Experimental determination of the elimination rate constant of riboflavin tablets by the urine drug concentration method
This experimental method was obtained from the official website of the Fourth Military Medical University
Operation method
Experimental determination of the elimination rate constant of riboflavin tablets by the urine drug concentration method
Principle
Drug in vivo process including absorption, distribution, metabolism, excretion and other processes, each of which is different, but also linked to the observation of a change in one aspect, often indirectly recognize the other side of the situation. So the speed of the drug in the body process change rule, both available blood concentration method to estimate, also available urine drug concentration method to estimate. Drug excretion pathway from the body, mainly for the renal excretion. Urinary drug excretion is not carried out at a constant rate, but with the blood drug concentration is proportional to the first level of speed process. In most cases, the urine drug concentration is higher than the blood drug concentration, and the quantitative analysis of urine drug method has good precision, the determination method is easier to establish, the sampling is convenient, and the drug users can be saved from the pain of multiple blood draws. Therefore, under the condition that there are more prophylactic drugs excreted from urine after drug administration, the urinary drug concentration method is usually used to estimate the elimination rate constant, biological half-life and other kinetic parameters. The rate process of urinary excretion of pro-drugs via the kidney can be expressed as follows: = KeX 28-1Ke is the rate constant of renal excretion at the first level, Xu is the cumulative amount of pro-drug excreted in the urine at time t, and X is the amount of drug in the body at time t. If the drug is administered intravenously, the transient time course of the amount of drug in the body can be expressed as X=Xoe-kt 28-2Xo is the dose administered and K is the first-order elimination rate constant. Substituting the value of X in equation 28-2 into equation 28-1 yields: =KeXoe-kt 28-3 Taking logarithms on both sides yields: lg =lgKeXo- 28-4 It can be seen from equation 28-4 that the logarithmic logarithmic time of the rate of excretion of the prodrug is plotted as a straight line, which has a slope of - , the same slope as that obtained from the logarithmic logarithmic time of the blood drug concentration plot. The slope of the line is used to find the elimination rate constant of the drug. If the drug is administered orally, the drug in the body via the time course can be expressed by the following equation: X= (e-Kt-e-Kat) 28-5Ka is the primary absorption rate constant. The instantaneous excretion rate of the drug in the urine in its original form can be substituted into the 28-1 equation with the 28-5 equation to obtain: = (e-Kt-e-Kat) 28-6 When Ka>K, t is sufficiently large, then e-kat → 0. The 28-5 equation is simplified to: = e-Kt 28-7 Taking logarithms of the two sides to obtain: lg = lg - 28-8 From the above equation, it can be seen that, if we use lgdXu/dt to plot against t, we can obtain A binomial exponential curve, from which the slope of the latter part of the line can be derived from the first elimination velocity constant K. Since the instantaneous rate of change of the urinary excretion rate of the pro-drug is impossible to measure experimentally, only the average urinary excretion rate can be derived experimentally, and set the amount of the pro-drug excretion at a certain time interval Δt for ΔXu, then the average rate of excretion, such as the mid-point of time for tc, so that the 28-4 or 28-4 formula can be rewritten as follows. -8 can be rewritten as follows: lg =lgKeXo- tc 28-9lg =lg - tc 28-10 so that lg → tc graph, due to the use of the average excretion rate instead of the instantaneous excretion rate in the experiments, the elimination rate constant K will be found in a large error. However, if the urine is collected at the same time intervals, which are not more than double the half-life of the drug, a deviation of less than 2% occurs.
Materials and Instruments
Rabbit Move 1. Drug administration and urine sample collection Caveat 1. Drink about 200ml of water after each urine collection to maintain urine output. 2. Collect urine at each bowel movement and do not lose it. 3. During the test period (including the day before taking the drug), control your diet and do not eat foods containing riboflavin, such as eggs, milk and milk sugar. And do not take drugs containing B vitamins. 4. After the measurement of A1 in the experiment, when adding the insurance powder, riboflavin is reduced to dihydroriboflavin, at this time the measurement of A2 should be carried out within one minute, in order to prevent the oxidation of dihydroriboflavin by the oxygen in the air and affect the results of the measurement. For more product details, please visit Aladdin Scientific website.
Riboflavin tablets Glacial acetic acid Powdered insurance
UV spectrophotometer Beaker Test tube Measuring cylinder Measuring cups
(1) Collect 24h urine one day before taking the medicine, measure the volume of urine after each collection, take 10ml and keep it, and pour out the rest.
(2) Empty the urine before taking the drug, take three riboflavin tablets (15mg) immediately after breakfast, swallow with warm water without chewing, and record the time of taking the drug.
(3) After taking the tablets, collect the urine according to the 2nd, 4th, 6th, 8th and 10th h of the time of taking the medicine, measure and record the volume of urine with a measuring cylinder, then, pour the urine into a graduated test tube containing 0.2 ml of glacial acetic acid up to 20 ml, shake it well, and store it in a cool place away from light.
2. Determination of riboflavin content in urine
(1) Preparation of standard solution
Precision weighing 105 ℃ dry 2h riboflavin control 50mg in 500ml volumetric flask, add 0.02mol / L acetic acid solution diluted to 300ml, placed in a water bath and heat to dissolve, cooled to room temperature, with 0.02mol / L acetic acid diluted to the scale, shaking well that is, each 1ml contains 100μg of riboflavin, and then add toluene to cover the top, placed in a cool, dark place to save.
(2) Preparation of standard curve
Precisely suck the standard solution 0.1ml, 0.3ml, 0.5ml, 1.0ml, 2.0ml, 3.0ml were placed in a 10ml volumetric flask, with acidified distilled water (1ml glacial acetic acid per 100ml distilled water) diluted to the scale, shaking well. Acidified distilled water was used as a blank, and the absorption was measured at 444 nm using UV spectrophotometer. Then, about 3 mg of insurance powder was added to each tube and shaken well. The absorbance was measured again within 1min. The difference between the two measurements is the absorbance of riboflavin, and the standard curve was plotted with this value as the vertical coordinate and the concentration as the horizontal coordinate.
(3) Determination of riboflavin content in urine samples
Determination of urine samples according to the method under the standard curve preparation. From "acidified distilled water as a blank", according to the law to determine the degree of absorption, the difference between the two measured values, from the standard curve to find out the content of riboflavin in urine.
The above procedures should be protected from light.
3. Data recording and results
(1) Record of results
The data of urine collection and measurement after drug administration are as follows:
(2) Plot the urinary drug excretion rate curve.
(3) Calculate the slope from the straight line portion of the latter part of the curve, thus calculating the elimination rate constant K and the biological half-life.
(4) Calculate the total excretion (mg), percent excreted.