Current Issue

Chinese Journal of Chromatography
2025, Vol. 43, No. 11
Online: 08 November 2025

Previous Issue   



For Selected:

Download Citations EndNote Ris BibTeX

Toggle Thumbnails

Reviews

Select

Research progress on paper-based microfluidic chips in pathogen detection LIU Xintong, SHI Jia, SHI Meng 2025, 43 (11):  1187-1199.  DOI: 10.3724/SP.J.1123.2025.04011 Abstract ( 75 )   HTML ( 16 )   PDF (787KB) ( 48 )   In the context of an increasingly severe global public health situation， diseases caused by bacteria and viruses have become a global threat， severely impacting human health worldwide. Rapid and accurate pathogen detection is crucial for preventing infectious diseases and plays a vital role in safeguarding public health through effective preventive measures. Against this backdrop， the development of efficient， simple， low-cost and widely applicable detection methods has become a key research direction in public health. As a novel detection platform， paper-based microfluidic devices （μPADs） open new pathways for pathogen detection due to their unique design and integrated properties， showing significant potential in medical diagnostics and beyond. Their low cost and ease of use make them particularly appealing for point-of-care pathogen testing， a field in which they are gaining increasing attention. These devices are user-friendly， require no complex equipment， and are easy to transport and store. This article provides an in-depth exploration of μPADs， discussing comprehensive material design considerations for microfluidic applications. It will also examine advanced fabrication techniques and innovative detection technologies in detail. Furthermore， practical detection cases for specific pathogens such as Escherichia coli （E. coli） and norovirus will be presented to demonstrate the significant advantages of these devices in applications. As scientific research advances， μPADs are expected to play an increasingly significant role and poised to greatly enhance global public health efforts. Their inherent simplicity and affordability make them ideally suited for regions with limited access to sophisticated laboratory infrastructure. They enable convenient on-site testing， which significantly reduces both time expenditure and operational costs. Furthermore， their portability makes them suitable for deployment in disaster relief scenarios and remote fieldwork. Rapid pathogen identification is crucial for effective outbreak containment. To advance this goal， μPADs can be integrated with high-sensitivity sensors and intelligent data processing modules. Advancements in material science have facilitated the development of highly sensitive and stable paper-based substrates for diverse applications. Simultaneously， nanotechnology enables the fabrication of miniaturized components. Meanwhile， bioengineering has contributed to highly sensitive detection probes. These probes can detect pathogens even at very low concentrations. In conclusion， continuous technological advancements are positioning μPADs at the forefront of diagnostic innovation. As research and development progresses， their role in global public health is anticipated to expand considerably. This progress offers considerable promise for enhancing global disease surveillance and response capabilities. The potential is largely due to the inherent simplicity of μPADs， which makes them accessible worldwide. Their low cost， portability， and ease of operation by personnel with minimal training make them particularly advantageous in low-resource settings and provide a robust platform for rapid， on-site diagnostic testing. The modular design of these devices facilitates extensive customization and scalability. This enables them to be tailored for the detection of a broad spectrum of pathogens. The technology is also readily scalable for mass production， facilitating widespread distribution. These attributes collectively make it a versatile and indispensable tool for global health initiatives. The integration of these devices with mobile technology represents a significant leap forward， enabling real-time digital tracking and monitoring of disease outbreaks. This capability significantly enhances rapid response and epidemic containment efforts. Furthermore， the collected data can also be leveraged for broader public health surveillance and epidemiological studies. The potential of this technology to revolutionize point-of-care diagnostics is immense. μPADs can deliver accurate results within minutes， a stark contrast to the hours or days required by conventional laboratory methods. This rapid turnaround time facilitates faster clinical decision-making and intervention， ultimately leading to improved patient outcomes. Moreover， μPADs hold considerable promise for the rapid detection of novel or emerging pathogens. As research and development continue unabated， the functional capabilities of μPADs are anticipated to expand significantly. Future iterations may incorporate cutting-edge detection methodologies and advancements in nanotechnology to further enhance sensitivity and accuracy. Such enhancements will further solidify the pivotal role of μPADs in global public health surveillance and infectious disease control strategies. Ultimately， μPADs hold immense potential to save lives worldwide by enabling rapid pathogen detection and effectively curbing the transmission of infectious diseases.

