group difference was observed for the other glycemic parameters (2-hour postmeal glucose and fasting plasma glucose levels). No issues were identified in drug allocation, dispensing or supply, patient compliance with trial medication, sample handling or analysis, or site trial conduct that explained the observed results. Measurement of drug levels from future biomedical research samples uncovered the use, with no investigator knowledge, of an antihyperglycemic agent that was prohibited by the protocol (ie, metformin) by 42.4% (39 of 92) of patients. Metformin was used by more patients in the placebo group (57% [25 of 44]) than in the omarigliptin group (29% [14 of 48])., Implications: The use of prohibited metformin in a trial of a dipeptidyl peptidase-4 inhibitor, omarigliptin, introduced a confounding factor that invalidated the results of the trial. This behavior may have been encouraged in the trial by protocol-specified self-monitoring of blood glucose levels. Use of prohibited medication may be an underappreciated confounder in clinical trial research., Trial Registrations: MK-3102-028 (US); ClinicalTrials.gov identifier, NCT01814748; EudraCT number, 2012-004303-12 (EU)., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

ACT-387042 and ACT-292706 are two novel bacterial topoisomerase inhibitors with broad-spectrum activity against Gram-positive and -negative bacteria, including methicillin-resistant Staphylococcus aureus and penicillin- and fluoroquinolone-resistant Streptococcus pneumoniae We used the neutropenic murine thigh infection model to characterize the pharmacokinetics (PK)/pharmacodynamics (PD) of these investigational compounds against a group of 10 S. aureus and S. pneumoniae isolates with phenotypic resistance to beta-lactams and fluoroquinolones. The in vitro activities of the two compounds were very similar (MIC range, 0.03 to 0.125 mg/liter). Plasma pharmacokinetics were determined for each compound by using four escalating doses administered by the subcutaneous route. In treatment studies, mice had 10(7.4) to 10(8) CFU/thigh at the start of therapy with ACT-387042 and 10(6.7) to 10(8.3) CFU/thigh at the start of therapy with ACT-292706. A dose-response relationship was observed with all isolates over the dose range. Maximal kill approached 3 to 4 log10 CFU/thigh compared to the burden at the start of therapy for the highest doses examined. There was a strong relationship between the PK/PD index AUC/MIC ratio (area under the concentration-time curve over 24 h in the steady state divided by the MIC) and therapeutic efficacy in the model (R(2), 0.63 to 0.82). The 24-h free-drug AUC/MIC ratios associated with net stasis for ACT-387042 against S. aureus and S. pneumoniae were 43 and 10, respectively. The 24-h free-drug AUC/MIC ratios associated with net stasis for ACT-292706 against S. aureus and S. pneumoniae were 69 and 25, respectively. The stasis PD targets were significantly lower for S. pneumoniae (P < 0.05) for both compounds. The 1-log-kill AUC/MIC ratio targets were ∼2- to 4-fold higher than stasis targets. Methicillin, penicillin, or ciprofloxacin resistance did not alter the magnitude of the AUC/MIC ratio required for efficacy. These results should be helpful in the design of clinical trials for topoisomerase inhibitors., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

The bark, seeds, fruits and leaves of the genus Fraxinus (Oleaceae) which contain a wide range of phytochemicals, mostly secoiridoid glucosides, have been widely used in folk medicine against a number of ailments, yet little is known about the metabolism and uptake of the major Fraxinus components. The aim of this work was to advance in the knowledge on the bioavailability of the secoiridoids present in a Fraxinus angustifolia Vahl seed/fruit extract using both targeted and untargeted metabolomic analyses. Plasma and urine samples from nine healthy volunteers were taken at specific time intervals following the intake of the extract and analyzed by UPLC-ESI-QTOF. Predicted metabolites such as tyrosol and ligstroside-aglycone glucuronides and sulfates were detected at low intensity. These compounds reached peak plasma levels 2 h after the intake and exhibited high variability among the participants. The ligstroside-aglycone conjugates may be considered as potential biomarkers of the Fraxinus secoiridoids intake. Using the untargeted approach we additionally detected phenolic conjugates identified as ferulic acid and caffeic acid sulfates, as well as hydroxybenzyl and hydroxyphenylacetaldehyde sulfate derivatives which support further metabolism of the secoiridoids by phase I and (or) microbial enzymes. Overall, the results of this study suggest low uptake of intact secoiridoids from a Fraxinus angustifolia Vahl extract in healthy human volunteers and metabolic conversion by esterases, glycosidases, and phase II sulfo- and glucuronosyl transferases to form smaller conjugated derivatives.

