31 January 2025, Volume 45 Issue 1
    

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    Special Column for Quality Research and Evaluation of Stem Cell Products
  • YANG Ying, RAO Chun-ming
    Chinese Journal of Pharmaceutical Analysis. 2025, 45(1): 4-11. https://doi.org/10.16155/j.0254-1793.2024-1299
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    Stem cells have varying degrees of proliferation, self-renewal, and differentiation potential, and can be applied in regenerative medicine and the treatment of various diseases. To ensure safety and effectiveness of stem cell products, it is important to establish quality control methods and standards. Herein, we review the regulations and guidelines for stem cell products, and provide an overview of the detection assays on the basic biological characteristics, microbiological safety, biological safety, biological effectiveness and other conventional testing methods and quality research methods for stem cells. We further describe the quality research methods for genetically modified stem cell products, functional cells derived from stem cells, and extracellular vesicles, etc.
  • WANG Yao, FANG Ji-qing, YUAN Zi-wei, LI Yao-ling, YANG Ying, RAO Chun-ming
    Chinese Journal of Pharmaceutical Analysis. 2025, 45(1): 12-19. https://doi.org/10.16155/j.0254-1793.2024-1325
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    Objective: To explore the biological function methods of mesenchymal stem cells(MSCs) for quality analysis. Methods: The surface markers of MSCs were detected by flow cytometry. MSCs osteogenic differentiation was induced by ascorbic acid and β-glycerophosphate sodium, etc., followed by Alizarin Red S staining. MSCs adipogenic differentiation was induced by IBMX, Rosiglitazone, etc., followed by Oil Red O staining. MSCs could differentiate into chondrocytes with treatment of ITS and TGFβ3, etc., followed by Alcian Blue staining. Cell co-culture of THP-1-macrophage with MSCs and ELISA assay were applied to detect the effects of MSCs on macrophage polarization. The expression levels of IL-10 and TNF α in the cell co-culture supernatant were detected by ELISA. To observe the effects of MSCs on lymphocyte proliferation, MSCs cultured with PBMCs, which were labeled with CFSE and activated by CD3/CD28, followed by flow cytometry. Results: The expression of MSCs surface markers, CD105, CD73, and CD90, was more than 95% respectively, while the expression of CD45, CD34, CD14, CD19, and HLA-DR expression was less than 2%. MSCs osteogenic differentiation assay showed red calcium nodules. Red lipid vacuoles were observed in MSCs adipogenic induction differentiation. Furthermore, MSCs have the differentiation potential to chondrocyte spheroids, and typical cartilage pits were observed. Co-culture of MSCs with THP-1 macrophages, an increase in IL-10 expression and downregulate TNFα secretion were observed. MSCs played inhibitory effects on the proliferation of PBMC activated by CD3/CD28, with an inhibition rate of 76.4%. Conclusions: This study established some of biological activity detection methods for MSCs, including MSCs surface markers, differentiation abilities, promotion of macrophage polarization, and inhibitory effects on lymphocyte proliferation. It provides a potential application for MSCs products quality control.
  • CHEN Xiao-fei, LI Hui-ting, DONG Ying-ying, CAO Yi-dan, FU Xin-yue, LIU Ming-yue, ZHANG Rui-rui, WANG Yan-hui, WANG Xin-yue, CUI Meng-shan, ZHANG Tong, PANG Lin, RAO Chun-ming
    Chinese Journal of Pharmaceutical Analysis. 2025, 45(1): 20-29. https://doi.org/10.16155/j.0254-1793.2024-1333
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    Objective: To establish a simple and efficient method for detecting telomerase activity for evaluating tumorigenic risk of cell products. Methods: Specific primers and probes were designed for the conserved domain of telomerase catalytic subunit (TERT), and the primers and probes of TERT gene were optimized and screened, and the primers and probes of internal reference genes were set up. Multiple quantitative PCR reaction was performed using two-color fluorescent probes in a reaction system, and RT-qPCR probe method was established. The expression of TERT gene in human mesenchymal stem cells HMSC was used to determine whether telomerase activity existed in the cells indirectly. Results: TERT gene of 293T/17 positive control cells could be stably and specifically detected by this method, with a mean Ct value of 23.96 and a RSD of 1.5. The internal reference gene GAPDH could be detected successfully, the mean Ct value was 14.13, the RSD was 1.3%. The reference gene GAPDH in MRC-5 could be detected in negative control cells, the mean Ct value was 12.81, the RSD was 0.46%, the TERT gene was not detected, and the telomerase activity was negative. The reference gene GAPDH in HMSC could be detected, the Ct mean value was 13.01, and the RSD was 3.8%, whereas, the telomerase activity of HMSC was negative. Conclusion: The real-time fluorescent quantitative RT-qPCR probe method established in this study can accurately detect the expression of catalytic subunit mRNA in telomerase positive cells with good repeatability and high specificity. It can be used to analyze telomerase activity of stem cells and indirectly evaluate tumorigenic risk of cell products derived from stem cells.
