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Chinese Journal of Chromatography
2025, Vol. 43, No. 9
Online: 08 September 2025

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Advances in the development of analysis techniques for organophosphate diesters in water CHEN Jie, LIU Mingliang, WU Lu, XU Feng, LYU Jing, CHEN Linhai, LI Wei, FU Jie, FU Jianjie 2025, 43 (9):  987-995.  DOI: 10.3724/SP.J.1123.2025.02007 Abstract ( 176 )   HTML ( 35 )   PDF (712KB) ( 68 )   Organophosphate triesters （tri-OPEs） are synthetic phosphate derivatives that are primarily used as flame retardants and plasticizers. Tri-OPEs have become significant aquatic contaminants owing to their large production volumes and wide range of applications. Organophosphate diesters （di-OPEs） are closely related to tri-OPEs. Aside from emissions resulting from the production and usage of di-OPEs themselves， tri-OPEs can become transformed into di-OPEs， which also provides a significant source of this environmental contaminant. The physicochemical properties of a di-OPE depend significantly on its structure， which provides challenges for their detection and analysis， including low extraction efficiencies， chromatographic separation difficulties， and a lack of highly sensitive quantitative methods for their analysis. An increasing number of studies have found that di-OPEs are present in industrial/domestic wastewater， surface water， and drinking water， with some concentrations in surface water and tap water close to or even higher than those of the corresponding tri-OPEs. Additionally， certain di-OPEs are somewhat more toxic than the corresponding tri-OPEs； hence， awareness that di-OPEs are present in aquatic environments has raised widespread concern. This review first systematically outlines the physicochemical properties of common di-OPEs and their potential sources based on previous research into di-OPEs in water matrices. In addition， the use of solid phase extraction （SPE） technology to extract， enrich， and purify di-OPEs from water matrices is summarized， while the advantages and limitations of SPE methodologies are critically evaluated. Furthermore， the use and distinctive features of reverse-phase chromatography， ion-pair reverse-phase chromatography， and hydrophilic interaction liquid chromatography （HILIC） for the chromatographic separation of di-OPEs are comprehensively summarized and compared. At the same time， advances in the quantitative analysis of di-OPEs using liquid chromatography-tandem triple quadrupole mass spectrometry （LC-MS/MS） and liquid chromatography-high-resolution mass spectrometry （LC-HRMS） are reviewed. Finally， in terms of efficient collection of water samples and high-throughput pretreatment of di-OPEs in water matrices， the prospect of developing novel sampling and on-site enrichment technologies for new pollutants in water matrices based on the principle of dispersed solid phase extraction is proposed. Additionally， the prospect of using liquid chromatography tandem high-resolution mass spectrometry for high-throughput screening and high-sensitivity detection of di-OPEs and unknown transformation products of tri-OPEs has been proposed.

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A large-scale method for the enrichment and identification of N-glycopeptides in microscale plasma samples YANG Xinyi, QIN Weijie 2025, 43 (9):  996-1004.  DOI: 10.3724/SP.J.1123.2025.04004 Abstract ( 73 )   HTML ( 9 )   PDF (1412KB) ( 27 )   Blood， which forms part of the systemic circulatory system， contains proteins from various tissues and organs. Hence， blood samples are ideal vehicles for studying diseases and physiological states. Plasma is an important component of blood and is essential for clinical proteomics research. Plasma contains rich physiological and pathological information； consequently， it is an ideal medium for discovering disease-related biomarkers. Protein N-glycosylation is a key post-translational modification route. This route is widely involved in biological processes such as intercellular communication， immune regulation， and signal transduction. Changes resulting from aberrant N-glycosylation are closely associated with various pathological conditions， including autoimmune and neurodegenerative diseases and tumors. Hence， N-glycosylation proteomics is highly valuable during biomarker and drug-target development. However， efficiently enriching N-glycopeptides in biological samples before detection by mass spectrometry （MS） is difficult. This is because the highly abundant unmodified peptides result in signal suppression. Consequently， achieving deep N-glycoproteomic coverage is a key challenge， particularly for trace plasma samples， for which in-depth studies are currently lacking. In this study， we developed a strategy for comprehensively profiling trace N-glycopeptides in plasma. This includes an efficient enrichment method in combination with highly sensitive MS. The developed approach integrates glycopeptide enrichment using advanced hydrophilic interaction liquid chromatography （HILIC） with state-of-the-art MS platforms. This significantly enhances detection depth and sensitivity during N-glycosylation analysis using minimal plasma volumes. Selectivity and efficiency during N-glycopeptide enrichment were maximized by systematically optimizing key