Ultra-performance Liquid Chromatography-tandem Mass Spectrometer Research Articles

Statins for the prevention of proliferative vitreoretinopathy: cellular responses in cultured cells and clinical statin concentrations in the vitreous

Proliferative vitreoretinopathy (PVR) with rhegmatogenous retinal detachment (RRD) is a complex inflammatory ocular disease. Statins are widely used cholesterol-lowering drugs with putative anti-inflammatory properties. In this study, we have explored their efficacy in controlling post-surgical PVR formation. Simvastatin (SIM), atorvastatin (ATV), or rosuvastatin (RSV) were added to cultures of human retinal pigment epithelial cells (ARPE-19) prior to exposure with the bacterial lipopolysaccharide (LPS), and the production of pro-inflammatory cytokines (IL-6, IL-8, MCP-1) was examined using an enzyme-linked immunosorbent assay. In addition, the concentrations of simvastatin, atorvastatin, rosuvastatin, and their metabolites were measured from the vitreal samples of 20 patients undergoing vitrectomy (16 of them receiving oral statin therapy) using an ultra-performance liquid chromatography-tandem mass spectrometer technique. All statins alleviated LPS-induced inflammation at 5 µM concentration in the ARPE-19 cell cultures. Statin levels in the vitreous samples ranged from 6 to 316 pg/mL (ca. 0.1–7 M−10). Vitreal statin concentrations were similar to the typical steady-state unbound statin concentrations in plasma, indicating that only the unbound drug distributes from the blood circulation into the vitreous. Pharmacokinetic simulations of the intravitreal delivery of statins indicate that the measured clinical statin concentrations could be maintained with existing drug delivery technologies for months. Our results suggest that intravitreal statin therapy may have the potential in alleviating the risk of post-surgical PVR.

Determination of acamprosate in human plasma by UPLC-MS/MS: Application to therapeutic drug monitoring.

Acamprosate is a medication used to treat alcohol dependence. Therapeutic drug monitoring is important in drugs for the treatment of substance-related disorders. Therefore, in this study, a new selective, very simple and rapid ultra-performance liquid chromatography-tandem mass spectrometer method was developed for the therapeutic drug monitoring of acamprosate. The developed method allows the determination of acamprosate in human plasma. The method was validated in terms of selectivity and linearity, which was in the range of 100-1,200 ng/ml for acamprosate. Intra-assay and inter-assay accuracy and precision were within the acceptable limits of the Eueopean Medicines Agency guideline. The lower limit of quantitation was 100 ng/ml for acamprosate. The developed method was successfully applied for therapeutic drug monitoring in patient plasma samples.

Estimation of the area under concentration-time curve of polymyxin B based on limited sampling concentrations in Chinese patients with severe pneumonia.

The efficacy and toxicity of polymyxin B (PB) are closely related to its pharmacokinetic/pharmacodynamic (PK/PD) index area under the concentration-time curve (AUC) to minimum inhibitory concentration (MIC) ratio. The purpose of this study was to obtain PK data for PB in Chinese severe pneumonia patients and establish appropriate blood sampling time points for the PB therapeutic drug monitoring (TDM). After treatment with at least four doses of PB (50 IU, q12h), the blood samples were collected immediately after the end of infusion (C0) and 1.5, 2, 4, 6, 8, and 12 h (C1.5, C2, C4, C6, C8, C12) after PB administration. The PB blood plasma concentrations were determined using an ultra-performance liquid chromatography-tandem mass spectrometer (UPLC-MS/MS). All 42 patients were randomly divided into modeling (n = 24) and validation (n = 18) groups. The relationship between AUCss,24h and PB plasma concentration at each time point in modeling group was analyzed using limited sampling strategy and a PK method based on one-compartment with correction model. C6 scheme was found to provide the most accurate prediction of AUCss,24h values (r2 = 0.984) with the target value of 1.9-4.2 μg/ml at steady state to reach the 50-100 μg h/ml criteria of AUCss,24h. C0 with target value of 1.0-2.8 μg/ml can be considered an alternative sampling scheme (r2 = 0.900) but prediction deviation may exist. C0 and Cmax sampling scheme also demonstrated good predicting ability of AUC values using PK model. This study provides a clear plan for the implementation of TDM of PB, which is useful for optimizing the dosing regimen and individualizing treatment in severe pneumonia patients.

