Determination of hydrogen sulfide in Blood by LC-MS/MS / 中国法医学杂志

Objective To establish a method for determining hydrogen sulfide(H2S)in blood and apply it to practical cases.Methods A delute solution was achieved by adding 0.8 mL saturated borax solution into 0.2 mL blood sample was diluted with.1 mL acetonitrile solution containing 0.1％formic acid was then taken in a test tube,followed by adding 0.1 mL dilute solution and 0.1 mL thiozine aqueous solution(1％).After thorough mixing,the mixture was left to stand for 30 minutes.Subsequently,the sample was subjected to liquid chromatography-tandem mass spectrometry(LC-MS/MS)analysis after centrifugation and membrane filtration.Results The results showed that H2S exhibited good linearity within the concentration range of 10～2 000 ng/mL,with the R2 value of 0.998 5.The detection limit was 5 ng/mL,and the quantification limit was 10 ng/mL.In three cases of H2S poisoning,sulfur ions were detected in the blood of the deceased individuals,with concentrations ranging from 0.17 to 0.56 μg/mL.Conclusion For the first time,this study established a LC-MS/MS method for determining H2S in blood,which can meet the detection needs of H2S poisoning cases.

Detecting Ketamine analogues in hair samples by QTRAP LC-MS/MS / 中国法医学杂志

Objective To develop a method for the determination of ketamine analogues in hair samples by liquid chromatography quadrupole linear ion trap mass spectrometry(QTRAP LC-MS/MS).Methods 20 mg of washed and dried hair was added to 1 mL extracting solution and then prepared using an ultrasonic extraction with frozen pulverization method.After centrifugation and purification with membrane,the supernatant was separated in a ACQUITY UPLC? HSS T3 column with gradient elution,finally tested with multiple reaction monitoring for the detection of 10 ketamine analogues.The above method was applied for quantitative analysis of ethylfluamine,F-norketamine and tiletamine in 20 positive samples.Results When the concentration ranged from 0.01 to 2.00 ng/mg,there was good linearity for 10 ketamine analogues with the correlation coefficients over 0.99.The recoveries ranged from 89.1％to 106.1％,and the matrix effects were between 88.3％and 106.0％.Among the 20 positive samples,the contents of ethylfluamine,F-norketamine and tiletamine in hair ranged between 0.02～8.35 ng/mg,0.01～0.94 ng/mg and 0.02～10.93 ng/mg,respectively.Their mean values were 1.59 ng/mg,0.28 ng/mg and 2.69 ng/mg.Their medians were 0.40 ng/mg,0.19 ng/mg and 2.11 ng/mg.Conclusion The established method was simple,efficient,reliable and suitable for the determination of ketamine analogues in hair.The data provided reference for the drug control and forensic science practice.

Simultaneous determination of bepotastine and hydroxychloroquine concentrations in human breast milk by LC-MS/MS / 中国药房

OBJECTIVE To establish a liquid chromatography-tandem mass spectrometry （LC-MS/MS） method for the simultaneous determination of bepotastine and hydroxychloroquine concentrations in human breast milk and apply it in clinical practice. METHODS The milk samples （50 μL） were precipitated with 200 μL methanol containing the internal standard （100 ng/mL chloroquine）， and the supernatant was taken for analysis after vortexing and centrifugation. The separation was performed on a Waters ACQUITY UPLC HSS T3 column with mobile phase consisted of 0.1% formic acid-10 mmol/L ammonium acetate solution （phase A） and methanol （phase B） at gradient elution of 0.35 mL/min. The injection volume was 2 μL， and the analysis time was 4 min. The detection of the analytes was performed by electrospray ionization in positive mode by multiple reaction monitoring with the transition of m/z 388.9→201.9 （bepotastine）， m/z 336.3→247.1 （hydroxychloroquine）， and m/z 320.2→247.2 （chloroquine）. The established LC-MS/MS method was researched in methodology and used to determine the drug concentrations in the breast milk of 1 case of lactating patient. RESULTS The linear range of bepotastine was 2-200 ng/mL（ r＝0.999）， and hydroxychloroquine was 50-1 000 ng/mL （r＝0.998）. The intra-assay and inter-assay precisions were both ≤15%， and the accuracy， extraction recovery， matrix effect， and stability all met the acceptance criteria for bioanalytical method validation. The concentration result of bepotastine and hydroxychloroquine in the breast milk of the lactating patient showed， after 2 h and 14 h， the concentrations of bepotastine in the breast milk of the patient were 34.95 ng/mL and 5.72 ng/mL； those of hydroxychloroquine were 211.92 ng/mL and 104.18 ng/mL， respectively. The relative infant doses were 1.83% and 0.56%， respectively. CONCLUSIONS The established method is simple， rapid， and sensitive. It is suitable for simultaneous determination of bepotastine and hydroxychloroquine concentrations in human milk and can provide reference for safe drug use during lactation.

