Investigation of Effect of Different Processed Products of Atractylodes chinensis Rhizoma on Rats with Spleen Deficiency Based on Pharmacodynamics and Metabolomics / 中国实验方剂学杂志

ObjectiveTo clarify the differences in the efficacy and mechanism of different processed products of Atractylodes chinensis rhizoma by the pharmacodynamics and metabolomics studies of raw， bran-fried and rice water-processed products on rats with spleen deficiency. MethodSixty male SD rats were randomly divided into blank group， model group， raw product group（3.75 g·kg-1）， bran-fried product group（3.75 g·kg-1）， rice water-processed product group（3.75 g·kg-1） and Shenling Baizhusan group（6.7 g·kg-1）， with 10 rats in each group. The method of excessive fatigue+improper diet was used to establish a spleen deficiency model in rats. After the end of modeling， except for the blank and model groups， each dosing group was given the corresponding drug suspension， the immune organ coefficients of each group of rats were examined， the levels of interleukin-6（IL-6）， tumor necrosis factor-α（TNF-α）， immunoglobulin G（IgG）， amylase（AMS）， motilin（MTL）， gastrin（GAS）， Na+-K+-adenosine triphosphatase（ATPase）， aquaporin 2（AQP2）， AQP3 and AQP8 in rats were measured by enzyme-linked immunosorbent assay（ELISA）. Ultra high performance liquid chromatography-quadrupole-time-of-flight tandem mass spectrometry（UPLC-Q-TOF-MS） combined with orthogonal partial least squares-discriminant analysis（OPLS-DA） were used to search for biomarkers in the plasma samples of spleen-deficient rats by using two criteria［P<0.05 and variable importance in the projection（VIP） value>1］， and to compare the different modulatory effects of the three decoction pieces on the splenic-deficient biomarkers， and metabolic pathway analysis was conducted through the Kyoto Encyclopedia of Genes and Genomes（KEGG） database. ResultCompared with the blank group， the thymus index and spleen index of rats in the model group were significantly decreased（P<0.05）， the levels of IL-6， TNF-α， IgG and AQP2 were significantly increased（P<0.05）， the levels of AMS， GAS， MTL， AQP3， AQP8 and Na+-K+-ATPase were significantly decreased（P<0.05）. Compared with the model group， raw products， bran-fried products and rice water-processed products all increased thymus index and spleen index（P<0.05）， decreased IL-6， TNF-α， IgG and AQP2 levels（P<0.05）， and increased AMS， GAS， MTL， AQP3， AQP8 and Na+-K+-ATPase levels to different degrees. A total of 176 differential metabolites were screened in the model group compared with the blank group， of which 75， 72 and 84 biomarkers were called back by the raw products， bran-fried products and rice water-processed products， respectively（P<0.05， P<0.01）. Raw products of A. chinensis rhizoma mainly affected glycine， serine and threonine metabolism. Bran-fried products mainly affected alanine， aspartate and glutamate metabolism， D-arginine and D-ornithine metabolism. Rice water-processed products mainly affected glycine， serine and threonine metabolism， alanine， aspartate and glutamate metabolism， citrate cycle， thiamine metabolism， D-arginine and D-ornithine metabolism. ConclusionRaw products， bran-fried products and rice water-processed products of A. chinensis rhizoma all have good spleen strengthening effects， among which the effects of bran-fried products and rice water-processed products were stronger. Meanwhile， raw products has the strongest dryness， followed by bran-fried products， and the weakest dryness of rice water-processed products. The three decoction pieces are able to significantly modulate metabolic abnormalities in spleen-deficient rats， and the mechanism may be related to amino acid metabolism such as glycine， serine and threonine metabolism as well as alanine， aspartate and glutamate metabolism.