Articles

Select

Liquid-liquid interface synthesized covalent organic framework films for efficient extraction of okadaic acid from seawater ZHANG Wenmin, FANG Min, ZHANG Lan 2025, 43 (11):  1200-1208.  DOI: 10.3724/SP.J.1123.2025.07007 Abstract ( 72 )   HTML ( 9 )   PDF (2065KB) ( 39 )   Okadaic acid （OA） is a fatty acid polyether biotoxin that poses risks to human health and the ecological environment. Determining the concentrations of OA in seawater not only allows for early warnings of potential toxin accumulation in marine organisms， but also helps clarify the actual impacts of OA on marine ecosystems. However， OA exists in seawater at extremely low concentrations， and the matrix has a high salt content. Thus， adequate sample pretreatment is necessary prior to high performance liquid chromatography-tandem mass spectrometry （HPLC-MS/MS） analysis. Film-based solid-phase extraction （F-SPE） has attracted considerable attention in sample pretreatment. Unlike conventional solid-phase extraction （SPE）， which relies on large quantities of adsorbent particles， F-SPE uses a thin film， thereby addressing common issues in SPE applications such as high column pressure， column clogging， and adsorbent leakage. Currently， most such films are prepared by modifying polymer film skeletons with a certain amount of adsorbent particles. Nonetheless， these adsorbent particles have inherent limitations， including insufficient adsorption capacity or irreversible adsorption， leading to poor extraction performance for specific targets. Therefore， developing new types of films with high extraction efficiency is highly significant， as it can facilitate the wider application and popularization of this technology. Covalent organic frameworks （COFs） are porous crystalline materials with adjustable pore size， high specific surface area， and excellent stability. COF films can not only inherit the high permeability of films， but also possess strong COF adsorption capacity. In this work， a COF film （TPB-BTCA） was synthesized through the liquid-liquid interface synthesis method under mild reaction conditions， and characterized in detail. The Fourier-transform infrared （FT-IR） spectrum， X-ray diffraction （XRD） pattern， and X-ray photoelectron （XPS） spectrum verify the successful preparation of the TPB-BTCA film using a Schiff base reaction. The scanning electron microscopy （SEM） images and water contact angle testing show the prepared TPB-BTCA film is a hydrophilic heterogeneous film with specific morphologies， which is beneficial to a more thorough and rapid contact between the film and the sample solution. The nitrogen adsorption-desorption experiment demonstrates the TPB-BTCA film has a high surface area （1 261.6 m2/g） and porosity （0.6 cm3/g）， providing sufficient and easily accessible sites to adsorb these OA molecules. Subsequently， the TPB-BTCA film-based F-SPE method was used for the extraction of OA in seawater. At the same time， the influence of potential factors on the extraction efficiency was also investigated， including the sample loading volume， the pumping speed for loading， the eluent type， the volume of the eluent， the pumping speed for elution， and the salt concentration. Under the optimal conditions， the TPB-BTCA film exhibited excellent extraction performance for OA. Finally， a new method for the detection and analysis of OA was established by combining F-SPE technology with HPLC-MS/MS instrument. The developed method has a wide linear range （0.8–500.0 pg/mL） with good linearity （r=0.999 0）， low limit of detection （0.2 pg/mL） and satisfactory precision （RSDs≤6.4%， n=5）. Meanwhile， seven seawater samples were used to evaluate the feasibility of the developed method for detecting OA in actual samples. Ultra-trace amounts of OA were detected in two seawater samples with concentrations of 5.4 pg/mL and 61.8 pg/mL， respectively. Compared with the reported HPLC-MS/MS methods， the developed method only requires a single layer of film to process large-volume （100 mL） seawater samples and achieve the lowest detection limit. This may be due to the advantages of the TPB-BTCA film， such as high permeability， high porosity， and high specific surface area， which enable it to efficiently extract OA from seawater. In conclusion， this study not only provides an effective analytical method for detecting ultra-trace OA in seawater， but also demonstrates the application potential of COF films in sample pretreatment.

