Objective: To prepare monoclonal antibodies (MAbs) that can specifically recognize genistein(Gen), and to establish a specific and rapid quantitative method for Gen using indirect competitive enzyme-linked immunosorbent assay (ELISA). Methods: Gen was coupled with carrier protein by the Mannich method to synthesize artificial antigens. Femal Balb/c mice aged 6-8 weeks old were immunized by Gen-BSA. After the booster immunization, splenocytes of the mice were collected and fused with SP2/0 myeloma cells under the action of 50% polyethylene glycol (PEG). Subclones were screened by the limiting dilution method to obtain specific MAbs and an indirect competitive ELISA method was established. Results: Two hybridoma cell lines (C1 and C3)were isolated successfully with the titers of 1 ： 32 000, 1 ： 40 000, respectively. Under the optimized conditions,the indirect competitive ELISA based on C3 for Gen showed a half maximum inhibition concentration (IC₅₀) of 16.15 ng · mL^-1 and detection ranges of 3.78-55.85 ng · mL^-1 with cross-reactivity for biochanin A and irisolidone of 4.96% and 2.40%, respectively, negligible cross-reactivity with other Gen analogs including apigenin and 4-oxo-4H-1-benzopyran-2-carboxylic acid. The recovery test showed that the recovery rate was between 85% and 110%, precision tests showed RSD of inter-assay and inter-assay was less than 10%. Conclusion: In this study,Gen-specific MAbs are prepared successfully. An sensitive and accurate indirect competitive ELISA based on MAb for Gen is developed. The developed method can meet the requirements for immunoassay.

Objective: To establish a two reference substances for determination of multiple components (TRSDMC)method for the simultaneous determination of scutellarin, rosmarinic acid, negletein, carvacrol and thymol in Moslae Herba, which was more economical and effective for the quality control of Moslae Herba. Methods: The retention times of the five components in Moslae Herba were determined using HPLC on 13 different C₁₈ columns. The average retention time of each component under each column was then used as the standard retention time for that component.Rosmarinic acid (peak 2) and carvacrol (peak 4) were chosen as linear calibration standards. The chromatographic peaks were localized by using liner calibration with two reference substances (LCTRS). Rosmarinic acid was used as the control substance to determine the content of each component using a relative correction factor. And the results were compared with those obtained from the external standard method. Chemometric analysis was used to excavate the differential quality markers of Moslae Herba. Results: LCTRS had accurate prediction results for the measured components and enjoyed a wide range of column applications. The correction factors of scutellarin, negletein, carvacrol and thymol to rosmarinic acid were 0.853 3, 0.475 2, 3.034 7 and 2.738 6, respectively, and their relative standard deviations were below 2.0%.There was no significant difference between contents obtained by the two methods. The contents of scutellarin, rosmarinic acid, negletein, carvacrol and thymol in 21 batches of Moslae Herba were 0.05-2.87 mg · g^-1, 0.73-9.73 mg · g^-1, 0.04-0.99 mg · g^-1, 0.36-8.52 mg · g^-1 and 0.61-10.2 7 mg · g^-1, respectively. The 24 batches of samples could be clustered into three categories, and thymol, carvacrol and negletein ether could be used as differential quality markers. Conclusion: TSDMC method for the simultaneous determination of the contents of 5 components in Moslae Herba is stable and reliable, and provides a new idea for the overall quality control of Moslae Herba.

Objective: To establish a method for the determination of veterinary drug residues in Galli Gigerii Endothelium Corneum by ultra high performance liquid chromatography-tandem triple quadrupole mass spectrometry (UPLC-QQQ MS/MS) with solid-phase extraction. Methods: The samples were evenly dispersed,extracted with acetonitrile and acetonitrile containing 0.5% formic acid, and cleaned up with Oasis PRiME HLB solid phase extraction columns. The extracts were separated on Agilent ZORBAX Eclipse Plus C₁₈ (3.0 mm×150 mm, 1.8 μm) using 5 mmol · L^-1 ammonium acetate and acetonitrile (containing 0.1% formic acid) as mobile phase by gradient elution. The detection of veterinary drug residues was detected by tandem mass spectrometry with positive electrospray ion source under multiple reaction monitoring (MRM) mode. The matrix-matched external standard method was used for the quantitation. Results: The method exhibited good linearities within a certain concentration range r≥0.995 8. The limits of detection (LODs) were in the range of 0.1-3 μg · kg^-1. The limits of quantitation (LOQs) were in the range of 0.2-10 μg · kg^-1. The good recovery values (61.9%-121.5%) were achieved for all the 68 veterinary drugs with RSDs ranging from 0.50% to 8.4% for spiking 3 different levels. Out of 50 batches of samples, 5 batches of Galli Gigerii Endothelium Corneum were found to contain veterinary drug residues,including amantadine, doxycycline, enrofloxacin, and florfenicol. Conclusion: This method proves suitable for the rapid determination of multiple veterinary drug residues in Galli Gigerii Endothelium Corneum, with a simple,quick, accurate and procedure.

