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This study aims to separate and characterize self-assembled nanoparticles(SAN) from Shaoyao Gancao Decoction(SGD) and determine the content of active compounds. Further, we aimed to observe the therapeutic effect of SGD-SAN on imiquimod-induced psoriasis in mice. The separation of SGD was performed by dialysis, and the separation process was optimized by single factor experiment. The SGD-SAN isolated under the optimal process was characterized, and the content of gallic acid, albiflorin, paeoniflorin, liquiritin, isoliquiritin apioside, isoliquiritin, and glycyrrhizic acid in each part of SGD was determined by HPLC. In the animal experiment, mice were assigned into a normal group, a model group, a methotrexate group(0.001 g·kg~(-1)), and SGD, SGD sediment, SGD dialysate, and SGD-SAN groups of different doses(1, 2, and 4 g·kg~(-1)) respectively. The psoriasis grade of mice was evaluated based on the pathological changes of skin lesions, the content of inflammatory cytokines, organ index and other indicators. The results showed that SAN obtained by centrifugation at 13 000 r·min~(-1) for 30 min was stable after dialysis for 4 times, which were uniform spherical nanoparticles with the particle size of(164.43±1.34) nm, the polydispersity index of(0.28±0.05), and the Zeta potential of(-12.35±0.80) mV. The active compound content accounted for more than 70% of SGD. Compared with the model group, SAN and SGD decreased the skin lesion score, spleen index, and inflammatory cytokine levels(P<0.05 or P<0.01) and alleviated the skin thickening and infiltration of inflammatory cells. However, the sediment group and the dialysate group had no obvious effect. SGD showed a good therapeutic effect on imiquimod-induced psoriasis in mice, and SAN demonstrated the effect equivalent to SGD in a dose-dependent manner. Therefore, we conclude that the SAN formed during decocting is the main active form of SGD, which can lower the levels of inflammatory cytokines, promote the normal differentiation of keratinocytes, and reduce the infiltration of inflammatory cells in the treatment of psoriasis lesions in mice.
Subject(s)
Mice , Animals , Imiquimod , Drugs, Chinese Herbal/pharmacology , Glycyrrhizic Acid , Chromatography, High Pressure Liquid/methodsABSTRACT
In order to comprehensively evaluate the quality of Viticis Fructus, this study established HPLC fingerprints and evaluated the quality of 24 batches of Viticis Fructus samples from different species by similarity evaluation and multivariate statistical analysis(PCA, HCA, PLS-DA). On this basis, an HPLC method was established to compare the content differences of the main components, including casticin, agnuside, homoorientin, and p-hydroxybenzoic acid. The analysis was performed on the chromatographic column(Waters Symmetry C_(18)) with a gradient mobile phase of acetonitrile(A)-0.05% phosphoric acid solution(B) at the flow rate of 1 mL·min~(-1) and detection wavelength of 258 nm. The column temperature was 30 ℃ and the injection volume was 10 μL. The HPLC fingerprint of 24 batches of Viticis Fructus samples was established with 21 common peaks, and nine peaks were identified. Similarity analysis was carried out based on chromatographic data of 24 batches of chromatographic data of Viticis Fructus, and the results showed that except for DYMJ-16, the similarity of Vitex trifolia var. simplicifolia was ≥0.900, while that of V. trifolia was ≤0.864. In addition, the similarity analysis of two different species showed that the similarity of 16 batches of V. trifolia var. simplicifolia was 0.894-0.997 and that of the eight batches of V. trifolia was between 0.990 and 0.997. The results showed that the similarity of fingerprints of these two species was different, but the similarity between the same species was good. The results of the three multivariate statistical analyses were consistent, which could distinguish the two different species. The VIP analysis results of PLS-DA showed that casticin and agnuside contributed the most to the distinction. The content determination results showed that there was no significant difference in the content of homoorientin and p-hydroxybenzoic acid in Viticis Fructus from different species, but the content of casticin and agnuside was significantly different in different species(P<0.01). The content of casticin was higher in V. trifolia var. simplicifolia, while agnuside was higher in V. trifolia. The findings of this study show that there are differences in fingerprint similarity and component content of Viticis Fructus from different species, which can provide references for the in-depth study of the quality and clinical application of Viticis Fructus.