Using Liquid Chromatography-Tandem Mass Spectrometry in Detecting Plasma Lyso-GL3 Levels in Patients with Fabry Disease and the Association Analysis of Phenotype-Genotype of the Disease / 罕见病研究

ObjectiveUsing the liquid chromatography-tandem mass spectrometry (LC-MS/MS) to determine the plasma level of Lyso-GL3 in patients with Fabry disease and to analyze the clinical application of the method.MethodsThirty-nine patients with a genetic diagnosis of Fabry disease were included, and plasma levels of Lyso-GL3 were measured by LC-MS/MS analysis, and detailed clinical information of the patients was obtained including: α-galactosidase A activity, genetic variants, quantification of urine protein, mean arterial pressure, and estimation of glomerular filtration rate, and the differences in the levels of Lyso-GL3 in different clinical phenotypes and genotypes were statistically analyzed, as well as the association with clinical indicators.ResultsLyso-GL3 showed good linearity within 0.7856-400 ng/mL(r=0.9992).Further analysis of 39 Fabry disease patients diagnosed in Ruijin Hospital, Shanghai Jiao Tong University School of Medicine showed a median Lyso-GL3 concentration of 23.6 ng/mL(4.3-92.9 ng/mL); Lyso-GL3 levels were significantly higher in patients with both the frameshift and the splicing mutations, as well as in patients with the nonsense mutations, than in patients with the missense mutations (median value 119.7 ng/mL vs. 11.9 ng/mL, P=0.006, and median value 97.0 ng/mL vs. 11.9 ng/mL, P=0.015, respectively). Whereas, association analysis revealed that Lyso-GL3 was not significantly associated with urinary protein, mean arterial pressure and estimated glomerular filtration rate.ConclusionsThe using of LC-MS/MS to quantify plasma Lyso-GL showed significant differences in Lyso-GL3 concentrations between classical and atypical phenotypes, suggesting that plasma Lyso-GL3 may help with clinical phenotypes. However, Lyso-GL3 levels is found to be overlapped between genotypes. No significant linear correlation was found between Lyso-GL3 and renal clinical indicators, suggesting the urgent need in finding a more accurate tool to assess renal involvement and prognosis in patients with Fabry disease.

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.

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.

Progress of newborn screening in China / 浙江大学学报·医学版

Newborn screening (NBS) plays a significant role in reducing the risk of birth defects. NBS in China began in the early 1980s. Under the protection of laws and regulations and the leadership of the national health administration, approved screening centers in public hospitals took the responsibility for publicity, screening, diagnosis, treatment, follow-up and management of birth defects. As of 2022, 31 provinces (autonomous regions and municipalities directly under the central government) have carried out NBS for phenylketonuria, congenital hypothyroidism, and hearing loss, 23 provinces have carried out screening for glucose-6-phosphate dehydrogenase (with a screening rate of 89.24%), and 24 provinces have carried out screening for congenital adrenal cortical hyperplasia (91.45% screening rate). Over the past four decades, screening techniques have evolved from bacterial inhibition, fluorescence analysis, and tandem mass spectrometry for the detection of biochemical markers to genetic testing, which has greatly contributed to the expansion of the types of diseases screened for. The combined use of metabolomics and genomics is currently being explored. Effective management and rigorous quality control of NBS are prerequisites for improving the quality and ensuring the accuracy of screening. The Quality Management System for Newborn Screening System Network (QMS-NBS), established by the National Center for Clinical Laboratories, covers all screening centers and related blood collection agencies. The operation of the QMS-NBS allows the quality and performance of screening to be transparent and measurable, ensuring the quality and efficiency of screening. This article provides an overview of the history of NBS, especially the evolution of policies for the NBS in China, the construction of screening institutions, the number of newborns screened, the incidence rates of screened diseases, the changes in screening technology, the expansion of new diseases screened for, and the quality control of NBS. Overall, the progress in NBS in China has not only benefited from the development and standardization at the technological level, but also benefited from the construction of policies, regulations and ethics.

