Materials and reagents
Ticagrelor tablets (90mg,Belinda, AstraZeneca AB) .Ticagrelor-d7 (Toronto Research Chemicals lnc, product number 282022). Epinephrine (product number 189652);histamine ( product number 168525);serotonin (product number 182552) purchased from China National Institute for Food and Drug Control; acetylcholine ( purity 98.5%, product number 195455) purchased from Beijing Standards Subsidiary; Pentobarbital sodium(product number: F20020694)purchased from China Pharmaceutical (Group) Shanghai Chemical Reagent Company. All other chemicals were of LC-MS grade.
Healthy male Sprague-Dawley rats(n=12, clean grade, weighed between 180 and 220g, 6 to 8 week old), purchased from the Experimental Animal Center of Hebei Medical University, license number SCXK (Ji) 2018-004, certificate number: 1907228. All procedures were performed in accordance to the Hebei Medical University’s Animal Care and Use Committee Guidelines. The rats were acclimatized for two weeks before the experiment. Animals were fasted for 12h prior to dosing with free access to water. All 12 rats received intraperitoneal injection of pentobarbital sodium (150 mg/kg) for euthanasia after this experiment.The ticagrelor suspensions were prepared using power of ticagrelor tablets with 3‰ Carboxymethyl Cellulose Sodium(90mg:10ml). The suspensions were fresh prepared, no earlier than an hour before administration, and were homogenized with vortex right before administration. Treatment modelled human medication intake. Right before the treatment, the body weights of the animals were measured individually in order to precisely calculate the amount of suspension to be given(Loading dose 24 mg/kg, Maintenance dose 12 mg/kg each rat). On the first day of the experiment, 3‰ Carboxymethyl Cellulose Sodium adout 0.6ml were given at 8:00am. From the second day, ticagrelor suspension was administered at a loading dose of 24 mg/kg for the first time, and then Maintenance dose 12 mg/kg, every 12h for 6 days.
Blood samples 0.5ml were collected at 9am and 4pm on the first day as blood samples before administration（each single animal is used as its own control）.On the second day of the experiment, after Loading dose of ticagrelor 40 min, 1.5 h, 3.5 h,10.5 h，and on the 3rd , 6th days blood was collected from the venous plexus of the fundus of the rats.Blood samples put into the heparin sodium anticoagulant centrifuge tube (containing 20μL heparin sodium ). It was immediately vortex mixed for 1 min, centrifuged for 5 min at 6500×g, and stored in the freezer at -40℃ until LC-MS/MS analysis. During our whole experimental period(7days), every rats were collected blood 8 times, about 0.5mL each time.
Quantification of adrenaline, histamine, serotonin, and acetylcholine in plasma samples
The preparation of samples was conducted as following: Plasma sample was thawed at room temperature, added 150 µL of the plasma into a 1.5 mL centrifuge tube, then added 400 μL (containing 50 ng / mL internal standard Ticagrelor-d7, 0.1% formic acid) acetonitrile solution, vortexed for 1 min and then centrifuged for 5 min at 12000g at 4℃, 10 μL supernatant was taken and injected directly into the LC-MS/MS apparatus.
The concentration of adrenaline, histamine, serotonin, and acetylcholine was determined using an LC-MS/MS on a liquid chromatograph system (ABSciex Shimadzu) ,which was equipped with an C18 column (150mm × 4.6mm, 3.5μm) maintained at 35°C. The mobile phase was composed of an aqueous solution containing 0.1% formic acid (A) and acetonitrile (B) at a flow rate of 0.6 mL/min. The gradient elution program was applied with the following program: 0.5min-5% B; 2.0min-20% B; 5.0min-100% B;6.5min-100% B; 6.6min-5% B; and then 5% B to equilibrate the column until 9min.
Mass spectrometric detection conducted on a Triple quadrupole mass spectrometer (model API4000 +, serial number BH20361205, ABSciex Shimadzu). The following parameter settings were used:Ionization method: electrospray ionization (ESI); monitoring method: positive ion monitoring; scanning method: multiple reaction monitoring (MRM); scanning time: 100ms; ion source spray voltage (ionspray voltage, IS) 5500 V; ion source temperature (TEM): 600 ° C; air curtain gas (CUR) pressure: 30 psi; atomizing gas (GS1, N2) pressure: 55 psi; auxiliary gas (GS2, N2) pressure: 60 psi ; Collision gas (CAD) pressure: 8 psi; The declustering potential (DP) and collision energy (CE) values of epinephrine, histamine, 5-hydroxytryptamine, acetylcholine and internal ticagrelor-d7 were 50eV and 20eV, respectively. Ev 50, 20 ev; 30 ev, 12 ev; 47 ev, 18 ev. The mass-to-charge ratio (m/z) of the ion formula used for quantitative analysis were: 184.2→166(adrenaline), 112→95 (histamine), 177→160.1 (serotonin), 146→87 (acetylcholine).
Results were expressed as mean ± standard deviation. The paired Student’s t-test for comparison between the concentration of before and after ticagrelor administration. A P value of < 0.05 was considered statistically significant. All calculations were performed with the SPSS22.0 software.