31 March 2025, Volume 45 Issue 3
    

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    Review & Monography
  • Research progress on extraction, isolation, structural identification and analytical methods of oligosaccharides from Chinese herbal medicine*
    GUO Hong, YAO Rui, FAN Jing, GUO Xiao-han, CHEN Jia, DUAN Bao-zhong, WANG Ying, YANG Jian-bo, CAI Wei, JING Wen-guang, CHENG Xian-long, WEI Feng
    Chinese Journal of Pharmaceutical Analysis. 2025, 45(3): 361-391. https://doi.org/10.16155/j.0254-1793.2024-1061
    Abstract ( ) Download PDF ( )   Knowledge map   Save
    Oligosaccharides are a class of bioactive components abundantly found in Chinese herbal medicine. They have clinical effects such as antidepressant and anti-Alzheimer’s disease activities, as well as pharmacological activities such as hypoglycemic, laxative, and immune-enhancing effects. The bioactivity of oligosaccharides is closely related to their structure. However, due to the diversity of glycosidic bond configurations, monosaccharide compositions and linkage patterns, it is difficult to accurately determine their structures by conventional analytical methods. Establishing rapid and reliable analytical methods remains a key challenge in carbohydrate research. Although studies on oligosaccharides are increasing, there is a lack of comprehensive reviews on the structure and analytical methods of oligosaccharides in Chinese herbal medicine. This paper systematically reviewed the research progress over the past decade on extraction, separation, structure identification and analytical methods of oligosaccharides in Chinese herbal medicine by consulting domestic and foreign literatures. This review aims to contribute to the development of evaluation method of traditional Chinese medicine based on oligosaccharide components, and provide reference for their application in the quality control of traditional Chinese medicine.
    • Ingredient Analysis
  • Qualitative determination of multiple components in Bletillae Rhizoma by linear calibration using two reference substances*
    LIN Chang, LIU Su-qing, ZHANG Jing, YANG Cong, QIAN Hai-bing, YANG Chang-fu, ZHAO Jun
    Chinese Journal of Pharmaceutical Analysis. 2025, 45(3): 392-399. https://doi.org/10.16155/j.0254-1793.2024-0317
    Abstract ( ) Download PDF ( )   Knowledge map   Save
    Objective: To analyze six components in Bletillae Rhizoma using high-performance liquid chromatography. Through the linear calibration using two reference substances method, the retention time of the components was predicted, and the feasibility and accuracy of this technique in the qualitative identification of chromatographic peaks were explored. This method was also compared with the relative retention time (RRT) method. Methods: High-performance liquid chromatography was employed using an Ultimate XB-C18 column (250 mm×4.6 mm, 5 μm). The mobile phase consisted of water (A) and acetonitrile (B), with a gradient elution as follows: 0-11 min,12% B; 11-58 min, 12% B to 68% B; 58-65 min, 68% B to 12% B. The flow rate was 0.9 mL · min-1, the injection volume was 5 μL, the column temperature was maintained at 30 °C, and the detection wavelength was set at 280 nm. By measuring the actual retention time of the six components on 16 different C18 chromatographic columns, the standard retention time was calculated. Gastrodin (Peak 1) and gymnadenin Ⅲ (Peak 4) were used as the double-standard compounds. The linear calibration using two reference substances method was used to predict the retention time of other components, and the prediction was verified on another three unknown C18 chromatographic columns. With the relative retention time (RRT) method, using bletilloside (Peak 5) as the reference compound, the retention times of the other five components were predicted. Results: A qualitative analysis method for the six components in Bletillae Rhizoma was established, which could accurately predict the retention times of each component in Bletillae Rhizoma. The standard retention times of the six components as as follows: gastrodin 4.279 min, gymnadenin Ⅲ 32.145 min, pleione bulbocodioides factor Ⅰ 27.220 min, 2-O-glucosylbletilloside 28.603 min, bletilloside 33.509 min, and batatasin Ⅲ 45.504 min. Conclusion: The linear calibration using two reference substances method can improve the prediction ability of the retention time of each component in Bletillae Rhizoma, thereby enhancing the accuracy and reliability of the analysis results.
