30 September 2026, Volume 46 Issue 9
    

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    Review & Monography
  • Recent advances in nanozyme-based pharmaceutical analysis*
    HUI Meng-ling, GONG Yu-ting, HE Meng-qi, LIANG Qiong-lin, AI Yong-jian
    Chinese Journal of Pharmaceutical Analysis. 2026, 46(9): 1561-1573. https://doi.org/10.16155/j.0254-1793.2025-0751
    Abstract ( )   Knowledge map   Save
    Nanozymes are a class of functional nanomaterials possessing enzyme-like catalytic activity. They offer advantages such as high stability, tunable activity, and low costs, and have demonstrated considerable potential in pharmaceutical analysis. This review systematically summarizes recent research progress in nanozyme-based pharmaceutical analysis. On the one hand, it summarizes the applications of nanozymes in pharmaceutical detection and analysis. The development of nanozyme-based analytical platforms and methods enables the interference-resistant detection of molecular drugs, antibody drugs, and nanomedicines with low detection limits and high sensitivity. These analytical platforms and methods have been widely applied in complex matrices such as urine, serum, and environmental samples. On the other hand, the review discusses the latest findings regarding the pharmacological activity of nanozymes, including their antibacterial, anti-inflammatory, and antitumor activities and therapeutic effects. Finally, the article explores the current challenges and future prospects in this field.
    • Ingredient Analysis
  • Changes in coumarin contents and production area differences in Peucedanum praeruptorum Dunn before and after bolting*
    GAN Xing-lin, YI Jian-huan, YAO Yi, ZHANG Xian-lin, SUN Le-ming, ZHENG Xian, WANG Huan, NI Liang
    Chinese Journal of Pharmaceutical Analysis. 2026, 46(9): 1574-1581. https://doi.org/10.16155/j.0254-1793.2025-0724
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    Objective: To establish an HPLC method for determining the contents of three coumarins, namely praeruptorin A, praeruptorin B, and praeruptorin E, in the roots, stems, and leaves of Peucedanum praeruptorum Dunn before and after bolting, and to analyze the differences in component contents among samples from different production areas and their accumulation patterns. Methods: An Amethyst C18-H column (250 mm×4.6 mm, 5 μm) was used for separation at a column temperature of 30 ℃. Methanol (A)-water (B) was used as the mobile phase for gradient elution. The detection wavelength was set at 321 nm, with a flow rate of 1.0 mL · min-1, and an injection volume of 10 μL. Results: The qualification rates of praeruptorin A and praeruptorin B in non-bolting roots were 50.0% and 42.8%, respectively. After bolting, they decreased to 21.4% and 28.6%, respectively, indicating that bolting affected the qualification rates of both components, with different degrees of change. The contents of individual coumarin components in the roots, stems, and leaves varied considerably at different growth and developmental stages. The total coumarin content ranked in the following order: non-bolting roots, bolting roots, bolting stems and leaves, non-bolting stems and leaves. Compared with non-bolting roots, the praeruptorin A content in roots after bolting decreased significantly [from (1.034 3±0.432 0)% to (0.669 8±0.384 5)%, P=0.004]. After bolting, the average praeruptorin B content decreased by 34.02% in roots but increased by 49.04% in stems and leaves, indicating that its response to bolting was weaker than that of praeruptorin A. The responses of praeruptorin A reduction varied among samples from 14 production areas, with a decrease range of 11.94%-70.77% and an RSD of 47.7%, indicating significant differences in its content among production areas. The total contents of the three coumarins in stems and leaves before and after bolting were equivalent to 53.09% and 83.76%, respectively, of those in roots, suggesting that stems and leaves have potential for comprehensive utilization. Conclusion: This study preliminarily revealed the changes in coumarin accumulation in P. praeruptorum at different growth and developmental stages. Bolting had a relatively pronounced effect on the praeruptorin A content and organ distribution in roots, providing data support for the traditional understanding that P. praeruptorum is unsuitable for medicinal use after bolting.
  • Content determination and methodological validation of reduced coenzyme Q10*
    WANG Jing, XU Shuo, XU Wen-feng, ZHANG Shan-shan, WANG Yue, GUO Si-rui, JIN Peng-fei
    Chinese Journal of Pharmaceutical Analysis. 2026, 46(9): 1582-1587. https://doi.org/10.16155/j.0254-1793.2025-0600
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    Objective: To establish a method for content determination of reduced ubiquind (coenzyme Q10, CoQ10) soft capsules based on high performance liquid chromatography (HPLC) by optimizing the conversion conditions of reduced CoQ10 to oxidized CoQ10. Methods: An Alltima C18 chromatographic column (150 mm×4.6 mm, 5 μm) was employed, column temperature at 35 ℃. The mobile phase consisted of methanol and anhydrous ethanol (50 ∶ 50) at a flow rate of 1.5 mL · min-1. The detection wavelength was set at 275 nm, and injection volume at 20 μL. The optimal conditions for the pretreatment were determined by screening the amount of oxidant (0.1% FeCl3 in ethanol) and the reaction time. The quantification was achieved by an external standard method through a calibration curve established with oxidized coenzyme Q10. The content of reduced coenzyme Q10 in the soft capsules was calculated based on the determined concentration of oxidized coenzyme Q10 in the sample solution after the oxidation treatment. Results: The optimal oxidation conditions were determined as follows: 0.5 mL 0.1% FeCl3 alcoholic solution was added to 50 mL reduced coenzyme Q10 test solution (0.2 mg · mL-1), and the reaction was conducted at room temperature (25 ℃) for 4 h. Under these conditions, reduced coenzyme Q10 in the system was completely converted to oxidized coenzyme Q10. Methodvalidation results demonstrated good linearity of reduced coenzyme Q10 in the range of 20.60 to 412.08 μg · mL-1. The method exhibited excellent precision and stability, with relative standard deviations (RSDs) of 0.011% and 0.44%, respectively. The average recovery rate of reduced coenzyme Q10 (n=9) was 100.1%, with an RSD of 1.2% (RSD<2.0%), indicating satisfactory recovery. The content of reduced coenzyme Q10 in soft capsules was determined by the established method to be 100.3%, with an RSD of 1.6%. Conclusion: The established HPLC method is accurate, simple, and rapid, which is suitable for the content determination of reduced coenzyme Q10 in soft capsules. It can provide a scientific basis for the quality control of reduced coenzyme Q10 in related products.
