Detection and Characterization of In Vitro Payload-Containing Catabolites of Noncleavable Antibody-Drug Conjugates by High-Resolution Mass Spectrometry and Multiple Data Mining Tools.

The formation and accumulation of payload-containing catabolites (PCCs) from a noncleavable antibody-drug conjugate (ADC) in targeted and normal tissues are directly associated with the therapeutic effect and toxicity of the ADC, respectively. Understanding the PCC formation is important for supporting the payload design and facilitating preclinical evaluation of ADCs. However, detection and identification of PCCs of a noncleavable ADC are challenging due to their low concentrations and unknown structures. The main objective of this study was to develop and apply a generic liquid chromatography-high-resolution mass spectrometry (LC-HRMS) method for profiling PCCs in vitro. Noncleavable ADCs, ado-trastuzumab emtansine (T-DM1) and ADC-1, were incubated in liver lysosomes, liver S9, and/or cancer cells followed by data acquisition using LC-HRMS. Profiling PCCs mainly relied on processing LC-HRMS datasets using untargeted precise and thorough background subtraction (PATBS) processing and targeted product ion filtering (PIF). As a result, 12 PCCs of T-DM1 were detected and structurally characterized in human liver lysosomal incubation, a majority of which consisted of 4-[N-maleimidomethyl]cyclohexane-1-carboxylate (MCC)-DM1 and a few amino acids. Additionally, the incubation of ADC-1 in human, rat, and monkey liver S9 and cancer cells generated one major and three very minor PCCs, verifying the payload design. The results demonstrate that PATBS enabled the comprehensive profiling of PCCs regardless of their molecular weights, charge states, and fragmentations. As a complementary tool, PIF detected specific PCCs with superior sensitivity. The combination of the in vitro metabolism systems and the LC-HRMS method is a useful approach to profiling in vitro PCCs of noncleavable ADCs in support of drug discovery programs. SIGNIFICANCE STATEMENT: Profiling in vitro payload-containing catabolites (PCCs) of a noncleavable antibody-drug conjugate (ADC) is important for optimization of the payload design and preclinical evaluation of ADC. However, currently used analytical approaches often fail to quickly provide reliable PCC profiling results. The work introduces a new liquid chromatography high resolution mass spectrometry method for comprehensive and rapid detection and characterization of PCCs released from a noncleavable ADC in liver lysosomes and S9 incubations.

Knockdown of circ_CLIP2 regulates the proliferation, metastasis and apoptosis of glioma cells through miR-641/EPHA3/STAT3 axis.

A great amount of reaches have confirmed that circular RNAs (circRNAs) are novel regulators in glioma progression. Here, our work aimed to probe the specific role of circ_CLIP2 in glioma. The mRNA and protein expressions were analyzed by qRT-PCR and western blot, respectively. Cell viability, migration, invasion and apoptosis were examined by MTT assay, tranwell and flow cytometry assays, respectively. Moreover, the binding relationships between circ_CLIP2, microRNA (miR)-641 and erythropoietin-producing human hepatocellular (Eph)A3 were verified by dual luciferase reporter gene assay and/or RIP assay. The following data showed that circ_CLIP2 and EPHA3 were markedly increased in glioma tissues and cells, while miR-647 was downregulated. Gain- and loss-of-function experiments discovered that circ_CLIP2 knockdown remarkably inhibited cell proliferation, migration and invasion and promoted cell apoptosis of glioma cells, while these effects of circ_CLIP2 knockdown were abolished by miR-641 inhibition. Circ_CLIP2 was proved as a sponge of miR-641 to competitively upregulate EPHA3 expression. In addition, EPHA3 overexpression could abolish the inhibitory effects of miR-641 overexpression on the malignant behaviors of glioma cells by activating the signal transducer and activator of transcription 3 (STAT3). These findings elucidated that circ_CLIP2 knockdown suppressed glioma development by regulation of the miR-641/EP HA3/STAT3 axis, which provided a novel mechanism for understanding the pathogenesis of glioma.

Exploring the mechanism of Chaihujia Longgu Muli decoction in the treatment of epilepsy in rats based on the RhoA/ROCK signaling pathway.