  • Study on UHPLC fingerprint and simultaneous determination of 4 components of hemp*
    LI Jun, LIU Xiao-dan, SUN Li-qiu, LAN Tao, WANG Dan, ZHAO ming
    Chinese Journal of Pharmaceutical Analysis. 2025, 45(3): 409-416. https://doi.org/10.16155/j.0254-1793.2024-0299
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    Objective: To establish the UHPLC fingerprint and quantitative analysis of CBDA, CBD, THC, and THCA for hemp, and provides a basis for the quantity control of hemp. Methods: UHPLC was launched on a Phenomenex C18 (150 mm×4.6 mm, 2.6 μm) with mobile phase of acetonitrile-0.2% phosphoric acid aqueous solution at a flow rate of 1.0 mL · min-1, column temperature of 35 ℃, detection wavelength of 220 nm, and an injection volume of 2 μL by gradient elution. 15 batches of hemp samples from Daxing ’anling region and Qiqihar were analyzed to establish the fingerprint. Cluster analysis, principal component analysis and orthogonal partial least squares discriminant analysis were adopted to assess of UHPLC fingerprint of hemp. Results: the UHPLC fingerprint of hemp was established, and 26 common peaks were selected the 26 common peaks were compared with the reference substance. The structures of four chromatographic peaks were identified and simultaneously analyzed for content determination. four chromatographic peaks were identified as CBDA, CBD, THC and THCA. The similarity of fingerprint of them was greater than 0.950. The samples from Daxing’anling were clustered into class Ⅰ, with the best quality. The results show that hemp samples from different regions were quite different. The variable importance projection (VIP) showed that the four compounds to be tested were the main components leading to the difference of hemp. Conclusion: The established method is stable and reliable and can be used for qualitative and quantitative analysis of hemp. The findings of this study are expected to provide a scientific basis for quality control of hemp.
  • Variation trend and evaluation of volatile components in hawthorn fruit at different growth stages
    LIU Tian-yi, WANG Yu-chun, LI Cun-man, REN Jie
    Chinese Journal of Pharmaceutical Analysis. 2025, 45(3): 417-425. https://doi.org/10.16155/j.0254-1793.2024-0238
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    Objective: To explore the growth trend of volatile components in hawthorn fruit at different growth stages. Methods: The volatile components in hawthorn fruits at different growth stages were quantitatively and qualitatively analyzed by gas chromatography-mass spectrometry (GC-MS), and their dynamic changes were investigated and statistically analyzed. The chromatographic column used was HP-5 (30 m×0.32 mm, 0.25 μm), while the column temperature was programmed (initial temperature 60 ℃, increased to 250 ℃ at a rate of 10 ℃ · min-1, maintained for 33 min), and the inlet temperature was 280 ℃. The ion source was an electron bombardment source (EI), with an ion source temperature of 230 ℃. Results: A total of 57 representative volatile components were detected, of which 16 components disappeared in the later stage of growth, and 12 components were not present in the early stage of growth. The content of 11-decyl-tetracosane was the highest and gradually increased, and the content of hexadecyl acrylate gradually decreased. Conclusion: The investigation of the dynamic changes of volatile components in hawthorn fruit during the growth period provides a reference for further rational development and utilization of hawthorn fruit.