Objective: To establish an ultra-performance liquid chromatography-triple quadrupole mass spectrometry(UPLC-QQQ MS/MS) method for the simultaneous determination of 14 active components in Qiyu Sanlong decoction,including L-argnine, monotropein, deacetyl asperulosidic acid, rutin, peimisine, calycosin-7-O-β-D-glucoside,caffeic acid, solasonine, solamargine, p-coumaric acid, ferulic acid, calycosin, astragaloside Ⅳ and astragaloside Ⅰ. Methods: The chromatographic separation experiment was performed on an ACQUITY UPLC BEH C₁₈ column(2.1 mm×100 mm, 1.7 μm), with gradient elution of 0.1% formic acid aqueous solution (A)-acetonitrile (B) as mobile phase at the flow rate of 0.2 mL · min^-1, injection volume of 5 μL, and column temperature of 35 ℃. The ion source was an electrospray ionization source (ESI), the scanning mode was simultaneous scanning of positive and negative ions, and the monitoring mode was multiple reaction monitoring. Results: The 14 active components revealed good linearity within their respective ranges (r>0.995 0), RSDs of precision and repeatability were below 2%, RSDs of stability were below 3%, and the average recoveries ranged from 88.4% to 108.5% with the RSDs ranging from 0.020%to 3.6%. The content ranges of the aforementioned 14 components in 10 batches of self-prepared Qiyu Sanlong decoction were as follows (in μg · g^-1): 667.28-785.78, 165.72-197.27, 196.32-275.60, 17.60-26.52, 4.68-10.75,279.12-388.05, 26.00-47.57, 385.52-442.77, 288.00-358.82, 629.88-839.02, 86.67-125.83, 51.58-65.83, 25.50-37.53, and 55.50-76.13. Conclusion: The UPLC-QQQ MS/MS method established in this study is rapid, accurate,sensitive and repeatable, which can provide a reference for the quality control of Qiyu Sanglong decoction.

The components of biological samples are complex, and the concentration range of target analytes varies significantly. Therefore, sample pretreatment is particularly important for subsequent analysis. With the continuous development of analytical techniques, the requirements for sample pretreatment are also increasing. Pretreatment technologies are evolving towards microscale, time-saving, high-efficiency, online, and environmentally friendly directions to meet the requirements of complex matrices and trace analysis. Online pretreatment technologies integrate sample pretreatment steps and subsequent detection processes directly into an automated system, which can reduce steps and errors, improve analysis efficiency and sensitivity, and achieve automatic, rapid, and efficient analysis of target compounds. This paper reviews the online pretreatment technologies for biological samples in China and abroad in the past 10 years, offering valuable insights to guide future research and innovation in the realm of biological sample pretreatment.

Objective: To identify and analyze the chemical components of Bufei Jianpi formula by ultra-high performance liquid chromatography-coupled with high-resolution mass spectrometry (UPLC-HRMS). Methods: A Hypersil GOLD (2.1 mm×100 mm, 2.6 μm) column was used. Methanol (A)-0.1% formic acid water (B) was used as the mobile phase with gradient elution, the flow rate was 0.2 mL · min^-1, and the column temperature was 30 ℃.The mass spectrometry data were collected using the Full MS/dd-MS² scanning mode of positive and negative ions, and the characteristic fragment ion peak information was analyzed by compound discoverer. Combined with Chemspider, mzCloud and other databases and existing reports of relevant chemical composition information,the chemical components of Bufei Jianpi formula was analyzed by using the cracking prediction of Mass Frontier and its cracking rule. Results: A total of 221 compounds were identified from Bufei Jianpi formula, including 65 flavonoids, 40 phenylpropanoids, 21 terpenoids, 16 alkaloids and 79 other compounds. Conclusion: UPLC-Orbitrap Fusion Lumos Tribrid-MS can quickly identify the chemical components of Bufei Jianpi formula and qualitatively analyze the material basis of Bufei Jianpi formula, which can be used for the quality control of Bufei Jianpi formula.