BACKGROUND: The Chinese herbal formula Chaihujia Longgu Muli Decoction (CD) has a good antiepileptic effect, but its mechanisms remain unclear. Therefore, in this study we explored the molecular mechanisms of CD against epilepsy. METHODS: Twelve-day-old SD rats were randomly divided into a normal group, model group, valproic acid group, and CD high, medium, and low groups. Except for the normal group, the other groups were given an intraperitoneal injection of pentylenetetrazol (PTZ) to establish epilepsy models, and the Racine score was applied for model judgment. After 14 consecutive days of dosing, the Morris water maze test was performed. Then, hippocampal Nissl staining and immunofluorescence staining were performed, and synaptic ultrastructure was observed by transmission electron microscopy (TEM). RhoA/ROCK signaling pathway proteins were detected. RESULTS: In PTZ model rats, the passing times were reduced, and the escape latency was prolonged in the Morris water maze test. Nissl staining showed that some hippocampal neurons swelled and ruptured, Nissl bodies in the cytoplasm were significantly reduced, and neurons were lost. Immunofluorescence detection revealed that the expression of PSD95 and SYP was significantly reduced. Electron microscopy results revealed that the number of synapses in hippocampal neurons was significantly reduced and the postsynaptic membrane length was significantly reduced. Western blot analysis showed that the RhoA/ROCK signaling pathway was activated, while SYP, SPD95, and PTEN expression was significantly decreased. After treatment with CD, neurobehavioral abnormalities and neuronal damage caused by epileptic seizures were improved. CONCLUSION: CD exerted an antiepileptic effect by inhibiting the activation of the RhoA/ROCK signaling pathway.

Influence of Triazole Pesticides on Wine Flavor and Quality Based on Multidimensional Analysis Technology.

Triazole pesticides are widely used to control grapevine diseases. In this study, we investigated the impact of three triazole pesticides-triadimefon, tebuconazole, and paclobutrazol-on the concentrations of wine aroma compounds. All three triazole pesticides significantly affected the ester and acid aroma components. Among them, paclobutrazol exhibited the greatest negative influence on the wine aroma quality through its effect on the ester and acid aroma substances, followed by tebuconazole and triadimefon. Qualitative and quantitative analysis by solid-phase micro-extraction gas chromatography coupled with mass spectrometry revealed that the triazole pesticides also changed the flower and fruit flavor component contents of the wines. This was attributed to changes in the yeast fermentation activity caused by the pesticide residues. The study reveals that triazole pesticides negatively impact on the volatile composition of wines with a potential undesirable effect on wine quality, underlining the desirability of stricter control by the food industry over pesticide residues in winemaking.

Population genomics demystifies the defoliation phenotype in the plant pathogen Verticillium dahliae.

Verticillium dahliae is a broad host-range pathogen that causes vascular wilts in plants. Interactions between three hosts and specific V. dahliae genotypes result in severe defoliation. The underlying mechanisms of defoliation are unresolved. Genome resequencing, gene deletion and complementation, gene expression analysis, sequence divergence, defoliating phenotype identification, virulence analysis, and quantification of V. dahliae secondary metabolites were performed. Population genomics previously revealed that G-LSR2 was horizontally transferred from the fungus Fusarium oxysporum f. sp. vasinfectum to V. dahliae and is exclusively found in the genomes of defoliating (D) strains. Deletion of seven genes within G-LSR2, designated as VdDf genes, produced the nondefoliation phenotype on cotton, olive, and okra but complementation of two genes restored the defoliation phenotype. Genes VdDf5 and VdDf6 associated with defoliation shared homology with polyketide synthases involved in secondary metabolism, whereas VdDf7 shared homology with proteins involved in the biosynthesis of N-lauroylethanolamine (N-acylethanolamine (NAE) 12:0), a compound that induces defoliation. NAE overbiosynthesis by D strains also appears to disrupt NAE metabolism in cotton by inducing overexpression of fatty acid amide hydrolase. The VdDfs modulate the synthesis and overproduction of secondary metabolites, such as NAE 12:0, that cause defoliation either by altering abscisic acid sensitivity, hormone disruption, or sensitivity to the pathogen.

Colorimetric Fluorescent Nanosensor Based on Hexamethylene Diisocyanate for Fluorescent Responses and Adsorption of Heavy Metal Ions.

