Objective: To verify the slope ratio method for determination of anticoagulant activity of leeches in laboratory and to investigate. The feasibility of slope ratio method for quality control of leech anticoagulant activity. Methods: Seven batches of leeches and Japanese medical leeches were used to prepare the extracts with different concentrations in parallel. The anticoagulant activity was determined by slope ratio method after the activated partial thromboplastin time(APTT) was measured. Specificity, linearity range, precision, accuracy and durability were systematically investigated in a laboratory. Results: The slope ratio method was used to measure the anticoagulant activity of leeches. The activity of leech extract decreased after heating at 100 ℃. The experiment was not established after adding low molecular weight heparin. The anticoagulant activity of Japanese medical leeches was determined by leeches as the control material, which indicated that the method had better specificity and selectivity. Accuracy inspection: the relative deviations of the 5 relative level values of 64%, 80%, 100%, 125% and 156% were all less than ±20%, and the logarithm of the theoretical value of potency (horizontal coordinate) and the logarithm of the corresponding value of potency (vertical coordinate) showed an obvious linear relationship, with the correlation coefficient greater than 0.95. The slope is between 0.95 and 1.05, indicating good accuracy of the method. The geometric coefficient of variation (GCV, %) of each relative potency level was less than 20%. RSDs of many batches of decoction pieces were all less than 20%, indicating that the precision of this method was good. Linearity and range test: the method could be used to accurately determine 64% to 156% relative to the control medicinal materials, and the linear range meets the requirements. Durability test: leech extract could be placed for 24 h and heated at 37 ℃ for 1 h without any change in activity, indicating that the durability of this method was good. Conclusion: The slope ratio method is precise, accurate and reproducible in the determination of anticoagulant activity of leeches, which is suitable for the quality control of leeches.
XIAO Si-ting, HU Yu-chi, YANG Wen-liang, LI Wei-ran, GUO Yu-dong, XU Hua-yu, LI Bo
. Validation of slope ratio method for determination of anticoagulant activity of Leeches*[J]. Chinese Journal of Pharmaceutical Analysis, 2023
, 43(3)
: 405
-413
.
DOI: 10.16155/j.0254-1793.2023.03.06
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