OBJECTIVE To preparet aurocholic acid modified PLGA nanospheres for oral delivery of semaglutide. METHODS The nanospheres were prepared by dobule emulsion solvent evaporation technique, and the preparation process was optimized by single-factor experiments; the nanospheres were characterized by scanning electron microscopy, Fourier transform infrared spectroscopy and laser particle size measurement; pharmacokinetic experiments were performed using SD rats; pharmacodynamic experiments were performed using db/db mice. RESULTS The FT-IR showed that taurocholic acid was successfully modified to the surface of the nanospheres. The particle size of the nanospheres was (185.9±3.31) nm, the ζ-potential was (-32.53±0.95) mV, and the drug loading and encapsulation rates were (11.15±0.07)% and (85. 51±0.01)%. The nanospheres showed good sustained release in vitro, with a cumulative release rate of 84. 96% within 192 h. Pharmacokinetic experiments were performed in SD rats, and the results showed that the bioavailability of nanospheres was 2.5%, and the slow release of semaglutide could be achieved within 192 h. The efficacy of nanospheres was verified in db/db mice, and the results showed that after gavage administration of nanospheres, the blood glucose of diabetic mice decreased rapidly and remained stable for about 3 d. CONCLUSION The oral delivery of semaglutide nanospheres prepared in this study has high drug loading and encapsulation efficiency, which can effectively control the blood glucose of diabetic mice within 3 d and improve the bioavailability.

OBJECTIVE To establish an ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method for simultaneous determination of three pyrrolizidine alkaloids and five aristolochic acids in Fufang Banxia Tablets, and carry out preliminary risk assessment. METHODS ACQUITY UPLC BEH C₁₈ column (2.1 mm×100 mm, 1.7 μm) was used. The mixed solution of methanol-acetonitrile (1∶1) was used as mobile phase A, and 0.1% formic acid solution and 5 mmol·L^-1 ammonium formate were used as mobile phase B. Gradient elution was performed at a flow rate of 0.3 mL·min^-1 and the column temperature was maintainedat 30 ℃. Electrospray ion source (ESI), multiple reaction monitoring mode (MRM) and positive ion scanning were used. RESULTS Three pyrrolizidine alkaloids showed a good linear relationship in the range of 0.48-482.60 pg, and five aristolochic acids showed a good linear relationship in the range of 0.51-538.51 pg. The correlation coefficients were higher than 0.99. The average recoveries were between 60.4%% and 120.5%, with RSDs of 2.1%-9.7%. Pyrrolizidine alkaloids and aristolochic acids were detected in all five batches, the total content of pyrrolizidine alkaloids was 5.42-5.46 mg·kg^-1, and the total content of aristolochic acids in positive samples were 1.04-1.70 mg·kg^-1. CONCLUSION The established method is simple and accurate, and can simultaneously determine the contents of three pyrrolizidine alkaloids and five aristolochic acids in Fufang Banxia Tablets. The preliminary risk assessment shows that the safety risk of Fufang Banxia Tablets is low.

OBJECTIVE To establish the HPLC fingerprints, determine the contents of chlorogenic acid, liquiritin, lobetyolin, liquiritigenin, glycyrrhetinic acid, atractylenolide Ⅲ, atractylenolide Ⅱ, atractylenolide Ⅰ, 10-gingerol in Lizhong Pills, and lay the foundation for its quality control. METHODS A Eclipse Plus C₁₈ column was used with acetonitrile-0.1% aqueous phosphoric acid as the mobile phase in gradient elution at a flow rate of 0.8 mL·min^-1. The detection wavelengths were set at 215 nm and 254 nm, and the column temperature was maintained at 30 ℃. Through similarity evaluation, combined with chemical pattern recognition, the fingerprints of 21 batches of Li Zhong Pills were evaluated and analyzed, and 9 index components were quantitatively determined. RESULTS The fingerprints of 21 batches of Lizhong Pills were established, with 29 peaks identified, the similarity of each batch was greater than 0.9, and 21 batches of samples from 3 manufacturers could be clustered into 3 categories, with 16 compounds differing between groups. Nine components showed good linear relationships within their respective linear ranges (r≥0.998 7), the average recoveries ranged from 95.34% to 104.41%, and the RSDs ranged from 0.95% to 2.95%. CONCLUSION The fingerprint and quantitative determination method of Lizhong Pills is simple, accurate and reproducible, and can be used for the evaluation of the overall quality of Lizhong Pills.

