The Correlation Between The Concentration Of Ticagrelor And Adenosine In Plasma And Dyspnea Owing To Ticagrelor

Part 1 Simultaneous determination of ticagrelor and adenosine in human plasma by HPLC-MS/MS method Objective:To establish an HPLC-MS/MS method for simultaneous determin-ation of ticagrelor and adenosine in human plasma.Methods:1.Chromatographic conditionsChromatographic separation was performed on a C18 column（150mm×4.6 mm,3.5μm）,the mobile phase was consisted of acetonitrile and water（containing 0.1%formic acid）using a gradient elution,the flow rate was set at 1.0 mL·min^-1,the column temperature was kept at 30℃,the injection volume was 10μL.2.Mass spectrometer conditionsThe detection was performed on a tandem mass system equipped with a turbo ion spray interface in positive modes.The turbo spray temperature was kept at 550℃.3.Pretreatment of plasma sampleStandard solution of mixed control of different volumes were added to100μL blank plasma,respectively mixed with the internal standard and the inhibitor 25μL,precipitated the protein with 300μL acetonitrile,and then the acetonitrile was supplied up to 500μL.Samples were mixed with a vortex mixer for 1 min,centrifuged with the speed of 10900r·min^-1 for 5 min,all that supernatant fluid was taken for test.Results:The standard curve of adenosine was Y=0.0013X-0.0155;the linear range was 12.55⁵020ng·mL^-1.The lowest detectable limit was 1.08ng·mL^-1;The lowest limit of quantitation was 2.68ng·mL^-1.The inter-day and intra-day precision of adenosine at low,medium and high concentration levels were19%,7.3%,5.6%and 15%,8.8%,9.4%,the accuracy（RE）were 8.9%,7.6%,-4.0%,the extraction recovery were（108.6±8.4）%,（100.3±11.4）%,（107.0±8.4）%.The standard curve of ticagrelor was Y=0.0008X+0.016;the linear range was 12.58⁵030ng·mL^-1.The lowest detectable limit was 0.99ng·mL^-1;The lowest limit of quantitation was 2.50ng·mL^-1.The inter-day and intra-day precision of ticagrelor at low,medium and high concentration levels were11%,5.1%,4.4%and 3.4%,4.5%,6.0%,the accuracy（RE）were 17%,-7.0%,-12%,the extraction recovery were（112.2±6.2）%,（94.82±4.15）%,（98.24±7.76）%.The post-preparative stability of samples were stabled at room tempe-rature for 4h.The short-term,long-term and freeze-thaw stability of plasma samples were stable when stored different conditions.Conclusion:An HPLC-MS/MS method was first established for simultaneously determination of ticagrelor and adenosine in human plasma,results showed that the method were accurate and reliable.Part 2 The correlation between the concentration of ticagrelor and adenosine in plasma and dyspnea owing to ticagrelor Objective:To explore the correlation between the degree of dyspnea caused by ticagrelor and the content of ticagrelor and adenosine in plasma.Methods:1.Patients selectionThe subjects were enrolled in the department of cardiology of the Second Hospital of Hebei Medical University from January to March,2018,we selected 30 patients from them who took ticagrelor.All patients received a loading dose of 180 mg ticagrelor followed by 90 mg BID.2.Blood sample collection and treatmentBlood samples（2ml）were collected before taking medicine and 7 h after taking loading dose,then be centrifuged with 4000 RPM for 5 min immediately,be separated and put at-40℃for test.The plasma samples（100μl）under test were collected on 1.5 ml centrifuge tube,mixed with internal standard solution 25μl and acetonitrile 375μl,centrifuged with 12000 RPM for5 min,the supernatant fluid was taken to sample bottle,10μl sample was measured.3.We recorded the occurrence of dyspnea within one week of the patient taking the loading dose of ticagrelor.4.Statistical analysisAnalyses were performed with SPSS software,version 22.Variables are imported to SPSS statistical software to generate measurement data,Spearman correlation was used for dealing the degree of dyspnea（no,mild,moderate,severe）.Results:No statistical difference was observed（P>0.05）in the correlation between the concentration of adenosine and the degree of dyspnea（64.90±9.78 ng/ml）after taking medicine.No statistical difference was observed（P>0.05）in the correlation between the concentration of ticagrelor and the degree of dyspnea（61.67±6.74ng/ml）after taking medicine.Significant statistical difference in adenosine concentration taking medicine before and after（P<0.01）.Conclusion:No statistically difference was observed（P>0.05）in the correlation between the degree of dyspnea caused by ticagrelor and the concentration of ticagrelor and adenosine in plasma.