Study of analytical procedure has revealed that linearity of creatinine calibration curve can be extended by using 0.03 molar picric acid solution made up in 70 percent ethanol instead of water. Three to five times more creatinine concentration can be encompassed within linear portion of calibration curve

Tom, H. Y.

English

NASA Technical Reports Server

January 1973
	 B73-10021 
•	 NASA TECH BRIEF 
NASA Pasadena Office 
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Increasing the Sensitivity of the Jaffe Reaction for Creatinine 
The classic Jaffe reaction for the determination of 
creatinine is predicated on the formation of a tran-
sient red-colored substance with alkaline picrate. In 
typical analytical procedures, the intensity of color is 
measured at a narrow bandwidth in the vicinity of 
490 nm. 
A plot of absorbance vs concentration of creatinine 
is linear over a narrow range of creatinine concentra-
tions; the plot characteristically curves downward at 
higher creatinine concentrations, and the position of 
.
the "break" in linearity is a function of procedural 
variables such as total concentration of base and 
picric acid, temperature, and the presence of other 
substances. 
The Jaffe reaction is the basis for an automated 
analysis of urine in the Biosatellite Program; the 
creatinine content of primate urine is used to assess 
the effects of weightlessness in space. Creatine is an 
alkaloid which is an end product of amino acid catab-
olism, and it exists in relatively large amounts in the 
muscle tissue of all vertebrates. The anhydride of 
creatine, creatinine, is a normal constituent of mam-
malian urine and its presence is an indicator of muscle 
waste. When creatine is heated in an acidic media for 
a short period of time, it is converted to creatinine. 
First a mixture of urine and alkaline picrate is pre-
pared; the color intensity of this mixture is related to 
the creatinine content of the urine sample. A second 
sample of urine is heated in an acidic (H2SO4) solution 
at 100°C for 25 minutes to convert creatine to creat-
mine and then made alkaline in the presence of pic-
rate; the incremental color intensity is linearly re-
lated to the creatine content of the urine sample. If 
the creatine content of the sample is too large, the 
absorbance of the creatinine-picrate reaction product 
falls on the nonlinear portion of-the calibration curve; 
in ordinary laboratory work, a smaller sample or a 
larger dilution is used to bring the absorbance into 
the linear region of the calibration curve. However, 
for automated analysis in space, it is desirable to use 
an analytical procedure which covers the expected 
range of creatinine concentrations. 
A systematic study of the analytical procedure has 
revealed that the linearity of the creatinine calibra-
tion curve can be extended by using a 0.03 molar 
picric acid solution made up in 70-percent ethanol 
instead of water; three to five times more creatinine 
concentration can be encompassed within the linear 
portion of the calibration curve. In a typical proce-
dure, the reference solution is made by mixing 0.100 
ml of urine sample, 2.00 ml of 0.03 M ethanolic picric 
acid solution, and 1.00 ml of 2.25 M sodium hydroxide 
solution, and then diluting to 10 ml with water. The 
creatinine conversion is accomplished by heating a 
mixture of 0.100 ml of urine sample and 0.54 ml of 2 
M sulfuric acid; then, the mixture is treated with 1.96 
ml of 2.25 M sodium hydroxide and 2.00 ml of 0.03 M 
ethanolic picric acid solution and diluted to 10 ml. 
With ethanolic picric acid, the color intensity of the 
Jaffe reaction product obeys Lambert-Beer's law at 
an absorbance as high as 3.4. 
Note: 
Picric acid of high purity must be used. 
(continued overleaf) 
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https://ntrs.nasa.gov/search.jsp?R=19730000021 2020-03-17T06:37:46+00:00Z
Patent status: 
NASA has decided not to apply for a patent. 
Source: Ho Yet Tom of 
Caltech/JPL 
under contract to 
NASA Pasadena Office 
(NPO-11587)
LI 
B73 .10021	 Category 04


Increasing the sensitivity of the Jaffe reaction for creatinine

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