31 October 2025, Volume 45 Issue 10
    

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    Special Column for Quality Research and Evaluation of Stem Cell Products (Continued)
  • Application of three molecular detection technologies in species identification of human stem cells and detection of cell cross-contamination*
    LIU Ming-yue, LI Hui-ting, FU Xin-yue, CAO Yi-dan, ZHANG Rui-rui, DONG Ying-ying, CHEN Xiao-fei, PANG Lin, RAO Chun-ming
    Chinese Journal of Pharmaceutical Analysis. 2025, 45(10): 1649-1659. https://doi.org/10.16155/j.0254-1793.2025-0166
    Abstract ( ) Download PDF ( )   Knowledge map   Save
    Objective: To explore the efficacy and practicality of the short tandem repeat (STR) method, the DNA barcoding method, and the species-specific polymerase chain reaction (PCR) method based on molecular techniques in the species identification of human stem cells and detection of cell line cross-contamination, for the quality control of corresponding cell products. Methods: The STR typing was used to identify human stem cells and detect cross-contamination by extracting the genomic DNA of samples and amplifying 20 STR loci and 1 sex-determining locus. The DNA barcoding method was used to identify the species of human stem cells and cross-contamination between species by amplifying the mitochondrial CO Ⅰ gene sequence and conducting sequence alignment analysis. The species-specific PCR method was used to identify the species of human stem cells and cross-contamination between species by specifically amplifying the conserved mitochondrial CO Ⅰ gene and cytochrome B gene. This study also compared the advantages and disadvantages of the three methods. Results: STR typing, DNA barcoding, and species-specific PCR, all based on molecular technologies, were able to accurately identify the species of human stem cells. When applied to the detection of human cell lines such as human umbilical cord-derived mesenchymal stem cells and human primary adipose-derived mesenchymal stem cells, the STR method was shown to generate distinct allele peaks and establish unique STR profiles. In terms of cell cross-contamination detection, the STR method was effective in detecting intra-species contamination among human stem cells, but could not detect inter-species contamination between human stem cells and other species, such as contamination with mouse cells or hamster cells. When used for detecting human cell lines including human umbilical cord-derived mesenchymal stem cells and human primary adipose-derived mesenchymal stem cells, the BOLD system identification engine enabled the identification of their corresponding species. In cell cross-contamination detection, the DNA barcoding method was able to detect contamination between human stem cells and cell lines of other species, but its sensitivity was contingent upon the species category. The detection sensitivity for cross-contamination between certain species was relatively low, and it could not detect intra-species contamination between human stem cells and other human cells. In the detection of human primary adipose-derived mesenchymal stem cells, the species-specific PCR method was found to yield the expected specific band size of 391 bp. In cell cross-contamination detection, the species-specific PCR method demonstrated efficiency in detecting contamination between human stem cells and cells of other species, exhibiting higher detection sensitivity compared to the DNA barcoding method. However, the range of detectable species was limited by the experimental throughput. Conclusion: All three methods can be used to identify the species of human stem cells. However, when it comes to detecting contamination within or between species of human stem cells, a comprehensive application of multiple detection methods should be considered. This approach ensures the identification of cross-species contamination between cell lines, providing an important guarantee for the quality and safety of human stem cells.
  • Establishment of the RT-qPCR method for HIV-1 virus detection in stem cell products*
    YUAN Zi-wei, WANG Yao, LI Yao-ling, FANG Ji-qing, YANG Ying, RAO Chun-ming
    Chinese Journal of Pharmaceutical Analysis. 2025, 45(10): 1660-1669. https://doi.org/10.16155/j.0254-1793.2025-0227
    Abstract ( ) Download PDF ( )   Knowledge map   Save
    Objective: To establish a reverse transcription real-time quantitative PCR (RT-qPCR) method using multiple primer pairs for HIV-1 detection, aiming at enhancing the quality control of viral safety in stem cell products. Methods: Using SnapGene software, multiple sets of template-specific primers and probes were designed targeting relatively conserved regions of the HIV-1 virus gag-pol gene to establish the RT-qPCR probe-based assay. The specificity, sensitivity, precision, and robustness of the method were validated. Results: The assay utilized six primer-probe sets, which demonstrated 83.0% coverage of HIV-1 variants. No detectable amplification curves were observed in human mesenchymal stem cells (hMSCs), and the spiked recovery rates ranged from 70% to 130%. A detection sensitivity of 2 copies · μL-1 was achieved. Repeatability was assessed by testing a plasmid standard at three concentrations (2×107 copies · μL-1, 2×105 copies · μL-1, and 2×103 copies · μL-1, respectively) in triplicate, with relative standard deviation (RSD) values ranging from 2.2% to 8.3%. Robustness was evaluated across different RT-qPCR platforms, yielding inter-system RSD values of 0.34%-2.3% for the same set of standards. Conclusion: The study develops an HIV-1 detection method that demonstrates superior specificity, excellent repeatability, and high sensitivity, and is validated for testing HIV-1 in biological products such as production-qualified cell substrates and stem cell products.