Select

Magnetic one-step purification-high performance liquid chromatography-triple quadrupole/composite linear ion trap mass spectrometry for the determination of 54 veterinary drug residues in carp WEN Yupeng, HUO Wendi, ZHANG Chaoying, LIU Huan, MU Yingchun, WU Lidong, LI Jincheng 2025, 43 (11):  1209-1221.  DOI: 10.3724/SP.J.1123.2024.12010 Abstract ( 90 )   HTML ( 18 )   PDF (1265KB) ( 43 )   Aquatic food products contain interfering substances， such as fats and proteins， which affect the analysis and detection of target compounds by significantly reducing detection efficiency and contaminating detection instruments. Effectively removing interfering substances is a key step in the field of food quality testing. To reduce the influence of interfering substances in aquatic products， this study established a detection method for 54 veterinary drug residues in carp by using magnetic one-step purification combined with high performance liquid chromatography-triple quadrupole/composite linear ion trap mass spectrometry （HPLC-QTRAP-MS/MS）. The purification process uses solvothermally synthesized spherical magnetite as the magnetic material. This material is strongly magnetic and enables impurities to be efficiently separated and collected. Additionally， a metal-organic framework material with an amino-rich surface （ZIF-67） was self-assembled to serve as the purifying agent. ZIF-67 is particularly effective at removing polar impurities during the magnetic one-step purification process. Scanning electron microscopy confirmed the successful synthesis of both materials， ensuring their structural integrity and suitability for the intended application.Acetonitrile was chosen as the extraction solvent owing to its excellent solubilizing properties. Furthermore， formic acid was added to enhance the extraction efficiencies of acidic veterinary drugs. Anhydrous sodium sulfate was used to remove water from the matrix， thereby further improving the purity of the extract. The extract underwent magnetic one-step purification following centrifugation， which effectively removed interfering substances while preserving the target compounds. Chromatographic separation was achieved using the C18 RRHD column （15 cm×4.6 mm， 2.7 µm）， with 5 mmol/L ammonium acetate solution （containing 0.2% formic acid） and 0.2% formic acid in methanol used as mobile phases. HPLC-QTRAP-MS/MS was then used to separate and determine the analytes， with data collected in multiple reaction monitoring （MRM） mode. The 54 veterinary drugs were quantitatively analyzed using matrix-matched standard curves， thereby ensuring accurate quantification even for complex matrices. Various parameters that affect the purification fillers were investigated to optimize the purification process. Octadecyl-bonded silica gel （C18） was used as an auxiliary purification material as it is known to effectively remove non-polar impurities. Combining 60 mg of ZIF-67 with 50 mg of C18 yielded the best purification results. All 54 compounds exhibited weak matrix effects after pretreatment and purification， with a value of only 18.11% recorded for the compound that exhibited the most significant matrix effect. The 54 veterinary drugs exhibited excellent linear relationships between response and concentration in the 0.5–50.0 µg/L range， with all coefficients of determination （r²） above 0.99. Detection and quantification limits of 0.5–1.0 and 1.0–2.0 µg/kg were recorded， respectively. The performance of the method was evaluated by spiking blank carp matrix samples at three concentrations （2.0， 5.0， and 20.0 µg/kg）. The recovery rates of the spiked samples ranged between 81.34% and 109.85%， and intra-day and inter-day relative standard deviations were less than 10%. The rapidity and accuracy of the developed method were assessed through comparisons with existing veterinary-drug detection methods， which revealed that the developed method is more sensitive and repeatable. Furthermore， the feasibility of the established detection method was verified by testing commercial aquatic products. No target veterinary drugs were detected in actual samples， which highlights the reliability of the developed method. In conclusion， the method developed in this study provides an effective means of monitoring veterinary drugs in carp. It meets the requirements of economy， simplicity， and efficiency while exhibiting high sensitivity and reproducibility. The developed approach represents a significant advancement in the field of aquatic food safety testing and is expected to ensure more accurate and reliable detection of veterinary drug residues.

