Comparison of two MALDI-TOF MS systems for theidentification of clinically relevant anaerobic bacteriain Argentina

Abstract The aim of this study was to compare the performance of two MALDI-TOF MS systems in the identification of clinically relevant strict anaerobic bacteria. The 16S rRNA gene sequencing was the gold standard method when discrepancies or inconsistencies were observed between platforms. A total of 333 isolates were recovered from clinical samples of different centers in Buenos Aires City between 2016 and 2021. The isolates were identified in duplicate using two MALDI-TOF MS systems, BD Bruker Biotyper (Bruker Daltonics, Bremen, Germany) and Vitek MS (bioMèrieux, Marcy-l'Etoile, France). Using the Vitek MS system, the identification of anaerobic isolates yielded the following percentages: 65.5% (n: 218) at the species or species-complex level, 71.2% (n: 237) at the genus level, 29.4% (n: 98) with no identification and 5.1% (n: 17) with misidentification. Using the Bruker Biotyper system, the identification rates were as follows: 85.3% (n: 284) at the species or species-complex level, 89.7% (n: 299) at the genus level, 14.1% (n: 47) with no identification and 0.6% (n: 2) with misidentification. Differences in the performance of both methods were statistically significant (p-values <0.0001). In conclusion, MALDI-TOF MS systems speed up microbial identification and are particularly effective for slow-growing microorganisms, such as anaerobic bacteria, which are difficult to identify by traditional methods. In this study, the Bruker system showed greater accuracy than the Vitek system. In order to be truly effective, it is essential to update the databases of both systems by increasing the number of each main spectrum profile within the platforms.

Resumen El objetivo de este estudio fue comparar el desempeño de dos sistemas MALDI-TOF MS en la identificación de bacterias anaerobias estrictas de interés clínico. La secuenciación del gen 16S ARNr fue el método de referencia utilizado cuando se observaron discrepancias o inconsistencias entre plataformas. Se recuperaron 333 aislados de muestras clínicas de diferentes centros de la Ciudad Autónoma de Buenos Aires entre 2016 y 2021. Los aislados se identificaron por duplicado mediante dos sistemas MALDI-TOF MS: el BD Bruker Biotyper (Bruker Daltonics, Bremen, Alemania) y el Vitek MS (bioMèrieux, Marcy-l'Etoile, Francia). A través del sistema Vitek MS, los mismos fueron identificados a nivel de especie o complejo de especies en un 65,5% (n: 218) y de género en un 71,2% (n: 237), mientras que no se identificaron en un 29,4% (n: 98) y fue incorrecta en el 5,1% (n: 17). Mediante el sistema Bruker Biotyper, dichos valores fueron del 85,3% (n: 284), del 89,7% (n: 299), del 14,1% (n: 47) y del 0,6% (n: 2), respectivamente. La diferencia entre ambos métodos fue estadísticamente significativa (p<0,0001). En conclusión, los sistemas MALDI-TOF MS aceleran la identificación microbiana. Son especialmente útiles para los microorganismos de crecimiento lento, como las bacterias anaerobias, que son difíciles de identificar con los métodos tradicionales. El sistema Bruker demostró ser más preciso que el Vitek MS. Para que estos métodos sean realmente efectivos es fundamental actualizar las bases de datos de ambos sistemas e incrementar el número de espectros de referencia dentro de las plataformas.

Physicochemical properties, metabolomic analysis, antioxidant and lipid - lowering activity of a functional beverage based on cocona (Solanum sessiliflorum Dunal) / Propiedades fisicoquímicas, análisis metabolómico, actividad antioxidante e hipolipemiante de una bebida funcional a base de cocona (Solanum sessiliflorum Dunal)

The physicochemical, microbiological and metabolomics analysis, antioxidant and lipid - lowering effect, and shelf life prediction of a functional beverage based on cocona pul p of SRN9 ecotype was to carry out. According to the results obtained, the beverage complies with all the characteristics of the Peruvian technical standard for juices, nectars and fruit beverages NTP 203.110:2009 and is within the limits established by th e sanitary technical standard NTS N° 071 - MINSA/DIGESA - V.01, with a shelf - life period of 4 months and 1 day. The metabolome regarding bioactive compounds showed the presence of 30 compounds, including several glycosylated flavonols, two flavanols, and two s permidines. Likewise, showed a lipid - lowering effect statistically significant (p < 0.05) about the serum levels of total cholesterol and triglycerides, with a mean reduction of 41.52 mg/dL for total cholesterol levels and 130.80 mg/dL for triglyceride lev els. This beverage could be an alternative for the treatment of atherosclerosis and prevention of cardiovascular diseases.