Subject(s)
Drugs, Chinese Herbal/chemistry , Chromatography, High Pressure Liquid/methods , Fruit/chemistry , Vitex/chemistryABSTRACT
@#TA method for the content determination of methionine sulfoxide and methionine sulfone in compound amino acid injection (18AA-II) was established in order to investigate their level in 155 batches of this product, and to explore the reason for the generation of these two impurities.The determination was performed on an Agilent Poroshell 120 EC-C18 column with mobile phases of sodium acetate/tetrahydrofuran solution (A) and sodium acetate solution -acetonitrile-methanol (B, 200∶400∶400) (gradient elution) at the flow rate of 0.5 mL/min.The excitation wavelength and the emission wavelength of the fluorescence detector were 233 nm and 441 nm, respectively.The column temperature was 40 °C, and the injection volume was 8 μL.The contents of methionine sulfoxide and methionine sulfone from 155 batches of compound amino acid injection (18AA-II) was determined using this method, and the residual oxygen content was detected by headspace gas analyzer.The results showed that the linear range of methionine sulfoxide and methionine sulfone were 0.128 1-10.250 0 μg/mL (r = 0.999 9) and 0.261 0-10.440 0 μg/mL (r = 0.999 8), respectively.The limits of quantitation were 0.13 μg/mL and 0.26 μg/mL, respectively; the limits of detection were 0.04 μg/mL and 0.09 μg/mL, respectively.RSDs of precision, stability and repetitive test were all lower than 1.3%.The recoveries ranged 98.00%-100.79% (RSD = 1.15%, n = 9) and 98.19%-102.31% (RSD = 1.33%, n = 9).The content level of oxidized related substances from different manufacturers showed significant difference, showing relevance with the residual oxygen content to some extent, yet no significant correlation with the added amount of antioxygen (sodium pyrosulfite).The method is validated to be useful for the content control of methionine sulfoxide and methionine sulfone in compound amino acid injection (18AA-II).It is quite necessary to include the determination of oxidized related substance into the quality specification.Manufacturers should strengthen the control of remaining oxygen in their products.
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@#A deuterated internal standard quantitative analysis method based on liquid-liquid extraction-ultra performance liquid chromatography-tandem mass spectrometry (LLE-UPLC-MS/MS) for simultaneous determination of 10 illicit drugs in wastewater was established.Wastewater samples were concentrated by liquid-liquid extraction with dichloromethane: ethyl acetate (1∶1), and separated by a linear gradient of 0.1% formic acid-5 mmol/L ammonium formate aqueous solution and acetonitrile on a C18 column. The samples were then detected by ESI positive ion mode and multiple reaction monitoring mode (MRM) for quantitative analysis.All analytes had a good linear relationship (r ≥ 0.995 7) within the range of their respective standard curves; the limit of quantification was 1 ng/L (except amphetamine at 2.5 ng/L); the relative recovery rate ranged from 96.36% to 106.43%, and the intra- and inter-day precisions were less than 4.70%.This method is accurate, reliable and reproducible, and is suitable for the quantitative determination of 10 illicit drugs in wastewater.It is also suitable for wastewater with complex matrixes that affect solid phase extraction and enrichment.It provides a new analytical method for real-time monitoring of drug abuse.
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@#An analytical liquid-liquid extraction-gas chromatography–mass spectrometry (LLE-GC-MS) method was established for the determination of genotoxic impurities including methyl methanesulfonate (MMS), ethyl methanesulfonate (EMS) and isopropyl methanesulfonate (IMS) in methanesulfonic acid. An Agilent HP-1MS capillary column (30 m × 0.32 m, 1 μm) was used for separating the analytes by programmed heating with the inlet temperature of 220 °C. Mass spectrometry was operated in positive ion mode, and selective ion monitors were set at m/z 80 for MMS, m/z 79 for EMS, m/z 123 for IMS and m/z 56 for internal standard butyl methanesulfonate (BMS). Results showed that the baseline separation of MMS, EMS and IMS was achieved, and the blank extraction solution had no interference; good linearity was achieved in the range of 37-1 480 ng/mL for three alkyl methanesulfonates; The mean recoveries of MMS, EMS, IMS were 104.99%, 107.26%,108.85%, respectively, with RSD ≤ 4.54%. The established method has the characteristics of specific, sensitive, accurate, stable and good versatility, and has been used for the detection and control of alkyl methanesulfonate impurities in methanesulfonic acid from a variety of manufacturers.