  • Study and application of the Adv vector PCV3-B19-TTSuV1 pseudovirus as positive control for PCR assays*
    LI Yao-ling, WANG Yao, FANG Ji-qing, YUAN Zi-wei, YANG Ying, RAO Chun-ming
    Chinese Journal of Pharmaceutical Analysis. 2025, 45(10): 1670-1679. https://doi.org/10.16155/j.0254-1793.2025-0025
    Abstract ( ) Download PDF ( )   Knowledge map   Save
    Objective: To evaluate the feasibility of polygenic pseudoviruses as positive controls in the detection of exogenous viruses by real-time quantitative PCR(qPCR) method and droplet digital PCR (ddPCR) method. Methods: Four gene segments (PCV3, B19-1, B19-2 and TTSuV1) of the Adv vector PCV3-B19-TTSuV1 pseudovirus (hereinafter referred to as PBT3) were amplified by qPCR and ddPCR, and the copy number was compared. The B19 gene copy number of PBT3 pseudovirus was measured by ddPCR. The PBT3 pseudovirus was subjected to 2-fold series dilution and 3 dilution factors were selected to evaluate the accuracy of gene copy number determined by ddPCR. The DNA copy number of pDC-PBT3 plasmid control was detected by ddPCR and further compared with the theoretical value calculated via the absorbance method. B19 virus in HMSCs was detected by qPCR, and the applicability of PBT3 pseudovirus as the positive control for PCR detection of exogenous viruses was evaluated. Results: The results of qPCR showed that the copy number of the four gene segments of PBT3 pseudovirus were (8.23±0.62)×105 copies · µL-1, (8.58±1.35)×105 copies · µL-1, (9.66±0.12)×105 copies · µL-1, and (8.17±0.36)×105 copies · µL-1, respectively, with the ratio of 1.00 ∶ 1.04 ∶ 1.17 ∶ 0.99, close to 1 ∶ 1 ∶ 1 ∶ 1. The copy number of the four gene segments of PBT3 pseudovirus detected by ddPCR was (4.62±0.23)×105 copies · µL-1, (4.84±0.01)×105 copies · µL-1, (4.92±0.04)×105 copies · µL-1, and (4.62±0.12)×105 copies · µL-1, respectively, with the ratio of 1.00 ∶ 1.05 ∶ 1.06 ∶ 1.00, close to 1 ∶ 1 ∶ 1 ∶ 1. The positive droplets ratio of diluted PBT3 pseudovirus B19 gene segments amplified by ddPCR was 4.00 ∶ 2.05 ∶ 1.16. The PBT3 pseudovirus gene copy number detected by ddPCR was 0.51-0.57 times of that detected by qPCR. The ratio of pDC-PBT3 DNA copy number measured by ddPCR to the theoretical copy number calculated via the classical absorbance method was between 0.47 and 0.75. Furthermore, the PBT3 pseudovirus was used as the positive control in the detection of two gene segments of B19 virus by qPCR in human mesenchymal stem cells and the recovery rates of B19-1 and B19-2 gene segments were 68.2% and 72.0%, respectively. Conclusion: The polygenic pseudovirus as the positive control of PCR assays for exogenous viruses provides a promising way with low costs, high safety, and controllable quality.
  • Establishment of a stem cell testing laboratory and quality control system*
    HAN Chun-le, ZHOU Jin, YANG Ying, CHEN Jia, LIAN Yi-bing, CAO Yi-dan, ZHANG Meng, PANG Lin, ZHAI Yong-zhao, RAO Chun-ming
    Chinese Journal of Pharmaceutical Analysis. 2025, 45(10): 1680-1689. https://doi.org/10.16155/j.0254-1793.2025-0161
    Abstract ( ) Download PDF ( )   Knowledge map   Save
    The establishment of a stem cell testing laboratory and its quality control system is a critical safeguard to ensure the safety and efficacy of stem cell products. Through scientific planning and efficient implementation, the laboratory can provide reliable technical support for stem cell research and production, guaranteeing the accuracy and traceability of experimental data as well as the safety and effectiveness of stem cell products. This article systematically reviews the construction of a stem cell testing laboratory from key aspects of site selection and layout, functional zoning, environmental control, safety protocols, and equipment configuration, while emphasizing the development of a quality control system. It serves as a reference for relevant enterprises and research institutions, promoting the clinical research and industrialization of stem cell products.
    • Review & Monography
  • Research advances in LC-MS/MS-based molecular networks in the characterization of chemical compounds*
    YANG Zhi-ming, HU Qian-nan, LI Xiang, ZHAN Zhi-lai, QIU Zi-dong, HUA Yu-tong, KANG Li-ping
    Chinese Journal of Pharmaceutical Analysis. 2025, 45(10): 1690-1699. https://doi.org/10.16155/j.0254-1793.2025-0083
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    Efficient characterization and identification of constituents in natural products are prerequisites for elucidating their pharmacodynamic material basis and mechanisms of action. In recent years, the molecular networking technology based on LC-MS/MS has been extensively applied in the qualitative research on natural products such as traditional Chinese medicine (TCM), microorganisms, marine organisms, fungi, and plants. This technology achieves more efficient discovery and identification of compounds than conventional methods, demonstrating great potential in rapid quantification. With molecular networking as the main thread, this article systematically reviews the literature of the past decade from both domestic and international sources. After briefly outlining the construction and development of molecular networking technology, it not only describes the application of a variety of molecular networking technologies but also introduces research progress in five areas: qualitative analysis, quantitative research, screening and identification of active ingredients, applications in metabolomics, and assistance in disease diagnosis. Furthermore, according to the current research status of molecular networking, the article proposes suggestions for future development.