  • Pyrrolizidine alkaloid content in different medicinal parts of Crotalaria assamica*
    ZHANG Rong-lin, WU Yun, LIN Que-yue, ZAN Ke
    Chinese Journal of Pharmaceutical Analysis. 2026, 46(9): 1588-1594. https://doi.org/10.16155/j.0254-1793.2025-0750
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    Objective: To analyze the content differences of pyrrolizidine alkaloids in different medicinal parts of Crotalaria assamica, providing references for its safe utilization and resource development. Methods: A quantitative analysis method was established using ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) for senkirkine, integerrimine, integerrimine N-oxide, monocrotaline, and monocrotaline N-oxide in various medicinal parts. Samples were ultrasonically extracted with 0.5% formic acid aqueous solution, purified through a mixed cation exchange column, and separated using a Waters ACQUITY UPLC HSS T3 chromatography column (2.1 mm×150 mm, 1.8 μm). Isocratic elution was performed with a mobile phase of 0.5% formic acid aqueous-methanol (30 ∶ 70), mass spectrometry was conducted in positive ionization multiple reaction monitoring (MRM) mode, and quantification was achieved by the external standard method. Results: In the stems, branches, leaves, and roots of C. assamica, the senkirkine content ranged from 0.011 to 7.115 μg · g-1, integerrimine from 0.005 to 0.28 μg · g-1, integerrimine N-oxide from 0.013 to 0.43 μg · g-1, monocrotaline from 0.137 to 39.24 μg · g-1, and monocrotaline N-oxide from 0.23 to 22.25 μg · g-1. The results showed that alkaloid content was the highest in the root, followed by branches, while the lowest content was observed in leaves. Conclusion: This study reveals the relationship between pyrrolizidine alkaloid content and medicinal parts in C. assamica. This provides a scientific basis for its quality evaluation and medication safety, beneficial for guiding the rational development and utilization of C. assamica resources.
  • Quality control of Qinggan Jieyu decoction based on fingerprint and quantitative analysis*
    ZENG Wei-jie, GUO Xia, HU Xia-feng, CHEN Ying
    Chinese Journal of Pharmaceutical Analysis. 2026, 46(9): 1595-1607. https://doi.org/10.16155/j.0254-1793.2025-0347
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    Objective: The purpose of this study was to establish HPLC fingerprint of Qinggan Jieyu decoction, and to determine the contents of 18 index components, so as to provide reference for its quality control. Methods: An Athena C18 column (250 mm×4.6 mm, 5 μm) was used with gradient elution using acetonitrile (A)-0.15% phosphoric acid aqueous solution (B) as the mobile phase at a column temperature of 40 ℃, a flow rate of 1 mL · min-1, and a detection wavelength of 210 nm. Twelve batches of Qinggan Jieyu decoction were analyzed. Similarity evaluation was performed using the Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System, and common peaks were identified by reference standards. Based on the fingerprint similarity analysis and literature, a Waters ACQUITY UPLC HSS T3 column (100 mm×2.1 mm, 1.8 μm) was employed with gradient elution using acetonitrile (A)-water (B) as the mobile phase. The mobile phase contained 0.1% formic acid in positive ion mode and 0.01% formic acid in negative ion mode, respectively. The column temperature was 40 ℃, and the flow rate was 0.4 mL · min-1. An electrospray ionization source was used, and the contents of rehmanidine D, geniposide, paeoniflorin, verbascoside, liquiritin, ferulic acid, hesperidin, ginsenoside Rg1, ginsenoside Rb1, paeonol, glycyrrhizic acid, saikosaponin A, saikosaponin D, ligustilide, atractylenolide Ⅱ, atractylenolide Ⅰ, dehydropachymic acid and pachymic acid were quantified using precursor ion detection in both positive and negative ion modes. Results: The HPLC fingerprint of 12 batches of Qinggan Jieyu decoction was established. The similarity was above 0.916. 40 common peaks were marked, and 18 peaks were confirmed. The linear relationship of 18 components was good in the concentration range, and the linear correlation coellicients were above 0.999 1. The precision, repeatability and stability of the method were good. The average recoveries were in the range of 97.4%-99.6% with RSDs≤2.7%. The contents of rehmanidine D, geniposide, paeoniflorin, verbascoside, liquiritin, ferulic acid, hesperidin, ginsenoside Rg1, ginsenoside Rb1, paeonol, glycyrrhizic acid, saikosaponin A, saikosaponin D, ligustilide, atractylenolide Ⅱ, atractylenolide Ⅰ, dehydropachymic acid and pachymic acid in 12 batches were 91.5-157.8 μg · g-1, 1 228.2-2 660.1 μg · g-1, 1 251.0-2 122.5 μg · g-1, 204.7-391.1 μg · g-1, 239.6-356.3 μg · g-1, 248.9-451.8 μg · g-1, 2 185.3-3 517.2 μg · g-1, 165.7-363.7 μg · g-1, 149.7-313.8 μg · g-1, 662.7-1 152.4 μg · g-1, 393.4-788.4 μg · g-1, 128.3-281.4 μg · g-1, 139.1-287.1 μg · g-1, 530.9-1 082.0 μg · g-1, 110.2-274.0 μg · g-1, 143.9-330.3 μg · g-1, 461.1-875.4 μg · g-1, and 357.7-861.7 μg · g-1, respectively. Conclusion: The established HPLC fingerprint and UPLC-MS methods are rapid, simple and reproducible, which can provide reference for the quality evaluation of Qinggan Jieyu decoction.
