OBJECTIVE To establish a liquid chromatography-tandem mass spectrometry(LC-MS/MS) method for the simultaneous determination of the concentrations of verapamil and norverapamil in human breast milk and apply it in clinical practice. METHODS Milk samples(100 μL) were precipitated with 300 μL of methanol containing internal standard(5 ng·mL^-1 verapamil-d₃), and the supernatant was taken for analysis after vortexing and centrifugation. The chromatographic separation was performed on a Waters ACQUITY BEH C₁₈ column with gradient elution at 0.4 mL·min^-1, where mobile phases A and B were 0.1% formic acid-10 mmol·L^-1 ammonium acetate-water and methanol, respectively. The injection volume was 2 μL, and the analysis time was 4 min. The detection of the analytes was performed by electrospray ionization in positive mode by multiple reaction monitoring with the transition of m/z 455.3→165.2(verapamil), m/z 441.5→165.3(norverapamil) and m/z 458.3→165.0(verapamil-d₃). The established LC-MS/MS method was validated, and the method was used to determine the drug concentrations in breast milk of lactating patients. RESULTS The calibration curves for verapamil and norverapamil exhibited good linearity within the concentration range of 0.5 to 100 ng·mL^-1 (r=0.999). The intra-assay and inter-assay accuracies were both within ±15% of the labeled values, and the relative standard deviations were less than 15%. The extraction recovery, matrix effect, and stability all met the acceptance criteria for bioanalytical method validation. This analytical method was effectively employed to quantify drug concentrations in the breast milk of a lactating patient. The verapamil concentration in the breast milk of the patient ranged from 1.80 ng·mL^-1 to 10.73 ng·mL^-1, while norverapamil concentrations varied from 2.15 ng·mL^-1 to 5.15 ng·mL^-1. The relative infant dose of verapamil in this patient was approximately 0.15%. CONCLUSION The established method is simple, rapid, and sensitive. It is suitable for monitoring drug concentrations in clinical milk and can provide reference for safe drug use during lactation.

OBJECTIVE To develop a new strategy for the value assignment of melting point reference standards based on optimized statistical methods, in order to enhance the accuracy and reliability of the assigned values. METHODS The development process included several key steps: structural confirmation, purity analysis, homogeneity evaluation, collaborative calibration of melting point, data preprocessing, factorial analysis, difference testing, equivalence testing, and robust statistical assignment. Using JMP software for statistical analysis, the process first involved removing outliers and conducted normality tests and homogeneity of variance tests on the melting point data. Subsequently, factorial analysis was employed to identify the main factors affecting the melting point. Each factor was then individually verified through difference testing. Equivalence testing was also conducted to determine whether the differences were within an acceptable range. Finally, based on the statistical results, the method and scope of robust statistical assignment were determined to value the melting point reference standards. RESULTS The reference materials and heating rates were identified as the main factors affecting the melting point. For each of the four model drugs, the calibration results of the same reference material were excluded because these results showed significant differences from the others and were not equivalent. The results from the remaining two reference materials and the two measurement methods were combined for statistical analysis, while the results from the two different heating rates were analyzed independently. CONCLUSION The accuracy of value assignment for melting point reference standards is improved through standardized experimental operations and optimized statistical methods, and the development process is systematically organized for the first time.

OBJECTIVE To develop and validate an HPLC analytical method for finerenone, based on the concept of analytical quality by design (AQbD). METHODS LC-MS was used to identify the impurity fractions of finerenone API, and then the HPLC chromatographic conditions of finerenone were screened and optimized by analytical factorial design (2^5-1), which examined several factors, such as the acetonitrile proportion at the beginning of the elution gradient, the acetonitrile proportion at the end of the elution gradient, the elution time, the column temperature and the concentration of phosphoric acid aqueous solution, to evaluate the relationship between the critical method attributes (CMAs) and the critical method parameters (CMPs), and to generate the method operable design region (MODR), and finally perform methodological validation. RESULTS The effects of each CMPs on CMAs were analyzed, and the optimized chromatographic conditions from MODR were column temperature 45 ℃, 0.06% phosphoric acid water, gradient elution starting with 5% acetonitrile and ending with 45% acetonitrile, running for 16 min. The established finerenone analysis method had good precision, stability, linearity and sample recovery results. CONCLUSION The chromatographic conditions established based on the AQbD concept can achieve a good separation of finerenone from impurities, accurately evaluate the content of finerenone, and effectively control the quality of the drug.

