Experimental methods for the determination of blood concentration and calculation of pharmacokinetic parameters of sulfonamides
Sulfonamides and compounds with free amino groups on the benzene ring undergo diazotization with sodium nitrite in acidic solutions to produce diazonium salts, in addition to coupling reactions with phenolic substances (e.g., muscimol) in alkaline solutions to produce reddish-orange azo compounds, which can be used for colorimetric determination of the concentration of sulfadiazine at a wavelength of 560 nm. The purpose of this experiment is to observe the changes of sulfadiazine concentration in blood at different times after intravenous injection of sulfadiazine sodium; to learn the methods of determining blood concentration and calculating pharmacokinetic parameters such as half-life.
Operation method
colorimetric method
Principle
Sulfonamides and compounds with free amino groups on the benzene ring undergo diazotization with sodium nitrite in acidic solutions to produce diazonium salts, in addition to coupling with phenolics (e.g., muscimol) in alkaline solutions to produce reddish-orange azo compounds, which can be used colorimetrically to determine the concentration of sulfadiazine at a wavelength of 560 nm.
Materials and Instruments
Rabbit Move 1. Anesthesia and fixation: A healthy rabbit was taken, weighed, and anesthetized intravenously at the margin of the ear with 1 ml/kg of 3% pentobarbital sodium; after anesthesia, the rabbit was fixed on the rabbit table in the supine position. Caveat 1. when separating the common carotid artery, separate the nerves and do not ligate the nerves when ligating. 2. be careful not to remove blood when removing supernatant with a fluid extractor. 3. When centrifuging, place the blood tubes symmetrically after weight balancing. 4. Do not touch the translucent surface of the cuvette with your hands; if there is any liquid, wipe it off with microscope paper. For more product details, please visit Aladdin Scientific website.
Heparin Sodium Sulfadiazine Trichloroacetic Acid Sodium Nitrite Sodium Pentobarbital Hydrochloride Muscimol Heparin Saline
Test tubes Centrifuge tubes Syringes Straws Centrifuges Spectrophotometers
2. Surgery: The skin of the middle of the neck was incised longitudinally, the tissues under the neck were separated, the trachea was found, the common carotid artery was separated on the side of the trachea, and two lines were threaded under the common carotid artery for use. Intravenous injection of sodium heparin 1 ml /kg from the ear margin, ligation of the distal end, the proximal end with an arterial clip, leaving a distance of 2 cm between the two ends, with ophthalmic scissors in the distal end of the proximal end of the direction of the 45-degree angle of the small cuts, and then from the direction of the heart inserted into the plastic tube, advancing 1 to 2 cm ligation and fixation, the plastic tube with hemostatic forceps clamped, loosened arterial clips.
3. Take blank blood: take 1 ml, put it in a dry test tube containing heparin and shake it.
4. Administration of blood: 20% sulfadiazine sodium solution (1.0 ml/kg) was injected intravenously from the margin of the ear, 1 ml of blood was taken 2, 5, 10, 20 and 30 minutes after administration, placed in a dry test tube containing heparin and shaken.
5. Deproteinization: 0.2 ml of blood was taken from each blood sample and placed in a centrifuge tube, 4.6 ml of 5% trichloroacetic acid was added and shaken well for a few minutes, and then centrifuged in a centrifuge to completely precipitate proteins in the blood.
6. Diazotization and coupling: take 1.5 ml of the supernatant and add 0.25 ml of 2N hydrochloric acid and 0.3 ml of 5% sodium nitrite in turn, shake thoroughly and then add 0.3 ml of musketophenol to make it reddish-orange after shaking.
7. Measurement: Pour the supernatant into the cuvette, put it into the spectrophotometer, use the blood sample before administration as blank control, perform colorimetric measurement at 560 nm, and record the optical density value.
8. Make the standard curve (the teacher will take the lead in drawing)
9. Calculate the plasma concentration of phenol red at different times by entering the optical density at different times into a calculator after administration. Plot the standard curve using concentration as the horizontal coordinate and optical density as the vertical coordinate and regress the equation. The time-volume relationship is often linear, using the logarithmic value of the plasma drug concentration as the vertical coordinate and time as the horizontal coordinate.
The equation for this straight line is logCt = lgCO-k/2.303-t
Drug plasma half-life T1/2 = 0.693/k (in h or min)
The plasma concentration of phenol red at different times was calculated using the FX-3600P calculator, using a linear regression arithmetic program. 