31 July 2024, Volume 44 Issue 7
    

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    Review & Monography
  • Research progress on the mechanism of action and bioactivity methods of human thyroid-stimulating hormone*
    GAO Jing, LIANG Cheng-gang, LI Jing
    Chinese Journal of Pharmaceutical Analysis. 2024, 44(7): 1105-1112. https://doi.org/10.16155/j.0254-1793.2024-0226
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    Thyroid-stimulating hormone (TSH) is a glycoprotein hormone produced by the anterior pituitary. It can regulate the synthesis and secretion of thyroid hormone in thyroid follicular cells, which has important physiological significance. As a drug, it has important application value. Biological activity detection is an effective and necessary to evaluate its quality. This article discusses the signal transduction mechanism of thyroid stimulating hormone, the clinical diagnosis and treatment of thyroid stimulating hormone, and the determination method of biological activity.
  • Application of LC-MS in the analysis of therapeutic oligonucleotide drugs*
    LI Li-li, WU Ni, XI Wan-lin, ZHAI Bao-qi, LI Xiao, LIU Ping-lan, SONG Hong-tao, ZHAO Qian
    Chinese Journal of Pharmaceutical Analysis. 2024, 44(7): 1113-1124. https://doi.org/10.16155/j.0254-1793.2023-0431
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    Therapeutic oligonucleotides (OGNs) drugs are artificially synthesized single or double stranded short nucleic acids, typically 15 to 30 base pairs in length. OGNs have been rapidly developed as new therapeutic drugs with increasing attention in the discovery and development of drugs concerning various disease fields. Compared with Europe and America, there are currently no other OGNs drugs listed in China, except for Spinraza, which has been approved for marketing as an orphan drug. The development of OGNs in China started relatively late and is still in its early stages of development. However, the OGNs drug market in China is anticipated to grow quickly due to the country's large population, high patient demand, ongoing support for the development of oligonucleotide drugs in the future, and the steady maturation of related technologies by domestic businesses. Because of their special physicochemical characteristics, OGNs drugs are challenging to design biological analysis techniques. Currently, there are few reports on quantitative analysis methods for oligonucleotide drugs in China. Therefore, the development of sensitive and reliable bioanalysis methods for oligonucleotides is the key to investigate oligonucleotides' pharmacokinetic and pharmacodynamic properties. Liquid chromatography-mass spectrometry (LC-MS) can quantify OGNs and their metabolites concurrently, compared with traditional ELISA approaches. Numerous benefits come with using LC-MS, in particular, the extensive use of high-resolution mass spectrometry allows for the identification of metabolites, which provides details on base composition and sequence structure, in addition to quantitative information about target oligonucleotides. It has now emerged as the go-to technique for OGN quantitative analysis. The application of LC-MS in the identification of therapeutic oligonucleotide medicines is the primary focus of this paper, which also discusses its benefits and drawbacks. Lastly, it looks at the LC-MS development trend for oligonucleotide detection, which includes a lower detection level and potential general methods.
    • Ingredient Analysis
  • HPLC fingerprint of substance benchmarks in Gualou Guizhi decoction and quantity transfer rule of key quality attributes*
    LIU Ke, LÜ Gui-jie, WANG Xi-lin, XIE Yu-he, XU Wen, YANG Yi, ZHANG Xun, LIN Yu
    Chinese Journal of Pharmaceutical Analysis. 2024, 44(7): 1125-1136. https://doi.org/10.16155/j.0254-1793.2023-0694
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    Objective: To establish a fingerprint of Gualou Guizhi decoction and a method for determination of multi-component to clarify the transfer rule of quantities and quality from decoction pieces and substance benchmarks. Methods: Fifteen batches of Gualou Guizhi decoction substance benchmarks were prepared and analyzed by the method of HPLC. Traditional Chinese medicine (TCM) Chromatographic Fingerprint Similarity Evaluation Software (2012) and hierarchical cluster analysis (HCA), principal component analysis (PCA) and orthogonal partial least squares-discriminant analysis (OPLS-DA) was used to evaluate the quality differences between batches of substance benchmarks, and screen the main chemical compositions that were led to quality discrepancy. The main differential components were determined by HPLC. And their contents, yields and transfer rates were analyzed for transfer rule of quantities and quality. Results: HPLC fingerprint of Gualou Guizhi decoction was established. A total of 30 common peaks in the fingerprint of Gualou Guizhi decoction substance benchmarks were assigned. Compared with the reference standards, 9 components were identified as gallic acid, albiflorin, paeoniflorin, liquiritin, ononin, liquiritigenin, cinnamic acid, isoliquiritigenin and glycyrrhizic acid. The similarities of 15 batches of Gualou Guizhi decoction substance benchmarks samples were all above 0.920. All of them were divided into two categories by three chemical recognition modes for 5 major differential components, including glycyrrhizic acid, liquiritigenin, paeoniflorin, iquiritin, and ononin. The HPLC was also applied to determine the contents of multi-components and the method validation results were good. The average transfer rates of five index components were 51.65%, 40.46%, 74.74%, 60.06%, and 34.54%, respectively and their average extraction rate was 14.20%. Conclusion: The critical quality properties of Gualou Guizhi decoction can be stably transferred from decoction pieces to substance benchmarks. The method of fingerprint and content determination is accurate and reliable, which is able to provide technique for quality control of Gualou Guizhi decoction formulation.
