Latest ArticlesObjective: 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.
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-1（r>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).
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.
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.
Objective: To establish the HPLC fingerprints for Zilian Shengji gel and to determine the contents of gallic acid, tableberberine, coptisine, palmatine, berberine and shikonin. Methods: The establishment of fingerprints was performed on a 25 ℃ thermostatic Agilent C18-WR（250 mm×4.6 mm, 5 μm）column with the mobile phase comprising of acetonitrile -0.15% phosphoric acid water at the flowing rate of 0.8mL·min-1 in a gradient elution manner, and the detection wavelength were set at 270 nm, 345 nm and 516 nm. Cluster analysis, principal component analysis(PCA) and orthogonal partial least squares discriminant analysis(OPLS-DA) were adopted in chemical pattern recognition. The six components identified were quantitatively determined. Results: The HPLC fingerprint of Zilian Shengji gel was constructed with twenty-six common chromatographic peaks, eleven batches of samples with the similarities of 0.828-0.997, six chromatographic peaks of gallic acid, tableberberine, coptisine, palmatine, berberine and shikonin were identified by reference substance comparison. Six constituents showed good linear relationships within their own ranges (r≥0.999 5) whose average recoveries were 98.7%-99.3%, with the RSDs of 1.3%-1.8%. The content ranges of the above mentioned six components in eleven batches of samples were 827.74-1 513.50 μg·g-1, 137.52-296.05 μg·g-1, 381.83-884.73 μg·g-1, 2 023.81-4 051.66 μg·g-1, 524.15-986.13 μg·g-1 and 47.65-549.46 μg·g-1. Conclusion: This simple, stable, reliable and reproducible method can be used for the quality control of Zilian Shengji gel.
To establish a method for the quantitative detection of bacterial endotoxins in human peripheral blood mononuclear cell (PBMC) suspensions and to validate the feasibility of the detection method.
According to the standards for photometric determination of bacterial endotoxin in General Notice 1143 of Chinese Pharmacopoeia (ChP) part Ⅲ (2020 edition) and 1085 of USP. A preliminary detection method of bacterial endotoxin content in PBMC suspension was established from standard curve reliability verification and primary interferential screening test. Bacterial endotoxins in three batches of PBMC suspensions were quantitatively detected by this method.
In the establishment of standard curve and reliability verification test，the linear regression equation was lgt= 2.910 9-0.300 1lgC，r=-0.999 8，negative control t>3 600 s，repeated reaction tube RSDs were less than 3%，the standard curve was established successfully. The results of the primary interferential screening test showed that the PBMC suspension had interference on limulus test at the dilution ratio of 10 and the recovery rate was close to 100% at the dilution ratio of 80，indicating that 80-fold was the best interference dilution ratio without interference. The results of the three batches of samples showed that the endotoxin contents were all less than 1.0 EU·mL-1 at the dilution ratio of 80，and the RSDs were less than 10%. The positive recoveries were 80.8%-100.3%，which complied with ChP 2020.
This method can quickly and quantitatively detect the content of endotoxin in PBMC suspension.
To establish a microbial limit test method and quality standard conforms to the USP for compound Kushen extract and its related components.
Comparative analysis was conducted on the standards for traditional Chinese medicine preparations and decoction pieces in the USP and the Chinese Pharmacopoeia 2020 Edition to determine the microbial limit standards for compound Kushen extract and its related components. Then，guided by the General Principles of the USP<61> and <62>，a microbial limit test method suitable for compound Kushen extract and its related components was developed. The plate pouring method was used to perform a methodological applicability test on the total aerobic bacterial count，mold and yeast count of compound Kushen extract and its two components Sophorae Flavescentis Radix and Heterosmilacis Rhizoma. The method applicability test was conducted on the control bacteria using the direct inoculation method. Finally，the MALDI/TOF MS microbial identification method and bacterial 16S rRNA gene sequence alignment analysis method were comprehensively used to identify the bacterial species of the contaminated microorganisms in the sample，and further analysis was conducted on the distribution and harm of the contaminated microbial population.
As a result，microbial limit standards for compound Kushen extract and its related components were established in accordance with the requirements of the USP. The recoveries of the counting methods for compound Kushen extract and its two components，Sophorae Flavescentis Radix and Heterosmilacis Rhizoma，were between 0.5 and 2.0. The positive control strains in the control bacterial method suitability test were able to grow，meeting the requirements of the USP. According to the identification results，40 contaminated microorganisms involved 13 species and 5 genera，mainly covering different types of Bacillus genus (87.5%)，Enterobacterium genus (10%)，and Pseudomonas genus (2.5%). Polluting microorganisms were all less harmful conditional pathogenic bacteria.