Simultaneous determination of bepotastine and hydroxychloroquine concentrations in human breast milk by LC-MS/MS / 中国药房

OBJECTIVE To establish a liquid chromatography-tandem mass spectrometry （LC-MS/MS） method for the simultaneous determination of bepotastine and hydroxychloroquine concentrations in human breast milk and apply it in clinical practice. METHODS The milk samples （50 μL） were precipitated with 200 μL methanol containing the internal standard （100 ng/mL chloroquine）， and the supernatant was taken for analysis after vortexing and centrifugation. The separation was performed on a Waters ACQUITY UPLC HSS T3 column with mobile phase consisted of 0.1% formic acid-10 mmol/L ammonium acetate solution （phase A） and methanol （phase B） at gradient elution of 0.35 mL/min. The injection volume was 2 μL， and the analysis time was 4 min. The detection of the analytes was performed by electrospray ionization in positive mode by multiple reaction monitoring with the transition of m/z 388.9→201.9 （bepotastine）， m/z 336.3→247.1 （hydroxychloroquine）， and m/z 320.2→247.2 （chloroquine）. The established LC-MS/MS method was researched in methodology and used to determine the drug concentrations in the breast milk of 1 case of lactating patient. RESULTS The linear range of bepotastine was 2-200 ng/mL（ r＝0.999）， and hydroxychloroquine was 50-1 000 ng/mL （r＝0.998）. The intra-assay and inter-assay precisions were both ≤15%， and the accuracy， extraction recovery， matrix effect， and stability all met the acceptance criteria for bioanalytical method validation. The concentration result of bepotastine and hydroxychloroquine in the breast milk of the lactating patient showed， after 2 h and 14 h， the concentrations of bepotastine in the breast milk of the patient were 34.95 ng/mL and 5.72 ng/mL； those of hydroxychloroquine were 211.92 ng/mL and 104.18 ng/mL， respectively. The relative infant doses were 1.83% and 0.56%， respectively. CONCLUSIONS The established method is simple， rapid， and sensitive. It is suitable for simultaneous determination of bepotastine and hydroxychloroquine concentrations in human milk and can provide reference for safe drug use during lactation.

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.

Aplicação da Toxicologia Analítica Verde no desenvolvimento de metodologias baseadas em LC-MS para detecção de Novas Substâncias Psicoativas em amostras biológicas / Application of Green Analytical Toxicology to the development of LC-MS-based methodologies for the detection of New Psychoactive Substances in biological samples

