28 February 2026, Volume 46 Issue 2
    

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    Ingredient Analysis
  • Simultaneous determination of seven components in Cistanches Herba formula by HPLC*
    YAN Luo-mei, Zakiyagul Gujahmat, Mourboul Ablise, YANG Zhao-jun, LI Zhen
    Chinese Journal of Pharmaceutical Analysis. 2026, 46(2): 197-203. https://doi.org/10.16155/j.0254-1793.2025-0618
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    Objective: To establish an HPLC method for the simultaneous determination of several key constituents, namely syringin, echinacoside, verbascoside, epimedin A, epimedin B, epimedin C and icariin in Cistanches Herba formula, aiming to provide experimental evidence for the quality control of the formula and serve as a material basis for research on its anti-fatigue efficacy. Methods: The analysis was performed on a ZORBAX Eclipse Plus C18 column (250 mm×4.6 mm, 5 μm) at 25 ℃, with the mobile phase comprising of acetonitrile-water flowing at 1.0 mL · min-1 in a gradient elution manner. The detection wavelengths were set at 210 nm, and the injection volume was 10 μL. Results: The content determination method for the signature constituents in the Cistanches Herba formula was established. The linear relationship of the seven constituents was excellent within their respective ranges (r≥0.999 7), and the RSD values of precision, repeatability, and stability were all below 5%. The average recovery rates ranged from 93.5% to 104.0%, with RSD values between 0.11% and 2.5%. The content ranges of the seven constituents in four batches of samples were as follows: syringin (0.12-0.18 mg · g-1), echinacoside (16.99-18.65 mg · g-1), verbascoside (1.35-1.76 mg · g-1), epimedin A (0.18-0.21 mg · g-1), epimedin B (0.83-0.98 mg · g-1), epimedin C (0.82-0.98 mg · g-1), and icariin (0.58-0.83 mg · g-1). Conclusion: This method demonstrates good sensitivity, strong specificity, and excellent repeatability. It can serve as a reliable basis for the quality evaluation and control of Cistanches Herba formula.
  • Screening of quality markers of raw Saposhnikoviae Radix and wine-processed Saposhnikoviae Radix based on HPLC fingerprint and network pharmacology*
    WANG Xiao-qing, JU Cheng-guo, AN Yue-yan, YANG Wu-jie, HUANG Si-hang, YANG Jian-hua, LÜ Ling, WANG Wei
    Chinese Journal of Pharmaceutical Analysis. 2026, 46(2): 204-217. https://doi.org/10.16155/j.0254-1793.2025-0420
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    Objective: To develop and quantify the quality markers (Q-markers) for the anti-inflammatory and pain-relieving effects of raw Saposhnikoviae Radix and wine-processed Saposhnikoviae Radix based on fingerprinting and network pharmacology, thus providing experimental evidence for the quality evaluation of Saposhnikoviae Radix before and after processing. Methods: Using a Shimadzu Shim-pack GIST C18 chromatographic column (250 mm×4.6 mm, 5 μm), acetonitrile-water solution as the mobile phase, gradient elution, detection wavelength at 254 nm, column temperature at 30 ℃, and flow rate at 1.0 mL · min-1, the chemical fingerprint chromatograms of raw and wine-processed Saposhnikoviae Radix were established. The common peaks were identified, and the potential Q-markers were screened through principal component analysis (PCA). Network pharmacology was employed to build a component-target-pathway network and predict Q-biomarkers, which were then validated through molecular docking. Finally, the content of Q-biomarkers was determined. Results: The fingerprints of raw Saposhnikoviae Radix and wine-processed Saposhnikoviae Radix were established, with 45 and 47 common peaks, respectively. Among them, 11 common peaks were identified. PCA showed that raw Saposhnikoviae Radix samples and wine-processed Saposhnikoviae Radix samples clustered separately, and 10 potential Q-markers were preliminarily obtained. Network pharmacology combined with molecular docking confirmed four Q-biomarkers: prim-O-glucosylcimifugin, cimifugin, 5-O-methylvisamminol, and sec-O-glucosylhamaudol. The average content of prim-O-glucosylcimifugin, cimifugin 5-O-methylvisammiol, and sec-O-glucosylhamaudol in 10 batches of raw Saposhnikoviae Radix was 1.942, 0.733 7, 1.716, and 0.350 6 mg · g-1, respectively. The average content in the corresponding 10 batches of wine-processed Saposhnikoviae Radix was 2.036, 0.775 3, 2.128, and 0.401 1 mg · g-1, indicating an increase in the content of the four Q-markers after wine processing. Conclusion: The established fingerprinting method is stable and reliable, and the combination of network pharmacology and molecular docking screens out four anti-inflammatory and pain-relieving Q-markers, providing an experimental basis for further research on the processed products of Saposhnikoviae Radix.