  • Determination of six active components in Tripterygii Radix from different habitats by RP-UPLC-PDA*
    ZHOU Guo-liang, SU Shu-lan, SHANG Er-xing, QIAN Da-wei, DUAN Jin-ao, YU Hao
    Chinese Journal of Pharmaceutical Analysis. 2024, 44(7): 1137-1144. https://doi.org/10.16155/j.0254-1793.2023-0298
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    Objective: To establish the RP-UPLC-PDA method for simultaneous determination of triptolide, triptonide, triptophenolide, wilforine, wilforlide A and celastrol in Tripterygii Radix. Methods: Tripterygii Radix were extracted with ethyl acetate and the extracts were dissolved and separated by methanol. The six components were determined by RP-UPLC-PDA method. The chromatographic column was AcquityTM UPLC BEH C18 column (100 mm×2.1 mm, 1.7 μm), the column temperature was 30 ℃, the injection volumn was 2 μL, the flow rate was 0.4 mL·min-1 and the mobile phase was acetonitrile (A)-0.1% formic acid (B). Results: The linear relationship of six components was good (0.999 2≤r≤0.999 7) in the concentration ranges. The average recoveries were 99.2%-103.1% and RSDs were 1.2%-2.9%. The contents in 10 batches of Tripterygii Radix from different habitat were determined. The results showed that the contents of Tripterygii Radix in prepared pieces from different producing areas were different. The highest content of triptolide was 140.2 μg·g-1, and the lowest content was 103.2 μg·g-1. The highest content of triptonide was 224.7 μg·g-1 and the lowest content was 112.2 μg·g-1. The highest content of triptophenolide was 306.7 μg·g-1 and the lowest content was 189.6 μg·g-1. The highest and lowest contents of wilforine were 283.2 μg·g-1 and 211.2 μg·g-1. The highest content of wilfhabitat was 31.2 μg·g-1 and the lowest content was 16.8 μg·g-1. The highest content of celastrol was 87.6 μg·g-1, and the lowest content was 52.1 μg·g-1. Conclusion: The RP-UPLC-PDA method can simultaneously determine six components in Tripterygii Radix. The method is reliable and stable, which is suitable for quantitative analysis and determination of Tripterygii Radix.
  • Analyze and optimize the stir-baked with vinegar technology of Genkwa Flos decoction pieces by orthogonal experiment and multi-index weighting method*
    ZHANG Ping, MI Hong-ying, YAN Hua, WEI Feng, GAO Hui-yuan, MA Shuang-cheng
    Chinese Journal of Pharmaceutical Analysis. 2024, 44(7): 1145-1153. https://doi.org/10.16155/j.0254-1793.2023-0555
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    Objective: To analyze and optimize stir-baked with vinegar technology of Genkwa Flos decoction pieces by multi-index weighted scoring method based on orthogonal experiment, and to provide technical index on standardization of stir-baked with vinegar technology of Genkwa Flos decoction pieces. Methods: The orthogonal experiment was employed to analyze and evaluate the four key factors related to stir-baked with vinegar technology including the amount of vinegar, dampening time, temperature and stir-baked time by the six main indicators including the total contents of chlorogenic acid, tiliroside, luteolin, apigenin, hydroxygenkwanin and genkwanin, the content of alcohol extractive and appearance characters. Results: The amount of vinegar and stir-baked temperature had notable effects, while the dampening time and stir-baked time had no notable effects on the test results. Considering comprehensively the effects of four key factors, the optimized stir-baked with vinegar technology was as follows: add 0.3 kg of vinegar to 1 kg of Genkwa Flos, and fry at 160 ℃ for 10 min after dampening for 15 min. Conclusion: This study offers guidance and reference for standardizing the stir-baked with vinegar technology of Genkwa Flos decoction pieces.
  • Optimization of HPLC with cyclodextrin as the mobile phase additive for the determination of ursolic acid and oleanolic acid in Corni Fructus by molecular design and response surface method*
    YANG Ruo-meng, TIAN Jun, LI Yun-zhe
    Chinese Journal of Pharmaceutical Analysis. 2024, 44(7): 1154-1160. https://doi.org/10.16155/j.0254-1793.2023-0789
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    Objective: To establish high-performance liquid chromatography method using cyclodextrin as a mobile phase additive for the determination of ursolic acid and oleanolic acid in Corni Fructus, providing a basis for quality control of Corni Fructus. Methods: Molecular design and response surface method were used to optimize the determination method of ursolic acid and oleanolic acid in Corni Fructus. Agilent Eclipse XDB C18 column (150 mm×4.6 mm, 5 μm) was adopted as the stationary phase, the mobile phase was 3 mmol·L-1 γ-cyclodextrin solution (contained 0.05% phosphoric acid)-acetonitrile (60∶40), the flow rate was 1.0 mL·min-1, the column temperature was 25 ℃ and the detecting wavelength was 210 nm. Results: The resolution of chromatographic peaks of ursolic acid and oleanolic acid was 3.81. Linearities were good in the range of 400-4 000 ng for ursolic acid and in the range of 200-2 000 ng for oleanolic acid. The RSDs for precision, reproducibility, and stability were less than 1.4%, 1.8%, and 1.7%, respectively. The recovery rates were 98.5% (RSD=1.7%) and 99.1% (RSD=2.0%). The contents of ursolic acid in three batches of Corni Fructus were 2.66 mg·g-1, 2.35 mg·g-1 and 2.91 mg·g-1, and those of oleanolic acid were 0.83 mg·g-1, 0.78 mg·g-1and 1.12 mg·g-1, respectively. Conclusion: This method is simple, accurate, reliable, and environmental friendly, and can be used as a quality control for Corni Fructus.