Se rea lizó el análisis fisicoquímico, microbiológico y metabolómico, efecto antioxidante e hipolipemiante, y vida útil de una bebida funcional a base de cocona ecotipo SRN9. De acuerdo a los resultados, la bebida cumple con las características de la norma técnic a peruana para jugos, néctares y bebidas de frutas NTP 203.110:2009 y se encuentra dentro de los límites establecidos por la norma técnica sanitaria NTS N° 071 - MINSA/DIGESA - V.01, con una vida útil de 4 meses y 1 día. Del perfil metabolómico se identificaro n 30 compuestos, entre ellos varios flavonoles glicosilados, dos flavanoles y dos espermidinas. Asimismo, mostró un efecto hipolipemiante estadísticamente significativo (p < 0,05) sobre los niveles séricos de colesterol total y triglicéridos, con una reduc ción media de 41,52 mg/dL y de 130,80 mg/dL para los niveles de colesterol total y de triglicéridos, respectivamente. Esta bebida podría ser una alternativa para el tratamiento de la aterosclerosis y prevención de enfermedades cardiovasculares.

Identification of cancer inhibitors from Hystrix brachyura bezoar extracts using LC - MS multivariate data analysis and in silico evaluation on Bcl - 2, cyclin B/CDK1, VEGF and NM23 - H1 / Identificación de inhibidores de cáncer a partir de extractos de bezoar de Hystrix branchyura usando análisis multivariado LC - MS y evaluación in silico en Bcl - 2, cyclin B/CDK1, VEGF, y NM23 - H1

Hystrix brach yura bezoar is calcified undigested material found in the gastrointestinal tract known for various medicinal benefits including as an anticancer agent. However, the H. brachyura population has been declining due to its demand and is under Malaysian law pro tection. Therefore, present study aimed to identify bezoar anticancer active compounds through metabolomics and in - silico approaches. Five replicates of bezoar powder were subjected to extraction using different solvent ratios of methanol - water (100, 75, 5 0, 25, 0% v/v). Cytotoxicity and metabolite profiling using liquid chromatography - mass spectrometry were conducted. Putative compounds identified were subjected to in - silico analysis with targeted anticancer proteins namely, Bcl - 2, Cyclin B/CDK1 complex, V EGF and NM23 - H1. The correlation of LC - MS and cytotoxicity profile pinpointed two compounds, mangiferin and propafenone. In - silico study showed both compounds exerted good binding scores to all proteins with hydrophobic interaction dominating the ligand - pr otein complex binding, suggesting the ligands act as hydrophobes in the interactions.

El bezpar de Hystrix branchyura es material calcificado sin digerir encontr ados en el tracto gastrointestinal, conocido por sus variados beneficios médicos, incluyendo propiedades anticancerosas. De todas formas, la población de H. Branchyura ha ido declinando debido a su demanda y está bajo la protección de la ley de Malasia. Po r esto, este estudio busca identificar los componentes activos anticancerosos del bezoar mediante abordajes metabolómico e in silico. Cinco réplicas de polvo de bezoar fueron sometidos a extracción usando solventes con diferentes proporciones metanol - agua (100, 75, 50, 25, 0% v/v). Se hicieron perfiles de citotoxicidad y de metabolitos usando cromatografía líquida - espectrometría de masa ( LC - MS ). Se identificaron compuestos putativos yse sometieron a a nálisis in silico, buscando las proteínas anticancerosas B cl - 2, complejo Cyclin B/CDK1, VEGF, y NM23 - H1. La correlación LC - MS y el perfil de citotoxicidad identificaron dos compuestos: mangiferina y propafenona. El estudio in silico mostró que ambos compuestos tenían buenos índices de enlace con todas las proteín as con interacción hidrofóbica dominando el enlace complejo proteína - ligando, sugeriendo que los ligandos actúan como hidrófobos en las interacciones

Effect of Atractylodis Macrocephalae Rhizoma on Short-chain Fatty Acids and Intestinal Barrier in Mice with Slow-transit Constipation / 中国实验方剂学杂志