    • Bioassay
  • Bioactivity assay method for bFGF preparations based on heparin environment optimization and its applicability evaluation
    WANG Jun-yan, SHI Jun-fang, TAN Ya-chao, YAN Ai-fen, JIANG Yu-hui
    Chinese Journal of Pharmaceutical Analysis. 2026, 46(2): 218-225. https://doi.org/10.16155/j.0254-1793.2025-0562
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    Objective: To establish an optimized bioassay for bFGF, namely the heparin sodium dilution method, aimed at improving the physiological relevance and stability of testing for heparin sodium-containing preparations. Methods: Based on the method described in General Chapter 3527 of the Chinese Pharmacopoeia (2025 Edition, Part IV), the assay was modified by introducing a maintenance medium supplemented with heparin sodium during the sample dilution step and shortening the cell starvation duration to 5 h. Both the optimized method and the standard pharmacopoeia method were employed to assay bFGF preparations with and without heparin sodium to compare their performance. Results: For heparin-containing preparations, the heparin sodium dilution method showed a significantly higher curve-fitting quality (0.998±0.001) and a wider dose-response interval (0.499±0.070) compared to the pharmacopoeia method (0.995±0.002, p<0.01, and 0.295±0.039, p<0.000 1, respectively). The EC50 value (0.419±0.088) was significantly lower than that of the pharmacopoeia method (0.627±0.154) (p<0.000 1). No significant difference was observed in labeled potency between the two methods, and the optimized method demonstrated a satisfactory linear range. For heparin-free preparations, both methods met the correlation coefficient requirement (r>0.9) and showed good parallelism; however, the labeled potency measured by the heparin sodium dilution method (146.78%±10.0%) was significantly higher than that of the pharmacopoeial method (90.65%±2.8%) (p<0.001). Conclusion: The heparin sodium dilution method improves the fitting quality, sensitivity, and method stability of the four-parameter regression assay for heparin sodium-containing preparations. It serves as a valuable supplement to the pharmacopoeia method. These findings provide a more scientific and reliable approach for the quality control of bFGF preparations.
  • Determination of denosumab potency and methodological validation using reporter gene assay
    HAN A-rong, SHEN Hong, DING Man-sheng, DAI Hu, FEI Qian-lan, XIA Xiao-yu, ZHAO Long-shan
    Chinese Journal of Pharmaceutical Analysis. 2026, 46(2): 226-236. https://doi.org/10.16155/j.0254-1793.2025-0288
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    Objective: To establish a cell line and detection method based on the reporter gene assay (RGA) for evaluating the biological activity of denosumab. Methods: The RANK-GloResponse NF-κB-RE-luc2P HEK293 cell line was constructed by transfecting HEK293 cells with plasmids carrying the RANK gene and the NF-κB response element (NF-κB-RE) coupled with a luciferase reporter gene (Luc2P) via transgenic technology. After optimizing experimental conditions including cell density, chromogenic assay conditions, quantity of RANK ligand (RANKL), and denosumab dilution gradient, the detection method was established. The accuracy, precision, linear range, specificity, and robustness of the method were validated in accordance with 9101 and 9401 of General Rules of the Pharmacopoeia of the People’s Republic of China, Four Volumes. Results: The RGA for detecting the biological activity of denosumab was successfully validated. Within the potency level range of 64%-156%, the relative bias (RB) of the method was within±12.0%. The regression equation had a slope of 1.047 6 and a correlation coefficient of 0.988 1. The maximum coefficient of variation (RSD) for precision and accuracy was 15.0%, and the maximum geometric coefficient of variation (GCV) for robustness across different cell passages, cell densities, and culture times was 7.9%. Cell passage stability tests confirmed that the probability (P) value exceeded 0.05 for up to 30 generations. Conclusion: This study successfully constructed a reporter gene-based cell line suitable for detecting the biological activity of denosumab. Methodological validation demonstrated its reliability, providing a robust technical support for the quality control of related biological products.