To study on the effects of Achyranthes bidentata on Tongsaimai pellets main active ingredients chlorogenic acid, isoliquiritin, harpagoside and glycyrrhizin in rats in vivo pharmacokinetic behaviors, a method for the simultaneous determination of chlorogenic acid, isoliquiritin, harpagoside and liquiritigenin in rat plasma was established by UPLC-MS/MS. The analysis was performed on a waters Acquity BEH C18 column (2.1 mm x 100 mm, 1.7 microm) with the mixture of acetonitrile and 0.1% formic acid/water as mobile phase, and the gradient elution at a flow rate of 0.3 mL x min(-1). The analytes were detected by tandem mass spectrometry with the electrospray ionization (ESI) source and in the multiple reaction monitoring (MRM) mode. It turned out that the analytes of Tongsaimai pellets groups C(max) and AUC(Q-infinity) values were higher than that with A. bidentata group, and the C(max) values of chlorogenic acid had significantly difference (P < 0.05), the AUC(0-infinity) values of chlorogenic acid and glycyrrhizin had significantly difference (P < 0.05); The T(max) and CL values of two groups had no significantly difference. Results showed that the established method was specific, rapid, accurate and sensitive for the studies of Tongsaimai pellets four main active ingredients in rat in vivo pharmacokinetic, and A. bidentata have varying degrees of effects on Tongsaimai pellets four main active ingredients in rat in vivo pharmacokinetic behaviors.

A simple and efficient liquid chromatography-mass spectrometry (LC-MS) method was developed and validated for simultaneous quantitation of catalpol and harpagide in normal and diabetic rat plasma. Protein precipitation extraction with acetonitrile was carried out using salidroside as the internal standard (IS). The LC separation was performed on an Elite C18 column (150 × 4.6 mm, 5 µm) with the mobile phase consisting of acetonitrile and water within a runtime of 12.0 min. The analytes were detected without endogenous interference in the selected ion monitoring mode with positive electrospray ionization. Calibration curves offered satisfactory linearity (r > 0.99) at linear range of 0.05-50.0 µg/mL for catalpol and 0.025-5.0 µg/mL for harpagide with the lower limits of quantitation of 0.05 and 0.025 µg/mL, respectively. Intra- and inter-day precisions (RSD) were <9.4%, and accuracy (RE) was in the -6.6 to 4.9% range. The extraction efficiencies of catalpol, harpagide and IS were all >76.5% and the matrix effects of the analytes ranged from 86.5 to 106.0%. The method was successfully applied to the pharmacokinetic study of catalpol and harpagide after oral administration of Zeng-Ye-Decoction to normal and diabetic rats, respectively., (Copyright © 2013 John Wiley & Sons, Ltd.)

In this paper, absorption and pharmacokinetic study of Radix Rehmanniae was studied by liquid chromatography coupled with mass spectrometry method after oral administration to rats. By comparing the chromatograms of ultraviolet, full scan, extracted ion and selective reaction monitoring (SRM) of standard solution, Radix Rehmanniae, blank plasma and rat plasma post drug administration, catalpol and ajugol were found to be the main compounds absorbed from Radix Rehmanniae. Plasma concentrations of aucubin, dihydrocatalpol, rehmannioside A (or rehmannioside B/ melittoside) and rehmannioside D were very low. Quantitative method for catalpol and aucubin and semi-quantitative method for other compounds in rat plasma were established. The pharmacokinetic study of those absorbed components was conducted after oral administration of 6 g x kg(-1) Radix Rehmanniae water extract to rats. Cmax, t(1/2) and AUC(0-infinity) of catalpol and ajugol were (2349.05 +/- 1438.34) and (104.25 +/- 82.05) ng x mL(-1), (0.86 +/- 0.32) and (0.96 +/- 0.37) h, (4407.58 +/- 2734.89) and (226.66 +/- 188.38) ng x h x mL(-1), respectively. tmax was at 1.00 h for catalpol and ajugol. Both catalpol and ajugol were absorbed and excreted rapidly.

Ethnopharmacological Relevance: Ajuga decumbens Thunb is a medicinal plant native to China popularly used to treat chronic pelvic inflammation and hysteromyoma. Its main bioactive components are iridoid glycosides, such as 8-O-acetylharpagide and harpagide that had presented antibacterial, anti-inflammatory, and antiviral activities., Aim of the Study: To establish a sensitive LC-MS/MS method and compare the pharmacokinetics of 8-O-acetylharpagide and harpagide in rats after oral administration of their pure forms and from compounds obtained from Ajuga decumbens extract., Materials and Methods: Rats received orally 15 mg/kg (equivalent of 6 mg/kg 8-O-acetylharpagide and 1.5mg/kg harpagide), 30 mg/kg and 60 mg/kg of Ajuga decumbens Thunb extract and were compared to animals that received 12 mg/kg of 8-O-acetylharpagide or 3mg/kg of harpagide p.o. Concentrations of 8-O-acetylharpagide and harpagide in plasma were determined by LC-MS/MS method at different time points and all pharmacokinetic parameters were estimated by non-compartmental analysis., Results: Results showed that the iridoid glycosides were quickly absorbed by oral route and showed a dose-dependence profile. Pharmacokinetic parameters of both glycosides were essentially the same except Tmax when dosed as the extract or pure forms., Conclusion: 8-O-acetylharpagide was metabolized to harpagide, which affected the pharmacokinetic profiles of harpagide when dosed as the extract. This pharmacokinetic study seems to be useful for a further clinical study of Ajuga decumbens Thunb extract., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