  • Establishment of the UPLC fingerprint of Jichuanjian and simultaneous determination of its main components
    CHEN Jiang-ping, YU Xin-tong, YE Ze-hao, CHEN Wei-mei, LIU Quan-zhen, WANG Mei, LI Guo-wei
    Chinese Journal of Pharmaceutical Analysis. 2026, 46(9): 1608-1617. https://doi.org/10.16155/j.0254-1793.2025-0506
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    Objective: To establish the UPLC fingerprint of Jichuanjian and conduct comprehensive quality evaluation by combining the chemical pattern recognition method and quantitative analysis of multi-components by a single marker (QAMS). Methods: The analysis was performed on a 30 ℃ thermostatic YMC Triart C18 Column (150 mm×2.1 mm, 1.9 μm), with the mobile phase comprising of acetonitrile 0.1% phosphoric acid flowing at 0.20 mL · min-1 in a gradient elution manner, and the detection wavelength was at 310 nm. The UPLC fingerprints of 12 batches of Jichuanjian were established and their similarity was analyzed. The chemical pattern recognition analysis of multiple batches was conducted via cluster analysis (CA), principal component analysis (PCA), and orthogonal partial least squares-discriminant analysis (OPLS-DA). A QAMS method was established with naringin as the internal reference substance to simultaneously determine the content of seven chemical components (caffeic acid, echinacoside, ferulic acid, isoferulic acid, verbascoside, neohesperidin, and tangeretin). The accuracy of the QAMS results was evaluated by comparing with the external standard method (ESM) for the quantification of these seven components. Results: The methodological validation results met the requirements. The UPLC fingerprints of 12 batches of Jichuanjian were calibrated and identified with 8 common peaks, showing similarity values of 0.954-0.999 compared with the reference fingerprint. CA classified 12 batches of Jichuanjian samples into 3 categories, while PCA extracted 3 principal components that explained 82.056% of the variance in Jichuanjian’s chemical information. OPLS-DA identified 5 differential markers. QAMS was established with naringin as the internal reference substance, demonstrating good robustness for 7 components in Jichuanjian. There was no significant difference in the results between QAMS and ESM. Conclusion: The established UPLC fingerprint is accurate and feasible. The integration of chemical pattern recognition with QAMS can be applied to the quality control and quantitative analysis of Jichuanjian, providing a comprehensive reference for its overall quality evaluation.
    • Bioassay
  • Application of imaging capillary isoelectric focusing electrophoresis in analysis of charge heterogeneity of recombinant vaccines against COVID-19*
    SUN Yu-fei, QIN Xi, HU Han, WANG Yang, LI Xiang, LIU Bin-lei
    Chinese Journal of Pharmaceutical Analysis. 2026, 46(9): 1618-1629. https://doi.org/10.16155/j.0254-1793.2026-0089
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    Objective: To establish an imaging capillary isoelectric focusing electrophoresis (iCIEF) method for analyzing the charge heterogeneity of recombinant vaccines against coronavirus disease-2019 (COVID-19). Methods: With a recombinant vaccine product against COVID-19 as the study subject, iCIEF was performed in the ultraviolet fluorescence detection mode. The sample tray temperature was set at 10 ℃. Pre-focusing (first stage) was performed at a voltage of 1.5 kV for 1 min, and focusing (second stage) at a voltage of 3 kV for 8 min. Results: This method exhibited excellent specificity and effectively separated 10 charge variants of the recombinant vaccine product. In repeatability tests, the RSDs of peak area ratios for charge variants did not exceed 4.0%, while those of isoelectric points ranged from 0.017% to 0.061%. For intermediate precision, the RSDs of peak area ratios for charge variants did not exceed 3.5%, and those of isoelectric points ranged from 0.022% to 0.082%. The total peak area showed a linear correlation with sample concentration within the range of 0.5 mg · mL-1 to 1.5 mg · mL-1. The accuracy at low (50%), medium (100%), and high (150%) concentrations was 102.8%, 99.9%, and 103.2%, respectively. The limit of quantification and limit of detection for the largest isomer were 0.125 mg · mL-1 and 0.063 mg · mL-1, respectively. In stability tests over 48 h, the RSDs of peak area ratios of charge isomers did not exceed 8.0%, and those of isoelectric points ranged from 0.041% to 0.13%. Stability tests demonstrated good consistency in measurement results across different concentrations of amphoteric electrolyte treatments, with minimal changes in isoelectric point (drift≤0.03) and peak area ratio (difference≤0.60%) for all variants, indicating excellent method stability. Deglycosylation treatment results suggested that N-glycosylation exerted a more significant impact on variant characterization. This method was then employed to screen six batches of recombinant vaccines against COVID-19. For each charge variant, the percentage of the main peak area and the isoelectric point were largely consistent across all batches, indicating good batch-to-batch consistency of the vaccine. Conclusion: The established method can be used for differential analysis of charge heterogeneity of recombinant vaccines against COVID-19, which has positive significance for characterization, quality control, and process stability monitoring of recombinant vaccine products against COVID-19.