  • HPLC determination of the content and the related substances of compound amino acid injection(3AA)*
    ZUO Li-min, Ruxianguli·Yiming, GUO Xin, XIAO Jing, XU Shi-jie, ZHAO Ting, LIAN Xiao-fang, LIU Hui-yi, ZHOU Yi, SHAN Guang-zhi
    Chinese Journal of Pharmaceutical Analysis. 2024, 44(7): 1161-1168. https://doi.org/10.16155/j.0254-1793.2023-0795
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    Objective: To establish an HPLC method of the content and related substances of compound amino acid injection(3AA). Methods: RP-HPLC was adopted to determine compound amino acid injection(3AA), combining with the use of two-dimensional column switching-LC/MSn method was applied to separate and identify the impurities. The determination was performed on Capcell PAK AQ C18(250 mm×4.6 mm, 3 μm) column with 0.2 mol·L-1 sodium dihydrogen phosphate solution (adjusted pH to 2.8 with phosphoric acid) -acetonitrile (98∶2) as mobile phase at the flow rate of 1.0 mL·min-1. The column temperature was 40 ℃, and the detection wavelength was 210 nm. And the injection volume was 20 μL. The LC/MSn method was performed on a Thermo Accucore AQ C18 (100 mm×4.6 mm, 2.6 μm) column with 0.1% formic acid solution as mobile phase A, 0.1% formic acid solution-acetonitrile as mobile phase B, at a flow rate of 0.4 mL·min-1, and at a column temperature of 40 ℃. The mass spectrometry conditions were performed using an ESI ionisation source in the positive-ion scanning mode with a scan range of m/z 100-1 000, and the secondary mass spectrum was carried out by data-dependent scanning. Results: The related substances were completely separated from the main constituents in RP-HPLC. The standard curve of valine was linear over the range of 1.263-5.050 mg·mL-1, with the average recovery of 99.0% (n=9). The standard curve of isoleucine was linear over the range of 1.350-5.402 mg·mL-1, with the average recovery of 99.4%(n=9). The standard curve of leucine was linear over the range of 1.647-6.588 mg·mL-1, with the average recovery of 99.5%(n=9). The main impurities in the three batches of samples were all process impurities introduced from the raw materials, with methionine content of 4.344 μg·mL-1, 3.751 μg·mL-1, 4.503 μg·mL-1, respectively, phenylalanine content of 4.636 μg·mL-1, 4.889 μg·mL-1, 4.753 μg·mL-1, respectively. The maximum single impurity contents were 0.01%, 0.02% and 0.01%, respectively. Conclusion: The method is proved by the methodology validation that it can be used for the quality control of compound amino acid injection(3AA).
  • Determination of vitamin D3 in vitamin D drops by high resolution sampling two-dimensional chromatography*
    WU Jue, GUO Wei-bin, QIU Xiao-feng, ZHENG Shu-feng
    Chinese Journal of Pharmaceutical Analysis. 2024, 44(7): 1169-1175. https://doi.org/10.16155/j.0254-1793.2023-0741
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    Objective: To establish an innovative analytical method based on high resolution sampling two-dimensional chromatography (HiRes 2D-LC) for determination of the content of vitamin D3 in vitamin D drops. Methods: Two-dimensional liquid chromatography was used. Thermo HYPERSIL Gold Silica (100 mm×2.1 mm, 1.9 μm) column was used in the first dimension with n-hexane-n-amyl alcohol (996∶4) as mobile phase. The flow rate was 0.2 mL·min-1. The samples were injected and tested at the wavelength of 265 nm. The column temperature was 40 ℃. In the second dimension liquid chromatography, ShimPack Velox Hilic (50 mm×2.1 mm, 2.7 μm) was used as the column with n-hexane-n-pentanol-isopropanol (98∶1∶1) as the mobile phase. The flow rate was 0.5 mL·min-1. The samples were injected and tested at the wavelength of 265 nm. The column temperature was 40 ℃. A six-position 14-way valve and was equipped with 2 multi-center cutting valves was equipped to make multiple consecutive cuts of the pre-vitamin D3 peak and vitamin D3 peak. Results: The calibration curves showed a good linearity at the range of 1.018 4-5.092 mg·mL-1(r≥0.999 8). The precision test showed the RSDs of the peak area of pre-vitamin D3 and vitamin D3 were 0.95% and 0.40%, respectively. The repeatability test showed the RSD of vitamin D3 content was 0.41%. The average recovery rate (n=9) was 101.4%. The test solution was stable at 4 ℃ and 10 ℃ for 12 h, and the RSDs were 0.58% and 0.66%, respectively. The contents of vitamin D3 in the samples of vitamin D drops measured by this method were 100.4%, 101.6%, 100.9%, 101.6%, 102.7% and 101.6%, which was basically consistent with the results measured by the fourth method in General Chapter 0722 of ChP 2020 Vol Ⅳ. Conclusion: This method has a good specificity and high sensitivity to accurately determine the content of vitamin D3 in vitamin D drops.