An inorganic-organic hybrid material based on magnetic Fe3O4@SiO2 nanoparticles was synthesized for fluorescent responses and removal of heavy metal ions, in which superparamagnetic Fe3O4@SiO2 nanoparticles were firstly prepared and modified with hexamethylene diisocyanate (HDI) instead of 3-isocyanatopropyltriethoxysilane (IPTES) as the organic coupling agent, and then a rhodamine derivative with spirolactam structure (Rho-en) was conjugated on the HDI functionalized Fe3O4@SiO2 nanoparticles through isocyanate groups. Both of functionalized Fe3O4@SiO2 nanoparticles based on IPTES and HDI were characterized by FT-IR and XPS, and the results indicated that HDI was a good alternative as chemical bridge for surface modification on the surface of Fe3O4@SiO2 nanoparticles. The inorganic-organic hybrid composites synthesized based HDI showed naked-eye color changes and fluorescent responses towards Zn2+, Cd2+, Mn2+, Pb2+, Hg2+ and Fe3+, which could serve as the available proofs for the qualitative analysis. Moreover, the as-obtained composites not only had excellent adsorption capability for Pb2+ and Hg2+, but also showed strong magnetic sensitivity, which could help to the removal and separation of functionalized magnetic nanocomposites after capturing the heavy metal ions. In addition, the plausible interaction mode of functionalized Fe3O4@SiO2 nanoparticles with heavy metal ions was discussed.

Upregulation of microRNA-196a and microRNA-196b cooperatively correlate with aggressive progression and unfavorable prognosis in patients with colorectal cancer.

BACKGROUND: Both microRNA (miR)-196a and miR-196b are implicated in normal cell differentiation, proliferation, and in tumorigenesis of various cancer types. Especially, miR-196a exerts a pro-oncogenic influence in colorectal cancer (CRC) cells and miR-196b expression is upregulated in CRC tissues. The aim of this study was to evaluate the associations of miR-196a and miR-196b dysregulation with clinicopathological characteristics and prognosis in patients with CRC. METHODS: Quantitative real time-PCR (qRT-PCR) was performed to detect the expression levels of miR-196a and miR-196b in 126 pairs of fresh tumor samples matched with adjacent colorectal mucosa obtained from 126 patients with CRC. RESULTS: miR-196a and miR-196b expression levels in CRC tissues were significantly higher than those in adjacent colorectal mucosa (both P < 0.002). Interestingly, the expression levels of miR-196a in CRC tissues were positively correlated with those of miR-196b. Then, high miR-196a expression and high miR-196b expression, alone or in combination, were all statistically linked to the presence of lymph node metastasis, the poor differentiation grade, and the advanced TNM stage of CRC. Moreover, overall and disease-free survivals of CRC patients with high miR-196a expression, high miR-196b expression and miR-196a-high/miR-196b-high expression tended to be shorter than the corresponding control groups (log-rank statistic, all P < 0.001). Furthermore, multivariate analysis indicated miR-196a and/or miR-196b expression as independent prognostic indicators for CRC patients (all P < 0.05). CONCLUSIONS: Both miR-196a and miR-196b may be correlated with aggressive progression and unfavorable clinical outcome in CRC patients. Combined expression of miR-196a and miR-196b may be a promising biomarker in identifying a poor prognosis group of CRC.

The effects of partially or fully linked boron with a cross-linking structure of an organic precursor on the purity and morphology of ZrB2 powder.

From a chemical infrastructure perspective, it is important to ensure that all ions constituting a target product, e.g., Zr and B ions for ZrB2, are fully linked with a cross-linking structure for synthesis via an organic precursor. In the present study, glycerol is used as a chelating ligand to prepare boron both partially and fully linked with the cross-linking structure of organic precursors by a sol-gel route. The results are far from expected, in that the more linked boron there is in the precursor, the purer the ZrB2 produced. In the case of a partially linked cross-linking structure, the carbothermic reduction reaction for ZrB2 is a multi-step process with an intermediary phase of ZrC, and then a high-purity prism-like ZrB2 powder with a larger size is obtained. A minimum of 0.26 wt% for the oxygen content of ZrB2 corresponds to a 0.67 molar ratio of glycerol to H3BO3. On the other hand, in the case with the boron fully linked, a single-phase of ZrB2 cannot be obtained, and instead a double-phase is obtained. Therefore, the amount of impurity is greater, even though the size is smaller. The carbothermic reduction reaction is direct, and has only one step.

Synthesis of C-N co-doped nano-CeO2 and dye degradation under compact fluorescent lamp irradiation.