  • ZHANG Tong, CHEN Xiao-fei, DONG Ying-ying, CAO Yi-dan, WANG Yan-hui, LI Hui-ting, LIU Ming-yue, WANG Xin-yue, CUI Meng-shan, FU Xin-yue, ZHANG Rui-rui, PANG Lin, RAO Chun-ming
    Chinese Journal of Pharmaceutical Analysis. 2025, 45(1): 30-38. https://doi.org/10.16155/j.0254-1793.2024-1301
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    Objective: To employ adeno-associated viruses as vectors to design and prepare pseudoviruses comprising multiple DNA viral gene fragments. These were used as positive controls for the detection of human viruses in the field of molecular testing, thereby compensating for the absence of wild-type viral controls in viral molecular testing methods. Methods: The adeno-associated viral vectors served as the backbone of the pseudoviruses, with a total of four target gene fragments from three viruses incorporated: human papillomavirus type 16 (HPV16), type 18 (including HPV18-1 and HPV18-2) and human polyomavirus (HPyV) were, respectively, inserted into one viral vector, and the adeno-associated viral pseudoviruses were packaged by multiple plasmid cotransfection. The infectious activity of the pseudoviruses was confirmed by a cell infection assay, and a quantitative analysis of the pseudoviruses genome was conducted using fluorescence quantitative PCR. Results: The titers of HPV16, HPV18-1, HPV18-2, and HPyV genomes in the pseudoviruses were 7.77×108 copies · mL-1, 6.77×108 copies · mL-1, 7.04×108 copies · mL-1, and 1.24×109copies · mL-1, respectively, as determined by fluorescence quantitative PCR. Following a 48 h period of infection in 293T/17 cells, the viral gene sequences of HPV16, HPV18-1, HPV18-2, and HPyV were successfully identified using fluorescence quantitative PCR with the intracellular pseudoviruses. The copy numbers were 1.30×106 copies · mL-1, 6.59×105 copies · mL-1, 6.27×105 copies · mL-1, and 4.17×106 copies · mL-1. Following the infection of 293T/17 cells with the reporter gene pseudoviruses for a period of 48 hours, a distinct green fluorescence was evident under a fluorescence microscope, thereby confirming the infectious activity of the pseudoviruses. The pseudoviruses was employed as a positive control for the verification of applicability in human mesenchymal stem cells, and the recoveries of the four viral gene fragments by fluorescence quantitative PCR for nucleic acid extraction were 94.4%, 70.7%, 83.1% and 90.9%, respectively. Conclusion: The present study demonstrates that the adeno-associated virus packaging method can be employed to produce a multiviral gene pseudoviruses with infectious activity. The pseudoviruses can be employed as a positive control for the replacement of multiple wild-type viruses in the viral fluorescence quantitative polymerase chain reaction (PCR) of stem cell samples.
  • Review & Monography
  • SI Zi-hao, KIM Mooseob, PAN Li, ZHANG Peng-sen, GU Li-hua, WU Li-hong, YANG Li, WANG Zheng-tao
    Chinese Journal of Pharmaceutical Analysis. 2025, 45(1): 39-50. https://doi.org/10.16155/j.0254-1793.2024-0471
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    Thin layer chromatography (TLC) has been widely used for the separation and analysis of various constituents across multiple fields, owing to its advantages of cost-effectiveness, flexibility, high-throughput, and intuitiveness. Among the factors influencing TLC results, the choice of stationary phase critical. Currently, the main stationary phases used in TLC for identifying Chinese materia medica and Chinese patent medicines recorded in Chinese Pharmcopoeia, are silica gel, polyamide, and cellulose. This article reviews the characteristics and application of these three stationary phases in the analysis of Chinese herbal medicines. Using Chrysanthemi Indici Flos, Descurainiae Semen and Lepidii Semen, Oroxyli Semen, Cistanches Herba, Typhae Pollen, and Catechu as examples where traditional method are suboptimal, silica gel plates were employed to develop TLC identification methods with well-defined and abundant spots. These results highlight the clear advantages and feasibility of silica gel as a stationary phase in analyzing flavonoids and phenolic acid components. The findings offer alternative approaches to improve the national standards of these traditional Chinese medicines. Additionally, the article discusses the future development trend of TLC technology.