    • Activity Analysis
  • Spectrum-effect relationship on antioxidant activity of Gardeniae Fructus before and after processing based on multivariate statistical analysis*
    HUI Bo-ping, XIAO Jing, TANG Ting, FENG Chuan-ping, HUANG Jian-hua, LI Jun-qiang
    Chinese Journal of Pharmaceutical Analysis. 2025, 45(3): 426-439. https://doi.org/10.16155/j.0254-1793.2024-0496
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    Objective: To establish the HPLC fingerprint of Gardeniae Fructus before and after processing, and to study the relationship between the fingerprint and antioxidant activity. Methods: Agilent 1200 high-performance liquid chromatograph was used, and the column was Agilent ZORBAX SB-C18 (250 mm×4.6 mm, 5 μm). The mobile phase system was 0.1% formic acid water-methanol solution. The ultraviolet detection wavelength was 254 nm. The column temperature was 25 ℃. The extraction solvent of the test solution was 70% methanol solution. The injection volume was 5 μL. The flow rate was 1 mL · min-1, and the fingerprints of six batches of gardenia before and after processing were established, and the antioxidant activity of gardenia before and after processing was evaluated by DPPH method, and the spectral effect relationship was studied by grey correlation analysis and partial least squares regression analysis. Results: 23 common peaks were identified in the fingerprint, and five chromatographic peaks were identified by comparison of reference substances, including geniposidic acid (peak 3), genipin gentiobiside (peak 9), gardenside (peak 11), crocetin I (peak 21), and crocetin Ⅱ (peak 22). The mean half inhibitory concentration (IC50) of DPPH radical in 5 different processed products (raw gardenia, fried gardenia, wine gardenia, gardenia charcoal and ginger gardenia) were 0.25 mg · mL-1, 28.61 mg · mL-1, 6.34 mg · mL-1, 11.79 mg · mL-1, and 0.68 mg · mL-1, respectively. Combined with the three statistical methods, peaks 3, 9, 11, 21, and 22 were the common peaks associated with DPPH radical scavenging. Conclusion: The antioxidant activity of 6 batches of Gardeniae Fructus before and after processing is the result of the interaction of multiple components, and peaks 3, 9, 11, 21, and 22 are closely related to antioxidant activity. The results of this study provide a reference for the basic research and clinical application of antioxidant substances in Gardeniae Fructus before and after processing.
  • Separation, purification, structural analysis, and comparison of the anti-inflammatory activity of polysaccharides in Green Fructus Forsythiae and Folium Forsythiae*
    ZHAO Xiao-yan, GAO Xiu-hua, SHEN Xiao-yun, LI Ke, LI Zhen-yu
    Chinese Journal of Pharmaceutical Analysis. 2025, 45(3): 440-451. https://doi.org/10.16155/j.0254-1793.2024-1320
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    Objective: To extract and isolate polysaccharides from Green Fructus Forsythiae and Folium Forsythiae for structural characterization and comparison of their anti-inflammatory activities. Methods: Crude polysaccharides were obtained through extraction and purification processes. Gel permeation chromatography and laser light scattering were used to determine their molecular weight. The MTT assay was employed to evaluate the cytotoxicity of the polysaccharides, while ELISA was used to measure levels of nitric oxide (NO), tumor necrosis factor-alpha (TNF-α), and interleukin-10 (IL-10). Results: The polydispersity index (PDI) indicated a relatively uniform molecular weight distribution. Monosaccharide composition analysis showed that both polysaccharides contained various monosaccharides, with Green Fructus Forsythiae polysaccharide having a higher content of galacturonic acid, while Folium Forsythiae polysaccharide had a higher glucose content. Methylation analysis revealed that polysaccharides in Green Fructus Forsythiae were primarily linked by 4-Gal (p)-UA, while those of Folium Forsythiae polysaccharide were mainly linked by 4-Glc (p). Both polysaccharides significantly inhibited the production of NO and TNF-α induced by LPS while promoting the generation of IL-10, with polysaccharide in Folium Forsythiae showing superior effects compared to Green Fructus Forsythiae polysaccharide. Conclusion: Polysaccharides from Green Fructus Forsythiae and Folium Forsythiae exhibit significant anti-inflammatory activities. This research lays the foundation for the development of Folium Forsythiae resources.