  • Separation, purification, and quality study of the degradation fragment of the recombinant human C1 esterase inhibitor
    YOU Xing-li, LI Chun-yang, XIE Jia-yi, DU Tian-fei, DENG Jie, GAO Ya-li, ZHAO Xiang-yu, YU Xiao-ping, LI Yan, CAI Feng
    Chinese Journal of Pharmaceutical Analysis. 2026, 46(9): 1630-1638. https://doi.org/10.16155/j.0254-1793.2025-0460
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    Objective: Degradation fragments are common product-related impurities in recombinant protein medicines, receiving significant attention from biopharmaceutical companies and regulatory agencies due to the fragments’ potential impact on product efficacy. Therefore, this study aimed to establish a strategy for purifying and characterizing trace degradation fragments using recombinant human C1 esterase inhibitor (rhC1INH) as an example. Methods: A late-eluting impurity peak (retention time longer than the main peak) detected by size exclusion chromatography-high performance liquid chromatography (SEC-HPLC) was enriched and purified from the rhC1INH bulk using preparative size-exclusion chromatography combined with ion-exchange chromatography. Additionally, physicochemical properties and activity of this impurity were analyzed by SDS-PAGE (sodium dodecylsulfate-polyacrylamide gel electrophoresis), activity assays, liquid chromatography-mass spectrometry (LC-MS), and Edman degradation. Results: The above impurity was successfully enriched and purified, with the purity increasing from the initial 0.5% to>99%. Identification results were as follows: SDS-PAGE confirmed a single band with a lower molecular weight than rhC1INH. Activity assays primarily confirmed the impurity as the rhC1INH degradation fragment with a specific activity reduced by approximately 40% reduction compared with the bulk. LC-MS analysis confirmed its identity as a degradation fragment, showing a sequence coverage from residues 85 to 478 of the theoretical sequence and an intact C-terminal sequence. Edman N-terminal sequencing matched residues 85 to 94 of the complete sequence of rhC1INH, consistent with the N-terminal sequence by the LC-MS. Conclusion: This study establishes the trace impurity preparation method and determines the impurity as a rhC1INH degradation fragment by multi-dimensional characterization techniques. Additionally, systematic analysis and identification were completed on the structural and physicochemical properties of rhC1INH degradation fragment, providing essential data for medicine quality control.
    • Metabolism Analysis
  • Analysis of the chemical constituents and serum-absorbed components of Shenzhu Qinghua mixture based on UHPLC-MS/MS*
    TANG Feng, DAI Zhen-yu, ZHANG Jia-lun, ZHAO Xiao-wei, YIN Ai-ling, ZHU Wei-na, TAN Yan-yan
    Chinese Journal of Pharmaceutical Analysis. 2026, 46(9): 1639-1651. https://doi.org/10.16155/j.0254-1793.2025-0792
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    Objective: To qualitatively analyze the chemical constituents and serum-absorbed components of the clinical empirical formula Shenzhu Qinghua mixture by ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). Methods: UHPLC-MS/MS was employed to identify the chemical constituents and serum-absorbed components of Shenzhu Qinghua mixture. A total of 30 C57BL/6J mice were randomized into a blank control group, and low and medium-dose Shenzhu Qinghua mixture groups. The low and medium-dose Shenzhu Qinghua mixture groups received an intragastric administration of the aqueous decoction at raw herb doses of 15 and 30 g · kg-1, respectively, while the blank group received an equivalent volume of normal saline. The administration in each group was performed for 7 consecutive days, and blood samples were collected 2 h after the final administration. Chromatographic separation was carried out through a Phenomenex Kinetex C18 column (2.1 mm×100 mm, 2.6 μm) at 40 ℃, with a mobile phase consisting of 0.1% formic acid in water (A) and acetonitrile (B) under gradient elution at a flow rate of 0.5 mL · min-1. The injection volume was 2 μL. Mass spectrometric detection was performed with an electrospray ionization (ESI) source over the mass range of m/z 70-1 200. The chemical constituents and serum-absorbed components of Shenzhu Qinghua mixture were characterized and identified based on the relative molecular mass, retention time, and MS/MS fragmentation patterns, by comparison with relevant literature data and reference standards. Results: A total of 80 chemical constituents were successfully characterized from Shenzhu Qinghua mixture, and 22 prototype compounds were detected as serum-absorbed components. Furthermore, the MS/MS fragmentation behavior of wogonoside, nobiletin, acacetin, L-tyrosine, oxyberberine, vidarabine, atractylenolide Ⅲ, and rutin was elucidated. Conclusion: The chemical constituents of Shenzhu Qinghua mixture are mainly flavonoids and alkaloids, with its primary serum-absorbed components also being flavonoids and alkaloids. UHPLC-MS/MS enables rapid, comprehensive, and accurate analysis, providing a reference for elucidating the clinical efficacy of traditional Chinese medicine.
  • Establishment and application of a method for determining the plasma concentration of zonisamide*
    LI Fa-shuang, LI Lin-bo, LI Hui-ying, YU Shao-chun, LEI Yan, LU Hong-min
    Chinese Journal of Pharmaceutical Analysis. 2026, 46(9): 1652-1660. https://doi.org/10.16155/j.0254-1793.2025-0558
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    Objective: To establish a method for determining the concentration of zonisamide in human plasma and employ it to monitor the plasma concentration of zonisamide in children with epilepsy. Methods: Phenobarbital was adopted as the internal standard. Plasma protein was precipitated with 7% perchloric acid, and the plasma concentration of zonisamide was determined by ultra-high performance liquid chromatography. Separation was performed through an Agilent InfinityLab Poroshell 120 EC-C18 column (50 mm×2.1 mm, 1.9 μm) at a column temperature of 40 ℃. Aqueous solution and acetonitrile solution were used as mobile phases for gradient elution at a flow rate of 0.25 mL · min-1. The detection wavelength was set at 200 nm, and the injection volume was 5 μL. This method was then employed to measure the plasma zonisamide concentrations in 48 pediatric epileptic patients treated with zonisamide, and the effects of gender, age, combined medication, liver and renal functions on plasma zonisamide concentrations were analyzed. Results: The plasma concentration of zonisamide showed a good linear relationship within the range of 0.50-100.00 μg · mL-1 (r=0.999 9), with a lower limit of quantification of 0.50 μg · mL-1. The intra-day precision and inter-day precision (relative standard deviations, RSDs) were both<12.2%, and the accuracy (relative error, RE) ranged from -4.48% to 11.34%. The recovery rate was between 91.3% and 103.6%, with RSD<12.9%. The plasma concentration of zonisamide in 48 children with epilepsy ranged from 5.57 to 17.23 μg · mL-1. Multivariate linear regression analysis showed that concomitant use of sodium valproate and age were factors influencing the plasma concentration of zonisamide (P<0.001). Conclusion: The established ultra-high performance liquid chromatography method can quickly and efficiently determine the plasma concentration of zonisamide in human plasma. When zonisamide is used to treat children with epilepsy, the effects of patient age and concomitant medications on the plasma concentration of zonisamide should be comprehensively considered, and the dosage of zonisamide should be adjusted reasonably.