No início dos anos 2000, as Novas Substâncias Psicoativas (NPS) emergiram de forma sem precedentes causando uma drástica mudança no mercado de drogas sintéticas mundial. Estas substâncias são sintetizadas para fins ilícitos e mimetizam o efeito psicoativo das drogas tradicionais. Até o momento, mais de 1000 substâncias foram reportadas mundialmente, representando um grande problema de saúde pública principalmente associado ao desconhecimento das suas propriedades toxicológicas. Por este motivo, métodos analíticos para detectar e quantificar estas substâncias em materiais biológicos são importantes nos casos de toxicologia analítica e forense. Contudo, a tendência de reduzir o impacto ambiental destas metodologias tem ganhado popularidade com a Toxicologia Analítica Verde (GAT). Portanto, o objetivo do presente trabalho foi desenvolver novas técnicas analíticas para analisar as principais classes de NPS em amostras biológicas enquanto aplicando os princípios sustentáveis estabelecidos pela GAT. Os resultados obtidos no presente trabalho são apresentados como coletânea de artigos científicos publicados em revistas. Estes estão descritos nos capítulos 4 a 8. No capítulo 4, uma revisão sobre os desafios no desenvolvimento de técnicas de preparo de amostra para fins forenses é abordada com foco no uso das matrizes secas. No Capítulo 5, está descrito a aplicação da microextração líquido-líquido dispersiva para catinonas sintéticas em amostras de sangue total e urina. No capítulo 6, o artigo descreve o desenvolvimento da técnica microextração líquido-líquido homogênea com solventes de hidrofilicidade comutável para canabinoides sintéticos em amostras de plasma. No capítulo 7, a microextração em fase líquida em placas de 96 poços, cunhada extração paralela em membranas artificiais líquidas, foi desenvolvida para diferentes classes de drogas de abuso, incluindo NPS. O capítulo 8 mostra o desenvolvimento de uma extração por eletromembrana também no formato de placa de 96 poços para catinonas sintéticas em amostras de sangue total. Em todos os trabalhos, as técnicas de extração foram desenvolvidas, otimizadas e validadas. Os princípios da GAT foram aplicados de diferentes formas, como reduzindo o volume de amostra, simplificando os procedimentos, evitando o uso de solventes orgânicos, dentre outros. Assim, alternativas mais sustentáveis para a análise de drogas de abuso em amostras biológicas foram apresentadas e estas ajudam a consolidar e difundir o conceito do desenvolvimento de métodos analíticos com consciência ambiental além de fornecer ferramentas para auxiliar o controle das NPS no país

In the early 2000s, New Psychoactive Substances (NPS) emerged and unprecedentedly changed the illicit drug market. These substances are synthesized for illicit purposes and mimic the psychoactive effect of traditional drugs of abuse. To date, more than 1000 substances have been reported worldwide, representing a major public health problem mainly associated with their mostly unknown toxicological properties. In this context, analytical methods able to detect and quantitate these new drugs in biological specimens are important in cases of analytical and forensic toxicology. However, reducing the environmental impact of these methodologies has recently gained popularity with Green Analytical Toxicology (GAT). Therefore, the aim of this work was to develop new analytical techniques to analyze the main classes of NPS in biological samples while applying the environmentally friendly principles established by GAT. The results obtained throughout the development of the present work were split into four papers (chapters 4-8). In chapter 4, a review of common challenges faced during the development of new sample preparation techniques for forensic applications is described focusing on the use of dried matrices. In chapter 5, the application of dispersive liquid-liquid microextraction for synthetic cathinones in whole blood and urine samples is described. In chapter 6, the application of the somewhat recent switchable hydrophilicity solvent-based homogenous liquidliquid microextraction to synthetic cannabinoids in plasma samples is reported. In chapter 7, liquid-phase microextraction in the 96-well plate format, termed parallel artificial liquid membrane extraction, for different classes of drugs of abuse, including NPS, in plasma samples is presented. In chapter 8, an electromembrane extraction in the 96-well plate format for synthetic cathinones in whole blood samples was developed. In this work, sample preparation techniques were developed, optimized and validated. The principles of sustainable chemistry in method development were applied in different ways, such as reducing the sample volume, simplifying procedures, avoiding the use of organic solvents, among others. Thus, greener alternatives were presented for the analysis of drugs of abuse in biological samples and contribute to consolidate and spread this trend of environmental consciousness during method development. Additionally, valuable techniques that can be used in the combat against NPS were provided

