Evaluation and optimization of content determination method of Chrysanthemi Flos in Chinese Pharmacopoeia / 中国中药杂志

This study discovered that the resolution of 3,5-O-dicaffeoylquinic acid(isochlorogenic acid A) in the content determination method of Chrysanthemi Flos in Chinese Pharmacopoeia(ChP)(2020 edition) was poor, which affected accurate quantification. We tested the method in ChP with chromatographic columns of seven brands to clarify the problems in the existing method, optimized the chromatographic conditions by adjusting the mobile phase composition and elution ratio and replacing the chromatographic column packing, and carried out the reproducibility assay for the new method. The two methods were compared for the content determination results of Chrysanthemi Flos prepared from six different varieties. As evaluated by the resolution based on different chromatographic columns of seven brands, the existing method failed to separate isochlorogenic acid A and isochlorogenic acid D well. The peaks of the two components were not completely separated on three chromatographic columns, and isochlorogenic acid A and isochlorogenic acid D generated a co-effluent peak in the other four columns. Isochlorogenic acid A and isochlorogenic acid D could be completely separated under the optimized chromatographic conditions. The difference in the peak areas of isochlorogenic acid A+isochlorogenic acid D obtained by the optimized method and the method in ChP was not significant, with deviation less than 3.0%, which further proved that the result measured by the method in ChP was the co-effluent of isochlorogenic acid A and isochlorogenic acid D. The optimized method can ensure the accurate quantification of isochlorogenic acid A. The existing content determination method of Chrysanthemi Flos has the problem of poor resolution. It is recommended to revise the chromatographic conditions for the content determination method of Chrysanthemi Flos to improve the resolution of isochlorogenic acid A and ensure its accurate quantification.

Optimization of pectin extraction from selected Philippine fruit peel wastes using Box-Behnken design

Background@#Pectin is a pharmaceutically relevant excipient that can be upcycled from selected Philippine fruit peel wastes. Method optimization of pectin extraction leads to maximizing yields from limited resources, while also reducing environmental wastes, and providing local alternative sources. @*Objectives@#This study aimed to optimize the method of extracting pectin from selected Philippine fruit peel wastes using the Box-Behnken design, by varying the acid extraction solvent, treatment time, and working temperature. @*Methodology@#The three-level (-1, 0, 1) Box-Behnken design (15 set-ups) was used to optimize the pectin extraction in each of the fruit peel samples (C. maxima; A. heterophyllus; ripe and unripe M. indica; D. zibethinus; and H. undatus). The three experimental factors were the type of 3N acid used as extracting solvent (HNO₃, H₂SO₄, and HCl); duration of treatment in minutes (60, 90, and 120); and temperature of treatment in C 60, 75, and 90). The %yield was computed in each set-up, and the projected yields were generated using multiple linear regression. The pectin samples obtained from the optimized conditions were subjected to the physicochemical characterization, with apple pectin as the standard. Degree of esterification (DE), equivalent weight (EW), methoxy content (MC), alkalinity of ash (AA), and anhydrouronic acid content (AUA) were performed. @*Results@#Maximum yields were extracted from C. maxima (28.96%), A. heterophyllus (20.12%), ripe M. indica (26.23%), and unripe M. indica (25.89%), using 3N H₂SO₄, for a treatment duration of 60 minutes, at a working temperature of 90 C, and H. undatus (25.03%) at 60 C, for a treatment duration of 120 minutes. @*Conclusion@#Optimum conditions were identified to extract pectin in each of the fruit peel samples. The 3N H₂SO₄ produced the highest pectin yields in all of the set-ups, while the treatment time and working temperature vary per fruit peel sample. Pectin extract from C. maxima, A. heterophyllus, and M. indica was comparable to the standard.

