Objective To develop a reversed-phase high-performance liquid chromatography(RP-HPLC) method for the determination of residual phenylmethanesulfonyl fluoride(PMSF) content in recombinant virus-like particle(VLP) vaccine stock solution,and to verify the method for the determination of PMSF residues in recombinant VLP vaccine stock solution.Methods A RP-HPLC method for the determination of PMSF residues was developed by screening detection wavelength,flow rate,injection amount and chromatographic column.The specificity,linearity and range,limit of quantitation(LOQ),limit of detection(LOD),accuracy,precision and durability of the method were verified.The developed method was used to detect PMSF residues in three batches of recombinant hepatitis E vaccine stock solution.Results The RP-HPLC method was developed,with ChromCore 300 C18(4.6 mm × 250 mm,5 μm) as the chromatographic column at the column temperature of 25 ℃,the detection wavelength of 210 nm,the injection amount of 100 μL,0.1% trifluoroacetic acid(TFA)/water and0.1% TFA/acetonitrile as mobile phase at a flow rate of 1 mL/min,and the detection time of 18 min.The absorption peaks of PMSF control and vaccine stock solution containing PMSF appeared around 5.0 min,while the vaccine stock solution and stock solution buffer showed no absorption peak.There was a good linear relationship between the concentration of PMSF control and peak area in the range of 0-5.0 μg/mL,with R~2 of 0.999.The LOQ and LOD of this method was 0.250 μg/mL and 0.125 μg/mL,respectively.The recovery rates of PMSF control with high,medium and low concentrations were all between 90% and 110%.The relative standard deviations(RSDs) of retention time,peak height and peak area for the determination of instrument repeatability,sample repeatability and intermediate precision were all less than 5%.The RSDs of retention time,peak height and peak area were all less than 5% under the TFA concentration of 0.09%,0.1% and 0.11% at column temperature of 20,25 and 30 ℃.PMSF was not detected in the three batches of recombinant hepatitis E vaccine stock solution.Conclusion The developed RP-HPLC method has good specificity,linearity and durability,high accuracy and precision,and can be used to detect residual PMSF content in recombinant VLP vaccine stock solution.
Phenylmethanesulfonyl fluoride(PMSF) ; Reversed-phase high-performance liquid chromatography(RP-HPLC) ; Virus-like particle(VLP) vaccine

OBJECTIVE

This study aimed to determine and quantify the presence of the active components in Thai propolis extracts using high performance liquid chromatography (HPLC). Moreover, the anti-caries potential of Thai propolis extract and its active ingredients were tested.

Fifty milligrams of Thai propolis were extracted using either 100%, 90%, 80%, or 70% ethanol and subsequently analyzed using HPLC with a mobile phase gradient system of 10-100% acetonitrile in 0.05% aqueous ortho-phosphoric acid, flow rate of 0.8 mL/min, and detection wavelength of 280 nm. Varying concentrations of Thai propolis extracts as well as four active ingredients were subjected to agar well diffusion test against the growth of Streptococcus mutans (S. mutans) or Lactobacillus caseii (L. caseii).

The concentrations of the four active ingredients: vicenin-2, vitexin, apigenin, and cinnamic acid, were significantly affected by ethanolic concentrations. The chromatographic peaks of all active ingredients from 70% and 80% ethanolic extracts appeared more defined, as compared to those which used higher concentrations of ethanol for extraction. Except for the absolute ethanolic extract, all of the examined propolis extracts, as well as its active ingredients inhibited both S. mutans and L. caseii.

Thai propolis extracts contain vicenin-2, vitexin, apigenin, and cinnamic acid as part of its active ingredients. These were found to be significantly affected by the increase in ethanol during its extraction. The presence of these active ingredients might have contributed to the anti-caries potential of Thai propolis extracts.