ObjectiveTo investigate the mechanism of Atractylodis Macrocephalae Rhizoma（AMR） in the treatment of slow-transmission constipation（STC） by observing the effects of AMR on short-chain fatty acids and intestinal barries in STC mice. MethodForty-eight male KM mice were randomly divided into blank group， model group， AMR low-， medium-， high-dose groups（2.5， 5， 10 g·kg-1） and mosapride group（2.5 mg·kg-1）. Except for the blank group， all groups were gavaged with loperamide suspension（5 mg·kg-1） twice daily for 14 d to construct the STC mouse model. At the same time， each drug administration group was given the corresponding drug by gavage for consecutive 14 d， the blank and model groups were gavaged with equal volume of distilled water. The effects of the treatment of AMR on body mass， defecation frequency， fecal water content and intestinal propulsion rate of mice were observed， the pathological changes of mouse colon were observed by hematoxylin-eosin（HE） staining and periodic acid-Schiff（PAS） staining， the levels of gastrin（GAS） and motilin（MTL） in serum were detected by enzyme-linked immunosorbent assay（ELISA）， gas chromatography-mass spectrometry（GC-MS） was used to detect the contents of short-chain fatty acids（SCFAs） in mouse feces， real-time fluorescence quantitative polymerase chain reaction（Real-time PCR） and Western blot were used to determine the mRNA and protein expression levels of zonula occludens-1（ZO-1）， Occludin， and Claudin-1 in the colon of mice. ResultCompared with the blank group， the body mass， defecation frequency， fecal water content and intestinal propulsion rate of mice in the model group were significantly decreased（P<0.05， P<0.01）， the arrangement of colonic tissues was disordered， and the number of goblet cells was reduced， the levels of GAS and MTL in serum were significantly decreased（P<0.01）， and the levels of SCFAs in the feces were on a decreasing trend， with the contents of acetic acid， propionic acid， butyric acid， isobutyric acid and valeric acid were significantly decreased（P<0.05， P<0.01）， the mRNA and protein expression levels of ZO-1， Occludin and Claudin-1 in the colonic tissues were significantly decreased（P<0.01）. The above results suggested that STC mouse model was successfully constructed. Compared with the model group， the body mass， defecation frequency， fecal water content and intestinal propulsion rate of mice in AMP administration groups all increased significantly（P<0.05， P<0.01）， the mucosal layer of the colonic tissues was structurally intact without obvious damage， and the number of goblet cells increased， serum levels of GAS and MTL were significantly increased（P<0.01）， the contents of SCFAs in the feces were all on a rising trend， with the contents of acetic， propionic， butyric and isobutyric acids rising significantly（P<0.05， P<0.01）， the mRNA and protein expression levels of ZO-1， Occludin and Claudin-1 in the colonic tissues were significantly increased（P<0.05， P<0.01）. ConclusionAMR is able to improve the constipation symptoms in STC mice， and its mechanism may be related to increasing the contents of SCFAs in the intestine as well as promoting the mRNA and protein expression levels of ZO-1， Occludin and Claudin-1 in the colon.

Qualitative and Quantitative Analysis of Chemical Constituents in Liu Junzitang by UPLC-Q-TOF-MS/MS and UPLC / 中国实验方剂学杂志

ObjectiveTo establish a qualitative and quantitative analysis method for chemical constituents in Liu Junzitang（LJZT）， and to clarify its material basis. MethodThe chemical constituents in LJZT were analyzed by ultra performance liquid chromatography-quadrupole-time-of-flight mass spectrometry（UPLC-Q-TOF-MS/MS）， and the resulting compounds were identified by using databases， such as MassBank， PubChem， ChemSpider， Traditional Chinese Medicine Systems Pharmacology Database and Analytical Platform（TCMSP）， and by combining with relevant literature. UPLC was used to establish a quantitative method for analysis of 9 compounds in LJZT， including liquiritin， hesperidin， lobetyolin， liquiritigenin， glycyrrhizic acid， nobiletin， tangeretin， atractylenolide Ⅱ and Ⅰ. ResultBy combining the relevant literature， database and MS information， a total of 79 compounds were identified from LJZT， including 31 flavonoids， 15 terpenoids， 14 nitrogen-containing compounds， 6 phenylpropanoids， 6 organic acids and 7 other compounds. The established quantitative analytical method for the nine representative components showed good linearity within their respective linear ranges， and the precision， stability， reproducibility and recovery were in accordance with the requirements. The quantitative results showed that the contents of liquiritin， hesperidin， lobetyolin， liquiritigenin， glycyrrhizic acid， nobiletin， tangeretin， atractylenolide Ⅱ and Ⅰ in LJZT were 0.376 5， 2.602 1， 0.082 6， 0.128 1， 1.778 6， 0.015 7， 0.006 7， 0.030 4， 0.003 2 mg·g-1， respectively. ConclusionThe established method can quickly， sensitively and accurately analyze the chemical constituents in LJZT， clarify that the material basis of LJZT is mainly flavonoids， terpenoids and nitrogen-containing compounds， and simultaneously determine the contents of the 9 components， which can lay a foundation for the research on quality control， mechanism and clinical application of LJZT.

