Studies on the Coumarin Anticoagulant Drugs : The Assay of Warfarin and its Biologic Application

Abstract

SummaryA spectrophotometric method is described for the determination of warfarin in biologic fluids. The warfarin is extracted from the specimen with acidified ethylene dichloride, washed with mild alkali and recovered from the organic phase with strong alkali. The extinction is then measured spectrophotometri-cally at the ultraviolet absorption maximum for warfarin. Results of UV absorption spectrophotometry and countercurrent distribution analysis showed that the assay is specific for unchanged warfarin in plasma; in addition, it measures a probable metabolite of warfarin in urine. Further studies indicated that the metabolite may be hydroxy-warfarin.The method was applied in determining the absorption and elimination of warfarin after oral and intravenous administration. Gastrointestinal absorption of the drug was rapid and complete. With both routes the plasma concentrations and prothrombinopenic response were similar, and the rate of disappearance from the plasma followed an exponential decay curve. A correlation was found between rate of elimination and biologic response — the slower the rate of elimination, the more pronounced the depression of prothrombin complex activity. The application of this dual approach, the simultaneous study of the metabolic fate of the drug and the prothrombinopenic response, should provide a more rational basis for the therapeutic use of anticoagulant drugs.

Studies on the Coumarin Anticoagulant Drugs: The Pharmacodynamics of Warfarin in Man*

“…No unchanged warfarin could be demonstrated in the stool at any time. We previously reported preliminary studies indicating that warfarin was present in the urine (16), but further investigation proved this to be almost entirely a metabolite of the drug (13). As shown in Table V, much larger quantities of metabolite were excreted for the two larger doses at all periods.…”

“…Samples of each were stored at -200 C. Plasma prothrombin time (prothrombin complex activity) Was determined by the method of Quick (14). Thromboplastin extract was prepared from dehydrated rabbit brain 2 Concentrations of warfarin in plasma, urine, and aqueous suspensions of homogenized stool were measured as described previously (13). Twenty ml of reagent-grade ethylene dichloride3 was placed in a 60-ml glass-stoppered bottle.…”

“…Onto this organic phase were poured in a layer exactly 2 ml of the test specimen, 1 ml of distilled water, and 0.5 ml of 3 N HCl. (13). The urine of subjects who had taken warfarin was extracted; the ultraviolet spectrum of these extracts was of similar configuration to pure warfarin, but the peak of maximal absorption was shifted from 308 to 320 m1A.…”

“…Recently, we described a spectrophotometric method for the estimation of warfarin in biologic fluids (13), and have used this method in the present study to investigate the pharmiacodynamics of warfarin in man. We determined the concentrations of warfarin in the plasma and of a warfarin metabolite in the urine of normal subjects after both oral and intravenous administration of the drug.…”

Enhanced elimination of warfarin during treatment with cholestyramine.

“…The percentage of prothrombin complex activity was determined from a standardization curve obtained from saline dilution of standard human plasma (Behringwerke AG, Marburg/Lahn, Germany) from 9 :10 to 1 : 10. For the determination of warfarin the assay procedure of O'Reillay, Aggeler, Hoag & Leong (1962) was used with the following modifications: warfarin was measured fluorimetrically in 0.5 N NaOH at 395 X using an excitation wavelength of 315 X in a Perkin-Elmer fluorimeter (Model 204). Plasma blanks obtained by this method were apparently zero when samples of 0.5 ml were extracted.…”