HILIC-packed stationary-phase parameters. These parameters include chemical composition， pore size， and surface modification. Additionally， the elution gradient was fine-tuned to improve glycopeptide recovery. This optimization process delivered high N-glycopeptide specificity， even in complex plasma matrices. To overcome the limitations of single-platform MS， we implemented a complementary dual-platform strategy. This strategy combines the high-speed， high-resolution capabilities of the Tims TOF Pro 2 instrument with the ultra-high mass accuracy and resolution of the Orbitrap Lumos spectrometer. The former instrument facilitates the rapid and sensitive identification of glycopeptides， particularly for low-abundance species. It exploits the trapped ion mobility spectrometry （TIMS） and parallel accumulated sequential fragmentation （PASEF） technology. The Orbitrap Lumos provides exceptional mass accuracy and high-resolution MS/MS spectra that enable confident glycopeptide structural characterization. This synergistic approach significantly expands the N-glycopeptide identification depth and ensures comprehensive glycosylation-site and glycan-composition coverage. The developed optimized workflow successfully identified 2 962 intact N-glycopeptides using only 20 μg of plasma peptides （equivalent to 0.5 μL of whole plasma）. This set a new benchmark for sensitivity in the micro-volume plasma glycoproteome field. This achievement addresses a critical gap， where conventional methods typically require much larger sample volumes. This limits their applicability to clinical and precision medicine settings where sample availability is restricted. The developed platform provides a robust and reliable analytical framework for plasma N-glycoproteomics with significant implications for precision medicine. This method facilitates large-scale clinical studies by enabling highly sensitive glycopeptide profiling from very small plasma volumes. This included the longitudinal monitoring of disease progression and therapeutic responses. Furthermore， it offers a powerful tool for discovering novel N-glycosylation-based biomarkers for use in early disease diagnosis， prognosis， and personalized treatment strategies. In summary， this study advances the technical capabilities of plasma N-glycoproteomics. Additionally， it facilitates the broader use of plasma N-glycoproteomics in biomedical research and clinical diagnostics.

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Determination of 26 perfluorinated and polyfluoroalkyl compounds in human serum by solid-phase extraction-high performance liquid chromatography-tandem mass spectrometry YUE Yingxiao, BIAN Yating, CHENG Yufan, HE Lu, WANG Dan, YAN Peixia, YAN Wei, LIU Guiying, SONG Huan, LIU Liangpo 2025, 43 (9):  1005-1013.  DOI: 10.3724/SP.J.1123.2024.10002 Abstract ( 128 )   HTML ( 11 )   PDF (643KB) ( 60 )   Perfluorinated and polyfluoroalkyl compounds （PFASs） represent a category of synthetic chemicals renowned for their environmental persistence. Owing to their hydrophobic， oleophobic， and high-temperature-resistant properties， PFASs are extensively utilized in industrial， agricultural， and civilian sectors， including applications in leather， textiles， flame-retardant materials， lubricants， and coatings， among others. PFASs can accumulate within the human body， exhibiting multi-organ toxicity. Continuous monitoring of PFASs with ambiguous toxicity profiles is vital for evaluating human exposure and associated health risks. Consequently， the establishment of a high-throughput and highly sensitive detection method is of paramount importance for accurately assessing the exposure levels of PFASs in the human body. In this study， a commercial high-throughput HMR-Lipid 96-well solid-phase extraction plate was adopted， combined with high performance liquid chromatography-tandem mass spectrometry （HPLC-MS/MS）， to establish a simple， efficient method that can simultaneously quantitatively detect 26 PFASs in human serum. Serum samples were extracted using the HMR-lipid 96-well solid-phase extraction plate. The Phenomenex C18 chromatography column （250 mm×4.6 mm， 5 μm） was used as the capture column and connected between the liquid chromatography mixer and the autosampler to avoid high background pollution. The target compounds were separated by the Accucore C18 chromatography column （100 mm×2.4 mm， 2.6 μm） and analyzed using the electrospray ionization with negative ion scanning mode and multiple reaction monitoring （MRM） mode. The methodological validation results indicated that the 26 PFASs had good linear relationships within the range of 0.2-100 ng/mL， with correlation coefficients （r） of 0.995 1-0.999 9. The limits of detection （LODs） and quantification （LOQs） were 0.01-0.15 ng/mL and 0.02-0.47 ng/mL， respectively. At three spiked levels of low， medium and high， the recoveries of the 26 PFASs ranged from 80.1% to 119.5%， and the relative standard deviations （RSDs） ranged from 0.5% to 11.9%. This method has the advantages of high sensitivity， good accuracy， simple operation， fast extraction speed， low reagent consumption and small sample volume required. It is suitable for large-scale population biological monitoring and provides a scientific method support for accurately assessing the exposure of PFASs in the human body and its potential health risks.