Establishment of UPLC-MS/MS method for the determination of plasma concentration of three common carbapenem antibiotics / 中国药房

OBJECTIVE To establish a UPLC-MS/MS method for the determination of plasma concentration of three carbapenem antibiotics， i.e. ertapenem （ETP）， imipenem （IPM） and meropenem （MEM）. METHODS After protein precipitation with methanol， the plasma samples were separated by ACQUITY UPLC BEH C18 column （2.1 mm×50 mm， 1.7 μm） using stable isotopes of three antibiotics （ETP-D4， IPM-D4， MEM-D6） as the internal standard. The mobile phases were 98% acetonitrile +2% water +0.1% formic acid and 98% water +2% acetonitrile +0.1% formic acid， by gradient elution. The flow rate was 0.3 mL/min and the column temperature was 40 ℃. Scanning analysis was performed in the positive ion and multiple reaction monitoring mode. RESULTS The method had good specificity， good linearity （r2≥0.993） in the range of 0.2-200， 0.1-100 and 0.1-100 μg/mL of ETP， IPM and MEM， and good intra-batch and inter-batch precision and accuracy （all RE≤5.14%， all RSD≤11.15%）， the matrix effect and extraction recovery were consistent （RSD≤12.99%）. CONCLUSIONS This study establishes the UPLC-MS/MS method to simultaneously quantify the plasma concentration of ETP， IPM and MEM. The method has the advantages of simple pretreatment， short detection time and small sample quantity to meet clinical requirement.

Study on metabolites derived from Zhideke granules in rats in vivo / 中国药房

OBJECTIVE To analyze the metabolites of Zhideke granules and speculate its metabolic pathway in rats in vivo. METHODS Male SD rats were randomly divided into blank group and administration group （Zhideke granules， 9.45 g/kg）； they were given ultrapure water or relevant medicine， twice a day， every 6-8 h， for 3 consecutive days. Serum， urine and feces samples of rats were collected， and their metabolites were identified by UPLC-Q-Exactive-MS technique after intragastric administration of Zhideke granules； their metabolic pathways were speculated. RESULTS After intragastric administration of Zhideke granules， 16 prototype components （i.g. irisflorentin， baicalin， chlorogenic acid） and 11 metabolites （i.g. hydration products of kaempferol or luteolin， methylation products of chlorogenic acid， and hydroxylation products of baicalin） were identified in serum， urine and feces of rats. Among them， 8 prototype components and 4 metabolites were identified in serum samples； 10 prototype components and 7 metabolites were identified in urine samples； 8 prototype components and 5 metabolites were identified in the fecal samples. CONCLUSIONS The metabolites of Zhideke granules in rats mainly include baicalin， irisflorentin，chlorogenic acid， and the main metabolic pathways included methylation， hydroxylation， glucuronidation.

Determination and Mechanism Prediction of Potential Active Ingredients in Erdongtang Based on UHPLC-QqQ-MS and Network Pharmacology / 中国实验方剂学杂志

ObjectiveTo establish an ultra-high performance liquid chromatography-tandem triple quadrupole mass spectrometry（UHPLC-QqQ-MS） for determination of the active ingredients in Erdongtang， and to predict the targets and pathways of anti-insulin resistance action of this formula. MethodThe analysis was performed on an ACQUITY UPLC BEH C18 column（2.1 mm×100 mm， 1.7 μm） with the mobile phase of 0.1% formic acid aqueous solution（A）-acetonitrile（B） for gradient elution（0-3 min， 90%-87%A； 3-6 min， 87%-86%A； 6-9 min， 86%-83%A； 9-11 min， 83%-75%A； 11-18 min， 75%-70%A； 18-19 min， 70%-52%A； 19-22 min， 52%A； 22-25 min， 52%-5%A； 25-27 min， 5%-90%A； 27-30 min， 90%A）. The contents of active ingredients in Erdongtang was detected by electrospray ionization（ESI） and multiple reaction monitoring（MRM） mode under positive and negative ion modes. On this basis， network pharmacology was applied to predict the targets and pathways of Erdongtang exerting anti-insulin resistance effect. ResultThe 20 active ingredients in Erdongtang showed good linear relationships within a certain mass concentration range， and the precision， stability， repeatability and recovery rate were good. The results of determination showed that the ingredients with high content in 15 batches of samples were baicalein（1 259.39-1 635.78 mg·L-1）， baicalin（1 078.37-1 411.52 mg·L-1）， the ingredients with medium content were mangiferin（148.59-217.04 mg·L-1）， timosaponin BⅡ（245.10-604.89 mg·L-1）， quercetin-3-O-glucuronide（89.30-423.26 mg·L-1）， rutin（46.91-1 553.61 mg·L-1）， glycyrrhizic acid（55.97-391.47 mg·L-1）， neomangiferin（37.45-127.03 mg·L-1）， nuciferine（0.89-63.48 mg·L-1）， hyperoside（6.96-136.78 mg·L-1）， liquiritin（30.89-122.78 mg·L-1）， liquiritigenin（26.64-110.67 mg·L-1）， protodioscin（58.57-284.26 mg·L-1）， the ingredients with low content were wogonin（7.16-20.74 mg·L-1）， pseudoprotodioscin（5.49-22.96 mg·L-1）， ginsenoside Rb1（7.31-23.87 mg·L-1）， ginsenoside Rg1（10.78-28.33 mg·L-1）， ginsenoside Re（7.78-24.76 mg·L-1）， ophiopogonin D（2.08-4.29 mg·L-1）， methylophiopogonanone A（0.74-1.67 mg·L-1）. The results of network pharmacology indicated that the mechanism of anti-insulin resistance exerted by Erdongtang might be related to the phosphatidylinositol 3-kinase/protein kinase B（PI3K/Akt） signaling pathway. ConclusionThe established UHPLC-QqQ-MS has the advantages of simple sample processing， strong exclusivity and high sensitivity， and can simultaneously determine the contents of the main ingredients from seven herbs in Erdongtang， which can lay the foundation for the development of Erdongtang compound preparations. The results of the network pharmacology can provide a reference for the mechanism study of Erdongtang in the treatment of type 2 diabetes mellitus.