    • Ingredient Analysis
  • Simultaneous determination of seven components in Bubai cream by UPLC-MS/MS*
    SUN Qi-yu, ZHANG Xiao-lin, WANG Qiang-li, LAI Mei-ling, TANG Ming-hui, YU Chen-hua, ZHANG Ping, WANG Ying
    Chinese Journal of Pharmaceutical Analysis. 2025, 45(10): 1700-1708. https://doi.org/10.16155/j.0254-1793.2025-0133
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    Objective: To establish an ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for the simultaneous determination of seven characteristic components, namely psoralenoside, isopsoralenoside, psoralen, isopsoralen, bakuchiol, imperatorin, and isointeratorin) in Bubai cream with carbamazepine and liquiritin as internal standards. Methods: The chromatographic separation was performed on an ACQUITY BEH C18 (150 mm×2.1 mm, 1.7 μm) column, with 0.05% acetic acid methanol as mobile phase A and a buffer solution consisting of 0.05% acetic acid and 1 mmol · L-1 ammonium acetate aqueous solution as mobile phase B for gradient elution. The mass spectrometry detection was carried out using an electrospray ionization source (ESI) and multiple reaction monitoring mode (MRM). Psoralen and liquiritin were detected in negative ionization mode, while other components were detected in positive ionization mode. Results: The linear relationships of the seven components were good within their respective concentration ranges (r≥0.993 5). The average spiked recovery rates were 96.2%-102.1%, with RSDs of 0.8%-4.4% (n=6). The contents of the 7 components in the Bubai cream range from 0.012 6 to 2.868 mg · g-1. The established method was specific, accurate and highly sensitive. The content of bakuchiol was the highest among the three batches of samples, followed by psoralenoside, isopsoralenoside and their aglycones, while the content of imperatorin and isoimperatorin was relatively low. Conclusion: The results can provide a basis for the quality control of Bubai cream.
  • Simultaneous determination of five components in Jiawei Xihuang pills by UFLC-MS/MS*
    YANG Yan, GONG Rui, WANG Xing, LI Yu-hua, YU Wen-jie, LIU Yang, LI Le-le, YU Chun-yu, GUAN Jiao, ZHU He-yun
    Chinese Journal of Pharmaceutical Analysis. 2025, 45(10): 1709-1716. https://doi.org/10.16155/j.0254-1793.2025-0241
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    Objective: To establish an ultra-fast liquid chromatography-tandem mass spectrometry (UFLC-MS/MS) method for simultaneous determination of 5 components (arenobufagin, bufotaline, bufalin, resibufogenin, and cinobufagin) in Jiawei Xihuang pills. Methods: UFLC-MS/MS was adopted, with Shimadzu Shim-Pack XR-ODS column (75 mm×3.0 mm, 2.2 μm) used for chromatographic separation and a mixture of 0.1% formic acid aqueous solution (A)-acetonitrile (B) as the mobile phase for gradient elution at a flow rate of 0.6 mL · min-1 and a column temperature of 35 ℃. Electrospray ionization source and positive ionization detection were used, and the multiple reactions monitoring mode was employed. Results: The mass concentrations of arenobufagin, bufotaline, bufalin, resibufogenin, and cinobufagin showed good linear relationships with the peak area in the ranges of 0.1-4 μg · mL-1 (r=0.999 3), 0.05-2 μg · mL-1 (r=0.998 3), 0.05-2 μg · mL-1 (r=0.998 7), 0.025-1 μg · mL-1 (r=0.999 6) and 0.1-4 μg · mL-1 (r=0.999 0), respectively. The average recovery rates (n=9) of the above-mentioned five components were 99.1%, 98.9%, 100.0%, 98.9%, and 99.7%, respectively. The content ranges of arenobufagin, bufotaline, bufalin, resibufogenin, and cinobufagin in 6 batches of Jiawei Xihuang pills samples were 19.28-23.58 μg · g-1, 3.31-4.04 μg · g-1, 3.64-4.30 μg · g-1, 1.58-1.93 μg · g-1, and 5.54-6.91 μg · g-1, respectively. Conclusion: The established method, being simple, rapid, and sensitive, can be used for the quality control of Jiawei Xihuang pills.