Select

Determination of 51 per- and polyfluoroalkyl substances in raw water and drinking water by online solid-phase extraction coupled with ultra performance liquid chromatography- tandem mass spectrometry CHEN Yongyan, LYU Jia, YE Bixiong, ZHANG Lan, WANG Yuanyuan, JIN Ning 2025, 43 (11):  1222-1234.  DOI: 10.3724/SP.J.1123.2024.12020 Abstract ( 132 )   HTML ( 12 )   PDF (963KB) ( 87 )   In this study， an online solid-phase extraction-ultra performance liquid chromatography-tandem mass spectrometry （online SPE-UPLC-MS/MS） method was established to rapidly screen and determine 51 per- and polyfluoroalkyl substances （PFASs） in raw and drinking water. Ammonium formate and 24 PFAS internal standards were added to each sample. The ammonium formate concentration in the sample was 2 mmol/L after mixing， and the contents of the PFAS internal standards ranged between 2.5 and 50 ng/L. Each sample was filtered through a 0.22-μm cellulose acetate filter， and a 5-mL aliquot was injected and adsorbed using an HLB online solid-phase extraction column and rinsed with 2 mmol/L ammonium formate. Acetonitrile and 2 mmol/L aqueous ammonium formate were used as mobile phases. Separation was performed on a BEH C18 chromatographic column with gradient elution. Electrospray ionization source negative ion mode and multiple reaction monitoring mode were used for detection， with quantification performed using the internal standard method. The method was validated by determining accuracies and precisions for the 51 PFASs using raw water and drinking water as matrices. Excellent linear relationships within their respective ranges were observed， with correlation coefficients （r2）>0.995. The method exhibited limits of detection （LODs， S/N=3） and quantification （LOQs， S/N=10） of 0.03–1.5 and 0.1–5.0 ng/L， respectively. The PFASs were spiked at levels of 1， 10 and 50 ng/L， with spiked recoveries of 60.2%–126.9% and 60.4%–122.6% obtained for raw- and drinking-water samples， respectively， along with corresponding relative standard deviations （RSDs， n=6） of 0.3%-17.9% and 0.4%-17.7%， respectively. The developed method was used to determine PFAS residues in raw and drinking water， with perfluoroalkyl carboxylic acids， perfluoroalkyl sulfonic acids， and perfluoroalkyl ether acids detected at relatively high rates with contents of 0.1–209.7 and 0.1–63.6 ng/L in raw water and drinking water， respectively. Compared to offline solid-phase extraction methods， the developed method requires fewer samples， is more convenient from a sample-collection perspective， and uses lower quantities of internal standards. It also analyzes rapidly and is highly sensitive and reproducible. Only 20 min was required to determine the 51 PFASs at the ng/L level， from online enrichment to detection. The developed method is suitable for the trace determination of various types of PFAS， such as perfluoroalkyl carboxylic acids， perfluoroalkyl sulfonic acids， perfluoroalkyl ether acids， fluorotelomers， and fluoroalkyl sulfonamides， in raw and drinking water， thereby effectively boosting the detection efficiency for perfluoroalkyl substances in water in a manner that has substantial practical applications value.

Select

Determination of eight per- and polyfluoroalkyl substances in seawater using dispersive membrane extraction-ultra-high performance liquid chromatography-tandem mass spectrometry REN Zhen, MA Jiping, LIU Yajing, ZHANG Lan, WU Gege, LI Shuang 2025, 43 (11):  1235-1243.  DOI: 10.3724/SP.J.1123.2025.04009 Abstract ( 99 )   HTML ( 15 )   PDF (1377KB) ( 52 )   Per- and polyfluoroalkyl substances （PFASs） are widely utilized in various industrial applications. Their persistence in ecosystems raises significant global environmental health concerns. Numerous studies have confirmed the toxicity of PFASs to human endocrine and immune systems. The carcinogenic risks associated with PFASs exposure increasingly alarm public health authorities worldwide. As a result， regulatory policies have been implemented to restrict both the production and environmental release of PFASs. In China， the GB 5749-2022 standard establishes stringent limits for PFASs concentrations in drinking water， capping levels of perfluorooctane sulfonic acid （PFOS） at 40 ng/L and perfluorooctanoic acid （PFOA） at 80 ng/L. Effective monitoring of PFASs requires advanced analytical techniques that exhibit exceptional sensitivity. Ultra-high performance liquid chromatography-tandem mass spectrometry （UHPLC-MS/MS） has emerged as the preferred technique for detecting these substances due to its superior selectivity and low detection limits. However， complex environmental matrices necessitate optimized sample pretreatment strategies to enhance analysis accuracy. Efficient extraction methods must effectively address matrix interference while ensuring analyte enrichment. Dispersive membrane extraction （DME） presents distinct advantages for the pretreatment of PFASs； its operational simplicity is complemented by high enrichment capabilities and efficient mass transfer. The selection of extraction materials critically influences DME performance parameters. Metal-organic frameworks （MOFs） are porous materials composed of organic ligands and metal clusters， featuring tunable pores， high surface areas， and rapid mass transfer. These unique properties enable their current use in sample pretreatment for environmental analysis. This study integrates DME with UHPLC-MS/MS utilizing cationic MOF membranes， enabling simultaneous detection of eight different PFASs within seawater matrices. Optimized parameters ensure reliable quantification of trace-level contaminants throughout this process. The experimental design assessed various parameters， including types of organic solvents （methanol and acetonitrile）， ammonium acetate concentrations in the aqueous phase （0.1， 0.5， 1.0， 2.0， and 5.0 mmol/L）， as well as ion source voltages （-2 500， -3 500， and -4 500 V）， all of which influenced detection efficacy. Chromatographic separation was performed using an ACQUITY UPLC BEH C18 column （100 mm×2.1 mm， 1.7 μm）. Gradient elution combined 1 mmol/L ammonium acetate with acetonitrile for optimal results. Detection was conducted utilizing electrospray ionization （ESI） in negative ion scanning mode coupled with multiple reaction monitoring （MRM）. The results indicated that all eight PFASs could be effectively separated within a timeframe of 12 min， exhibiting favorable peak shapes and high response values. Under optimal conditions， the eight PFASs demonstrated strong linearity across their respective concentration ranges； the correlation coefficients （r²） were all not less than 0.990 7. Recoveries of PFASs at spiking levels of 10， 50， and 100 ng/L ranged from 50.4% to 116.4%， while intra-day and inter-day relative standard deviations （RSDs） varied from 1.0% to 19.2% and from 2.2% to 19.5%， respectively. The method’s limits of detection （LODs） ranged from approximately 0.07 ng/L to 0.49 ng/L， whereas the limits of quantification （LOQs） varied from around 0.22 ng/L to 1.63 ng/L. Jiaozhou Bay serves as a typical semi-enclosed bay along the Pacific coast where terrestrial runoff converges nutrients alongside diverse organic/inorganic pollutants that pose potential health risks for aquatic organisms inhabiting these waters. This method was utilized for the detection of the eight PFASs in the surface seawater of Jiaozhou Bay， and seven types were successfully identified. Among these， potassium 11-chloroeicosafluoro-3-oxaundecane-1-sulfonate （Minor F-53B） exhibited the highest detection concentration， with an average mass concentration of 17.11 ng/L. When compared to the 2018 detection results of PFASs in the surface seawater of Jiaozhou Bay， the average mass concentration of perfluorooctanoic acid （PFOA） has significantly decreased. Additionally， potassium 9-chlorohexadecafluoro-3-oxanonane-1-sulfonate （F-53B）， one of the newly emerging PFASs， was detected in the surface seawater of Jiaozhou Bay， which may be attributed to the recent shift in PFASs production. The widespread use of these new PFASs may introduce environmental risks. These risks resemble those of traditional PFASs and require urgent attention. In summary， this method is operationally straightforward， rapid， and highly sensitive； it is suitable for analyzing the eight PFASs in seawater. This approach can provide a valuable data foundation and scientific basis for research and analysis concerning PFASs in environmental water bodies.