  • HUANG Ying, LIANG Cheng-gang
    Chinese Journal of Pharmaceutical Analysis. 2025, 45(1): 51-58. https://doi.org/10.16155/j.0254-1793.2024-0197
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    Human chorinonic gonadotropin (hCG) is an important regulator of reproductive and metabolic processes in the human body. hCG can be used for assisted reproduction and treatment of infertility. It is also used to treat sexual dysfunctions such as impotence, cryptorchidism, and dwarfism. hCG plays an irreplaceable role as an endocrine system drug. This article briefly describes the applications, molecular structure, receptor structure, signaling pathways and bioactivity assays of hCG. In addition, an outlook on the development of hCG drugs and bioactivity detection menthods is presented.
  • Ingredient Analysis
  • LIU Xu-xia, LIU Xiao-ling, MA Hai-tang, WANG Xin, CHEN Zheng-jun, LUO Wen-rong, YANG Fu-de
    Chinese Journal of Pharmaceutical Analysis. 2025, 45(1): 59-71. https://doi.org/10.16155/j.0254-1793.2024-0372
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    Objective: To establish a new method for in situ visualization of spatial distribution characteristics of various secondary metabolites in the root of Wendang (Codonopsis pilosula Nannf. var. modesta (Nannf.) L. T. Shen), so as to realize the tissular localization of secondary metabolites, and to provide a reference for the in-depth excavation of the Wendang. Methods: Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) was used for the mass spectrometry imaging analysis of the root metabolites in Wendang. The matrix was DHB+/DHB- (30 mg · mL-1), the substrate flow rate was 20 μL · min-1, the nitrogen flow rate was 5 L · min-1, the nozzle movement speed was 3 mm · s-1, the nozzle temperature was 60 ℃, and the spraying time of each slice was 50 min. The laser energy intensity in positive ion detection mode was 60%, and that in negative ion detection mode was 40%. The detection mass range was m/z 70-1 050, the mass resolution was 70 000, the spatial resolution was 50 μm, and the pixel size was 420 px×200 px. At the same time, the pathway enrichment analysis was also carried out for the metabolites. Results: A total of 214 metabolites were detected, and the spatial distribution characteristics of 40 representative metabolites were visualized. The distribution patterns of different kinds of secondary metabolites in the cork - phloem - xylem varied considerably, with flavonoids mainly distributed in xylem, alkaloids, phenolics, and carboxylic acids mainly in the cork and phloem, phenylpropanoids and quinones mainly in the cork, amino acids were abundant in the phloem, and nucleotides were distributed throughout the root tissues, the indicator components atractylenolide Ⅲ and syringoside were distributed in the cork, and lobetyolin was distributed in both the cork and the xylem. Pathway enrichment analysis showed that metabolites were significantly enriched in metabolic pathways, biosynthesis of secondary metabolites, flavonoid biosynthesis, biosynthesis of amino acids, and carbon metabolism pathways. Conclusion: This study visualizes the spatial distribution characteristics of metabolites in the roots of Wendang. The results of this study can provide certain theoretical support for the quality control, the identification, the extraction and separation of active ingredients, and the metabolic pattern of Wendang.
  • GUO Xiao-han, DING Yi-ming, ZHANG Yu, YANG Jian-bo, KANG Shuai, JING Wen-guang, CHENG Xian-long, WEI Feng
    Chinese Journal of Pharmaceutical Analysis. 2025, 45(1): 72-80. https://doi.org/10.16155/j.0254-1793.2024-0323
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    Objective: To establish the ultra performance liquid chromatography (UPLC) characterization profile of Styrax, and to determine the contents of three components (cinnamic acid, cinnamyl cinnamate, 3-phenylpropyl cinnamate) in Styrax, the aim was to study the quality of commercially available sulforaphane of different origins and forms, and to provide a reference for the quality control of imported medicinal herbs Styrax. Methods: The UPLC method was performed on an Acquity UPLC BEH C18 (100 mm×2.1 mm, 1.7 µm) column with acetonitrile-1% acetic acid solution as the mobile phase in a gradient elution. The volume flow rate was 0.3 mL · min-1. The detection wavelength was 277 nm. and the temperature of the column was 35 ℃. Characteristic chromatograms of styrax were established, and the contents of three major components of Styrax from different sources were determined and the data were statistically analyzed by hierarchical cluster analysis (HCA) and principal component analysis (PCA) with ChemPattern software. Results: The results showed that the UPLC characteristic chromatograms of Styrax was established with 10 common peaks and 4 identified components. The linear relationship of 3 components in their respective ranges was good (r≥0. 999), and the average recoveries (n=6) were 101.8% (RSD=1.3%), 105.8% (RSD=1.2%), 99.2% (RSD=1.8%), respectively. The content of 3 components were 2.74%-3.69%, 28.21%-30.63%, 16.89%-20.98%, respectively. Conclusion: The method is simple, accurate and reproducible, and can provide an overall quality control basis for the quality standard of Styrax. Combined with the information of origin research, it has important reference significance for the expansion of the origin of Styrax, as well as the harmonization and improvement of the standards of Styrax in different countries.