OBJECTIVE To establish HPLC fingerprint and multi-component content determination method for Linderae Radix, and explore the quality of Linderae Radix from different origins by combining chemical pattern recognition method, providing scientific basis for the quality control and further development of Linderae Radix. METHODS High-performance liquid chromatography(HPLC) was employed, using acetonitrile-0.1% triethylamine aqueous solution(adjusted to pH=3.0 with acetic acid) as the mobile phase for gradient elution, to establish the fingerprint of Linderae Radix. The quality of Linderae Radix from different areas was explored through chemical pattern recognition methods, such as similarity evaluation, cluster analysis(HCA), principal component analysis(PCA), and orthogonal partial least squares discriminant analysis(OPLS-DA). RESULTS The established fingerprint method meets the methodological requirements. A total of 17 common peaks were calibrated in the fingerprint spectra of 44 batches of Linderae Radix, and the similarity was greater than 0.8. Seven components were identified, including norisoboldine, boldine, reticuline, linderalactone, linderane, linderene and lindenanolide H. The HCA analysis results showed that the 44 batches of Linderae Radix were classified into five categories. PCA analysis indicated that the cumulative variance contribution rate of the first four principal components was 80.123%. OPLS-DA analysis showed that linderane, linderene, norisoboldine, and linderalactone were identified as differential chemical components of Linderae Radix. The content determination results indicate that “Tiantai Linderae Radix” has significantly higher levels of linderane (P<0.001), linderene (P<0.001), and linderalactone (P<0.01) compared with Linderae Radix from other regions. CONCLUSION The established fingerprint and content determination method is reliable and stable, providing a reference for the quality evaluation of Linderae Radix and the multi-index quality control of related preparations.

OBJECTIVE To prepare lutein microcapsules using yeast cells as the wall material and lutein as the core material by the freeze-drying method. METHODS One-way tests and response surface method optimization were employed to explore the effects of different temperatures, times and core ratios on the embedding rate of the microcapsules, thereby determining the optimal preparation process. Additionally, the microchemistry of the microcapsules, storage stability, and in vitro simulated digestion were analyzed to evaluate the impacts on the antioxidant activity of the microcapsules, in vitro digestive release behavior, and the digestive effect under a fluorescence microscope. RESULTS The results indicated that the optimal preparation process involved a temperature of 40 ℃, a time of 1.5 h, and a core material ratio of 1∶3(g∶g). Under these optimized conditions, the embedding rate of lutein microcapsules reached 81.77%. Scanning electron microscopy results demonstrated that the microcapsule particles were intact, with a smooth surface, dense structure and an excellent embedding effect. Compared with the control group, microencapsulation treatment could significantly enhance the physiological activity and storage stability of lutein. Under light conditions, it increased by 29.40%, showing good light resistance. Under acidic conditions, it increased by 14.70%-48.07%, and under neutral or weak alkaline conditions, it increased by 45.33%-58.44%, which improved the pH stability of lutein and enabled it to better resist the destruction of intestinal fluid. Investigations of the storage environment of lutein microcapsules revealed that the activity of lutein microcapsules increased by 7.06% under light-avoidance conditions. Lutein microcapsules stored under neutral or weak alkaline conditions had the best effect. They could be stored at room temperature for 28 d, during which the antioxidant activity decreased by 23.74%. Reducing the temperature could increase the antioxidant activity and significantly extend the shelf life. In the simulated gastrointestinal digestion test, the yellow-green fluorescence signal under the fluorescence microscope was first enhanced and then weakened, indicating that lutein was digested and utilized in the gastrointestinal tract. The microcapsules had favorable release properties, and exhibited the highest antioxidant activity and release rate during 2 h gastrointestinal digestion, which could effectively reduce the loss of lutein and improve its bioavailability. CONCLUSION This paper broadens new perspectives for the application of yeast cells and also provides a new reference basis for the development of the lutein industry.