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@#A reversed phase HPLC method for determination of hydroxylsafflower yellow A in safflower W/O cream was established. The column was Zorbax Eclipse C18 column(4. 6 mm×250 mm, 5 μm), and the mobile phase was composed of methanol, acetonitrile and 0. 02% phosphoric acid solution(26 ∶2〓 ∶72). The flow rate of mobile phase was set at 1. 0 mL/min, and the column temperature was kept at 55 °C. The detection wavelength was 403 nm. Safflower W/O cream was successively demulsified with methanol at high temperature and followed by the addition of purified water for the extraction. The results showed that the excipients did not interfere with the chromatographic peak of hydroxylsafflower yellow A. Hydroxylsafflower yellow A presented a good linear relationship in the range of 1. 236- 12. 36 μg/mL(y=156. 17x+1. 198 3, r=0. 999 5), and the detection limit was 23. 6 ng/mL with the quantitative limit of 118 ng/mL. The percentage of extracting recovery was in the range of 99. 7% to 103. 3%. The precision RSD was 0. 12%(n=6), and the sample stability was acceptable when being stored at room temperature for 24 h. The developed method in this study was simple, rapid, accurate and reproducible, and can be used for the determination of hydroxylsafflower yellow A in safflower W/O cream.
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@#To develop a rapid and sensitive liquid chromatography-tandem mass spectrometry(LC-MS/MS)method for the determination of endogenous glutathione in rat plasma. Glycyltyrosine was used as the internal standard(IS)and 4-(N-maleimido)phenyl trimethylammonium iodide(MPTA)was used as the derivation reagent. Chromatographic separation was achieved on a Zorbax HILIC PLUS column(4. 6 mm×100 mm, 3. 5 μm)and the mobile phase consisted of acetonitrile and 0. 1% formic acid(75 ∶25)pumped at a flow rate of 1. 0 mL/min. Detection was carried out on a triple quadrupole tandem mass spectrometer by selected reaction monitoring(SRM)in the can meet positive ion mode. The linearity ranged from 3. 000 to 2 000 ng/mL(r=0. 997 1); and the limit of detection of glutathione in rat plasma was 10 pmol/L. Matrix effect, stability, precision and accuracy of the method met the requirements. The proposed method was proved to be selective and sensitive, which is suitable for the quantification of endogenous glutathione in rat plasma.
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This study aimed to develop an HPLC method for simultaneous determination of the 3 components in Ziziphora bungeana. The optimum HPLC condition was as follows：ZORBAX SB-C₁₈ column（4.6 mm×250 mm, 5.0 μm）with gradient elution of methanol (A)-0.2% glacial acetic acid (B), detection wavelength 340 nm,column temperature 30 °C, flow rate 1 mL·min⁻¹. There were good linearity between peak areas and injection quantity of caffeic acid, rosmarinic acid, and linarin in the range of 2-40 mg·L⁻¹（=0.999 9）, 3-60 mg·L⁻¹（=1）, 7-140 mg·L⁻¹（r=0.999 9）, respectively. The average recoveries were 100.9%（RSD 1.3%）,98.25%（RSD 2.0%）,and 98.73%（RSD 1.5%）, respectively. The HPLC method was stable and accurate, which could be used to detect caffeic acid,rosmarinic acid, and linarin in Ziziphora bungeana.
