Objective: To study the heavy metals and harmful elements and the arsenic (As) speciation in Rukuaixiao preparations and evaluate the As risk. Methods: Residues of Cr, Ni, Cu, As, Cd, Hg, and Pb in 78 batches of Rukuaixiao preparations were determined by microwave digestion-inductively coupled plasma-mass spectrometry (ICP-MS). High performance liquid chromatography coupled with inductively coupled plasma-mass spectrometry (HPLC-ICP-MS) was employed to analyze six As forms. The hazard index (HI) was adopted to evaluate the risk of As in 10 batches of samples. Results: The two methods demonstrated good linear relationship, precision, repeatability, and recovery. The content of seven heavy metals varied greatly among the samples from different manufacturers. The As speciation and valence states in 10 batches of samples from E company were analyzed. The results showed that total As content was high. HPLC-ICP-MS results showed that trivalent and pentavalent As exhibited high toxicity. The HI<1 indicated that the As risk was controllable. Conclusion: The established method for the determination of seven heavy metals and harmful elements and six forms of As demonstrates high efficiency, rapidity, accuracy, and stability. This study provides data support for the formulation of the limit standards for the heavy metals and harmful elements in Rukuaixiao preparations.

Objective: This paper aims to establish a single bioanalytical method applicable for detecting anti-drug antibodies to insulin glargine and its biosimilar. Methods: The enzyme-linked immunosorbent assay was established by using biosimilars as both the capture antigen and the competitive antigen in the confirmation assay, as well as peroxidase-conjugated recombinant protein A/G as the detection reagent. The method validation was carried out from the aspects of antigen equivalence, screening cutoff, titer cutoff, confirmatory cutoff, sensitivity, precision, selectivity, hook effect, stability, and system adaptability. Results: Antigen equivalence (titration similarity and inhibition similarity) cross validation showed that the binding ability of biosimilars and reference drugs exhibited similar binding ability to positive antibodies. The screening cutoff factor was 1.186; the titer cutoff factor was 1.454; the confirmatory cutoff was 10.7%. The sensitivities of the screening assay and confirmatory assay were 8.9 ng · mL^-1 and 7.7 ng · mL^-1, respectively. The intra-plate and inter-plate relative standard deviations (RSDs) were less than 20% (The RSD of intra-plate precision of inhibition rate in the negative samples in the confirmation assay was 20.0%-22.8%, excluded). There was no hook effect in the range of 0.8-6.4 μg · mL^-1. The samples were stable at room temperature for 3 h, refrigerated (2-8 ℃) for 1 d, and at -70 ℃ after four freeze-thaw cycles. Selectivity, drug tolerance, and system suitability were satisfactory. Conclusion: In this study, a single bioanalytical method applicable for detecting anti-drug antibodies to insulin glargine and its biosimilar is successfully established by the antigen equivalence verification of biosimilar and reference drugs.

Objective: To establish an ultra-performance liquid chromatography-triple quadrupole mass spectrometry (UPLC-QQQ MS/MS) method for the simultaneous determination of nine blood-absorbed components in Qijiao Shengbai capsules, including 7-hydroxy-4-benzopyrone, calycosin, formononetin, medicarpin, p-coumaric acid, daidzein, maackiain, emodin, and icaritin. Methods: Chromatographic separation was performed on a Waters Acquity UPLC HSS T₃ column (150 mm×2.1 mm, 1.8 μm) using gradient elution, with 0.1% formic acid in water (A) and acetonitrile (B) as the mobile phase and column temperature at 30 ℃. The flow rate was 0.2 mL · min^-1,and the injection volume was 2 μL. Mass spectrometry was carried out using an electrospray ionization (ESI) source, and both positive- and negative-ion scanning were performed in multiple reaction monitoring (MRM) mode. Results: The established method showed good linear relationships (r≥0.998 1) and demonstrated good precision, repeatability, and stability (RSD<5.0%). The average recoveries (n=6) of the nine blood-absorbed components ranged from 97.3% to 107.2% with RSDs under 5.0%. The content ranges of the nine compounds in three batches of Qijiao Shengbai capsules were 0.271 2-0.275 3 μg · g^-1, 139.9-146.5 μg · g^-1, 42.31-43.80 μg · g^-1, 5.817-5.946 μg · g^-1, 6.740-7.145 μg · g^-1, 1.206-1.246 μg · g^-1, 42.83-44.78 μg · g^-1, 0.364 4-0.387 0 μg · g^-1, and 2.787-2.967 μg · g^-1. Conclusion: The established method is specific, sensitive, and stable. It can be used to simultaneously determine the contents of nine blood-absorbed components in Qijiao Shengbai capsules, providing a scientific basis for its quality control and safe medication.