  • CHEN Wen-long, HE Jian, TAO Qi-qi, CHEN Zhi-chao, ZHOU Ling-jia, CHEN Zhi-xu, JIANG Zheng-jin, ZHANG Ting-ting
    Chinese Journal of Pharmaceutical Analysis. 2025, 45(1): 81-91. https://doi.org/10.16155/j.0254-1793.2024-0404
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    Objective: To study the bioactive components of the hypoglycemic effect of the traditional Chinese medicine Polygoni Cuspidate Rhizoma et Radix through its ability to inhibit α-glucosidase activity. Methods: Four modules: high-performance liquid chromatography system, the nanofraction collector, the channel of bioassay and high-resolution mass spectrometry were integrated to build a high-throughput bioassay profiling analysis platform. The preparative liquid chromatography was introduced to apply segmented enrichment to the extract of Polygoni Cuspidate Rhizoma et Radix, and the rapid screening of α-glucosidase inhibitors from Polygoni Cuspidate Rhizoma et Radix was realized by optimizing the chromatographic separation conditions, microfluidic fractionation collection parameters and bioassaying of enzyme activity in multi-well plates. Results: 28 α-glucosidase inhibitors were identified from Polygoni Cuspidate Rhizoma et Radix. The inhibitory activities of catechin-3-O-galloyl and procyanidin B2-3''-O-gallate were determined with the IC50 values of (9.49±1.93) μmol · L-1 and (69.94±8.14) μmol · L-1, respectively. Conclusion: This study has enabled high-throughput and high-resolution screening of α-glucosidase inhibitors in traditional Chinese medicine, providing a valuable tool for elucidating the active components and mechanisms of Polygoni Cuspidate Rhizoma et Radix to lower hyperglycemia.
  • Metabolism Analysis
  • LI Sai, LI Shou-lin, WANG Dong-liang, SU Ling-ying
    Chinese Journal of Pharmaceutical Analysis. 2025, 45(1): 92-98. https://doi.org/10.16155/j.0254-1793.2024-0300
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    Objective: To establish an HPLC method for simultaneous determination of vancomycin, caffeine and aminophylline in serum of children patients and apply it to clinical detection. Methods: 6% perchloric acid was used as protein precipitator and acetanilide was used as internal standard. The determination was performed on Nucifera C18 column (250 mm×4.6 mm, 5 μm) with mobile phase methanol-0.05 mol · L-1 potassium dihydrogen phosphate (18 ∶ 82, v/v). The detection wavelength was 236 nm, the flow rate was 1 mL · min-1, the column temperature was 25 ℃, and the sample size was 20 μL. Results: The serum concentrations of vancomycin and aminophylline were in the range of 1.0-100 μg · mL-1, and the standard curves were Y=0.010 6X+0.006 9 (r=0.998 8), Y=0.012 7X+0.008 4 (r=0.998 9), respectively. The serum concentration of caffeine had a good linear relationship in the range of 1.0-160 μg · mL-1, and the standard curve was Y=0.015 4X-0.010 1 (r=0.999 1). Quantitative downlines were all 1.0 μg · mL-1. The average recovery rates of the three compounds ranged from 97.8% to 103.1%, and the relative standard deviations of intraday and daytime precision were less than 10%, indicating good sample stability. Ten cases of children receiving vancomycin, aminophylline or caffeine were collected for method verification, which met the needs of clinical detection. Conclusion: The method is simple, rapid, accurate, and is suitable for the simultaneous determination of serum vancomycin, caffeine and aminophylline in children.
