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  • Ling-run LIU, Shi-ling TIAN, Chang-jian WU, Rui CHEN, Yuan-qing YE, Yan-hua QIN, Zhi-qi YIN, Yi CAO, Jian ZHANG
    Chinese Journal of Pharmaceutical Analysis. 2024, 44(9): 1485-1496.

    Objective: To investigate the flavor components of Epimedium extract and analyze pyrolysis products of the extracts from Epimedium. Methods: Ultra fast gas phase electronic nose was adopted to analyze volatile components in ethanol extracts of Epimedium and the alcohol extracting process of Epimedium was optimized by orthogonal experiment. Epimedium extract was pyrolysed to simulate cigarette smoking by TG-GC-MS. The lysates of Epimedium extract were analysed in a nitrogen environment, and the possible lysate mechanism of the products was reasonably speculated. Results: 22 volatile components were detected in the ethanol extracts of Epimedium at different concentrations. Concentration of Epimedium extracted with 60% ethanol was superior than others. Analyzing pyrolysis products of Epimedium extract, 78 compounds were identified at 150, 300 and 450 ℃, including aldehydes, ketones, alcohols, phenols, furans and benzene series. Conclusion: Ethanol extraction of Epimedium contains many aromatic volatile components. A large number of ketones, alcohols, phenols, furans and other volatile aroma compounds are produced after pyrolysis of Epimedium extract.

  • Yue WANG, Yan-jie YIN
    Chinese Journal of Pharmaceutical Analysis. 2024, 44(9): 1624-1631.

    Objective: To develop an HPLC method for determination of naphazoline hydrochloride, diphenhydram hydrochloride and lidocaine hydrochloride added in nasal cold compress gel (dew), and to establish an HPLC-triple quadrupole mass spectrometry (HPLC-MS) method to confirm the positive samples. Methods: The samples were extracted with acetonitrile, detected by high performance liquidchromatography, quantified by external standard method and confirmed by HPLC-MS. The separation was performed on a XTerra RP18 (150 mm×4.6 mm, 5 μm) column with the mobile phase consisting of 50 mmol·L-1 ammonium acetate (the pH value was adjusted to 7.8 with acetic acid or ammonia solution)-acetonitrile (72∶28) at the flow rate of 1.0 mL·min-1 and the detection wavelength was 230 nm. The analysis was performed on a BEH C18 (100 mm×2.1 mm, 1.7 μm) column with a gradient elution of 0.1% formic acid aqueous solution-0.1% formic acid acetonitrile solution. The column temperature was set at 40 ℃ and the flow rate was 0.4 mL·min-1. Electrospray ionization source was applied and operated in positive electrospray ionization and the multiple reaction monitoring mode. Results: The method showed the lowest detection concentrations of naphthazoline hydrochloride, diphenhydramine hydrochloride and lidocaine hydrochloride were 2.4 ng·mL-1, 50 ng·mL-1 and 50 ng·mL-1. The sample recoveries ranged from 93.9% to 104.6%. Good linearities were found in the concentration range of 10-200 μg·mL-1r>0.999 0). A total of 42 batches of samples were detected and the total positive rate was 70% (36/42). Naphthazoline hydrochloride were found in 34 batches. Diphenhydramine hydrochloride and lidocaine hydrochloride were found in 2 batches simultaneously. Conclusion: The established method is specific, sensitive, simple, accurate and reliable. It can be used for the qualitative and quantitative determination of naphthazoline hydrochloride, diphenhydramine hydrochloride and lidocaine hydrochloride in nasal cold compress gel (dew).

  • Bu-ren-man-da, Jie-si WU, Ao-dun-ge-ri-le, Huang-ge-er-zhu-la, A-rong HU, Qi-ri-ge-er, La-xi-na-mu-ji-la
    Chinese Journal of Pharmaceutical Analysis. 2024, 44(9): 1497-1503.