Blueberry and Banana Consumption Mitigate Arachidonic, Cytochrome P450 Oxylipin Generation During Recovery from 75-Km Cycling

Abstract Objectives Oxylipins are bioactive lipid oxidation products that have vital regulatory roles in numerous physiological processes including inflammation. This study determined if 2-weeks blueberry and/or acute banana ingestion influenced generation of in n-6 and n-3 PUFA-derived oxylipins during recovery from a 75-km cycling time trial. Methods Cyclists (n = 59, 38.6 ± 1.5 years of age) were randomized to freeze-dried blueberry or placebo groups, and ingested 26 grams/d (1 cup/d fresh blueberries equivalent) for 2 weeks. Cyclists reported to the lab in an overnight fasted state and engaged in a 75-km cycling time trial (185.5 ± 5.2 min). Cyclists from each group (blueberry, placebo) were further randomized to ingestion of water-only or water with Cavendish bananas (carbohydrate intake, 0.2 g/kg every 15 min) during exercise. Blood samples were collected pre- and post-2- weeks supplementation, and then 0 h-, 1.5 h-, 3 h-, 5 h-, 24 h-, 48 h-post-exercise. Plasma oxylipins and phenolic blueberry and banana metabolites were measured with ultra-performance liquid chromatography-tandem mass spectrometer (UPLC–MS/MS) using reference standards. Results Significant time × treatment effects (8 time points, 4 groups) were found for 24 blueberry-related and seven banana-related gut-derived metabolites in plasma (fdr P-value < 0.05). Significant and substantial post-exercise increases were measured for 64 of 67 identified plasma oxylipins. Oxylipins were grouped according to relevant fatty acid substrates [arachidonic acid (ARA), eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), α-linolenic acid (ALA), linoleic acid (LA), and enzyme systems [cytochrome P450 (CYP), lipoxygenase (LOX)]. Banana and blueberry ingestion were independently associated with significant post-exercise reductions in pro-inflammatory ARA-CYP HETEs and DiHETrEs (treatment effect, fdr P-value = 0.003). These trial differences were especially apparent within the first three hours of recovery from the 75-km cycling bout. Conclusions Prolonged and intensive exercise evoked a transient but robust increase in plasma levels of oxylipins, with a strong attenuation effect of ARA-CYP oxylipins linked to acute carbohydrate intake from bananas and 2-weeks ingestion of blueberries. Funding Sources Dole Foods, Westlake Village, CA.

Determination of pethidine of abuse and relevant metabolite norpethidine in urine by ultra-performance liquid chromatography-tandem mass spectrometry

Pethidine is an opiate agonist used orally and parenterally. Pethidine-containing drugs abuse is frequently encountered on health workers and patients. The analysis methods used to determine the abuse of pethidine are important for forensic toxicology. Pethidine is metabolized to norpethidine by the liver. Therefore, the determination of pethidine and norpethidine in urine is one of the methods to determine the abuse of pethidine. In this study, we have developed a precise, simple and rapid ultra-performance liquid chromatography-tandem mass spectrometer method for the determination of pethidine and norpethidine simultaneously. The developed method was validated in terms of selectivity and linearity which was in the range of 9–1800 ng/mL for both pethidine and norpethidine. The intra-assay and inter-assay accuracy and precision were found within acceptable limits of the EMA guideline. Lower limits of quantitation were 9 ng/mL for both pethidine and norpethidine. The developed method was successfully applied for the determination of both analytes in the real samples.