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Determination of 17 bisphenol compounds in human urine by solid supported liquid-liquid extraction-ultra-high performance liquid chromatography-tandem mass spectrometry JIN Yu’e, ZHANG Lange, ZHOU Jingxian, MA Jinjing, YUAN Lili, XIAO Ping, WANG Guoquan 2025, 43 (9):  1014-1024.  DOI: 10.3724/SP.J.1123.2024.11032 Abstract ( 89 )   HTML ( 12 )   PDF (1492KB) ( 65 )   Bisphenol A （BPA） and its analogs are collectively termed bisphenol compounds （BPs）， which are predominantly utilized in the manufacturing of polycarbonate plastics and epoxy resins. BPs are ubiquitous in diverse environmental matrices， human tissues， and metabolic products. Extensive research has demonstrated that BPs exert adverse effects on the nervous， reproductive， immune， and metabolic systems. After exposure in humans， BPs are primarily excreted in urine. Consequently， the development of efficient and robust analytical methods for BPs quantification in urine is essential for assessing population exposure levels. In this study， solid supported liquid-liquid extraction （SLE） was combined with ultra-high performance liquid chromatography-tandem mass spectrometry （UHPLC-MS/MS） technology to establish a high-throughput determination method for 17 BPs in human urine. After enzymatic hydrolysis treatment of the samples， the steps of fully automatic SLE purification， nitrogen blowing concentration and redissolution were carried out successively. Separation was performed using a CAPCELL PAK ADME chromatography column （100 mm×2.1 mm， 2 μm）， and gradient elution was carried out with 0.05 mmol/L ammonium fluoride aqueous solution and 0.05 mmol/L ammonium fluoride methanol solution as the mobile phases. MS detection was carried out using the electrospray ionization （ESI） negative ion scanning mode under the multi-reaction monitoring （MRM） mode. Qualitative analysis was conducted based on retention time and ion abundance ratio， and quantitative analysis was performed using the internal standard method. Under the optimized conditions， 17 BPs can be effectively separated. The linear relationships of the 17 BPs within the corresponding mass concentration ranges were good， and the correlation coefficients （r） were ≥0.998 6， the limits of detection （LODs） and quantification （LOQ） were 0.002-0.489 μg/L and 0.005-0.986 μg/L， respectively. Children’s mixed urine samples with low background content were selected as the matrix， and then spiked recovery tests were conducted at three spiked levels （low， medium and high）. The results showed that the recoveries of 17 BPs were 61.1%-121.7%， the intra-day RSDs were 1.3%-11.2%， and the inter-day RSDs were 3.7%-19.0%. This method was used to determine 50 random urine samples， and the results showed that a total of 11 BPs were detected. Among them， bisphenol S （BPS） and BPA had the highest detection rates， which were 98.0% and 86.0% respectively， and the median detection levels were 0.075 μg/L and 0.829 μg/L respectively. This method is simple to operate， sensitive and reliable. It is suitable for the rapid quantitative analysis of 17 BPs in human urine and can provide effective technical support for the risk assessment of BPs exposure in the population.