    • Metabolism Analysis
  • Analysis of drug-derived components of Pinus yunnanensis Franch. pinecone ethanol extract in rat plasma and feces*
    WANG Zhong-ju, ZHANG Hai-peng, LIU Guang-ming, WANG Zu-ding, WANG Fei-fei, LEI Ting
    Chinese Journal of Pharmaceutical Analysis. 2026, 46(2): 237-250. https://doi.org/10.16155/j.0254-1793.2025-0242
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    Objective: The ethanol extract of Pinus yunnanensis Franch. pinecone exhibits various pharmacological activities, including antiviral and antitumor effects, but its in vivo metabolic characteristics and pharmacodynamic material basis remain unclear. This study aimed to quantitatively clarify the metabolic and excretory patterns of the ethanol extract in mammals, thereby providing a foundation for its clinical application and innovative drug development. Methods: Using Sprague-Dawley (SD) rats as models, the ethanol extract of Pinus yunnanensis Franch. pinecone was orally administered, Combined with ultra-high-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS), an Agilent SB-C18 column (1.8 µm, 2.1 mm×100 mm) was employed. Gradient elution was performed using a mobile phase consisting of 0.1% formic acid in water and 0.1% formic acid in acetonitrile at a flow rate of 0.35 mL · min-1. The column temperature was set at 40 ℃, and the injection volume was 2 μL. The system was used for the qualitative analysis of drug-derived components in plasma and feces, including the prototype components and their metabolites. Results: Eight prototype compounds were identified in drug-containing plasma, while 18 prototype compounds were detected in feces. Among them, 2,3-dihydrobenzofuran and phyllanflexoid A were found in both plasma and feces, suggesting partial absorption followed by bile or direct excretion. The discovery of key components such as 2,3-dihydrobenzofuran and 7-hydroxydehydroabietic acid was identified, providing insights into the pharmacodynamic basis for their pharmacological action. Conclusion: This study, for the first time, clarifies the metabolic characteristics of P. yunnanensis pinecone ethanol extract in mammals, filling a gap in its pharmacokinetic research. The findings offer critical evidence for further development and clinical utilization of pinecone resources.
  • Determination of five new psychoactive nitazenes in blood of mice by QuEChERS combined with UPLC-MS/MS*
    LI Xiang, XIA Xin-xin, ZHANG Rui-wen, SONG Hui, ZHU Yu
    Chinese Journal of Pharmaceutical Analysis. 2026, 46(2): 251-260. https://doi.org/10.16155/j.0254-1793.2025-0502
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    Objective: A method was established for the simultaneous determination of five nitazenes in the blood of mice using ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) based on QuEChERS pretreatment technology. Methods: The samples were extracted with a mixture of methanol-acetonitrile (1 ∶ 3, volume ratio) and dehydrated with MgSO4. The samples were separated on an XSelect HSS T3 column (2.1 mm×100 mm) by positive ion scanning in multiple reaction monitoring (MRM) mode, with gradient elution using a mobile phase composed of methanol-0.1% formic acid aqueous solution. Results: Five nitazenes exhibited good linearities within the concentration range of 0.1-100 ng · mL-1, with correlation coefficients all exceeding 0.999 0. The limits of detection (LOD) of five nitazenes were within the range of 0.4-2.0 pg · mL-1, and the limits of quantitation (LOQ) were within the range of 1.3-6.3 pg · mL-1. The spiked recovery rates (n=6) ranged from 89.4% to 110%, with relative standard deviations (RSD) of 3.9%-6.3%. Conclusion: This method offers advantages including high sensitivity, accuracy, and strong operability. It can be effectively applied to detect five nitazenes in blood samples, providing a scientific basis for drug-related cases.