Melatonin is a major chronobiological regulator involved in circadian phasing, sleep, and numerous other functions. The novel melatonin agonist Neu-P11 is used in treatment of physiological insomnia. In animal studies Neu-P11 showed sleep-promoting effect. In a phase 1 study Neu-P11 was administered to cohorts of healthy young male volunteers in an ascending single dose study. Up to now no method for measurement of the new drug in human plasma was described and validated. The aim of this study was to develop a suitable analytical procedure. With solid phase extraction (SPE) for sample preparation and liquid chromatography tandem mass spectrometry (LC/MS/MS) a sensitive method with a lower limit of quantification of 0.39 ng/ml was found. For SPE samples were put into Strada C18 cartridges and automatically extracted by a Gilson Automatic Sample Processor ASPEC XL. The HPLC was carried out with a Purospher C18 column and mobile phase gradient with acetonitrile, and ammonium acetate. The precision ranged from 0.4% to 9.5%. The deviation of the measured from the true value, of the standard samples and QCSs was lower than 10%. The method was specific, precise and accurate and the analyte was stable under the conditions of measurement. Results show that the method is suitable for the quantification of NeuP11 in human plasma after a single dose of 5, 20, 50 or 200mg., (Copyright © 2012 Elsevier B.V. All rights reserved.)

A sensitive and specific HPLC-UV method was developed and validated for the determination of xyloketal B in rat plasma. Following liquid-liquid extraction, the separation was performed using an isocratic mobile phase of methanol-acetonitrile-water (30/30/40, v/v/v) on a Phenomenex C(18) column (4.6mm×250mm, 5μm). The eluent was monitored at 220nm and at a flow rate of 0.8mlmin(-1). A linear curve over the concentration range of 1-128μg/ml (r>0.999) was established. The LLOQ of the method was 1μg/ml. Good precision and accuracy at concentrations of 2.5, 25 and 100μg/ml were obtained. The recovery of xyloketal B in plasma was >87.91%. The validated method was found to be specific, precise and accurate in the study. The analytic method was satisfactorily applied to perform preclinical pharmacokinetic study of xyloketal B in rat plasma., (Copyright © 2011 Elsevier B.V. All rights reserved.)

The current study describes the pharmacokinetic parameters of two carboxylic polyether ionophores: monensin in turkeys and salinomycin in chickens. These data can be used to understand and predict the occurrence of undesirable residues of coccidiostats in edible tissues of these animal species. Special attention is paid to the distribution of residues between the different edible tissues during and at the end of the treatment period. For the bioavailability studies, monensin was administered to turkeys intravenously, in the left wing vein, at a dose of 0.4 mg /kg and orally at a dose of 20 mg /kg. Salinomycin was administered to chickens intravenously, in the left wing vein, at a dose of 0.25 mg /kg and orally at a dose of 2.5 mg /kg. Residue studies were carried out with supplemented feed at the rate of 100 mg /kg of feed for monensin in turkeys and 70 mg /kg for salinomycin in chickens, respectively. Coccidiostats had a low bioavailability in poultry (around 30% for monensin in chickens, around 1% for monensin in turkeys and around 15% for salinomycin in chickens). Monensin in chickens had a longer terminal half-life (between 3.07 and 5.55 h) than both monensin in turkeys (between 1.36 and 1.55 h) and salinomycin in chickens (between 1.33 and 1.79 h). The tissue /plasma partition coefficients showed a higher affinity of both monensin and salinomycin for fat, followed by liver and muscle tissue. The depletion data showed a fairly rapid elimination of coccidiostats in all the tissues after cessation of treatment. According to the results of depletion studies, a withdrawal period of 1 day seems sufficient to avoid undesirable exposure of consumers., (© 2011 Blackwell Publishing Ltd.)

Salinomycin is a polyether ionophore antibiotic that is widely used in poultry and livestock. Exposure of humans to salinomycin via inhalation or ingestion can cause severe toxicity. The aim of the present work was to develop a simple and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the rapid identification and quantification of salinomycin in human plasma. After removing protein using methanol, plasma samples were eluted from a Waters Xterra(®) MS C18 column with an isocratic mobile phase. Detection and quantification of the drug were performed with a triple-quadruple mass spectrometer by monitoring for two specific transitions in the electrospray, positive-ion, multiple-reaction monitoring mode. Assay validation showed good linearity (r(2) = 0.998). The detection and quantification limits of the method were 0.6 and 16 pg/mL, respectively. The inter- and intraday coefficients of variation for the assay were both <15%. Twelve authentic plasma samples from intoxicated patients were analyzed using this method. Salinomycin was detected in six samples, at concentrations of between 0.6 and 46.5 pg/mL. The described assay method allows the sensitive and rapid identification and quantification of salinomycin in human plasma, and thus provides a valuable tool for the specific diagnosis of salinomycin intoxication in clinical and emergency rescue practice.

Oleuropein (OE) is the cardinal bioactive compound derived from Olea europaea and possesses numerous beneficial properties for human health. However, despite the plethora of analytical methods that have studied the biological fate of olive oil-derived bioactive compounds, no validated methodology has been published to date for the simultaneous determination of OE, along with all its major metabolites. In this study, a liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI MS/MS) method has been developed and validated for the quantification of OE, simultaneously with its main metabolites hydroxytyrosol, 2-(3,4-dihydroxyphenyl)acetic acid, 4-(2-hydroxyethyl)-2-methoxy-phenol or homovanillyl alcohol, 2-(4-hydroxy-3-methoxyphenyl)acetic acid or homovanillic acid, and elenolic acid in rat plasma matrix. Samples were analyzed by LC-ESI MS/MS prior to and after enzymatic treatment. A solid-phase extraction step with high mean recovery for all compounds was performed as sample pretreatment. Calibration curves were linear for all bioactive compounds over the range studied, while the method exhibited good accuracy, intra- and inter-day precision. The limit of detection was in the picogram range (per milliliterof plasma) for HT and OE and in the nanogram range (per milliliter of plasma) for the other analytes, and the method was simple and rapid. The developed methodology was successfully applied for the simultaneous quantification of OE and its aforementioned metabolites in rat plasma samples, thus demonstrating its suitability for pharmacokinetics, as well as bioavailability and metabolism studies., (2009 John Wiley & Sons, Ltd.)