    • Bioassay
  • Establishment and validation of a double antibody sandwich ELISA method for the in vitro relative potency test of recombinant SARS-CoV-2 vaccine (CHO cells)*
    CAO Sha-sha, HE Peng, LIU Xiao-ya, LIU Ying, LIU Yu, MA Zhi-tao, WEI Fen, WANG Jia-ji, HU Zhong-yu
    Chinese Journal of Pharmaceutical Analysis. 2025, 45(3): 452-459. https://doi.org/10.16155/j.0254-1793.2024-1276
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    Objective: To establish and validate a double antibody sandwich ELISA method for the in vitro relative potency test of severe acute respiratory syndrome coronavirus 2, (SARS-CoV-2) recombinant protein vaccine (CHO cells). Methods: Human monoclonal antibodies GH4 and CB6 against the receptor binding domain (RBD) of SARS-CoV-2 spike protein were prepared by genetic recombinant technology. A double antibody sandwich ELISA method was established using GH4 as coating antibody and CB6 labeled with HRP enzyme (CB6-HRP) as detection antibody. The working concentrations of GH4 and CB6-HRP were determined. Methodological validation was carried out, including linearity and range, specificity, precision (repeatability, intermediate precision), accuracy, and robustness. The established method was used to detect the in vitro relative potencies of three batches of process-validated and twenty-two batches of continuous process verification of SARS-CoV-2 recombinant protein vaccines (CHO cells). Results: The optimal working concentrations of GH4 and CB6-HRP were 1 000 ng · mL-1 and 31.25 ng · mL-1, respectively. The vaccine reference had a good log-linear relationship with A450 in the concentration range of 0.312 5-5 ng · mL-1, and the slopes of the regression equations were all in the range of 0.80-1.25 and the R2 values were all>0.99. The established method could specifically detect the SARS-CoV-2 recombinant protein vaccine (CHO cells), and there was no cross-reactivity with the MERS Vaccine (CHO cells), influenza virus vaccine, rabies virus vaccine and CHO host cell proteins. For precision validation, the geometric coefficient of variation (GCV) of repeatability was in the rang of 1.6% to 2.4%, and the GCV of intermediate precision was in the range of 1.2% to 2.6%. The R2 value of the regression equation was 0.994 5, and the slope was 0.999 5 which was in the range of 0.80-1.25. For accuracy validation, the relative bias (RB) was in the range of -4.04% to 7.36%. In the robustness validation, the in vitro relative potency under different test conditions ranged from 0.96 to 1.08. The geometric mean in vitro relative potency of the three batches of process-validated SARS-CoV-2 recombinant protein vaccines (CHO cell) was 1.02, with an GCV of 4.8%; the geometric mean in vitro relative potency of twenty-two batches of continuous process verification SARS-CoV-2 recombinant protein vaccines (CHO cells) was 1.04, with an GCV of 4.4%. Conclusion: The established double antibody sandwich ELISA method has good specificity, precision, accuracy and robustness. It can be used for the detection of the in vitro relative potency and the quality control of SARS-CoV-2 recombinant protein vaccine (CHO cells).
  • Primary structure characterization of the active national standard candidate of infliximab*
    LI Wei-yu, ZHANG Jia-ling, LI Meng, YU Xiao-juan, WU Gang, YU Chuan-Fei, WANG Lan
    Chinese Journal of Pharmaceutical Analysis. 2025, 45(3): 460-474. https://doi.org/10.16155/j.0254-1793.2024-1266
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    Objective: To characterize the primary structure of the active national standard candidate of infliximab to provide a technical basis for this monoclonal antibody. Methods: The complete relative molecular mass and the relative molecular masses of the light and heavy chain subunits of the active national standard candidate of infliximab were detected based on ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS). The relative molecular masses of the light and heavy chain subunits of the active national standard candidate of infliximab were analyzed by UPLC-MS/MS. UPLC-MS/MS was used to analyze the sequence coverage, glycosylation sites and disulfide bond localization of infliximab activity candidate national standard products. Results: The complete relative molecular mass of infliximab was 148 515. The relative masses of its light and heavy chain subunits were 23 435 and 50 827 respectively. The sequence coverage of infliximab was 100% for light chain and 100% for heavy chain. The glycosylation sites of infliximab were analyzed by UPLC-MS/MS; and the disulfide bonds were localized. The glycosylation site of the monoclonal antibody candidate was located at N300 of the heavy chain; there were 16 pairs of disulfide bonds, 4 pairs within each of the two heavy chains, 2 pairs between chains, 2 pairs within each of the 2 light chains, and 1 pair between each of the two groups of light and heavy chains. Conclusion: The primary structural confirmation of the national standard candidate of infliximab monoclonal antibody activity provides a technical guarantee for the establishment of the monoclonal antibody standard substance, the standardization of analytical methods and the control of product quality.