Carbon and nitrogen (C-N) co-doped nano-CeO2 was synthesized by the solvothermal method using hexamethylenetetramine (HMT) as a precipitator at 140 degrees C for 24 h. We found that the degradation of acid orange 7 (AO7) was 94.4% and 98.8% with C-N co-doped nano-CeO2 upon irradiation with a 100-watt high-pressure mercury lamp (HML) and a 10-watt compact fluorescent lamp (CFL), respectively. By comparison, TiO2 degraded 68.4% and 43.0% of the AO7 irradiated by HML and CFL, respectively. We found that the degradation efficiencies of AO7 upon irradiation with the 10-watt CFL in the presence of the samples synthesized using different precipitators decreased as follows: CeO2(HMT)> CeO2-TiO2(HMT) > TiO2(HMT) >> CeO2(NaHCO3) > CeO2(Na2CO3).

The angiotensin receptor and neprilysin inhibitor, LCZ696, in heart failure: A meta-analysis of randomized controlled trials.

BACKGROUND: LCZ696 is a novel neuroendocrine inhibitor that has been widely used in heart failure (HF). However, its advantage over other neuroendocrine inhibitors, such as angiotensin-converting enzyme inhibitors (ACEis) and angiotensin-receptor blockers (ARBs) has not been fully elucidated. This study aimed to provide the latest evidence regarding the efficacy and safety of LCZ696 as compared to other ACEis and ARBs with regards to the treatment of HF. METHODS: We systematically searched databases, including PubMed, Embase, and the Cochrane Library, for relevant randomized controlled trials (RCTs). The outcome measures included all-cause mortality, rate of hospitalizations for HF, rate of death from cardiovascular causes, change in N-terminal pro-brain natriuretic peptide (NT-proBNP) levels, and decline of renal function. RESULTS: Five RCTs involving 19,078 patients were identified. The meta-analysis indicated that LCZ696 was associated with a significant reduction in all-cause mortality (hazard ratio [HR] = 0.84; 95% confidence interval [CI], 0.76-0.93; P = .0005), rate of hospitalizations for HF (HR = 0.80; 95% CI, 0.73-0.87; P < .00001), reduction in NT-proBNP levels (rate ratio = 0.78; 95% CI, 0.70-0.88; P < .0001), and decline in renal function (odds ratio = 0.77; 95% CI, 0.68-0.88; P < .0001) compared with ACEis and ARBs. However, there was no statistical difference in the rate of death from cardiovascular causes (HR = 0.86; 95% CI, 0.72-1.03; P = .09) between LCZ696 and ACEis and ARBs. CONCLUSION: LCZ696 is superior to ACEis and ARBs in the treatment of HF. Hence, it should be more widely used clinically.

Residues and enantioselective behavior of cyflumetofen from apple production.

The effect of food processing on the level and fate of chiral pesticide residues in apple products has rarely been investigated. In this study, we used ultra-performance convergence chromatography coupled with triple quadrupole mass spectrometry to determine the content of the novel chiral acaricide cyflumetofen. The matrix-matched calibration lines were constructed for apple slices, juice, wine and vinegar, and the determination coefficients (r2) exceeded 0.9954. Acceptable average recoveries were within 81.1% to 119.9%, and the relative standard deviations (RSDs) ranged from 0.8% to 11.0%. Processing effectiveness is represented by the processing factor (PF). The results indicated that the PFs of different procedures (washing, peeling, enzymolysis, fermentation, among others.) were generally less than 1. The reduction of cyflumetofen enantiomers during fermentation was in accordance with first-order kinetics, and stereoselective behavior was observed. This study provides reliable references for the risk assessment of cyflumetofen in the processing of apple products.

Screening of pesticide residues in Traditional Chinese Medicines using modified QuEChERS sample preparation procedure and LC-MS/MS analysis.

A robust and high-throughput method was developed for the determination of 108 pesticide residues in Traditional Chinese Medicines (TCMs) simultaneously using a combination of UHPLC-MS/MS analysis and the modified QuEChERS method. Extraction was carried out in acetonitrile containing 0.75% (v/v) acetic acid with ultrasonication for 15 min; MgSO4 and C18 were used as the dispersive-solid phase extraction sorbents. The method exhibited good linearity (r2 > 0.9901), in addition to good selectivity, precision and repeatability. More than 92% of pesticides exhibited high rates or recovery in the 70-120% range. This method showed high sensitivity, with Limits of Quantitation in the 0.01-20 ng/mL range in Cortex Moutan, and 0.01-50 ng/mL in the other TCMs. The method was employed for the analysis of 39 real samples from different habitats, and pesticides were detected in 92.3% of the samples, with 26 pesticides being detected in these three TCMs. More than four pesticides were detected in a third of the samples. Among them, tebuconazole was detected in all the three TCMs with 0.22-22.02 µg/kg concentration, which was lower than the provisions in GB 2763-2019 (50 µg/kg). In addition, the paclobutrazol detection rate in Ophiopogon japonicus was 100%, and the detected concentrations of 9 samples exceeded the Maximum Residue Levels defined for vegetables (50 µg/kg). Considering there are no regulations that govern the limits of pesticide residues in the three TCMs in China, we recommend the acceleration of efforts to introduce appropriate regulations.