  • Determination of 18 benzodiazepines in hair based on UPLC-QE-Orbitrap MS*
    LI Xiang, LIN Jia-yu, ZHU Yu, XU Ying-jian
    Chinese Journal of Pharmaceutical Analysis. 2026, 46(9): 1661-1669. https://doi.org/10.16155/j.0254-1793.2025-0597
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    Objective: To establish an analytical method for the qualitative and quantitative determination of 18 benzodiazepines in hair based on ultra-high performance liquid chromatography-quadrupole/electrostatic field orbitrap mass spectrometry (UPLC-QE-Orbitrap MS). Methods: Hair samples were extracted with 1.0 mL methanol. Under parallel reaction monitoring (PRM) mode, the gradient of the mobile phase and the collision voltage were optimized to ensure good separation and high sensitivity for 18 benzodiazepines. The Hypersil GOLD Vanquish column (100 mm×2.1 mm, 1.9 μm) was used with a column temperature of 30 ℃ and the mobile phase consisted of 0.1% formic acid aqueous solution and 0.1% acetonitrile in formic acid. Four structural types of benzodiazepines were analyzed by mass spectrometry in ESI positive ion mode, and the ESI-MS/MS fragmentation patterns of 1,4-benzodiazepines, triazolobenzodiazepines, thienotriazolodiazepines, and thienodiazepines were deduced. Results: The 18 benzodiazepines exhibited good linear relationships, with correlation coefficients greater than 0.999 0. The method showed the limits of detection ranging from 0.000 5 to 0.003 ng · mg-1 and the limits of quantification from 0.002 to 0.01 ng · mg-1. Recovery rates at three addition levels ranged from 78.9% to 95.3%, and the matrix effect ranged from 83.2% to 106.5%. The intra-day and inter-day relative standard deviations were 1.1%-6.5% and 2.4%-8.9%, respectively. Conclusion: This method is simple to operate and has good reproducibility. The results provide a theoretical basis and methodological reference for drug abuse and drug-assisted crime cases.
    • Quality Control
  • Quality evaluation of the Xizang medicinal herbs Gentiana veitchiorum and Gentiana trichotoma based on fingerprinting and multi-component quantification*
    LI Yue, PENG Si-rong, WANG Jun-jun, WULU Jian-sang, GAO Wen-hong, WANG Wei, ZHANG Zhi-feng
    Chinese Journal of Pharmaceutical Analysis. 2026, 46(9): 1670-1680. https://doi.org/10.16155/j.0254-1793.2025-0678
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    Objective: To establish the fingerprints of the Xizang medicinal herbs Gentiana veitchiorum Hemsl. and Gentiana trichotoma Kusnez. and establish the method for determining the content of seven constituents (loganic acid, swertiamarin, gentiopicroside, sweroside, isoorientin, isovitexin, and isoscoparin), thereby providing reference for the quality control and evaluation of the two medicinal herbs. Methods: The fingerprints of 22 batches of Gentiana were established via the UPLC method. Specifically, gradient elution was performed through the ACQUITYUPLC®HSS C8 column (100 mm×2.1 mm, 1.7 μm) with the mobile phase composed of methanol and 0.2% formic acid aqueous solution at the column temperature of 30 ℃, the flow rate of 0.1 mL · min-1, and the injection volume of 1 μL. The detection wavelength was 254 nm. Cluster analysis (CA), principal components analysis (PCA), and orthogonal partial least squares-discriminant analysis (OPLS-DA) were performed for quality evaluation. Results: The fingerprints contained 19 common peaks, of which 7 peaks were identified as loganic acid, swertiamarin, gentiopicroside, sweroside, isoorientin, isovitexin, and isoscoparin. The similarity among the 22 batches of samples ranged from 0.797 to 0.957. Methodological investigation showed that the seven compounds exhibited good linear relationships (r>0.999), and the mean spiked recovery rates ranged from 96.6% to 130.4%. The results of CA showed that G. trichotoma samples clustered into one group and G. veitchiorum samples clustered into two groups. The first three principle components of PCA showed a cumulative contribution rate of 86.83%, explaining most information of the raw data. OPLS-DA screened out 10 differential constituents. Conclusion: This study predicts the potential Q-markers of G. veitchiorum and G. trichotoma based on fingerprinting and establishes the content determination method. The findings provide a scientific basis for evaluating the quality of G. veitchiorum and G. trichotoma and lay a theoretical foundation for the study of quality standards of traditional Chinese medicine.
