I am performing the USP assay for denatonium benzoate in rubbing alcohol. This is my first solo project at my new job, so any help would be greatly appreciated. My analytical results are horrible and I think it may just be a math error. The procedure is given below.
The USP monograph indicates that the quantity of denatonium benzoate is calculated as shown below.
Calculate the quantity, in mg,of denatonium benzoate (C28H34N2O3H2O) in 100 mL of Rubbing Alcohol taken by the formula:
0.025 C (AU / AS)
If possible, could someone explain where the 0.025 multiplier is derived?
I think it comes from the evaporation of 200 mL of alcohol, reconstituted to 50 mL, 10 mL aliquot taken and extracted with a final extract volume of 100 mL.
50/200 * 10/100 = 0.025, but the standard is prepared at a concentration of 50 ug/mL and it is extracted concomitantly with the sample (10 mL aliquot, final extract volume of 100 mL), yet USP indicates that the standard concentration in the formula should be 50 ug/mL). To me, it looks like the standard concentration is 5 ug/mL.
NOTE: The assay preparation originates from a 200 mL volume of alcohol that is taken to dryness and then reconstituted in 50 mL of buffer. Ten (10) mL of the reconstituted solution is then transferred to separator funnel as described below. The limit is 1.4 mg / 100 mL. The concentration of the standard (C in the above equation is 54.9 ug/mL). My standard response is 0.2410 and my sample response is 0.0382 to produce a result of 0.22 mg/100 mL.
Procedure Treat the Standard preparation, Assay preparation, and blank similarly and concomitantly. Transfer 10.0 mL each of the Standard preparation, Assay preparation,and Buffer solution to individual 250-mL separators,and add to each 40 mL of Buffer solution, 10 mLof a 1in 1000solution of bromophenol blue in chloroform, and 60mLof chloroform. Shake the separators vigorously for 2 minutes,allow to stand for 15minutes, then withdraw the chloroform layers through chloroform-washed cotton into 100-mLvolumetric flasks. Repeat the extraction with 20 mLof chloroform,adding the filtered chloroform extracts to the respective volumetric flasks, dilute with chloroform to volume, and mix. Without delay, concomitantly determine the absorbances of the solutions in 1-cm cells at the wavelength of maximum absorbance at about 410 nm, with a suitable spectrophotometer, using the blank to set the instrument. Calculate the quantity,in mg,of denatonium benzoate (C28H34N2O3H2O)in 100 mLof Rubbing Alcohol taken by the formula: 0.025 C (AU / AS)
As mentioned the limit is 1.4 mg / 100 mL. The concentration of the standard (C in the above equation is 54.9 ug/mL). My standard response is 0.2410 and my sample response is 0.0382 to produce a result of 0.22 mg/100 mL.
The vendors CofA indicates >1.4 mg/100 mL
Thanks for any help. Billie