Objective: To establish a method for determining the content of poloxamer 188 added in panitumumab based on high performance liquid chromatography-evaporative light scattering detector (HPLC-ELSD). Methods: The content of poloxamer 188 was determined through an Oasis wax column (20 mm×2.1 mm, 30 µm/80 Å) at a column temperature of 30 ℃. Gradient elution was performed with 0.2% formic acid aqueous solution as mobile phase A and acetonitrile containing 0.2% formic acid as mobile phase B at a flow rate of 1.0 mL · min^-1. The ELSD was used. Results: The specificity of the method was strong. In the range of 5 to 80 μg · mL^-1, the logarithm of the content of poloxamer 188 was linearly correlated with the logarithm of the peak area, with a coefficient of determination r²>0.99. The recovery rates of the six validation solutions with different poloxamer 188 content (5 μg · mL^-1,10 μg · mL^-1, 20 μg · mL^-1, 40 μg · mL^-1, 60 μg · mL^-1, and 80 μg · mL^-1) were in the range of 90.0%-110.0%. Three independent measurements were carried out on three different working days for each of the six validation solutions mentioned above, and the RSDs for their concentrations were 4.4%, 5.5%, 2.3%, 0.66%, 1.6%, and 1.6%, respectively. The injection was repeated six times for the validation solution at 40 μg · mL^-1, and the RSD of their poloxamer 188 content was 0.60%. The RSD of the poloxamer 188 content was 0.80% for the validation solution at 40 μg · mL^-1, which was assayed after storage for 0 h and 24 h at room temperature, respectively. Conclusion: An HPLC-ELSD method for content deter mination of poloxamer 188 in monoclonal antibody drugs was successfully developed, demonstrating good reproducibility, specificity, precision, and accuracy, which can provide a reliable technical basis for the quantitation and quality control of poloxamer 188 in monoclonal antibody drugs.

Objective: To establish an ultra-high performance liquid chromatography-triple quadrupole mass spectrometry (UPLC-QQQ MS/MS) method for the simultaneous determination of 10 aminoglycoside antibiotics in water. Methods: Following alkaline desorption of particulates in the water sample, heptafluorobutyric acid was added to form an ion pair with the target analytes, and then the ion pair was determined by the UPLC-QQQ MS/MS method. The analytes were separated on a Poroshell 120 SB-C₈ chromatographic column (100 mm×3.0 mm, 2.7 μm) by gradient elution, with the mobile phase of 1 mmol · L^-1 ammonium acetate and 0.1% (volume fraction) formic acid aqueous solution and acetonitrile. Then, the target analytes were ionized by electrospray ionization in positive ion mode, detected in multiple reaction monitoring mode, and quantified by the internal standard method. Key influencing factors, including sample stability, storage container, particulate matter in samples, solution pH, filter membranes, residual chlorine content in samples, and liquid chromatographic conditions, were systematically evaluated, and spiked water samples of five types of matrices were determined. Results: Samples can be stored sealed, refrigerated, and protected from light at below 4 ℃ for at least seven days, and plastic containers shall be used for storage. Aminoglycoside antibiotics tended to be adsorbed onto particulate matter, so alkaline desorption was required, and the filtrate must be adjusted to neutral before determination. A 0.22 μm polypropylene filter membrane shall be used for sample filtration. Residual chlorine can cause loss of the target analytes, and ascorbic acid must be added immediately after water sample collection. After optimizing instrumental conditions, limits of detection ranged from 1.0 to 1.7 μg · L^-1. Recovery tests were conducted using the standard addition method. Except for spectinomycin, which showed slightly lower recoveries in complex matrices (with average recoveries of 42.8% and 41.0% in domestic sewage and industrial wastewater, respectively), the recoveries of the other compounds in all matrices, as well as those of spectinomycin in pure water, groundwater, and surface water matrices, ranged from 60.0% to 127.0%. The relative standard deviations (RSDs) (n=6) were between 1.3% and 16.0%. Conclusion: All performance parameters of this method satisfy current quality-control requirements for environmental antibiotic monitoring and thus are of practical referential significance.

In recent years, the abuse of etomidate has become increasingly serious. With etomidate being listed in the control catalog, the synthesis of non-scheduled analogues by modifying the chemical structure has become an emerging abuse trend to evade regulation, posing a serious threat to public health and safety. In the face of the increasing types of abuse, it is particularly important to strengthen the regulation and detection of etomidate and its analogues. Based on a systematic summary of the pharmacological and toxicological properties of etomidate, this paper focuses on the structure-activity relationships and detection methods of etomidate and its analogues, with the expectation of providing references for the establishment of control strategies and analytical methods for etomidate.