    • Quality Control
  • Quality evaluation of Artemisia rupestris L. based on fingerprints and QAMS combined with chemometrics, EW-TOPSIS method and WRSR method*
    DING Man, CHENG Jiang-nan, ADINA Abuduaini, MAO Yan
    Chinese Journal of Pharmaceutical Analysis. 2025, 45(3): 475-488. https://doi.org/10.16155/j.0254-1793.2024-0395
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    Objective: To establish a method combining high performance liquid chromatography (HPLC) fingerprint with quantitative analysis of multi-components with a single marker (QAMS), for simultaneous determination of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, cynaroside, isochlorogenic acid B, isochlorogenic acid A, isochlorogenic acid C, buddleoside, rupestonic acid, chrysosplenetin and artemisetin in Artemisia rupestris L.. The comprehensive quality evaluation model of different producing areas was established to provide reference for the overall quality evaluation. Methods: HPLC method was used to determine the fingerprints of 15 batches of Artemisia rupestris L. from different origin. Stationary phase was YMC-Pack ODS-A C18 column (250 mm×4.6 mm, 5 μm) was adopted, and the mobile phase was acetonitrile-water (containing 0.2% formic acid) with gradient elution, the detection wavelength was segmented changes, the column temperature was 30℃, the flow rate was 1.0 mL·min-1. The information of fingerprinting spectrum was analyzed by cluster analysis (CA), principal component analysis (PCA) and orthogonal partial least-squares discrimination analysis (OPLS-DA). At the same time, the entropy weight technique for order preference by similarity to ideal solution (EW-TOPSIS), the weighted rank sum ratio (WRSR) and the fuzzy combination of the two methods to construct the evaluation model. With buddleoside as the internal standard, the relative correction factors (RCF) of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, cynaroside, isochlorogenic acid B, isochlorogenic acid A, isochlorogenic acid C, rupestonic acid, chrysosplenetin and artemisetin were determined and their contents were calculated to establish QAMS method. Results: A total of 18 common peaks were calibrated and ten of them were identified by the established fingerprint of Artemisia rupestris L.. The study of stoichiometric model showed that there were obvious differences among different producing areas of Artemisia rupestris L.. Eleven different components were selected by OPLS-DA method. The comprehensive quality evaluation model of EW-TOPSIS method, WRSR method and their fuzzy combination showed the consistent quality evaluation ranking results of different producing areas. The resolution and linear relationship of ten components in quantitative analysis were good. The average recovery rates were 92.6%-107.2% with RSD<3.0%. There was no significant difference between the results of QAMS with chlorogenic acid as internal standard and the results of external standard (P>0.05). Conclusion: The established HPLC fingerprint combined with QAMS method is simple, reliable and has good repeatability. The results of the comprehensive quality evaluation model established are comprehensive and objective, which can be used to evaluate the overall quality of Artemisia rupestris L..
  • Quality evaluation research of Vernonia amygdalina Del. based on HPLC fingerprint combined with chemical pattern recognition and multi-component determination*
    YANG Jing, QIN Hua-liang, FU Chuan-wu, HU Shi-hua, QIU Qin, LAN Yu-qing, QIN Dong-jie, HUANG Min-tao, ZHONG Wen-jun, XU Jia, QIN Zi-long
    Chinese Journal of Pharmaceutical Analysis. 2025, 45(3): 489-503. https://doi.org/10.16155/j.0254-1793.2024-0478
    Abstract ( ) Download PDF ( )   Knowledge map   Save
    Objective: To establish a multi-component determination method and HPLC fingerprint for Vernonia amygdalina Del., combined with chemical pattern recognition to comprehensively evaluate the quality of Vernonia amygdalina Del. from different producing, laying a foundation for the future development of quality control standards for Vernonia amygdalina Del. and other related research on Vernonia amygdalina Del.’s components. Methods: The HPLC using Welch Ultimate® AQ-C18 column (250 mm×4.6 mm,5 µm) and 0.2% phosphoric acid aqueous solution - acetonitrile as moblie phase by gradient elution at the flow rate of 1 mL · min-1, wavelength was set at 258 nm and the column temperature was 35 ℃. Simultaneously establish 24 batches of fingerprint spectra of Vernonia amygdalina Del. from different origins and a multi-component HPLC determination method for 9 components of Vernonia amygdalina Del.. Combine chemical pattern recognition