Urine is hydrolyzed by acid, changing bound 17-ketosteroids to free 17-ketosteroids. The latter can react with m-dinitrobenzene in alkaline solution to give a red color.
Operation method
Urine 17-ketosteroids assay
Principle
Urine is hydrolyzed by acid, changing bound 17-ketosteroids to free 17-ketosteroids. The latter can react with m-dinitrobenzene in alkaline solution to give a red color. Move I.. Experimental reagents: Caveat 1. The ethanol used needs to be refined. 2. commercially available m-dinitrobenzene needs to be purified. 3. The ethanol used must be analytically pure. 4. When the urine is mixed with ether by shaking, if it is found that the ether and urine are not obvious, several ml of saturated NaCL solution can be added to continue shaking. 5. hydrolysis, the use of nitric acid is more gentle and safer hydrolysis. 6. The required test tubes and pipettes should be dry and free of water, otherwise the results will be affected. 7， the method is not stable enough to show the color, color compounds sensitive to light, so the color development and colorimetry should be carried out in the dark, colorimetry step should be completed after adding reagents 6-12min, a large number of specimens should be divided into batches of color development. For more product details, please visit Aladdin Scientific website.
1. anhydrous ethanol
2. 10/1000 m-dinitrobenzene solution
3. 5 mol/L KOH
4. 70% de-formalized ethanol
5. Androstenone standard solution (1ml=0.2mg)
II. Experimental Methods:
l. Take a clean container and add 10ml of concentrated HCL as preservative. Then collect 24h urine and record the total urine volume accurately.
2. Mix the urine and absorb 5ml in a triangular beaker, add ice ester acid 5ml heating boiling, reflux hydrolysis 10min, running water cooling.
3. The hydrolyzed urine is put into a separatory funnel, add about 20ml of ether (AR) and shake for 2-3min, and then dispose of the lower layer of urine after stratification.
4. Add lmol/L NaOH 10 ml and gently invert 20 times, discard the NaOH layer, then add 15 ml of distilled water and gently invert 20 times, discard the NaOH layer, then add 15 mL of distilled water and gently invert 20 times, discard the aqueous layer.
5. Put the washed ether solution into a large test tube with 20ml scale, wash the funnel wall with a small amount of ether, this washing ether into a large test tube, then add ether to add to the 20ml scale mixing.
6. Take 2 large test tubes each with 5ml of ether solution, one tube for the measurement, the other tube for the measurement of the blank, placed in a 37 ℃ drying oven so that the ether evaporation dry.
7. Take a standard tube prepared above, containing androstenedione 0.02mg.
The above tubes are operated according to the table: 
Mix well and read the absorbance of each tube at 530nm with 70% de-aldehyde ethanol solution.
Calculation:
(Absorbance of measurement tube - Absorbance of measurement blank tube - Absorbance of test tube blank)/(Absorbance of standard tube - Absorbance of test tube blank)×0.02×Total amount of 24h urine (ml)/[(5/20)×5]= 17-ketosteroids mg/24h urine