  • Geographical origin traceability of Desmodium caudatum (Thunb.) DC. by UPLC MS/MS coupled with BP neural network
    YANG Jing, FU Chuan-wu, QIN Hua-liang, QIN Dong-jie, QIN Zi-long
    Chinese Journal of Pharmaceutical Analysis. 2024, 44(7): 1176-1185. https://doi.org/10.16155/j.0254-1793.2023-0464
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    Objective: To establish the UPLC-MS/MS method for simultaneous determination of 9 components (nicotinic acid, kaempferol, swertisin, quercetin, luteolin, rutin, vitexin, spinosin, salicylic acid) in Desmodium caudatum (Thunb.) DC. and construct a back propagation(BP) neural network model to predict the origin of Desmodium caudatum (Thunb.) DC. from different habitats. Methods: The chromatographic separation was achieved on an Agilent Zorbax SB C18 column (50 mm×3.0 mm,1.8 μm). The mobile phase consisted of methanol-0.1% acctic acid (containing 0.02 mol·L-1 ammonium acetate) at a flow rate of 0.3 mL·min-1 with gradient elution, the MS analysis were performed by multiple reaction monitoring (MRM) under ESI+ and ESI. A correlation analysis was conducted on the contents of each component, and a BP neural network model was constructed to distinguish Desmodium caudatum (Thunb.) DC. from different habitats. Results: Under the optimized conditions, 9 components(nicotinic acid, kaempferol, swertisin, quercetin, luteolin, rutin, vitexin, spinosin, salicylic acid) showed good linear relationships in the ranges of 0.388 8-38.88 ng·mL-1, 10.07-1 006.6 ng·mL-1, 34.22-34 221.6 ng·mL-1, 3.944-394.4 ng·mL-1, 2.124-212.4 ng·mL-1, 4.344-434.4 ng·mL-1, 46.50-4 650.1 ng·mL-1, 1.649-164.9 ng·mL-1, 4.880-488.0 ng·mL-1, respectively (r>0.995 1), whose average recoveries were 96.9%-103.9% (RSDs<1.9%). The contents of the above nine components in 40 batches of Desmodium caudatum (Thunb.) DC. were 1.657-7.407 μg·g-1, 15.801-64.488 μg·g-1, 1 068.348-4 270.780 μg·g-1, 10.608-123.228 μg·g-1, 3.897-16.802 μg·g-1, 1.269-97.834 μg·g-1, 405.285-1 955.796 μg·g-1, 13.614-36.124 μg·g-1, 4.417-87.509 μg·g-1, respectively. According to correlation analysis, four components (swertisin, rutin, spinosin, and luteolin) in Desmodium caudatum (Thunb.) DC. showed a highly linear positive correlation, indicating that these four components had a certain synergistic effect in Desmodium caudatum (Thunb.) DC.. The BP neural network model was constructed to predict Desmodium caudatum (Thunb.) DC. from different habitats, and the accuracy of the test set reached 92.3%. Conclusion: The method is simple, sensitive and efficient, and can be used for the rapid determination of the components in Desmodium caudatum (Thunb.) DC.. Using the BP neural network model to predict the habitats plays a significant role in tracing the origin of Desmodium caudatum (Thunb.) DC..
  • Simultaneous determination of nine effective components in Solidaginis Herba by QAMS
    YANG Qun, XIAO Wen-tao, LIU De-hong
    Chinese Journal of Pharmaceutical Analysis. 2024, 44(7): 1186-1194. https://doi.org/10.16155/j.0254-1793.2024-0386
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    Objective: To establish a quantitative analysis of multi-components by single marker (QAMS) method for the simultaneous determination of nine effective components including phenolic acids and flavonoids (neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, rutoside, isoquercitrin, kaemperol-3-O-rutinoside, 3,4-O-dicaffeoylquinic acid, 3,5-O-dicaffeoylquinic acid, 4,5-O-dicaffeoylquinic acid), in Solidaginis Herba. Methods: Ultra-high performance liquid chromatography was developed on an ACQUITY UPLC® HSS T3 (100 mm×2.1 mm, 1.8 μm) column with acetonitrile (A)-0.1% phosphoric acid solution (B) as mobile phase in gradient elution. The flow rate was 0.3 mL·min-1, the detection wavelength was 327 nm, the column temperature was 30 ℃, and the injection volume was 1 μL. Chlorogenic acid and rutoside were chosen as the internal standards for phenolic acids and flavonoids respectively to establish the determine correction factors of neochlorogenic acid, cryptochlorogenic acid,3,4-O-dicaffeoylquinic acid, 3,5-O-dicaffeoylquinic acid, 4,5-O-dicaffeoylquinic acid isoquercitrin and kaemperol-3-O-rutinoside, respectively, by multi-point correction method and their contents were calculated. The results were compared with the results of external standard method to verify their accuracy. Results: Nine components showed good linear relationships within their own ranges(r≥0.999 5), whose average recoveries were 98.2%-101.7% with the RSDs of 1.0%-1.7%. The contents of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, rutoside, isoquercitrin, kaemperol-3-O-rutinoside, 3,4-O-dicaffeoylquinic acid, 3,5-O-dicaffeoylquinic acid and 4,5-O-dicaffeoylquinic acid, in nine batches of samples were 0.008%-0.014%, 0.055%-0.097%, 0.010%-0.019%, 0.191%-0.412%, 0.018%-0.035%, 0.116%-0.50%, 0.028%-0.048%, 0.111%-0.235% and 0.113%-0.182%. The results obtained by QAMS were consistent with those of external standard method(relative average deviations less than 2.8%). Conclusion: The simple,accurate and reproducible method can be used for the quality control of Solidaginis Herba.