Analysis of genotypes and biochemical phenotypes of neonates with abnormal metabolism of butyrylcarnitine / 浙江大学学报·医学版

OBJECTIVES@#To investigate the genotypes and biochemical phenotypes of neonates with abnormal metabolism of butyrylcarnitine (C4).@*METHODS@#One hundred and twenty neonates with increased C4 levels detected by tandem mass spectrometry in the neonatal screening at Children's Hospital, Zhejiang University School of Medicine from January 2018 to June 2023 were included. The initial screening data and recalled data of C4 and C4/C3 were collected and converted into multiples of C4 reference range. Next generation sequencing was performed and the exons with adjacent 50 bp regions of ACAD8 and ACADS genes were captured by liquid phase capture technique. Variant information was obtained by bioinformatic analysis and the pathogenicity were classified according to the American College of Medical Genetics and Genomics criteria. The Wilcoxon rank sum test was used to analyze the differences in C4 levels among neonates with different variation types.@*RESULTS@#In total, 32 variants in ACAD8 gene were detected, of which 7 variants were reported for the first time; while 41 variants of ACADS gene were detected, of which 17 variants have not been previously reported. There were 39 cases with ACAD8 biallelic variations and 3 cases with ACAD8 monoallelic variations; 34 cases with ACADS biallelic variations and 36 cases with ACADS monoallelic variations. Furthermore, 5 cases were detected with both ACAD8 and ACADS gene variations. Inter group comparison showed that the multiples of C4 reference range in initial screening and re-examination of the ACAD8 biallelic variations and ACADS biallelic variations groups were significantly higher than those of the ACADS monoallelic variations group (all P<0.01), while the multiples in the ACAD8 biallelic variations group were significantly higher than those in the ACADS biallelic variations group (all P<0.01). The multiples of C4 reference range in the initial screening greater than 1.5 times were observed in all neonates carrying ACAD8 or ACADS biallelic variations, while only 25% (9/36) in neonates carrying ACADS monoallelic variations.@*CONCLUSIONS@#ACAD8 and/or ACADS gene variants are the main genetic causes for elevated C4 in newborns in Zhejiang region with high genotypic heterogeneity. The C4 levels of neonates with biallelic variations are significantly higher than those of neonates with monoallelic variations. The cut-off value for C4 level could be modestly elevated, which could reduce the false positive rate in tandem mass spectrometry neonatal screening.

UPLC-MS/MS Method for Detection of Etomidate and Its Metabolite Etomidate Acid Quantity in Blood / 法医学杂志

OBJECTIVES@#To establish a method for the simultaneous quantitative analysis of etomidate and its metabolite etomidate acid in blood, and to discuss its application value in actual cases.@*METHODS@#Acetonitrile precipitate protein method was used, and C18 column was selected. Gradient elution was performed with acetonitrile and 5 mmol/L ammonium acetate within 6 min. Electrospray ionization source in positive ion mode was used. The internal standard etomidate acid-d5 was obtained by etomidate-d5 alkaline hydrolysis reaction. Ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was used for quantitative analysis. The methodological verification was conducted.@*RESULTS@#Etomidate and etomidate acid in blood showed good linear relationship in the quantitative linear range (r>0.999), with the lower limit of quantification was 2.5 ng/mL and 7.5 ng/mL, respectively. The accuracy, precision, recovery rate, and matrix effect of the method met the professional verification standards. The practical application results showed that etomidate and etomidate acid could be detected in the blood of the abusers, and their mass concentrations ranged from 17.24 to 379.93 ng/mL.@*CONCLUSIONS@#The method established in this study can simultaneously quantify etomidate and etomidate acid in blood, which is simple and convenient to operate with accuracy. It can meet the detection needs of actual cases and provide technical support for law enforcement to crack down on etomidate abuse.

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.