The chemically induced mouse colitis model is the most commonly used animal model for human inflammatory bowel disease, among which dextran sulfate sodium induced colitis model is the most widely used。In the preparation of a dextran sulfate induced model, different concentrations of dextran sulfate and different strains of mice showed varying degrees of lesion severity in the intestinal segments of different mice. Therefore, it is unreasonable to take a sample of a certain segment of the colon for pathological evaluation. The entire colon should be made into a “Swiss roll” and the pathological changes should be comprehensively evaluated by dividing it into upper, middle and lower segments of the colon.The anal canal lesion is severe, it should be evaluated separately. Only by comprehensively and correctly evaluating the intestinal lesion can the therapeutic effect of drugs be correctly evaluated.

OBJECTIVE To establish a quantitative nuclear magnetic resonance coupled with high performance liquid chromatography (qNMR-HPLC) technique for the rapid determination of cilostazol impurity Ⅰ correction factor. METHODS The mixture of cilostazol and cilostazol impurity Ⅰ was dissolved in deuterated dimethyl sulfoxide. A portion of the solution was determined by qNMR, while the other portion of the solution was diluted with water-acetonitrile (60:40) and analyzed by HPLC. The correction factor of cilostazol impurity Ⅰ was calculated with the response signals from qNMR and the peak areas from HPLC. The correction factor of cilostazol impurity Ⅰ was also determined by HPLC standard curve method. A mixed solution containing residual solvent was prepared to simulate the effect of solvents in determining correction factors. When the content of cilostazol impurity Ⅰ was inaccurately assigned due to residual solvent, difference between qNMR-HPLC method and standard curve method was compared. RESULTS When the contents of cilostazol and cilostazol impurity Ⅰ were assigned accurately, the correction factors for cilostazol impurity Ⅰ by qNMR-HPLC method and HPLC standard curve method were 1.74 and 1.76, respectively, which were basically consistent with the pharmacopoeial results. When cilostazol impurity Ⅰ contained residual solvents, and there was an error in the content assignment, the correction factor of the determination by the qNMR-HPLC technique was still 1.72, and the result was not affected by the accuracy of the content. While the correction factor of HPLC standard curve was 2.01, which was deviated from the actual results. CONCLUSION Compared with the HPLC standard curve method, the qNMR-HPLC coupling technique is independent of the accuracy of the content of the substance to be measured and the weighing volume, and does not require purification to prepare a high purity compound. qNMR-HPLC is a powerful tool in the determination of impurity correction factors.

OBJECTIVE To prepare risedronate sodium (RIS)-loaded dissolving microneedle (DMN) and evaluate its efficacy in preventing postmenopausal osteoporosis. METHODS The preparation process of RIS-DMN was optimized by Box-Behnken design of response surface methodology. The appearance, solubility, mechanical property, safety and transdermal effect of RIS-DMN were characterized by scanning electron microscope, intradermal dissolution test, puncture test, skin barrier recovery test and in vitro permeation test. The pharmacodynamic evaluation of RIS-DMN was performed in ovariectomized osteoporosis model rats. RESULTS The optimal formulation were determined to be 45% for solute (mixed with 1:0.86 PVP K30 and CS) and 55% for solvent. It was found that the RIS-DMN have good physical characteristics and properties, and showed great effects in regulating the level of Ca²⁺, P³⁺and alkaline phosphatase (ALP). Meanwhile, the RIS-DMN showed great effects in repairing bone microstructure and improving bone density in ovariectomized osteoporosis model rats, as similar as oral administration. CONCLUSION The RIS-DMN has stable quality, convenient use and precise efficacy, shows great potential in the treatment of postmenopausal osteoporosis.

OBJECTIVE To investigate the feasibility of performing identification of Citri Sarcodactylis Fructus (CSF) and Citri Fructus (CF) based on high performance liquid chromatography (HPLC) fingerprint coupled with multivariate data analysis. METHODS HPLC method was applied to establish the fingerprints of CSF and CF. Similarity evaluation of 12 batches of CSF samples and 10 batches of CF samples was performed by Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2004A edition). Hierarchical cluster analysis (HCA), principal component analysis (PCA) and orthogonal partial least squares-discriminant analysis (OPLS-DA) were used for data analysis. RESULTS HPLC fingerprints of CSF and CF were established, respectively. The fingerprint similarities of CSF samples, as well as the fingerprint similarities of CF samples, were all greater than 0.9. The similarities between the fingerprints of CSF samples and the control fingerprint of CF, as well as the similarities between the fingerprints of CF samples and the control fingerprint of CSF, were all less than 0.9, which indicated significant differences in chemical composition between CSF and CF. The total 22 samples were divided into two groups by HCA, PCA and OPLS-DA, which was consistent with the two varieties. Three differential markers were screened by OPLS-DA and two of them were identified as 5, 7-dimethoxycoumarin and hesperidin by reference substances. The statistical analysis indicated that the ratio of absolute peak area (APA) of hesperidin and 5, 7-dimethoxycoumarin can distinguish CSF and CF from each other simply and accurately. CONCLUSION HPLC fingerprint coupled with multivariate data analysis can be used to identify CSF and CF.

