The Components Of Qingreliangxue Recipe Were Analyzed By Ultra Performance Liquid Chromatography-tandem Mass Spectrometry

Psoriasis is a common chronic inflammatory dermatosis.The skin lesions are characterized by erythema,papules,or plaques,covered with multilayered silvery white scales.Modern studies have found that it is related to genetic factors and abnormal immune responses,and it is difficult to treat.TCM which has the characteristics of alleviating comprehensive symptoms,shortening the course of disease and reducing the recurrence has good effects on the treatment of this disease.Psoriasis is called "Baibi" in TCM.TCM believes that "blood-heat" is the main pathogenesis of psoriasis vulgaris,psoriasis of blood-heat syndrome is the most common type of psoriasis,so drugs of heat-clearing and blood-cooling are usually used to treat psoriasis of blood-heat type in clinic.Prescription for heat-clearing and blood-cooling is from experiences of Professor Bai Yanping,specializes in treating psoriasis of blood-heat type.Its curative effect has been confirmed through clinical trials,and it has been used in animal experiments and cell experiments.But the components of Chinese Medicine Prescription are complex,so specific mechanism of the prescription is still in the study.Ultra high performance liquid chromatography(UPLC)is a new scientific separation technology which has super high degree of separation,sensitivity and speed of analysis launched by Waters in March 2004.Mass spectrum(MS)which forms spectrogram based on mass charge ratio(m/z)can measure component and structure of compounds.The combination of UPLC and MS can give full play to their advantages.Compared with the traditional high performance liquid chromatography and mass spectrometry(HPLC-MS),UPLC has significantly improved the accuracy,repeatability and reliability of quantitative analysis,and become an important method of drug analysis.Ultra-high performance liquid chromatography-tandem mass spectrometry(UPLC-MS)technology was used to identify components of prescription for heat-clearing and blood-cooling,we can further explore and reveal the pharmacological basis of the treatment of psoriasis vulgaris.Important components can be developed new Chinese Medicine used in clinic,bring benefits to broad masses of patients.Objective:To analyze components of prescription for heat-clearing and blood-cooling and determine content of them,we can explore the material basis for the treatment of psoriasis vulgaris.Methods:Using Ultra-high performance liquid chromatography-tandem mass spectrometry(UPLC-MS)to detect the prescription for heat-clearing and blood-cooling,and investigate methodology of precision,repeatability,stability and recovery rate,etc.,finally detecte six different batches of extract and compare the difference in the content of each component.Results:(1)Chromatographic conditions:HPLC column 2.1 ×100mm,1.7um BEH C18 column(Waters,USA),column temperature 40?,mobile phase 0.5%formic acid water(A)-acetonitrile(B).Gradient elution as follows:0 to 5 min,10%B;5 to 6.5 min,25%B;6.5 to 7.0 min,40%B;7.0 to 8.0 min,90%B;10%B for 1 min.Flow rate 0.3 mL/min.Automatic injection of luL at 4?.(2)Mass spectrum conditions:(3)The components of prescription for heat-clearing and blood-cooling include Astilbin,Catalpol,chlorogenic acid and p-CoumaricAcid.(4)Specific investigation:The retention time of the five components of astilbin,catalpol,chlorogenic acid,gallic acid and p-coumaric acid in the test solution respectively was 4.50,0.83,2.15,1.00,3.70 min.High responsiveness and good resolution.(5)Linear relationship investigation:Linear relationship is good of astilbin,catalpol,chlorogenic acid,gallic acid and p-coumaric acid respectively in the range of 12.50-800.00ng-mL-1,50.00-1600.00ng·mL-1,3.13-200.00ng.mL-1,48.83-3125.00ng·mL-1 and 6.25-400.00ng·mL-1,r2 is greater than or equal to 0.999.(6)Precision investigation:The RSD values of astilbin,catalpol,chlorogenic acid,gallic acid and p-coumaric acid in the mixed control products were less than or equal to 1.38%;indicating that the instrument has good precision.(7)Repeatability investigation:The RSD values of astilbin,catalpol,chlorogenic acid,gallic acid and p-coumaric acid in the same batch of test solutions respectively were 0.76%,1.06%,4.31%,1.79%and 3.53%,the results show that the repeatability of this method is good.(8)Stability investigation:The RSD values of astilbin,catalpol,chlorogenic acid,gallic acid and p-coumaric acid in the same batch of test solutions respectively were 1.26%,5.30%,1.82%,3.15%and 3.17%,the results show that the method has good stability.(9)Review of sample recovery:The average recoveries of astilbin,catalpol,chlorogenic acid,gallic acid and p-coumaric acid respectively were 101.16%,100.92%,103.74%,99.59%and 100.39%;RSD values respectively were 4.67%,0.66%,4.42%,0.88%and 3.82%.The results show that the method has good recovery rate.(10)Determination of sample content:The average content of astilbin,catalpol,chlorogenic acid,gallic acid and p-coumaric acid in six different batches of prescription for heat-clearing and blood-cooling respectively were 50.25ug/mL,957.34ug/mL,5.73ug/mL,175.87ug/mL and 7.82ug/mL;the RSD values respectively were 0.93%,2.71%,2.03%,2.81%and 2.67%.The results showed the contents of catalpol,gallic acid and astilbin are relatively high;the contents of each component in different batches of prescription for heat-clearing and blood-cooling have little differences.Conclusion:(1)UPLC-MS technique was used to identify the four components of prescription for heat-clearing and blood-cooling,namely,Astilbin,Catalpol,Chlorogenic acid and p-Coumaric Acid.(2)The contents of catalpol,gallic acid and astilbin in prescription for heat-clearing and blood-cooling(containing 1g/mL crude drug)are relatively high,and they may be the main active component.The contents of each component in different batches of prescription for heat-clearing and blood-cooling have little differences.The result can provide dose basis for the clinical and experimental research of the components in prescription for heat-clearing and blood-cooling in the future.(3)This experiment has established a method for simultaneous determination of astilbin,catalpol,chlorogenic acid,gallic acid and p-coumaric acid in prescription for heat-clearing and blood-cooling,and the UPLC-MS technique is rapid and accurate,with good methodological findings.It can be used to study pharmacokinetics and control quality of prescription for heat-clearing and blood-cooling in the future.