Development Of Quality Fluctuation Monitoring Methods Of Yiqifumai Injection (Lyophilized) Based On Bio-Response

Traditional Chinese medicine injection (TCMI) has definite therapeutic effect, however, adverse reactions about which occur occasionally. The main reason of ADR events is that the current quality control approaches have no direct correlate to the clinical safety and effectiveness, which makes it harder to guide clinical reasonable prescription. It is urgent to development more efficient quality control method TCMI.The current research takes the quality fluctuation as starting point, the Yiqifumai Injection (lyophilized) as a model. This study is aimed at developing quality fluctuation monitoring and early warning methods by studying clinical samples (safety samples and special samples) using biological thermal activity fingerprinting and electrochemiluminescence. We hope that though our efforts; we might reduce the occurrence of ADR of TCMI and provide technical support to the safety of clinical drug usage of traditional Chinese medicine.The main research contents are as follows:1. Sample collection and quality analysis with routine methodsThe manufacturer provides31batches of "Yiqifumai Injection". Among which,21batches are normal,2are expired validity,2are specially made samples and6are double-blind samples. All the samples are judged as eligible according to the inspection standard of drugs and the appendix IU of Chinese Pharmacopoeia (2010). This result shows the normal methods are hard to distinguish the quality fluctuated samples.2. Chemical composition analysis and quality evaluation.A rapid analysis method of Yiqifumai injection has been established using HPLC. Compared with current methods, the detection time is shortened from70min to45min; the sensitivity is doubled to the previous methods. It can rapidly detect22common peaks. Discriminant analysis shows, the current method can detect all the expired samples,50%of the specially made samples and83%of the normal samples. The results show that chemical composition analysis could distinguish samples with different properties to some extent.The determination and dynamic stability monitoring of5-hydroxymethyl furfural (5-HMF) in Yiqifumai Injection (lyophilized) is carried on HPLC. The results showed that the5-HMF degraded quickly in glucose in0-90min and then stabilized after210min. The FTIR results revealed the shift of characteristic wavelength of5-HNF at769cm-1,808cm-1,1521cm-1,1668cm-1. C=C, C=O characteristic absorption peak disappeared, which indicated that the aromatic structure splitting to form new product after interacting with glucose. The result indicated that the glucose solution may not be suitable for those who might contain5-MHF for the safety hidden trouble.3. Quality evaluation methods on biological responseQuality evaluation methods are established about Yiqifumai Injection based on biological response using E.coli bio-thermodynamics fingerprint. We established the evaluation methods of dynamic fluctuation of Yiqifumai Injection using E. coli growth rate constant and heat power as parameters. The result showed that, the established method could recognize the samples with different properties in42h. The rate of is100%for normal and specially made samples.However, the method could not recognize the expired samples. Thus, E.coli bio-thermodynamics fingerprint could identifies samples with different properties to some degree.The current pyrogen test, either rabbit or endotoxin test, has limitations when applied to TCMI. The pyrogen could stimulate the immune cells such as lymphocyte to secrete cytokines and then induce fever in human body. We establish a new detection method about pyrogen in Yiqifumai Injection by analysis of cytokines. The results showed the LPS concentration ranges from0.25EU/ml-6EU/ml and is correlated with IL-6(r=0.9362). The sensitivity is0.25EU/ml, consistent with the sensitivity of limulus test. Compared to the method in Chinese Pharmacopoeia, the new method contains technical advantages of being easy and decreasing other interferences, which helps to eliminate the laborious rabbit pyrogen test.4. Development of quality evaluation method based on ElectrochemiluminescenceIn the current study, the electrochemiluminescence method was first introduced in the evaluation of quality fluctuation in TCMI. We established the evaluation method of quality fluctuation in Yiqifumai Injection based on the properties of electrochemiluminescence, in which the redox reaction of chemical substances can be triggered by electrode, then produce rhythmic electrical signal and luminescent characteristics. The results showed that the accuracy rate of quality determination is100%in21batches of normal samples,2batches of special samples and6batches of double-blind samples. The detection time is as quickly as5min, little sample (200μl) is required. However, the electrode is prone to be inactivated, as well as the circumstances (such as temperature and humidity) have certain effect on the results. Further study is required.5. Evaluation methods for quality volatility and early warning about Yiqifumai InjectionAll the methods contain both advantages and disadvantages. The chemical method was used widely because of its reproducibility and requirement of common equipments. The biological response method could detect both quantitatively and qualitatively with high throughput and it is safer and more effective. The electrochemiluminescence method is quick and sensitive. All of the three methods are complementary with each other. This paper established the evaluation methods of traditional Chinese medicine infection by using biological response as the core and chemical analysis as the supplement. The current study aims to provide the research foundation for reasonable usage of traditional Chinese medicine infection and safe medication.