Development of a LC-MS/MS Method for a New Ortho-aryl Chalcone Compound of OC26 and its Application to Bile

Background OC26 and its pro-drug BOC26P, both ortho-aryl chalcone compounds, showed a well-definedantitumor activity in various cancer cells especially in drug-resistant tumor cell lines.Aim The purpose of this study was to investigate the bile excretion characteristics of OC26 after OC26 and BOC26Padministered in rats respectively.Method An ultra-performance liquid chromatography/tandem mass spectrometry (UPLC-MS/MS) method wasdeveloped and validated for OC26 in rat bile. Liquid-liquid extraction with ethyl acetate method was use to pretreatthe bile samples. After that, a gradient mobile phase at a flow rate 0.5mL/min of acetonitrile and 2mM CH3COONH4with 0.1% aqueous ammonia solution (v/v) and the positive ion alternate mode separated and quantified OC26.Bile samples were collected from rats after intravenous injection (i.v) 12.5mg/kg of OC26 and BOC26P, respectively.Results For method validation, the method showed high extraction recovery. The assay showed a good linearitywith correlation coefficient >0.99 at the concentration ranges of 20-2000ng/mL. All data were within the requiredlimits. The bile excretion results showed that the excretion amount of OC26 was gradually stabilized after 2h. Theaccumulative excretion percentage of OC26 after i.v 12.5mg/kg BOC26P was significantly higher than that of OC26after i.v 12.5mg/kg OC26. Significant gender differences were also observed in bile excretion of OC26.Conclusion This method was selective, sensitive and reliable and successfully applied to the bile excretion of OC26.This study provided theoretical basis for OC26 further research.

Effects of all-trans retinoic acid on goat dermal papilla cells cultured in vitro

Background: All-trans retinoic acid (ATRA), a vitamin A-derived active metabolite, exerts important functions in hair biology. Previous studies indicated that excess ATRA hampered hair follicle morphogenesis and cyclic regeneration in adulthood, but other studies stated that ATRA promoted hair growth. Dermal papilla (DP), a cluster of specialized fibroblasts, plays pivotal roles in controlling development and regeneration of hair follicle. Several lines of evidence indicated that DP might be the target cells of ATRA in the hair follicle. To confirm this hypothesis, the present study was performed to explore the biological effects of ATRA on goat dermal papilla cells (DPCs) and clarify the roles of ATRA in hair biology. Results: Our experimental results indicated that key signaling transducers of ATRA were dynamically expressed in distinct stages of goat cashmere growth cycle, and high-dose ATRA treatment (10-5 M) significantly impaired the viability of goat DPCs and lowered the ratio of proliferating cells. Otherwise, goat DPCs were stimulated to enter apoptosis and their cell cycle progression was severely blocked by ATRA. Moreover, the expression of fibroblast growth factor 7 (Fgf7), one of the potent hair growth stimulators secreted by DPCs, was transcriptionally repressed following ATRA treatment. Conclusion: DPCs are the targets of ATRA in the hair follicle, and ATRA negatively regulates hair growth by the targeted suppression of cell viability and growth factor expression of goat DPCs. Through these observations, we offer a new mechanistic insight into the roles of ATRA in hair biology.

Simultaneous Determination of Clopidogrel and Its Metabolites in Human Plasma by UPLC-MS/MS / 中国药房

OBJECTIVE:To establish the method for the simultaneous determination of clopidogrel (CLO) and its active metabolites (CATM) and inactive metabolites (CCAM),and to conduct pharmacokinetic study. METHODS:The plasma sam-ple had been derivatized by 2-bromine-3′-methoxy acetophenone(MPB),and was precipitated by acetonitrile. Using carbam-azepine as internal standard,UPLC-MS/MS was adopted. The separation was performed on Waters ACQUITY UPLC HSS T3 column with mobile phase consisted of water(containing 0.1% formic acid)-acetonitrile(containing 0.1% formic acid)using a gradient elution program at the flow rate of 0.50 ml/min. The ESI was equipped and quantitative analysis was operated in posi-tive ion and MRM mode. The mass transition ion-pairs were followed as m/z 322.1→211.8(CLO),m/z 504.1→155.0(the alkyl-ation derivatives of CATM,CATMD),m/z 308.3→198.0(CCAM),m/z 273.2→194.3(internal standard). RESULTS:The lin-ear calibration curves for CLO,CATMD and CCAM were obtained in the concentration range of 0.03-20.00 ng/ml,0.30-200.00 ng/ml and 10.00-10 000.00 ng/ml in plasma,respectively;intra-day and inter-day RSD for them were all less than 15%,and relative error(RE)ranged from -3.5% to 5.7%. Main pharmacokinetic parameters of CLO,CATMD and CCAM in 5 healthy volunteers after oral administration of CLO 300 mg were as follows:cmax were(7.89±5.46),(15.58±8.08),(8 023.33± 1 047.39)ng/ml;tmax were(1.25 ± 0.43),(1.25 ± 0.43),(1.67 ± 0.29)h;t1/2 were (2.31 ± 0.61),(0.64 ± 0.08),(6.53 ± 2.55)h;AUC0-t were(17.19±14.59),(21.39±9.58),(30 648.85±8 026.63)ng·h/ml. CONCLUSIONS:The established method is sensi-tive,rapid and convenient,which is suitable for pharmacokinetic study and plasma concentration determination of CLO and its metabolites.

Uncertainty Evaluation on Determination of Clopidogrel Active Metabolite Derivative in Human Plasma by LC-MS/MS / 中国药房

OBJECTIVE:To evaluate the uncertainty of the determination of clopidogrel active metabolite derivative(CAMD) in human plasma by LC-MS/MS. METHODS:The course of CAMD determination in human plasma by LC-MS/MS were analyzed to evaluate the uncertainty caused by evaluate repeatability,weighing,standard solution preparation,biological sample prepara-tion,recovery,instrument precision and calibration curve fitting in CAMD assay;the uncertainty were calculated and expanded. RESULTS:The expanded uncertainty for low(0.107 ng/ml),medium(4.467 ng/ml)and high(155.667 ng/ml)levels of CAMD were 0.010 2 ng/ml,0.188 ng/ml and 6.413 ng/ml,respectively(k=2,P=95%). CONCLUSIONS:The uncertainty of the CAMD determination in human plasma by LC-MS/MS at low concentration is mainly caused by calibration curve fitting;at medium and high concentrations is mainly caused by biological sample preparation,extraction procedure,standard solution preparation and in-strument precision.