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Determination of N-（1，3-dimethylbutyl）-N′-phenyl-p-phenylenediamine-quinone in urine and dust by ultra performance liquid chromatography-tandem mass spectrometry LU Zhuliangzi, DENG Fenfang, BAI Zhijun, PENG Rongfei, TAN Lei 2025, 43 (9):  1025-1033.  DOI: 10.3724/SP.J.1123.2025.02010 Abstract ( 130 )   HTML ( 8 )   PDF (1110KB) ( 64 )   An ultra performance liquid chromatography-tandem mass spectrometry （UPLC-MS/MS） method was established to determine N-（1，3-dimethylbutyl）-N′-phenyl-p-phenylenediamine-quinone （6PPD-Q） in human urine and dust in order to understand the internal and external exposure levels in humans. The sample preparation conditions were systematically investigated and the chromatographic conditions and MS parameters were optimized. Briefly， internal standard 13C6-6PPD-Q （0.1 ng） was added to a urine sample. Glutathione and NaCl were added after equilibration for 30 min. The mixture was then twice ultrasonically extracted with ethyl acetate. Internal standard 13C6-6PPD-Q （1.0 ng） was also added to a dust sample and the mixture was twice ultrasonically extracted with n-hexane. The combined organic phases were concentrated to near-dryness and then redissolved for instrumental determination， which was performed on a Phenomenex Kinetex F5 column （100 mm×3 mm， 2.6 μm）， with the target analyte gradient eluted using 10 mmol/L ammonium acetate aqueous solution containing 0.01% formic acid and acetonitrile. Positive electrospray ionization （ESI+） and multiple reaction monitoring （MRM） modes were used for identification purposes， and an isotope-labeled internal standard was added for quantification. Good linearities were achieved under the optimized conditions within the 0.01–4.00 and 0.01–20.0 μg/L ranges for urine and dust， respectively， with correlation coefficients of 0.999 9 and 0.999 3， respectively. Limits of detection （LODs） were 0.6 ng/L （urine） and 0.018 ng/g （dust）. Spiked recoveries of 6PPD-Q were 90.3%–94.1% at low， medium， and high spiked levels， with intra-day and inter-day precisions of 0.9%–5.9% and 1.1%–6.3%， respectively. Matrix-effect investigations revealed that 6PPD-Q exhibited weak matrix effects in urine and dust after correction with the isotopic internal standard. The developed method was used to analyze 120 human urine samples， which led to a 6PPD-Q detection frequency of 74.2%， with mass concentrations ranging from <LOD to 13 ng/L， with average and median mass concentrations of 2 and 1 ng/L， respectively. However， 6PPD-Q was detected with a frequency of 100% in 31 indoor dust samples with contents of 1.8–24.9 ng/g， and average and median contents of 5.23 –3.05 ng/g， respectively. The developed method is accurate， reliable， highly sensitive， and it is suitable for the rapid determination of 6PPD-Q in human urine and dust samples.

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Rapid screening of 111 pesticides and veterinary drugs in livestock and poultry meat by ultra performance liquid chromatography-quadrupole-time of flight mass spectrometry WANG Yiming, LI Xiaotong, CHU Kun, WANG Qianqian, WU Shuai, CHEN Chen 2025, 43 (9):  1034-1044.  DOI: 10.3724/SP.J.1123.2024.10026 Abstract ( 121 )   HTML ( 9 )   PDF (1124KB) ( 50 )   The consumption of agricultural products has increased in recent years owing to abundant production and improved living standards. Veterinary drugs are highly commercialized and widely used in animal husbandry to ensure animal health and production performance. Moreover， pesticides can become enriched during animal breeding， resulting in animal-derived food pollution through foraging， drinking， and environmental disinfection that can potentially damage human health. Consequently， food-safety issues associated with pesticide and veterinary-drug residues have attracted considerable attention. However， few reports on the multi-residue analysis of livestock and poultry meat using ultra performance liquid chromatography-quadrupole time of flight mass spectrometry （UPLC-Q-TOF/MS） have been published. Therefore， developing high-throughput and efficient screening methods for monitoring various illegal and/or restricted drugs in animal-derived foods is imperative. In this study， we developed a protocol for simultaneously examining seven pesticide and veterinary-drug types that uses an accurate mass-spectral library. UPLC-Q-TOF/MS was then employed to screen 111 such compounds， including quinolones， macrolides， cephalosporins， and antiviral drugs. The developed protocol was subsequently used to establish a method for quantitatively analyzing more than 90 compounds in livestock and poultry meat. The formula， theoretical exact mass， experimental exact mass， and retention time of each analyte were recorded and used for identification purposes. The main factors affecting the response and sensitivity of the method， such as the LC separation conditions （chromatographic column and mobile phases） and MS parameters， were optimized during instrumental analysis. Pork and chicken samples were extracted with an 80% acetonitrile aqueous solution， after which the supernatant was purified using an Oasis PRiME HLB solid-phase extraction column. The 111 target analytes were separated on a Waters HSS T3 analytical chromatographic column （100 mm×2.1 mm， 1.8 μm） after being blown with nitrogen and redissolved， with gradient elution performed using mobile phases composed of 0.1% formic acid aqueous solution and methanol. The analysis process included a flow rate of 0.4 mL/min， a column temperature of 40 ℃， and an injection volume of 5 μL， with positive electrospray ionization （ESI） and time of flight mass spectrometry full scan information-dependent acquisition-product ion （TOF MS-IDA-Product Ion） scanning modes used. The method was validated in terms of linearity， limits of screening and quantification （SDLs and LOQs， respectively）， matrix effects， accuracy， and precision. Quantification was performed using matrix-matched external-standard calibration. All target compounds exhibited good linearities in their corresponding concentration ranges， with all correlation coefficients （r2） above 0.99. The SDLs of all analytes were in the range of 0.5-10 μg/kg， and the proportion of LOQs within the range of 0.5-10 μg/kg were 88.3% and 86.5%， respectively. The compounds quantified in pork and chicken exhibited recoveries of between 60.2% and 100.2%， and 61.1% and 116.7%， respectively， at spiked levels of LOQ， 2×LOQ and 10×LOQ， with relative standard deviations （RSDs） ranging from 1.1% to 13.9% and 1.0% to 14.1%， respectively. Simulated positive samples and commercial livestock and poultry meat samples were screened using an in-house-constructed mass spectrometry database. Commercial samples were screened while enrofloxacin was detected in two pork samples and tilmicosin was detected in one chicken sample， with content in the range of 4.94-29.1 μg/kg. The method developed in this study is advantageous because it involves simple sample processing and is less time consuming than existing methods； consequently， it is suitable for the rapid and high-throughput screening of pesticides and veterinary residues in livestock and poultry meat.