  • Bioassay
  • KANG Zhao-fei, WANG Ke-xin, HAN Chun-le, PANG Lin, RAO Chun-ming
    Chinese Journal of Pharmaceutical Analysis. 2025, 45(1): 99-103. https://doi.org/10.16155/j.0254-1793.2024-0285
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    Objective: To optimize an LC-Q TOF MS method for the determination of target proteins expressed in recombinant cells from a cell bank. Methods: The recombinant Chinese hamster ovary (CHO) cell line expressing antibodies was selected. The supernatant of recombinant CHO cell suspensions was retained after 12 500 r · min-1centrifuging for 10 min and treated with 50 mmol · L-1 NH4HCO3 solution. Chymotrypsin was added for enzymatic digestion, and the comparative analyses were carried out using two LC-Q TOF MS with Advance Bio Peptide (100 mm×2.1 mm, 2.7 μm) column, the Sciex TRIPLE TOF 5600+ and the Agilent Q TOF G6545, respectively. To establish the database, the amino acid sequences of the characteristic peptides of the protein heavy and light chains were entered into SCIEX BioPharmaView Version 3.0 and Agilent MassHunter BioConfirm 12.0 software, respectively. The peptides detected in the samples were searched to identify the target peptides. Results: The samples were analysed by a Sciex TRIPLE TOF 5600+ instrument to obtain 8 characteristic peptides with 100% amino acid sequence coverage and the samples were analysed by an Agilent Q TOF G6545 instrument to obtain 12 characteristic peptides with 100% amino acid sequence coverage. Conclusion: The method established in this study is simple to operate, has high coverage and good stability, can be used to identify the expression of target proteins in recombinant CHO cell lines. It also provides a reference for the expression identification of target proteins in similar cell lines.
  • YANG Li-hong, YUE Guang-zhi, XU Hong-shan, LIU Xin-yu, YE Qiang
    Chinese Journal of Pharmaceutical Analysis. 2025, 45(1): 104-109. https://doi.org/10.16155/j.0254-1793.2024-0466
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    Objective: To confirm the validity of solvent/detergent (S/D) treatment and dry heating for inactivation of virus in C1 esterase inhibitor (C1-INH). Methods: The Sindbis virus in samples with S/D was inactivated by the S/D method, and the virus titer before and after inactivation was detected using the plaque assay. Dry heating method was used to inactivate encephalomyocarditis virus (EMCV) and porcine parvovirus (PPV), and cytopathic assay was used to detect the virus titer before and after inactivation. Results: After inactivation by the S/D method, the reductions of Sindbis virus in three batches of smaples with S/D were > 4.35 lgPFU · mL-1, > 4.51 lgPFU · mL-1, > 4.64 lgPFU · mL-1, respectively. After dry heating, the reductions of EMCV in three batches of samples without S/D were ≥5.38 lgTCID50/0.1 mL、≥5.12 lgTCID50/0.1 mL、≥5.25 lgTCID50/0.1 mL, respectively, and the reductions of PPV in three batches of samples without S/D were 4.57 lgTCID50/0.1 mL、4.18 lgTCID50/0.1 mL、4.68 lgTCID50/0.1 mL, respectively. Conclusion: With evaluation on the inactivation capability of the indicator viruses, it is proved that the S/D treatment and dry heating has good inactivation and removal effect on the virus in C1-INH.
  • ZHANG Ting, DING Rui, WANG Jing, ZHOU Chang-ming
    Chinese Journal of Pharmaceutical Analysis. 2025, 45(1): 110-115. https://doi.org/10.16155/j.0254-1793.2024-0426
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    Objective: To establish a atomic absorption spectroscopy (AAS) method for detecting calcium content in recombinant human coagulation factor Ⅷ for injection, and evaluate its uncertainty. Methods: A calcium hollow cathode lamp light source was used. Air acetylene flame atomizer. Detection wavelength 422.7 nm. 8.9% lanthanum oxide solution was used as an ionization inhibitor. Conduct inspections on specificity, accuracy, precision, quantification limit, linearity and range, stability, etc. in accordance with the validation guidelines for drug quality standard analysis methods in Part 9101 of the 2020 edition of the Chinese Pharmacopoeia. The established method was applyed to detect the content of 6 batches of samples. Results: There was a good linear relationship between calcium element and absorbance in the concentration range of 0.0-5.0 μg · mL-1, with r=0.999 8. The detection line was 0.071 6 μg · mL-1; The quantification limit was 0.216 9 μg · mL-1. The recovery rate of the spiked sample was 100.8% to 103. 2%. The repeatability RSD was 0.64%, and the solution was stable within 8 hours. The content determination results of 6 batches of samples all complied with the regulations. The uncertainty assessment result was (117.12±4.82) μg · mL-1, k=2. Conclusion: AAS method is established for the detection of calcium content in recombinant human coagulation factor Ⅷ for injection.This method has the advantages of high sensitivity, fast analysis speed, good repeatability, high accuracy, and cost savings. At the same time, the uncertainty of this method is evaluated, which can provide quantitative evaluation indicators for the quality control of this variety.