The relationship between rat pharmacokinetics and physicochemical parameters [the partition coefficient between octanol and buffer at pH 7.4 (log D((7.4))) and pK(a)] was studied for a series of tetrahydropyran compounds. Sixteen compounds ranging in log D((7.4)) 0.1 to 1.8 were administered intravenously to rats, and the pharmacokinetic parameters were determined from blood concentration time curves. Across the series, a weak correlation was observed between log D((7.4)) and blood clearance, suggesting that log D((7.4)) values less than 0.5 were required to prevent clearance at hepatic blood flow. In terms of the volume of distribution (V(d)), the compounds fell into three distinct subseries characterized by the number of basic centers and differences in ionization of each basic center at physiological pH. These were referred to as the monobasic, weak second base, and strong second base subseries. All the compounds exhibited V(d) greater than body water, as would be expected from their lipophilic and basic nature. For a given clog P, the strong second base subseries showed higher V(d) than the weak second base subseries, which in turn exhibited higher values than the monobasic subseries. In addition, for the weak second base subseries, V(d) could be tuned by modulating the pK(a) of the second basic center. This relationship was rationalized in respect to the interactions of the ionizable centers with phospholipid heads in the cell membrane and/or lysosomal trapping. Compounds in the weak second base subseries showed optimal V(d), and when combined with a log D((7.4)) of 0.1, driving to moderate blood clearance, one compound showed the optimal pharmacokinetic profile.

Harpagoside, a major bioactive iridoid glucoside in genus Scrophularia, has been widely used in clinical practice for the treatment of pain in the joints and lower back for its neuroprotective and anti-inflammation activities. To investigate the pharmacokinetics and hepatobiliary excretion, an in vivo microdialysis method coupled with high performance liquid chromatography was developed to monitor the concentration of harpagoside in blood and bile. The harpagoside bile-to-blood distribution ratio (AUC(bile)/AUC(blood)) up to 986.28+/-78.46 significantly decreased to 6.41+/-0.56 or 221.20+/-18.92 after co-administration of cyclosporin A or verapamil. The results indicated that harpagoside went through concentrative elimination from the bile which was probably regulated by P-glucoprotein, providing possible clinical trials of co-administration of transporter inhibitors to decrease drug efflux, thus to enhance the curative effects.

A simple HPLC method with ultraviolet detection has been developed and validated for the simultaneous determination of haplamine and its metabolites (trans/cis-3,4-dihydroxyhaplamine) in rat. A liquid-liquid extraction was used to extract the compounds from rat plasma. The analysis was performed on a C(18) Nucleosil Nautilus column. The mobile phase consisted of water (A) and a mixture of methanol and acetonitrile (85:15; v/v) (B) used in gradient mode (38-40% B for 10 min, 40-58% B for 49 min, 58-38% B for 1 min, and 38% for 5 min) pumped at 1 mL/min. The calibration curves showed good linearity with correlation coefficients greater than 0.999 for the analytes in the investigated concentration range. The lower limit of detection was 0.007, 0.008 and 0.009 microg/mL and the lower limit of quantification was 0.014, 0.017 and 0.018 microg/mL for haplamine, and trans/cis-3,4-dihydroxyhaplamine, respectively. The method was applied to a preliminary pharmacokinetic study in rats. This method proved to meet fully the standards required of experimental pharmacokinetic studies and should be used in further preclinical investigation., ((c) 2007 John Wiley & Sons, Ltd.)

Solid-phase extraction cartridges among those usually used for screening in horse doping analyses are tested to optimize the extraction of harpagoside (HS), harpagide (HG), and 8-para-coumaroyl harpagide (8PCHG) from plasma and urine. Extracts are analyzed by liquid chromatography coupled with multi-step tandem mass spectrometry. The extraction process retained for plasma applies BondElut PPL cartridges and provides extraction recoveries between 91% and 93%, with RSD values between 8 and 13% at 0.5 ng/mL. Two different procedures are needed to extract analytes from urine. HS and 8PCHG are extracted using AbsElut Nexus cartridges, with recoveries of 85% and 77%, respectively (RSD between 7% and 19%). The extraction of HG involves the use of two cartridges: BondElut PPL and BondElut C18 HF, with recovery of 75% and RSD between 14% and 19%. The applicability of the extraction methods is determined on authentic equine plasma and urine samples after harpagophytum or harpagoside administration.