Select

Quantitation of cow’s milk allergen in hypoallergenic infant formulas using ultra-high performance liquid chromatography- tandem mass spectrometry XU Daokun, YANG Yang, HUANG Haolun, SUN Xiaojie, HU Wenyan, LIU Xinmei, YANG Jun 2025, 43 (11):  1244-1251.  DOI: 10.3724/SP.J.1123.2024.12030 Abstract ( 71 )   HTML ( 10 )   PDF (798KB) ( 36 )   Cow’s milk protein allergy （CMPA） has become a common public health concern and hypoallergenic formula （partially or extensively hydrolyzed formula） is considered as an alternative during infancy. Alcalase and papain are widely used in industrial production of hydrolysates， as they can modify or destroy the linear and conformational epitopes of cow’s milk allergens （CMAs）， thus leading to decreased allergenicity. However， despite considerable emphasis has been laid on the manufacturing technique and nutritional value of hypoallergenic formulas， whether there are traces of CMAs in hypoallergenic formulas， remains unknown. Consequently， the need for a quantitative method of detecting multiple CMAs at a time is rising. Traditionally， enzyme-linked immunosorbent assay （ELISA）-based methods have proved to be advantageous with a wide availability of commercial kits for CMAs detection. Moreover， polymerase chain reaction （PCR）， as one of DNA-based methods， has shown exceptional specificity and high sensitivity. However， they both may encounter false-positive results and high-throughput detection is challenging. In the past two decades， mass spectrometry （MS）-based proteomic technology has advanced， including improvements in computational capability， algorithm model， as well as mass spectrometric platforms. MS is gaining more and more attention in the field of food allergen detection， allowing multiplex allergen detection with high sensitivity， accuracy and selectivity in a single run. In this study， an analytical method was developed for the simultaneous quantitation of six CMAss （namely， α-lactalbumin， β-lactoglobulin， αS1-casein， αS2-casein， β-casein， and κ-casein） in hypoallergenic formulas using ultra-high performance liquid chromatography-tandem mass spectrometry （UHPLC-MS/MS）. Proteins of spiked hypoallergenic formula were extracted with ammonium bicarbonate and digested with trypsin. After digestion， the samples were purified with a C18 spin column， and analyzed by UHPLC-MS/MS （AB SCIEX Triple Quad 6500+）. Separation was achieved on a Waters ACQUITY UPLC Peptide CSH C18 column （150 mm×2.1 mm， 1.7 μm）. The mobile phase consisted of 0.1% formic acid aqueous solution and 0.1% formic acid in acetonitrile， and the flow rate was 0.3 mL/min. The sample injection volume was 10 μL， and the column temperature was set to 40 ℃. Mass spectrometer was operated in positive electrospray ionization （ESI+） mode， and multiple reaction monitoring （MRM） mode was adopted. Precursor ions and product ions of marker peptides were predicted by the Skyline software. Mass spectrometric parameters such as declustering potential （DP） and collision energy （CE） were also optimized. Afterwards， by taking allergen epitopes and mass spectrometric responses into consideration， six peptides were retained for quantitative analysis. Calibration curves were constructed with good linearity in the range of 0.05-500 mg/kg， and correlation coefficients （r2） greater than 0.99. The limits of detection （LODs） were 0.05-5.0 mg/kg， and limits of quantification （LOQs） were 0.1-10.0 mg/kg， respectively. The recoveries of the developed method in incurred hypoallergenic formulas ranged from 74.8% to 93.4%. The intraday and interday accuracies ranged from -25.09% to -6.09% and from -24.0% to -5.41%， with precisions between 1.08%-5.05% and 1.35%-4.85%， respectively. Screening of commercial hypoallergenic formula samples confirmed existence of CMAs in three partially hydrolyzed formulas （pHF） and two extensively hydrolyzed formulas （eHF）， with contents ranging from 0.48 to 27.36 mg/kg. The analytical method proposed in this study exhibited high sensitivity， high-throughput capability， and can be used to monitor traces of CMAs in hypoallergenic formulas.