Objective: To determine the content of five heavy metals and harmful elements—copper (Cu), arsenic (As), cadmium (Cd), mercury (Hg), and lead (Pb)—in eight Mongolian medicinal materials (Heibingpian, Oxytropis myriophylla (Pall.) DC., Scabiosa comosa Fisch. ex Roem. et Schult., Lomatogonium rotatum (L.) Fries ex Nym., Syringa pinnatifolia Hemsl. var. alashanensis Ma et S. Q. Zhou, Liquidambar formosana Hance, Vladimiria souliei (Franch.) Ling, Dracocephalum moldavica L., 40 batches in total) circulated in Hohhot, analyze their pollution status, and establish a risk assessment model. Methods: The content of heavy metals and harmful elements in the 40 batches of Mongolian medicinal materials was measured through inductively coupled plasma mass spectrometry (ICP-MS). Element concentrations and pollution indices were determined to assess pollution levels, and the target hazard quotient (THQ) and carcinogenic risk (CR) were adopted to evaluate non-carcinogenic and carcinogenic risks, respectively. Results: Among the 40 batches of Mongolian medicinal materials, Heibingpian exhibited the highest total content of heavy metals and harmful elements, which ranged from 21.478 mg · kg^-1 to 33.330 mg · kg^-1.The Cu, As, and Pb content was ND (not detected)-21.936 mg · kg^-1, ND-2.351 mg · kg^-1, and 0.035-8.834 mg · kg^-1,respectively. The content of Cd and Hg in all the samples did not exceed the limit standards. The average Pb content in Heibingpian was 8.035 mg · kg^-1, and the content of As and Cu in Heibingpian was close to the threshold limits. The pass rate of heavy metal and harmful element tests for the other seven tested Mongolian medicinal materials except Heibingpian reached 100%. Non-carcinogenic risk assessment revealed that the THQ values of the five elements in Heibingpian and As in O. myriophylla exceeded the standard limits, indicating potential health risks. The CR values of all tested medicinal materials were far below the safety threshold (1×10^-6), suggesting negligible carcinogenic risks. Conclusion: Mongolian medicinal materials circulated in Hohhot in this study are generally safe. Special attention should be paid to the health risks posed by heavy metals in individual medicinal materials such as Heibingpian and O. myriophylla, and stricter supervision on raw material traceability and processing procedures is required. The multi-element analytical method and multi-index evaluation system constructed in this study can provide scientific references for pollution monitoring and risk management of heavy metals in Mongolian medicinal materials.

Objective: To establish a method for identifying Epiphyllum oxypetalum (DC.) Haw. and its adulterant Hylocereus undatus (Haw.) Britt. et Rose to ensure the accuracy and safety of clinical medication. Methods: E. oxypetalum and its adulterant H. undatus were compared by original plant identification, morphological characterization, and optical microscopy and pathological section scanning imaging for microscopic characteristics of roots, stems, flowers, and powder. Results: Significant differences exist between E. oxypetalum and H. undatus in terms of original plant features, macroscopic characteristics of medicinal materials, and microscopic characteristics. The two species can be distinguished by the morphological characteristics of their roots, stems, flowers, hypanthia, and fruits, and they exhibit obvious differences in texture, odor, and color of medicinal materials. Furthermore, distinct differences were observed in the transverse sectional tissue structures of roots, leaf-like stems, hypanthia, styles, and perianths, as well as in the powder microscopic characteristics of the two medicinal materials. Conclusion: E. oxypetalum and its adulterant H. undatus can be effectively identified by the differences in traits and microscopic characteristics, which can provide reference for the safe medication and further development and utilization of E. oxypetalum.

Objective: To conduct a qualitative study on the chemical components of Ganlu Xiaodu Dan and its blood- and brain-entering components using ultra-high-performance liquid chromatography-quadrupole/electrostatic field Orbitrap high-resolution mass spectrometry (UHPLC-Q Exactive Orbitrap HRMS). Methods: UHPLC-Q Exactive Orbitrap HRMS was employed to identify the chemical components of Ganlu Xiaodu Dan. Twelve male Sprague-Dawley (SD) rats were randomly divided into a blank group and a Ganlu Xiaodu Dan group. The Ganlu Xiaodu Dan group received an intragastric administration of the aqueous decoction at a raw herb dose of 1.8 g · kg^-¹, while the blank group received an equivalent volume of purified water. Plasma samples were collected 1.5 h post-administration. Using an ACQUITY UPLC HSS T₃ column (100 mm×2.1 mm, 1.8 µm) at a column temperature of 40 ℃. The mobile phase consisted of 0.1% formic acid in water and acetonitrile with gradient elution at flow rate of 0.30 mL · min^-1. The injection volume was 6 µL. Electrospray ionization (ESI) was applied with a scanning range of m/z 100-1 500. Combined with information from a reference database, qualitative analysis of blood-entering components was performed in both positive and negative ion modes. Results: A total of 195 compounds were identified in the 80% methanol extract of Ganlu Xiaodu Dan, and 66 prototype components were detected in serum. The fragmentation patterns of baicalin, apigenin-7-glucoside, isorhamnetin-3-O-galactoside, biochanin A, 28-deacetylbelamcandal, plantamajoside A, phillyrin A, aurantio-obtusin, neochlorogenic acid, chrysosplenol, mangiferin, and chuanbeiketonine were analyzed. Conclusion: This study enabled the rapid characterization and identification of multi-category in vitro chemical components and blood-entering components of Ganlu Xiaodu Dan, providing a reference for research on its pharmacodynamic material basis and quality control.