This study establishes a high-performance liquid chromatography (HPLC) method for the determination of acetate content in hemodialysis solutions and dialysis concentrates. In this study, Synergi Polar-RP column is utilized. Phosphate buffered saline (50 mmol/L, pH=2.5) is used as a mobile phase. The flow rate is 1.0 mL/min. The wavelength of detection is 212 nm. Results show that the linear relationship of acetate is good in the range of 0.1~20 mmol/L, r =0.999 9 and the spike recoveries are from 98.9%~99.5%, RSD<0.5% ( n=3). This method can easily and accurately determine the acetate content in hemodialysis solutions and dialysis concentrates, and can be applied to quality control in the production and use of such products.
Chromatography, High Pressure Liquid/methods* ; Acetates/analysis* ; Hemodialysis Solutions/analysis* ; Dialysis Solutions/analysis* ; Renal Dialysis

A method for determining volatile organic compounds (VOCs) emitted from medical molecular sieve oxygen concentrators was developed using thermal desorption-gas chromatography-mass spectrometry (TD-GC-MS). The oxygen concentrator gas was sampled at a flow rate of 0.5 L/min through a branched sampling system onto Tenax GR/carbopack B adsorption tubes. The adsorbed compounds were desorbed and introduced using a programmed temperature vaporization inlet system, followed by chromatographic separation on an SH-I-624Sil MS column. Four VOCs (BHT-Q, PTBP, BHT-quinol, and EHB) were detected in the medical oxygen concentrator using this method. Calibration curves for these compounds exhibited excellent linearity ( R ²>0.99) within the range of 3~100 ng. With a sampling volume of 20 L, the detection limit of the four VOCs ranged from 0.003 9 to 0.022 2 μg/m ³. Spike recovery rates for the four VOCs were between 95% and 115%, with relative standard deviations (RSDs) below 5% ( n=6). The method is simple, rapid, highly sensitive, and accurate, making it suitable for VOCs detection in medical molecular sieve oxygen concentrators.
Volatile Organic Compounds/analysis* ; Gas Chromatography-Mass Spectrometry/methods* ; Oxygen

OBJECTIVES: Tiletamine, a veterinary anesthetic, has emerged as a novel psychoactive substance and has been abused in many parts of the world, causing great harm to public health. However, the sensitivity of existing detection methods cannot meet the needs of forensic practice. This study aims to establish an ultra-high-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for the determination of tiletamine and its metabolite desethyltiletamine in human biological samples, and to verify its applicability in forensic practice. METHODS: SKF_525A was used as the internal standard. Biological samples were extracted with acetonitrile containing 1 ng/mL SKF_525A, vortexed for 10 min, ultrasonicated for 20 min, centrifuged at 10 000 r/min for 10 min, and 500 μL of the supernatant was filtered through a 0.22 μm membrane. Analyses were performed using an ACQUITY UPLC H-Class PLUS system and an XEVO TQ-S Micro triple quadrupole mass spectrometer. An ACQUITY UPLC^® BEH C18 (1.7 µm, 2.1 mm×100 mm) column at a flow rate of 0.3 mL/min was used, and four mobile phase systems were tested to optimize separation. Detection used positive electrospray ionization (ESI+) in multiple reaction monitoring (MRM) mode, with quantifier ion transitions of mass to charge 224.043→179.016 for tiletamine and mass to charge 196.08→151.06 for desethyltiletamine. Calibration curves were established over 0.1-200 ng/mL in spiked blood samples. The linear range, limit of detection (LOD), and limit of quantification (LOQ) were determined. Low (5 ng/mL), medium (20 ng/mL), and high (100 ng/mL) concentrations of tiletamine were spiked into blood, liver, and kidney to evaluate precision, accuracy, matrix effect, recovery, and stability. Finally, actual forensic case samples were tested to validate applicability. RESULTS: The established UPLC-MS/MS method achieved simultaneous detection of tiletamine and desethyltiletamine in human biological samples, with retention times of 3.42 min and 2.82 min, respectively. Using mobile phase A (20 mmol/L ammonium acetate and 0.1% formic acid in water) and mobile phase B (acetonitrile) produced the best separation. In blood, tiletamine showed good linearity from 0.1-200 ng/mL (r=0.992, R²=0.983), LOD 0.03 ng/mL, LOQ 0.1 ng/mL, recovery 92%-107%, and matrix effect 71%-99%. In liver and kidney, recoveries were 91%-98% and 93%-104%, and matrix effects were 69%-96% and 72%-100%, respectively. Intra- and inter-day precision [expressed as relative standard deviation (RSD)] and accuracy [expressed as relative error (RE)] were within 15%, and samples were stable at -20 ℃. Tiletamine was detected in actual case samples at 0.37 μg/mL (blood), 0.15 μg/g (liver), 0.11 μg/g (kidney) in case 1, and 8.75 ng/mL (blood) in case 2; desethyltiletamine was also detected in blood. CONCLUSIONS The UPLC-MS/MS method is efficient, accurate, and sensitive, and is suitable for detecting tiletamine and desethyltiletamine in human biological samples.
Tandem Mass Spectrometry/methods* ; Humans ; Chromatography, High Pressure Liquid/methods* ; Substance Abuse Detection/methods* ; Liquid Chromatography-Mass Spectrometry