Pollution Characteristics and Risk Assessment of Typical Organophosphate Esters in Beijing Municipal Wastewater Treatment Plant and the Receiving Water

Organophosphate esters (OPEs) are ubiquitous in the environment and pose a potential threat to ecosystems and human health. A method for the determination of eight OPEs by ultra-performance liquid chromatography-tandem mass spectrometer (UPLC-MS/MS) was established. The recovery rates of eight target compounds with different solid-phase extraction columns, different eluents, and different eluent volumes were compared. The results showed that using ENVI-18 column enrichment, OPEs were eluted with 8 mL acetonitrile containing 25% (volume fraction) dichloromethane, and the labeled recovery rate of the target compound was 92.5%-102.2%. The recoveries of different matrix samples were 88.5%-116.1% and relative standard deviation was 1.7%-9.9%. The concentration range of 8 different detectable organophosphate esters in the effluent of sewage treatment plant is 85.9-235.4 ng·L-1 during the six-day sampling process, permissive river downstream of the six-day ΣOPEs average total concentration was 130.3 ng·L-1, higher than the 119.4 ng·L-1 upstream water concentration, but lower than the sewage treatment plant effluent concentration of total 162.5ng·L-1. The study shows that the sewage treatment plant cannot completely remove OPEs; for triethyl phosphate (TEP) and 3 (2-ethyl hexyl) phosphate ester (TEHP) there exists a negative removal phenomenon, whereas for other OPEs the removal rate was between 14.1% and 84.9%, and the total ΣOPEs removal rate by the sewage plant was 50.0%. The TPhP in the effluent of the sewage treatment plant has medium environmental risk (RQ>0.1), and other organophosphates have low environmental risk (RQ<0.1); however, the long-term mixing effects of organophosphate esters on the ecosystem of the receiving river should not be ignored.

Juvenile hormone III skipped bisepoxide, not its stereoisomers, as a juvenile hormone of the bean bug Riptortus pedestris

Juvenile hormone (JH) plays a pivotal role in many aspects of insect physiology. Although its presence was first reported in a blood-sucking bug belonging to the suborder Heteroptera (true bugs), JH species in the group has long been controversial. Although some recent studies proposed a putative JH molecular species in several Heteropteran species, it is not conclusive because physicochemical analyses were insufficient in most cases. Here, we studied this issue with an ultraperformance liquid chromatography–tandem mass spectrometer (UPLC-MS/MS) equipped with C18 and chiral columns in the bean bug Riptortus pedestris (Heteroptera, Alydidae), in which the JH species has long been controversial. Although a recent study describes JHSB3 as the major JH of this species, that finding was not conclusive because its chirality has not been clarified. In the present study, we detected methyl (2R,3S,10R)-2,3;10,11-bisepoxyfarnesoate, commonly named juvenile hormone III skipped bisepoxide (JHSB3), in the culture media of the corpora cardiaca-corpus allatum (CC-CA) complex and in the hemolymph of this species by a chiral ultraperformance liquid chromatography- tandem mass spectrometer (UPLC-MS/MS). Other JHSB3 stereoisomers were not detected. Topical application of JHSB3 effectively averted diapause. These results indicate that JHSB3 is the major JH of R. pedestris. The present study further revealed that JHSB3 and its (2R,3S,10S) isomer are more potent than (2S,3R,10R) and (2S,3R,10S) isomers, which suggests that there is a significance to the configuration of the 2,3-epoxide moiety in JH action. We further found a supplemental significance to the configuration of the 10-position.

Determination and pharmacokinetics and bioavailability of O-demethyl nuciferine in mice by UPLC–MS/MS

In this study, a precise, rapid, and accurate ultra-performance liquid chromatography–tandem mass spectrometer (UPLC–MS/MS) method for the quantitation of O-demethyl nuciferine in mouse blood was developed, and pharmacokinetics of O-demethyl nuciferine was studied for the first time after sublingual injection and gavage. The study was performed with an UPLC ethylene bridged hybrid (UPLC BEH) (2.1 mm × 50 mm, 1.7 μm) column at 30 °C, using diazepam as the internal standard (IS). The mobile phase consisted of acetonitrile–10 mmol/L ammonium acetate (containing 0.1% formic acid), with a flow rate of 0.4 mL/min for 4 min run time. Multiple reaction monitoring (MRM) modes of m/z 282.1→219.0 for O-demethyl nuciferine and m/z 296.2→265.1 for IS were utilized to conduct quantitative analysis. Protein in mouse blood was directly precipitated with acetonitrile for sample preparation. The linear range was 1–500 ng/mL with r &gt; 0.995, and the lower limits of quantification (LLOQ) was 1 ng/mL. The intra- and inter-day precision of O-demethyl nuciferine in mouse blood were RSD &lt; 14% and RSD &lt; 15%, respectively.r The accuracy ranged from 89.0% to 110.7%, with a recovery higher than 88.9%, while the matrix effect was between 103.1% and 108.7%. We further applied this UPLC–MS/MS method to the pharmacokinetic study on O-demethyl nuciferine after sublingual injection and gavage and determined the bioavailability to be 6.4%.