Objective: To identify impurities in the starting material of paliperidone by LC-MS, analyze the impurities in combination with the synthetic process, and establish a HPLC method for the determination of related substances in the starting material of paliperidone. Methods: LC-MS was performed with an Agilent Eclipse XDB-C₁₈ column (250 mm×4.6 mm, 5 μm) at a column temperature of 40 ℃. Gradient elution was performed with 0.1% formic acid solution as mobile phase A and water-acetonitrile (10 ∶ 90) as mobile phase B at a flow rate of 1.0 mL · min^-1. The detection wavelength was set at 238 nm, the injection volume at 20 μL, and the sample tray temperature at 10 ℃. For HPLC, an Agilent Eclipse XDB-C₁₈ chromatographic column (250 mm×4.6 mm, 5 μm) at a column temperature of 40 ℃ was used for gradient elution with the mobile phase A consisting of 3.4 g · L^-1 potassium dihydrogen phosphate solution (adjusted to pH 4.0 with phosphoric acid), methanol, and acetonitrile at a ratio of 90 ∶ 5 ∶ 5 and the mobile phase B being a mixture of acetonitrile and methanol (50 ∶ 50) at a flow rate of 1.0 mL · min^-1. The detection wavelength was set as 238 nm, the injection volume as 20 μL, and the sample tray temperature as 10 ℃. Results: Five impurities in the starting material of paliperidone were detected by LC-MS. The five known impurity reference standards showed good linear relationships within a certain concentration range in the HPLC method. The resolution between peaks was greater than 1.5. The limits of detection (LOD) of each component ranged from 0.009 2 to 0.032 8 μg · mL^-1. The recovery rates of all impurities were between 90% and 108%. The relative standard deviations (RSDs) of impurity recovery rates in the precision test were no more than 6%. The test solution remained stable when being stored at 10 ℃ for 4 h. The method had satisfactory robustness. Five batches of samples were determined. The detected content of impurity ii-1, impurity ii-2, impurity ii-3, impurity ii-4, and impurity ii-5 was 0.04%-0.26%, 0-0.16%, 0-0.24%, 0, and 0-0.24%, respectively. The content of other single impurity was 0.13%, and the total impurity content was 0.17%-0.53%. Conclusion: This method possesses strong specificity, high sensitivity, and high accuracy, being suitable for the determination of related substances in the starting material of paliperidone.

Objective: To establish a high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method for the simultaneous determination of the content of 5 genotoxic impurities in vonoprazan fumarate, thereby meeting the detection requirements for trace genotoxic impurities in this bulk drug. Methods: An Agilent ZORBAX Eclipse XDB-C₁₈ column (4.6 mm×150 mm, 3.5 μm) was used for linear gradient elution with 0.1% formic acid aqueous solution as mobile phase A and acetonitrile containing 0.1% formic acid as mobile phase B at the flow rate of 0.5 mL · min^-1, the column temperature of 30 ℃, and the injection volume of 10 μL. The mass spectrometry adopted electrospray mass ionization (ESI) and the multiple reaction monitoring (MRM) in the positive ion mode. Results: The linear relationships of various impurities were good within a certain concentration range (r: 0.995 0-0.999 7). The limit of detection and the limit of quantification reached 0.02-0.09 ng · mL^-1 and 0.04-0.27 ng · mL^-1, respectively. The accuracy test showed that the average recovery rates of various impurities were between 89.0% and 109.4%, and the relative standard deviations (RSDs) were all no more than 6.3%. The content of genotoxic impurities in multiple batches of samples were all considerably lower than 30% of the acceptable limit specified in ICH M7 (R2). Conclusion: The established HPLC-MS/MS method exhibits high sensitivity and strong specificity, enabling the simultaneous determination of five genotoxic impurities in vonoprazan fumarate. This method can be employed for the routine quality control of the above five genotoxic impurities in vonoprazan fumarate.

Objective: To establish a ultra high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method for simultaneous determination of 13 chemical components (terrestrosin K, phellodendrine hydrochloride, imperatorin, isoimperatorin, prim-O-glucosylcimifugin, buddleoside, verbascoside, liquiritin, wogonin, geniposide, luteolin-7-O-glucoside, paeoniflorin, and baicalein) in Mingmu Jili pills. Methods: The separation was performed through a Waters Acquity BEH C₁₈ column (100 mm×2.1 mm, 1.7 µm) with a column temperature at 40 ℃. The mobile phase consisted of acetonitrile-5 mmol · L^-1 ammonium formate (containing 0.1% formic acid) with gradient elution at a flow rate of 0.3 mL · min^-1. The injection volume was 1 µL. The detection was carried out by electrospray ionization (ESI) source combined with multiple reaction monitoring (MRM) in positive and negative ion modes. Results: The 13 components showed good linear relationships in their respective concentration ranges (r>0.999 0). The method demonstrated high precision, repeatability, and stability. The spiked recovery rates ranged from 93.7% to 110.0%, with the relative standard deviation (RSD) of 0.28%-9.7%. The content of 13 components in 13 batches of Mingmu Jili pills was 11.94-75.55, 66.88-156.32, 57.18-87.83, 18.57-27.68, 20.20-196.20, 230.73-485.25, 539.71-3 061.53, 24.30-87.02, 314.32-973.51, 297.45-1 736.38, 61.45-473.48, 397.44-1 275.18, and 331.81-4 091.77 µg · g^-1. Conclusion: The established method is efficient, simple, accurate, and reproducible, which can provide a reference for the quality control of Mingmu Jili pills.