Neuroprotective effect of Capparis ovata Desf. var. palaestina Zoh. on H2O2­induced neurotoxicity in SH­SY5Y cells / Efecto neuroprotector de Capparis ovata Desf. var. palaestina Zoh. sobre la neurotoxicidad inducida por H2O2 en células SH ­SY5Y

The neuroprotective effect of flower and fruit parts of Capparis ovata Desf. var. palaestina Zoh. plant was investigated in H2O2-induced cytotoxicity in SH-SY5Y cells. The cells were treated with H2O2 alone or pretreated with flower (COMFL) and fruit extract (COMFR) of C. ovatavar. palaestina. MTT, xCELLigence, and qualitative and quantitative determination of phytochemical constituents in the extracts by LC-MS/MS methods were employed. COMFL and COMFR had a neuroprotective effect and this effect was stronger when the presence of oxidative stress. The mass spectrums revealed the presence of flavonoids and phenolic acid derivatives in the extracts. According to quantitative analyses, the main compounds were myristoleic acid, apigenin, caffeic acid, caffeic acid-3-glucoside, and 5-cynapoil quinic acid in both COMFL and COMFR and rutin was found in COMFL. The extracts could inhibit H2O2induced neuronal cell death which might be beneficial for the pretreatment of oxidative stress in neurodegenerative diseases.

Se investigó el efecto neuroprotector de flores y frutos de Capparis ovata Desf. var. palaestina Zoh sobre la citotoxicidad inducida por H2O2en células SH-SY5Y. Las células se trataron con H2O2solo o se pretrataron con extracto de flores (COMFL) y frutos (COMFR) de C. ovatavar. palaestina. Se emplearon MTT, xCELLigence y determinación cualitativa y cuantitativa de constituyentes fitoquímicos en los extractos mediante LC-MS/MS. COMFL y COMFR que tuvieron un efecto neuroprotector y este efecto fue mayor cuando hubo estrés oxidativo. Los espectros de masas revelaron la presencia de flavonoides y derivados del ácido fenólico en los extractos. Según los análisis cuantitativos, los compuestos principales fueron ácido miristoleico, apigenina, ácido cafeico, ácido cafeico-3-glucósido y ácido quínico 5-cinapoil tanto en COMFL como en COMFR y se encontró rutina en COMFL. Los extractos podrían inhibir la muerte celular neuronal inducida por H2O2, lo que podría ser beneficioso para el pretratamiento del estrés oxidativo en enfermedades neurodegenerativas.

Click chemistry-based enrichment strategy for tracing cellular fatty acid metabolism by LC-MS/MS / 药物分析学报

Fatty acids(FAs),which were initially recognized as energy sources and essential building blocks of biomembranes,serve as the precursors of important signaling molecules.Tracing FA metabolism is essential to understanding the biochemical activity and role of FAs in physiological and pathological events.Inspired by the advances in click chemistry for protein enrichment,we herein established a click chemistry-based enrichment(CCBE)strategy for tracing the cellular metabolism of eicosapentaenoic acid(EPA,20:5 n-3)in neural cells.Terminal alkyne-labeled EPA(EPAA)used as a surrogate was incubated with N2a,mouse neuroblastoma cells,and alkyne-labeled metabolites(ALMs)were selectively captured by an azide-modified resin via a Cu(I)-catalyzed azide-alkyne cycloaddition reaction for enrichment.After removing unlabeled metabolites,ALMs containing a triazole moiety were cleaved from solid-phase resins and subjected to liquid chromatography mass spectrometry(LC-MS)analysis.The proposed CCBE strategy is highly selective for capturing and enriching alkyne-labeled metabolites from the complicated matrices.In addition,this method can overcome current detection limits by enhancing MS sensitivity of targets,improving the chromatographic separation of sn-position glycerophospholipid regioisomers,facilitating structural characterization of ALMs by a specific MS/MS fragmentation signature,and providing versatile fluorescence detection of ALMs for cellular distribution.This CCBE strategy might be expanded to trace the metabolism of other FAs,small molecules,or drugs.