  • Botanical origin authentication of Astragali Radix based on the concept of “digital identity” for traditional Chinese medicine*
    LI Wen, LI Ming-hua, JING Wen-guang, YU Kun-zi, YANG Jian-bo, YIN Meng-qi, WANG Xian-rui, CHEN Jia, WEI Feng, CHENG Xian-long, LIU Xiao-qiu
    Chinese Journal of Pharmaceutical Analysis. 2026, 46(9): 1681-1689. https://doi.org/10.16155/j.0254-1793.2025-0769
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    Objective: To establish an ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q TOF MS) method for the accurate, objective, and digital authentication of the botanical origin of Astragali Radix. Methods: A Waters ACQUITY UPLC HSS T3 column (100 mm×2.1 mm, 1.7 μm) at a column temperature of 35 ℃ was used for gradient elution with the mobile phase composed of methanol (A) and 0.05% formic acid in water (B) at a flow rate of 0.3 mL · min-1. The injection volume was 2 μL, and detection was performed in electrospray positive/negative ion switching multiple reaction monitoring (MRM) mode. UPLC-Q TOF MS was employed to perform mass spectrometry analysis on Astragalus membranaceus (Fisch.) Bge.var. mongholicus (Bge.) Hsiao and Astragalus membranaceus (Fisch.) Bge. from different production areas and batches. Peak extraction and quantification were conducted by Progenesis QI software to obtain a data matrix comprising retention time, exact mass, and ion abundance. Then, the “digital identities” of A. membranaceus mongholicus and A. membranaceus were constructed and obtained based on the concept of “digital identity” for Chinese materia medica. Finally, the digital matching was performed based on the “digital identities” of A. membranaceus mongholicus and A. membranaceus, thereby providing feedback on matching credibility and enabling evaluation. Results: The matching credibility of A. membranaceus mongholicus and A. membranaceus were both greater than 76% when compared with their own “digital identities,” but no more than 10% when compared with non-own “digital identities.” Nonparametric statistical analysis revealed a P-value less than 0.01, indicating a statistically significant difference. Evaluation analysis based on confusion matrix showed that all assessment parameters including accuracy, precision, and specificity achieved 100%. Conclusion: The “digital identity” of A. membranaceus mongholicus and A. membranaceus can realize the efficient and accurate digital botanical origin authentication of Astragali Radix, providing support for the quality control of Astragali Radix.
  • Study on the dissolution determination method for vitamin D3 soft capsules with lauryl dimethylamine oxide as the medium
    CHEN You-zhong, WANG Juan, CHEN Yu-xiang, JIANG Ning-yu, CHEN Wei-bin, WEI Zong-you, ZHOU Jing-ming
    Chinese Journal of Pharmaceutical Analysis. 2026, 46(9): 1690-1696. https://doi.org/10.16155/j.0254-1793.2026-0003
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    Objective: To establish a novel method for the dissolution profiling of vitamin D3 soft capsules. Methods: With lauryl dimethylamine oxide (DDAO) aqueous solution as the dissolution medium, the effects of different DDAO concentrations (0.5%-1.5%), alkali concentrations (0-1.0 mol · L-1), and agitation speeds (100-200 r · min-1) on the dissolution behavior of vitamin D3 soft capsules were investigated. The dissolution samples were analyzed by high performance liquid chromatography (HPLC), and the cumulative dissolution percentage was calculated. An Agilent Zorbax Eclipse XDB-C8 chromatographic column (150 mm×4.6 mm, 5 μm) was used, with a column temperature of 35 ℃, a mobile phase of methanol-water (96 ∶ 4), a flow rate of 1 mL · min-1, a detection wavelength of 265 nm, and an injection volume of 100 μL. Results: The optimal dissolution conditions were determined as follows, 1.0% DDAO aqueous solution as the dissolution medium, an agitation speed of 150 r · min-1, and a temperature of 37 ℃. The dissolution study demonstrated that this method could effectively differentiate the dissolution behaviors of vitamin D3 soft capsules of different manufacturers, different strengths (400/800 IU), and different batches, showing good discriminatory power. Conclusion: The established dissolution method with 1.0% DDAO as the medium is simple to operate and exhibits high analytical sensitivity. It provides a reliable analytical means for the process optimization and quality improvement of vitamin D3 soft capsules.
    • Process Evaluation·Standard Deliberation
  • Optimization of extraction process for nucleoside components from processed Pinelliae Rhizoma products using AHP-entropy weight method combined with Box-Behnken response surface methodology*
    WANG Bin, LI Yu-zhen, XIAO Huai-qiu, XIE Lian-wu, LIAO Qiong, LING Xian-hua
    Chinese Journal of Pharmaceutical Analysis. 2026, 46(9): 1697-1706. https://doi.org/10.16155/j.0254-1793.2025-0728
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    Objective: To optimize the accelerated solvent extraction (ASE) process of nucleoside components from processed Pinelliae Rhizoma products. Methods: Using ginger-processed Pinelliae Rhizoma as the representative sample, a high-performance liquid chromatography (HPLC) method was established. The chromatographic separation was performed on a Shim Nex CS-C18 chromatographic column (250 mm×4.6 mm, 5 μm) with methanol (A)-0.1% acetic acid aqueous solution (B) as the mobile phase in gradient elution at a flow rate of 1.0 mL · min-1 and a column temperature of 30 ℃. The detection wavelength was set at 260 nm, and the content of eight nucleoside components, namely uracil, cytidine, hypoxanthine, uridine, inosine, guanosine, thymidine, and adenosine, was simultaneously determined. The comprehensive weight values were determined by combining the analytic hierarchy process (AHP) and the entropy weight method. On the basis of single-factor experiments, the Box-Behnken response surface methodology (RSM) was employed to comprehensively optimize the key extraction parameters, including extraction temperature, time, pressure, and number of extraction cycles. Results: The optimal process conditions were determined as follows: extraction temperature 91.0 ℃, extraction time 8 min, pressure 10 MPa, and 3 extraction cycles. Under these conditions, the overall desirability (OD) for extraction efficiency reached 95.984 9, which was close to the model-predicted value (96.070 0), with a relative deviation of only 0.09%. Conclusion: The combination of AHP-entropy weight method and Box-Behnken response surface methodology can accurately and rapidly optimize the extraction process of nucleoside components from Pinelliae Rhizoma. This process provides universal reference value for different processed products, including ginger-processed Pinelliae Rhizoma, licorice-processed Pinelliae Rhizoma, and alum-processed Pinelliae Rhizoma, and offers efficient and green technical support for the development of extracts and related preparations from processed Pinelliae Rhizoma products.