  • Metabolism of etomidate, metomidate, propoxate, and isopropoxate in vitro*
    YANG Li, QIU Wen-pu, QI Guan, CHAI Yi-lin, LONG Jiao, XU Bu-yi
    Chinese Journal of Pharmaceutical Analysis. 2026, 46(2): 261-273. https://doi.org/10.16155/j.0254-1793.2025-0284
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    Objective: To determine the metabolic profiles of etomidate and its structural analogues metomidate, propoxate, and isopropoxate in human liver microsomes (HLMs). Methods: In a human liver microsome incubation model, four target compounds were separately added and incubated at 37 ℃ for 1 hour. The reaction was terminated by adding ice-cold acetonitrile. After centrifugation, the supernatant was collected, evaporated to dryness under nitrogen, and reconstituted. The processed samples were analyzed using ultra-high performance liquid chromatography coupled with high-resolution mass spectrometry in ESI+ mode. The mobile phases consisted of 0.1% formic acid in water and 0.1% formic acid in acetonitrile, with a Waters HSS T3 column used for separation. The IDA scan mode was employed to detect the parent compounds and their metabolites, aiming to investigate the metabolic pathways. Results: The biotransformation of the four compounds primarily involved pathways such as dealkylation, dehydrogenation, oxidation, and glucuronidation. In three real hair samples with positive results, the unchanged forms of etomidate, metomidate, and isopropoxate were detected, along with the same dealkylation metabolites and carboxylation products. Additionally, the characteristic metabolites of two compounds were detected (metabolites M3 yielded from etomidate and isopropoxate after the loss of the phenylethyl group). Conclusion: It is recommended that the unchanged form of etomidate and its metabolite M3, the unchanged form of metomidate, the unchanged form of propoxate and its metabolite M3, as well as the unchanged form of isopropoxate and its metabolite M3 can be used as biomarkers for the intake of the four target compounds.
  • Comparison of in vitro permeation, release, and rheological properties of commercial indometacin cataplasms
    XIE Li, LI Ping, LIAN Xiang-jin, ZHANG Rong-qin, CHEN Hong, ZHENG Ping
    Chinese Journal of Pharmaceutical Analysis. 2026, 46(2): 274-284. https://doi.org/10.16155/j.0254-1793.2025-0560
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    Objective: To conduct an in vitro permeation test (IVPT) of indometacin cataplasms via Franz diffusion cells, which was combined with the in vitro release test (IVRT) and rheological analysis to compare the consistency between imported and domestic products. Methods: IVPT was performed with Franz diffusion cells for the skin of miniature Bama pigs. LC-MS/MS was employed to determine the permeation amount, and HPLC was adopted to measure the skin surface residue and intradermal retention. IVRT was carried out with Franz diffusion cells and HPLC. Rheological properties, including storage modulus (G'), loss modulus (G''), and glass transition temperature (Tg), were evaluated by a rotational rheometer. Results: The maximum flux (126.66±10.11 ng · cm-2 · h-1) and cumulative permeation amount (4 322.96±718.41 ng · cm-2) of the imported product AA01 were significantly higher than those (62.98±11.37 ng · cm-2 · h-1 and 1 674.63±162.87 ng · cm-2) of the domestic product BB02. The IVRT results showed that the release rates were 29.50±0.84 μg · cm-2 · h-1/2 and 16.50±0.61 μg · cm-2 · h-1/2 for AA01 and BB02, respectively. Rheological analysis results showed that both G' and G'' of AA01 were higher than those of BB02. Conclusion: Significant differences exist in transdermal performance, release rate, and rheological properties between imported and domestic indometacin cataplasms. This study provides a reference for in vitro quality evaluation of such preparations.