An eco-friendly route for template-free synthesis of high specific surface area mesoporous CeO2 powders and their adsorption for acid orange 7.

An eco-friendly route was developed for the synthesis of mesoporous CeO2 powders without any additional template. The original cerium precursors were separated from Ce3+ aqueous solution by (NH4)2CO3 or Na2CO3 via a chemical precipitation method, then H2O2 was introduced to induce the phase transformation from original cerium precursors to CeO2 precursors with initial porous structures, finally the crystallinities of CeO2 precursors were improved by a hydrothermal treatment, meanwhile the mesoporous structures of final CeO2 powders were formed. The BET surface areas of mesoporous CeO2 powders synthesized using (NH4)2CO3 and Na2CO3 as precipitants were 106.1 and 76.9 m2 g-1, respectively. Moreover, a mesoporous CeO2 sample with BET surface area of 100.0 m2 g-1 was also synthesized using commercial Ce2(CO3)3·xH2O as an existing cerium precursor under the same conditions as control, which could shorten experimental processes and reduce costs. The oxidation-induced phase transformation from original cerium precursors to CeO2 precursors with initial porous structures was the precondition for further forming of mesoporous structures of final CeO2 powders during the hydrothermal process. These mesoporous CeO2 powders showed the rapid and effective adsorption for acid orange 7 dye from simulated wastewater without pH pre-adjustment at room temperature. Furthermore, the adsorption capacities of these mesoporous CeO2 powders for removal of acid orange 7 dye were determined according to the Langmuir linear fits.

A simple and sensitive competitive bio-barcode immunoassay for triazophos based on multi-modified gold nanoparticles and fluorescent signal amplification.

A simple and highly sensitive immunoassay based on a competitive binding and bio-barcode amplification was designed for detection of small molecules, triazophos. The gold nanoparticles (AuNPs) were modified with monoclonal antibodies and 6-carboxyfluorescein labeled single-stranded thiol-oligonucleotides (6-FAM-SH-ssDNAs); the fluorescence of 6-FAM was quenched by AuNPs. Ovalbumin-linked haptens were coated on the bottom of microplate to compete with the triazophos in the sample for binding to the antibodies on the AuNP probes. The fluorescence intensity was inversely proportional to analyte concentration. Parameters of AuNP probes preparation and immune reaction were optimized. At the optimal conditions, the salting process was shortened to 1 h and 166 ± 9 ssDNAs were loaded onto a single AuNP. The competitive fluorescence bio-barcode immunoassay was performed on water, rice, cucumber, cabbage and apple samples. The linear range of the method was 0.01-20 µg L-1and the limit of detection (LOD) was 6 ng L-1. The recovery and relative standard deviations (RSDs) ranged from 85.0 to 110.3% and 9.4-17.4%, respectively. Good correlations were obtained between the results of the developed method and liquid chromatography-tandem mass spectrometry (LC-MS/MS). In conclusion, it is suggested that the competitive fluorescent bio-barcode immunoassay had the potential to be used as a sensitive method for detection of a variety of small molecules in various samples.

Metabolite identification of bentysrepinine (Y101), a novel anti-HBV agent in rats using a five-step strategy based on a combined workflow with two different platforms of liquid chromatography-tandem mass spectrometry.

