Measurement of creatinine by Jaffe's reaction--determination of concentration of sodium hydroxide required for maximum color development in standard, urine and protein free filtrate of serum.
Creatinine in serum or urine is determined by Jaffe's reaction where creatinine produces quantitatively an orange color with picric acid in alkaline medium. After allowing an incubation time of 15 min at room temperature for color development the color is measured at 520 nm. Without taking into consideration the acidic nature of standard, protein free filtrate (PFF) of serum and urine, 1% picric acid and 0.75N NaOH are used in this reaction for color development in standard, PFF of serum and urine. An investigation was thought to be necessary to determine the optimum alkali concentration required in standard, PFF of serum and urine. The results show that 0.25, 0.75 and 1N NaOH give maximum color in urine, standard and PFF of serum respectively. A standard solution of creatinine is prepared in 0.1N HCl and the PFF of serum is obtained by addition of fresh tungstic acid. Alkali is consumed to neutralise the acids in both these cases. For urine creatinine measurement, a direct diluted urine sample is used. The difference in the requirement of NaOH is conceivable. The routine use of 0.75N NaOH irrespective of the nature of specimen as is done in all biochemical laboratories, for creatinine measurement needs modification in the light of this investigation.