  • Quality evaluation of Mori Fructus from different areas of Xinjiang based on fingerprint and multi-component determination combined with chemometrics and entropy weight-TOPSIS method*
    TAN Mei-e, LUO Yong-dong, PAYIMAN · Haimity, YU Ning
    Chinese Journal of Pharmaceutical Analysis. 2025, 45(10): 1717-1729. https://doi.org/10.16155/j.0254-1793.2024-1211
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    Objective: To establish an HPLC fingerprint of Mori Fructus and a method for simultaneous determination of eight chemical components (protocatechuic acid, neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, cyanidin-3-O-glucoside, hyperoside, rutin, and isoquercitrin) and to evaluate the quality of Mori Fructus from different origins and batches in Xinjiang by combining chemometrics and entropy-weighted TOPSIS, thus providing a reference for the quality control of Mori Fructus. Methods: A SHIMADZU VP-ODS C18 column (250 mm×4.6 mm, 5 μm) was used for gradient elution with acetonitrile-0.2% phosphoric acid aqueous solution as the mobile phase at the flow rate of 1.0 mL · min-1. The detection wavelengths were set at 260 nm for protocatechuic acid, hyperoside, rutin, and isoquercitrin, 330 nm for neochlorogenic acid, chlorogenic acid, and cryptochlorogenic acid, and 520 nm for cyanidin-3-O-glucoside, and the column temperature was maintained at 30 ℃. The content of protocatechuic acid, cryptochlorogenic acid, chlorogenic acid, neochlorogenic acid, cyanidin-3-O-glucoside, hyperoside, rutin, and isoquercitrin in Mori Fructus samples from different origins and batches in Xinjiang was determined, and the HPLC fingerprints were established. Common peaks were identified by comparison with reference standards. Cluster analysis (CA), principal component analysis (PCA), orthogonal partial least squares-discriminant analysis (OPLS-DA), and entropy-weighted TOPSIS were employed to analyze the quality differences among 15 batches of Mori Fructus, and a comprehensive quality evaluation model was constructed. Results: The similarity of fingerprints for the 15 batches of Mori Fructus ranged from 0.962 to 0.998, with 20 common peaks marked and 8 chromatographic peaks identified. Chemometric analysis classified the samples into two categories. Eleven batches (S1-S3, S5, S7-S11, S14, and S15) were grouped into Class I, while 4 batches (S4, S6, S12, and S13) were classified as Class II. OPLS-DA screened out five major differential components. The content determination results showed that the content ranges of protocatechuic acid, neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, cyanidin-3-O-glucoside, hyperoside, rutin, and isoquercitrin were 0.033 5-0.240 2 mg · g-1, 0.293 1-0.443 4 mg · g-1, 0.128 1-0.393 7 mg · g-1, 0.322 8-0.580 0 mg · g-1, 1.831 9-5.476 3 mg · g-1, 0.007 0-0.024 8 mg · g-1, 0.528 1-1.211 0 mg · g-1, and 0.209 4-0.396 6 mg · g-1, respectively, indicating certain differences in chemical composition among different batches. The entropy-weighted TOPSIS method was used to evaluate and rank the quality of the 15 batches of Mori Fructus. Conclusion: The established fingerprint and multi-component content determination method, combined with chemometrics and entropy-weighted TOPSIS, is stable, feasible, accurate, and reproducible, and can be employed for the quality control of Mori Fructus.
  • Differential analysis of Spatholobi Caulis from different origins based on chemometrics combined with multi-component quantification*
    ZHANG Yu, GUO Xiao-han, WANG Xian-rui, WU Zhe, ZHANG Xin-yue, LI Ling-xi, CHENG Xian-long, WEI Feng
    Chinese Journal of Pharmaceutical Analysis. 2025, 45(10): 1730-1741. https://doi.org/10.16155/j.0254-1793.2025-0266
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    Objective: To compare the chemical composition between different origins of Spatholobi Caulis based on chemometrics, search for differential components for identification, and clarify the differences in the content of chemical components of Spatholobi Caulis from different origins through multi-component quantitative analysis, so as to provide a scientific basis for evaluating the quality of Spatholobi Caulis from different origins. Methods: Separation was performed by gradient elution through a Waters XTerra C18 (250 mm×4.6 mm, 5 μm) column with 0.1% formic acid aqueous solution (A) and acetonitrile (B) as the mobile phases at a flow rate of 1 mL · min-1, a detection wavelength of 260 nm, a column temperature of 25 ℃, and an injection volume of 25 μL. The fingerprints of Spatholobi Caulis were then established. Chemometrics was employed for difference analysis, and the content of multiple components in Spatholobi Caulis from different origins was determined simultaneously by high performance liquid chromatography (HPLC). Results: The similarity of the fingerprints of Spatholobi Caulis was in the range of 0.713-0.979, and seven components—epicatechin, catechin, formononetin, protocatechuic acid, genistein, calycosin, and daidzein—were identified. Both cluster analysis and principal component analysis indicated that Spatholobi Caulis samples from Guangxi and Yunnan of China and Vietnam were clustered into one group, which was clearly distinguished from those from Laos. On the basis of the OPLS-DA, the components such as daidzein with VIP>1.0 were the key components distinguishing the Spatholobi Caulis samples of Laos from those of other origins. The results of multi-component quantification showed that catechin and epicatechin presented high content, with the mass fraction ranges of 0.064 8-0.555 9 mg · g-1 and 0.198 8-0.894 5 mg · g-1, respectively. Conclusion: Chemometric and multi-component quantitative analyses provide a scientific basis for the quality control study and standardization of the evaluation system of Spatholobi Caulis. Follow-up studies can explore the standards with catechins and epicatechins as quality control markers and take daidzein and formononetin as the markers for distinguishing different origins of Spatholobi Caulis.