    • Safety Monitoring
  • Exploration of non-carcinogenic and carcinogenic probabilistic risk assessment of toxic elements in Houttuyniae Herba based on Monte Carlo simulation*
    ZUO Tian-tian, LIU Jia-lin, LIAO Chang, JIN Hong-yu, LIU Jing, CHENG Xian-long, WEI Sheng, LIN Yong-qiang, WEI Feng
    Chinese Journal of Pharmaceutical Analysis. 2026, 46(2): 285-291. https://doi.org/10.16155/j.0254-1793.2025-0149
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    Objective: To establish a probabilistic risk assessment method for non-carcinogenic and carcinogenic risks of heavy metals and harmful elements in Houttuyniae Herba, thus providing guidance for the safe use of traditional Chinese medicine. Methods: The content of lead (Pb), cadmium (Cd), arsenic (As), mercury (Hg), and copper (Cu) in 40 batches of Houttuyniae Herba were measured by inductively coupled plasma mass spectrometry (ICP-MS). On the basis of Monte Carlo simulation, the hazard index (HI) and carcinogenic risk (CR) were employed to assess the non-carcinogenic and carcinogenic probabilistic risks caused by exposure to heavy metals and harmful elements in Houttuyniae Herba. Results: The mean content of Pb, Cd, As, Hg, and Cu in Houttuyniae Herba was 2.62, 0.48, 0.58, 0.04, and 7.95 mg · kg-1, respectively. The maximum HI values for males and females were 1.44 and 1.39, respectively, indicating that the total non-carcinogenic health risks for high-exposure populations could not be ignored. The carcinogenic risk assessment results indicated that the carcinogenic health risks associated with Pb, Cd, and As in Houttuyniae Herba should be of concern for both general and high-exposure populations. Conclusion: This study explores a probabilistic health risk assessment method for heavy metals and harmful elements in Houttuyniae Herba based on Monte Carlo simulation, considering the characteristics of traditional Chinese medicine consumption. It provides technical support for the safety evaluation of traditional Chinese medicine and the formulation and revision of related limit standards.
  • Detection of toluene diisocyanate in medicinal composite membranes*
    LI Sha
    Chinese Journal of Pharmaceutical Analysis. 2026, 46(2): 292-298. https://doi.org/10.16155/j.0254-1793.2025-0636
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    Objective: To establish a gas chromatography-mass spectrometry (GC-MS) method for the determination of toluene diisocyanate content in medicinal composite membranes, and to test the medicinal composite membranes from different manufacturers and of varying compositions available on the market. Methods: The samples were cut into small pieces, shaken with ethyl acetate (pre-dried with molecular sieve to remove water), and then passed through a membrane before loading onto the instrument. The chromatographic condition was as follows: a TG-5MS column (60 m×0.25 mm, 0.25 μm) was used, with an injection port temperature of 260 ℃, a split ratio of 10 ∶ 1, a flow rate of 1.0 mL · min-1, and an injection volume of 1 μL. The ion source was an electron impact ionization (EI), with a transmission line temperature of 300 ℃ and an ion source temperature of 300 ℃. Results: The calibration curve for toluene diisocyanate showed a good linear relationship within the concentration range of 6.65-266.07 ng · mL-1 (r=0.999 6). The detection limit and quantification limit were 2.66 ng · mL-1 and 6.65 ng · mL-1. The spiked recovery rate was 92.7%-104.4%. Among the 90 batches of samples, toluene diisocyanate was detected in 89 batches (accounting for 98.9%), with concentrations ranging from 0 to 9 162.8 ng · mL-1. Conclusion: This method reduces the detection limit, simplifies the sample pretreatment process, and is easy to operate while providing accurate results with high sensitivity and good reproducibility. It can serve as an analytical method for determining the content of toluene diisocyanate in medicinal composite membranes, offering a reference for the detection of toluene diisocyanate.