A simple and sensitive method was developed for the simultaneous quantification of harpagoside and cinnamic acid in rat plasma using high-performance liquid chromatography system coupled to a negative ion electrospray mass spectrometric analysis. The plasma sample preparation was a simple deproteinization by the addition of two volumes of acetonitrile. The analytes were separated on an Intersil C8-3 column (2.1 mm i.d.x250 mm, 5 microm) with acetonitrile-5 mm ammonium formate aqueous solution (60:40, v/v) as mobile phase at a flow-rate of 0.2 mL/min. Detection was performed on a quadrupole mass spectrometer equipped with electrospray ionization (ESI) source operated under selected ion monitoring (SIM) mode. [M+HCOO]- at m/z 539 for harpagoside, [M-H]- at m/z 147 for cinnamic acid and [M-H]- at m/z 137 for salylic acid (internal standard) were selected as detecting ions, respectively. The method was validated over the concentration range 7-250 ng/mL for harpagoside and 5-500 ng/mL for cinnamic acid. The lower limits of quantitation for harpagoside and cinnamic acid were 7 and 5 ng/mL, respectively. The intra- and inter-day precisions (RSD%) were within 9.5% and the assay accuracies (RE%) ranged from -5.3 to 3.0% for both analytes. Their average recoveries were greater than 86%. Both analytes were proved to be stable during all sample storage, preparation and analysis procedures. The method was successfully applied to the pharmacokinetic study of harpagoside and cinnamic acid following oral administration of Radix Scrophulariae extract to rats., (Copyright (c) 2007 John Wiley & Sons, Ltd.)

Radix Scrophulariae (xuanshen) is one of the famous Chinese herbal medicines widely used to treat rheumatism, tussis, pharyngalgia, arthritis, constipation, and conjunctival congestion. Harpagoside and cinnamic acid are the main bioactive components of xuanshen. The purpose of this study was to develop an HPLC-UV method for simultaneous determination of harpagoside and cinnamic acid in rat plasma and investigate pharmacokinetic parameters of harpagoside and cinnamic acid after oral administration of xuanshen extract (760 mg kg(-1)). After addition of syringin as internal standard, the analytes were isolated from plasma by liquid-liquid extraction. Separation was achieved on a Kromasil C18 column, and detection was by UV absorption at 272 nm. The described assay was validated in terms of linearity, accuracy, precision, recovery, and limit of quantification according to the FDA validation guidelines. Calibration curves for both analytes were linear with the coefficient of variation (r) for both was greater than 0.999. Accuracy for harpagoside and cinnamic acid ranged from 100.7-103.5% and 96.9-102.9%, respectively, and precision for both analytes were less than 8.5%. The main pharmacokinetic parameters found for harpagoside and cinnamic acid after oral infusion of xuanshen extract were as follows: Cmax 1488.7 +/- 205.9 and 556.8 +/- 94.2 ng mL(-1), Tmax 2.09 +/- 0.31 and (1.48 +/- 0.14 h, AUC(0-24) 10,336.4 +/- 1426.8 and 3653.1 +/- 456.4 ng h mL(-1), AUC(0-infinity) 11,276.8 +/- 1321.4 and 3704.5 +/- 398.8 ng h mL(-1), and t(1/2) 4.9 +/- 1.3 and 2.5 +/- 0.9 h, respectively. These results indicated that the proposed method is simple, selective, and feasible for pharmacokinetic study of radix Scrophulariae extract in rats.

The pharmacokinetics and bioavailability of gentiopicroside (GPS), an active component of the Gentian plant species, from orally administered decoctions of Gentianae (DG), or in combination with other plants in the prescription of Longdan Xiegan Tang (LXT), was compared in rats with oral administration of GPS alone, using doses adjusted to deliver equivalent amounts of GPS (150 mg/kg). Changes in plasma levels of GPS following oral administration of GPS or DG could be fitted to a one compartment open model with elimination half times (T(1/2)Ke) of 3.35+/-0.76 h and 6.21+/-3.07 h, respectively. Kinetics of plasma GPS following oral administration of LXT could be fitted to a two compartments open model with an elimination half time (T((1/2)beta)) of 3.83+/-1.54 h. The bioavailability of GPS from DG was markedly better, and that from LXT markedly worse, compared with GPS alone, as judged by the area under concentration-time curve (AUC) values of 70.0+/-13.9 microgh/ml (DG), 32.7+/-12.9 microgh/ml (GPS) and 19.1+/-5.9 microgh/ml (LXT). The study demonstrates the marked variability in pharmacokinetics and bioavailability of an active component from different herbal preparations.

Fast and sensitive liquid chromatography-tandem mass spectrometric assays for the determination of salinomycin in mouse plasma, liver, brain and small intestinal contents and in OptiMEM cell culture medium, were developed and validated using simple sample pre-treatment procedures. Tissue samples were homogenized with phosphate buffered saline or, for high levels in liver, with human plasma. After addition of monensin as the internal standard to plasma, homogenate or culture medium and acetonitrile extraction for tissue and plasma, the diluted medium or the supernatant was directly injected into the isocratic chromatographic system using a polar embedded reversed-phase column and formic acid in water-acetonitrile as the eluent. The eluate was completely led into an electrospray interface with positive ionization and the analytes were quantified using triple quadrupole mass spectrometry. The assays were successfully validated in the ranges 10-2000 ng/ml for OptiMEM cell culture medium, 1-2000 ng/ml for plasma and 3-2000 ng/g in liver brain and small intestinal contents. At the lowest levels, the intra-day precisions were < or =9%, inter-day precisions were < or =14% and accuracies were between 91 and 112%. The analytes were chemically stable under all relevant conditions and the assays were applied in different in vitro transport studies and in pharmacokinetic and tissue distribution studies with salinomycin in mice.