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An HPLC method was developed for the determination of iridoid glycosides (loganin acid, loganin, sweroside) and saponins (asperosaponin Ⅵ) in the wild Dipsacus asper. A total of 108 samples consecutive growing 12 month were collected in 9 plots in Wulong district of Chongqing. Subsequent analysis of the content of loganin acid, loganin, sweroside and asperosaponin Ⅵ was performed by HPLC to evaluate the quality. In addition, 20 climate data provided by the world climate database (http://www.worldclim.org/) was analyzed to deduce the correlation between the growing environment factors and the active ingredient content accumulation of D. asperoides and choose the apposite growing environment for D. asper. The range of active ingredient content in wild D. asper were 0.01%-3.80%(loganin acid), 0.08%-0.62%(loganin), 0.12%-0.78%(sweroside), 0.64%-5.26%(asperosaponin Ⅵ). The highest content of these active ingredients was concentrated from February to April, with 2.64% of loganin acid, 0.36% of loganin), 0.57% of sweroside, and 3.09% of asperosaponin Ⅵ. The method used for determination of the active ingredient content in D. asper was simple and convenient with accurate result. The selection of the quadrats is scientific and reasonable and can be used for the analysis of the contents of the wild D. asper, thus provide a reference for quality evaluation of D. asper and protection of D. asper resources.
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OBJECTIVE:To establish the method for simultaneous determination of 9 components in Huichun yuzi granules. METHODS:LC-MS/MS method was adopted. The determination was performed on Shim-pack XR-ODS C18column with column temperature of 30 ℃. The sample size was 5 μL,and ion source as electrospray ion source;MRM mode was adopted. The acetonitrile-water was used as mobile phase for ferulic acid,rutin,paeonol,icariin and schisandrin(gradient elution);positive ion mode monitoring was conducted. The methanol-0.1% fomic acid water was used as mobile phase for naringin,verbascoside, amygdalin and protocatechuic acid(gradient elution);negative ion mode monitoring was conducted. RESULTS:The linear ranges of ferulic acid,rutin,paeonol,icariin,schisandrin,naringin,verbascoside,amygdalin and protocatechuic acid were 0.499 5-500, 25-1 000,0.245 9-250,5.185 1-1 000,0.981 9-1 000,0.248 1-125,2.510 4-250,10-2 500,4.999 7-1 000 ng/mL(r≥0.997 8), respectively. The limits of detection were 0.075,0.30,0.072,0.015,0.015,0.075,0.15,0.30,0.15 ng/mL,respectively;the limits of quantitation were 0.25,1.00,0.24,0.51,0.49,0.25,0.50,0.99,0.49 ng/mL,respectively. The recoveries rate were 91.39%-103.56%(RSD=1.03%-3.67%,n=6).RSDs of stability test ranged 1.4%-3.4%(n=6)within 48 h at room temperature. CONCLUSIONS:The method is rapid,sensitive and reproducible,and can be used for content determination of 9 components in Huichun yuzi granules.It can be used for quality control of Huichun yuzi granules.
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A method based on in situ formed ionic liquids microextraction-ultra performance liquid chromatography(ISFILM-UPLC) was established for the determination of vancomycin in human serum.The samples were pretreated by vortex and centrifugation.[C6MIM] [Br] and NaPF6 were applied as the extracting agent,and Metronidazole as the internal standard.Methanol was applied to redissolve the precipitate.The separation was carried out on a BEH C18 column,and the mobile phase consisted of methanol and 0.05 mol/L monopotassium phosphate solution.The flow rate was 0.2 mL/min,the injection volume was 5 μL,and the column temperature was 18 ℃.The key parameters of this method such as the specificity,accuracy and precision were validated.This method showed good correlation with the immunoassay.It shows great potential for the application in clinical practice.
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In order to research the related substances of dabigatran etexilate mesylate,seven related compounds were synthesized and the related detection methods were established (Using XBridge C18 as the column,methanol-0.01mol/L potassium dihydrogen phosphate as the mobile phase Gradient elution,detection wavelength of 310 nm).The solvents and the injection volume were also screened.Results showed that the established method could separate the dabigatran etexilate mesylate and seven compounds completely,and the reproducibility was good and the accuracy was high,which was suitable for the detection of the related substances of dabigatran etexilate mesylate.