Detection and Analysis of Blood Dexmedetomidine in Drug-Facilitated Cases / 法医学杂志

OBJECTIVES@#To establish a simple and rapid qualitative and quantitative detection method of dexmedetomidine in blood.@*METHODS@#Blood was separated on the Allure PFP Propyl liquid chromatography column with isocratic elution after it was precipitated by acetonitrile and filtered. Qualitative and quantitative analysis of dexmedetomidine was performed using positive ion scan mode and multi-reaction monitoring mode.@*RESULTS@#The limit of detection of dexmedetomidine in blood was 0.2 ng/mL and the limit of quantification was 0.5 ng/mL. The linearity of the method was good in the range of 0.5-1 000 ng/mL, and the correlation coefficient was greater than 0.99. The accuracy of the method was 90.34%-112.67% and the extraction recovery was 50.05%-91.08%, with no significant matrix effect.@*CONCLUSIONS@#This method is simple, selective and suitable for the qualitative and quantitative analysis of dexmedetomidine in blood, which can provide a reference for drug-facilitated cases involving dexmedetomidine.

Rapid Determination of Bucinnazine in Blood by UPLC-MS/MS / 法医学杂志

OBJECTIVES@#To establish a rapid method for the analysis of bucinnazine in blood by UPLC-MS/MS and to apply the method to the practical case.@*METHODS@#After the internal standard was added to blood, the protein was precipitated with 900 μL mixed solution (Vacetonitrile∶Vwater=8∶2). After vortex and centrifugation, the protein was measured through 0.22 μm filter membrane. The separation was performed on C18 chromatography column, with acetonitrile and 5 mmol/L ammonium acetate containing 0.1% formic acid aqueous as mobile phase gradient elution at the flow rate of 0.4 mL/min. Multiple reaction monitoring scan was performed in electrospray positive ion mode, quantitative measurement was performed by internal standard method, and methodological verification was carried out.@*RESULTS@#The linear relationship of bucinnazine in blood was good in the range of 0.5-200 μg/L, the correlation coefficient (r) was 0.999 7, the limit of detection was 0.1 μg/L, the limit of quantitation was 0.5 μg/L, and the recovery was 78.3%-83.8% at 1, 10 and 100 μg/L mass concentration levels. The matrix effect was 69.4%-73.8%, the intra-day precision was 1.9%-2.8%, and the inter-day precision was 2.8%-3.2%, the accuracy was 3.1%-3.5%. The stability test results of 1 and 100 μg/L mass concentrations at -25 ℃ showed that the accuracy (bias) of 10 d was less than 4.5%.@*CONCLUSIONS@#This method has the advantages of simple pre-treatment process, fast sample processing speed, high sensitivity of instrument analysis, good stability of content determination and reliable identification results, and can meet the needs of case identification.

Establishment and application of detection methods of dicamba in drinking water / 环境与职业医学

Background Dicamba is widely used in agricultural production in China, but it is extremely soluble in water and can be harmful to human health when it enters the body via water drinking. It is necessary to establish an accurate, sensitive, and rapid detection method to determine the residues of dicamba in domestic drinking water. Objective To establish two methods for the determination of dicamba residues in drinking water by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) and gas chromatography-tandem mass spectrometry (GC-MS/MS) respectively. Methods The conditions of the proposed method using HPLC-MS/MS included CAPCELL PAK ST chromatographic column, ammonium formate water solution and methanol as the mobile phase, and isocratic elution. The system was operated under multiple reaction monitoring mode and electrospray negative ionization mode. Trimethylsilylated diazomethane was used as a derivatizing agent for GC-MS/MS, and an external standard curve was used to evaluate the system. The residues of dicamba in seven water samples of tap water or secondary water supply from six regions in Chengdu were detected by the established systems to evaluate their applicability and to understand the status quo of dicamba residues in drinking water. Results For the HPLC-MS/MS, the linear range of dicamba was 1.00-100 μg·L−1, the regression equation was \begin{document}$\hat Y $\end{document}=1250.9X+2681.5, the correlation coefficient was 0.9988, the relative standard deviations were 1.23%-26.3%, the limit of detection was 0.95 μg·L−1, and the spiked recoveries were 91.8%-111%. For the GC-MS/MS, the linear range of dicamba was 0.200-10.0 μg·L−1, the regression equation was \begin{document}$\hat Y $\end{document}=190597X+40911, the correlation coefficient was 0.9993, the relative standard deviations were 0.64%-3.90%, the limit of detection was 0.18 μg·L−1, and the spiked recoveries were 97.3%-105%. No dicamba residue was identified in the seven water samples of tap water or secondary water supply from six regions in Chengdu by the proposed methods. Conclusion The two detection methods established in this study are sensitive and rapid, meet the requirements from the detection of dicamba residues in drinking water, and provide an experimental basis for subsequent research on the detection of dicamba residues. In the future, it is necessary to continue to pay attention to the pollution of dicamba in drinking water in Chengdu.

