Development and validation of an LC-MS/MS method for pharmacokinetic study of lobetyolin in rats

Abstract A simple and selective liquid chromatography tandem with mass spectrometry (LC-MS/ MS) method for quantification of lobetyolin in rat plasma was developed and validated. Chromatographic separation was achieved on a Thermo ODS C18 reversed-phase column using 0.1% aqueous formic acid-methanol (50:50, v/v) in an isocratic elution mode at a flow rate of 0.4 mL.min-1. LC/MS performance was done in a positive ion ESI mode and the MS/MS transitions were monitored at m/z 419.3 [M+Na]+ → m/z 203.1 for lobetyolin and m/z 394.9 [M+Na]+ → m/z 231.9 for IS, respectively. The assay exhibited a linear dynamic range over 1.0-500 ng.mL-1 for lobetyolin in plasma. Both the precision (%RSD) and accuracy (RE%) were within acceptable criteria (<15%). Recoveries ranged from 87.0% to 95.6%, and the matrix effects were from 91.0% to 101.3%. After oral administration, the peak plasma concentration of lobetyolin was obtained as 60.1 ng.mL-1 at 1.0 h. The proposed LC-MS/MS method could be applied to a pharmacokinetic study employing 66 samples from 6 Wistar rats

Determination of Calycosin-7-O-β-D-Glucopyranoside and Lobetyolin in Danqi Xinmaikang boiled powders and pieces by quantitative analysis of multi-components by single maker / 国际中医中药杂志

Objective:To establish a quality evaluation method for the simultaneous determination of Calycosin-7-O-β-D-Glucopyranoside and Lobetyolin in Danqi Xinmaikang boiled powders and pieces.Methods:Quantitative analysis of multi-components was performed to determine contents of Calycosin-7-O-β-D-Glucopyranoside and Lobetyolin with Calycosin-7-O-β-D-Glucopyranoside as the reference substance by single-maker (QAMS). The chromatogram conditions were established, with C18 column as solid phase, acetonitrile-water as flowing phase, 268 nm as detecting wavelength, 1.0 ml/min as flowing rate, 30 ℃ as column temperature, and 10 μl as injection volume.Results:The relative correction factor between Calycosin-7-O-β-D-Glucopyranoside and Lobetyolin was 1.14. There was no significant difference of measured values between the external standard method and QAMS ( P>0.05). With Calycosin-7-O-β-D-Glucopyranoside retention time of 1.00, the relative retention time of Lobetyolin was 1.51 and RSD was less than 5%. Conclusion:It is feasible and accurate to evaluate the quality of Danqi Xinmaikang boiled powders and pieces by QAMS.

Interaction Between Lobetyolin and Bovine Serum Albumin by Steady-State Fluorescence and Molecular Docking / 中国实验方剂学杂志

Objective:The interaction between lobetyolin and bovine serumal bumin（bovine serum albumin，BSA）. Method:By the steady-state fluorescence analysis method，the molecular-docking，ultraviolet absorption spectrum and fluorescence quenching were used to calculate quenching constant and binding constant，the number of sites，the position，the force and the distance of lobetyolin-BSA system. In addition， the effect of metalionson quenching constant of the lobetyolin-BSA system was studied. Result:The quenching constant was 1.25×104 L·mol-1（37 ℃），the binding constant was 2.95×104 L·mol-1（37 ℃），and the number of sites was 1 and bound with site 1 in ⅡA of BSA， thermodynamic meters were ΔH=-19.374 kJ·mol-1，ΔS=23.1 J·mol-1·K-1， the interaction distance was 3.2 nm. Meta lions could accelerate the quenching. Conclusion:By the steady-state fluorescence technique，molecular-docking and ultraviolet absorption spectrum，the quenching mechanism of Lobetyolin-BSA is quiescent quenching，and the interactive force is electro static force. The Lobetyolin-BSA can be well combined. At the same time，it also shows that the molecular docking results are similar to the experimental results obtained by steady-state fluorescence analysis.

Effect of Different Processing Methods on Types and Contents of Chemical Constituents in Codonopsis Radix from Shanxi / 中国实验方剂学杂志

Objective:To establish the HPLC fingerprint of raw products,rice-processed products and honey-processed products of Codonopsis Radix from Shanxi,and establish determination of their chemical constituents,which was used to analyze the changes of types and contents of chemical constituents in Codonopsis Radix from Shanxi before and after processing. Method:Agilent ZORBAX SB-C18 column(4.6 mm×250 mm,5 μm) was adopted,the separation process was carried out using a binary gradient elution system composed of 0.5% phosphoric acid aqueous solution and acetonitrile,the column temperature was 30℃ and the detection wavelength was 220 nm. Result:Compared with the corresponding reference fingerprint,the similarities of HPLC fingerprint of 10 batches of raw products and processed products were >0.90.In raw products,rice-processed products and honey-processed products of Codonopsis Radix from Shanxi,the contents of lobetyolin were (0.33±0.049),(0.24±0.034),(0.18±0.047) mg·g-1,the contents of atractylenolide Ⅲ were (0.20±0.046),(0.40±0.046),(0.31±0.060) mg·g-1,the contents of 5-hydroxymethylfurfural(5-HMF) were (0.74±0.16),(1.45±0.19),(1.54±0.12) mg·g-1,respectively. Conclusion:Different processing methods have little effect on types of chemical constituents in Codonopsis Radix from Shanxi,but have great effect on the contents of some chemical constituents.

