Development and validation of a method for quantitation of cefepime/avibactam in M-H broth: application to antibacterial activity using in vitro PK/PD Model / 中国临床药理学与治疗学

AIM: To establish a method for quantitation of cefepime and avibactam in M-H broth, and applicated in the in vitro dynamic PK/PD model. METHODS: The cefepime was also determined using the high-performance liquid chromatography method (HPLC), the avibactam was also determined using the liquid chromatography-mass spectrometry (LC-MS/MS), an in vitro dynamic PK/PD model was established to study the PK/PD relationship of cefepime/avibactam against carbapenem resistant Klebsiella pneumoniae (CRKP). RESULTS: The linear ranges of cefepime and avibactam were good at (0.5-20) and (0.1-25) μg/mL (r=0.999), and the lower limit concentrations were 0.5 and 0.1 μg/mL. The extraction recoveries of cefepime and avibactam in M-H broth were 88.0%-101.7% and 90.9%-95.2%, the relative standard deviation of intra-day precision and inter-day precision were less than 5.2%. The concentration-time curves were well simulated by the PK/PD model. All observed concentrations in each experiment were in the range of 20% of the targeted values. For the CRKP of MIC=8 μg/mL and MIC=16 μg/mL, the colony decreased to 2.783Log10 CFU/mL and 1.325Log10 CFU/mL at the cefepime/avibactam 2.5 g q8 h administration after 24 h. CONCLUSION: The determination method of cefepime and avibactam in broth established in this study has high sensitivity and good stability. For the CRKP with MIC≤8 μg/mL,cefepime/avibactam showed that good anti-CRKP activity under routine administration in vitro dynamic PK/PD model.

LC-MS/MS method for the determination of an anti-tumor compound C17 in nude mice plasma and its application to a pharmacokinetic study / 药学学报

C17 is an orally available anti-tumor compound inhibiting cancer stem cell (CSC). In this study, a stable, sensitive and simple liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was established and validated, and was further applied to a pharmacokinetic study in nude mice receiving C17 by gavage. Using propranolol as the internal standard, the plasma samples were pre-treated by precipitation with methanol and analyzed on an Intersil C8-3 column (100 mm × 2.1 mm, 3 μm), and gradient elution was performed with a mobile phase consisting of 0.1% formic acid aqueous and solution mixed up by 90% isopropanol and 10% acetonitrile. The analyte was detected by a triple quadrupole tandem mass spectrometer, and multiple reaction monitoring was employed to select C17 at m/z 439.3/247.1 and propranolol at m/z 260.2/116.2 in the positive ion mode. The calibration curves were linear (r > 0.995) over the range of 5-800 ng·mL-1. The intra- and inter-day precisions and accuracies were 7.42%-13.22% and -8.99%-8.81% respectively. The method was successfully applied to a PK study in nude mice administered with a single oral dose of 50 mg·kg-1 C17, and the PK data were analyzed with non-linear mixed effect model (NONMEM). Two separated absorption peaks were found in the PK curve of C17, and a two-compartment model with two sequential first-order absorption rate was utilized to describe the PK properties of C17, and the model could provide insights into the physiological process and exposure of C17 in nude mice. All animal experiments were in strict accordance with the regulations of the Biomedical Ethics Committee of Peking University.

Pharmacokinetic study of small molecule inhibitor SYHA1809 in Beagle dogs / 中国药房

OBJECTIVE To study the pharmacokinetics of small molecule inhibitor SYHA1809 in Beagle dogs. METHODS LC-MS/MS method was adopted. Beagle dogs were randomly divided into single intravenous administration group （3.75 mg/kg）， single low-dose intragastric administration group （3.75 mg/kg）， single medium-dose intragastric administration group （7.5 mg/kg）， single high-dose intragastric administration group （15 mg/kg） and multiple intragastric administration group （7.5 mg/kg， once a day， for 7 consecutive days）， with 6 dogs in each group， half male and half female. The plasma samples of Beagle dogs were collected in each group according to the set time point， and underwent LC-MS/MS quantitative analysis after preprocessing. The pharmacokinetic parameters were calculated by using Phoenix WinNonlin 8.0 software using obtained data. RESULTS After intravenous injection， CL of SYHA1809 in Beagle dogs was （2.70±0.48） mL/（min·kg）， steady-state distribution volume was 0.757 L/kg， and t1/2 was （3.35±1.36） h； after single intragastric administration of low-dose， medium-dose and high-dose of SYHA1809， average tmax was （0.53±0.02） h， and the blood drug concentration increased with the increase of dose； after single intragastric administration of 3.75 mg/kg SYHA1809， the absolute bioavailability was 83.5%； within the dose range of 3.75-15 mg/kg， the increase in cmax and AUC of SYHA1809 was positively correlated with the dose； after intragastric administration of 7.5 mg/kg SYHA1809 for 7 consecutive days， the pharmacokinetic parameters of SYHA1809 were comparable to those of a single intragastric administration of the same dose， with no statistically significant difference （P＞0.05）. CONCLUSIONS SYHA1809 is absorbed rapidly in Beagle dogs， shows the dose-dependent blood concentration， high bioavailability， no obvious accumulation after multiple intragastric administration， and good pharmacokinetic behavior.