Dynamic changes in the physiochemical, structural, and flavor characteristics of ginger-juice milk curd were explored by texture analysis, scanning electron microscopy, rheometry, electronic tongue, and gas chromatography-mass spectrometry (GC-MS). Protein electrophoresis showed that ginger juice could hydrolyze α_s-, β-, and κ-casein. Curd formation was initiated at 90 s, marked by significant changes in intensity detected via intrinsic fluorescence. The contents of soluble protein and calcium decreased rapidly during coagulation, while the caseinolytic activity, storage moduli, loss moduli, hardness, adhesiveness, and water-holding capacity increased, resulting in a denser gel structure with smaller pores and fewer cavitations as observed by scanning electron microscopy. Electronic tongue analysis indicated that milk could neutralize the astringency and saltiness of ginger juice, rendering the taste of ginger-juice milk curd more akin to that of milk. Approximately 70 volatile components were detected in ginger-juice milk curd. α‍-Zingiberene, α‍-curcumene, β‍-sesquiphellandrene, and β‍-bisabolene were the predominant volatile flavor compounds, exhibiting an initial decrease in content followed by stability after 90 s. Decanoic acid, γ-elemene, and caryophyllene were identified as unique volatile compounds after mixing of milk and ginger juice. Understanding the dynamic changes in these characteristics during coagulation holds significant importance for the production of ginger-juice milk curd.
Zingiber officinale/chemistry* ; Milk/chemistry* ; Animals ; Taste ; Gas Chromatography-Mass Spectrometry ; Caseins/chemistry* ; Microscopy, Electron, Scanning ; Rheology ; Flavoring Agents

Aconiti Lateralis Radix Praeparata (Fuzi) represents a significant traditional Chinese medicine (TCM) that exhibits both notable pharmacological effects and toxicity. Various processing methods are implemented to reduce the toxicity of raw Fuzi by modifying its toxic and effective components, primarily diterpenoid alkaloids. To comprehensively analyze the chemical variations between different Fuzi products, ultra-high performance liquid chromatography-linear ion trap quadrupole Orbitrap mass spectrometry (UHPLC-LTQ-Orbitrap MS) was employed to systematically characterize Shengfuzi, Heishunpian and Baifupian. A total of 249 diterpenoid alkaloids present in Shengfuzi were identified, while only 111 and 61 in Heishunpian and Baifupian were detected respectively, indicating substantial differences among these products. An untargeted metabolomics approach combined with multivariate statistical analysis revealed 42 potential chemical markers. Through subsequent validation using 52 batches of commercial Heishunpian and Baifupian samples, 8 robust markers distinguishing these products were identified, including AC1-propanoic acid-3OH, HE-glucoside, HE-hydroxyvaleric acid-2OH, dihydrosphingosine, N-dodecoxycarbonylvaline and three unknown compounds. Additionally, the MS imaging (MSI) technique was utilized to visualize the spatial distribution of chemical constituents in raw Fuzi, revealing how different processing procedures affect the chemical variations between Heishunpian and Baifupian. The distribution patterns of different diterpenoid alkaloid subtypes partially explained the chemical differences among products. This research provides valuable insights into the material basis for future investigations of different Fuzi products.
Diterpenes/chemistry* ; Alkaloids/chemistry* ; Chromatography, High Pressure Liquid/methods* ; Aconitum/chemistry* ; Drugs, Chinese Herbal/chemistry* ; Metabolomics ; Mass Spectrometry/methods* ; Plant Roots/chemistry* ; Molecular Structure