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Simultaneous identification and detection of four novel isoxazoline drugs in bovine-origin foods using ultra-high performance liquid chromatography coupled to quadrupole/linear ion trap mass spectrometry YANG Cheng, ZHU Weixia, LIU Yafeng, WEI Wei, ZHAO Fang, HU Kai, ZHAO Wenjie 2025, 43 (9):  1045-1052.  DOI: 10.3724/SP.J.1123.2024.11012 Abstract ( 83 )   HTML ( 10 )   PDF (859KB) ( 45 )   Isoxazoline drugs （ISOs） are a class of five-membered heterocyclic compounds containing N and O atoms. They can inhibit γ-aminobutyric acid gated chloride channels and are widely used in the treatment of parasitic diseases in poultry. The intake of animal-derived foods by humans is an important way to come into contact with ISOs. Excessive use of ISOs can lead to their residues in animal-derived foods， thereby threatening human health and causing neurotoxicity and hepatotoxicity. To address the safety issues caused by ISO residues in animal-derived foods， an ultra-high performance liquid chromatography-quadrupole/linear ion trap mass spectrometry （UHPLC-Q/Trap MS） analytical method for four novel ISOs （fluralaner， sarolaner， afoxolaner， lotilaner） in bovine-origin foods （including milk， beef and bovine liver） was established. The sample was first extracted with acetonitrile and then purified with PRiME HLB solid phase extraction （SPE） column. Using 5 mmol/L ammonium acetate aqueous solution and acetonitrile as the mobile phase， after separation by Shim-pack GIST C18-AQ （100 mm×2.1 mm， 2.7 μm） chromatography column， the analysis was carried out in the multi-reaction monitoring （MRM） mode by information-dependent acquisition （IDA）， enhanced product ion scanning （EPI） and spectral library retrieval， and quantification was performed using the external standard method. The results showed that the four ISOs had good linear relationships within their respective mass concentration ranges. The correlation coefficients （r） were all ≥0.993 6， and the limits of detection （LODs） and quantification （LOQs） were 0.2-0.5 μg/kg and 0.5-1.0 μg/kg， respectively. Under the low， medium and high spiked levels （1， 2 and 10 μg/kg）， the recoveries of the four ISOs ranged from 67.6% to 118.9%， and the relative standard deviations （RSDs） ranged from 2.0% to 20.0%. In addition， in this study， qualitative screening and analysis of the target compounds were conducted through MRM-IDA-EPI combined with spectral library retrieval. Dual qualitative analysis of the target compounds was carried out based on information such as retention time and EPI fragment ions， which improved the accuracy of qualitative analysis and effectively eliminated the interference of false positive results. This method features low LODs and good recoveries. It is also simple and rapid to operate， with high sensitivity and accuracy. It can achieve qualitative and quantitative analysis of new ISOs residues in bovine-origin foods. This study can provide technical support for food safety agencies to implement preventive measures against new ISOs in animal foods.