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In order to compare the effect of sulfur fumigation processing and direct hot air heating technology on puerarin contents and efficacy of Puerariae Thomsonii Radix, the fresh roots of Pueraria thomsonii were cut into small pieces and prepared into direct sunshine drying samples, direct hot air drying samples, and sulfur fumigation-hot air drying samples. Moisture contents of the samples were then determined. The puerarin contents of different samples were compared by HPLC method. Moreover, the models of drunkenness mice were established, and then with superoxide dismutase (SOD) content as the index, aqueous decoction extracts of Puerariae Thomsonii Radix samples with sulfur fumigation processing and non-sulfur fumigation processing methods were administrated by ig; the effects of sulfur fumigation on contents of SOD in mice liver and serum were determined, and the sulfur fumigation samples and non-sulfur fumigation samples were investigated for moth and mildew under different packaging and storage conditions. Results showed that the sulfur fumigation samples significantly changed the puerarin content from Puerariae Thomsonii Radix. The content of puerarin was decreased gradually when increasing the times of sulfur fumigation and amount of sulfur. SOD content in drunken mice liver and serum was significantly decreased when increasing the times of sulfur fumigation, showing significant difference with both direct sunshine drying group and direct hot air drying group. Moth and mildew were not found in the sulfur fumigation samples and direct hot air drying samples whose moisture contents were lower than the limit in Pharmacopoeia. Research showed that sulfur fumigation can significantly reduce the content of main active ingredients and reduce the efficacy of Puerariae Thomsonii Radix, indicating that the quality of Puerariae Thomsonii Radix was significantly decreased after sulfur fumigation. However, the contents of the main active ingredients, efficacy and storage results of the direct hot air drying samples were similar to those in direct sunshine drying samples, so the hot air drying process was a nice drying technology which could be promoted for use.
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To establish an HPLC-ELSD method for the quantification of triterpenoids in the fruits of Buddleja lindleyana. The RP-HPLC-ELSD method was used for the determination of triterpenoids in B. lindleyana fruits, which were collected from different habitats. The column used was a packed with 5 μm stationary phase Waters SunFireTM C₁₈ (4.6 mm×150 mm, 5 μm). The mobile phase consisted of Methanol-water(82∶18) at a flow rate of 1 mL•min⁻¹. Column temperature： 30 ℃. ELSD conditions： drift tube temperature： 106 ℃; carrier gas (nitrogen) flow rate： 1.5 L•min⁻¹; amplification factor： 1. The calibration curves showed good linear relationship on a range from 0.702 to 28.08 μg(r=0.999 2) for Clinoposaponin III, 0.390 to 15.60 μg(r=0.998 9) for Desrhamnoverbascosaponin and 0.192 to 7.68μg(r=0.999 0) for Mimengoside I. The average recovery rate(n=6) were 99.41%, 99.08% and 98.67% and it's RSD were 0.86%, 1.56% and 1.80%. This method can be used to determine the contents of triterpenoids in the fruits of Buddleja lindleyana for its simplicity, accurateness and reliability.
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@#An ultrasensitive electrochemical immunosensor modified with graphene and dienestrol(DE)was developed for the detection of dienestrol through indirect competition with K3Fe(CN)6 acting as the redox probes. The results revealed that under optimized conditions a calibration for DE was obtained with a linear range of 500-5 000 ng/mL and the detection limit was up to 0. 2 ng/mL, showing that the proposed electrochemical immunosensor had excellent sensitivity and wide detection range. The immunosensor was examined in real samples for the analysis of DE. A good recovery in the range of 83. 8%-97. 7% was obtained in pork samples.
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@#Carbon dots(CDs)were firstly prepared via a simple pyrolysis route using citric acid as the carbon precursor and glycine as modifier. The obtained CDs were further purified by n-butyl alcohol, which made the particle size more uniform and the fluorescence intensity stronger. When excited at 380 nm, the maximum emission wavelength of the CDs was about 480 nm, with a quantum yield of 47%. Based on fluorescence quenching, a novel method for chloramphenicol assay was developed with glycine-passivated CDs. This method is simple and rapid. A linear relationship between the change of fluorescence intensity and the concentration of chloramphenicol was obtained with a relation coefficient of 0. 999 7. The recovery was in the range of 99% to 101% with a relative standard deviation of 0. 3%, which shows CDs′ potential application in drug detection.