OBJECTIVE To identify the composition of glyceryl mono-and distearate (GMD) from different sources using an LC-TOF-MS method and compare the composition and proportion of glycerides in samples. METHODS A Kinetex C₁₈ column (2.1 mm×100 mm, 2.6 μm) was used as analysis column. Methanol-acetonitrile-water(1∶1∶1, containing 5 mmol·L^-1 ammonium acetate) was used as mobile phase A, and isopropanol(containing 5 mmol·L^-1 ammonium acetate) was used as mobile phase B, gradient elution was performed at a flow rate of 0.3 mL·min^-1. The analysis was carried out in ESI positive and negative ion full scanning(TIC) mode and quantified by area normalization method. RESULTS The composition and proportion of 19 fatty acid glycerides in six different sources of GMD showed significant differences in the composition and proportion of glycerides. CONCLUSION This method can be used to determine the composition and proportion of glycerides from different sources, thus contributing to the revelation of the internal mechanism affecting the quality equivalence of GMD.

OBJECTIVE To optimized the extraction process of Ganoderma lucidum crude polysaccharide using Ganoderma lucidum fruiting body as experimental material, and discuss its antioxidant activity. METHODS In this study, the extraction conditions of crude polysaccharide from Ganoderma lucidum were optimized by single factor experiment and response surface methodology. The polysaccharide was extracted by hot water extraction method, protein was removed by Sevag method, and purified by anion exchange column chromatography and gel column chromatography. Its structure was preliminarily determined by UV spectrum, FT-IR spectrum and atomic force microscope. The antioxidant activity of Ganoderma lucidum polysaccharide was evaluated by detecting DPPH radical, ABTS radical, hydroxyl radical and superoxide anion scavenging ability. RESULTS The results showed that the best extraction conditions of Ganoderma lucidum crude polysaccharide was extraction temperature 92.4 ℃, extraction time 5 h 5 min, liquid-solid ratio 21∶1, and the yield was 2.967±0.228%. Ganoderma lucidum polysaccharide obtained by extraction and purification conform to the structural characteristics of polysaccharide, and did not contain nucleic acids and proteins. Atomic force microscopy showed that most of them presented a chain like conformation. Ganoderma lucidum polysaccharide can significantly scavenge DPPH radical, ABTS radical, hydroxyl radical and superoxide anion. CONCLUSION Ganoderma lucidum polysaccharide has good antioxidant ability, this study provides a basis for further development and application of Ganoderma lucidum.