Effect of Sulfuring on Time-quality Relationship of Codonopsis Radix in the Process of Storage / 中国药学杂志

OBJECTIVE: To investigate the advantages and disadvantages of sulfuring to Codonopsis Radix in its process of storage. METHODS: Sulfur dioxide residual amount was determined by distillation. The character was observed referring to Ch.P. The content of polysaccharide was measured by ultraviolet spectrophotometry. The contents of lobetyolin and rhizome atractylodis inner fat III were measured by high performance liquid chromatography, and fingerprints were established. RESULTS: Under the natural storage conditions, the contents of effective components decreased with the extension of storage time. After being stored for 30 m, the Codonopsis Radix samples without sulfuring treatment had darkened color and were damaged by worms seriously, the contents of polysaccharide, lobetyolin, and rhizome atractylodis inner fat III reduced by more than half, and yellow aspergillus toxin were detected; however, few sulfured samples were damaged by worms and the magnitude of content decrease of the three compounds was significantly smaller than that of the non-sulfured ones. CONCLUSION: Storage time is negatively correlated with the medicinal quality of Codonopsis Radix and proper sulfuring can slow the rate of quality deteriorating of Codonopsis Radix.

Effects of Chitosan Flocculation Clarification Process and Alcohol Precipitation Process on Water Extract of Codonopsis Radix / 中国中医药信息杂志

Objective To explore the effects of chitosan flocculation clarification process and alcohol precipitation process on the principal chemical constituents of water extract of Codonopsis Radix. Methods The influence of two purification processes on water extract of Codonopsis Radix was investigated through lobetyolin contents, extract yield, and relative apparent content of each component in HPLC fingerprint as evaluation indexes. Results Chitosan flocculation clarification process showed a significantly higher extract yield of water extract compared with alcohol precipitation process, and it has a markedly better retention effect for strong polarity constituents; alcohol precipitation process exhibit a little better retention effect for lobetyolin and a better retention effect for weak polarity constituents. Conclusion The above two processes have some regularity in the influence on the main chemical constituents in the water extract of Codonopsis Radix, which can provide some guidance for the reasonable choice of the purification process for water extracts of Codonopsis Radix, and other TCM water extracts.

Effect of different fertilization treatments on yield and secondary metabolites of Codonopsis pilosula / 中国中药杂志

The research studies the effect of different fertilization treatments on yield and accumulation of secondary metabolites of Codonopsis pilosula by using single factor randomized block design, in order to ensure reasonable harvesting time and fertilization ratio, and provide the basis for standardized cultivation of C. pilosula. According to the clustering results, the nitrogen fertilizer benefitted for the improvement of root diameter and biomass of C. pilosula. The phosphate fertilizer could promote the content of C. pilosula polysaccharide. The organic fertilizers could increase the content of lobetyolin. With the time going on, C. pilosula's yield, polysaccharide and ehanol-soluble extracts increased while the content of lobetyolin decreased. According to various factors, October is a more reasonable harvest period. Organic fertilizers are more helpful to the yield and accumulation of secondary metabolites of C. pilosula.

Determination of lobetyolin, scopolin, scopoletin, quercetin, kaempferol and isorhamnetin in Shenqi Jiu by HPLC with gradient elution method / 国际药学研究杂志

Objective To develop an HPLC method with gradient elution for determination of lobetyolin, scopolin, scopoletin, quercetin, kaempferol and isorhamnetin in Shenqi Jiu. Methods A Shiseido C18 column (4.6 mm×250 mm, 5μm) was adopted, the mobile phase was acetonitrile (A)-0.5% acetic acid solution (B) with gradient elution at a flow rate of 1.0 ml/min, the column temperature was set at 30 ℃, the sample quantity was 20 μl, and the detection wavelengths were 269 (lobetyolin), 346 (scopolin and scopoletin) and 360 nm (quercetin, kaempferol and isorhamnetin). Results The linear ranges of above mentioned 6 ingredients in order fell within the range of 4.59-91.80 (r=0.9999), 2.62-52.40 (r=0.9996), 1.95-39.00 (r=0.9998), 3.34-66.80 (r=0.9995), 2.30-46.00 (r=0.9991), and 2.86-57.20 μg/ml (r=0.9999), respectively. The average recoveries and RSD (n=9) were 98.27% (1.33%), 97.62% (1.48%), 99.17% (0.99%), 98.50% (1.37%), 97.35% (1.35%), and 98.86% (1.70%), respectively. Conclusion The established HPLC gradient elution method can simultaneously determine the above mentioned 6 ingredients Shenqi Jiu. The method is simple, accurate, with good reproducibility. The method is helpful for the quality control of Shenqi Jiu.