  • Simultaneous determination of 13 components in Yankening tablets by UPLC
    DONG Xiao-qian, YAN Chang-yu, MA Jin
    Chinese Journal of Pharmaceutical Analysis. 2024, 44(7): 1195-1201. https://doi.org/10.16155/j.0254-1793.2023-0639
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    Objective: To establish an UPLC quantitative analysis method for the simultaneous determination of 13 components in Yankening tablets, including phellodendrine, coptisine, baicalin, palmatine, berberine, wogonoside, baicalein, aloe-emodin, rhein, wogonin, emodin, chrysophanol, physcion. Methods: Agilent Eclipse Plus C18(100 mm×2.1 mm, 1.8 μm) column was used with acetonitrile-0.1% phosphoric acid as mobile phase, and gradient elution at a flow rate of 0.3 mL·min-1. The detection wavelengths were 210 nm and 254 nm. The column temperature was 40 ℃. Results: Phellodendrine hydrochloride, coptisine hydrochloride, palmatine hydrochloride, berberine hydrochloride, baicalin, wogonoside, baicalein, aloe-emodin, rhein, wogonin, emodin, chrysophanol, physcion showed good linear relationships within their concentration range of 0.97-48.63 μg·mL-1, 0.95-47.52 μg·mL-1, 0.86-43.15 μg·mL-1, 0.86-43.19 μg·mL-1, 0.89-44.37 μg·mL-1, 1.00-49.84 μg·mL-1, 1.02-51.01 μg·mL-1, 0.97-48.31 μg·mL-1, 0.99-49.50 μg·mL-1, 1.04-51.80 μg·mL-1, 1.00-50.04 μg·mL-1, 1.00-49.80 μg·mL-1, 1.01-50.64 μg·mL-1. The average recoveries(n=6) were 95.2%, 96.7%, 95.9%, 98.3%, 94.1%,97.6%, 99.2%, 96.6%, 95.5%, 97.2%, 97.0%, 97.8%, 98.7%, RSD values were all less than 2.0%. The contents of the 13 chemical components in 3 batches of samples were 1.367-1.488 mg·g-1(calculated as phellodendrine hydrochloride), 0.378-0.412 mg·g-1(calculated as coptisine hydrochloride), 4.611-5.505 mg·g-1, 0.324-0.407 mg·g-1(calculated as palmatine hydrochloride), 3.665-3.878 mg·g-1(calculated as berberine hydrochloride), 1.107-1.682 mg·g-1, 0.392-0.941 mg·g-1, 0.076-0.105 mg·g-1, 0.097-0.116 mg·g-1, 1.059-1.213 mg·g-1, 0.149-0.167 mg·g-1, 0.213-0.239 mg·g-1, 0.047-0.059 mg·g-1. Conclusion: The method is accurate, high analysis efficiency, good repeatability, it can be used to control the quality of Yankening tablets.
    • Metabolism Analysis
  • Comparative pharmacokinetics of characteristic components of Scutellariae Radix in rats plasma after oral administration of Scutellariae Radix and Niuhuang Jiedu tablets*
    LIU Yue-xin, SONG Min, HANG Tai-jun, WU Xiao
    Chinese Journal of Pharmaceutical Analysis. 2024, 44(7): 1202-1211. https://doi.org/10.16155/j.0254-1793.2023-0302
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    Objective: To establish an HPLC-MS/MS method for the simultaneous determination of six characteristic components of Scutellariae Radix including baicalein, baicalin, wogonin, wogonoside, chrysin and oroxylin A in rat plasma, and to compare their pharmacokinetic profiles after oral administration of Scutellariae Radix and Niuhuang Jiedu tablets (NHJDT) to rats. Methods: Rats were given 250 mg·kg-1 of NHJDT suspension or the prescribed equivalent amount of Scutellariae Radix aqueous extract, and plasma samples were collected at different time intervals. Naringenin was used as internal standard. After precipitated with methanol, the plasma samples were separated on an Inertsil C8-3 (150 mm×4.6 mm, 5 μm) column by linear gradient elution with 0.1% formic acid solution(A)-0.1% formic acid-methanol(B) as mobile phase. The flow rate was 1.0 mL·min-1, and the column temperature was 35 ℃. The analytes were detected by tandem mass spectrometry with the electrospray ionization (ESI) source combined with multiple reaction monitoring(MRM) mode in positive ion mode. The pharmacokinetic parameters were calculated and statistically analyzed. Results: The six characteristic components of Scutellariae Radix were all in good linear relationships in the range of 5-500 ng·mL-1. The RSDs for accuracy test were in the range of 88.43%-108.6%, and the RSDs for inter-batch and intra-batch precision tests were all below 15%. The matrix effect and plasma stability met the requirements of methodology validation in biological sample analysis. Baicalin and wogonoside were the major components detected in rat plasma after oral gavage of Scutellariae Radix aqueous extract and NHJDT suspension. The AUC0-t of wogonoside was significantly increased in NHJDT group compared with Scutellariae Radix group. Furthermore, the Cmax of wogonoside and baicalin were significantly increased while the Tmax was decreased after NHJDT suspension administration. Conclusion: This method is specific and sensitive for the determination of six characteristic components of Scutellariae Radix, and suitable for pharmacokinetic study of rat plasma. NHJDT with co-existing components enhances the absorption and influences the pharmacokinetic behaviors of active ingredients of Scutellariae Radix.