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Determination of 79 pesticide residues in fruits and vegetables by QuEChERS-ultra performance liquid chromatography-tandem mass spectrometry ZHAO Le, LIU Xianjun, ZHANG Hao, LI Jian, CAI Liang, FAN Xiang, LI Tanyao, CHEN Dongyang 2025, 43 (9):  1053-1062.  DOI: 10.3724/SP.J.1123.2025.02015 Abstract ( 120 )   HTML ( 17 )   PDF (927KB) ( 70 )   Pesticide residues in fruits and vegetables are becoming a serious issue. These residues can affect the quality of agricultural products and people’s health. Therefore， it has become crucial to effectively monitor and control pesticide residues in the food safety field. In this study， a rapid and effective QuEChERS-ultra performance liquid chromatography-tandem mass spectrometry （QuEChERS-UPLC-MS/MS） method was established for the simultaneous determination of 79 typical pesticides in vegetables and fruits， including organophosphates， carbamates， and pyrethroids. The pretreatment， UPLC， and MS/MS conditions were optimized. The fruit and vegetable samples were extracted with frozen acetonitrile after pulverization and homogenization， cleaned up by the QuEChERS method， filtered through a centrifugal membrane， and analyzed by UPLC-MS/MS. The separation was carried out on an ACQUITY UPLC HSS T3 column （100 mm×2.1 mm， 1.8 μm） with gradient elution. The aqueous and organic phases were water-methanol （98∶2， v/v） and methanol-water （98∶2， v/v） respectively， both with 5 mmol/L ammonium acetate and 0.1% formic acid. A triple quadrupole mass spectrometer was used in positive-ion electrospray ionization （ESI+） scanning mode， with target pesticide residues quantified using the matrix-matched standard-curve method. The results showed that under the optimized conditions， the 79 target compounds were determined with good linearities in the range of 0.1-200 μg/L， and the correlation coefficients （r） were all greater than 0.990. The limits of detection （LODs） and limits of quantification （LOQs） of the 79 compounds were in the range of 0.01-4.0 μg/kg and 0.03-13.0 μg/kg. The recoveries at three spiked levels ranged from 78.2% to 119.8%， with relative standard deviations （RSDs） all less than 15.8%. The established method was successfully applied to 80 samples of fruits and vegetables from Hunan province. As a result， 19 pesticides were detected in 31 samples， and thiamethoxam， acetamiprid and clothianidin being the most highly detected with a content range of 0.012-2.62 mg/kg； According to the data of the Hunan province survey yearbook， the percentages of acceptable daily intake （%ADI） for chronic dietary exposure of the detected neonicotinoid insecticides （thiamethoxam and clothianidin） have been calculated. The results indicate that the %ADI of clothianidin in fruits and vegetables ranged from 5.74% to 0.36%， respectively， and the %ADI of thiamethoxam in fruits and vegetables ranged from 0.40% to 19.50%. The %ADI of both pesticides were found to be less than 100%， indicating they are within acceptable limits. The method is simple， sensitive， accurate， and suitable for the simultaneous determination of multiple pesticide residues in fruits and vegetables.

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Rapid determination of five antipyretic analgesics in surface water by online solid phase extraction and ultra-high performance liquid chromatography-tandem mass spectrometry HE Ping, WANG Liqun, ZHOU Shan, ZHANG Baofeng, JIA Xuan, CHI Yi, XU Zhenqi, TANG Wei 2025, 43 (9):  1063-1069.  DOI: 10.3724/SP.J.1123.2024.10018 Abstract ( 128 )   HTML ( 18 )   PDF (886KB) ( 61 )   Antipyretic analgesics are typical pharmaceutical and personal care products （PPCPs） that are widely used in our daily life because they relieve fever and pain， and have anti-inflammatory and anti-rheumatic properties. These drugs inhibit the synthesis and release of prostaglandins （PGs） in the neurons of the anterior hypothalamus and exert therapeutic effects as a consequence. However， these drugs are relatively commonly misused and abused， often owing to a lack of proper medication guidance. As a result， these drugs enter the environment via various pathways， including wastewater treatment plants， agricultural runoff， and improper disposal， thereby posing potential threats to human health and ecosystems. The presence of these contaminants in surface water has become an environmental safety concern that necessitates the development of rapid， accurate， and high-throughput analysis methods. In this study， an analytical method was established for the determination of five antipyretic analgesics （ibuprofen， aminophenazone， antipyrine， phenacetin， and naproxen）. The developed method is based on online solid phase extraction coupled with ultra-high performance liquid chromatography-tandem mass spectrometry （online SPE-UHPLC-MS/MS）， which provides a high degree of automation and efficiency. Water samples were collected and filtered through 0.2-μm regenerated cellulose （RC） filter membranes， after which Na2EDTA and an internal standard were added. An aliquot （0.9 mL） of each sample was injected into the online SPE system using an automatic sampler. Samples were first adsorbed on a PLRP-S online SPE column， washed with 0.05% formic acid aqueous solution， and finally gradient-eluted with a