External contamination of antineoplastic drug containers from a Canadian wholesaler.

Contamination of hospitals' surfaces with antineoplastic drugs is documented despite safe handling practices. The exterior of commercial containers is often contaminated during the manufacturing process and can cross-contaminate hospitals' surfaces. The aim was to investigate the contamination of the exterior of antineoplastic drug vials available in Canada in 2018. Cross-sectional study. All available antineoplastic drugs vials from a single wholesaler were targeted. Containers were sampled upon their receipt by the pharmacy staff, before they were cleaned. One wipe was used to sample the external surface of five vials from a single batch from the same manufacturer. Nine antineoplastic drugs were quantified by ultra-performance liquid chromatography-tandem mass spectrometer: cyclophosphamide, docetaxel, 5-fluorouracil, gemcitabine, ifosfamide, irinotecan, methotrexate, paclitaxel, vinorelbine. Twenty-one samplings were done (105 containers from nine different manufacturers): cyclophosphamide = 2, docetaxel = 1, gemcitabine = 2, 5-fluorouracil = 2, ifosfamide = 2, irinotecan = 3, methotrexate = 6, paclitaxel = 2, vinorelbine = 1. One of these samplings was done on blister packaging, the remainder were done on glass vials. A total of 15/21 samples (71%) were positive to at least one drug (docetaxel, 5-fluorouracil, ifosfamide, and vinorelbine). A maximum of 272 ng/vial was quantified (gemcitabine). Cross contamination with other antineoplastic drugs was detected on 16/21 (76%) samples. The majority of samples were positive to at least one antineoplastic drug, confirming that the exterior of antineoplastic drugs containers is still an important source of contamination. Manufacturers should reduce this contamination. Vials should be washed upon receipt, before they are stored in pharmacy. Gloves must be worn at all times to avoid occupational exposure.

Phenolic and Antioxidant Analysis of Olive Leaves Extracts (Olea europaea L.) Obtained by High Voltage Electrical Discharges (HVED).

Background: The aim of this study was to evaluate high voltage electrical discharges (HVED) as a green technology, in order to establish the effectiveness of phenolic extraction from olive leaves against conventional extraction (CE). HVED parameters included different green solvents (water, ethanol), treatment times (3 and 9 min), gases (nitrogen, argon), and voltages (15, 20, 25 kV). Methods: Phenolic compounds were characterized by ultra-performance liquid chromatography-tandem mass spectrometer (UPLC-MS/MS), while antioxidant potency (total phenolic content and antioxidant capacity) were monitored spectrophotometrically. Data for Near infrared spectroscopy (NIR) spectroscopy, colorimetry, zeta potential, particle size, and conductivity were also reported. Results: The highest yield of phenolic compounds was obtained for the sample treated with argon/9 min/20 kV/50% (3.2 times higher as compared to CE). Obtained results suggested the usage of HVED technology in simultaneous extraction and nanoformulation, and production of stable emulsion systems. Antioxidant capacity (AOC) of obtained extracts showed no significant difference upon the HVED treatment. Conclusions: Ethanol with HVED destroys the linkage between phenolic compounds and components of the plant material to which they are bound. All extracts were compliant with legal requirements regarding content of contaminants, pesticide residues and toxic metals. In conclusion, HVED presents an excellent potential for phenolic compounds extraction for further use in functional food manufacturing.