    Objective: To establish an UHPLC-MS/MS method for determining eight primary components (catechin, epigallocatechin, rutin, quercitrin, epicatechin, (+)-dihydromyricetin, myricitrin and dihydroqurcetin) in the young branches and leaves of Xanthoceras sorbifolia Bunge, a medicinal plant from Mongolia, and to compare their contents in samples at different growth stages. Methods: A Waters CORTECS C18 (100 mm×2.1 mm, 1.6 μm) chromatographic column was adopted using the mobile phase comprised of water containing 0.1% formic acid (A) and acetonitrile (B) with gradient elution(0-1 min, 5% B; 1-10 min, 5% B→28% B; 10-11 min, 28% B→95% B; 11-14 min, 95% B; 14-15 min, 95% B→5% B) at a flow rate of 0.3 mL·min-1. The temperature of the column was set at 40 ℃. Injecting volume was 2 μL. Detection was conducted using electrospray ionization (ESI) in negative ion mode with multiple reaction monitoring (MRM). Results: The linearity of the eight chemical components was found to be excellent in the tested concentration ranges, with correlation coefficients above 0.997 6. Precision, repeatability and stability were satisfactory and the average recoveries were between 97.4% and 106.0% with RSDs≤5.0%. In six batches of leaves, contents of catechin, epigallocatechin, rutin, quercitrin, epicatechin, (+)-dihydromyricetin, myricitrin and dihydroqurcetin were in the ranges of 0.090-0.904 mg·g-1, 0.093-2.258 mg·g-1, 0.001-0.005 mg·g-1, 0.530-6.176 mg·g-1, 0.158-1.561 mg·g-1, 0.002-0.056 mg·g-1, 4.008-10.218 mg·g-1 and 1.049-16.990 mg·g-1, respectively. In six batches of young branches, the contents ranged from 0.384-1.025 mg·g-1, 0.911-2.427 mg·g-1, 0.008-0.127 mg·g-1, 0.870-2.295 mg·g-1, 0.659-1.746 mg·g-1, 0.125-1.079 mg·g-1, 2.296-4.681 mg·g-1 and 1.958-4.946 mg·g-1, respectively. The contents of eight components varied a lot in samples from different parts. The contents of myricitrin, rutin and quercitrin in the leaves exhibited noticeable changes with the growth cycle, suggesting their potential as quality control markers for leaves of Xanthoceras sorbifolia. Conclusion: The method is accurate, sensitive, stable, repeatable, and suitable for simultaneous determination of eight components in Xanthoceras sorbifolia Bunge, offering reference for quality control of its leaves and branches.

  • Xia YAN, Tao ZHU, Song-hua HE, Yi LUO
    Chinese Journal of Pharmaceutical Analysis. 2024, 44(9): 1567-1577.

    Objective: To establish a multi-component content determination and characteristic chromatogram method for Jieyu Anshen granules, combined with chemometrics, to comprehensively evaluate the quality of Jieyu Anshen granules. Methods: The separation was performed on a CAPCELL PAK MG Ⅱ C18(250 mm×4.6 mm, 5 μm) column using acetonitrile (A) and 0.1% phosphoric acid aqueous solution (B) as the mobile phases with gradient elution at a flow rate of 1.0 mL·min-1. The column temperature was 30 ℃ and the detection wavelengths were set at 237 nm and 335 nm. The analysis method was validated. Based on the multi-component content determination method, characteristic chromatogram was established. The samples were analyzed and evaluated through chemometrics to identify the main factors affecting their quality. Results: A multi-component content determination method was established for Jieyu Anshen granules. The five components had good linear relationships within their respective ranges, with recovery rates ranging from 95.6% to 100.0%. And 145 batches of samples were tested, and a total of 54 batches were found to be unqualified, with a total unqualified rate of 37.2%. Establishing a characteristic chromatogram of Anshen granules, 12 characteristic peaks were attributed to 6 medicinal herbs. Through chemometric analysis, it was found that there were certain differences in the quality of samples from 10 manufacturers. The quality of fried gardenia and stir-baked licorice was the key factor. Conclusion: The established multi-component content determination method and characteristic chromatogram method for Jieyu Anshen granules are convenient and reliable, and can be used for comprehensive evaluation of the quality of Jieyu Anshen granules.

  • Wei-tao CHEN, Yang-xue LI, Jie-yi JIANG, Can-hui XIE, De-zheng JIA, Xiao-tong LIU, Hui-na LIAO, Su-mei LI
    Chinese Journal of Pharmaceutical Analysis. 2024, 44(9): 1529-1534.

    Objective: To establish an authentication method for Eupolyphaga sinensis based on loop-mediated isothermal amplification (LAMP). Methods: Two pairs of primers (outer primer DB-F3, DB-B3 and inner primer DB-FIP, DB-BIP) were designed according to Eupolyphaga sinensis COⅠspecific loci. The reaction system containing template, primers, Bst DNA polymerase and methyl red-phenol red indicator amplified at 63 ℃ for 90 min. The LAMP results were determined by visual observation and compared with DNA barcoding and polymerase chain reaction (PCR) methods to investigate the specificity and sensitivity. Results: All 11 batches of the authentic Eupolyphaga sinensis sample tubes colour changed from purple red to orange yellow, while sample tubes of 1 batch of Steleophaga plancyi, 9 batches Opisthoplatia orientalis and 1 batch of the adulterant, Cybister tripunctatus orientalis, remained purple red after the reaction. The LAMP results were consistent with those of DNA barcoding and PCR. The detection limit of LAMP was 0.984 ng·mL-1. Conclusion: The established LAMP method is accurate, sensitive and easy to operate, low equipment required, and can be applied to the rapid screening of the authentication of Eupolyphaga sinensis.