mobile phase composed of 0.2 mmol/L ammonium fluoride solution and methanol-acetonitrile （1∶1， v/v）. Analytes were separated on a ZORBAX Eclipse Plus C18 column， detected by multiple reaction monitoring with electrospray ionization in both positive- and negative-ion modes， and quantified using the internal-standard method.The five antipyretic analgesics were effectively separated under the optimized experimental conditions and showed good linearities within their respective concentration ranges， with correlation coefficients （r） greater than 0.998. The method detection limits （MDLs） ranged from 0.05 to 0.20 ng/L， and the method quantification limits （MQLs） ranged from 0.20 to 0.80 ng/L. The five antipyretic analgesics exhibited average recoveries of between 64.2% and 112%， with relative standard deviations （RSDs， n=6） of 2.06%-8.99% at low， medium， and high spiked levels. Furthermore， the method was successfully used to analyze water samples from the Hangzhou section of the Qiantang River， in which four target compounds were detected， with antipyrine found to have the highest mass concentration. This newly developed method features a high degree of automation， facilitates the injection of large volumes， and enables online enrichment， purification， and quantitative analysis in an integrated process， with minimal operational errors， high repeatability， and high sensitivity. The developed method shortens sample-analysis times， is more cost-effective， and meets the needs of large-scale sample testing， as it requires only 15 min to completely determine a sample， thereby significantly improving detection efficiency. Overall， the method features low detection limits， analyzes rapidly， and is easy to operate； hence it is suitable for rapid risk screening and the quantitative determination of antipyretic analgesics in surface water.

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A protein-specific quantitative detection method based on polyacrylamide gel electrophoresis and online fluorescence imaging ZOU Rui, YU Zixian, GUO Zehua, DAI Haozheng, ZHANG Qiang, LIU Weiwen, CAO Chengxi 2025, 43 (9):  1070-1077.  DOI: 10.3724/SP.J.1123.2024.12017 Abstract ( 102 )   HTML ( 6 )   PDF (943KB) ( 34 )   Specific protein detection plays a crucial role in biological analysis and clinical diagnostics， serving as an essential tool for disease diagnosis， therapeutic monitoring， and biological research. However， conventional methods such as immunofixation electrophoresis （IFE） and western blotting （WB） suffer from complex workflows， time-consuming operations， and limited quantification capabilities owing to intricate staining and de-staining procedures. In addition， these traditional immunological detection methods require extensive manual handling and specialized expertise， while low levels of automation restrict their applicability to high-throughput or large-scale analysis scenarios. Moreover， the multistep nature of these methods increases the risk of experimental errors and compromises quantification accuracy.Herein， we present a quantitative protein immune polyacrylamide gel electrophoresis （PAGE） detection method that combines immune-recognition principles with online fluorescence imaging technology， thereby offering a rapid and specific approach for quantifying target proteins. The developed method exploits the specificity of fluorescently labeled antibodies and the separation capability of PAGE， with formaldehyde crosslinking used to stabilize antigen-antibody complexes under the denaturing conditions of electrophoresis， thereby ensuring reliable quantification. The entire experimental workflow can be completed within 1.5 h and consists of three main steps. Firstly， the target protein is incubated with fluorescently labeled antibodies at room temperature for 0.5 h to form immune complexes， after which they are crosslinked using formaldehyde. The cross-linked samples are then loaded onto polyacrylamide gels and separated under optimized electrophoresis conditions （120 V， 15 min； 150 V， 15 min； 200 V， 15 min）. Electrophoretic separation is finally monitored in real-time using an online fluorescence imaging system， which enables direct visualization of protein migration and eliminates the need for post-separation processing. Three distinct bands are observed on the precast gel following immune PAGE separation： the immune complexes at the uppermost position， the free fluorescent antibodies in the middle， and other proteins at the bottom. ImageJ software is used to analyze the electrophoresis pattern， and quantification is achieved based on the linear relationship between the fluorescence intensity of the free antibody and the mass concentration of the target protein. We systematically validated the performance of the method using human transferrin （TRF） as the model antigen protein and fluorescein-isothiocyanate-labeled （FITC-labeled） anti-TRF IgG antibody （anti-TRF IgG-FITC） as the detection probe， which involved analyzing three key aspects： the necessity of the formaldehyde-crosslinking step for maintaining immune complex stability， antibody recognition specificity in complex samples， and the linear correlation between the fluorescence signal and the mass concentration of the target protein. The method exhibited excellent analytical