  • Application and influencing factors of dynamic vapor sorption (DVS) in drug-water interactions*
    YU Shao-wen, XIONG Jing, ZHANG Yuan-yuan, CHEN Ying, NING Bao-ming, ZHANG Xuan
    Chinese Journal of Pharmaceutical Analysis. 2026, 46(2): 299-308. https://doi.org/10.16155/j.0254-1793.2025-0189
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    Objective: To systematically investigate the application of dynamic vapor sorption (DVS) technology in the interactions between drugs/pharmaceutical excipients and water, analyze the key factors that affect the test results, and propose strategies for optimizing experimental design. Methods: DVS technology was used to monitor the mass changes of materials in real-time under varying humidity conditions. Combined with techniques such as X-ray diffraction, particle size analysis, and pore size analysis, this study systematically examined the effects of sample characteristics, including crystallinity, solvation state, particle size, pore size, initial moisture content, and sample quantity on the moisture absorption behavior. Results: This study clarified the significant impact of sample characteristics (e.g., crystallinity, particle size, pore size) and experimental conditions (e.g., equilibrium criteria, sample quantity, initial state) on the DVS test results were clarified. Additionally, optimization recommendations for sample pretreatment, instrument parameter settings, and experimental design were proposed. Conclusion: DVS technology is an effective means for studying drug/excipient-water interactions. By systematically analyzing the influencing factors and optimizing the experimental protocol, the accuracy and reproducibility of the data can be improved, thereby providing reliable support for drug stability studies and formulation development.
  • Construction, multidimensional analysis, and application of in-house library of microbial isolates for pharmaceutical manufacturing environment*
    WANG Jian-dong, YANG Hong-xia, LIU Cheng-zhi, CHEN Huan, HE Lu-ping
    Chinese Journal of Pharmaceutical Analysis. 2026, 46(2): 309-319. https://doi.org/10.16155/j.0254-1793.2025-0589
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    Objective: To address the limitations of trend analysis methods in microbiological monitoring of pharmaceutical manufacturing environment, this study focuses on constructing an in-house library of environmental microorganisms and integrating analytical methods. Methods: Based on the 2022 environmental microbiological identification data from a pharmaceutical enterprise, biological and sampling information of microorganisms was integrated using structured information tables. Python and R were used for data aggregation, calculation of diversity indices, analysis of phenotypic and species-level changes, and construction of an early-warning model, combined with multidimensional analyses such as Sankey diagrams and PCA. Results: A traceable in-house library was successfully established. Sphingomonas was identified as the dominant genus (14.50%), with its detection increasing from January to June and exceeding the alert level in June. Core microbes, persistent microbes (e.g., Ralstonia), and transient microbes were identified, with persistent microbes mainly originating from water systems. Microbial diversity and phenotypic proportions varied over time, and the microbial community structure differed significantly across sampling sources. Conclusion: This in-house library of microbial isolates enables dynamic monitoring and risk early warning of microorganisms, supporting proactive prevention and control in pharmaceutical enterprises and ensuring drug quality.
  • Development of an enrichment broth formulation for detecting Burkholderia cepacia complex in pharmaceutical water systems
    ZHENG Xiao-ling, ZHANG Xu-hong, WANG Yin-huan, CHEN Shuang, LI Jue, WANG Zhi-jian
    Chinese Journal of Pharmaceutical Analysis. 2026, 46(2): 320-327. https://doi.org/10.16155/j.0254-1793.2025-0512
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    Objective: To develop an enrichment broth for detecting Burkholderia cepacia complex (Bcc) in pharmaceutical water systems. Methods: Representative standard strains and wild-type strains of Bcc were selected for experimental analysis. Carbon source screening was first conducted, followed by single-factor tests and orthogonal tests on the broth to determine the optimal Bcc enrichment broth (designated as BCB) for the pharmaceutical water systems. Subsequently, 16 strains of Bcc (10 standard strains and 6 wild-type strains), 2 wild-type Burkholderia microorganisms, and 2 common water system microorganisms were used to investigate the enrichment ability and anti-interference ability of the BCB culture medium. Results: For both standard strains of Bcc and wild-type strains isolated from pharmaceutical water systems, the enrichment capacity of BCB was far superior to that of 1/10 TSB and R2A liquid media, with a maximum difference of 6.42-fold. BCB effectively improved the detection rate of Bcc in pharmaceutical water,and the anti-interference ability of BCB was superior to that of 1/10 TSB. Conclusion: To a certain extent, BCB demonstrated advantages in mitigating the issue of missed detections of Bcc in pharmaceutical water systems.