  • HPLC fingerprint and multi-component content analysis of chemical components in Chebulae Fructus before and after processing with Amygdali Dulcis Oil*
    FAN Chen-yang, YELXAT Danabek, GUO Qiao-hui, DILINUER Tuerxunjiang, HE Jiang
    Chinese Journal of Pharmaceutical Analysis. 2026, 46(9): 1707-1718. https://doi.org/10.16155/j.0254-1793.2025-0802
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    Objective: To investigate the effects of processing with Amygdali Dulcis Oil on the chemical components in Chebulae Fructus and establish a quality evaluation method for the processed product. Methods: HPLC fingerprints of Chebulae Fructus were established. A Caprisill C18-B chromatographic column (250 mm×4.6 mm, 5 μm) was used for gradient elution with the mobile phase composed of 0.05% phosphoric acid aqueous solution and acetonitrile at a flow rate of 0.6 mL · min-1. The detection wavelength was set at 270 nm, the column temperature was maintained at 30 ℃, and the injection volume was 3 μL. Principal component analysis (PCA) and orthogonal partial least squares-discriminant analysis (OPLS-DA) were adopted for pattern recognition of the chromatographic data, and the differential components were screened. On this basis, a method was established for the simultaneous quantification of five components—gallic acid, corilagin, chebulagic acid, chebulinic acid, and 1,2,3,4,6-penta-O-galloyl-β-D-glucose. On the basis of chromatographic conditions for fingerprinting, the flow rate was adjusted to 0.8 mL · min-1, and the column temperature to 35 ℃. Meanwhile, the gradient elution program was optimized. The chemical composition was compared between crude Chebulae Fructus and its Amygdali Dulcis Oil-processed product. Results: HPLC fingerprints of crude and oil-processed Chebulae Fructus were established, with 16 common peaks identified. PCA and OPLS-DA effectively distinguished the crude from the oil-processed samples, and 10 components with VIP>1 were identified as differential markers. Component quantitative analysis revealed that the content of gallic acid was significantly increased after processing, reaching approximately 4.66 times that of the crude product, while the content of corilagin, chebulagic acid, chebulinic acid, and 1,2,3,4,6-penta-O-galloyl-β-D-glucose decreased by 57.32%, 44.31%, 46.54%, and 34.92%, respectively. The five markers all showed good linearity, precision, stability, repeatability, and spiked recovery (r≥0.999 7, RSD≤2.9%). Conclusion: Processing with Amygdali Dulcis Oil significantly alters the chemical profile of Chebulae Fructus. The established fingerprint and multi-component quantification method can be used for the quality control of the processed product, providing a scientific basis for the clinical application and further research on the processing mechanism of Chebulae Fructus.
    • Safety Monitoring
  • Related substances in methotrexate and its preparations*
    WU Jing-fang, SHI Ying, ZHANG Juan, LIU Yi
    Chinese Journal of Pharmaceutical Analysis. 2026, 46(9): 1719-1726. https://doi.org/10.16155/j.0254-1793.2026-0303
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    Objective: To establish a high performance liquid chromatography (HPLC) method for the determination of related substances in methotrexate and its preparations, to identify the sources of impurities through impurity profiling, and to propose control strategies. Methods: A Waters XBridge C18 column (250 mm×4.6 mm, 5 μm) at a column temperature of 30 ℃ was used for gradient elution with a mobile phase consisting of 0.34% sodium dihydrogen phosphate anhydrous solution (pH 5.6) and acetonitrile at a flow rate of 1.0 mL · min-1. The detection wavelength was set at 280 nm, and the injection volume was 20 μL. Results: Baseline separation was achieved between methotrexate and six known impurities. Forced degradation studies indicated that impurities B and C were degradation impurities, while impurity D, impurity E, impurity I, and impurity H were process impurities. Good linearity was observed within the concentration range from the limit of quantification (LOQ) to 200% of the specification limit (r>0.999 9, n=7). The mean recovery (n=9) ranged from 98.5% to 102.1%. The limit of detection (LOD) was 4-9 ng · mL-1. The method demonstrated good precision, stability, and robustness. Impurity content was calculated through the main component self-reference method without correction factor. Impurity C was detected in all 16 batches of active pharmaceutical ingredient (API) and 7 batches of preparations, and the impurity content in preparations showed a positive correlation with the impurity content in the corresponding APIs used. Conclusion: The HPLC method established in this study for the determination of related substances is simple, accurate, and efficient. On the basis of impurity profiling, a “source-oriented” control strategy is proposed, providing technical support for the revision of standards and quality control of methotrexate and its preparations.