Simultaneous determination of 15 bile acids in Tongren niuhuang qingxin pills by UPLC-MS/MS / 中国药房

OBJECTIVE To establish a method for simultaneous determination of 15 bile acids in Tongren niuhuang qingxin pills， and to determine the contents of 15 batches of samples. METHODS Using dehydrocholic acid as internal standard， the determination was performed by ultra-high performance liquid chromatography-tandem mass spectrometry （UPLC-MS/MS） method. The determination was performed on Hypersil GOLD C18 column with methanol-0.1% formic acid solution as the mobile phase by gradient elution at the flow rate of 0.2 mL/min. The column temperature was 40 ℃ ， and the sample size was 2 µL. Using heated electrospray ion source， parallel reaction monitoring mode scanning was performed in negative ion mode. SPSS 24.0 software was used for chemical pattern recognition analysis of content determination results. RESULTS The 15 bile acid components had a good linear relationship with peak area （all R2≥0.998 9）； their precision， repeatability and stability were all good （all RSD≤5.49%）； the average recoveries were 93.8%-105.7% （RSD was 0.5%-5.8%）. The average contents of taurocholic acid， 7-oxodeoxycholic acid， 12-dehydrocholic acid， glycocholic acid， 3-oxo-7α， 12α-hydroxy-5β-cholanoic acid， taurochenodeoxycholic acid， 3α-hydroxy- 7-oxo-5β -cholanic acid， hyocholic acid， taurodeoxycholic acid sodium salt hydrate， hyodeoxycholic acid， cholic acid， glycochenodeoxycholic acid， glycodeoxycholic acid， chenodeoxycholic acid and deoxycholic acid were 670.56， 25.97， 10.54， 280.12， 4.04， 29.81， 182.98， 813.55， 120.95， 220.31， 797.37， 18.37， 68.59， 30.13， 59.82 μg/g， respectively. Both cluster analysis and principal component analysis divided 15 batches of Tongren niuhuang qingxin pills into 2 categories， S1-S12 as one category and S13-S15 as the other category. CONCLUSIONS The established method is accurate， sensitive and specific， and can determine many types of bile acids. It also can quickly achieve the quantitative analysis of 15 bile acids in Tongren niuhuang qingxin pills， which is suitable for the quality control of this drug.

The PK bioanalysis method study of c-Met antibody-drug conjugate (RC108) in cynomolgus monkey / 药学学报