The metabolism and disposition of KR31378 (a benzopyran derivative and a novel neuroprotective agent) were investigated following single oral or intravenous administration of [(14)C]-KR31378 to rats. [(14)C]-KR31378 was rapidly absorbed after oral dosing with an oral bioavailability of greater than 71%. The maximum plasma concentration and area under the curve of total radioactivity in rat plasma increased proportionally to the administered dose. KR31378 was distributed over all organs and tissues except for brain, eyeball and testis, and declined by first order kinetics up to 24 h after dosing. Excretion of the radioactivity was 29.5% of the dose in the urine and 58.5% in the feces within 2 days after oral administration. Biliary excretion of the radioactivity in bile duct-cannulated rats was about 66.0% for the first 24 h. KR31378 was extensively metabolized by ring hydroxylation, O-demethylation, oxidation and reduction with subsequent N-acetylation and O-glucuronide conjugation. N-acetylated conjugates (M2, M10, M11, M12, M14, and M15) were identified as the predominant metabolites in rats.

This study describes a chemometric analysis of 1H NMR spectra of canine plasma following oral single dose administrations of two food components (lycopene and oleuropein) and of two drug products, Fungoral (ketoconazole) and Adalat (nifedipine). Due to the high interday physiological variation, 1H NMR plasma data were first filtered (by applying orthogonal signal correction) and then subjected to principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA). A distinct discrimination was achieved between samples obtained with and without administration of xenobiotics using both techniques. According to distance to model criterion and as shown by the Cooman's plots there was no overlap between the four models which proved to be specific for each xenobiotic. Moreover, it was shown that this approach has the potential to recognize subtle variations in the metabolic profile even if the administered xenobiotic itself could not be detected in the canine plasma by conventional HPLC methods.

A simple, rapid, and specific analytical method for simultaneous determination of geniposide, baicalin, cholic acid and hyodeoxycholic acid in 50 microL samples of rat serum was developed by high performance liquid chromatography-tandem mass spectrometry. The quantification of the target compounds was determined by multiple reaction monitoring (MRM) mode using electrospray ionization (ESI). The correlation coefficients of the calibration curves were better than 0.997. The intra- and inter-day accuracy, precision, and linear range had been investigated in detail. This method was subsequently applied to pharmacokinetic studies of geniposide, baicalin, cholic acid and hyodeoxycholic acid in rats successfully.

A new HPLC method for the determination of geniposide in rat serum with solid-phase extraction (SPE) for preconcentration is described. Geniposide and an internal standard (paeoniflorin) were extracted from serum by SPE using C18 cartridges. Analysis of the extract was then performed on a reversed-phase C18 column using acetonitrile-water (16:84, v/v) as the eluting solvent system, and UV detection at 238 nm was used to measure the analyte with a limit of quantitation about 0.1 microg/mL. The calibration curve for geniposide was linear (r = 0.9993) in the concentration range 0.1-16.0 microg/mL. The intra- and inter-day precision of the geniposide were determined and their RSD did not exceed 10%. The validated method has been successfully applied for pharmacokinetic studies of geniposide from rat serum after oral administration of Yin-Zhi-Ku decoction., (Copyright 2005 John Wiley & Sons, Ltd.)

A new iridoid, gardaloside (1), and a new safranal-type monoterpene, jasminoside G (2), together with 10 known compounds including nine iridoids and a second safranal-type monoterpene, were isolated from the fruits of Gardenia jasminoides. The structures of 1 and 2 were established on the basis of spectroscopic evidence. Of these compounds, geniposide (3), 6alpha-hydroxygeniposide (5), ixoroside (7), and shanzhiside (8) showed significant inhibition of IL-2 secretion by phorbol myristate acetate and anti-CD28 monoclonal antibody co-stimulated activation of human peripheral blood T cells.

A method based on high-performance liquid chromatography (HPLC) with a diode array detection system was developed and validated aiming at the simultaneous determination of oleuropein (OE) and its metabolites, hydroxytyrosol (HT) and tyrosol (T), in human plasma. These phenolic components are believed to play a vital role in the prevention of coronary artery disease and atherosclerosis. The proposed method includes a clean-up solid-phase extraction procedure (using a C(18) column) with high recovery efficiency (85-100%). The statistical evaluation of the method reveals good linearity, accuracy and reproducibility for all the compounds analyzed with RSD values less than 6.5%, while the detection limit is 50 ng/ml for both OE and T and 75 ng/ml for HT. This assay can be employed in bioavailability studies of olive oil phenolic compounds, thus assisting the evaluation of their pharmacological role.