    • Safety Monitoring
  • Study on bioequivalence exemption for levofloxacin tablets post-excipient modification*
    WANG Wen-li, ZHANG Xiao-yan, WANG Xiao-jing, YU Li-ju, ZHANG Xiao-ming, SUN Ying
    Chinese Journal of Pharmaceutical Analysis. 2024, 44(7): 1212-1221. https://doi.org/10.16155/j.0254-1793.2024-0089
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    Objective: To investigate the feasibility of applying for bioequivalence exemption based on parallel artificial membrane permeability assay (PAMPA) data by evaluating the solubility and in vitro permeability of levofloxacin, comparing the prescription differences between the generic and reference formulations, assessing the impact of disparate excipients on the permeation behavior of the active pharmaceutical ingredient (API), and predicting the bioequivalence of the two formulations. Methods: The dissolution of the raw material at pH 1.0 to pH 6.8 was determined using high-performance liquid chromatography. The μFluxTM system was employed to determine the permeability of the raw material and the mixture of raw material with sodium stearyl fumarate in various media. pH 5.0 fed-state intestinal fluid, pH 6.5 fasted-state intestinal fluid, and pH 7.4 phosphate buffer (16 mL) were precisely added to the donor chamber, while 16 mL of Accepter Sink Buffer was added to the receptor chamber. The rotor speed was set at 200 r·min-1, and the collection time was 180 min. Determine the permeability of the raw material and the mixture of raw material with sodium stearyl fumarate in various media, as well as the permeability of the raw material in the reference and generic formulations powders. The impact of newly added excipients and other changed excipients on API was evaluated through a two-tailed t-test. The Macro FluxTM system was used to measure the dissolution-permeation curves of the reference and generic formulations, intestinal simulation fluids at pH 5.0 and pH 6.5 (1 000 mL) were added to the dissolution cup as dissolution media, with a stirring speed of 75 r·min-1 using a paddle method, 12 mL of Accepter Sink Buffer was added to the receptor chamber, and the stirring speed of the micro-stirring rod was set at 450 r·min-1, one tablet of each reference or generic formulation was placed in the dissolution cup, and the dissolution-permeation curves of the formulations were measured. The similarity of dissolution curves was compared, and the permeability rate (JFlux) and cumulative drug permeation amount (AMT) were calculated to predict the bioequivalence of the two formulations, ensuring that the 90% confidence interval for the geometric mean ratio of JFlux and AMT of the two formulations fell within the range of 80% to 125%. Results: Levofloxacin solubility ranged from 16.4 mg·mL-1 to 62.7 mg·mL-1 across different mediums, its permeability in pH 5.0 fed-state simulated intestinal fluid, pH 6.5 fasted-state simulated intestinal fluid, and pH 7.4 phosphate-buffered saline was 2.92×10-6 cm·s-1, 1.01×10-5 cm·s-1, and 1.07×10-5 cm·s-1, respectively. The addition of sodium stearyl fumarate showed no significant difference in permeability compared to the original API (P＜0.05), and there were no significant differences between the powder API of the reference and generic formulations (P＜0.05). The dissolution curves of both formulations were similar, with the 90% confidence interval for JFlux and AMT within the predefined range. Conclusion: Levofloxacin tablets, classified as BCS Class Ⅰ, demonstrated that the altered excipients in the formulation did not impact on the API's permeability, confirming bioequivalence between the reference and generic formulations. The bioequivalence exemption study based on PAMPA can be utilized for permeability studies of raw materials, excipient screening and optimization, and prediction of formulation bioequivalence, effectively reducing drug development costs and time. This study provides reference data for pharmaceutical companies applying for bioequivalence exemptions.
  • Establishment of identification method for Fritillariae Cirrhosae Bulbus and its adulterant Fritillariae Ussuriensis Bulbus based on chemometrics
    SHI Yan, LIU Wei, WEI Feng, MA Shuang-cheng
    Chinese Journal of Pharmaceutical Analysis. 2024, 44(7): 1222-1232. https://doi.org/10.16155/j.0254-1793.2023-0616
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    Objective: To establish a method for identifying Fritillariae Cirrhosae Bulbus and its adulterant Fritillariae Ussuriensis Bulbus. Methods: Chemometrics techniques were used for the analysis of the sample data from determination with Q TOF MS, and characteristic ion pairs were selected as m/z 578.3→164.14 and m/z 578.3→398.31 with triple quadrupole mass spectrometry applied, respectively. Then a specific approach was developed which used Waters Acquisition UPLC CSH (75 mm×2.1 mm, 1.7 μm) as column, and with acetonitrile and 0.1% formic acid solution as the mobile phase, and a flow rate of 0.4 mL·min-1. Two ion pairs: m/z 578.3→164.14 and m/z 578.3→398.31 were detected by triple quadrupole mass spectrometer with MRM mode. Results: The validation results through 108 batches of samples indicated that the two characteristic ion pairs, m/z 578.3→164.14 and m/z 578.3→398.31, could effectively distinguish between Fritillariae Cirrhosae Bulbus and its adulterant Fritillariae Ussuriensis Bulbus. Conclusion: The method had good specificity and sensitivity, and could be used for the detection of adulterated Fritillariae Cirrhosae Bulbus with Fritillariae Ussuriensis Bulbus.