to evaluate the quality of 24 batches of Vernonia amygdalina Del. from different origins. Results: The similarities of the fingerprint spectra of 24 batches of Vernonia amygdalina Del. were above 0.9. The established control fingerprint spectra could stably and effectively distinguish Vernonia amygdalina Del. qualitatively. There were 10 common peaks in the fingerprint spectra, and 9 peaks were identified. The mass fractions of 24 batches of Vernonia amygdalina Del. components that were uracil, chlorogenic acid, luteolin, luteolin-7-O-β-D-glucuronide, isochlorogenic acid B, 3,5-O-dicaffeoyl quinic acid, 1,5-dicaffeoyl quinic acid, apigenin-7-O-β-D-glucopyranoside, 4,5-O-dicaffeoyl quinic acid were 0.007 314-0.084 30 mg · g-1,0.619 6-9.763 mg · g-1,0.303 8-4.031 mg · g-1,0.984 6-8.146 mg · g-1,0.043 29-0.438 7 mg · g-1,0.537 5-11.57 mg · g-1,0.437 6-13.78 mg · g-1,0.032 19-0.720 1 mg · g-1,0.190 0-1.931 mg · g-1. Through cluster analysis, 24 batches of Vernonia amygdalina Del. were divided into 2 categories. Vernonia amygdalina Del. from Guangdong and Hainan were classified as one category, while Vernonia amygdalina Del. from Guangxi were classified as one category alone. The classification results of principal component analysis were consistent with cluster analysis. Further research on the differences in leaf quality among different regions of Vernonia amygdalina Del. was conducted through principal component scores, and it was found that the P5, P24 and P22 regions ranked in the top three with better quality than other regions. Through orthogonal partial least squares discriminant analysis, it was found that the reasons for the differences in leaf quality among different regions of Vernonia amygdalina Del. were ranked in descending order: 1,5-dicaffeoyl quinic acid, chlorogenic acid, and luteolin-7-O-β-D-glucuronide, 3,5-O-dicaffeoyl quinic acid. Conclusion: The fingerprint and multi-component determination methods of Vernonia amygdalina Del. are stable and effective through methodological detection, which can fill the gap in quality control standards for Vernonia amygdalina Del.. Combined with chemical pattern recognition methods, the high-quality production areas of Vernonia amygdalina Del. and the components that should be emphasized in controlling the quality of Vernonia amygdalina Del. from different production areas are pointed out. A comprehensive evaluation of the quality of Vernonia amygdalina Del. has been conducted, providing a solid foundation for future quality control and evaluation of Vernonia amygdalina Del..
  • Study on quality evaluation of Yankening tablets based on fingerprint combined with multivariate statistical analysis and multi-component quantification
    WANG Hong-ming, WANG Xiao-bing, XU Xue-li, LIANG Xiu-kun
    Chinese Journal of Pharmaceutical Analysis. 2025, 45(3): 504-513. https://doi.org/10.16155/j.0254-1793.2024-0450
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    Objective: To establish a method for determining the fingerprint of Yankening tablets and evaluate their quality combined with multivariate statistical analysis and multi-component quantification. Methods: The seperation was performed on a 30 ℃ thermostatic InertSustain C18(250 mm×4.6 mm, 5 μm) column,with the mobile phase comprising of methanol-acetonitrile-0.2% phosphoric acid flowing at 1.0 mL · min-1 in a gradient elution manner, and the detection wavelength was set at 254 nm to establish HPLC fingerprint of Yankening tablets, contents of 10 components (berberine hydrochloride, baicalin, wogonoside, baicalein, aloe emodin, wogonin, rhein, emodin, chrysophanol, emodin methyl ether) in 17 batches of Yankening tablets were determined, and similarity evaluation, cluster analysis, entropy weight TOPSIS method, and entropy weight grey correlation analysis were used to comprehensively evaluate the quality of Yankening tablets. Results: There were significant differences in the quality of Yankening tablets of the current market. 33 common peaks were obtained by matching in the fingerprints of 17 batches of samples, and the similarity evaluation and cluster analysis results showed differences among different samples. The comprehensive quality ranking and multi-component content determination revealed that the important factors affecting drug quality were the quality and process control level of the three medicinal herbs, Phellodendri Chinensis Cortex, Coptidis Rhizoma and Scutellariae Radix. Conclusion: The fingerprint established by this method, combined with a multivariate statistical model, can provide reference for the quality evaluation research and products quality improvement of Yankening tablets.