Established a microbial limit test method and quality standard conforms to the USP for compound Kushen extract and its related components. Defined the distribution and harm analysis of the pollution microorganism poulation of the above products. Improved the microbial quality control of the Chinese medicine preparation and its components.
To establish an integrated model of biosensing technology for the study of zebrafish，and to study and verify the interaction between cetirizine hydrochloride，fexofenadine hydrochloride，mizolastine，and ebastine，with the key target of allergic rhinitis，macrophage migration inhibitory factor（MIF）.
A MIF-high electron mobility transistor(HEMT) biosensor was constructed using AlGaAs/GaAs HEMT as the sensing element and MIF as the biological element. The interaction strength between cetirizine hydrochloride，fexofenadine hydrochloride，mizolastine，and ebastine and MIF was characterized. Furthermore，a zebrafish immune inflammation model was established. The number of neutrophil migration in the blank control group，model group，positive drug group，and low，medium，and high-dose drug groups were recorded. And the drug inflammation inhibition rate were calculated.
Cetirizine hydrochloride exhibited the strongest binding ability to MIF，with a dissociation constant of 7.05×10-13 mol·L-1. The KD for the interactions between fexofenadine hydrochloride，mizolastine，and ebastine with MIF were 2.34×10-8 mol·L-1，1.94×10-7 mol·L-1 and 2.44×10-8 mol·L-1，respectively. All of them had binding effects with MIF. Further the validation using the zebrafish immune anti-inflammatory model revealed that all four antihistamines significantly reduced the number of neutrophil migrations，among which 100 μmol·L-1 of cetirizine hydrochloride achieved a neutrophil migration inhibition rate of 68.5%.
This study explores the interaction between antihistamines and the key protein target of AR through the integration of biosensing technology and zebrafish experiments. It was found that MIF is a potential target for antihistamine drugs to exert their pharmacological effects. It provides an important reference for research on drug efficacy and its association with targets.
To establish the first batch of national drug reference standard of L-methionine sulfoxide.
The molecular structure of methionine sulfoxide was confirmed by the methods of IR，NMR and high resolution MS. Moreover，the moisture，residual solvents，residual ignition and related substances of L-methionine sulfoxide were determined，and then，its hygroscopicity，homogeneity and stability were also observed. Finally，mass balance method，HPLC and quantitative NMR were applied to determine and calculate its contents.
The results of experiments showed the structure of L-methionine sulfoxide were confirmed，its contents of moisture，residual ignition，metal elements and related substances were 0.23%，0.20%，10.87 μg·g-1 and 5.09%. The residual solvents were not detected in the samples. The final content of methionine sulfoxide was 94.5% by mass balance method. The contents of this batch of L-methionine sulfoxide were uniform，and it is stable in short-terms.
The first batch of national drug reference standard of L-methionine sulfoxide was established，and it is used for the inspection for related substances of amino acid drug，which is helpful in normalizing drug quality and guaranteeing drug safety.
To establish an inductively coupled plasma mass spectrometry (ICP-MS) method for the determination of migration Co，As，Br，Cd，Sb，Ba and Pb in recombinant human coagulation factor Ⅷ-Fc fusion protein for injection，and to quantitatively analyze the compatibility of long-term stored samples.
After adding 0.75 mL of water to the sample for 4 h，the purified water was filled to 5 mL，and the contents of 7 elements in the solution were determined by standard addition method. The working parameters of ICP-MS：radio frequency (RF) power of 1 550 W，sampling depth of 10.0 mm，carrier gas flow rate of 0.50 L·min-1，atomizer pump speed of 0.30 r·s-1，atomization chamber temperature of 2 ℃，dilution gas flow rate of 0.74 L·min-1，repeated sampling times of 3 times，analysis mode of full quantitative.
The linear relationship of each element was good in the concentration range of the detected mass，and the recoveries of the element added were 92.0%-101.8%. When applied to the determination of the samples at each accelerated placement point，the increased migration amounts were 43.2，68.7 and 66.8 ng·mL-1，respectively，except the element content of Br increased slightly. The contents of the remaining 6 elements were far below the prescribed limit.
The method is reliable and can be used for the determination of migration of 7 elements in recombinant human coagulation factor Ⅷ-Fc fusion protein for injection.
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