  • Safety Monitoring
  • FAN Yi-lei, ZHAO Sen, YANG Yi-xuan, KE Xing, XU Yu, CHEN Xian-xin
    Chinese Journal of Pharmaceutical Analysis. 2025, 45(1): 116-124. https://doi.org/10.16155/j.0254-1793.2024-0289
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    Objective: To explore the fragmentation patterns of etomidate analogues by mass spectrometry to provide reference for the identification and structural analysis of these substances. Methods: Etomidate, metomidate, propaxate and isopropaxate were analyzed by gas chromatography-quadrupole time-of-flight mass spectrometry (GC-Q TOF MS) and liquid chromatography high-resolution mass spectrometry (LC-Q Orbitrap MS) under EI and ESI positive ionization modes. And the EI-MS and ESI-MS/MS fragmentation patterns of etomidate analogues were deduced. Results: Etomidate and its analogues had similar fragmentation pathways. In EI-MS mode, the fragmentation pathway was mainly focused on the breaking of carbon-nitrogen bonds, and the specific fragment ions (m/z 105 and m/z 77) were formed. In ESI-MS/MS positive ion mode, the fragmentation pathway was mainly focused on the fracture of carbon-nitrogen bond, resulting in the formation of the characteristic fragment ion with styrene as neutral loss, Subsequently, the neutral fragments R1 and H2O were lost in turn, and the specific fragment ions (m/z 113 and m/z 95) was formed. Moreover, the neutral loss of R1 in ESI-MS/MS positive ion mode could more intuitively indicate the substituents on the parent nucleus and further determine the structure of etomidate and its analogues. Conclusion: The fragmentation patterns of etomidate and its analogues are helpful to analyze and infer the structure of these substances, and provide reference for the identification and structural analysis of these substances in forensic laboratory.
  • DING Ying, WEN Hong-liang, LE Jian, LIU Hao, LIAN Xiang-jin
    Chinese Journal of Pharmaceutical Analysis. 2025, 45(1): 125-134. https://doi.org/10.16155/j.0254-1793.2024-0403
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    Objective: To establish a method to determination of the related substances and polymer impurities in cefminox sodium for injection. Method: Cefminox sodium was degraded in high temperature to prepare degradation solution. An RP-HPLC method for the related substances analysis was established with a Kromisil C18 column (250 mm×4.6 mm, 5 μm), using 10 mmol · L-1 phosphate buffer solution (pH 2.0)-acetonitrile (98∶2) (A)-acetonitrile (B) with gradient elution at a flow rate of 1.0 mL · min-1. The column temperature was maintained at 25 ℃, the detection wavelength was set at 254 nm, and the injection volume was 20 μL. The specificity of RP-HPLC method and identification of unknown impurities was researched by 2D HPLC-MS/MS. Results: 14 main impurities were characterized in the degradation solution, including 3 cefminox dimmers and isomers which were characterized firstly. The impurities were determined in 7 batches of samples by principal component self-control with correction factor, the contents of impurity 3 were 0.01%-0.13%, the contents of impurity 4 were 0.10%-0.16%, the contents of impurity 5 were 0.02%-0.07%, the contents of impurity 6 were 0.04%-0.07%, the contents of polymer impurities were 0.03%-0.06%, the maximum single impurity contents were 0.01%-0.03%, while the total impurity contents were 0.28%-0.63%. Conclusion: Cefminox degradation solution in high temperature can be used to identify related impurities and polymer peaks as the systematic suitability testing solution. The RP-HPLC method was suitable for related substances as well as polymer impurities in cefminox sodium for injection. This work provides useful information for the quality control of cefminox sodium, which can contribute to establishment of reasonable impurity limits.