Antibody-drug conjugate (ADC) has the characteristics of low toxicity and high efficiency, and plays an important role in cancer treatment. However, due to the complexity of its structure, it brings difficulties in pharmacokinetic (PK) bioanalysis. This study established an analytical method for the detection of ADC (RC108) in cynomolgus monkey plasma by ligand-binding assay (LBA) and liquid chromatography tandem mass spectrometry (LC-MS/MS), which was used to analyze and quantify the total antibody, bound antibody and free drug in cynomolgus monkey plasma. Based on the LBA method, rabbit anti-RC108 Fab and mouse anti-MMAE (monomethyl auristatin E) mAb were pre-coated in 96-well plates as the total antibody and antibody binding reagents, respectively. The samples to be tested were added, and then the detection reagents were added in turn. Goat anti-human IgG (H+L)-HRP, chromogenic solution tetramethylbenzidine (TMB), H2SO4 terminate the reaction, read data at 450 nm/630 nm wavelength of microplate reader; LC-MS/MS analysis method quantifies MMAE concentration, and refer to relevant regulations for methodological validation. The analytical method for quantifying total antibody, bound antibody and free drug of RC108 drug obtained good accuracy and precision, and the selectivity, dilution linearity, hook effect, parallelism and stability were verified. Meet the requirements of biological analysis. Finally, a bioanalytical method for the determination of the concentration of the test substance RC108 (total antibody, conjugated antibody, free MMAE) in cynomolgus monkey plasma with high sensitivity and high throughput was established by LBA and LC-MS/MS method. Subsequent non-clinical research on PK research in cynomolgus monkeys will provide technical support.

Simultaneously quantitative analysis of 35 components in gualoupi injection using hydrophilic interaction liquid chromatography tandem mass spectrometry / 药学学报

A hydrophilic interaction chromatography tandem mass spectrometry method was developed for simultaneous quantification of 35 components in gualoupi injection. The analytes were separated with an ACQUITY XBridge Amide column using 20 mmol·L-1 ammonium formate aqueous solution (pH 3.0) as mobile phase A and 20 mmol·L-1 ammonium formate (pH 3.0)∶acetonitrile (1∶9) as mobile phase B for gradient elution. Mass spectrometry with dynamic multiple reaction monitoring and external standard method were used for quantitative analysis. A total of 35 components were determined in 10 batches of gualoupi injection. The results showed that the 35 compounds had a good linear relationship within their respective concentration ranges with the correlation coefficients (R2 > 0.998 0), the recoveries ranged from 76.6% to 118.5%. The results showed that γ-aminobutyric acid, trigonelline, alanine, threonine, homoserine, citrulline, and leucine were abundant in gualoupi injection, while nicotinamide, methylsuccinic acid, cytosine and choline account for a low percentege. The present study provides an important reference for elucidation of the effective material basis and the improvement of quality standard of gualoupi injection.

Study on Clinical Application and Supervision of LC-MS in in Vitro Diagnosis Test / 中国医疗器械杂志

From the perspective of technical evaluation, this study reviewed the current situation of application and clinical application of medical device products were detected by liquid chromatography-tandem mass spectrometry in the market in recent years. The regulatory requirements of these products in China, USA, EU and Japan were compared and analyzed, and the monitoring situation of adverse events after listing, the standards for reference and the domestic and foreign regulatory documents were combined, the clinical application and regulatory risks of the product were analyzed. The problems such as pre-treatment, system matching, adequacy of performance index requirements, inter-room consistency, reference interval and registration unit were discussed and suggestions for supervision were given, with a view to the field of product R&D and production, review and approval of supervision to provide technical reference.

Determination of bongkrekic acid in corn flour by direct extraction ultra-high performance liquid chromatography tandem mass spectrometry / 公共卫生与预防医学

Objective To establish a direct extraction ultra-high performance liquid chromatography tandem mass spectrometry method for the determination of bongkrekic acid in corn flour. Methods Bongkrekic acid was directly extracted with 80% methanol from corn flour samples, and the supernatant after vortex and centrifugation was determined after passing through membrane filtration. At the same time, the corn flour samples were extracted by solid phase extraction. The determination results of the two methods were compared. Results The linearity of standard series was good within the range of2-20 μg/L, and the linearity coefficient was＞0.999. The determination result of the positive sample by direct extraction method was 193.40 mg/kg (n=6). Adding the standard to the blank sample at the levels of 2, 6, and 10 μg/L, the calculated recovery rate was 75.82% - 99.33%, and the relative standard deviation was 3.54 % - 8.45%. The detection limit of the method reached 6 μg/kg. After extraction by solid phase extraction, the determination result of the positive sample was 196.84 mg/kg (n=6). The recovery rate was 77.12% -100.83%, with a relative standard deviation of 8.32% - 9.54%. Conclusion Compared with the solid phase extraction, the direct extraction method for the extraction of bongkrekic acid from corn flour has the advantages of rapidity, simplicity, and cost savings.

