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.

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 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 meet the needs of gadolinium-based contrast agent (GBCA) analysis and testing, and to ensure drug quality and safety, a new strategy for inductively coupled plasma tandem mass spectrometry (ICP-MS/MS) determination of 12 impurity elements (Cr, Fe, Co, Ni, Cu, V, Zn, As, Cd, Ba, Sb, and Pb) in GBCA was proposed. Methods: GBCA was diluted with 2% HNO₃ and directly analyzed by ICP-MS/MS. Spectral interferences from V, Cr, Fe, Co, Ni, and Cu were eliminated using cool plasma/NH₃/He reaction mode. In addition,spectral interferences from Zn, As, Cd, Ba, and Sb were eliminated using hot plasma/O₂/H₂ reaction mode. To evaluate the accuracy and reliability of the method, the standard reference material (NIST SRM 1643f) was analyzed by comparing the method with high-resolution ICP-MS (HR-ICP-MS), and the spiked recovery experiment was conducted. Results: Under optimized conditions, the limits of detection (LOD) of the analytes were 0.014-0.86 ng · L^-1, the spiked recoveries were 96.7%-105.8%, and the RSDs were 1.6%-4.0%. The HR-ICP-MS determination values were consistent with reference values of NIST SRM 1643f. Statistical analysis showed that there was no significant difference between the measured values of the two methods at a 95% confidence level. Conclusion: The analysis method is accurate, reliable, stable and high precision. The combination of ICP-MS/MS and various reaction modes under different plasma conditions has shown promotion value for multi-element analysis in GBCA and can be extended to other fields.

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 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.

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 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 HPLC method for the simultaneous determination of chlorogenic acid,puerarin, 3’-methoxy puerarin, puerarin apioside, liquiritin, forsythoside A, 3,5-O-dicaffeoylquinic acid,4,5-dicaffeoylquinic acid, forsythin, honokiol and magnolol in Jinlian Quwen capsules. Methods: The analysis was performed on a Waters Atlantis^TM T3 (150 mm×4.6 mm, 3 μm) column, and the mobile phase was comprised of acetonitrile-0.1% phosphoric acid, with the flow rate of 1.0 mL · min^-1 in a gradient elution manner. The column temperature was set at 35 ℃. The injection volume was 4 μL and the UV detection wavelengths were set at 225 nm (detecting forsythin), 250 nm (detecting puerarin, 3’-methoxy puerarin and puerarin apioside), 276 nm(detecting liquiritin), 290 nm (detecting honokiol and magnolol) and 325 nm (detecting chlorogenic acid,3,5-O-dicaffeoylquinic acid, 4,5-dicaffeoylquinic acid and forsythoside A). Results: All the 11 constituents showed good linearity within their detection ranges (r≥0.999 5), whose average recoveries (n=6) were 100.1%-108.1%, with the RSDs of 2.8%-4.1%. The content ranges of 11 components in three batches of samples were 7.505-7.606 mg · g^-1, 3.485-3.920 mg · g^-1, 0.969-1.068 mg · g^-1, 0.837-0.955 mg · g^-1, 1.009-1.436 mg · g^-1,3.037-3.602 mg · g^-1, 5.259-5.371 mg · g^-1, 0.931-1.012 mg · g^-1, 1.428-2.053 mg · g^-1, 0.939-1.018 mg · g^-1 and 2.744-2.827 mg · g^-1, respectively. Conclusion: The method is simple, sensitive and reliable. It can be used as a reference for the establishment of quality standard of Jinlian Quwen capsules.

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.