Select

Determination of 29 organic acids in tobacco based on isopropyl esterification derivatization by gas chromatography-mass spectrometry YU Jie, GAO Yang, WU Da, CAO Yuqi, QI Dawei 2025, 43 (11):  1252-1261.  DOI: 10.3724/SP.J.1123.2025.01010 Abstract ( 70 )   HTML ( 12 )   PDF (1337KB) ( 44 )   Organic acids are important components of dry tobacco and significantly affect its sensory properties. Therefore， determining the organic-acid composition and content in tobacco is greatly significant for evaluating its quality. Organic acids in tobacco are classified according to volatility as either volatile， semi-volatile， or non-volatile， with semi-volatile and non-volatile organic acids typically methylated for identification purposes. However， volatile acids cannot be determined in this manner owing to their low molecular weights. Therefore， a comprehensive， efficient， and accurate method for rapidly and simultaneously determining volatile， semi-volatile， and non-volatile organic acids in tobacco needs to be established. Herein， an innovative gas-chromatography–mass-spectrometry-based （GC-MS-based） isopropyl-esterification method was developed for simultaneously determining 29 volatile， semi-volatile， and non-volatile organic acids in tobacco. The developed method provides esterified small-molecule acids of lower volatility than those obtained using classical methylation and avoids interference from byproducts obtained via the esterification reaction. Experimental conditions and GC parameters were optimized， which ultimately led to 10% sulfuric acid in isopropanol （containing 0.1 g/mL p-toluenesulfonic acid） as the esterification reagent， with the reaction performed for 3 h at 70 °C and 500 r/min. The mixture was then cooled to room temperature， 10 mL of deionized water and 2 mL of n-hexane were added， and the mixture was vortexed for 30 s （4×）， after which the supernatant was collected for analysis. The DB-FASTFAME （30 m×0.25 mm×0.25 μm） and DB-5MS （30 m×0.25 mm×0.25 μm） chromatographic columns were compared， with the former finally selected for GC-MS separation and quantitative detection. All method-validation experiments were subsequently performed under the established instrumental and derivatization conditions. The 29 organic acids exhibited good linearities within the detection range， with all correlation coefficients （r2） exceeding 0.99. All recoveries at three spiked levels ranged from 81.8% to 118.7%. The intra-day repeatabilities （RSD， n=6） ranged from 0.5% to 8.1%， and the inter-day repeatabilities （RSD， n=6） ranged from 0.9% to 8.2%. The limits of detection （S/N=3） were 0.01-49.02 μg/g， and the limits of quantification （S/N=10） were 0.03-163.41 μg/g， respectively. Comparative experimental results revealed that the developed method is highly consistent with the classical methylation-based derivatization method for the detection of semi- and non-volatile organic acids in tobacco. Furthermore， the new method is able to simultaneously determine volatile organic acids that cannot be analyzed using the classical methylation method. Therefore， the developed method is more widely applicable for determining acids in tobacco. The newly developed method was used to determine three types of tobacco sample： flue-cured tobacco， cigar tobacco， and burley tobacco， with all 29 organic acids successfully detected. Cigar tobacco leaves were found to contain significantly more volatile organic acids than flue-cured tobacco， especially 3-methylvaleric acid； they also contain less semi-volatile organic acids and more non-volatile organic acids than flue-cured tobacco. The developed method is simple， accurate， and suitable for the large-scale simultaneous determination of volatile， semi-volatile， and non-volatile organic acids in tobacco； it also provides additional technical support for the study of organic acids in tobacco and evaluating tobacco quality.