Objective: To conduct microbial monitoring and analysis of pharmaceutical water systems from three enterprises, and preliminarily explore culture media suitable for subculture of microorganisms isolated from these systems. Methods: A total of 130 microbial strains were isolated from the pharmaceutical water systems of three enterprises. Bacterial and fungal isolates were identified by 16S rDNA and ITS rDNA sequencing, respectively. Phylogenetic trees were constructed for frequently detected genera to analyze potential contamination sources. Furthermore, subculture studies were performed on all the 130 strains with three different culture media and the modified sterilization method. Results: The microorganisms most frequently detected in the water systems of the three pharmaceutical enterprises were Methylobacterium, Ralstonia, and Bradyrhizobium. Subculture results of the 130 strains showed that R3A and R2A media performed better than Tryptic Soy Agar (TSA). The sterilization method was modified as autoclaving phosphate and agar components separately before mixing in the R3A medium, which improved the growth rate of some slow growing microorganisms on RXA media. Conclusion: Microorganisms in pharmaceutical water systems show certain commonality. RXA media may be more suitable for subculture of microorganisms from pharmaceutical water systems. For some microorganisms susceptible to oxidative stress, modifying the sterilization method for the culture medium can promote their growth.

Objective: To develop a LC-MS method for the quantitation of three free fatty acids—lauric acid, myristic acid, and oleic acid—in antibody pharmaceuticals based on the analytical quality by design (AQbD) principles outlined in the ICH Q14 guideline on analytical procedure development, and to perform subsequent method validation. Methods: The analytical target profile (ATP) was initially established. Based on literature review and laboratory experience, a 1290 UHPLC (ultra-high performance liquid chromatography) system coupled with an SQ- MS (single quadrupole mass spectrometer) was selected to develop the method. All operational steps, spanning from sample preparation to LC-MS detection, that could potentially influence analytical method performance were comprehensively identified, and their potential impact on method accuracy and precision was systematically assessed. With respect to the control strategy, factors associated with high risk or high operational cost were subjected to stringent control. Experimental investigations were conducted on chromatographic column selection, mobile phase composition, gradient or isocratic elution modes, column temperature, as well as mass spectrometric parameters including capillary voltage, fragmentor voltage, and nebulizer pressure. A quantitative analytical method suitable for the degradation products of polysorbate (Tween) was developed and subsequently validated in accordance with ICH Q2 requirements. Results: Methodvalidation demonstrated that the established LC-MS method for free fatty acid quantification exhibited spike recoveries ranging from 82.3% to 106.6% for lauric acid, myristic acid, and oleic acid. The linear range spanned from 0.01 to 2.00 μg · mL^-1, with a lower limit of quantitation (LLOQ) below 0.01 μg · mL^-1. No interference from proteinaceous components or excipients within the antibody formulation was observed. The method was applied to analyze the concentrations of lauric acid, myristic acid and oleic acid in both within-specification and expired antibody preparations, as well as in forced degradation samples from two investigational antibody formulations. Notably, significantly elevated levels of lauric acid, myristic acid, and oleic acid were detected in the forced degradation samples, indicative of polysorbate degradation. The method performance met the predefined requirements specified in the ATP, rendering it suitable for the quantitative detection of free fatty acids in diverse antibody pharmaceuticals. Conclusion: This study presents a case study of LC-MS method development employing AQbD principles. Through rigorous control of experimental materials, measurement procedures, and operational protocols, and via experimental verification of permissible ranges for mass spectrometer gas flow and voltage parameters, the final method specifies mass spectrometric parameters as acceptable operational ranges rather than discrete fixed values. This approach holds substantial value for the method lifecycle, particularly in the context of change management. The developed method is applicable to the quantification of free fatty acids derived from polysorbate degradation in antibody products. Furthermore, this case provides a meaningful reference for the application of LC-MS methodology in pharmaceutical quality control.

Objective: To establish an ultra-performance liquid chromatography-triple quadrupole mass spectrometry (UPLC-QQQ MS) method for the rapid and simple qualitative identification of Qizhi Tongluo capsules. Methods: Gradient elution was performed through a Waters ACQUITY^TM UPLC Hss T3 C₁₈ column (100 mm×2.1 mm, 1.8 μm) with the mobile phase composed of acetonitrile and 0.1% formic acid aqueous solution at the flow rate of 0.3 mL · min^-1 and the column temperature of 35 ℃. The chromatographic effluents were detected by mass spectrometry in positive and negative ion modes. Results: The specific chromatograms showed 32 common characteristic peaks from 13 Chinese herbal medicines. The structures of 23 characteristic components were confirmed by comparison with reference substances. RSDs of the relative retention time of 32 characteristic peaks in 10 batches of samples were all less than 1.0%, indicating that the 32 characteristic peaks were stable. Conclusion: The established liquid chromatography-mass spectrometry (LC-MS) specific chromatogram method is simple, sensitive, and accurate, comprehensively reflecting the chemical composition characteristics of Qizhi Tongluo capsules and serving as a new method for rapid identification of Qizhi Tongluo capsules.