Evaluation of A Liver Yin Deficiency Mouse Model Based on Untargeted Metabolomics / 中国实验方剂学杂志

ObjectiveBased on ultra performance liquid chromatography-quadrupole-time-of-flight tandem mass spectrometry（UPLC-Q-TOF-MS）， to evaluate the establishment of a mouse model of liver Yin deficiency by thyroid tablet suspension combined with 10% carbon tetrachloride（CCl4） from the perspective of non-targeted metabolomics， in order to lay the foundation for the establishment of a traditional Chinese medicine（TCM） syndrome model. MethodA total of 24 mice were randomly divided into blank group and model group. The model group was given thyroid tablet suspension（0.003 2 g·kg-1） by gavage for 14 consecutive days， and 10% CCl4（5 mL·kg-1） was intraperitoneally injected once a week to establish a liver Yin deficiency model， while the blank group was injected with an equal amount of olive oil intraperitoneally and gavaged with an equal amount of distilled water， and was fed with normal feed. After the modeling was completed， 6 mice in each group were randomly selected， the levels of alanine aminotransferase（ALT）， aspartate aminotransferase（AST）， cyclic adenosine monophosphate（cAMP）， cyclic guanosine monophosphate（cGMP）， interleukin（IL）-6， IL-10， tumor necrosis factor-α（TNF-α）were measured in the mice serum， and malondialdehyde（MDA）， superoxide dismutase（SOD）， total protein（TP）， hydroxyproline（HYP） and other indicators were measured in the mice liver. Liver tissue sections were taken for hematoxylin-eosin（HE） staining and observing pathological changes. The remaining 6 mice in each group were subjected to UPLC-Q-TOF-MS combined with principal component analysis（PCA） and orthogonal partial least squares-discriminant analysis（OPLS-DA） were used to screen differential metabolites in the liver Yin deficiency mouse model， Kyoto Encyclopedia of Genes and Genomes（KEGG） database was used to analyze the corresponding metabolic pathways of differential metabolites. ResultCompared with the blank group， mice in the model group showed liver Yin deficiency manifestations such as reduced body weight， fatigue and sleepiness， disheveled and lusterless hair， irritability. The levels of ALT， cAMP/cGMP， IL-6， AST， MDA， cAMP， TNF-α significantly increased（P<0.05， P<0.01）， while the levels of SOD， IL-10 and cGMP significantly decreased（P<0.05， P<0.01）， and the changes of HYP and TP were not statistically significant. Hepatic steatosis and distortion of the radial arrangement of the liver plate cells were seen in the section images of the model group， endogenous substances were clearly separated， and 252 differential metabolites were identified in the serum samples， which were mainly involved in the metabolic pathways of purine metabolism， steroid hormone biosynthesis and pyrimidine metabolism. A total of 229 differential metabolites were identified in the liver samples， mainly involving nucleotide metabolism， purine metabolism， steroid hormone biosynthesis， pyrimidine metabolism， antifolate resistance， insulin resistance， primary bile acid biosynthesis， prostate cancer， sulfur relay system， arachidonic acid metabolism and other metabolic pathways. ConclusionThe successful establishment of liver Yin deficiency model in mice by CCl4 combined with thyroid hormone is evaluated through the investigation of serum and liver metabolomics， combined with biochemical indicators， which provides a biological basis and experimental foundation for the Yin deficiency syndrome model of TCM.