  • HAO Jun-ju, CHEN Li, NING Man-ru, LU Yu-ting, SONG Min, HANG Tai-jun
    Chinese Journal of Pharmaceutical Analysis. 2025, 45(1): 135-145. https://doi.org/10.16155/j.0254-1793.2024-0195
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    Objective: To identify the related substances of progesterone sustained-release vaginal gel by liquid chromatography mass spectrometry (LC-MS). Methods: The separation was carried out on YMC-Triart C18 (150 mm×4.6 mm, 3 μm) column by gradient elution using acetonitrile and water as the mobile phases. The accurate mass and elemental composition of the parent ions and their product ions of related substances were determined by electrospray-ionization quadrupole time-of-flight high resolution mass spectrometry (ESI Q TOF MS) at collision energies of 5-45 eV. The structures of the related substances were identified by spectral analysis. Results: Under the established analytical condition, progesterone and its related substances were adequately separated, and 18 major related substances were detected and identified by hyphenated techniques in progesterone sustained-release vaginal gel and their stressed samples. Among them, 7 were the impurities listed in the EP or previous reported, and the others were unknown related substances identified for the first time in this paper. The established LC-MS technique was effective for the separation and identification of the related substances of progesterone sustained-release vaginal gel. Conclusion: Progesterone is unstable under acid, alkaline, and high-temperature stress conditions, and prone to produce oxidation degradants. The research results can provide a reference for the quality control of progesterone sustained-release vaginal gel.
  • YIN Kang-ming, HAO Jun-ju, LU Yu-ting, SONG Min, HANG Tai-jun
    Chinese Journal of Pharmaceutical Analysis. 2025, 45(1): 146-155. https://doi.org/10.16155/j.0254-1793.2024-0269
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    Objective: To identify the related substances in desloratadine using two-dimensional liquid chromatography-mass spectrometry (2D LC-MS) technology. Methods: A Waters Symmetry C8 column (250 mm×4.6 mm, 5 μm) was employed. The mobile phase A consisted of a mixture containing 1% triethylamine and 10 mmol · L-1 potassium dihydrogen phosphate solution adjusted to pH 2.0 with phosphoric acid, acetonitrile, and methanol (80∶10∶10), while mobile phase B contained 1% triethylamine, 10 mmol · L-1 potassium dihydrogen phosphate solution (pH adjusted to 2.0 with phosphoric acid), acetonitrile, and tetrahydrofuran (30∶70∶5). A linear gradient elution was used for the first-dimension liquid chromatography separation of related substances in desloratadine. Individual separated components were captured through a multi-channel switching valve into retention vessels, then transferred to a Thermo BDS Hypersil C18 column (100 mm×4.6 mm, 3 μm). After a second-dimension gradient elution with a mobile phase composed of 10 mmol · L-1 ammonium acetate solution and methanol to achieve rapid desalting, electrospray ionization positive ionization-quadrupole-time-of-flight high-resolution mass spectrometry was employed to measure the accurate masses and elemental compositions of parent ions and their daughter ions of the related substances. Structural identification was achieved through spectral analysis. Results: Under the established analytical conditions, desloratadine and its related substances were well separated. A total of 12 related substances were detected and identified in desloratadine and its stressed degradation samples. Among these 12 degradants, three were known impurities, while the others are newly identified impurities. Conclusion: 2D LC-MS can effectively provide specific identification of desloratadine’s related substances under non-volatile mobile phase separation conditions. The findings from this study serve as a reference for controlling the quality of desloratadine.
  • SONG Jin-hong, SUN Guo-xiang
    Chinese Journal of Pharmaceutical Analysis. 2025, 45(1): 156-163. https://doi.org/10.16155/j.0254-1793.2021-0040
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    Objective: To establish an in vitro permeation method for crisaborole ointment and to provide a reference for the bioequivalence of crisaborole ointment. Methods: The HPLC method was used to determine the permeability of crisaborole ointment, and the vertical Franz diffusion pool method was used to study the release characteristics of the ointment in vitro. The chromatographic column was Thermo BDS Hypersil-C18(150 mm×4.6 mm, 5 μm), with 0.1% phosphoric acid solution-acetonitrile (60∶40) as the mobile phase, flow rate of 1.5 mL · min-1, column temperature of 30 ℃, detection wavelength of 254 nm, and injection volume of 10 μL. Results: The peak shape of crisaborole in the chromatogram of the test sample was good. There was no peak at the position of crisaborole in the negative sample chromatogram, which did not interfere with the determination. The linear relationship of crisaborole was good within the range of 0.026-21.02 μg · mL-1 (r≥0.999). In six repeated experiments, the mean maximum transmittance (Jmax) of the self-made product and the reference formulation were compared (odds ratio: 0.94). Simultaneously calculate the confidence interval for Jmax, with a 90% confidence interval ranging from 87.9% to 102.0%. The mass conservation percentage of each sample was 94.1%-101.1% (n=12). The reference solution was left at room temperature for 26 h, with a peak area RSD of 0.44%. The self-made product and reference preparation were left in a water bath at room temperature and 32 ℃ for 26 h, with peak area RSD ranging from 0.78% to 1.1%, indicating good stability of the solution. For the three batches of samples tested, the ratio of the mean Jmax values of the self-made product and the reference formulation was within the range of 0.92-0.97. Simultaneously calculate the confidence interval for Jmax, with a 90% confidence interval ranging from 84.8%-100.7%. Conclusion: This method is simple and convenient to operate, with high selectivity, strong exclusivity and high accuracy.