  • Absolute configurations of dexzopiclone and levozopiclone*
    ZHOU Xiao-li, ZHAO Chen, LI Li, YIN Li-hui
    Chinese Journal of Pharmaceutical Analysis. 2025, 45(10): 1742-1748. https://doi.org/10.16155/j.0254-1793.2025-0055
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    Objective: To study the absolute configurations of dexzopiclone and levzopiclone by electronic circular dichroism (ECD) and vibrational circular dichroism (VCD) spectroscopy. Methods: ECD spectra of dexzopiclone and levozopiclone were characterized by a JASCO-1500 circular dichroism spectrometer, with the wavelength range of 400-190 nm, the data pitch of 0.5 nm, the optical path length of 1 mm, the digital integration time of 1 s, the slit width of 1.0 nm, and the scanning speed of 100 nm · min-1. VCD spectra of dexzopiclone and levozopiclone were characterized by a FVS-6000 vibrational circular dichroism spectrometer, with the wavenumber range of 1 850-950 cm-1, the data pitch of 0.96 cm-1, the optical path length of 150 µm, and the number of accumulations of 5 000. Time-dependent density functional theory calculations were performed with Gaussian 16 software to predict theoretical ECD spectra, which were compared with experimental ECD spectra to determine the absolute configurations of this enantiomeric pair. Results: The ECD and VCD behaviors of dexzopiclone and levzopiclone were characterized. In their ECD and VCD spectra, the positions of Cotton effects were identical with the opposite sign and similar intensities, confirming their opposite absolute configurations. The absolute configurations of the dexzopiclone and levozopiclone reference standards were verified through quantum chemical calculations, with dexzopiclone exhibiting (S)-configuration and levozopiclone exhibiting (R)-configuration. Conclusion: The combination of ECD spectroscopy and quantum chemical calculations confirms the absolute configurations of dexzopiclone and levzopiclone, providing effective assistance in the confirmation of the absolute configurations of chiral compound reference standards during calibration.
  • Determination of sodium sulfite content in ketoconazole cream by liquid-liquid extraction and ion chromatography*
    DING De-min, XIN Chang-ying, XUN Yan-bin, YU Xin-ying, ZHAO Long-shan, WANG Chang-yu, YANG Li-hong
    Chinese Journal of Pharmaceutical Analysis. 2025, 45(10): 1749-1755. https://doi.org/10.16155/j.0254-1793.2024-1183
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    Objective: To establish an ion chromatography method for the determination of sodium sulfite in ketoconazole cream, and to evaluate the differences in the content of antioxidant sodium sulfite among ketoconazole cream products from different manufacturers. Methods: After dissolution in ethyl acetate, the samples were sequentially extracted with 0.1% acetaldehyde solution and n-hexane. Separation was carried out on a Dioneslon Pac AS11 anion-exchange column (250 mm×4 mm, 9 µm), using potassium hydroxide solution as the eluent under gradient condition. The flow rate was 1.0 mL · min-1, the injection volume was 25 μL, and the column temperature was 35 ℃. The detection was done with a suppressed conductivity detector. Results: A good linear relationship was observed for sodium sulfite in the concentration range of 0.001-0.04 mg · mL-1, with a correlation coefficient of 0.999 1. The average recovery rate from spiked samples (n=9) was 93.9%. The test solutions remained stable for up to 8 h under light-protected conditions. Among 85 batches of ketoconazole cream samples containing sodium sulfite as an antioxidant from 10 manufacturers, the sodium sulfite content ranged from 2% to 83% of the labeled formulation amount. In 14 batches, the content of sodium sulfite was below 40% of the labeled amount. Conclusion: This method can be used for the determination of the antioxidant sodium sulfite in ketoconazole cream and provides a reference for establishing the shelf life of ketoconazole cream products and analyzing the origins of related substances.
    • Bioassay
  • Development of a biological activity reference material for recombinant COVID-19 vaccine (CHO cell)*
    CAO Sha-sha, HE Peng, WANG Xian-xiang, LIU Xiao-ya, WEI Fen, LI Jing-jing, LIU Yu, WANG Ting-ting, WANG Chen-fei, WANG Jia-ji, HU Zhong-yu
    Chinese Journal of Pharmaceutical Analysis. 2025, 45(10): 1756-1764. https://doi.org/10.16155/j.0254-1793.2024-1237
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    Objective: To develop a biological activity reference material for recombinant COVID-19 vaccine (CHO cell) for the evaluation of in-vitro relative potency and in-vivo potency in mice for recombinant COVID-19 vaccine (CHO cell). Methods: One qualified bulk of recombinant COVID-19 vaccine (CHO cell) was selected. After calibration of protein content, an aluminum adjuvant was added in the bulk to prepare one batch of vaccine as the candidate biological activity reference material. After passing the finished product test, the candidate biological activity reference material was subjected to homogeneity tests. Five independent experiments using one batch of phase Ⅲ clinical vaccine as the biological activity reference material were conducted to calibrate the in-vitro relative potency and in-vivo potency in mice for candidate biological activity reference material. The stability of the candidate biological activity reference material was analyzed by storage at (37±2) ℃ and (5±3) ℃. The phase Ⅲ clinical batch vaccine and the candidate biological activity reference material were used to test the applicability of the candidate biological activity reference material by evaluation of the in-vitro relative potency and in-vivo potency of six batches of vaccines. Results: All test items of the biological activity reference material for the recombinant COVID-19 vaccine (CHO cell) complied with the requirements. The filling volume was not less than the labeled amount, with the filling precision within ±1%. The RSD values of D10, D50, and D90 reflecting particle size distribution were 0.45%, 0.39%, and 0.54%, respectively, and that of antigen content was 2.7%. The phase Ⅲ clinical vaccine, when being calibrated as a candidate reference material, demonstrated the in vitro relative potency and in-vivo potency in mice no less than 0.5, with the geometric mean values of 1.0 and 1.0 and the geometric coefficients of variation of 8.9% and 31%, respectively. After storage at (37±2) ℃ for 28 days and (5±3) ℃ for 36 months, the in-vitro relative potency and the in-vivo potency in mice showed no significant upward or downward trends, indicating good stability. The results of the in-vitro relative potency and in-vivo potency in mice tests for the six batches of vaccines using phase Ⅲ clinical vaccine and the candidate biological activity reference material ranged from 0.9 to 1.0 and 0.7 to 1.4, respectively, indicating former and candidate biological activity reference materials had good consistency. Conclusion: The biological activity reference material for recombinant COVID-19 vaccine (CHO cell) can be used for evaluation of the in-vitro relative potency and the in-vivo potency in mice for this vaccine.