Technical Notes

Select

Determination of genotoxic impurity in the bulk drug of crizotinib by high performance liquid chromatography-electrospray ionization tandem mass spectrometry ZHAN Xiaobing, QIN Yi, YAN Ying, ZHOU Jie, ZHAO Longshan 2025, 43 (11):  1262-1267.  DOI: 10.3724/SP.J.1123.2025.04017 Abstract ( 48 )   HTML ( 6 )   PDF (589KB) ( 30 )   A method based on high performance liquid chromatography-electrospray ionization tandem mass spectrometry （HPLC-ESI-MS/MS） was developed for the determination of the potential genotoxic impurity （WHT1408-Q2H） in the bulk drug of crizotinib. An Agilent Eclipse XDB C8 chromatographic column （150 mm×4.6 mm， 3.5 µm） was used for chromatographic separation. The mobile phases were 0.1% formic acid aqueous solution and 0.1% formic acid acetonitrile solution at a flow rate of 0.4 mL/min. The column temperature was maintained at 40 ℃ and the sample size was 5 μL. In the mass spectrometry section， the electrospray positive ion （ESI+） mode with multiple reaction monitoring （MRM） scanning was adopted. The accurate mass of the ［M+H］+ parent ion of WHT1408-Q2H was m/z 205.3， and the accurate mass of the extracted fragment ion was m/z 121.0. The results of methodological validation demonstrated that the established method exhibited excellent specificity. The peak area and mass concentration of WHT1408-Q2H exhibited a good linear relationship within the range of 2-40 ng/mL， with a correlation coefficient （r） of 0.999 9. The limit of detection （LOD） and limit of quantitation （LOQ） for WHT1408-Q2H were 0.396 9 ng/mL and 1.984 6 ng/mL， respectively. The recoveries of WHT1408-Q2H at low， medium， and high levels were in the range of 95.6%-102.7%， while the relative standard deviations （RSDs） were between 0.4% and 0.7%. Finally， the proposed method was successfully applied to analyze three independent batches of the bulk drug of crizotinib. The results revealed that WHT1408-Q2H was not detected in all samples， indicating that the current production process can effectively control the content of this genotoxic impurity. In conclusion， the developed HPLC-ESI-MS/MS method is highly specific， sensitive， and simple， making it suitable for the stringent quality control of WHT1408-Q2H in the bulk drug of crizotinib. According to the M7 guideline on genotoxic impurities， this method is capable of accurately quantifying trace amounts of genotoxic impurities and will further ensure compliance with regulatory requirements and safeguard drug safety. Future applications may extend this analytical framework to similar genotoxic impurities assessment in other therapeutic compounds， thereby advancing the field of pharmaceutical impurity profiling and control.

Teaching Research

Select

Undergraduate innovation training experiment： zooarchaeology by mass spectrometry for species identification of paleontological remnants XU Yang, JIANG Liyan, YANG Shasha 2025, 43 (11):  1268-1274.  DOI: 10.3724/SP.J.1123.2025.03003 Abstract ( 92 )   HTML ( 5 )   PDF (936KB) ( 33 )   Against the educational backdrop of deep interdisciplinary integration and empowering new models for cultivating composite talents， this innovative training experiment focuses on the hot interdisciplinary field of molecular archaeology， targeting the fragmented state of paleontological remains that do not have morphological identification characteristics and cannot be accurately identified for species. We developed a zooarchaeology by mass spectrometry （ZooMS） method based on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry （MALDI-TOF MS）， utilizing collagen peptide mass fingerprinting （PMF）. This approach was successfully applied to species identification of late pleistocene fragmented paleontological samples. ZooMS offers significant advantages including operational simplicity， high identification accuracy， broad applicability， low testing costs， high-throughput analysis and stringent contamination control， making it highly valuable for species identification and the extraction of “hidden information” from fragmented paleontological remains. Through this innovation training experiment， we enhanced interdisciplinary collaboration between humanities and sciences， stimulated students’ scientific thinking， and improved their research competencies， laying a solid foundation for cultivating interdisciplinary and innovative talents.