Serum Metabonomics Study of Difference in Lymph Node Metastasis of Pancreatic Ductal Adenocarcinoma Based on LC-MS / 肿瘤防治研究

Objective To investigate the differential metabolites of lymph node metastasis in pancreatic ductal carcinoma (PDAC) and provide new ideas for the pathogenesis, early diagnosis and treatment of metastatic pancreatic cancer. Methods Forty serum specimens of patients with pancreatic ductal carcinoma were collected and divided into lymph node metastasis group (18 cases) and non-metastasis group (22 cases). Thirty-one serum specimens were also collected from the healthy control group. Liquid chromatographytandem mass spectrometry was used to analyze the differential metabolites and metabolic pathways between patients with PDAC and healthy controls as well as between lymph node metastasis and non-metastasis groups. Results Principal component analysis and partial least squares-discriminant analysis revealed statistically significant differences in metabolites and metabolic pathways between patients with PDAC and the healthy controls and between lymph node metastasis and non-metastasis groups. The differences in profiles were also statistically significant. Seventy-six different metabolites and 11 metabolic pathways were screened between patients with PDAC and the healthy controls, among which phenylalanine metabolism and histidine metabolism were the two most influential metabolic pathways. Four different metabolites were screened between lymph node metastasis and non-metastasis groups, and the expression of ethopropazine and phenylalanine were upregulated but the expression of tetrahydrodeoxycorticosterone and oxprenolol were downregulated. Conclusion Metabolites are significantly altered in the lymph node metastasis group of patients with PDAC compared with the non-metastasis group. Ethopropazine, phenylalanine, tetrahydrodeoxy corticosterone, and oxprenolol are potential biomarkers of lymph node metastasis in patients with PDAC.

Determination of pentachlorophenol in wooden chopping boards with gas chromatography-tandem mass spectrometry / 预防医学

Objective@#To optimize the determination of pentachlorophenol in wooden chopping boards through pretreatment of miniaturized samples.@*Methods@# The pretreated wooden chopping board samples were subjected to ultrasound extraction (1 mL of 0.5 mol/L K2CO3 added in 5 mL extraction solution) in 8 mL acetone and 2 mL water, followed by derivatization with 0.3 mL acetic anhydride, extraction with n-hexane and separation with DB-5ms column (30 m×0.25 mm, 0.25 μm). Gas chromatography tandem mass spectrometry (GC-MS/MS) was performed in multiple reaction monitoring (MRM) mode with quantitative analysis using the internal standard method.@*Results@#The GC-MS/MS assay showed a good linear relationship within the range of 0.01 to 0.2 µg/mL (R2>0.999), with a 0.003 mg/kg limit of detection and 0.01 mg/kg limit of quantitation. The mean recovery rates were 84.2% to 96.7% at spiked concentrations of 0.003, 0.01 and 0.03 mg/kg, with relative standard deviation of 2.2% to 6.1%.@*Conclusions@#The established GC-MS/MS assay is easy to perform, environment-friendly, highly accurate and sensitivity, which is feasible for determination of pentachlorophenol in wooden chopping boards.