Simple Method For Optimization Of Heparin And Enoxaparin Detection Using Agarose Gel Electrophoresis

Objective: A simple gel electrophoresis method for low-molecular-weight heparins (LMWH) is required for use in a variety of laboratories to allow further identification and purification. This study aimed to optimize the detection of heparin and enoxaparin (low-molecular-weight heparin by gel electrophoresis. Methods: Several gel electrophoresis conditions were tested to optimize the detection of enoxaparin by using a simple method with a modified Volpi’s approach. Multiple gel thicknesses, voltage settings, and enoxaparin concentrations were tested in the optimization procedure. Enoxaparin was purchased from a local supplier as pre-filled pharmaceutical injections. Highly purified 0.5% and 1.0% agarose gels were prepared and a series of enoxaparin concentrations was added to both gels for comparison and optimization. The 0.2% toluidine blue stain was prepared by the addition of 1 ml in an ethanol-water-acetic acid mixture (50:49:1; v/v/w). The staining process comprised two steps: first, toluidine blue was added for 30 min and destained overnight in the solvent mixture. Subsequently, the following morning, the second step was conducted, in which the gel was restained for 30 min with the same concentration of toluidine blue. We continued to stain the gel until the bands were visible. Results: The gel electrophoresis results showed that clearest and sharpest bands were obtained using 65–75 mAh and 85 V settings. At 95 mAh, the bands were slightly washed out. Conclusion: This study successfully facilitated the detection of enoxaparin, a LMWH, and heparin in the laboratory by using simple tools and techniques available in most laboratories.

Optimization of determination methods of astragaloside Ⅳ and analysis of contents of astragaloside Ⅳ in different origin and grade / 中国中药杂志

A rapid and accurate method for determination of astragaloside Ⅳ was established,which was further applied to determine the contents of astragaloside Ⅳ in 87 batches of different origin and different grade of Astragali Radix. The ROC curve was used to analyze the contents of astragaloside Ⅳ in different origin. Simultaneous contents of astragaloside Ⅳ in different grade were compared with chemometrics. HPLC-ELSD method was used to determine the contents of astragaloside Ⅳ. A Vensil MP C18 column( 4. 6 mm×250 mm,5 μm) was used with acetonitrile-water( 32 ∶68) as the mobile phase at a flow rateof 1 m L·min-1. The column temperature was 25 ℃ with ELSD parameters as follows: gas flow rate was 2. 5 L·min-1,the drift tube heating temperature was set to 105 ℃,and the gain value was 4. 0. The optimized method avoided the problem that the consumable quality unstable and the recovery rate was not high. The contents determined by the optimized method were higher than the pharmacopoeia method,with less time and high recovery rate. The ROC curve analysis showed that there was no significant difference of contents of astragaloside Ⅳ between the top grade of Shanxi wild-simulated Astragali Radix top and the first grade of Gansu cultivated Astragali Radix. The contents of astragaloside Ⅳ in the second,third and fourth grade of Shanxi wild-simulated Astragali Radix was significantly higher than those of produced from Gansu.There was a significant negative correlation between the contents of astragaloside Ⅳ and grade in Shanxi Astragali Radix. While there was no correlation for Gansu Astragali Radix. This study provided the basis for the quality grade standard of Astragali Radix.

Establishment of Capillary Zone Electrophoresis Method for Charge Heterogeneity Analysis of IgG2 Monoclonal Antibody / 中国药学杂志

OBJECTIVE: To establish a capillary zone electrophoresis(CZE) -based method for charge heterogeneity analysis of IgG2 monoclonal antibody. METHODS: The concentrations of TETA and EACA, pH of separation buffer and temperature of capillary were optimized, to obtain a method which can efficiently separate the charge variants of IgG2 mabs. The reproducibility and repeatability of the optimized CZE method were validated and a battery of mabs were evaluated. RESULTS: The separation effect was significantly influenced by TETA/EACA concentration, pH and separation temperature. Through a series of optimization, a CZE method which showed good resolution and precision(RSD of area percentage was below 3.0% was established and the method parameters were as below: 400 mmol•L-1 EACA/0.05% TETA, pH 6.2, separation under 30 kV voltage at 35℃. This method was also suitable for IgG1 and IgG4 mabscharge heterogeneity analysis. Based on the specific electrophorogram and migration time, it could also be used as an identification method for monoclonal antibodies. CONCLUSION: The optimized CZE method can efficiently separate the charge variants of IgG2 mabs and shows good precision and specificity, which can be used to analyze the charge heterogeneity and identification of monoclonal antibodies.