  • Simultaneous determination of 10 sulfonamides and retinoids illegally added in anti-acne cosmetics by solid-phase extraction-high performance liquid chromatography-tandem mass spectrometry*
    WANG Ren, WU Yuan-yang, LI Ze-hua
    Chinese Journal of Pharmaceutical Analysis. 2026, 46(2): 328-337. https://doi.org/10.16155/j.0254-1793.2025-0399
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    Objective: To establish a solid-phase extraction-high performance liquid chromatography-tandem mass spectrometry (SPE-HPLC-MS/MS) method for the simultaneous determination of ten illegally added drugs, including sulfapyridine, sulfamerazine, sulfamethizole, sulfamethoxypyridazine, sulfachloropyridazine, sulfamethoxazole, tazarotene, acitretin, etretinate and adapalene, in three matrix types (aqueous, emulsion, and cream) of anti-acne cosmetics. Methods: Cosmetic samples were extracted with methanol, purified using an HLB solid-phase extraction column, and analyzed by SPE-HPLC-MS/MS. The samples were separated by ZORBAX-C18 column with a mobile phase consisting of 0.1% formic acid aqueous solution and 0.1% formic acid in methanol by gradient elution. Ionization was performed in positive and negative modes via electrospray ionization (ESI), with quantification in multiple reaction monitoring (MRM) mode. The external standard method with matrix-matched calibration curves was used for quantification. Results: The ten target compounds had excellent linearity in their respective concentration ranges, with correlation coefficients (r)≥0.998. The limits of detection ranged from 0.02 to 0.92 mg · kg-1, and the limits of quantification from 0.06 to 2.8 mg · kg-1. The average recovery rates were 85.5%-100.6%, with relative standard deviations(RSD)less than 10%. Conclusion: The method is simple, accurate, and highly sensitive, enabling reliable detection of ten illegally added drugs in commercially available anti-acne cosmetics.
  • Characterization of suspicious structures of phosphocholine chloride by nuclear magnetic resonance
    WANG Li-juan, ZHANG Xiu, FU Hui
    Chinese Journal of Pharmaceutical Analysis. 2026, 46(2): 338-344. https://doi.org/10.16155/j.0254-1793.2025-0607
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    Objective: To characterize the suspicious structures of several commercially available phosphocholine chloride reagents by nuclear magnetic resonance (NMR). Methods: The suspicious structures of phosphocholine chloride reagents were analyzed by 1H NMR, 31P NMR, 13C NMR, 1H-1H COSY, 1H-13C HSQC, 1H-13C HMBC, and 1H-15N HMBC. Results: The suspicious structures were successfully identified as choline, betaine, and taurine, respectively. The 1H NMR and 13C NMR spectra of the suspicious structures and their corresponding reference standards were compared to further confirm their identity and the peaks were assigned for each compound. Conclusion: This study provides a reliable analytical approach and novel insights for the structural verification and quality control of similar compounds.