Objective: To establish a rapid and sensitive method for determining As (Ⅲ) in Realgar, a commonly used Chinese medicinal material with safety mainly influenced by As (Ⅲ), thereby ensuring its safe use. Methods: An iron-cobalt layered double hydroxide/transition metal carbide (Fe-Co-LDH/MXene) nanocomposite was synthesized by a hydrothermal method. A glassy carbon electrode (GCE) was modified with this nanocomposite to fabricate a sensor (Fe-Co-LDH-MXene/GCE). The materials were characterized by scanning electron microscopy (SEM) and X-ray diffraction (XRD). The sensor performance under different solution conditions was systematically evaluated by electrochemical methods. Furthermore, the stability and repeatability were assessed. Results: A nanocomposite with a distinct layered structure was successfully prepared. In the neutral aqueous solution, the sensor demonstrated excellent electrochemical responses to As (Ⅲ). A good linear relationship was observed between the response signal and As (Ⅲ) concentration within the range of 100-500 μg · mL^-1. The sensor showed good stability and repeatability. Moreover, it performed well in the determination of As (Ⅲ) in Realgar samples. Conclusion: The electrochemical sensor enables rapid and accurate determination of As (Ⅲ) in Realgar under neutral conditions, with simple operation and high sensitivity. It offers a new method for quality control of Realgar-containing Chinese medicine preparations.

Objective: To establish a method for determining the particle size and particle size distribution of dapagliflozin based on laser diffraction. Methods: The Malvern Mastersizer 3000 laser size analyzer with the Malvern SV sample dispersion unit was used to determine the particle size and particle size distribution of dapagliflozin by the wet method. The Isopar G containing 2% of Span 80 was used for pre-dispersion. The dapagliflozin samples had the refractive index of 1.614 and the absorbance of 0.01. The Isopar G was used for dispersion, with laser obscurations of 8%-15%. The samples were determined after sonicating for 60 s at the pump speed of 2 500 r · min^-1. Results: The particle size and particle size distribution of dapagliflozin products could be analyzed accurately by the laser diffraction wet method. The D[4,3] could show the size differences between different products. The RSDs of methodological survey for d(0.1), d(0.5), and d(0.9) were lower than 5%, which indicated good repeatability. The particle size distribution d(0.9) values of 12 batches of dapagliflozin were less than the standard limit (30 μm) in quality control. Conclusion: The method is accurate and repeatable, and suitable for the particle size determination of dapagliflozin. Moreover, it provides reference for the development of particle size determination methods for other drugs prone to agglomeration and moisture absorption.

Objective: To establish a rapid coculture and detection method for six pathogenic bacteria (Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus, Salmonella paratyphi B, Pseudomonas aeruginosa, and Burkholderia cepacia) in cosmetics and raw materials based on the gene membrane chip technology and improve the biological safety assurance capability and risk screening efficiency of the product. Methods: The co-culture medium was used for co-culture of the six pathogenic bacteria, and specific primers and probes for the six pathogenic bacteria were designed. Multiplex PCR amplification was performed on the sample nucleic acid, and the gene membrane chip technology was employed for further product hybridization and analysis. Results: Specificity tests showed that the six pathogenic bacteria developed color only at their corresponding target spots, while the four negative template bacteria (Listeria monocytogenes, Bacillus cereus, Vibrio vulnificus, and Shigella spp.) showed no color development, indicating no cross-reaction and good probe specificity. At primer concentrations of 0.20/0.30 μmol · L^-1 (internal control) and 0.067-0.75 μmol · L^-1 (for the six targets), the color development intensities of all targets were uniform. The limit of detection (LOD) test showed that the established method achieved an LOD of 1.0×10⁵ copies · mL^-1 for all six pathogenic bacteria. The test results of nine batches of samples were all negative, consistent with those obtained by conventional methods. The established method specifically detected the corresponding targets in the spiked samples without cross-interference, meeting the requirements for routine testing. Conclusion: A co-culture method for six pathogenic bacteria in cosmetics and a multiplex detection method based on PCR-gene membrane chip technology were established. This method exhibits strong specificity, good stability, and high sensitivity, ensuring the quality and safety of cosmetics and raw materials. It can be further applied to the detection of cosmetics in factories and markets, enriching the detection methods for pathogenic bacteria in the cosmetics industry and providing a rapid and powerful technical means for detection.