OBJECTIVE To study the accumulation pattern of effective components in Cistanche deserticola in different months, and to provide theoretical support for the harvesting, processing and quality control of Cistanche deserticola. METHODS Fifty-four batches of Cistanche deserticola samples were collected from April to December 2023, and a high performance liquid chromatography(HPLC) method was established for the simultaneous determination of six components in Cistanche deserticola, such as echinacoside, acteoside, tubuloside A, isoacteoside, poliumoside and geniposide, etc. The content of 54 batches of herbs were determined to analyze the trend of content changes. 54 batches of Cistanche deserticola herbs were identified and classified by chemometrics analysis, hierarchical cluster analysis (HCA), principal component analysis (PCA), and orthogonal partial least squares-discriminant analysis (OPLS-DA). RESULTS The established HPLC method was able to achieve a good separation of six components in the samples. The contents of the echinacoside, acteoside, tubuloside A, and isoacteoside, decreased gradually with the blooming of the herbs in April-August and increased in September-December while the contents of poliumoside decreased in the blooming stage of Cistanche deserticola. The content of geniposide reached its highest in the full bloom period; the content of the cyclic enol ether terpene constituent geniposide was higher in November-December than in the other periods. From the HCA and PCA analyses, it was found that 54 batches of Cistanche deserticola samples could be clearly differentiated into four groups(elongation period, full bloom period, fruit splitting period, and dormant period), among which all of them were clustered together in one group during the spring harvesting period, and the main feature was that the content of echinacoside was the highest; the samples of Cistanche deserticola samples were clustered together in one group during the autumn harvesting period, and the main feature was the lowest content of acteoside; most of the Cistanche deserticola samples at the full bloom stage were clustered into one group; all the samples at the dormant stage were clustered into one group, and the main characteristic was the highest content of geniposide.The results showed that the contents of the constituents in different growth stages of the herbs of Cistanche deserticola were significantly different. From the results of OPLS-DA analysis, it can be concluded that echinacoside and acteoside are the two components that contribute more to the different growth periods, which are the key components to distinguish the different growth periods, and can be used as the index components for evaluating the quality of Cistanche deserticola herbs, so as to further differentiate the different growth periods. CONCLUSION The HPLC method established in this study has good accuracy, and combined with chemometric analysis, it can be used to distinguish the differences in the quality of the herbs of Cistanche deserticola in different months, and provide a reference for its harvesting and quality control.

OBJECTIVE To investigate the current research status and trends of gut microbiota in diabetes both domestically and internationally, thereby providing a reference for advancing related studies. METHODS By systematically retrieving literature on diabetes and gut microbiota published in the Web of Science (WOS) and China National Knowledge Infrastructure (CNKI) databases from 2000 to 2024, visual analyses were performed using specialized tools to examine the number of publications, authors, institutions, journals, citation frequencies, and keywords. RESULTS A total of 1 514 Chinese and 2 780 English articles were retrieved. Since 2010, the number of published articles has shown an upward trend. Domestically and internationally, high-impact journals include the Chinese Journal of Microecology and Nutrients, respectively. High-frequency keywords revealed key areas such as metabolomics, insulin resistance, and bifidobacteria (Chinese), research hotspots including diabetic nephropathy, intestinal nitrogen homeostasis disorder, and probiotic supplementation (English). Future research will focus on specific strains, metabolites, and traditional Chinese medicine therapies. CONCLUSION Significant progress has been made in the study of gut microbiota and diabetes; however, disparities between domestic and international research remain. In the future, efforts should be directed toward deepening mechanistic research, enhancing interdisciplinary collaboration, and promoting clinical translation.

OBJECTIVE To evaluate the safety of intra-articular injection of α2-macroglobulin(α2-M) derived from human plasma Cohn fraction Ⅳ. METHODS α2-M was prepared from human plasma Cohn fraction Ⅳ and its purity and activity were tested. A rabbit red blood cell hemolysis test was conducted using an in vitro test tube method. The local irritancy was evaluated by injecting α2-M into the knee joint cavity of rats. The abnormal toxicity test was conducted by injecting α2-M into the abdominal cavity of mice and guinea pigs. RESULTS The purity of fraction Ⅳ derived α2-M was 95%, with an activity of (6.835±0.104) μg·mL^-1. In the in vitro hemolysis test, no hemolysis or aggregation was observed macroscopically, but slight hemolysis was detected by spectrophotometry. The irritancy test showed no obvious abnormalities at the injection site of the rats after administration, with good general condition, appearance, behavior, and spirit, and no abnormal changes in routine blood and blood biochemistry indicators except for PCT and Bas. In the abnormal toxicity test, no abnormal reactions were observed within 30 min after intraperitoneal injection of the test article in mice and guinea pigs, and the average body weight of mice and guinea pigs increased by the end of the observation period. CONCLUSION α2-M derived from human plasma Cohn fraction Ⅳ is considered to be safe in vivo.