Processing for deodorization is widely used in the production of animal-derived Chinese medicinal materials. In this study, Heracles Neo ultra-fast gas-phase electronic nose combined with chemometrics was employed to analyze the overall odor difference of Cervi Cornu Pantotrichum(focusing on that derived from Cervus nippon Temminck in this study) before and after processing. The results showed that the electronic nose effectively distinguished between the medicinal materials and decoction pieces of Cervi Cornu Pantotrichum. HS-GC-MS was used to identify and quantify the volatile components in the medicinal materials and decoction pieces of Cervi Cornu Pantotrichum, and 35 and 37 volatile components were detected in the medicinal materials and decoction pieces, respectively. The medicinal materials and decoction pieces contained 28 common volatile components contributing to the odor of Cervi Cornu Pantotrichum. The odor activity value(OAV) of each volatile component was calculated based on the olfactory threshold and relative content. The results showed that there were 17 key odor substances such as isovaleraldehyde, 2-methylbutanal, isobutyraldehyde, hexanal, and methanethiol in the medicinal materials and decoction pieces of Cervi Cornu Pantotrichum. All of them had bad odor and were the main source of the odor of Cervi Cornu Pantotrichum. The results of principal component analysis(PCA) and orthogonal partial least squares-discriminant analysis(OPLS-DA) showed that there were significant differences in volatile components between the medicinal materials and decoction pieces of Cervi Cornu Pantotrichum. Based on the thresholds of P<0.05 and Variable Importance in Projection(VIP)>1, 21 differential volatile odor components were screened out. Among them, isopentanol, isovaleraldehyde, 2-methylbutanal, n-nonanal, and dimethylamine were the key differential odor compounds between the medicinal materials and decoction pieces of Cervi Cornu Pantotrichum. The odor compounds and their relative content reduced, and some flavor substances such as esters were produced after processing with wine, which was the main reason for the reduction of the odor after processing of Cervi Cornu Pantotrichum.
Odorants/analysis* ; Electronic Nose ; Gas Chromatography-Mass Spectrometry/methods* ; Animals ; Volatile Organic Compounds/analysis* ; Deer ; Drugs, Chinese Herbal/chemistry*