  • Jian JIAO, Yang DENG, Ya-jun LI, Yu-juan NIU, Xi-qin MAO, Jin-peng LIU
    Chinese Journal of Pharmaceutical Analysis. 2024, 44(9): 1524-1528.

    Objective: To develop a method to assess bioactivity of trasfer factor (TF) based on their protective effcets on Jurkat E6-1 cells. Methods: Proliferative effects of different concentrations of TF on 6-mercaptopurine(6-MP) treated Jurkat E6-1 cells detected by CCK-8 assay and the precision was validated. Results: TF exhibited protective activity to change the inhibitory concentration (IC50) value of 6-MP from(0.46±0.10)μg·mL-1 to (1.11±0.30)μg·mL-1 when treated with 20 μg·mL-1 TF. When cells were treated with 6-MP, there is a good linear relationship between the concentration of TF and cell survival rate, r>0.95. At each concentration level of the dose-response curve, the RSD was less than 20%. The median effective concentration (EC50) of TF was (28.49±9.60)μg·mL-1, and the confidence limit was less than 20%. Conclusions: TF significantly improved Jurkat E6-1 cell survival rate in dose dependent manner when challenged with 6-MP. It may be suitable for evaluate bioactivity of TF.

  • Xin LIU, Rui-li LIN, Lin NI, Ping-shun SONG, Ling-xia YANG
    Chinese Journal of Pharmaceutical Analysis. 2024, 44(9): 1613-1623.

    Objective: To investigate the source and quality of commercially available Epimedium based on HPLC fingerprint and multi-index assay, so as to provide reference for its quality evaluation and resource development. Methods: Forty batches of commercially available epimedium were collected. Fingerprint analysis and determination of the total flavonoid glycosides were performed by HPLC method. The total flavonoid glycosides were determined by UV method. And the contents of their extracts were determined. Stoichiometric analysis was employed to perform the comprehensive quality evaluation of different medicinal materials of Epimedium. Results: There were 9 different plant sources of Epimedium in the market. The five authentic Epimedium were of good quality, and the local varieties were coarse Epimedium. The contents were high in Epimedium from Hunan, while the contents were low in Epimedium from Qianling and Sichuan. The comprehensive quality ranking of Epimedium indicated that Epimedium Fletchum and Epimedium Fletchum ranked first by the principal component analysis method. The ranking of the same variety varied greatly with different habitats, and the Qianling Epimedium and adulterated Epimedium ranked last. The results of cluster analysis and principal component analysis were basically consistent. The cluster analysis showed distinguishing significance in plant sources, habitats, processing methods and contents. Based on the common pattern of E. brevicornu, a total of 8 common peaks were identified for 9 species of E. Brevicornu, and their similarities ranged from 0.066 to 0.979. Differences were observed among different species. Conclusion: Both the fingerprint similarity and the chemical model of content determination can be used to evaluate the species and quality of Epimedium.

  • Fu-kang JIA, Jian-hui FENG, Bi-ying WANG, Rong-wei LIU, Sui-qing CHEN, Yu FU
    Chinese Journal of Pharmaceutical Analysis. 2024, 44(9): 1535-1550.

    Objective: To study the difference of chemical composition between ancient and modern clinical prescriptions of Baihe Dihuang decoction by multi-component analysis. Methods: The polysaccharide extract of Baihe Dihuang decoction was determined by anthrone-sulfuric acid-ultraviolet spectrophotometer under 580 nm ultraviolet light. Six monosaccharides and oligosaccharides in the ancient and modern Baihe Dihuang decoction were determined by UPLC-CAD. An Agilent InfinityLab Poroshell 120 HILIC-Z (3.0×100 mm, 2.7 μm) column was used. The mobile phase was consisted of A 0.2% ammonia in acetonitrile and 0.2% ammonia in water with gradient elution. The flow rate was 0.8 mL·min-1, the column temperature 35 ℃ and the CAD detector nebulizer temperature was 70 ℃. Data acquisition frequency was 10 Hz and the filter was 1.05 s. The injection volumn was 2 μL. The non-polar components were qualitatively and quantitatively analyzed by high performance liquid-quadrupole tandem time-of-flight mass spectrometry (UPLC-Q TOF MS/MS) and high performance liquid-phase tandem triple quadrupole mass spectrometry (UPLC-QQQ MS), respectively. The differential components of ancient and modern Baihe Dihuang Decoction were analyzed and 9 components were determined. Results: The linear relationship of 6 mono-oligosaccharides/polysaccharides and 9 differential components was good (r>0.999). The average recoveries ranged from 97.7% to 104.9% with RSDs≤3.1%. Precision, repeatability and stability met the requirements. The results showed that the conents of all the analytes in ancient and modern Baihe Dihuang decoction were significantly different (P<0.05). Conclusion: The methods used in this experiment are accurate, sensitive, stable and reproducible, which can provide reference for the quality control and pharmacodynamic material basic research of Baihe Dihuang Decoction.