    • Safety Monitoring
  • Determination of residual human factor Ⅻ and prekallikrein in high concentration human immunoglobulin for intravenous injection by enzyme-linked immunosorbent assay*
    YU Yu-rong, LIU Yong, YANG Long, TANG Liang-yu, XIAO Chun-qiao, LI Dan, JIAN Chang-yong
    Chinese Journal of Pharmaceutical Analysis. 2025, 45(3): 514-521. https://doi.org/10.16155/j.0254-1793.2024-0235
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    Objective: To establish, validate and apply an enzyme-linked immunosorbent assay (ELISA) method for determination of residual human factor Ⅻ(FⅫ) and prekallikrein (PK) in high concentration human intravenous immunoglobulin (10%IVIG). Methods: Microplates were pre-coated with specific antibodies for FⅫ and PK, respectively, and then blocked. The FⅫ and PK in 10%IVIG were allowed to bind with the pre-coated antibodies on the plates. After appropriate washing steps, biotinylated detection antibodies specific for FⅫ and PK were added. Following a second washing, HRP/Streptavidin-Peroxidase conjugates were added and unbound conjugates were washed away with another washing step. After the addition of a chromogenic substrate and a stop solution, the absorbance was measured at 450 nm using a microplate reader. The detection method was validated according to the 2020 edition of Chinese Pharmacopoeia. Then the validated method was used to determine the residual levels of FⅫ and PK in process intermediates and final products 10%IVIG. Results: The dilution linearity results showed a matrix effect and a hook effect in FⅫ and PK detection methods, respectively, which could be solved by diluting 5-20 times and 80-120 times, respectively. The average recoveries in the spiked experiments were 110.3% and 99.1% for FⅫ and PK respectively, with RSD of 3.6% and 2.7%. indicating good accuracy of the method. The RSDs of the repeatability validation were 8.3%、7.7% for both methods, and the RSD of intermediate precision validation were 7.0% and 9.2% for FⅫ and PK, respectively, indicating good precision of the two methods. The limits of quantitation of the two methods were 1.0 ng · mL-1 and 1.25 ng · mL-1 respectively. Both methods exhibited good linearity (r = 0.999 9). The robustness validation, which included incubation time, the effective period of the opened reagent kit, and different batches of reagent kits, showed good robustness of the method. The residues of high concentration human immunoglobulin FⅫ and PK maintained at low levels by using the method, indicating that octanoic acid precipitation combined with two-step anionic exchange chromatography process could effectively remove FⅫ and PK in 10%IVIG. Conclusion: The detection method shows good applicability and can be used for the determination of FⅫ and PK residues in 10%IVIG.
  • Determination of 10 kinds of elemental impurities in pharmaceutical excipients sodium succinate by ICP-MS*
    LIU Kai-shuang, LI Mei-fang, ZHANG Xiang, WANG Ping, WANG Xiao-wei
    Chinese Journal of Pharmaceutical Analysis. 2025, 45(3): 522-529. https://doi.org/10.16155/j.0254-1793.2024-0434
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    Objective: To establish an inductively coupled plasma mass spectrometry (ICP-MS) method to determine the contents of 10 elemental impurities in the pharmaceutical excipients sodium succinate according to the ICH Q3D elemental impurity guideline, and to provide a basis for the comprehensive evaluation of potential risks. Methods: According to the types of elements that needed to risk assessment and the permitted daily exposure (PDE) of the injection route, the single-component limit calculation method was used to calculate the limit and control threshold of each element in sodium succinate with 10 g · d-1 as the maximum daily dose. Samples were preprocessed by direct dissolution and the iCAP RQ ICP-MS was used for the simultaneous determination of the residual amounts of 10 impurities in sodium succinate, using Ge, In and Bi as the internal standards. Results: All elemental impurities showed good linear relationship (r>0.99). The limits of detection of Cd, Pb, As, Hg, Co, V, Ni, Li, Sb and Cu were 0.27,19.22,8.86,16.63,0.12,0.28,1.48,6.91,0.35,9.26 ng · g-1, respectively. The recoveries (n=3) of each concentration were between 82.4% and 130.9%, and the RSD (n=6) of the repeatability test was not more than 7.1%, with all findings meeting the requirements for methodological validation. The