A Novel PL9 Pectate Lyase from Paenibacillus polymyxa KF-1: Cloning, Expression, and Its Application in Pectin Degradation.

Pectate lyases play an important role in pectin degradation, and therefore are highly useful in the food and textile industries. Here, we report on the cloning of an alkaline pectate lyase gene (pppel9a) from Paenibacillus polymyxa KF-1. The full-length gene (1350 bp) encodes for a 449-residue protein that belongs to the polysaccharide lyase family 9 (PL9). Recombinant PpPel9a produced in Escherichia coli was purified to electrophoretic homogeneity in a single step using Ni2+-NTA affinity chromatography. The enzyme activity of PpPel9a (apparent molecular weight of 45.3 kDa) was found to be optimal at pH 10.0 and 40 °C, with substrate preference for homogalacturonan type (HG) pectins vis-à-vis rhamnogalacturonan-I (RG-I) type pectins. Using HG-type pectins as substrate, PpPel9a showed greater activity with de-esterified HGs. In addition, PpPel9a was active against water-soluble pectins isolated from different plants. Using this lyase, we degraded citrus pectin, purified fractions using Diethylaminoethyl (DEAE)-sepharose column chromatography, and characterized the main fraction MCP-0.3. High-performance gel permeation chromatography (HPGPC) analysis showed that the molecular mass of citrus pectin (~230.2 kDa) was reduced to ~24 kDa upon degradation. Ultra-performance liquid chromatography - tandem mass spectrometer (UPLC-MS) and monosaccharide composition analyses demonstrated that PpPel9a worked as an endo-pectate lyase, which acted primarily on the HG domain of citrus pectin. In vitro testing showed that the degradation product MCP-0.3 significantly promotes the growth of Lactobacillus plantarum and L. rhamnosus. In this regard, the enzyme has potential in the preparation of pharmacologically active pectin products.

Increased BBB permeability contributes to EGCG-caused cognitive function improvement in natural aging rats: pharmacokinetic and distribution analyses.

Previous studies report that (-)-epigallocatechin-3-gallate (EGCG), the most abundant polyphenolic ingredient in green tea, has high efficacy against Alzheimer's disease (AD) in various in vivo and in vitro models. However, as a water-soluble component, how EGCG exerts its anti-AD effects in the brain was not elucidated. In the present study, we investigated the anti-AD mechanisms of EGCG in natural aging rats with cognitive impairments (CIs) assessed using Morris water maze. The rats were treated with EGCG (100 mg/kg per day, intragastrically) for 4 weeks. The expression of β-amyloid (Aβ1-42) in the brain was detected with immunohistochemical staining. We showed that EGCG administration significantly ameliorated the CI in the aging rats with CI and decreased Aβ1-42 plaque formation in their brains. Then we used an efficient ultra-performance liquid chromatography-tandem mass spectrometer method to evaluate EGCG concentrations in rat plasma and tissue distribution. We found that EGCG absorption was significantly increased in the aging with CI group compared with control young rats. After oral administration of EGCG (100 mg), EGCG could not be detected in the brain tissues of control young rats, but it was found in the brain tissue of aging rats with CI. By using Evans Blue assay, transmission electron microscopy, and Western blotting assay, we demonstrated that the permeability of blood-brain barrier (BBB) was significantly increased in aging rats with CI. These results suggest that the permeability change of BBB is the physiological structural basis for EGCG treatment to improve learning and memory, thus providing a solid evidence for EGCG druggability in anti-AD therapeutic field.