OBJECTIVE To carry out screening of pesticide residues in Panax notoginseng for the pesticide residues commonly used in Panax notoginseng,understand the pesticide residues situation and carry out the related risk assenssment study. METHODS GC-MS/MS and LC-MS/MS methods were used to establish detection methods for commonly used pesticides in Panax notoginseng, as well as those regulated by laws and regulations; the methods were used to conduct a comprehensive screening and risk assessment of the collected Panax notoginseng samples; and the indexes of the transformed pesticides were confirmed in accordance with the pesticide residue limits of Panax notoginseng in the GB 2763 standard. RESULTS In this study, the residue determination methods for about 40 commonly used pesticides and pesticides regulated by regulations in Panax notoginseng were successfully established, and the corresponding limits were set according to the GB 2763 standard. Eight pesticides including tebuconazole, phenyl ether metronidazole and carbendazim were finally recognized as transformed pesticide indicators. CONCLUSION The pesticide residue detection method for Panax notoginseng established in this study is characterized by simple and rapid operation, high specificity and high sensitivity, and the related risk assessment and limit setting provide technical support for the improvement of the quality standard of Panax notoginseng.

OBJECTIVE To establish an analysis method of the charge heterogeneity of human urinary kininogenase (HUK) by using image capillary isoelectric focusing (iCIEF) and complete the methodological verification. METHODS The 35 μL 1% methyl cellulose, 10 μL 200 mmol·L^-1 IDA, 4 μL pharmalyte electrolyte, 48 mg urea, 0.5 μL pI marker with pI 6.15 and 7.05 were added in the sample solution. The focusing condition was pre-focusing voltage 1 500V, duration 1 min, focusing voltage 3 000 V, duration 6 min. RESULTS The optimized method has stable baseline, and the target protein was significantly different from the unrelated protein, The recovery rate of accuracy verification was within 90%-110%, and the linearity verification result had r² of 0.995 7, The RSD of each isomer pI in the repeatability verification was less than 0.2%. The limit of quantification was 0.013 mg·mL^-1. The concentration of urea durability, IDA durability and the electrolyte pharmalyte durability are good. Using this method, the charge isomers of HUK from different manufactures were analyzed effectively. CONCLUSION The developed iCIEF method has good specificity, precision, linearity and durability, and can solve the problem of charge heterogeneity evaluation of protein products, which is of great significance to the quality control of such products from the perspective of charge heterogeneity.

OBJECTIVE To develop and validate a peptide mapping method of an anti-CD33 monoclonal antibody. METHODS Different types of chromatographs (HPLC, UPLC) and different mobile phase systems (formic acid, trifluoroacetic acid) were used for peptide map detection of anti-CD33 antibody. The signature peptide segments were localized using synthetic CDR peptide of the antibody and the localization results were confirmed by mass spectrometry. Based on the relative retention time (RRT), the specificity, precision, and robustness of the method were validated according to the Pharmacopoeia of the People's Republic of China (ChP, 2020). RESULTS The separation time of the peptides by UPLC was shorter than that by HPLC, and the degrees of separation with trifluoroacetic acid in the mobile phase were higher than that with formic acid. The identification results of the signature peptide segment using the maps of synthetic peptide segments were consistent with the results of mass spectrometry. The specificity validation demonstrated that the formulation blank and sample solution blank did not interfere with the detection of signature peptide segments, and there were significant differences between peptide mapping results of different antibodies. The repeatability validation showed that the RSDs (RRT) of signature peptide segments between six parallel samples were 0.01%-0.05%; the intermediate precision validation proved that the RSDs (RRT) of signature peptide segments for different analysts were 0.04%-0.32%; the robustness validation exhibited that the RSDs (RRT) of signature peptide segments were 0.02%-0.09% under different enzyme treatment conditions and 0.36%-1.43% under different chromatographic conditions. Within 25 h in detection, the RSDs (RRT) of the signature peptide segments were 0.01%-0.04%. CONCLUSION This study uses synthetic peptide segments for peptide localization in peptide mapping detection and uses relative retention time to determine the results, which provide a new approach for biopharmaceutical peptide mapping detection.