  • Thoughts on determination method of the related substances in halometasone/triclosan cream
    ZHAO Jing-dan, CHEN Yang, LIU Hao, JIN Wei, LE Jian
    Chinese Journal of Pharmaceutical Analysis. 2024, 44(7): 1233-1237. https://doi.org/10.16155/j.0254-1793.2024-0045
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    Objective: To analyze the structure of the maximum impurity in halometasone/triclosan cream and discuss the rationality of the current registered standard for imported drugs. Methods: The related substances in halometasone/triclosan were detected according to JX20080304, and dioxins were determined according to USP. Finally, the possible structures of the maximum impurity were characterized by LC-Q TOF/MS. Results: The results showed that the maximum unknown impurity(RRT 0.67), calculated according to halometasone was OOS(1.0%) at 2.5%. The peak was related to triclosan through comparing with the chromatography behavior of triclosan in reference substance solution. MS information indicted the process impurities of triclosan. Conclusion: For compound preparations, to ensure the stability and safety of the product, the impurities relevant to different components should be controlled separately at rational limit based on safety data.
  • Detection of related substances in vitamin D drops (soft capsules) by SPE-HPLC
    XIE Zhi-cai, YANG Li-na, PAN Han, XU Qing-jun, LU Da-feng, LI Shu-ying, XIE Qiang-sheng
    Chinese Journal of Pharmaceutical Analysis. 2024, 44(7): 1238-1245. https://doi.org/10.16155/j.0254-1793.2023-0441
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    Objective: To establish a solid-phase extraction-high performance liquid chromatography(SPE-HPLC) method for the rapid detection of related substances of vitamin D3, trans vitamin D3 (impurity A), 7-dehydrocholesterol (impurity B), lumisterol 3 (impurity C), and isotachysterol 3 (impurity D), in vitamin D drops (soft capsules). Methods: The content, which was approximately equivalent to vitamin D3 1 548 IU, was taken in about 1.2 g, weighed accurately, put into a test tube, 4 mL of isooctane was added to it, and it was swirled and mixed well. Purification was performed using solid phase extraction columns, with n-hexane-ethyl acetate(85∶15) as the elution agent and anhydrous ethanol as the desorption agent. A Luna Silica(2) 100 Å silica gel column(150 mm×4.6 mm, 3 μm) was used, with n-hexane-n-pentanol (996.5∶3.5) as the mobile phase. The content of vitamin D3 impurities was calculated using the adding standard calibration factors principal component self-control method. Results: The separation degree between pre-vitamin D3 and vegetable oil was greater than 1.5. The linear range of vitamin D3 was 0.02-0.80 μg·mL-1, with r=0.999 5. The linear range of impurity A was 0.02 -0.80 μg·mL-1, with r=0.999 9. The linear range of impurity B was 0.02-0.80 μg·mL-1, with r=0.999 9. The linear range of impurity C was 0.02-0.80 μg·mL-1, with r=0.999 9. The linear range of impurity D was 0.02-0.80 μg·mL-1, with r=0.999 9. The average recovery rate of impurities A-D (n=9) was 93.2%-102.9%. The detection results of 3 batches of vitamin D drops (soft capsules) showed that the content of known impurities and other maximum single impurities were less than 0.5%, and the total content of impurities was less than 1.0%. Conclusion: The results indicate that the established method is suitable for the detection of vitamin D3 related substances in vitamin D drops (soft capsules). It has the advantages of being easy to operate, providing rapid and accurate results, and providing technical assurance for the quality control of fat-soluble vitamin D3 preparations.
    • Standard Deliberation
  • Studies on the pharmacognosic and digital characters of four medicinal seeds,such as Plantaginis Semen*
    MA Min, KANG Shuai, MA Xin, NING Hong-ting, LUO Jin-ping, ZHANG Nan-ping, MA Shuang-cheng
    Chinese Journal of Pharmaceutical Analysis. 2024, 44(7): 1246-1254. https://doi.org/10.16155/j.0254-1793.2023-0530
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    Objective: To explore the method of pharmacognosic identification of four medicinal seeds, such as Plantaginis Semen from two kinds of original plant,Allii Fistulosi Semen, Allii Tuberosi Semen, and to digitize the identification characteristics. Methods: The seeds of the four medicinal plants were studied by means of type microscope and light microscope, including the appearance(positive view, bottom view, hilum view and surface feature), internal morphology(cross profile and longitudinal profile) and the characters of micromorphology(transverse section, powder). The characteristics of the four medicinal seeds were summarized, and the identification points were characterized by digital method. Results: The seeds of the four medicinal plants could be identified according to the appearance, internal morphology and the characters of micromorphology. Conclusion: This study lays a foundation for the variety identification and provides reference for the standard formulation and quality control of the four medicinal seeds. It also provides ideas for digital characterization of seed medicinal materials.