Objective: To optimize and validate the specific neutralization test method for the Corynebacterium diphtheriae strain C7 (β197) (strain C7), thereby improving the clarity of results and shortening the detection time. Methods: Strain C7 was inoculated in both wide and narrow patterns. A filter paper strip was placed either on the surface of culture medium or embedded within the culture medium. Seven types of culture media—peptone A Elek’s medium, peptone B Elek’s medium, peptone C Elek’s medium, peptone D Elek’s medium, peptone E Elek’s medium, nutrient agar medium, and commercially available Elek’s medium—were screened. Diphtheria antitoxin was prepared at four concentrations: 2 000, 1 500, 1 000, and 500 Lf · mL^-1. Petri dishes were poured with 15, 25, and 35 mL of culture medium, and strain C7 was inoculated at distances of 3, 5, 7, 9, and 11 mm from the filter paper strip. Equine serum or bovine serum was added to the basal culture medium. The key conditions for the specific neutralization test of strain C7 were systematically optimized and screened. Additionally, the specificity and repeatability of the optimized method were validated. Results: The optimal conditions for the specific neutralization test were determined as follows: strain C7 inoculated in a narrow pattern, filter paper strip placed on the surface of culture medium, peptone C Elek’s medium used as the neutralization medium, filter paper strip saturated with diphtheria antitoxin at 1 500 Lf · mL^-1, petri dish poured with 15 mL medium, strain C7 inoculated at a distance of 3 mm from the filter paper strip, and bovine serum added to the basal medium. The validation results demonstrated that the optimized method had good specificity and repeatability. Conclusion: The specific neutralization test method optimized in this study is stable and reliable. It significantly improves the clarity of results and detection efficiency for strain C7, providing more efficient and reliable technical support for the laboratory identification of this bacterium and the quality control of related vaccines.

Objective: This study aimed to establish a high-speed countercurrent chromatography (HSCCC) method for the separation of catechin (CAT) and epicatechin (EC) from Catechu, and to investigate the effects of CAT and EC on skin pruritus and vascular permeability in mice. Methods: High-purity CAT and EC were successfully isolated from Catechu by HSCCC with optimized separation parameters. An Agilent TC-C₁₈ column (250 mm×4.6 mm, 5 μm) was adopted for high-performance liquid chromatography analysis. Methanol-water (30 ∶ 70) served as the mobile phase under isocratic elution conditions, with a flow rate of 1.0 mL · min^-1, injection volume of 10 μL, column temperature of 30 ℃, and detection wavelength set at 280 nm. Pruritus mouse models induced by histamine (HA), chloroquine (CQ) and 2,4-dinitrofluorobenzene (DNFB) were established to evaluate the antipruritic efficacy of CAT and EC as well as their influences on cutaneous vascular permeability. Results: The purity of CAT and EC separated by HSCCC was 95.2% and 96.4%, with recovery rates of 83.4% and 84.1%, respectively. In mouse models of acute and chronic pruritus, the low-dose CAT group (CAT-L, 200 mg · kg^-1), high-dose CAT group (CAT-H, 400 mg · kg^-1), low-dose EC group (EC-L, 50 mg · kg^-1), and high-dose EC group (EC-H, 100 mg · kg^-1) all significantly reduced the frequency of scratching, decreased skin vascular permeability, and inhibited the release of inflammatory factors in a clear dose-dependent manner. The high-dose groups exhibited significantly better effects on antipruritus and improving vascular permeability than the low-dose groups. Conclusion: This study is the first to establish and optimize an HSCCC system for the separation of CAT and EC from Catechu, and the first to systematically evaluate their antipruritic activities and characteristics in acute and chronic pruritus models. The method is efficient, stable, simple, and yields high-purity products, making it suitable for the separation and preparation of CAT and EC from Catechu. Both CAT and EC show significant therapeutic effects on acute and chronic pruritus, providing new directions and experimental references for the research and development of natural drugs against skin pruritus.

Objective: To establish a high-performance liquid chromatography (HPLC) method for the determination of diosmetin in thermosensitive liposomes and investigate its in vitro release behavior. Methods: HPLC was conducted with a Supersil ODS₂ column (250 mm×4.6 mm, 5 μm), the detection wavelength of 343 nm, and the injection volume of 20 μL. Single-factor experiments were carried out to investigate the influences of mobile phase methanol-water ratio by volume, flow rate, and column temperature and optimize the chromatographic conditions. With 1% Tween 80 aqueous solution as the release medium, the in vitro release behavior of diosmetin-loaded thermosensitive liposomes at different temperatures was investigated via dialysis coupled with constant-temperature shaking. The cumulative release rate was calculated, and Origin software was adopted to fit drug release models. Results: The optimized chromatographic conditions were as follows: a mobile phase composed of methanol and water at a volume ratio of 75 ∶ 25, a flow rate of 1 mL · min^-1, and a column temperature of 35 ℃. Diosmetin exhibited a good linear relationship between peak area and concentration within the range of 0.1-50 μg · mL^-1, and the regression equation was A=86.489C-2.142 2 (r=1.000). The limit of detection was 0.6 ng, and the limit of quantitation was 1.8 ng. The spiked recoveries at high, medium, and low concentrations were all above 95%, and the RSD values were all less than 2%. In vitro release experiments revealed that diosmetin-loaded thermosensitive liposomes exhibited obvious temperature responsiveness. Compared with free diosmetin, the liposomes showed slow drug release at 37 ℃, whereas the release rate rose significantly at 42 ℃ and 53 ℃. The release behavior at 37, 42, and 53 ℃ all complied with the first-order kinetic model. Conclusion: This method is simple, stable, and efficient, and is thus suitable for the in vitro release evaluation of diosmetin-loaded thermosensitive liposomes. Furthermore, the prepared liposomes exhibit heat-triggered drug release behavior and sustained-release properties, and their release behavior is primarily dominated by diffusion.