Determination of lobetyolin,scopolin,scopoletin,quercetin,kaempferol and isorhamnetin in Shenqi Jiu by HPLC with gradient elution method / 国际药学研究杂志

Objective To develop an HPLC method with gradient elution for determination of lobetyolin,scopolin,scopole?tin,quercetin,kaempferol and isorhamnetin in Shenqi Jiu. Methods A Shiseido C18 column(4.6 mm×250 mm,5μm)was adopted, the mobile phase was acetonitrile(A)-0.5%acetic acid solution(B)with gradient elution at a flow rate of 1.0 ml/min,the column tem?perature was set at 30℃,the sample quantity was 20μl,and the detection wavelengths were 269(lobetyolin),346(scopolin and sco?poletin)and 360 nm(quercetin,kaempferol and isorhamnetin). Results The linear ranges of above mentioned 6 ingredients in order fell within the range of 4.59-91.80(r=0.9999),2.62-52.40(r=0.9996),1.95-39.00(r=0.9998),3.34-66.80(r=0.9995),2.30-46.00 (r=0.9991),and 2.86-57.20μg/ml(r=0.9999),respectively. The average recoveries and RSD(n=9)were 98.27%(1.33%),97.62%(1.48%),99.17%(0.99%),98.50%(1.37%),97.35%(1.35%),and 98.86%(1.70%),respectively. Conclusion The established HPLC gradient elution method can simultaneously determine the above mentioned 6 ingredients Shenqi Jiu. The method is simple,ac?curate,with good reproducibility. The method is helpful for the quality control of Shenqi Jiu.

Simultaneous Determination of Six Components in Shenqi Fuzheng Injection by HPLC / 中国药房

OBJECTIVE:To establish a method for the contents determination of astragalosidesⅠ,Ⅱ,Ⅲ,Ⅳ,lobetyolin and for-mononetin in Shenqi fuzheng injection. METHODS:HPLC was performed. The detector was evaporative light scattering detector, column was Ultimate XB-C18 with mobile phase of acetonitrile-0.5% formic acid (gradient elution) at a flow rate of 1.2 ml/min, column temperature was 30 ℃,and injection volume was 10 μl. RESULTS:The linear range was 0.62-5.67 μg for astragalosidesⅠ(r=0.999 6),0.78-6.31 μg for astragalosidesⅡ(r=0.999 6),0.36-3.48 μg for astragalosidesⅢ(r=0.999 7),0.81-6.72 μg for as-tragalosides Ⅳ(r=0.999 5),0.82-7.03 μg for lobetyolin(r=0.999 8)and 0.58-6.62 μg(r=0.999 7)formononetin;limit of quan-titation was 1.21,0.15,0.12,0.03,0.12,0.17 μg/ml;limit of detection was 0.35,0.35,0.04,0.01,0.03,0.04 μg/ml;RSDs of pre-cision,stability and reproducibility tests were lower than 2%;recoveries were 95.1%-101.1%(RSD=2.0%,n=9),95.2%-100.7%(RSD=1.9%,n=9),95.8%-100.2%(RSD=1.4%,n=9),96.2%-100.6%(RSD=1.7%,n=9),96.6%-101.2%(RSD=1.4%, n=9) and 95.9%-99.5%(RSD=1.2%,n=9),respectively. CONCLUSIONS:The method is simple and accurate,and can be used for the simultaneous determination of astragalosidesⅠ,Ⅱ,Ⅲ,Ⅳ,lobetyolin and formononetin in Shenqi fuzheng injection.

Simultaneous Determination of Four Components in Huangqi Shengmai Decoction by HPLC-DAD / 中国中医药信息杂志

Objective To establish a method for simultaneous determination of contents of schisandrin B, schisantherin A, lobetyolin and ruscogenin in Huangqi Shengmai Decoction with HPLC method. Methods The chromatographic column was an Agilent Eclipse C18 column (4.6 mm × 250 mm, 5 μm) that was maintained at a constant temperature of 30 ℃. The mobile phase was 0.1% acetic acid solution-acetonitrile, with gradient elution. The flow rate was 1.0 mL/min. The detection was at wavelength of 280 nm. Results The standard curves of schisandrin B, schisantherin A, lobetyolin and ruscogenin had good linearity in the ranges of 0.862 5-21.562 5μg (r 2=0.999 1), 0.737 5-18.437 5μg (r 2=0.999 4), 0.095 2-2.380 0 μg (r 2=0.999 6), and 0.810 0-20.250 0 μg (r 2=0.999 0), respectively. The average recoveries of the four components were 98.06%(RSD=1.64%), 99.61%(RSD=2.72%), 97.98%(RSD=1.45%), and 99.30%(RSD=1.25%), respectively. Conclusion This method is accurate, rapid and specific, achieving the stable results with high reproducibility.