Determination of tobacco alkaloid enantiomers using reversed phase UPLC/MS/MS

Nʹ-Nitrosonornicotine (NNN), a carcinogenic tobacco-specific Nʹ-nitrosamine (TSNA), is on the FDA list of harmful and potentially harmful constituents (HPHCs). Nornicotine, a product of the demethylation of nicotine, is the immediate alkaloid precursor for NNN formation. Nicotine, nornicotine and NNN are optically active. The accumulation of the isomers of nicotine, nornicotine, and NNN impacts their biological activity. In this paper, we report the determination of tobacco alkaloid enantiomers (including nicotine, nornicotine, anabasine, and anatabine) in samples of different tobacco lines using a reversed phase ultra-performance liquid chromatography-tandem mass spectrometer (UPLC/MS/MS) method. Current method demonstates excellent detection capability for all alkaloid enantiomers, with correlation coefficients (r2) > 0.996 within their linear dynamic ranges. The limit of detection (LOD) and limit of quantitation (LOQ) of all analytes are less than 10 ng/mL and 30 ng/mL, respectively. In addition, their recovery and coefficient of variation (CV%) are within 100–115% and 0.2–3.7%, respectively. The method validated in this paper is simple, fast, and sensitive for the quantification of alkaloid enantiomers in tobacco leaf and has been applied to investigations of tobacco alkaloid enantiomer ratios in different tobacco lines and tobacco products.

Comparative effect of black, green, oolong, and white tea intake on weight gain and bile acid metabolism.

The beneficial effects of tea on health, including obesity, are well known. However, the comparative effects of black, green, white, and oolong teas, which are prepared from the same fresh leaves, on weight gain and the potential mechanisms involved are not yet fully understood. Bile acids (BAs) are shown to be powerful regulators of metabolism; however, to our knowledge, no studies have investigated the effect of tea on BA metabolism. The aim of this study was to investigate the modulatory effects that green, black, white, and oolong teas that were prepared from the same raw tea leaves have on the plasma BA profile. Female rats were dosed with the aforementioned tea types as their sole source of drinking fluid for 28 d. We then investigated their weight and effect on BA metabolic profile using advanced ultra-performance liquid chromatography-tandem mass spectrometer (UPLC-MS/MS)-based metabolomics. The UPLC-MS/MS analysis of the plasma show that the levels of murocholic acid, glycochenodeoxycholic acid, glycocholic acid, glycodeoxycholic acid, taurochenodeoxycholic acid, tauroursodeoxycholic acid, taurodeoxycholic acid, tauromuricholic acid, and taurocholic acid were increased; whereas levels of taurolithocholic acid and isolithocholic acid were decreased after drinking green, oolong, and white tea types compared with control. Surprisingly, oolong tea significantly influenced reduction in relative weight compared with control, black, and green tea; whereas black, green, and white teas had no effects on weight compared with control. Green, black, oolong, and white teas altered the BA metabolism. This change in BA metabolism could be associated with the health benefit effects of tea. Oolong tea was most effective in reducing weight.

Rapid, On-Site, Ultrasensitive Melamine Quantitation Method for Protein Beverages Using Time-Resolved Fluorescence Detection Paper

To ensure protein beverage safety and prevent illegal melamine use to artificially increase protein content, a rapid, on-site, ultrasensitive detection method for melamine must be developed because melamine is detrimental to human health. Herein, an ultrasensitive time-resolved fluorescence detection paper (TFDP) was developed to detect melamine in protein beverages within 15 min using a one-step sample preparation. The lower limits of detection were 0.89, 0.94, and 1.05 ng/mL, and the linear ranges were 2.67-150, 2.82-150, and 3.15-150 ng/mL (R2 > 0.982) for peanut, walnut, and coconut beverages, respectively. The recovery rates were 85.86-110.60% with a coefficient of variation <7.80% in the spiking experiment. A high specificity was observed in the interferent experiment. When detecting real protein beverage samples, the TFDP and ultraperformance liquid chromatography-tandem mass spectrometer (UPLC-MS/MS) results were consistent. This method is a promising alternative for rapid, on-site detection of melamine in beverages.