Pharmacokinetic study of HMS-01 in mice / 药学实践杂志

Objective To study the pharmacokinetics of HMS-01 in mice and provide support for subsequent studies. Methods Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to establish a sensitive and specific method for the determination of the concentration of HMS-01 in plasma and other biological samples. The pharmacokinetics of HMS-01 in C57BL/6J mice were studied by the established method. To obtain the basic pharmacokinetic parameters, three doses of HMS-01 were given orally and one dose of HMS-01 was given intravenously. Results The pharmacokinetic results of mice showed that the intestinal absorption of HMS-01 was fast, the oral bioavailability of HMS-01 in mice was moderate (50% to 70%). The exposure levels (AUC and cmax) of HMS-01 in mice increased with the increase of dosage, while the AUC was linearly correlated with the increase of dosage. After intravenous administration of HMS-01, the half-life period in mice was about 1 h which was not long. The plasma clearance rate (CLtotal.p) was 2.8 L/h·kg, which was similar to the hepatic blood flow of mice. The apparent volume of distribution (VSS) was 5 L/kg, which was much larger than the total mouse fluid. There were significant differences in AUC and F (P<0.05), but no significant differences in parameters such as cmax,AUC0−∞,t1/2,CLtot,p,MRT,Vss in male and female mice which were given 30 and 60mg/kg HWS-01 orally. Conclusion The pharmacokinetic process of HMS-01 in mice showed gender differences, and the area under the curve of blood concentration time and bioavailability of female mice were higher than that of male mice. As oral bioavailability was reasonable, further in vivo studies on HMS-01 in mice with heart failure by oral administration could be considered to provide evidence.

Structure identification and content analysis of active components in Xiaoyao pills / 药学学报

Xiaoyao pills are a famous traditional Chinese medicine collected in Welfare Pharmacy, which is a classic prescription for treating liver depression and spleen deficiency. However, its composition is complex. In order to better control the quality of Xiaoyao pills, in this study, HPLC-ion-trap time-of-flight mass spectrometry (LC-IT-TOF/MS) was used to identify the main ingredients of Xiaoyao pills, paeoniflorin, albiflorin, glycyrrhizic acid, saikosaponin A and saikosaponin B2. Then a liquid chromatography tandem mass spectrometry (LC-MS/MS) was developed for simultaneous determination and quantification of the main compounds. Fragmentation pathways of five active components were obtained. The method was validated. Five active ingredients in Xiaoyao pills had a good linear relationship, and the values of RSD (%) of repeatability were all less than 5%, the recovery ranges were between 90% and 115%, and the values of RSD (%) of each substance were less than 10% after the sample solution is placed for 24 hours. Three batches of Xiaoyao pills (concentrated pellets) and two batches of Xiaoyao pills (water pellets) were determined, the contents of paeoniflorin in concentrated pills were more than 4.0 mg·g-1, and those in water pills were more than 2.5 mg·g-1, which was accordance with Chinese Pharmacopoeia. However, other compounds behave differently. This method has high sensitivity and reliable measurement results, which provides basis for quality control of Xiaoyao pills and material basis for pharmacology research.

