A Rapid Micro Method for Determ nation of Ascorbic Acid in Urine by Ferric Reduction

A new method for the determination of ascorbicacid in urine is based on the quanti-tative reduction of ferric to ferrous ion by ascorbicacid and the colorimetric measure-ment of the ferrous ion through its formation of a colored complex with bathophenan-throline. The complex is quantitatively extracted into chloroform, and its absorbance is measuredin this solvent. The method is simple, very rapid, and has good specificity for clinical purposes. Only 0.05-0.25 ml. of urine is required per determination. All reagentsusedin the method, except the ascorbicacid standard, are stable indefinitely. 0 UR LABORATORY required a method to measure the urinary ascorbic acid of a large number of specimens of very small volume. The ascorbic acid reduction of 2,6-dichlorophenol-indophenol lacks specificity (1, 2) and, though more specific when used in conjunction with a cation ex-change resin (3), still requires too large a volume of urine. The oxida-tion of ascorbic acid through the use of Norit * and condensation of the dehydroascorbic acid formed with 2,4-dinitrophenyihydrazine (4) is ap-parently specific when 3-hr. incubation periods are used (5) and can be adapted for use with small urine volumes. However, the method is too long and requires too much manipulation when large numbers of samples are to be run. Recently a new method has been described (6, 7) which appears to be very specific. It is based on the ascorbic acid reduction of mercuric chloride to mercurous chloride, separation of the mercurous chloride, and its measurement by iodometric metho(1s. However, it requires 15-23 ml. of urine for each determmatioii. iron in the ferric form is only a slightly stronger oxidizing agent than mercuric chloride, the standard oxidation potentials being-0.74 and-0.63 v, respectively (a). Hence it was believed that it might 1)e possible to develop an ascorbic acid method based on the ascorbic acid reduction of ferric to ferrous ion