  • Establishment of peptide mapping method for human papillomavirus type 16 L1 antigen and analysis of chromatographic characteristic peak identification*
    LI Tao, LIU Bing, TANG Mao-ling, HU Rong, WANG Meng, LIANG Hao-yu, XU Nan, ZOU Han-yan, HUANG Wei-jin, NIE Jian-hui
    Chinese Journal of Pharmaceutical Analysis. 2025, 45(10): 1765-1781. https://doi.org/10.16155/j.0254-1793.2025-0264
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    Objective: To establish and optimize a peptide mapping assay for human papillomavirus type 16 L1 antigen applicable to different expression systems, and to identify the peptide fragments of the characteristic peaks of the peptide mapping to ensure the applicability and scientificity in the quality control of HPV type 16 L1 antigen. Methods: A ZORBAX SB-C18 column (250 mm×4.6 mm, 5 µm) was employed with a gradient elution using 0.1% trifluoroacetic acid-water (A) and 0.1% trifluoroacetic acid-acetonitrile (B) as the mobile phase. The flow rate was set at 1.0 mL · min-1, the detection wavelength was performed at 214 nm, and the column temperature was maintained at 30 ℃. This method was used to screen for the optimal sample preparation approach for examining L1 antigen peptide profiles from different expression systems. Identification and analysis were performed using an electrospray ionization (ESI+) source in positive-ion mode using data-dependent scanning. The full MS scan range was m/z 150-2 000 at a resolution of 70 000 with an AGC target of 3.0×106, and MS/MS scans were acquired at a resolution of 17 500 with an AGC target of 1.0×105. These mass spectrometry parameters were used to identify characteristic peaks exhibiting differences and the characteristic peaks containing effective epitope peptides. Results: The optimal protease digestion volume for the human papillomavirus type 16 L1 antigen peptide map analysis method established in this study, applicable to different expression systems, was 400 μg. The protease-to-protein content ratio was 1 ∶ 50 with a 20-hour digestion at 37 ℃. In the repeatability evaluation, the RSDs of the retention times for the fourteen common characteristic peaks with the highest peak heights did not exceed 0.15%, and the cosine similarity of the peptide maps was not lower than 0.987. For inter-day precision, the maximum RSD of the retention times of common characteristic peaks among samples from four manufacturers was≤0.34%, with similarity values≥0.911. Mass-spectrometric analyses were performed to elucidate the effects of amino-acid sequence variations and characteristic peak differences on peptide distribution, and the positions of the characteristic peaks corresponding to the major antigenic epitope peptides of the L1 antigen were identified. Conclusion: This study established a method for analyzing peptide profiles of HPV type 16 L1 antigen derived from different expression systems. The peptide maps reveal the composition of differential characteristic peaks and precisely identify the peaks that contain peptides corresponding to critical antigenic epitopes of L1.