Analysis of Forty-Two Psychoactive Substances in a Single Hair by Micro-Segmental Technique / 法医学杂志

OBJECTIVES@#To establish an LC-MS/MS method based on single hair micro-segmental technique, and verify the detection of 42 psychoactive substances in 0.4 mm hair segments.@*METHODS@#Each piece of single hair was cut into 0.4 mm segments and extracted by sonication and the segments were immersed in dithiothreitol-containing extraction medium. Mobile phase A was the aqueous solution containing 20 mmol/L ammonium acetate, 0.1% formic acid, and 5% acetonitrile. Mobile phase B was acetonitrile. An electrospray ionization source in positive ion mode was used for data acquisition in multiple reaction monitoring (MRM) mode.@*RESULTS@#The 42 psychoactive substances in hair had a good linear relationship within their respective linear ranges (r>0.99), the limits of detection were 0.2-10 pg/mm, the limits of quantification were 0.5-20 pg/mm, the intra-day and inter-day precisions were 1.5%-12.7%, the intra-day and inter-day accuracies were 86.5%-109.2%, the recovery rates were 68.1%-98.2%, and the matrix effects were 71.3%-111.7%. The method was applied to hair samples collected from one volunteer at 28 d after a single dose of zolpidem, with zolpidem detected in 5 hairs was 1.08-1.60 cm near the root tip, and the concentration range was 0.62-20.5 pg/mm.@*CONCLUSIONS@#The micro-segmental technique of single hair analysis can be applied to the investigation of drug-facilitated sexual assault cases.

Pharmacokinetic study of five ingredients from Chitong xiaoyanling granules in rats / 中国药房

OBJECTIVE To study the pharmacokinetics of paeoniflorin， hesperidin， naringenin， formononetin and enoxolone from Chitong xiaoyanling granules in rats. METHODS Six male SD rats fasted but not deprived of water for 12 h were given Chitong xiaoyanling granules （5.0 g/kg） intragastrically at one time. Blood was collected from inner canthus of rats at different time points after administration. Plasma samples were pretreated with acetonitrile precipitated protein （sulfamethoxazole as internal standard）， and the concentrations of 5 ingredients in plasma were determined by liquid chromatography-tandem mass spectrometry （LC-MS/MS）. The pharmacokinetic parameters of each ingredient were calculated using DAS 3.0 software. RESULTS After intragastric administration of Chitong xiaoyanling granules， tmax and t1/2 of formononetin were the shortest among 5 ingredients （0.25 h and 0.39 h， respectively）. For naringenin and enoxolone， the tmax was shorter， but t1/2 was longer， and there was an obvious double-peak phenomenon in the drug-time curve of naringenin. Compared with the other three components， cmax and AUC of paeoniflorin and hesperidin were not directly proportional to the content of in vitro components. CONCLUSIONS Formononetin， naringin and enoxolone in Chitong xiaoyanling granules were absorbed rapidly in rats， while paeoniflorin and hesperidin were absorbed slowly.

Development of national secondary reference materials of urea and creatinine in frozen human serum / 中华检验医学杂志

Objective:To develop a national secondary reference material of Urea and Creatinine in frozen human serum as a standard for metrological traceability.Methods:According to JJF1343-2012 "General and Statistical Principles for Characterization of Reference Materials" and JJF 1006-1994 " Technical Norm of Primary Reference Material ", the homogeneity, stability, and commutability were evaluated;Using the JCTLM recommended methods, the value of the reference materials was assigned through collaboration with 6 accredited reference laboratories from Guangdong Provincial Hospital of Chinese Medicine, Beijing Aerospace General Hospital, Shenzhen Mindray Bio-Medical Electronics, Maccura Biotechnology, Beijing Leadman Biochemistry, and Zhejiang MedicalSystem Biotechnology. Uncertainty components including inhomogeneity, stability and value assignment were evaluated.Results:The results of one-way analysis of variance of homogeneity for the reference materials showed P>0.05, and the stability evaluation was less than the critical value of the t-test. The measured values were in the 95% confidence interval in the four conventional detection systems for commutability, and the certified values and expanded uncertainties were urea:(14.7±0.3) mmol/L ( k=2),Cr:(313.9±14.5) μmol/L ( k=2). Conclusion:The prepared secondary reference materials of urea and creatinine had promising homogeneity, stability, and commutable, the values of urea and creatinine concentration in reference materials were accurate and reliable.