Oleuropein, the main glycoside present in olives, and hydroxytyrosol, the principal degradation product of oleuropein present in olive oil, have been linked to reduction of coronary heart disease and certain cancers. In the present study a direct and sensitive reversed-phase high-performance liquid chromatographic assay was developed for simultaneous quantification of both oleuropein and hydroxytyrosol. The plasma protein was precipitated with acetonitrile, samples were then centrifuged and supernatants were dried, and reconstituted with water prior to injection. The chromatographic analysis was carried out using a phenyl column and an isocratic elution of acidified water and acetonitrile with fluorescence detection at 281 and 316 nm for excitation and emission, respectively. The calibration curve was linear and limits of quantification were 30 ng/ml and 3 microg/ml for hydroxytyrosol and oleuropein, respectively. The method has been successfully applied to monitor oleuropein and hydroxytyrosol plasma levels in the rat.

We describe a liquid chromatography-electrospray ionisation tandem mass spectrometry method for the qualitative and quantitative determination of the secoiridoid oleuropein and its bioactive metabolite hydroxytyrosol in rat plasma and urine. Samples were prepared by liquid-liquid extraction using ethyl acetate with a recovery for both compounds of about 100% in plasma and about 60% in urine. The chromatographic separation was performed with a RP-ODS column using a water-acetonitrile linear gradient. The calibration curve was linear for both biophenols over the range 2.5-1000 ng/ml (LOD 1.25 ng/ml) for plasma and 5-1000 ng/ml (LOD 2.5 ng/ml) for urine. Plasma concentrations of oleuropein and hydroxytyrosol were measured after oral administration of a single dose (100 mg/kg) of oleuropein. Analysis of treated rat plasma showed the presence of unmodified oleuropein, reaching a peak value of 200 ng/ml within 2 h, with a small amount of hydroxytyrosol, whereas in urine, both compounds were mainly found as glucuronides.

An LC/MS/MS method for the simultaneous determination of a neuroprotective agent for ischemia-reperfusion damage, KR-31378 and its N-acetyl metabolite KR-31612 in human plasma was developed. KR-31378, KR-31612 and the internal standard, KR-31543 were extracted from human plasma by liquid-liquid extraction. A reverse-phase HPLC separation was performed on Luna phenylhexyl column with the mixture of acetonitrile-5 mM ammonium formate (55:45, v/v) as mobile phase. The detection of analytes was performed using an electrospray ionization tandem mass spectrometry in the multiple reaction monitoring mode. The lower limits of quantification for KR-31378 and KR-31612 were 2.0 ng/ml. The method showed a satisfactory sensitivity, precision, accuracy, recovery and selectivity.

Purpose: Harpagophytum extract and its marker substance harpagoside were shown to exert anti- inflammatory effects by interacting with the eicosanoid biosynthesis. In this study, different Harphagophytum extracts were tested with respect to inhibition of leukotriene and thromboxane biosynthesis in vitro and ex vivo. In addition, pharmacokinetic parameters of Harpagophytum extracts were investigated in vivo., Methods and Subjects: Different fractions of Harpagophytum extracts were tested in vitro in human whole blood samples for effects on basal and ionophore A23187-stimulated cysteinyl-leukotriene (Cys-LT) and thromboxane synthesis. Furthermore, in 3 independent studies with different numbers of human male volunteers, a Harpagophytum extract was administered orally and tested in whole blood samples for Cys-LT and thromboxane B2 (TXB2) biosynthesis and for the determination of pharmacokinetic parameters of harpagoside., Results: The special Harpagophytum extract WS1531 had a stronger inhibitory effect on ionophore A23187-stimulated Cys-LT levels compared with pure harpagoside or other extract fractions. Fractions without harpagoside had no pronounced inhibitory effect. When Cys-LT levels were measured after oral intake of Harpagophytum extract, a biphasic but dose-independent decrease of 28% and 58%, respectively, in basal Cys-LT formation was observed. Pharmacokinetic studies with the Harpagophytum extract WS1531 showed that the maximum levels of plasma harpagoside were reached after 1.3 to 2.5 hours. A linear relationship between dose and the first maximal concentration (Cmax) or area under the curve (AUC) (0-1)/AUC(0-infinity) was observed., Conclusions: Our observations strongly indicate a close relation between serum harpagoside levels and the inhibition of leukotriene biosynthesis.

A high-performance liquid chromatographic method was developed for the determination of a neuroprotective agent for ischemia-reperfusion damage, KR-31378, in human plasma and urine and in rat tissue homogenates. The method involved deproteinization of the the biological samples with 0.5 volumes of saturated Ba(OH)2, 0.5 volumes of 0.04 M ZnSO4 and 1 volume of acetonitrile. A 80-microl aliqout of the supernatant was injected onto a reversed-phase C18 column. The mobile phase, 50 mM triethylamine acetate : acetonitrile : tetrahydrofuran (65:30:5, v/v/v), was run at a flow rate of 1.0 ml/min. The column effluent was mornitored by a ultraviolet detector set at 310 nm. The retention time of KR-31378 was approximately 6.5 min. The detection limits of KR-31378 in human plasma and urine and rat tissue homogenates were 0.2, 0.5 and 0.5 microg/ml, respectively. The coefficients of variation (within-day and between-day) were below 13.6% for human plasma and urine and rat homogenates. No interferences from endogenous substances were found.