Select

Comprehensive analytical chemistry experiment： analysis of non-covalent interactions between double-stranded deoxyribonucleic acid and a natural drug by electrospray ionization mass spectrometry MA Lei, WANG Yinghui, YUAN Yunlan, WANG Shanshan 2025, 43 (11):  1275-1283.  DOI: 10.3724/SP.J.1123.2024.12026 Abstract ( 91 )   HTML ( 8 )   PDF (1349KB) ( 40 )   Analytical chemistry experiments are essential foundational courses for first- and second-year undergraduates in chemistry， chemical engineering， materials science， and pharmacy. These courses provide students with principles and operational skills of analytical instruments， alongside training in qualitative and quantitative analysis. However， current teaching practices face three main challenges： （1） insufficient focus on instrumental analysis； （2） outdated experimental content misaligned with modern scientific advancements； and （3） limited experimental hours due to curriculum constraints. To systematically address these issues， we proposed educational objectives， competency goals， and course objectives based on outcome-based education （OBE） philosophy. Building upon this backdrop， a comprehensive experiment utilizing electrospray ionization mass spectrometry （ESI-MS） was designed to investigate non-covalent interactions between double-stranded deoxyribonucleic acid （DNA） and naringin， a flavonoid natural drug. The experiment is offered to third-year undergraduate students as an elective. In the implementation process， a blended teaching model combining online and offline methods is adopted. The experimental teaching process is structured into three stages： pre-class preparation， in-class practice， and post-class review. Pre-class tasks include literature reviews， artificial intelligence （AI）-assisted summaries， pre-lab report writing and group discussions. This part is mainly conducted online without occupying class hours. During in-class practice， students synthesized double-stranded DNA by annealing single-stranded DNA （heated at 90 ℃ for 15 min， followed by slowly cooling to （25±1） ℃ overnight and stored at -20 ℃）. The resulting DNA was incubated with naringin at a 1∶4 concentration ratio in ammonium acetate for 15 min. The mixture was then analyzed by ESI-MS on a linear ion trap mass spectrometer. Both negative and positive ion modes were employed with optimized parameters encompassing spray voltage， capillary voltage， tube lens offset， heated capillary temperature， nitrogen sheath and auxiliary gas flows. Data acquisition involved 150 averaged scans using Xcalibur software. ESI-MS under negative ion mode was used to detect the non-covalent complexes. Secondary mass spectrometry （MS/MS） of 5-charged complex ions showed guanine base loss and minimal drug dissociation， indicating strong non-covalent interactions. In positive ion mode， MS yielded lower complex abundance， likely due to charge redistribution during ionization. The results reveal that naringin binds DNA predominantly via π-π stacking and hydrogen bonding， with a 1∶1 stoichiometry （relative abundance 60.91%） and a relative binding affinity of 39.20%. Post-class， students were required to process data， write formal lab reports， create presentations for defense， and design a feasible extension experiment. At the same time， a grading system was established for these three phases. The evaluation system emphasizes formative assessment， focusing on aspects such as compliance with experimental procedures， workflow efficiency， safety measures， teamwork， problem-solving skills， and experimental data handling. This multidimensional approach ensures equitable grading and pedagogical validity. This curriculum bridges research and education by introducing MS-based non-covalent interaction analysis into undergraduate curricula. The extended experimental design permits curricular expansion of the course. Some students designed structure-activity relationship （SAR） investigations of flavonoids （e.g.， naringenin vs. naringin）， revealing the role of glycosylation in DNA binding affinity. In addition， students designed a fluorescence quenching spectroscopy experiment， demonstrating interdisciplinary problem-solving skills. It closely aligns with the OBE philosophy， a student-centered framework that fosters innovation. Feedback indicates that 96% of undergraduates perceived significant improvements in their research capabilities and interdisciplinary integration skills. However， some challenges were noted， including students’ initial hesitancy with advanced instrumentation and limited instruments and drugs. We plan to improve teaching in these areas in the future. In conclusion， this OBE-driven experiment successfully modernized analytical chemistry education in mass spectrometry applications by integrating theoretical knowledge with cutting-edge research skills. The project establishes a comprehensive teaching platform and constructs a holistic teaching system， featuring operational safety and accessibility while offering strong demonstrative value in fostering interest， extensibility and innovation. The experiment not only enriches the content of analytical chemistry courses， inspires students’ research interests， and hones their critical thinking abilities， but also enhances their safety awareness and lays a solid foundation for future research endeavors， thus achieving the comprehensive educational goals of experimental teaching.