Bentysrepinine (Y101), a derivative of repensine (a compound isolated from Dichondrarepens Forst), is a novel phenyalanine dipeptide inhibiting DNA-HBV and cccDNA activities and is currently under development for the treatment of hepatitis B virus (HBV)-infected hepatitis. Our previous study implied that there might be an existence of extensive metabolism of Y101 in rats. Therefore, it is necessary to perform metabolic profiling study to further evaluate its safety and drug-like properties. In this study, the metabolism of Y101 in rats was investigated by a convincible five-step strategy to characterize metabolites in plasma and that excreted into urine, bile and feces. The five-step strategy was realized by using an combined workflow on two different MS platforms, including various scan modes of liquid chromatography with hybrid quadruple-linear ion trap mass spectrometry (LC-QTRAP-MS/MS) and various post-acquiring data mining tools of liquid chromatography-quadrupole time-of-flight tandem mass spectrometry (LC-QTOF-MS/MS). QTOF MS/MS was employed as a powerful complementary tool to enable high confidence of metabolites identification using its functions of accurate MS and MS/MS fragmentation. As a result, a total of 30 metabolites were detected, including 25 phase I and 5 phase II metabolites. Among them, four primary metabolites (M6-M9) were further identified by comparing with the authentic standards chemically synthesized. The possible metabolic pathways of Y101 in rats were proposed to be amide hydrolysis, monohydroxylation, dihydroxylation, N-oxidation, demethylation, methylation, glucosidation and glucuronidation. This is the first study of the metabolism of Y101 in rats. The five-step strategy was successfully used to systematically characterize metabolites of Y101 in rats, and it would be generally applied for metabolite identification of new drug candidate.

Adsorption Kinetics of Acid Orange 7 on Nano-CeO2-TiO2 in Water.

To investigate the application of nano-CeO2-TiO2 as a sorbent in wastewater treatment, CeO2-TiO2 powder was prepared by the solvothermal method and then characterized. The adsorption kinetics of the adsorption of acid orange 7 (AO7) on CeO2-TiO2 were investigated under various conditions, such as initial concentration, temperature, and pH of the AO7 solution. Kinetic analyses were conducted with both Lagergren pseudo-first and pseudo-second order models. The results showed that the CeO2-TiO2 powder was composed of cubic CeO2 and anatase TiO2 with a specific surface area of 140.42 m2 x g(-1). The adsorption capacity of AO7 on CeO2-TiO2 increased with increasing starting concentration of AO7, but decreased with increasing temperature. The most favorable pH range of the A07 solution was 3-8 for the adsorption of AO7 on CeO2-TiO2. The results revealed that the adsorption kinetics of AO7 on CeO2-TiO2 matched the pseudo-second order model very well. The results indicate that CeO2-TiO2 has a potential application in the removal of AO7 from wastewater.

Synthesis of ZrB2 and ZrB2-SiC Powders Using a Sucrose-Containing System.

ZrB2 and ZrB2-SiC powders are synthesized by a sol-gel method from zirconium n-propoxide, tetraethyl orthosilicate (only for ZrB2-SiC), boric acid, and sucrose. After reduction at 1550 degrees C, both ZrB2 and ZrB2-SiC are unconsolidated, soft gray powders. The ZrB2-SiC particles have an equiaxed shape with a diameter of about 800 nm and a uniform size distribution. The SiC may be very finely distributed, because we barely find SiC among ZrB2 particles when using energy dispersive X-ray spectroscopy (EDS), although both ZrB2 and SiC are identified by X-ray diffractometry (XRD).

Quantitation of bentysrepinine (Y101) in rat plasma by liquid chromatography tandem mass spectrometry: application to pharmacokinetic study.

A simple, accurate and sensitive liquid chromatography tandem mass spectrometry (LC-MS/MS) method was developed and validated for quantitation of bentysrepinine (Y101) in rat plasma. After the addition of diphenhydramine (internal standard, IS), plasma samples were pretreated by protein precipitation. Chromatographic separation was carried out on an Atlantis(®) analytical column (4.6 mm × 100 mm, 5 µm, Waters) with methanol: 20 mM ammonium formate consisting of 1.0% formic acid (65:35, v/v) as the mobile phase at an isocratic flow rate of 0.4 mL/min for 7.5 min. The multiple reaction monitoring (MRM) transitions were performed at m/z 490.2→339.5 for Y101 and m/z 256.0→167.0 for IS on a SCIEX API 4000 mass spectrometer in the positive ion mode with electrospray ionization (ESI) source. Good linearity was achieved over the concentration range of 1-2500 ng/mL. The intra- and inter-day precisions were less than 8.3%, and the accuracy ranged from -4.0% to 2.8%. Y101 was stable during the analysis and the storage period. The pharmacokinetic profiles of Y101 at three dose levels were successfully studied for the first time in rats by this method. After single intra-gastric administration of Y101 at the doses of 25, 50 and 100 mg/kg, C(max) and AUC(0-t) were proportional to the doses given.