EGFR Methylation in Peripheral Blood of Driver Gene-Positive Lung Cancer and its Clinical Correlation / 医学研究杂志

Objective To explore the methylation status of EGFR promoter region in peripheral blood of EGFR driver gene-positive lung adenocarcinoma patients and its clinical significance.Methods The methylation of EGFR promoter region in peripheral blood DNA of 32 EGFR driver gene-positive lung adenocarcinoma patients and 24healthy controls were detected by(matrix-assisted laser desorp-tion/ionisation,time-of-flight mass spectrometry,MALDI-TOF MS)technique to screen the differential methylation of CpG sites with lung cancer and analyze its correlation with clinicopathological characteristics and EGFR mutant subtype.Results The methylation levels of EGFR were significantly lower in the lung cancer group than in the normal control,EGFR#29 CpG_2,EGFR#30 CpG_8.9.10.11,CpG_25,CpG_40.41.42methylation of lung cancer group(25.7％±2.1％,4.6％±0.4％,2.8％±0.7％,4.40±0.4％)were low-er than the normal control(36.5％±4.6％,6.0％±0.6％,4.8％±0.9％,7.1％±0.4％),the difference was statistically signifi-cant(P＜0.05).No significant differences in the methylation levels within EGFR CpGs were associated with age,cancer differentiation,disease location,TNM stage with lung cancer(P＞0.05).The methylation level of EGFR#30 CpG_25 in the female group was signifi-cantly lower than that in the male group(P＜0.05),and the methylation level of EGFR#30 CpG_25 in the smoking group was higher than that in the non-smoking group(P＜0.05),the methylation level of EGFR#30 CpG_8.9.10.11 in the lymph node metastasis group was significantly lower than that in the non-lymph node metastasis(P＜0.01).There was no significant difference in methylation of EGFR between EGFR 19del and 21 L858R mutant subtypes.Conclusion Our findings suggest that EGFR methylation may be associated with the occurrence,development,gender,and smoking history of patients with EGFR driving gene-positive lung cancer,which may serve as a biomarker for the diagnosis and prognosis prediction of lung cancer.

Monitoring Concentration of Nirmatrelvir and Ritonavir by Ultra High Performance Liquid Chromatography-tandem Mass Spectormetry Method / 医药导报

Objective To establish a highly sensitive,stable,and universally applicable ultra-high-performance liquid mass spectrometry tandem method(UPLC-MS/MS)for simultaneous determination of nirmatrelvir and ritonavir blood concentrations in human plasma.Methods The separation was performed on an ACQUITY UPLC BEH C18 column(2.1 mm× 50 mm,1.7 μm)with gradient elution,and the mobile phase consisted of 0.1％formic acid-water and 100％acetonitrile at the flow rate of 0.3 mL·min-1.The column temperature was 45℃,and the injection volume was 2 μL.Electrospray ionization as ion source(ESI+)was used as the ion source and multiple reactions monitoring mode(nirmatrelvir m/z 500.20→319.10,nirmatrelvir-D9 m/z 508.59→328.10,ritonavir m/z 721.30→426.10,13C,2H3-ritonavir m/z 725.30→426.10)was adopted.Thirty patients with coronavirus disease 2019(COVID-19)treated with nirmatrelvir and ritonavir at the People's Hospital of Changxing County in Jan.2023 were selected to measure their steady-state trough concentrations of nirmatrelvir and ritonavir after 3 days of treatment.Results The linear range of nirmatrelvir was 0.100-10.0 μg·mL-1(R2=0.997 2),and the linear range of nirmatrelvir was 0.050-5.00 μg·mL-1(R2=0.995 2).The recovery rates of nirmatrelvir and lopinavir were both＞90％and the intra-batch and inter-batch precision relative standard deviations(RSDs)were both＜10％.Additionally,the recovery ranges for nirmatrelvir and lopinavir were 91.5％-97.0％,and the matrix effects ranged from 92.4％to 97.7％.The results of clinical samples showed that the plasma concentrations of nirmatrelvir and ritonavir in patients with COVID-19 varied greatly among individuals.Conclusion The method for simultaneous determination of nirmatrelvir and ritonavir concentrations in human plasma established in this study is convenient,highly specific,highly accurate,with high precision,which is suitable for monitoring the concentrations of nirmatrelvir and ritonavir in patients.

Effects of Different Processing Methods on the Volatile Components of Amomi Fructus Based on GC-MS and Multivariate Statistical Analysis / 医药导报

Objective To analyze the influence of different processing methods,including frying,ginger frying,and salt frying,on the volatile components of A.fructus.Methods The volatile components in different processed products of A.fructus were detected and analyzed by gas chromatography-mass spectrometry(GC-MS)based on multivariate statistical analysis.After OPLS-DA analysis,the different components were screened under the conditions of VIP＞1.5 and P＜0.05 and were qualitatively searched using the NIST 11 spectral library.Results A total of 49 different components were identified,with 14 components only changing in the seed mass and 22 components changing in the peel.The content of camphor could be significantly reduced in the seed mass after A.fructus was processed and the content of bornyl acetate significantly increased in the peel of frying A.fructus.Salt frying had a great influence on the alkanes in A.fructus,and ginger processing did not only increase the volatile components in ginger,which reflected the complexity of the processing mechanism.Conclusion At present,the specific processing mechanism is not clear,but the experimental results provide theoretical data for the "detoxification and efficiency enhancement" effect of A.fructus processing,reflecting the scientific nature of the processing,enriching the processing theory of A.fructus,and providing a reference for further in-depth research on the activity of different processed products of A.fructus.

