Aminophylline injection blood drug concentration measurement experiment
This experimental method was obtained from the official website of the Fourth Military Medical University
Operation method
Aminophylline injection blood drug concentration measurement experiment
Principle
Absorption spectra overlap a, b components coexist, if you want to determine the b component, the use of dual-wavelength spectrophotometry can eliminate the interference of the a component. The method is to select two wavelengths λ1 and λ2, so that the component a in these two wavelengths at the degree of absorption is equal; and to measure the b component, in the two wavelengths at the degree of absorption has a significant difference. With such two wavelengths measured the difference between the absorbance of the mixture △ A, only with the concentration of component b is proportional to the concentration of component a is not related to the concentration of component a. Therefore, the interference of a can be eliminated. Therefore, the interference of a can be eliminated. Aminophylline, a commonly used clinical asthma-alleviating drug, is produced by combining theophylline and ethylenediamine. Its theophylline is quickly isolated in body fluids, and under acidic conditions, theophylline can be raised from serum with organic solvents. And at the same time precipitate serum proteins; and then the theophylline is raised from the organic solvent with alkaline solution. Determine the absorbance (A) of alkaline extract at λ274 and λ298, A274 is the theophylline and background (solvent, serum) absorbance, A298 is the absorption of the background, the absorption of theophylline is △A = A274 - A298, you can determine the aminophylline blood concentration.
Materials and Instruments
Rabbit Move 1. Animal preparation and administration Caveat 1. Preheat the UV spectrophotometer for 30min before use and stimulate the hydrogen lamp. 2. Prevent spattering and add samples accurately when shaking and mixing the solution. 3. The sample should be leveled before centrifugation. 4. If you change the wavelength during measurement, you must readjust 0 and 100%. For more product details, please visit Aladdin Scientific website.
Aminophylline injection Theophylline standard Isopropyl alcohol chloroform solution HCl solution NaOH solution
External spectrophotometer Centrifuge Syringe Straw Test tube Blade
One healthy rabbit weighing 2.5-3 kg was taken, and 15 mg/kg of aminophylline was injected intravenously into one ear (finished in 2 min), and the other ear was cut with a sharp blade to get blood.
2. Blood collection
2 ml of venous blood was collected by ear vein cutting method at 0 min, 10 min, 20 min, 30 min, 60 min, 120 min, 180 min, 300 min and 420 min after drug administration.
3. Theophylline standard curve
Take 5 graduated test tubes, add the theophylline standard solution 20 μl, 40 μl, 60 μl, 80 μl, 100 μl, each with 0.1 mol/L NaOH solution 4.0 ml, the final concentration of 0.5 μg / ml, 1.0 μg / ml, 1.5 μg / ml, 2.0 μg / ml, 2.5 μg / ml, in order to the 0.1 mol/L NaOH solution as a blank, the final concentration of 0.5 μg / ml, 1.0 μg / ml, 1.5 μg / ml, 2.5 μg / ml, and the final concentration of the theophylline standard solution 0.5 μg / ml, 0.1 mol / LaOH solution as a blank. solution as blank, the absorbance was measured at 274 nm and 298 nm, and the ΔA~C curves were plotted.
4. Determination of blood concentration
Aspirate 0.5 ml of serum into a graduated test tube, add 0.2 ml of 0.1 mol/LHCl solution, 5 ml of 5% isopropanol chloroform solution, shake, mix, and centrifuge (2500r/min) for 10 min, aspirate 4.0 ml of the chloroform solution (the lower layer) and put it into another test tube, add 4.0 ml of 0.1 mol/LNaOH solution, shake, centrifuge (2500r/min) for 10 min. Pipette 3~3.5 ml of alkaline solution (upper layer) and measure the absorbance at 274 nm and 298 nm with 0.1 mol/L NaOH as blank, and calculate ΔA.
5. Substitute the measured ΔA of the serum sample into the equation of the standard curve to find the corresponding C.