    • Quality Control
  • Research on the dynamic accumulation patterns of chemical components in Paeoniae Radix Alba harvested from Bozhou in different periods based on HPLC combined with chemometrics*
    RONG Yu, CHENG Lu, CHEN Xiao-tong, DENG Chen-yang, LUO Yun, JIN Chuan-shan, LIU Jun-ling, WANG Xiao-li
    Chinese Journal of Pharmaceutical Analysis. 2025, 45(10): 1782-1793. https://doi.org/10.16155/j.0254-1793.2025-0156
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    Objective: To investigate the dynamic accumulation patterns of chemical components in Paeoniae Radix Alba harvested from Bozhou in different periods, thereby determining the best harvesting time and providing reference for the reasonable harvesting of Paeoniae Radix Alba in the authentic producing area of Bozhou. Methods: The HPLC fingerprint of Paeoniae Radix Alba was established. Gradient elution was carried out through the Thermo C18 chromatographic column (250 mm×4.6 mm, 5 μm), with acetonitrile-0.1% phosphoric acid water as the mobile phase, the detection wavelength of 230 nm, the column temperature of 30 ℃, the flow rate of 1.0 mL · min-1, and the sample injection volume of 10 μL. The similarity of fingerprints for Paeoniae Radix Alba harvested in different periods was evaluated via the Similarity Evaluation System for Chromatograghic Fingerprint of TCM (version 2012). Chemometric software was employed to perform discriminant and correlation analyses. Results: The fingerprints of Paeoniae Radix Alba harvested from April to December had 7 common peaks, with a similarity range of 0.925-0.996. By comparison with reference standards, seven chromatographic peaks were identified as gallic acid, catechin, albiflorin, paeoniflorin, paeoniflorin gallate, 1,2,3,4,6-O-pentagalloyglucose, and benzoylpaeoniflorin, and the content of five of these components were determined. The content of gallic acid, catechin, albiflorin, paeoniflorin, and paeoniflorin gallate in Paeoniae Radix Alba harvested in different periods varied within the ranges of 0.620-1.074 mg · g-1, 0.637-1.253 mg · g-1, 4.144-9.896 mg · g-1, 26.480-39.191 mg · g-1, and 0.343-0.913 mg · g-1, respectively. The content showed large differences during different growth periods. The content of chemical components in Paeoniae Radix Alba decreased during the flowering period, while it gradually increased after the above-ground part began to wither, being higher and stable in September and October. The cluster analysis and principal component analysis classified Paeoniae Radix Alba samples harvested from April to December into four categories: April; May, November, and December; June, July, and August; and September and October. Conclusion: The best harvesting period is from September to October. A significantly positively correlation exists between albiflorin and catechin, as well as between paeoniflorin and paeoniflorin gallate, in different harvesting periods. Orthogonal partial least squares-discriminant analysis demonstrates that albiflorin and paeoniflorin can used as key marker components to evaluate the quality of Paeoniae Radix Alba harvested in different periods.
  • Adulteration analysis of Rubiae Radix et Rhizoma based on UHPLC-Q TOF MSE and machine learning algorithms*
    WANG Xian-rui, WU Zhe, HAN Ting, GUO Xiao-han, LI Ming-hua, JING Wen-guang, CHENG Xian-long, WEI Feng, AN Fu-dong
    Chinese Journal of Pharmaceutical Analysis. 2025, 45(10): 1794-1804. https://doi.org/10.16155/j.0254-1793.2024-1158
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    Objective: To realize the digital identification of the adulteration of Rubiae Radix et Rhizoma by combining UHPLC-Q TOF MSE with machine learning algorithms. Methods: Firstly, Rubiae Radix et Rhizoma, Rubiae Schumannianae Rhizoma, Rubiae Radix et Rhizoma Magna, Rubiae Yunnanensis Radix et Rhizoma, Rubiae Tibeticae Radix et Rhizoma, and adulterated samples of Rubiae Radix et Rhizoma were subjected to ultrasonic treatment at 500 W and 40 kHz for 0.5 h, and then separated by Waters ACQUITY UPLC BEH C18 (100 mm× 2.1 mm, 1.8 μm) column, with acetonitrile-0.1% formic acid water as the mobile phase for gradient elution. Electrospray positive ionization, Q TOF/MSE, and m/z 100-1 200 were used for scanning and digital quantization. Then, the features were filtered by the Fast Correlation-Based Filter (FCBF). Finally, the screened feature variables were combined with different machine learning algorithms to construct the identification models. The best model was selected based on parameters such as accuracy, precision, and recall, and the prediction results were validated by identification based on external features. Results: Through FCBF, 153 feature variables were screened out. Moreover, the identification model constructed by combining the support vector machine algorithm had the best identification performance, with the accuracy, precision, and recall not less than 0.990. The correct rate of the model identification results validated by identification based on external features was as high as 100%. Conclusion: The UHPLC-Q TOF MSE and support vector machine algorithms contribute to the digital identification of the adulteration of Rubiae Radix et Rhizoma, which has practical significance.
    • Safety Monitoring
  • Application of cyclic ion mobility mass spectrometry in the analysis of topological isomers of marketed peptide drugs*
    ZHU Shao-zhou, FAN Li-jiao, ZHANG Tuo, SUN Jia-bei, YAO Jing, HUANG Hai-wei
    Chinese Journal of Pharmaceutical Analysis. 2025, 45(10): 1805-1815. https://doi.org/10.16155/j.0254-1793.2024-1291
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    Objective: To explore the feasibility of using cyclic ion mobility mass spectrometry (cIM-MS) for the qualitative detection of topological isomers of marketed peptide drugs in the spatial dimension, and to develop quality control strategies for drug topological isomers. Methods: Methoddevelopment was carried out using the active pharmaceutical ingredients of three marketed peptide drugs: terlipressin, motixafortide, and tirzepatide. The three drugs were treated for 4 hours at 0, 50, and 80 ℃, respectively. The processed samples were analyzed by cIM-MS using a direct injection method, and the conformational changes of these drugs under different conditions were detected. Results: Under untreated conditions, topological isomers were observed in all three marketed drugs. In accelerated stability experiments, no significant conformational changes were observed for terlipressin under the three temperature conditions, which indicated that the topological structure of small-molecular-weight drugs was relatively stable. In contrast, significant conformational changes were observed for motixafortide under these conditions, and separable topological isomers were generated under high-temperature conditions. For the high-molecular-weight tirzepatide, topological isomers were observed at 0 ℃ and 50 ℃, whereas degradation was observed at 80 ℃. The cIM-MS was employed to precisely identify conformational changes, including folding and disassembly, in these three marketed drugs. Conclusion: cIM-MS provides precise analysis of the spatial conformations of complex, marketed peptide drug active pharmaceutical ingredients. It detects and separates subtle conformational changes in these complex drugs. The method operates simply and accurately, making it suitable for the conformational characterization and quality control of marketed complex peptide drugs.