Metabonomic Profiling Reveals Difference in Altered Metabolic Pathways Between Chronic Kidney Disease and High-Fat-Induced Insulin Resistance in Rats

Background/Aims: Chronic kidney disease (CKD) is closely correlated with the development of insulin resistance (IR). Until now, the underlying molecular mechanisms remain to be elucidated. This study aimed to identify metabolites and molecular pathways unique to CKD-induced IR. Methods: Ultra-performance liquid chromatography-tandem mass spectrometer (UPLC-MS) analysis coupled with orthogonal partial least square discriminant analysis (OPLS-DA) were performed to profile metabolites in the serum, liver, and muscle tissues and to analyze molecular pathways in relation to CKD- and high fat diet (HFD)-induced IR in the rats. Results: At 18 weeks after the 5/6 Nx operation, CKD induction was demonstrated by renal histology and biochemical tests. Furthermore, both CKD-induced IR and HFD-induced IR rats showed significantly greater levels of fasting insulin and homeostasis model assessment of insulin resistance (HOMA-IR). In the UPLC-MS in combination with OPLS-DA analysis, we identified 101, 59, and 41 differential metabolites in the serum, liver, and muscle, which were associated with the CKD-induced IR, while 58, 38, and 17 differential metabolites in the serum, liver, and muscle were revealed in the HFD-induced rats compared to controls. Moreover, compared to HFD-induced IR rats, those with CKD-induced IR exhibited abnormal pathways primarily in the tryptophan metabolism, arginine metabolism, and trimethylamine oxide metabolite. Interestingly, altered metabolites in the CKD-induced IR and HFD-induced IR displayed an opposite direction. Conclusion: Alterations in metabolites and relevant pathways were significantly different between the CKD- and HFD-induced IR rats. These findings may offer important information regarding the pathogenesis specific to IR caused by the decline in the renal function.

Evaluation of the pharmacokinetics, tissue distribution and excretion studies of YMR-65, a tubulin polymerization inhibitor with potential anticancer activity, in rats using UPLC-MS/MS

1. YMR-65, 5-(5-bromo-1-methyl-1H-indol-3-yl)-3-(3-methoxyphenyl)-4, 5-dihydro-1H-pyrazole-1-carboxamide, is a new tubulin polymerization inhibitor with encouraging anticancer activity.2. The validated ultra-performance liquid chromatography-tandem mass spectrometer (UPLC-MS/MS) method was successfully applied to the pharmacokinetics, tissue distribution and excretion study of YMR-65 after oral and intravenous administration. The area under concentration-time curve (AUC0-∞) for YMR-65 were 151.67 ± 54.48 and 459.45 ± 49.23 ng/ml*h for oral and intravenous administration at the dosage of 1.5 mg/kg, respectively and the oral bioavailability was about 33.01%. Moreover, YMR-65 was extensively distributed in heart, liver, spleen, lung, kidney, stomach, intestine and testis and the highest were detected in heart, followed by stomach, intestine and liver. The majority of YMR-65 was excreted via feces and its accumulative excretion ratio during the period of 96 h was 19.83 ± 3.01%, but only 1.54 ± 0.37 and 0.215 ± 0.026% for urine within 96 h and bile within 10 h after intravenous administration, respectively, though the fecal and urine excretion were incomplete within 96 h.3. In summary, this study defined the pharmacokinetic characteristics of YMR-65 in vivo and the important data can be a useful resource for further research and development.

Simultaneous Determination of Multiple Components in Guanjiekang in Rat Plasma via the UPLC-MS/MS Method and Its Application in Pharmacokinetic Study.

Guanjiekang (GJK) that is formed by five medicinal herbs including Astragali Radix, Aconiti Lateralis Radix Praeparaia, Glycyrrhizae Radix et Rhizoma, Corydalis Rhizoma and Paeoniae Radix Alba was used for the treatment of rheumatoid arthritis (RA). However, the pharmacokinetic (PK) profile of active components in GJK remains unclear. This study aims to evaluate the pharmacokinetic behavior of seven representative active constituents in GJK (i.e., benzoylhypaconine, benzoylmesaconine, paeoniflorin, tetrahydropalmatine, calycosin-7-glucoside, formononetin and isoliquiritigenin) after oral administration of GJK in rats. A rapid, sensitive and reliable ultra-performance liquid chromatography-tandem mass spectrometer (UPLC–MS/MS) method has been successfully developed for the simultaneous determination of these seven constituents in rat plasma. Chromatographic separation was achieved on a C18 column with a gradient elution program that consists of acetonitrile and water (containing 0.1% formic acid) at a flow rate of 0.35 mL/min. Detection was performed under the multiple reaction monitoring (MRM) in the positive electrospray ionization (ESI) mode. The calibration curves exhibited good linearity (R2 > 0.99) over a wide concentration range for all constituents. The accuracies ranged from 92.9% to 107.8%, and the intra-day and inter-day precisions at three different levels were below 15%. Our PK results showed that these seven compounds were quickly absorbed after the administration of the GJK product, and Tmax ranged from 30 min to 189 min. The in vivo concentrations of paeoniflorin and isoliquiritigenin were significantly higher than the reported in vitro effective doses, indicating that they could partly contribute to the therapeutic effect of GJK. Therefore, we conclude that pharmacokinetic studies of representative bioactive chemicals after administration of complex herbal products are not only necessary but also feasible. Moreover, these seven compounds that were absorbed in vivo can be used as indicator standards for quality control and for determining pharmacokinetic behavior of herbal medicines in clinical studies.