The trend of changes in serum vitamin A, D, and E levels during pregnancy / 中华检验医学杂志

Objective:High performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) was used to quantify the levels of vitamins A, D and E in pregnant women during the second trimester, and to investigate the change trends of serum vitamins A, D and E levels during pregnancy.Method:A total of 720 pregnant women with an average age of (29.7±4.4) years and 12-22 weeks of gestation were included from October 1, 2021 to October 30, 2022 in the obstetrics department of the People′s Hospital of Wuhan University. The concentrations of vitamins A, D and E were determined by HPLC-MS/MS. The concentration levels of each group were statistically analyzed and the deficiency rate were calculated.Results:The distribution range of vitamin A, D and E (95% CI) was 0.74-2.74 μmol/L, 2.88-25.37 ng/ml and 6.18-35.08 μmol/L, with the deficiency rates were 9.30%, 93.76% and 35.83%, respectively. Vitamin A, D and E levels in the twin group were (1.67±0.51) μmol/L, (13.18±7.44) ng/ml and 11.97 (8.85, 14.60) μmol/L, respectively. They were significantly higher than those in the singlet group (1.45±0.36) μmol/L, (10.87±5.26) ng/ml and 10.46 (6.99, 14.11) μmol/L, with statistical significance by independent sample t-test ( P<0.001). The concentration of vitamin D in the lower BMI group (<22 kg/m 2) was (12.54±5.74) ng/ml, significantly higher than that in the fat group (≥22 kg/m 2) (10.46±4.90) ng/ml, and the rank-sum test was statistically significant ( P<0.001). Conclusion:In this study, the levels of three vitamins were monitored in mid-pregnancy using HPLC-MS/MS, and the changes of serum vitamin A, D, and E levels during pregnancy were analyzed.

Development and validation of an LC-MS/MS method for the determination of 12 ceramides in human plasma / 中华检验医学杂志

Objective:To establish and validate a reliable and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the detection of 12 ceramides in human plasma.Methods:From October 2021 to October 2022, 438 apparently healthy individuals were enrolled in the Affiliated Hospitals of Zunyi Medical University for reference intervals of 12 ceramides in this population. Plasma samples were collected, and separated using the ACQUITY UPLC BEH C18 (2.1×50 mm, 1.7 μm) column, deuterated isotopes were used as internal standards. The mobile phase is water (containing 0.1% formic acid) and isopropanol: acetonitrile (1∶1, v/v, containing 0.1% formic acid) at a flow rate of 0.4 ml/min with gradient elution. The detection method was established using the Qlife Lab 9000 Plus triple quadrupole mass spectrometer. The performance of the method was evaluated in terms of linearity, the lower limit of quantification, precision, recovery, and stability.Results:The method passed the performance evaluation in terms of linearity, the lower limit of quantification, recovery, precision, and stability. The intra-and inter-batch precision of the 12 ceramides ranged from 1.3% to 14.3%, the correctness was verified by spiked recovery experiments, and the recoveries ranged from 91.9% to 111.0%. The lower limit of quantification ranged from 0.001 to 0.100 μmol/L. Standard curve showed good linearity (correlation coefficient r>0.990). Stability tests showed that the 12 ceramides were stable in the biological matrix and after processing under different conditions for a specified period of time. The corresponding biological reference intervals were established for each of the 12 ceramides: 0.103-0.326 μmol/L for Cer(d18∶1/16∶0), 0.018-0.098 μmol/L for Cer(d18∶1/18∶0), 0.933-3.919 μmol/L for Cer(d18∶1/24∶0), 0.243-1.072 μmol/L for Cer(d18∶1/24∶1), 0.001-0.007 μmol/L for Cer(d18∶1/14∶0), 0.022-0.095 μmol/L for Cer(d18∶1/20∶0), 0.185-0.835 μmol/L for Cer(d18∶1/22∶0), 0.003-0.022 μmol/L for Cer(d18∶0/16∶0), 0.001-0.016 μmol/L for Cer(d18∶0/18∶0), 0.017-0.156 μmol/L for Cer(d18∶0/24∶0), 0.008-0.074 μmol/L for Cer(d18∶0/24∶1), and 0.106-0.721 μmol/L for LacCer(d18∶1/24∶1). Conclusion:Our study shows that the newly established LC-MS/MS method for the determination of 12 ceramides in human plasma is reliable, and suitable for clinical application.