Fragmentation stability and retention time-shift obtained by LC-MS/MS to distinguish sialylated N-glycan linkage isomers in therapeutic glycoproteins / 药物分析学报

Sialylated N-glycan isomers with α2-3 or 42-6 linkage(s)have distinctive roles in glycoproteins,but are difficult to distinguish.Wild-type(WT)and glycoengineered(mutant)therapeutic glycoproteins,cyto-toxic T lymphocyte-associated antigen-4-immunoglobulin(CTLA4-Ig),were produced in Chinese ham-ster ovary cell lines:however,their linkage isomers have not been reported.In this study,N-glycans of CTLA4-Igs were released,labeled with procainamide,and analyzed by liquid chromatography-tandem mass spectrometry(MS/MS)to identify and quantify sialylated N-glycan linkage isomers.The linkage isomers were distinguished by comparison of 1)intensity of the N-acetylglucosamine ion to the sialic acid ion(Ln/Nn)using different fragmentation stability in MS/MS spectra and 2)retention time-shift for a selective m/z value in the extracted ion chromatogram.Each isomer was distinctively identified,and each quantity(＞0.1％)was obtained relative to the total N-glycans(100％)for all observed ionization states.Twenty sialylated N-glycan isomers with only α2-3 linkage(s)in WT were identified,and each isomer's sum of quantities was 50.4％.Furthermore,39 sialylated N-glycan isomers(58.8％)in mono-(3 N-glycans;0.9％),bi-(18;48.3％),tri-(14;8.9％),and tetra-(4;0.7％)antennary structures of mutant were obtained,which comprised mono-(15 N-glycans;25.4％),di-(15;28.4％),tri-(8;4.8％),and tetra-(1;0.2％)sialy-lation,respectively,with only α2-3(10 N-glycans;4.8％),both α2-3 and α2-6(14;18.4％),and only α2-6(15;35.6％)linkage(s).These results are consistent with those for α2-3 neuraminidase-treated N-glycans.This study generated a novel plot of Ln/Nn versus retention time to distinguish sialylated N-glycan linkage isomers in glycoprotein.

An emerging analytical tool for highly sensitive quantitative analysis based on liquid chromatography-multiple-reaction monitoring cubed (MRM3) / 中国药科大学学报

@#Liquid chromatography-multiple-reaction monitoring (LC-MRM) has been widely recognized as the golden standard for multiple components-targeted quantitative analysis of complicated matrices,with extensive applications for analysis in such fields as chemical drugs, traditional Chinese medicines and foods.Unfortunately, when facing the task of quantitatively analyzing trace chemical components in complex matrices, MRM suffers dramatically from the background noise or matrix interference, leading to undesirable sensitivity and selectivity in terms of the lower limits of quantification (LOQ) and detection (LOD).In recent years, MRM cubed (MRM3), also known as MS3 scan, has received much attention because of its unique ability to significantly improve detection selectivity and sensitivity attributing to the successive ion filtering function, enabling LC-MRM3 as an emerging analytical tool.In this review,our attention is devoted to: 1) the illustration of the principle for MRM3; 2) parameter settings; and 3) the application progress of LC-MRM3 in such fields as the pursuit of biomarkers, pharmaceutical analysis, forensic analysis, toxicological analysis, food chemistry, and environmental analysis, aiming to provide a promising analytical tool of LC-MRM3 advantageous in the quantification analysis of trace chemical components in complex matrices.

Bioequivalence study of cinacalcet hydrochloride tablets in healthy Chinese volunteers / 中国临床药理学与治疗学

AIM: To evaluate the bioequivalence of cinacalcet hydrochloride tablets in healthy Chinese volunteers. METHODS: A randomized, open, double-period and crossover trial was conducted, 48 healthy volunteers were administered a single dose of cinacalcet test tablets or reference tablets orally under each fasting and fed condition. The concentration of cinacalcet was determined by validated LC-MS/MS method. Pharmacokinetic parameters were calculated by Phoenix WinNonlin 8.0 to study its bioequivalence. RESULTS: The main pharmacokinetic parameters of test tablets and reference tablets under fasting condition were as follows: C

Determination of 22 phospholipids in serum by ultra performance liquid chromatography-tandem mass spectrometry / 中华劳动卫生职业病杂志

Objective: To establish ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for the determination of 22 phospholipids in serum. Methods: In September 2022, Using synthetic non endogenous phospholipids as internal standard, phospholipids in serum were extracted by methanol-dichloromethane (2∶1, V/V) protein precipitation method. Chromatographic separation was achieved on an ACQUITY UPLC BEH shield RP18 column, and the mobile phase was methanol/water (5∶95, V/V) containing 10 mM ammonium formate and methanol. Detection was performed in multiple reaction monitoring mode with ion mode switching. And the method was applied by analyzing phospholipids in the serum of coal workers' pneumoconiosis patients. Results: The 22 phospholipids showed good linear relationships in their respective concentration ranges and the correlation coefficients were higher than 0.990. The spiked recoveries of the 22 phospholipids were 81.03%-121.63% at the three spiked levels. The intra-assay were less than 14.52%, and the inter-assay were less than 15.00%. Conclusion: The method with the advantages of simplicity, stability and high sensitivity, and it can be used for the analysis of phospholipids in serum.