Identification and quantitation of mifepristone and its N-demethyl metabolite in the plasma of an aborted fetus by liquid chromatography–quadrupole–time-of-flight–mass spectrometry (LC–Q–TOFMS) and ultra-performance liquid chromatography–tandem mass spectrometry (UPLC–MS–MS)

Mifepristone, also known as RU 486, is a derivative of norethindrone, a synthetic 19-nor-steroid; it acts as an antagonist to progestational and glucocorticoid functions by its binding to progesterone as well as glucocorticoid receptors [1]. Mifepristone is useful for terminating early pregnancy and for emergency contraception [1–4]. Mifepristone is approved in the US, EU countries, Israel, and China, but is not approved in Japan. In Japan, mifepristone has been ‘privately’ or secretly imported; the Ministry of Health, Labour and Welfare warned about its private import in 2004 because of possible complications, e.g., massive hemorrhaging [5]. Despite the warning, it has still been imported and used. It is, thus, important to detect mifepristone and its metabolites with high sensitivity. Several analytical methods have been reported for the measurement of mifepristone, such as the radioimmunoassay [6, 7], the radioreceptor assay [8, 9], voltammetry [10] and high performance liquid chromatography (LC) with an ultraviolet detector [11–17]. Recently, LC– tandem mass spectrometry (MS/MS) [18, 19] has been applied to detect and identify mifepristone and its metabolites from body fluids. In this Letter, we describe the determination of mifepristone and its metabolite in a plasma sample of an aborted fetus using a novel LC–quadrupole–time-of-flight– mass spectrometer (Q–TOFMS) with high sensitivity and specificity and an ultra-performance liquid chromatography–tandem mass spectrometer (UPLC–MS–MS). A woman in her twenties consulted an ER due to her poor health conditions; she then confessed her delivery and that she discarded the dead fetus. She also admitted that she had administered mifepristone and misoprostol prior to her delivery, although the time of administration and the amounts of the compounds were not clear. Around one day after delivery, autopsy of the fetus was performed. Autopsy findings were as follows: a male fetus whose height and weight were 30 cm and 505 g, respectively. No apparent injuries or malformations were observed. A cephalic hematoma-like finding was obtained. The lungs seemed liver-like, although histopathological observations detected some alveolar enlargement. Based on the above findings, the possibility that the fetus had been alive at the delivery was not fully excluded. The fetus was estimated to be at approximately 20–24 weeks gestation. Mifepristone and misoprostol were purchased from Sigma (St. Louis, MO, USA); N-demethyl mifepristone and alfaxalone were from Toronto Research Chemicals (Ontario, Canada) and Tocris (Bristol, UK), respectively. Drug free plasma was purchased from Tennessee Blood Services (Memphis, TN, USA). Other chemicals used were of the highest purity commercially available. A plasma specimen was collected from the femoral blood of the fetus. Mifepristone and its metabolites were analyzed based on the method by Homer et al. [19] with appropriate modifications. Screening of mifepristone and quantitation of the drug and its metabolites by UPLC–MS–MS were employed on an ACQUITY UPLC-TQD system (Waters, & Akira Ishii akishii@med.nagoya-u.ac.jp