Simultaneous detection of 34 emerging contaminants in tap water by HPLC-MS/MS and health risk assessment / 中国法医学杂志

Objective To establish a simultaneous detection approach for 34 emerging contaminants(ECs)in tap water by liquid chromatography-tandem mass spectrometry(HPLC-MS/MS).Human health risk assessment was performed according to the detection results from 43 tap water samples.Methods Tap water samples were concentrated and extracted by solid phase extraction,and then blown to near dry by nitrogen at 40℃.The sample extracts were dissolved in methanol-water solution(95:5,VN)to 0.5 mL for analyzing.Agilent Jet Stream Electrospray Ionization(AJS ESI)and the multiple reaction monitoring(MRM)mode were performed for MS to acquire the data of 34 ECs.A database including precursor ion,product ion and retention times was established accordingly.Results The average linear correlation coefficients(r)of 34 kinds of ECs was 0.995 9.The limits of detection were 0.01～0.60 ng/L and the recoveries were between 60.7％and 119.8％.The intra-group precisions were between 0.05％～9.89％and the intra-day precisions were between 0.20％～14.40％for the spiked samples.The method was applied to analyze 43 tap water samples and a total of 15 ECs were detected.According to the results,the detection rate of caffeine was the highest(84％),and the concentration range was ND～74.42 ng/L.Among all the ECs detected,1,2,3-benzotriazole had the highest concentration(ND～361.15 ng/L),where detection rate was 44％.Humans may be exposed to these ECs by drinking the tap water.The human health risk assessments of 12 kinds of ECs were carried out,however,the estimated risk was negligible(risk quotient＜0.01).Conclusion The method is simple,highly sensitive and selective,and could meet the detection needs of ECs at trace level in tap water.There was no human health risk posed for ECs identified in 43 tap water samples analyzed by this method.

Effects of l-tetrahydropalmatine on different stages of conditioned place preference in ketamine dependent rats and the mechanism of DA / 中国法医学杂志

Objective To observe the effects of Levo-tetrahydropalmatine(l-THP)on the expression,regression and relapse of conditioned place preference(CPP)in ketamine induced rats,and to detect the content of dopamine(DA)in the striatum(caudate putamen,CPu)of the rat brain at different time points.Methods Ketamine addiction rat model was established by CPP.The effects of l-THP on the expression,regression and relapse of ketamine induced rat CPP were investigated using CPP score as the index.The content of DA in CPu of rats was determined by ultra-performance liquid chromatography coupled to tandem mass spectrometry(UPLC-MS/MS)after ketamine administration and l-THP intervention at 30 min,60 min,90 min,120 min and 150 min.Results It indicated that 1-THP could decrease the expression of CPP in ketamine induced rats,promote the process of CPP resolution and inhibit the process of relapse.In addition,l-THP combined with ketamine administration significantly inhibited the ketamine-induced increase in DA content in the CPu of the rats.Conclusion The mechanism of l-THP inhibiting the reward effect of ketamine may be related to blocking DA receptors and reducing the release of DA neurotransmitters.l-THP has potential implications for the treatment of ketamine addiction.

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.

Clinical application of MALDI-TOF MS for homology analysis of Acineto-bacter baumannii / 中国感染控制杂志

Objective To evaluate the clinical application value of matrix-assisted laser desorption ionization time-of-flight mass spectrometry(MALDI-TOF MS)in analyzing the homology of Acinetobacter baumannii(AB).Methods After excluding repetitive strains from multiple specimens of the same patient or environment,a total of 46 AB strains isolated from patients'sputum and environmental specimens of neurological intensive care unit(ICU)in a tertiary first-class general hospital from May 2020 to February 2021 were collected.Strains were detected by VITEK-MS mass spectrometer.Cluster analysis was performed by SARAMIS Premium software,and verified by multilocus sequence typing(MLST).Results Cluster analysis and comparison of MALDI-TOF MS and MLST found that among the 46 AB strains,39 were the type MS-a of MALDI-TOF MS,of which 22 strains were the clus-ter MT-A of MLST,including ST208(n=3),ST540(n=3),ST195(n=8),ST369(n=5),ST136(n=1),ST436(n=1)and ST1893(n=1);16 strains were MT-B,including type ST381(n=4),type ST469(n=11),and type ST938(n=1);one strain was cluster MT-C(ST1821);one strain of type MS-b was ST381;two strains of type MS-c were ST369;one strain of type MS-d was ST195;two strains of type MS-e were ST540 and ST369,respectively;one strain of type MS-f was STN1.Conclusion As a homology analysis method,MALDI-TOF MS still has certain limitations such as low consistency with MLST results,low resolution and specificity,thus cannot replace MLST technology.