Rapid determination of acetaminophen in plasma by LC-MS/MS / 中华劳动卫生职业病杂志

Objective: To establish a method for the rapid determination of acetaminophen (APAP) in human plasma by LC-MS/MS. Methods: The plasma samples were extracted by methanol and acetonitrile (1: 1) and purified directly. C(18) column was used for sample separation. The mobile phase were methanol (5 mmol/L ammonium acetate) and water (5 mmol/L ammonium acetate). Samples were analyzed by LC MS/MS with the electrospray ionization multi reaction monitoring (MRM) mode. Results: The calibration curves of APAP was linear in the concentration range of 0~10 mg/L, the correlation coefficient (r) was greater than 0.999 0. The relative standard deviation within and between batches was less than 10%. The recovery rate were 96.81%~101.7%. The detection limit of the method was 0.1 μg/L and the lower limit of quantification was 0.3 μg/L. Conclusion: This method has strong specificity, high sensitivity and reliable determination results. It is suitable for the rapid analysis of clinical plasma samples.

Detection of Carbamazepine and Its Metabolites in Blood Samples by LC-MS/MS / 法医学杂志

OBJECTIVES@#To establish a method for the detection of carbamazepine and its metabolites 10,11-dihydro-10,11-epoxycarbamazepine and 10,11-dihydro-10-hydroxycarbamazepine in blood samples by liquid chromatography-tandem mass spectrometry (LC-MS/MS).@*METHODS@#The blood samples were treated with 1-butyl-3-methylimidazolium hexafluorophosphate as an extraction solvent. The samples were extracted by ultrasound-assisted extraction and separated by ZORBAX Eclipse Plus C18, 95Å column. The mobile phase A aqueous solution containing 0.1% formic acid and 10 mmol/L ammonium acetate, and mobile phase B mixed organic solvent containing acetonitrile/methanol (Vacetonitrile∶Vmethanol=2∶3) were used for gradient elution at the flow rate of 1.00 mL/min. An electrospray ion source in positive mode was used for detection in the multiple reaction monitoring.@*RESULTS@#The linearities of carbamazepine and its metabolites 10,11-dihydro-10,11-epoxycarbamazepine and 10,11-dihydro-10-hydroxycarbamazepine in blood samples were good within the corresponding range, with correlation coefficients (r) greater than 0.995 6. The limits of detection were 3.00, 0.40 and 1.30 ng/mL, respectively. The limit of quantitation were 8.00, 1.00 and 5.00 ng/mL, respectively. The extraction recoveries ranged from 76.00% to 106.44%. The relative standard deviations of the intra-day and inter-day precisions were less than 16%. Carbamazepine and its main metabolite 10,11-dihydro-10,11-epoxycarbamazepine were detected in blood samples of death cases with a mass concentration of 2.71 μg/mL and 252.14 ng/mL, respectively.@*CONCLUSIONS@#This method has high sensitivity and good selectivity, which is suitable for the detection of carbamazepine and its metabolites in blood samples, and can be used for carbamazepine-related forensic identifications.

Proteomic Difference Analysis of Whole Blood and Bloodstains / 法医学杂志

OBJECTIVES@#To study the changes of protein levels in peripheral blood after it dried.@*METHODS@#The proteins from whole blood and bloodstains were detected by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and normalized by the label-free quantification (LFQ) method. The differential proteins were analyzed by using R 4.2.1 software, limma and edgeR package. The analysis of biological function, signaling pathway and subcellular localization for the differential proteins was then performed.@*RESULTS@#A total of 623 and 596 proteins were detected in whole blood and bloodstains, respectively, of which 31 were statistically significant in the quantitative results, including 10 up-regulated and 21 down-regulated proteins in bloodstains.@*CONCLUSIONS@#The protein abundances in whole blood and bloodstains are highly correlated, and the variation of protein abundances may be related to the changes of endogenous and structural proteins in cells. The application of proteomics technology can assist the screening and identification of protein biomarkers, thereby introducing new biomarkers for forensic research.