Objective: To investigate the histological structural differences and the spatial distribution characteristics of saponins in the roots and rhizomes of Pulsatilla chinensis (Bunge) Regel, Pulsatilla cernua (Thunb.) Bercht. & J. Presl, and Pulsatilla turczaninovii Krylov & Serg. by combining microscopy and matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI). Methods: The histological structural characteristics of the roots and rhizomes of three Pulsatilla species were observed and compared by microscopy. An OLYMPUS microscopic imaging system and a BX-51 biological microscope equipped with a DP-72 microscope digital camera and polarizing filters were used for observation, and the microscopic features were documented. For MALDI-MSI analysis, tissue sections were prepared through a Leica CM1950 cryostat, and DHB matrix was applied with a TM-Sprayer matrix sprayer. The matrix-coated ITO slides were placed on the mass spectrometer target plate, and tissue regions of interest were selected by DataImaging. Under identical laser energy, the TimsTOF fleX mass spectrometry system was used to identify and detect tissue samples, and the in situ spatial distribution of 21 saponins, including the marker component pulchinenoside B₄ specified in the Pharmacopoeia of the People’s Republic of China, was visualized across different tissue regions of the roots and rhizomes. Results: Microscopic observations revealed that the roots of all the three species possessed a distinct endodermis (with prominent casparian strips), while the rhizomes contained a pith or fissures. Significant interspecies differences were observed in the number of xylem bundles and the distribution of phloem fibers. MALDI-MSI results revealed that among the 21 saponins, the majority (17) of saponins were predominantly enriched in the cortical tissue, while 4 were undetected. Some saponins (pulchinenoside D and pulchinenoside E₂) exhibited a unique ring-shaped distribution pattern in the endodermal region of the roots or rhizomes, most prominently in the roots of P. chinensis and P. cernua. Pulchinenoside E showed higher distribution in the xylem. Conclusion: This study reveals the specific spatial distribution patterns of saponins (cortex enrichment and endodermal ring distribution) in the roots and rhizomes of these three Pulsatilla species and clarifies the interspecies differences in their microscopic tissue structures. This study reveals histochemical differences among three Pulsatilla species and between their roots and rhizomes, providing a histochemical basis for species identification and the scientific evaluation and development of medicinal resources of this genus.

Objective: To establish a high performance liquid chromatography (HPLC) method for the determination of enantiomer in dapagliflozin. Methods: The determination was performed through a CAPCELL PAK MG Ⅱ C₁₈ column (250 mm×4.6 mm, 3.0 µm) at the column temperature of 30 ℃, with the mobile phase consisting of acetonitrile and water at a ratio of 43 ∶ 57 at the flow rate of 0.7 mL · min^-1. The detection wavelength was set at 225 nm, and the injection volume at 10 μL. Results: The peaks of dapagliflozin and enantiomer were well separated. The calibration curve of enantiomer was linear in the range of 0.051 8-10.36 μg · mL^-1 (r=0.999 9). The limit of quantification was 0.05 μg · mL^-1, and the limit of detection was 0.02 μg · mL^-1. The average recovery of enantiomer was 98.8%, with the RSD of 2.1%. The test results of three batches of dapagliflozin samples showed that the content of enantiomer and other unknown impurities was 0.6 mg · g^-1 and 0.1 mg · g^-1, respectively, and the total amount of impurities was less than 1.0 mg · g^-1. Conclusion: The established method is accurate and reliable. It can be used for the quality control of dapagliflozin.

Objective: To determine the content of eight aristolochic acid components in Liujing Toutong tablets, thus providing reference for the quality control. Methods: An ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was established to determine the content of aristolochic acidⅠ, aristolochic acid Ⅱ, aristolochic acid Ⅳa, aristolochic acid Ⅶa, aristolactamⅠ, aristolactam AⅡ, aristolactam BⅡ, and aristolactam FⅠ in Liujing Toutong tablets. Gradient elution was carried out through an Agilent ZOBARX SB C₁₈ column (50 mm×2.1 mm, 1.8 μm), with methanol as mobile phase A and 0.1% formic acid solution (containing 1 mmol · L^-1 ammonium acetate) as mobile phase B. The electrospray ionization (ESI) in positive ion multi-reaction monitoring mode was adopted. The content of the eight aristolochic acid components in Liujing Toutong tablets was calculated with the standard curve method. Results: Aristolochic acid Ⅱ and aristolactam BⅡ were detected in none of the samples. The content of aristolochic acid Ⅰ, aristolochic acid Ⅳa, and aristolactam Ⅰ varied within the ranges of 0.010-0.055 μg · g^-1, 0.064-0.998 μg · g^-1, and 0.028-0.454 μg · g^-1, respectively. Aristolochic acid Ⅶa, aristolactam FⅠ, and aristolactam AⅡ were detected in some samples but all below the limits of quantitation. Conclusion: The UPLC-MS/MS method is established for determining the content of eight aristolochic acid components in Liujing Toutong tablets, with high specificity, high sensitivity, good repeatability, which can provide reference for the quality control and safety supervision of Liujing Toutong tablets.