Fluorophotometric method for the estimation of salicylate in blood.

While salicylates are among the most frequently prescribed drugs, methods for their determination in blood have been neither sufficiently sensitive nor precise. Brodie, Udenfriend, and Coburn (1) have devised a procedure suitable for the clinical measurement of blood salicylate levels if large doses are given. In their method the salicylate is extracted from acidified plasma with ethylene dichloride. A portion of the ethylene dichloride is removed and shaken with an aqueous ferric nitrate solution to produce a colored iron complex. Coburn (2) advises the use of quantities of plasma larger than 1 ml. if the salicyl “radical” is less than 100 y per ml. Peters (3,4) has drawn attention to the low sensitivity of the method. Volterra and Jacobs (5) applied the xanthoproteic reaction to trichloroacetic acid filtrates for the determination of salicylates. However, their serum blanks in normal subjects were relatively large, corresponding to 3 mg. per cent. In the method to be presented a simple physical phenomenon, the bluish violet fluorescence of the salicylate ion on exposure to ultraviolet light, will be used for its measurement. Under suitable conditions a high degree of specificity and sensitivity can be attained. Pr~nciple-Salicylates are quantitatively separated from proteins by precipitation of the latter with a dilute tungstic acid reagent. Strong alkali is added to increase the fluorescence of the salicylate ion about g-fold. The blank fluorescence of plasma without salicylate is negligible. The fluorescence is measured directly in a fluorophotometer with the same hlters as in the vitamin B1 determination. ReagentsI.. Standard salicylate solution (equivalent to 109 mg. of salicylic acid per 100 ml.). 116 mg. of sodium salicylate are dissolved in exactly 100 ml. of water. Working standards are prepared by dilution with water. Store in the refrigerator. 2. 19 per cent HCl. 3. Ethylene dichloride. Do not pipette. 4. 40 per cent NaOH. 5. Dilute tungstic acid reagent. 10 per cent sodium tungstate, 1 volume,