  • Impurity profile study of domestic famciclovir*
    GUO Wei-bin, XU Yong-bin, HU Meng-shen, ZHENG Shu-feng
    Chinese Journal of Pharmaceutical Analysis. 2026, 46(9): 1727-1734. https://doi.org/10.16155/j.0254-1793.2025-0542
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    Objective: To investigate and establish the impurity profile of domestic famciclovir drug substance. Methods: Preparative liquid chromatography was performed using a YMC-Actus Triart C18 column (250 mm×20 mm, 5 μm), with water-acetonitrile as the mobile phase and a flow rate of 5 mL · min-1 linear gradient elution to enrich the related impurities. The related impurities of famciclovir were identified by high-performance liquid chromatography-high-resolution Orbitrap mass spectrometry (HPLC-HR Orbitrap MS) using a Phenomenex Kinetex C18 column (150 mm×2.1 mm, 1.7 μm), with the column temperature maintained at 35 ℃, water (pH adjusted to 4.0 with formic acid)-acetonitrile as the mobile phase, linear gradient elution, and a flow rate of 0.4 mL · min-1. Mass spectrometry was performed using an electrospray ionization source in positive ion mode. Impurity profile analysis was performed using a Phenomenex Luna C18(2) column (250 mm×4.6 mm, 5 μm), with the column temperature maintained at 35 ℃, 0.02 mol · L-1 potassium dihydrogen phosphate solution (pH adjusted to 4.0 with phosphoric acid)-acetonitrile as the mobile phase, linear gradient elution, and a flow rate of 1.0 mL · min-1. Results: The impurity profile of domestic famciclovir was established. Twenty related impurities were identified, of which four were reported for the first time, and a typical chromatogram of related substances was obtained. Conclusion: This study establishes the impurity profile of domestic famciclovir drug substance, providing a basis for improving the standards for famciclovir.
  • Determination of 21 elemental impurities in levofloxacin lactate and sodium chloride injection packaged with different packaging materials by ICP-MS*
    DING Fang-fang, WU Si, AI Jie, SHAO Peng, GU Xiao
    Chinese Journal of Pharmaceutical Analysis. 2026, 46(9): 1735-1741. https://doi.org/10.16155/j.0254-1793.2026-0025
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    Objective: To develop an inductively coupled plasma-mass spectrometry (ICP-MS) method for the determination of 21 elemental impurities in levofloxacin lactate and sodium chloride injection and investigate the migration of elemental impurities from different packaging materials (five-layer coextrusion infusion bags, polypropylene plastic bottles, and soda-lime glass bottles). Methods: Samples were diluted with a mixed solution of 5% nitric acid and 1 μg · mL-1 gold and directly analyzed by ICP-MS with Sc, Ge, In, and Bi as internal standards. The RF power was set at 1 550 W and the carrier gas flow rate was set at 1.05 L · min-1. Results: All the 21 elemental impurities exhibited good linear relationships (r>0.999). Limits of detection were between 0.001 ng · mL-1 and 2.919 ng · mL-1. The average recovery rates were between 94.2% and 105.6% (n=9). The RSDs of the repeatability test were between 2.6% and 6.0% (n=6). The content of Mg, Al, and Si in the injection samples packaged with soda-lime glass bottles from the same manufacturer was higher than that in the injection samples packaged with five-layer co-extrusion infusion bags. The results of the accelerated test showed that the content of Al and Si in the injection samples packaged with soda-lime glass bottles exhibited an increasing trend over storage time. The content of 21 elemental impurities in all the tested samples packaged with different packaging materials and the accelerated test samples complied with ICH requirements. Conclusion: The established method is simple, rapid, sensitive, accurate, and applicable for the elemental impurity control of levofloxacin lactate and sodium chloride injection and can be extended to the analysis of elemental impurities in other drugs with similar properties.
  • Determination methods of related substances in propranolol hydrochloride raw materials and tablets*
    LU Li, HUANG Qing, SHAO Fang-xian, XU Hui, HUANG Chao-yu, LI Rui, YAN Fei
    Chinese Journal of Pharmaceutical Analysis. 2026, 46(9): 1742-1753. https://doi.org/10.16155/j.0254-1793.2025-0591
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    Objective: To establish a high performance liquid chromatography (HPLC) method for the determination of related substances in propranolol hydrochloride raw materials and tablets and investigate the impurity profiles of these products, thus providing a reference and basis for formulating scientific and rigorous quality standards and quality control strategies. Methods: A pentafluorophenyl-bonded silica Agilent Poroshell 120 PFP column (150 mm×4.6 mm, 4 μm) was used as the stationary phase. The mobile phase consisted of 0.1% trifluoroacetic acid aqueous solution (phase A) and acetonitrile-methanol (9 ∶ 1) (phase B). Gradient elution was carried out with the flow rate of 0.8 mL · min-1, the column temperature of 20 ℃, the injection volume of 20 μL, and the detection wavelength of 272 nm. The impurity content was calculated by the main component self-control method without correction factors. Results: Simultaneous separation and accurate quantification of 11 organic impurities (two pairs of positional isomeric impurities and four impurities with genotoxic alert structures) in propranolol hydrochloride were achieved. Among 178 batches of national drug sampling samples of propranolol hydrochloride tablets and 10 batches of raw materials in 2024, impurity A, impurity B, impurity C, β-propranolol, and 1-naphthol were detectable. Impurity B was the known impurity with the highest detected amount, and photodegradation impurity Ⅰ was detected in individual batches. For all generic tablets, the content of each individual impurity was below 0.2%, and the total impurity content was below 0.5%. The forced degradation tests were conducted to infer the sources of each impurity. The genotoxicity (mutagenicity) of the impurities was predicted by Derek and Sarah software. Impurities with genotoxic alert structures (α-etherate, β-etherate, 1,4-naphthoquinone, and impurity Ⅰ) were not detected. On the basis of the sample detection results, impurity toxicity assessment, and the types and limits of impurities controlled by various standards, the control limits for each impurity were proposed. The limits for genotoxic impurities α-etherate, β-etherate, 1,4-naphthoquinone, and impurity I were 0.000 65%, and that for each of the remaining impurities was 0.2%. Conclusion: The established method has high sensitivity, strong specificity, and good robustness, with accurate and reliable experimental results, being suitable for the inspection of related substances in propranolol hydrochloride raw materials and tablets. This study provides a scientific reference for the impurity control of propranolol hydrochloride raw materials and tablets.
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