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@#To determine the contents of p-coumaric acid and ferulic acid in Rhizama Phragmitis by HPLC for quality evaluation. Rhizama Phragmitis alkaline alcoholic solution exact of p-coumaric acid and ferulic acid was refluxed. An HPLC method was developed by using a Diamonsil C18(4. 6 mm×250 mm, 5 μm)column, and the mobile phase of acetonitrile and 0. 3% acetic acid with linear gradient elution at a flow of 1. 0 mL/min and UV detection at 310 nm. Calibration curvers of p-coumaric acid and ferulic acid were linear over the range of 0. 2-200 μg/mL and 0. 1-120 μg/mL respectively. . Average recoveries were(96. 9±2. 91)% and(98. 7±1. 78)%, respectively. The average contents of p-coumaric acid and ferulic acid expressed as mean±sd(max-min)were(0. 88±0. 16)%(1. 21%-0. 70%)and(0. 41±0. 035)%(0. 49%-0. 36%)in 15 batches of Rhizama Phragmitis. The established method will be beneficial to the quality evaluation of Rhizama Phragmitis.
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Objective To establish a high performance liquid chromatography(HPLC) method for quantitative determination of S-clopidogrel and its related substance R-clopidogrel in clopidogrel tablets, and to compare their contents between clopidogrel tablets produced by three different manufacturers. Methods An ULTRON ES-OVM column (4.6 mm × 150 mm,5 μm) was used in this study. The mobile phase consisted of a mixture of acetonitrile and 0.01 mol/L potassium dihydrogen phosphate solution (20:80). The flow-rate was 1 mL/min, with the column temperature being 17°C and the UV detection wave lenth being 220 nm. Results S-clopidogrel and its related substance R-clopidogrel were in good linear relationship within 50-117 mg/L and 1.52-30.04 mg/L, respectively (both r=0.999 8). The precision of our method was satisfactory and the average recoveries were 99.7% and 98.8%, respectively. The test solution remained stable for 12 h. S-clopidogrel and R-clopidogrel in clopidogrel tablets produced by three manufacturers were in line with the quality requirements of pharmacopoeia. Conclusion Our method is accurate and has good specificity. It can be used for the quality control of clopidogrel tablets.
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OBJECTIVE:To study the feasibility of the quality standard of Potassium sodium pehydroandroandrographolide succinate for injection. METHODS:The contents of dehydroandyograpolide succinate in Potassium sodium dehydroandrograpolide succinate for injection from 13 manufacturers were determined by HPLC and UV spectometry.The related substances of all the samples were determined by HPLC and TLC,respectively. The feasibility of different assaying methods was compared. RESULTS:Determined by HPLC assay,the principal constituent and the degradation products could be separated. There were significant differences between different assaying methods in the determination results of the related substances of samples; TLC assay was inferior to HPLC in respect of the limit of quantitation and accuracy. CONCLUSION:The quality in a few enterprises remains to be improved and the quality standard of Potassium sodium pehydroandroandrographolide succinate for injection remains to be further improved.
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OBJECTIVE:To develop a RP-HPLC method for the simultaneous determination of Notoginsenoside R1 and Ginsenoside Rg1 in Danqi tablets. METHODS:The chromatographic separation was performed on a Kromasil C18(250mm?4.6mm,5?m) column with column temperature at room temperature. The mobile phase consisted of acetonitrile-water-H3PO4(20.5∶79.5∶0.02) at a flow rate of 1.0mL?min-1.The detection wavelength was set at 203nm. RESULTS:The linear range of Notoginsenoside R1 and Ginsenoside Rg1 were 4.67～46.68?g?mL-1 (r=0.999 0) and 4.62～115.50?g?mL-1 (r=0.999 9), respectively; with the average recover of Notoginsenoside R1 at 97.93% and that of Ginsenoside Rg1 at 97.98%, and RSD at 1.31%(n=6) and 1.38%(n=6), respectively. CONCLUSION:The method is simple, rapid and accurate, and suitable for the quality control of Danqi tablets.