Chemical constituent identification of Zhuangxi-6 powders based on HPLC-Q-Exactive-MS / 中国药师

Objective To quickly analyze and identify the chemical constituents in Zhuangxi-6 powders by HPLC-Q-Exactive-MS.Methods The separation was performed on Shim-pack GIST C18 column(150 mm×4.6 mm,5 μm)with the mobile phase of methanol-0.1%formic acid aqueous solution(gradient elution).The flow rate was 0.35 mL/min,column temperature was 35℃,and the volume of sample injection was 10 μL.Electrospray ionization was applied for scanning under positive and negative ion modes with the scanning range of m/z 110-1 200.Results Combining mass spectrometry data and literature information,a total of 65 components were identified in Zhuangxi-6 powders,including 16 alkaloids,11 flavonoids,14 organic acids,6 chromones,7 sesquiterpenoids,4 fatty acids,3 amino acids,2 tannins,1 phenolic acids,and 1 phenylpropanoid.Conclusion HPLC-Q-Exactive-MS method can quickly identify the chemical components of Zhuangxi-6 powders,laying a foundation for the further research on the pharmacodynamics substances of Zhuangxi-6 powders.

Establishment and evaluation of HPLC-MS/MS method in determining serum H2S / 中国医学装备

Objective:To establish a determination method of high performance liquid chromatography-tandem mass spectrometry(HPLC-MS/MS)for serum hydrogen sulfide(H2S),so as to determine serum H2S.Methods:This study collected serum samples of 30 patients who admitted to Beijing Jishuitan Hospital affiliated to Capital Medical University from April 2023 to May 2023,and they were divided into osteoporosis group and control group according to whether existed osteoporosis,with 15 cases in each group.HPLC-MS/MS and enzyme-linked immunosorbent assay were used respectively to determine serum H2S.And then,the precision,accuracy and correlation between the two methods were evaluated.Results:HPLC-MS/MS can fast detect the content of serum H2S through detecting methylene blue in the serum,which analysis time was only 1.5 minutes,and its specificity was higher.The relative standard deviation(RSD)value of quality control plasma was 8.77%,and that of quality control plasma with the standard and pure water with standard were respectively 4.58% and 8.23%.The precisions of them met the requirement of detection(less than 20%).The recovery was 103.5% through used the above data,and the accuracy accorded with the requirements of quantitative detection(recovery was 103.5%).Conclusion:HPLC-MS/MS method is rapid and accurate in detecting H2S,which can accurately detect the content of serum H2S.This method has a series of advantages include fast,high throughput,high sensitivity and favorable stability,which contributes to conduct basic research of the content of serum H2S in the cellular pathways of human.

Optimization of Extraction Process of Weile Prescription Based on Multi-index Comprehensive Evaluation of UPLC-MS/MS and G1-entropy Weight Method / 中国中医药信息杂志

Objective To establish the method for simultaneous determination of six index components in the water extract of Weile Prescription;To optimize the water extraction process.Methods UPLC-MS/MS was used with Waters CORTECS C18 column(2.1 mm×100 mm,1.6 μm)as the chromatographic conditions;the mobile phase was 0.1%formic acid water-acetonitrile with gradient elution;the flow rate was 0.25 mL/min;the column temperature was 40℃;the sample volume was 2 μL.Electrospray negative ion source,positive and negative ion switching multi-reaction monitoring(MRM)mode were detected.Taking the content of six index components(gallic acid,vitexin,paeoniflorin,naringin,hesperidin and glycyrrhizic acid)and extraction rate as evaluation indexes,the weight coefficient of each index was determined by G1-entropy weight method,and the optimum parameters of extraction process were determined by orthogonal experiment design with the amount of water,extraction time and extraction times as investigation factors.Results There was a good linear relationship of the six components in the water extract of Weile Prescription in the concentration range(r>0.999),and the average recovery rate was 96.83%-102.56%,RSD<4.0%.The best technological parameters were as follows:Chinese decoction pieces were soaked in 12 times of water for 2 h,and extracted twice,each time for 1.5 h.Conclusion The UPLC-MS/MS method established in the study for simultaneous determination of six components in Weile Prescription is rapid,simple and sensitive,and the optimized extraction process is stable and feasible,which provides experimental basis for the development and research of the preparation.