Objective: To develop the first national reference standards for the quantitative determination of bivalirudin,in order to effectively control the product quality of bivalirudin injections, and to explore alternative methods for the quantitative characterization of synthetic peptide reference materials. Methods: Infrared (IR) spectroscopy, UV spectroscopy, HPLC, MS were used to confirm the structure of bivalirudin. Related substance were analyzed by HPLC.The mass balance method was used to determine the content of bivalirudin, which was further verified by a peptide content assay corrected for related peptide impurities. In addition, the stability and uniformity of the candidate reference material were evaluated. Results: The content of the first batch of bivalirudin national reference standard was 88.8%,calculated on C₉₈H₁₃₈N₂₄O₃₃, and the stability and uniformity tests met the required specifications. Conclusion: Based on the structural characteristics of the synthetic peptides, multiple qualitative and quantitative methods were used to ensure the accuracy of the content assignment for the national reference standard of bivalirudin.

Objective: To clarify the differences between the “Room temperature” definitions in the European Pharmacopoeia and the Chinese Pharmacopoeia, to emphasize that storage temperature was closely related to the design of long-term stability studies and product labeling, and to investigate the effects of storage temperature variations on the critical quality attributes (CQAs) of terlipressin for injection, so as to raise awareness among generic drug manufacturers regarding this critical issue. Methods: Given the discrepancies in storage temperature recommendations across different manufacturers’ product inserts, the original drug’s long-term stability studies and storage conditions were traced. Validated methods for related substances and polymer content were employed to assess the impact of temperature differences on the product’s CQAs. Results: Samples stored at 30 ℃exhibited a significantly higher increase in related substances compared to those stored at 15 ℃. In one manufacturer’s product, polymer levels exceeded specification limits within just five months of storage at 30 ℃. The divergence among manufacturers stems from some companies misinterpreting the original drug’s labeling by directly translating without considering the differences between Chinese and European Pharmacopoeias. Conclusion: Storage temperature has a significant impact on the levels of related substances and polymer content in terlipressin for injection. To ensure product quality, the storage temperature in the labeling should be restricted to “not exceeding 25 ℃.”

Objective: To establish a selective culture medium screening method for counting viable bacteria from multi-strain microecological preparation based on gene and metabolic pathway analysis, in order to solve the problem of mutual interference in the measurement of counting viable bacteria. Methods: By analyzing the genomic scale metabolic network, the antibiotic sensitivity and resistance of Bacillus cereus, Enterococcus faecalis,Lactobacillus acidophilus, and Bifidobacterium infantis were quickly predicted. Based on the prediction results,suitable types and concentrations of antibiotics with resistance differences among the four microorganisms were screened and added to the culture medium, effectively inhibiting the growth of interfering microorganisms and ensuring accurate and effective counting results. Simultaneously, conventional drug sensitivity experiments results were used to ensure and compare with this method. Results: In the process of viable counting for multi-strain microecological preparation, this method effectively eliminated the interference of Enterococcus faecalis on the counting of Lactobacillus acidophilus live bacteria and the interference of Lactobacillus acidophilus and Enterococcus faecalis on the counting of Bifidobacterium infant, and the counting results were accurate and reliable of each microorganism. Conclusion: The method has strong foresight and specificity. Genomic analysis and metabolic pathway analysis can effectively predict microbial antibiotic resistance, and experimental design based on the predicted results will greatly accelerate experimental efficiency, making the results more predictive and accurate,and has broad application prospects in selective culture medium screening for counting viable bacteria from multi-strain microecological preparation.

Objective: To investigate the effect of adsorbed moisture on drug purity determination by differential scanning calorimetry (DSC). Methods: The DSC analysis for drug purity was performed with a heating rate of 0.5℃ · min^-1 under a nitrogen atmosphere (drying gas flow rate: 50 mL · min^-1). The optimized method reduced by 10 ℃ than the conventional method and included an additional heating segment at 1 ℃ · min^-1 for 10 min, while other parameters remained unchanged. Results: Without pre-drying, the conventional DSC method accurately determined the purity of imidazole, phenylephrine hydrochloride, and neostigmine methylsulfate samples with adsorbed moisture below 7%. After optimization, the method further eliminated moisture interference, allowing accurate analysis of samples with adsorbed moisture below 8%. Conclusion: Adsorbed moisture may affect DSC-based purity analysis. For general samples with a melting point above 80 ℃ and adsorbed moisture below 7%, pre-drying is unnecessary. The optimized method further reduces the impact of adsorbed moisture.