Pharmacokinetic study about compatibility of Eucommia ulmoides and Psoralea corylifolia / 中草药·英文版

OBJECTIVE@#The compatibility of Eucommia ulmoides (Eu) and Psoralea corylifolia (Pc) on the pharmacokinetic (PK) properties in the rat was explored in this study.@*METHODS@#Eu extract, Pc extract and the combined extracts (crude drug ratio was 2:1) was administered by gavage, respectively. Two PK experiments were conducted. In first one, the blood samples were collected via the occuli chorioideae vein to get the PK properties of the components. In second one, the blood samples were simultaneously collected via the internal jugular vein or portal vein at different time points and the concentrations of target ingredients were detected by LC/MS/MS to clear the location where the interaction of Eu and Pc took place in vivo.@*RESULTS@#Eight of 11 ingredients in Eu and Pc extract were determined in rat plasma. The exposure levels of geniposidic acid (GPA), aucubin (AU), geniposide (GP), pinoresinol diglucoside (PDG), psoralen glycosides (PLG) and isopsoralen glycosides (IPLG) were decreased 1/5-2/3 after administration of combined extracts. Comparing to the combined administration, the exposure of GPA and AU in plasma of single Eu administration collected via the portal vein were decreased 1/3-2/3, and the values of AUC0-24h and AUC0-∞ of GP collected from the portal vein or internal jugular vein were double increased. The other components' parameters were not significantly changed.@*CONCLUSION@#In summary, the Pc and Eu combined administration could affect the exposure of the main components of Eu extract in rats due to the changed intestinal absorption. The research on the compatibility of Pc and Eu was helpful to guide the clinical administration of Eu and Pc simultaneously.

Establishment and application for simultaneous determination method of atorvastatin and its active/toxic metabolites in rat plasma / 中国药房

OBJECTIVE To establish a method for simultaneous determination of atorvastatin （ATV） and its active metabolites 2-hydroxy atorvastatin acid （2-HAT）， 4-hydroxy atorvastatin acid （4-HAT） and toxic metabolite atorvastatin lactone （ALT） in rat plasma and apply it for pharmacokinetic study. METHODS LC-MS/MS method was adopted for analysis. The one-step precipitation method was used for processing plasma samples （plasma samples were pretreated by acidification to adjust pH value so as to prevent inversion of configuration）， gradient elution was used to analyze the samples， and the analysis time was 5 min. Electrospray positive ionization was adopted， and positive ion scanning was performed in multi-reaction monitoring. The m/z of quantified ion pairs of ATV and its metabolites such as 2-HAT， 4-HAT and ATL， and internal standard pitavastatin were 559.3→ 440.2， 575.2→440.3， 575.0→440.2， 540.9→448.2 and 422.2→290.0， respectively. After conducting a comprehensive methodological investigation of the analytical method， the concentrations of ATV and its metabolites 2-HAT， 4-HAT，and ATL were determined， and the pharmacokinetic parameters of ATV and its metabolites were calculated using the non- compartment model of WinNonlin 6.1. RESULTS The results of methodological validation showed that endogenous substances in blank plasma did not interfere with the determination of the components to be tested， and the standard curve had a good linear relationship； the lower limits of quantification for ATV， 2-HAT， 4-HAT and ATL were 0.5， 0.5， 0.25 and 0.063 nmol/L， respectively. The precision， accuracy， recovery， matrix effect and stability investigation were all in line with the requirements of biological analysis. Pharmacokinetic analysis showed that after intragastric administration in rats， ATV calcium metabolized rapidly， and was mainly exposed to blood circulation in the form of ATV and 2-HAT， with the lowest concentration of lactone-type metabolites. CONCLUSIONS The established method is precise， rapid and accurate for plasma concentration analysis of ATV and its active/toxic metabolites. The application of the method could help to fully elucidate the pharmacokinetic characteristics of atorvastatin calcium in rats.