  • Pharmacognostical studies on medicinal herbs of Baijiangcao
    YU Kun-zi, HAN Shi-kai, MA Si-yu, KANG Shuai, CHENG Xian-long, WEI Feng
    Chinese Journal of Pharmaceutical Analysis. 2024, 44(7): 1255-1266. https://doi.org/10.16155/j.0254-1793.2023-0508
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    Objective: To find out identification characteristics of Baijiangcao from different origins by pharmacognostical study. To discuss the establishment of whole herbs Chinese medicine standard. Methods: Principles of plant taxonomy, macroscopic identification and microscopic identification were used to compare the differences among Baijiangcao from different origins and commercial counterfeit. Results: The macroscopic characteristics, such as the surface of stem, the cross section of stem, the shape of leaves, hairy on inflorescence and odour. And the microscopic characteristics of transverse section of stem could be used to distinguish Baijiangcao from different origins and commercial counterfeit. Conclusion: This research establishes the identification method of Baijiangcao from different origins and commercial counterfeit, and provides references for the improvement of quality standard of Baijiangcao from different origins.
    • Quality Control
  • Molecular DNA identification of adulteration in processed Chinese herbal medicine containing whole scorpions*
    DUAN Bao-li, YOU Chun-xue, JIANG Chao
    Chinese Journal of Pharmaceutical Analysis. 2024, 44(7): 1267-1275. https://doi.org/10.16155/j.0254-1793.2023-0482
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    Objective: To establish an effective method for detecting the components of Lychas mucronatus and investigating instances of adulteration with whole scorpions in Chinese patent medicines. Methods: Thirty Chinese patent medicines containing whole scorpion components, as documented in the Pharmacopoeia of the People's Republic of China, were collected. DNA was extracted, and differences in the mitochondrial cytochrome C oxidase subunit Ⅰ(COⅠ) gene sequences were compared. Specific primers for the identification of Mesobuthus martensii and Lychas mucronatus DNA were designed. Annealing temperature, cycle number, Taq polymerase, and DNA concentration were optimized to establish the most suitable PCR identification system and conditions. The optimal PCR conditions for distinguishing the sharp-tailed wolf scorpion were finally determined to be an annealing temperature of 52 ℃, 36 cycles, 2×PCR Mix Taq enzyme, and 1 μL of DNA template. The developed method was applied to identify the presence of Centruroides gracilis components in 30 commercially available Chinese patent medicines. Results: Gel electrophoresis revealed the presence of authentic DNA bands specific to the Mesobuthus martensii in all 30 whole scorpion-containing Chinese patent medicines. Eighteen medicines (60%) showed a single specific DNA band between 100-250 bp, indicative of Lychas mucronatus, while the blank control exhibited no bands. Sequencing of the PCR products containing the identified Lychas mucronatus -specific bands, after TA cloning, revealed sequences with high similarity (92%-98%) to the reference sequences of Lychas mucronatus. Phylogenetic analysis indicated that the obtained sequences belonged to the same lineage as Lychas mucronatus and were distinct from the Mesobuthus martensii. Therefore, the presence of a single band around 120 bp indicates the identification of Lychas mucronatus. Conclusion: The presence of Lychas mucronatus components mixed with authentic scorpion materials in Chinese patent medicines indicates a need for strengthened supervision. The method established in this study enables the rapid and accurate detection of Lychas mucronatus components in Chinese patent medicines. This is beneficial for improving the quality control of whole scorpion-containing Chinese patent medicines, providing assurance for their clinical applications.
    • Rapid Analysis
  • Study on molecular identification methods and reagents “genetic marker”-based DNA fingerprinting-test strips of human placentophagy*
    WANG Yan-shuang, WANG Yi-tong, MU Run-hong, ZHANG Li-hua
    Chinese Journal of Pharmaceutical Analysis. 2024, 44(7): 1276-1284. https://doi.org/10.16155/j.0254-1793.2023-0435
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    Objective: To establish a rapid molecular identification method for DNA fingerprinting-immunocolloidal gold test strips of Chinese herbal medicine human placentophagy genetic markers and to develop an integrated quality control gene detection kit. Methods: Self-developed method and reagents could be for template DNA extraction from human placentophagy. Species-specific primers were designed according to the human mtDNA cytb gene, and the 5' ends of the primers were labeled with FAM and Biotin, respectively. The optimal annealing temperature and cycle number of PCR were screened to establish a DNA fingerprinting molecular identification method. To develop PCR gene detection reagent premixes, to prepare DNA clones of control herbs using molecular cloning and gene sequencing techniques and to utilize immunocolloidal gold test strips for result detection. Finally, a rapid gene detection kit integrating DNA extraction reagent, PCR gene detection reagent premixes, control herb DNA clone solution, blank control solution and test strips was developed, and evaluated for specificity, reproducibility, sensitivity and stability. Results: The template DNA extracted by self-developed DNA extraction reagents were all in the concentration of 150-280 ng·μL-1, the purities were all in the range of 1.8-1.9, and there were no trail in the agarose gel electrophoresis. And the immunocolloidal gold test strips showed two red bands for human placentophagy control herb and authentic product, and one red band for easy-mix product and blank control at annealing temperature of 59 ℃ and 20 cycles for human placentophagy specific primers. The agarose gel electrophoresis was verified to be correct. The DNA sequencing results of the control herbs were 100% homologous with human mtDNA cytb gene. The self-developed integrated rapid gene detection kit was specific, reproducible, stable and sensitive at 0.1 ng·μL-1. Conclusion: The DNA fingerprinting-immunocolloidal gold test strip molecular identification method can specifically, accurately, rapidly and visually identify the authenticity of human placentophagy, and the integrated rapid gene detection kit provides a regulated and standardized testing method for quality control of human placentophagy. Therefore, it is more suitable for on-site field testing and universal promotion.
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