  • ZHANG Chun-li, ZHOU Xiao-hua, SHI Hai-wei, YUAN Yao-zuo, WANG Ya, TANG Hui, WANG Bao-cheng
    Chinese Journal of Pharmaceutical Analysis. 2025, 45(1): 164-174. https://doi.org/10.16155/j.0254-1793.2024-0252
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    Objective: To identify the structure and determine the source of unknown impurities present at levels greater than 0.1% in representative pharmaceutical excipient benzalkonium chloride samples, in accordance with ICH Q3A guidelines. Methods: A new LC-MS method suitable for the structure prediction of related substances of benzalkonium chloride was established. The separation was performed on an Acclaim 120 C18 column (250 mm×4.6 mm, 5 μm) using a mobile phase consisting of methanol (mobile phase A) and 20 mmol · L-1 ammonium formate aqueous solution (pH 3.5, mobile phase B) with gradient elution at a flow rate was 1 mL · min-1. The post-column split was set to 1 ∶ 3. MS data were collected in positive ion mode using an electrospray ionization (ESI) source. The structures of unknown impurities were inferred using a “Diagnostic fragment ion extension strategy” and confirmed by comparing the chromatographic and mass spectrometric behaviors of the impurities with those of reference substances. Additionally, the source attribution and genotoxicity prediction of the detected impurities were performed. Results: A total of five unknown impurities were detected in a representative sample of benzalkonium chloride by the newly established LC-MS method, and their structures were inferred. The structures of four impurities were confirmed using two commercially available reference substances and two reference substances synthesized in a directionally oriented manner. Impurity D was identified as N-benzyl-N,N-dimethyl-1-phenylmethanaminium chloride. Impurity E as N-benzyl-N-methyl-1-phenylmethanamine. The impurity G as N-benzyl-N-methyldodecan-1-amine, and impurity H as N,N-dibenzyl-N-methyldodecane-1-ammonium chloride. Impurity F was hypothesized to be an isomer of the formula C15H17N. Predictions using Nexus 2.6.0 software indicated that all the above impurities fell into category 5 and had no genotoxic potential. Furthermore, the method successfully located four unknown impurities detected by the USP method in benzalkonium chloride related substances using impurity reference standards. Conclusion: This study systematically examines the structure and safety risks of potential impurities in benzalkonium chloride, providing valuable insights for enhancing quality control standards and pharmacopoeia criteria both domestically and internationally.
  • JIN Yun, ZHANG Xian-hua, WANG Jun, LI Jin-xia, WU Yao-li, SHEN Meng-jie, ZHAO Long-shan
    Chinese Journal of Pharmaceutical Analysis. 2025, 45(1): 175-180. https://doi.org/10.16155/j.0254-1793.2024-0155
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    Objective: To establish an HPLC-MS method for determination of three diastereoisomers in nirmatrelvir API. Methods: The analytical column was an InfinityLab Poroshell SB-C18 (150 mm×3.0 mm,2.7 μm), the two columns connected in series in series. The mobile phase was buffer (0.1% formic acid) (A) -acetonitrile (B), the diluent was water-acetonitrile-methanol (61 ∶ 19.5 ∶ 19.5). The whole run carried out by gradient elution. The column temperature was 80 ℃,the injection volume was 2 μL, and the sample temperature was 5 ℃. Results: Nirmatrelvir was separated completely from three diastereoisomers (the resolution>1.5). The test solution was stable for at least 3 d. The LOQ of diastereoisomer 1 was 0.04%, the LOQ of diastereoisomer 2 was 0.04%, the LOQ of diastereoisomer 3 was 0.05%. The linear correlation coefficient of diastereoisomer 3 was >0.99. The linear range was LOQ-150% of specification. The average recovery (n=9) of diastereoisomer 3 was 97.2%, RSD = 3.1%. The repeatability and intermediate precision completely met the requirements. The three diastereoisomers contents in three batches of nirmatrelvir API 24 months long-term stability test were all completely met the requirements, respectively. Conclusion: This method is simple, rapid, sensitive and specific to be used for the determination of three diastereoisomers in nirmatrelvir API.