Development and validation of liquid chromatography-tandem mass spectrometry method to quantify dasatinib in plasma and its application to a pharmacokinetic study

Abstract Dasatinib, a potent oral multi-targeted kinase inhibitor against Src and Bcr-Abl, can decrease inflammatory response in sepsis. A simple and cost-effective method for determination of an effective dose dasatinib was established. This method was validated in human plasma, with the aim of reducing the number of animals used, thus, avoiding ethical problems. Dasatinib and internal standard lopinavir were extracted from 180 uL of plasma using liquid-liquid extraction with methyl tert-butil ether, followed by liquid chromatography coupled to triple quadrupole mass spectrometry in multiple reaction monitoring mode. For the pharmacokinetic study, 1 mg/kg of dasatinib was administered to mice with and without sepsis. The method was linear over the concentration range of 1-98 ng/mL for DAS in mice and human plasma, with r2>0.99 and presented intra- and interday precision within the range of 2.3 - 6.2 and 4.3 - 7.0%, respectively. Further intra- and interday accuracy was within the range of 88.2 - 105.8 and 90.6 - 101.7%, respectively. The mice with sepsis showed AUC0-t = 2076.06 h*ng/mL and Cmax =102.73 ng/mL and mice without sepsis presented AUC0-t = 2128.46 h*ng/mL. Cmax = 164.5 ng/mL. The described analytical method was successfully employed in pharmacokinetic study of DAS in mice.

Performance evaluation of automated magnetic beads extraction method for the measurement of catecholamine metabolites analyzed by liquid chromatography tandem mass spectrometry / 中华检验医学杂志

Objective:To evaluate the performance of magnetic beads extraction method (MGE) for the measurement of catecholamine metabolites by liquid chromatography tandem mass spectrometry.Methods:This is a methodological evaluation study. The linearity, limit of quantitation, recovery, precision, and matrix effect of catecholamine metabolites 3-methoxyepinephrine (MN), 3-methoxynorepinephrine (NMN) and 3-methoxytyramine (3-MT) extracted by MGE method were evaluated according to CLSI C62-A. Consensus of method development and validation of liquid chromatography-tandem mass spectrometry in clinical laboratories and other guidelines, 132 clinical residual plasma samples were collected and extracted by automated MGE and traditional solid phase extraction (SPE) method to compare the harmonization of the two extraction methods.Results:The linearity of MN, NMN and 3-MT extracted by automated MGE was>0.99, and the LOQ for MN, NMN and 3-MT were 0.033 5 nmol/L, 0.054 7 nmol/L and 0.011 0 nmol/L, respectively. The repeatability of MN, NMN and 3-MT were 1.3%-5.1%, 2.2%-5.6% and 1.7%-7.1%, respectively. The total imprecision in the laboratory were 1.5%-8.2%, 2.2%-7.7%, 2.1%-11.2%. Although the absolute recovery is low, the average relative recoveries of MN, NMN and 3-MT were 91.5%-108.5%, 92.0%-108.6%, and 89.3%-104.1%, respectively, and the percentage deviation from the expected concentration was within 15%. After isotope internal standard correction, the relative matrix effect is close to 100%, which can compensate for the potential matrix effect. The results of MGE and SPE of MN, NMN and 3-MT showeda good correlation (correlation coefficient r>0.99). The average relative deviations of MN, NMN and 3-MT were 0.2%, -1.4% and 1.0%, respectively. Conclusion:The automatic MGE method hasa good performance in extracting catecholamine metabolites, and is expected to be used in high-throughput analysis of samples in clinical in the future.

Orthogonal quantification of soluble inducible T-cell costimulator(ICOS)in healthy and diseased human serum / 药物分析学报

Inducible T-cell costimulator(ICOS),a homodimeric protein expressed on the surface of activated T-cells,is being investigated as a potential therapeutic target to treat various cancers.Recent studies have re-ported aberrant increases in the soluble form of ICOS(sICOS)in human serum in disease-state patients,primarily using commercial ELISA kits.However,results from our in-house immunoassay did not show these aberrant increases,leading us to speculate that commercial sICOS ELISAs may be prone to inter-ference.We directly tested that hypothesis and found that one widely used commercial kit yields false-positives and is prone to human anti-mouse antibody interference.We then analyzed a panel of healthy,cancer,chronic hepatitis C virus,systemic lupus erythematosus,and diffuse cutaneous systemic sclerosis human serum using our in-house immunoassay and reported the measured sICOS concentrations in these populations.Since even well characterized immunoassay methods are prone to non-specific interference,we also developed a novel sICOS LC-MS/MS method to confirm the results.Using these orthogonal approaches,we show that sICOS is a low abundance soluble protein that cannot be measured above approximately 20 pg/mL in human serum.