performance， with a linear range that extended between 5.0 and 200.0 mg/L and a correlation coefficient （R2） of 0.993 0. Triplicate measurements of fluorescence intensity at all mass concentration points revealed a maximum relative standard deviation （RSD） of 1.65%. The limit of detection （LOD） reaches 0.5 mg/L， with recoveries between 98.2% and 105.0%. Repeatability experiments revealed maximum intra- and inter-day RSDs of 1.21% and 1.58%， respectively. Specificity testing confirmed that the developed method accurately quantified TRF without interference from other proteins in complex samples. These results highlight the good accuracy， excellent consistency， high sensitivity， and robust specificity of the developed method， thereby confirming its reliability for use in precise protein-quantification applications. Compared to traditional PAGE methods， the immune PAGE method introduced herein provides the ability to selectively quantify specific target proteins online using real-time fluorescence imaging technology. The method exhibits several notable advantages， including high resolution， a simple workflow， strong specificity， rapid analysis， and good reproducibility. The online fluorescence imaging system eliminates the need for complex gel-dismantling， membrane-transfer， fixation， staining， and de-staining steps while effectively preventing protein band broadening， thereby enabling highly sensitive quantitative analyses with superior resolution. The cost-effectiveness of the developed method is achieved through economical fluorescently labeled antibodies and low sample consumption. Moreover， the fundamental principle of the immune PAGE method suggests that this approach is readily adaptable to the quantification of other proteins through the judicious selection of specific fluorescently labeled antibodies. Consequently， the versatility of the developed method makes it a comprehensive analytical platform suitable for pharmaceutical preparations and clinical diagnostics， in which rapid and accurate protein quantification is essential for decision-making processes. Additionally， this method is an ideal choice for high-throughput applications in both academic research and industrial settings owing to the integration of automated analysis and short operation times. The protein immune PAGE method represents a significant advancement in specific protein quantification methodology with great potential as a promising tool that is expected to be widely adopted in various biological analysis scenarios.

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Development and practice of an interactive chromatography learning tool for beginners based on GeoGebra： a case study of plate theory ZHANG Yuhan, HE Junyao, LIN Shujing, AI Bingjian, SHI Zhihong, ZHANG Hongyi 2025, 43 (9):  1078-1085.  DOI: 10.3724/SP.J.1123.2025.03008 Abstract ( 199 )   HTML ( 32 )   PDF (1155KB) ( 129 )   This study developed a GeoGebra platform-based interactive pedagogical tool focusing on plate theory to address challenges associated with abstract theory transmission， unidirectional knowledge delivery， and low student engagement in chromatography teaching in instrumental analysis courses. This study introduced an innovative methodology that encompasses theoretical model reconstruction， tool development， and teaching-chain integration that addresses the limitations of existing teaching tools， including the complex operation of professional software， restricted accessibility to web-based tools， and insufficient parameter-adjustment flexibility. An improved mathematical plate-theory model was established by incorporating mobile-phase flow rate， dead time， and phase ratio parameters. A three-tier progressive learning system （single-component simulation， multi-component simulation， and retention-time-equation derivation modules） was developed on a cloud-based computing platform. An integrated teaching chain that combined athematical modeling （AI-assisted “Doubao” derivation）， interactive-parameter adjustment （multiple adjustable chromatographic parameters）， and visual verification （chromatographic elution-curve simulation） was implemented. Teaching practice demonstrated that： （1） The developed tool transcends the dimensional limitations of traditional instruction， elevating the classroom task completion rate to 94% and improving the student accuracy rate for solving advanced problems to 76%. （2） The dynamic-parameter-adjustment feature significantly enhances learning engagement by enabling 85% of the students to independently use the tool in subsequent studies and experiments. （3） The AI-powered derivation and regression-analysis modules enable the interdisciplinary integration of theoretical chemistry and computational tools. The process of deriving chromatographic retention-time equations through this methodological approach proved more convincing than the current textbook practice of directly presenting conclusions. The developed innovative “theoretical-model visualizable-model-parameter adjustable-interactive-knowledge generating” model provides a new avenue for addressing teaching challenges associated with chromatography theory， and its open-source framework and modular design philosophy can offer valuable references for the digital teaching reform in analytical chemistry.