content of elemental impurities in sodium succinate was less than 30% of the PDE, indicating that the elemental impurities in sodium succinate had no potential safety risk with the medicinal product. Conclusion: The method is simple in sample pretreatment, with high sensitivity and good accuracy. It is suitable for monitoring and risk assessment of elemental impurities in sodium succinate, which is conducive to the quality control. It also provides a reference for the determination of elemental impurities in other products. Keywords: sodium succinate; ICH Q3D; inductively coupled plasma mass spectrometry (ICP-MS); elemental impurities; permitted daily exposure（PDE）;content determination;internal standard method;risk assessment
  • Determination for the bacterial endotoxin of recombinant typeⅢhumanized collagen solution for injection by the recombinant C-factor method
    LI Ling, CHENG Xiao, WANG Jian, LIAN Kai-na, ZHANG Hong, FAN Yu-hui, SUN Dan-dan, YU Yu-feng
    Chinese Journal of Pharmaceutical Analysis. 2025, 45(3): 530-536. https://doi.org/10.16155/j.0254-1793.2024-0498
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    Objective: To establish a recombinant C-factor method to detect the content of bacterial endotoxin in recombinant type Ⅲ humanized collagen solution for injection. Methods: Three batches of injectable recombinant type Ⅲ humanised collagen solutions were determinedand method validation by using the BIOMERIEUX recombinant C-factor kit and the lonza recombinant C-factor kit,respectively. Results: The results showed that about the two kits the concentration points of the standard curve were ≥ 3, and the linear correlation coefficient was r > 0.980, the ΔRFU value of the negative control was smaller than that of the lowest point of the standard curve; the bacterial endotoxin contents were all < 6 EU · mL-1. The reproducibility was good, the recoveries of bacterial endotoxin were in the range of 50%-200%, which was in accordance with the standard requirements of the ChP. Conclusion: The bacterial endotoxin content in the solution of recombinant type Ⅲ humanized collagen solution for injection is determined by the two kits. The results were < 0.05 EU · mL-1 and < 0.005 EU · mL-1, which are in accordance with the standard requirements for bacterial endotoxin in the product, respectively. The method can be used for the determination of bacterial endotoxin content in the solution of recombinant type Ⅲ humanized collagen solution for injection. This study provides a reference for the research related to the determination of endotoxin in recombinant protein products using recombinant C-factor method.
    • Process Evaluation
  • Study on taste masking of ambroxol hydrochloride direct oral granules based on electronic tongue technology*
    LIU Meng, ZHANG Na, XU Hui, SHEN Li-ping, ZHU Guang-hui, CHEN Hua, YU Li-ju
    Chinese Journal of Pharmaceutical Analysis. 2025, 45(3): 537-542. https://doi.org/10.16155/j.0254-1793.2024-1281
    Abstract ( ) Download PDF ( )   Knowledge map   Save
    Objective: To investigate the improvement in bitter taste of ambroxol hydrochloride by the corrective materials and weight gain of the taste-masking layer, and to establish an objective and scientific taste evaluation method. Methods: The taste-masking effects of corrective materials and taste-masking layer weighting were evaluated using electronic tongue technology, and the palatability between ambroxol hydrochloride direct oral granules and 15 marketed preparations of ambroxol hydrochloride were compared. The electronic tongue datas were subjected to principal component analysis and loadings analysis. Results: The corrective materials were able to mask the bitter taste of ambroxol hydrochloride. The bitter value of the sample solution of ambroxol hydrochloride direct oral granules decreased as the masking layer gained weight. In terms of bitterness and sweetness, ambroxol hydrochloride oral direct granules had a clear advantage over 15 marketed formulations. Conclusion: Using the electronic tongue technology, the taste correction effect and the taste difference between different samples can be accurately assessed, and an evaluation method for the bitter taste masking effect of ambroxol hydrochloride direct oral granules has been established, which provides new ideas and methods for the taste masking study of oral preparations for children.
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