  • Analysis of related substances in ivabradine hydrochloride by two-dimensional liquid chromatography-mass spectrometry techniques
    JIN Ya-wen, ZHU Qiong, ZHENG Zhong-hui, CHAO Rui-rui, LIAN Xun-yu, LI Fu-xiang, WANG Yi-yun, HANG Tai-jun, YIN Li-fang
    Chinese Journal of Pharmaceutical Analysis. 2025, 45(10): 1816-1825. https://doi.org/10.16155/j.0254-1793.2024-1319
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    Objective: To identify the structures of the related substances in both the raw material and the forced degradation samples of ivabradine hydrochloride by two-dimensional liquid chromatography quadrupole time-of-flight tandem mass spectrometry. Methods: The first-dimensional separation was performed on an ODS column (150 mm×4.6 mm, 5 μm) using gradient elution with a mobile phase composed of phosphate buffer solution (prepared by dissolving 4.54 g of potassium dihydrogen phosphate, dissolved in 1 000 mL of water, adjusted to pH 3.0 with 85% phosphoric acid) and acetonitrile. The separated related substances were automatically fractionated and injected into the second-dimensional gradient elution, which was conducted on another ODS (150 mm× 4.6 mm, 5 μm) column with 0.1% formic acid in water and methanol as the mobile phases. Each of the related substance was trapped in the second column with rapid removal of the residual non-volatile salts. Thereafter, each related substance was eluted using 0.1% formic acid in methanol and introduced into a quadrupole time-of-flight high resolution mass spectrometry using positive electrospray ionization. The accurate masses and elemental compositions of the parent and product ions of each related substance were determined. Finally, all the structures of the related substances were identified through MS elucidation. Results: Under the established two-dimensional liquid chromatography-mass spectrometry analysis conditions, ivabradine hydrochloride and its related substances were effectively separated. A total of 23 main related substances in both the ivabradine fractionated raw material and forced degradation samples were detected and identified. Structures of these related substances was determined through chromatographic retention behavior, elemental composition analysis, and mass spectrometry characterization. Conclusion: The results provide a reference for the quality control of ivabradine hydrochloride fractionated.
  • Determination of genotoxic impurities in urapidil hydrochloride by HPLC-MS/MS method
    ZHONG Ao, WANG Han, HUANG Peng-mian, PENG Yuan-jian, LI Ming, WU Mu-lin, HUANG Zi-han, ZHENG Wen-chao, MA Xiao-ning
    Chinese Journal of Pharmaceutical Analysis. 2025, 45(10): 1826-1832. https://doi.org/10.16155/j.0254-1793.2024-1146
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    Objective: To establish an HPLC-MS/MS method for the determination of genotoxic impurities bis (2-chloroethyl) amine hydrochloride and O-anisidine hydrochloride in urapidil hydrochloride. Methods: Chromatographic separation was performed on a Horizon Amide 18 column (100 mm×2.1 mm, 3 μm) using a mobile phase consisting of 0.1% formic acid aqueous solution (A) and acetonitrile (B) with gradient elution. The flow rate was 0.3 mL · min-1 and the column temperature was 30 ℃. Detection was carried out on a Vanquish-TS Quantis Plus triple quadrupole mass spectrometer equipped with an electrospray ionization source (ESI), operating in positive ion mode, using multiple reaction monitoring. Results: The linear range of bis (2-chloroethyl) amine hydrochloride was 0.47-120 ng · mL-1 (r=0.999 7). The limit of detection was 0.24 ng · mL-1, the limit of quantitation was 0.47 ng · mL-1. The recovery rates at three concentration levels ranged from 75.7% to 110.2% with RSDs of 2.9%-7.8% (n=3). The calibration curve for O-anisidine hydrochloride was linear over the range of 2.34-120 ng · mL-1 (r=0.999 9). The limit of detection was 0.59 ng · mL-1, and the limit of quantitation was 2.34 ng · mL-1. The recovery rates at three concentration levels ranged from 79.6% to 110.9% with RSDs of 1.8% to 7.9% (n=3). In three batches of urapidil hydrochloride samples, the contents of bis (2-chloroethyl) amine hydrochloride were 0.19, 0.17, and 0.27 μg · g-1, while the contents of O-anisidine hydrochloride were 1.54, 1.57, and 2.35 μg · g-1 all of which complied with the specification limit (<5.48 μg · g-1). Conclusion: The method is rapid, specific, sensitive and accurate, and can be effectively applied to the determination of bis (2-chloroethyl) amine hydrochloride and O-anisidine hydrochloride in urapidil hydrochloride.
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