Based on ultra-high performance liquid chromatography-quadrupole-Exactive Orbitrap mass spectrometry(UPLC-Q-Exactive Orbitrap-MS) technology and network pharmacology, this study explored the pharmacodynamic substances and potential mechanisms of Huazhuo Sanjie Chubi Decoction in the treatment of gouty arthritis(GA). UPLC-Q-Exactive Orbitrap-MS technology was used to identify the components in Huazhuo Sanjie Chubi Decoction, and the qualitative analysis of its active ingredients was carried out, with a total of 184 active ingredients identified. A total of 897 active ingredient targets were screened through the PharmMapper database, and 491 GA-related disease targets were obtained from the OMIM, GeneCards, CTD databases. After Venn analysis, 60 intersecting targets were obtained. The component target-GA target network was constructed through the Cytoscape platform, and the STRING database was used to construct a protein-protein interaction network, with 16 core targets screened. The core targets were subjected to Gene Ontology(GO) and Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway enrichment analyses, and the component-target-pathway network was constructed. It was found that the main active ingredients of the formula for the treatment of GA were phenols, flavonoids, alkaloids, and terpenoids, and the key targets were SRC, MMP3, MMP9, REN, ALB, IGF1R, PPARG, MAPK1, HPRT1, and CASP1. Through GO analysis, it was found that the treatment of GA mainly involved biological processes such as lipid response, bacterial response, and biostimulus response. KEGG analysis showed that the pathways related to the treatment of GA included lipids and atherosclerosis, neutrophil extracellular traps(NETs), IL-17, and so on. In summary, phenols, flavonoids, alkaloids, and terpenoids may be the core pharmacodynamic substances of Huazhuo Sanjie Chubi Decoction in the treatment of GA, and the pharmacodynamic mechanism may be related to SRC, MMP3, MMP9, and other targets, as well as lipids and atherosclerosis, NETs, IL-17, and other pathways.
Drugs, Chinese Herbal/therapeutic use* ; Network Pharmacology ; Arthritis, Gouty/metabolism* ; Chromatography, High Pressure Liquid/methods* ; Humans ; Mass Spectrometry/methods* ; Protein Interaction Maps/drug effects*

This study aims to investigate the correlations of the appearance traits, total antioxidant capacity, and component content of Forsythiae Fructus processed by different methods, explore the effects of different processing methods on the abovementioned three aspects of Forsythiae Fructus, and screen out the internal and external indicators that have important effects on its quality. It determined the length, diameter, stem length, chroma value L~*, a~*, b~*, and other appearance indexes and antioxidant activity of Forsythiae Fructus processed by different methods. The content of forsythiaside A, rutin, forsythin, pinoresinol, and phillygenin was determined by ultra performance liquid chromatography(UPLC). Correlation analysis, principal component analysis(PCA), orthogonal partial least squares discriminant analysis(OPLS-DA), and independent sample t-test analysis were performed on the appearance indexes and the component content. The correlation analysis showed that there were differences in the appearance traits and the component content. L~* and E~* had highly significant negative correlations with pinoresinol and phillygenin(P<0.01) and significant positive correlations with forsythiaside A(P<0.05). There were a highly significant negative correlation between a~* and forsythiaside A(P<0.01) and highly significant positive correlations of a~* with pinoresinol and phillygenin(P<0.01). There were a highly significant positive correlation between b~* and forsythiaside A(P<0.01) and highly significant negative correlations of b~* with pinoresinol and phillygenin(P<0.01). The total antioxidant capacity had highly significant negative correlations with pinoresinol and phillygenin(P<0.01). The PCA results showed that there were differences among Forsythiae Fructus samples processed by different methods. OPLS-DA marked five important indicators, which were forsythiaside A, stem length, E~*, L~*, and b~*. The results of independent sample t-test showed that the content of forsythiaside A, pinoresinol, and phillygenin, the total antioxidant capacity, and the appearance traits such as L~*, a~*, b~*, and E~* were significantly different between the Forsythiae Fructus samples processed by steaming and boiling(P<0.05). According to content determination and a related biological activity analysis, steaming is a good choice from the perspective of improving the stability of chemical constituents and antioxidant activity of Forsythiae Fructus. From the point of view of improving the stability of chemical constituents and anti-inflammatory and anti-cancer activities of Forsythiae Fructus, it is recommended to use boiling as the processing method. Based on the above analysis methods, the main indexes for the appearance traits of Forsythiae Fructus processed by different methods are powder chroma value(L~*, a~*, b~*, E~*), stem length, and total antioxidant capacity, and those for chemical constituents are the content of forsythiaside A, pinoresinol, and phillygenin. This study provides reference for seeking scientific processing methods of Forsythiae Fructus.
Forsythia/chemistry* ; Drugs, Chinese Herbal/isolation & purification* ; Fruit/chemistry* ; Antioxidants/analysis* ; Chromatography, High Pressure Liquid ; Glycosides/analysis* ; Principal Component Analysis ; Furans ; Lignans