The blood partition of KR-31378 between plasma and blood cells and the factors influencing the binding of the drug to 4% human serum albumin (HSA) using an equilibrium dialysis technique were evaluated. KR-31378 reached an equilibrium rapidly between plasma and blood cells of rabbit blood. The equilibrium plasma/blood cells concentration ratios were independent of initial rabbit blood concentrations of KR-31378, 1, 10 and 50 microg/ml; the values were in the range of 1.42-2.33. It took approximately 12-h incubation to reach an equilibrium between plasma and isotonic Søresen phosphate buffer of pH 7.4 containing 3% dextran ('the buffer'). The binding of KR-31378 to 4% HSA was dependent on HSA concentrations (the binding values were 25.3 and 32.0% for HSA concentrations of 2 and 5%, respectively), incubation temperature (the binding values were 48.8, 29.0 and 25.8% for 4, 22 and 37 degrees C, respectively), pHs of isotonic Sørensen phosphate buffer containing 3% dextran (the binding values were 17.7, 20.6, 22.8, 25.6 and 29.5% for buffer pHs of 5.8, 6.4, 7.0, 7.4 and 8.0, respectively) and alpha-1-acid glycoprotein (AAG) concentrations (the binding values were 25.6, 29.9, 34.4 and 50.3% for AAG concentrations of 0, 0.08, 0.16 and 0.32%, respectively).

Objective: To study the metabolism of gentiopicroside by rat intestinal flora in vitro and time profiles of gentiopicroside and its metabolites in rat serum after oral administration of gentiopicroside., Method: Using high performance liquid chromatography (HPLC) for determination of gentiopicroside, gentianine and gentianal content of rat intestinal flora culture medium in vitro and rat serum sample in vivo., Result: Gentiopicroside was metabolized quickly in vitro by rat intestinal flora, consequently gentianine and gentianal content appeared. Gentianine and gentianal were observed after gentiopicroside oral administration 4 times in rat serum sample, and gentiopicroside content decreased after oral administration 6 times., Conclusion: Gentianine and gentianal were the important bioactive metabolites of gentiopicroside in rats.

Salinomycin is widely used as a feed additive to control coccidiosis in the broiler industry world-wide. EC legislation on veterinary drug residues will soon be extended to cover poultry. This will require the development of rapid assays to screen for the presence of residual concentrations of this and other drugs in poultry meat. This study describes the development of an enzyme-linked immunosorbent assay for salinomycin that has a limit of detection of approximately 0.2 ng/g. The antibody cross-reacts with narasin, but is not subject to interference from lasalocid, maduramicin or monensin. Residual concentrations of salinomycin were measured in the tissues of broilers following feeding of medicated feed containing 60 mg/kg salinomycin. Salinomycin residues were present only at very low concentrations in liver and muscle, and fell below the limit of decision of the assay within 2 days of withdrawal of the medicated feed.

Salinomycin is a polyether antibiotic used to promote growth in cattle and poultry. Workers may be exposed to salinomycin through handling of animal feeds that contain the drug and it is necessary to monitor plasma samples from these workers for salinomycin to ensure safety. A method for analysis of salinomycin in plasma samples was therefore developed. Salinomycin and the internal standard narasin are extracted into iso-octane then subjected to silica gel solid-phase extraction in which the sample is washed with methylene chloride-methanol (98.5:15) then eluted with a 90:10 proportion of the same mixture. Both salinomycin and narasin are oxidized with pyridinium dichromate to form a chromophore absorbing at 225 nm. The concentrated product was injected onto a C18 pre-column and heart cut from 1.85 to 3.65 min onto a C18 analytical column. The method was shown to be selective for salinomycin and narasin in six blank plasma samples. The method was linear over a range of 15-300 ng ml-1 with a detection limit of approximately 5 ng ml-1. The mean absolute recovery was found to be 93.4 and 97.9% for salinomycin and narasin, respectively. The method was accurate to within 5% at all concentrations studied. Within-run and between-run precision were both less than 8% RSD at all concentrations studied and the method was suitable for the purpose of monitoring plasma from exposed agricultural workers.

A method is described for the determination of the novel hydroxymethyl glutaryl coenzyme A reductase inhibitor RP 61969 (I) and its hydrolysis product, the dihydroxy acid RP 62420 (II), in human plasma. A structural isomer of I is used as internal standard. Both I and II were extracted from acidified plasma with diethyl ether. The dried residues were reconstituted in the high-performance liquid chromatography mobile phase and chromatographed on a 5 microns ODS2 column. The mobile phase used was aqueous dipotassium phosphate +tetra-n-butyl ammonium bromide (both 10 mM)-acetonitrile-methanol (60:40:5, v/v). At a flow-rate of 1.5 ml min-1 and ambient temperature, the retention time of II is 3.5 min, that of the internal standard is 5 min, and that of I is 8 min. The method has been validated and applied to the assay of plasma samples resulting from a cell-plasma distribution experiment in human whole blood.

The enzyme system which synthesizes prostaglandins and thromboxanes in extracts of washed human platelets has been characterized with respect to kinetic parameters, pH and cofactor dependence, and inhibitor potencies. Arachidonate and dihomo-gamma-linolenate were shown to be mutually competitive substrates, thus providing biochemical support for the assumption that both substrates are metabolized by the same cyclooxygenase, although they are ultimately metabolized to different patterns of products. Products of the synthetase of human leucocytes qualitatively resemble those obtained with human platelets. The prostaglandin synthetase of bovine seminal vesicles was studied under similar conditions, and kinetic parameters and inhibitor potencies were compared with those of platelet extracts.