Determination of pharmacokinetic parameters
Sulfonamides containing p-aminobenzene in their structure can react diazotically with sodium nitrite in an acidic environment to produce a diazonium salt. This salt reacts with phenolic compounds (muscimol) in an alkaline environment to form an orange-colored azo compound. The shade of color of the azo compound correlates with the concentration of sulfa drugs. The drug concentration can be deduced by determining its optical density. The present experiment was designed to determine the pharmacokinetic parameters of sulfamethoxazole (SMZ) in rabbits after a single rapid intravenous injection.
Operation method
calculation method
Principle
Sulfonamides containing p-aminobenzene in their structure can react diazotically with sodium nitrite in an acidic environment to produce a diazonium salt. This salt reacts with phenolic compounds (muscimol) in an alkaline environment to form an orange-colored azo compound. The shade of color of the azo compound correlates with the concentration of sulfa drugs. The concentration of the drug can be deduced by measuring its optical density.
Materials and Instruments
Rabbit Move I. Methodology Caveat 1. Blood can also be taken by carotid artery bloodletting or arterial blood sampling at the edge of the ear. 2. The amount of blood and various liquids taken should be accurate. 3. The blood sample should be added to the TCA test tube and shaken immediately, otherwise blood clots will easily appear. 4. Each reagent must be mixed immediately after addition, and the order of reagents added must not be reversed, otherwise the results will be affected. 5. The calculation of blood concentration can also be based on the standard curve equation made separately. For more product details, please visit Aladdin Scientific website.
Sulfamethoxazole standard solution Sulfamethoxazole solution Trichloroacetic acid solution Sodium nitrite solution Muscimol solution Heparin saline solution Pentobarbital sodium solution
Model 722 Spectrophotometer Ordinary Centrifuge Infant Scale Rabbit Operating Table Surgical Instruments Centrifuge Tubes Suction Tubes Syringes Test Tube Racks Balance Pipette Balls Glass Pencil Gauze Filter Paper
1. 标记试管：取5 ml 离心管8 支，5 ml 试管16 支，用记号笔分别标记“0”“1”“2”“3 "Add trichloroacetic acid: add trichloroacetic acid to the tubes.
2. Add TCA: Take the labeled centrifuge tubes and add 3.8 ml of 75 mg/ml TCA solution to each tube.
3. Anesthesia and fixation of animals: take 1 healthy rabbit, weigh it, inject 1 ml/ Kg of 30 mg/ml sodium pentobarbital solution intravenously from the ear margin, and fix the rabbit supine on the operating table after anesthesia.
4. Premedication blood sampling: cut open the neck skin and expose the external jugular vein. Use a 1 ml syringe (first flush with 500 U/ml heparin saline solution) to take 0.2 ml of blood from the external jugular vein, put it into a centrifugal tube of No. 0 and mix well. 5.
5. Drug administration: 0.2 ml/Kg (20 mg/Kg) of 100 mg/ml sulfamethoxazole solution was injected intravenously from the ear margin and the time was noted.
6. Blood collection after drug administration: Blood was collected at 1 min, 5 min, 10 min, 15 min, 30 min, 45 min and 60 min in the same way as in Method 4, and put into centrifuge tubes No. 1 to No. 7, respectively, and mixed well.
7. Centrifugation: Put the centrifuge tubes into centrifuge tubes, level two by two, and then put them into the centrifuge, centrifuge at 15 rpm for 5 min, and then take out the centrifuge tubes after stopping the centrifugation.
8. Transfer the supernatant from the centrifuge tube to a corresponding 5 ml test tube.
9. Preparation of standard tube: Take another test tube and add 1.5 ml of 20 μg/ml sulfamethoxazole standard solution. 10.
10. Color development: Pipette 1.5 ml of supernatant from each tube with a 2 ml pipette (the pipette should be replaced one by one) into the corresponding test tube. Add 0.5 ml of 5 mg/ml sodium nitrite solution to each tube (including the standard tube) with a 1 ml filler, mix well, then add 1 ml of 5 mg/ml muscimol solution and mix well again.
11. Colorimetry: Zero the test tube No. 0, and determine the optical density of the test tubes No. 1 to 7 and the standard tube at 525 nm with the spectrophotometer No. 722.
Results
Results of SMZ pharmacokinetic parameters 
1. The blood drug concentration at each time (t) was calculated according to the following formula and logC. sample tube concentration (μg/ml) = sample tube optical density/standard tube optical density × standard tube concentration
2. determine the atrial model of SMZ by plotting the drug-time curve of SMZ on semi-logarithmic paper using time as the horizontal coordinate and concentration as the vertical coordinate.
3. Perform linear regression of logC on t to obtain the equation: logC = a + bt.
4. Calculate the pharmacokinetic parameters K, C0, t 1/2 , Vd, and CL with the following equations: C0=log-1 a, K=2.303b t 1/2= 0.693/K,Vd= X0/C0 ( X0 is the amount of drug used) CL = Vd-K