  • Gao-yan LI, Yi-di YANG, He-fei YUAN, Fan-na QU, Hui-yong LI, Lei SUN, Shuang-cheng MA, Xue-yan BI
    Chinese Journal of Pharmaceutical Analysis. 2024, 44(9): 1560-1566.

    Objective: To establish an HPLC method for the simultaneous determination of six components (uridine, adenine, adenosine (R, S)-goitrine, guanosine, clemastanin B) in Radix Isatidis, and to investigate the linear calibration with two reference substances (LCTRS) method for the qualitative analysis of multiple components in Radix Isatidis. Methods: HPLC method was used, with methanol as mobile phase A and water as mobile phase B. Gradient elution (0-3 min, 3% A; 3-18 min, 3% A→14% A; 18-25 min, 14% A→26% A; 25-34 min, 26% A; 34-40 min, 26% A→46% A; 40-60 min, 46% A→90% A) was performed at a flow rate of 0.8 mL·min-1. The column temperature was 30 ℃, the detection wavelengths were 254 nm (0-32 min) and 230 nm (32-60 min). The injection volume was 10 μL. The actual retention time of 6 components in Radix Isatidis was determined on 13 C18 chromatographic columns of different brands and models. Guanosine and clemastanin B were used as double reference compounds, and LCTRS method was used to locate the chromatographic peak of each component. Three unknown chromatographic columns were used for method validation. Using guanosine as a reference substance, the relative retention time method was used to predict the retention time of the other 5 components. The predictive accuracy and column coincidence of these two methods were compared. Results: The LCTRS method could effectively predict and qualitatively analyze the retention time of six indicator components. Compared with the relative retention time method, the LCTRS method had higher accuracy in predicting results and better column universality. Conclusion: The LCTRS method for simultaneous determination of multiple components in Radix Isatidis is feasible and accurate, with simple operation and good durability, and has promotional value.

  • Jie-min WANG, Hao-chuan GUO, Meng-wei ZHAO, Hui-gai SUN, Yong-xing SONG, Yu-guang ZHENG, Dong-lai MA
    Chinese Journal of Pharmaceutical Analysis. 2024, 44(9): 1475-1484.

    Objective: To analyze the fractions and relative contents of volatile oils of Magnoliae Flos at different harvesting periods, to elucidate the dynamic pattern of changes in the chemical composition of Magnoliae Flos at five harvesting periods, and to evaluate its antioxidant and antimicrobial activities. Methods: The volatile oils of Magnoliae Flos at five harvesting periods was extracted by water vapour distillation, and the chemical composition was analyzed by gas chromatography-mass spectrometry (GC-MS) technique, and the relative content of each constituent was calculated. The constituents of Magnoliae Flos at the five harvesting periods were analyzed by PLS-DA analysis, which was used in combination with the VIP value to screen out the differential compounds. The antioxidant activity of the volatile oil of Magnoliae Flos was determined by ferric ion reducing antioxidant power (FRAP) method, and its in vitro antimicrobial activity was investigated by 96-well plate method. Results: The total volatile oils content of Magnoliae Flos was the highest in samples at the 4th harvesting period (10 February 2023). Thirty-eight components were identified in the volatile oils of Magnoliae Flos, and 12 differential compounds were screened, including γ-muurolene, elemene, δ-cadinene and α-terpineol, etc. The relative contents of γ-muurolene, alloaeromadendrene, borneol, camphor and cis-4-thujanol were the largest in samples at the 4th harvesting period, which was basically in line with the trend of the change of volatile oil content. The volatile oils in samples at five harvesting period showed certain antioxidant and antibacterial activities. And that in samples at the 4th harvesting period showed the strongest antioxidant activity and the inhibition ability against all five species of bacteria. Conclusion: The chemical composition of the volatile oils in Magnoliae Flos was basically the same in in samples at five harvesting periods, but there is a significant difference in the relative content of its volatile components in each harvesting period, and it is presumed that the beginning of February is the optimal harvesting period for Magnoliae Flos.