  • Nuclear magnetic resonance spectroscopic analysis of impurities in alfentanil hydrochloride injection*
    HUANG Tao, LÜ Zhen-xing, LIU Chen-xi, ZHANG Xu, ZENG Dan-yun, DU Tian-peng
    Chinese Journal of Pharmaceutical Analysis. 2026, 46(2): 345-353. https://doi.org/10.16155/j.0254-1793.2025-0603
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    Objective: To establish a new method for detecting impurities in alfentanil hydrochloride injection by nuclear magnetic resonance (NMR) techniques. Methods: A small sharp peak at δH 2.24 was shown on one-dimensional high-efficiency water peak suppression and 13C-filtered 1H-NMR spectra of the alfentanil hydrochloride injection, which implies an impurity. The impurity structure was identified by 1H-13C HSQC (1H-13C heteronuclear single-quantum correlation) and 1H-13C HMBC (1H-13C heteronuclear multiple-bond correlation). Further traceability analysis confirmed that the impurity originated from the bulk drug. The content of the impurity was quantified by quantitative proton nuclear magnetic resonance (1H-qNMR) with water suppression. Results: The impurity was identified as acetone. The separation between the acetone peak and the internal standard peak was satisfactory, with high precision and excellent reproducibility. The measured content of the impurity ranged from 0.10% to 0.17%. Conclusion: For high-risk injectable drugs lacking unified standards, the application of NMR techniques offers the advantages of non-differential response, enabling rapid identification of chemical components in the injection solution. This method features simple pretreatment of samples, comprehensive detection of chemical components, and the capability to perform both qualitative and quantitative analysis simultaneously.
  • Determination of optical isomers in BEBT-808 API
    WANG Li, HUANG Cai-li, HE Jie, CAI Xiong
    Chinese Journal of Pharmaceutical Analysis. 2026, 46(2): 354-368. https://doi.org/10.16155/j.0254-1793.2024-0250
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    Objective: To study the chiral separation of four optical isomers in the BEBT-808 project by high-performance liquid chromatography and achieve baseline separation of the four isomers, thus providing a basis for the quality evaluation of the project. Methods: The Lux Cellulose-4 column (150 mm×4.6 mm, 5 μm) was used, with a column temperature of 30 ℃. Gradient elution was carried out with ethanol-n-hexane-acetonitrile-trifluoroacetic acid-diethylamine (300 ∶ 700 ∶ 10 ∶ 1 ∶ 0.1, v/v) as the mobile phase A and ethanol-n-hexane-acetonitrile-trifluoroacetic acid-diethylamine (470 ∶ 530 ∶ 10 ∶ 1 ∶ 0.2, v/v) as the mobile phase B at a flow rate of 1 mL · min-1. The UV detection wavelength was 286 nm. Results: The method validation results showed that the minimum resolution between adjacent peaks of the four optical isomers in BEBT-808 API was 1.5, and Z0 had a good linear relationship in the concentration range of 1.448-28.95 μg · mL-1 (r=1.000 0). The minimum detectable amount was 0.438 7 μg · mL-1. The recovery rates of the three optical isomers were all greater than 91.9%, and the relative standard deviations of precision were within the range of 0.64%-1.0%. The test solution was stable at room temperature for 27.5 h. Conclusion: This method has high specificity, resolution, precision, accuracy, and stability, thus being suitable for the determination of four optical isomers in BEBT-808 API.
  • Helium mass spectrometry leak detection method (vacuum mode) for the container-closure integrity testing of injection vials at low temperature condition
    ZHANG Fang-fang
    Chinese Journal of Pharmaceutical Analysis. 2026, 46(2): 369-376. https://doi.org/10.16155/j.0254-1793.2025-0360
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    Objective: To establish a helium mass spectrometry leak detection method (vacuum mode) to test the container-closure integrity of the injection via at low temperature conditions. Methods: The helium mass spectrometry leak detection method (vacuum mode) was used, the total test time was 110 s and the methodology was verified to test the closure integrity of the vials. Results: The helium mass spectrometry leak detection method (vacuum mode) was established, and the detection limit was 1 μm, and it had good system adaptability、accuracy、precision, and durability. The container closure integrity test results of the injection vial were all below the established acceptance criteria 1×10-6 mbar · L · s-1. Conclusion: The helium mass spectrometry leak detection method (vacuum mode) established in this paper could effectively test the container-closure integrity of the vial at low temperature conditions.
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