Rapid determination of acteoside, lobetyolin and another eleven effective components of Shengan Guanxin Mixture by LC-MS/MS / 中国药学杂志

OBJECTIVE: To establish an LC-MS/MS method to determine eleven components(paeoniflorin, acteoside, ferulic acid, naringin, salvianolic acid B, lobetyolin, hesperidin, liquiritin, isoliquiritoside, liquiritigenin, isoliquiritigenin) in Shengan Guanxin Mixture(traditional Chinese medicines) simultaneously. METHODS: The HPLC analysis was performed on an Agilent SB-C18 column(2.1 mm × 50 mm, 1.8 μm) with gradient elution of 0.1% formic acid and methanol containing 0.1% formic acid, the flow rate was 0.3 mL · min, and the column temperature was room temperature. Electrospray ionization (ESI) source was used. Quantitation was performed by using selected multiple reaction monitoring (MRM) mode. RESULTS: The method had good linearity in the ranges of 0.7580-30.32 μg · mL-1 (r =0.9949) for paeoniflorin, 0.3040-12.16 μg · mL-1 (r =0.9995) for acteoside, 0.2480-9.920 μg · mL-1 (r = 0.9969) for ferulic acid, 0.5035-20.14 μg · mL-1 (r = 0.9983) for naringin, 0.9100-36.40 μg · mL-1 (r = 0.9994) for salvianolic acid B, 0.0975-3.900 μg · mL-1 (r = 0.9989) for lobetyolin, 0.9965-39.86 μg · mL-1(r = 0.9982) for hesperidin, 0.2535-10.14 μg · mL-1 (r =0.9975) for liquiritin, 0.2480-9.920 μg · mL-1 (r = 0.9967) for isoliquiritoside, 0.2770-11.08 μg · mL-1 (r = 0.9950) for liquiritigenin, and 0.1530-6.120 μg · mL-1 (r = 0.9928) for isoliquiritigenin. The average recoveries of the eleven active components were 98.47%-101.91% with RSDs of less than 2.5% (n=6). CONCLUSION: The method is convenient, rapid, sensitive and reliable. It may provide a basis for the quality control of Shengan Guanxin Mixture.

Determination of lobetyolin in Shengmai Granules by HPLC / 国际中医中药杂志

Objective To establish an HPLC method for the determination of lobetyolin in Shengmai Granules. Methods ZORBAX SB-C18(250mm×4.6 mm,5μm) column was used, the mobile phase consisted acetonitrile:0.1% acetic acid (22:78, v/v), the flow rate was 0.8 ml/min, the column temperature was 30℃ and the detecting wavelength was 267 nm. Results The cablibration curve showed good linear relation within a range of 0.082～ 1.640 mg/ml, the average recovery was 98.5% and the RSD was 0.70%.Conclusion The method was simple, repeatable and accurate. It can be applied in quantitative determination of lobetyolin in Shengmai Granules.

The standard of quality control of Yiqi Zhixue Granule / 中成药

AIM:To improve the standard of quality control of Yiqi Zhixue Granule(Radix Codonopsis,Radix Astragali,Rhizoma Atractylodis macrocephalae,etc.) METHODS: In the prescription,both Radix codonopsis and Radix Astragali were identified by HPLC.Folium mahoniae was distinguished by TLC.Rhizoma Bletillae was authenticated by microscope.Morever,RP-HPLC method was adopted to determine the astragaloside content in the productions,using a column of C_(18),the column temperature was at 35(?C),acetonitrile-water(32∶68) as a mobile phase,he flow rate was 1.0 mL/min,and ELSD as detection in which the tube temperature was set at 102(?C) and the air flow was at 2.8 mL/min,the impactor was chosen off. RESULTS: The established identification methods were simple and proper,reflecting a good specificity,while the determination method was accurate and reliable,which showed the recovery of 97.95%,RSD=2.21%(n=9),the intermediate precision of RSD=2.67%(n=3),the robustness of RSD=3.67%(n=3) between columns,and the LOQ of 0.539 2 ?g,RSD=2.92%(n=5). CONCLUSION: The improved standard of quality control of Yiqi Zhixue Granule is not only practical but capable of effectively controlling the quality of the medicine as well.