Quantitative analysis of polymyxin B in human serum by LC-MS/MS / 药物流行病学杂志

Objective To develop a simple and sensitive LC-MS/MS method for the determination of polymyxin B in human serum.Methods Polymyxin E2 was used as an internal standard and acetonitrile(0.1%formic acid)was used for protein precipitation.Chromatographic separation was performed on a Hypersil GOLDTM C18 column.Mobile phase A was 0.1%formic acid in water,and mobile phase B was methanol.The flow rate was 0.4 mL·min-1,with gradient elution.The column temperature was 40℃.The injection volume was 2 μL and the analysis time was 3.3 min.Multiple reaction monitoring and electrospray ion source positive ion mode were applied for quantitative analysis.The quantitative analysis ion pairs were m/z 402.10>101.10(polymyxin B1),m/z 397.45>101.10(polymyxin B2),m/z 578.50>101.10(polymyxin E2).Results The linear relationship of polymyxin B1 was at 22-15 000 ng·mL-1 and B2 was at 5-1 700 ng·mL-1 in human serum,which achieved excellent linearity,and the correlation coefficient was higher than 0.999 0.The lower limit of quantification of polymyxin B1 and B2 were 22 ng·mL-1 and 5 ng·mL-1,respectively.The accuracies were 95.98%-104.60%for polymyxin B1,98.11%-105.59%for polymyxin B2,respectively.The RSDs of intra-and inter-day precision were less than 15%.The recoveries of polymyxin B1 and B2 were 97.26%-103.31%and 95.81%-101.22%,respectively,and the matrix effects were 96.52%-109.54%and 93.29%-109.95%,respectively.The RSDs of the samples were all less than 15%.The mean AUC0-24h was(72.85±17.87)mg·h·L-1 in six patients.Conclusion The established LC-MS/MS method for determining polymyxin B in human serum meets the requirements of biological sample analysis.It is suitable for determining polymyxin B in human serum.

Postmortem redistribution of amantadine in rats / 中国法医学杂志

Objective To establish an animal model of postmortem redistribution of amantadine,and to study its postmortem redistribution in rats,so as to provide experimental evidence for forensic identification.Methods One hundred and twenty-six male SD rats were randomly divided into 3 groups and subjected to intragastric administration according to the maximum dose of treatment(L),LD50(M)and 2LD50(H).Those who did not die were killed according to the average time of death of LD50.Heart-blood,peripheral blood,heart,liver,spleen,lung,kidney,brain,muscle and testis were collected at 0 h,6 h,12 h,24 h,48 h,72 h and 96 h after death,and amantadine content was detected.Results For the rats in the L group,the concentration of amantadine decreased within 6 h after death and then increased in the heart-blood,heart and liver,unchanged within 48 h and reached the peak at 96 h in the spleen,kidney,brain,muscle and testis,while decreased in the lung.For the rats in the M group,the concentration of amantadine decreased within 24 h after death and then increased in all samples,and it reached the peak at 48 h after death in the peripheral blood,spleen,kidney and muscle tissues,at 72 h after death in the heart-blood and testis,and at 96 h after death in the liver,lung and brain tissues.For the rats in the H group,the concentration of amantadine showed a downward trend within 12 h after death in the heart and liver tissue,showed a downward trend within 48 h after death in the lung,brain and muscle tissue,and reached the peak at 96 h after death in the heart,liver,spleen,muscle and testicle tissues.Conclusion The postmortem redistribution was found in amantadine poisoning dead rats,which could provide experimental evidence for the forensic identification of death cases caused by amantadine poisoning.

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.

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.

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

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.

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.

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.

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.

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.

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.

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.