Acta pharmacol. et toxicol. 1964, 21, 299-306.

From the Institute of Hygiene, University of Aarhus, (Professor B. Lambert, M.D.), and The Department of Biochemistry of The Royal Dental College, Aarhus, Denmark.

(Professor S. Darling, Ph.D.)

Paper Chromatographic Determination of Dicoumarol in Biological Materials

BY

Flemming Christensen (Received June 8, 1964)

A quantitative method for determining dicoumarol on paper chromato- grams has recently been described (CHRISTENSEN 1964 a). This method has been further developed to make possible determinations of dicoumarol in biological materials. In this paper we have described the extended method, with which we have compared ultraviolet spectrophotometric methods for determining dicoumarol.

Experimental methods Principles.

Dicoumarol is extracted from biological materials by an organic solvent from which it is re-extracted with dilute sodium hydroxide. After acidifica- tion it is extracted into chloroform, which is evaporated. The residue is dissolved in a small volume of chloroform and submitted to paper chromatography. This procedure has been described in a previous paper (CHRISTENSEN 1964 a).

Generalprocedure in detail. Place 4 ml of the biological material (e.g. plasma, urine or tissue homogenate) in a 100 ml glass-stoppered flask. Homogenates should not contain more than 35 g of tissue in a total of 100 ml. Add 0.5 ml of 2 M-(NH&S04, adjust pH to 4 5 by means of 0.1 or 1.0 M-HCl, and bring the volume to 5 ml with water.

Add 25 ml of ethylene chloride (Merck, extra pure) to the flask, and shake mechan- ically for one hour at room temperature. Transfer to a centrifuge tube and centrifuge. Aspirate the two upper layers, consisting of water phase and precipitated protein. Transfer 20 ml of the organic phase to a 100 ml glass-stoppered flask, add 4 ml of 2.5 M-NaOH and shake mechanically for 10 min. Transfer to a centrifuge tube and

Zla

300 FLEMMLNG CHRISTENSEN

centrifuge. Pipette 3 ml of the sodium hydroxide into a 25 mlglass-stoppered test tube. (A preliminary check of the amount of dicoumarol in the sample may be achieved at this point by determining the absorption of the sodium hydroxide solution at 314 mfi). Next, place the tube in a water-ice bath for 5 min., add conc. HCl(1 ml) and 8 ml of chloroform (Merck, pro analysi). Shake the tube mechanically for 15 min., and allow the two phases to separate completely. Aspirate the water phase carefully and pipette 7 ml of the chloroform into a cylindrical glass vessel measuring 2.5 cm in diameter and 5 cm in height. Evaporate the extract to dryness, eventually using a warm air stream.

Dissolve the residue in chloroform (e.g. 1 ml), and apply a known fraction (e.g. 750 pl) to the chromatogram by means of the applicator mentioned below.

Application of the sample to the paper chromatogram. Because of the low solubility of dicoumarol in organic solvents, it was necessary to apply as much as 750 p1 of chloroform to the paper. A simple and inexpensive device has been developed for applying such quantities of chloroform to chromatograms.

The applicator is prepared from a glass tube (outer diameter about 8 mm, wall thickness about 2 mm), which is drawn to a capillary at one end, the other end re- maining open. The broad end of the tube should be made 3-4 cm in length and the capillary 12-16 cm. The applicator is by means of a clamp fixed in a vertical position, the end of the capillary being in contact with the paper at the point where the spot is to be applied. It is important that the tip of the capillary be also fixed in its position, for example by means of a small wire loop.

A measured volume of the sample is placed in the larger part of the applicator, which serves as a reservoir. By capillary force fluid runs from the reservoir on to the paper, forming a spot, whose evaporation rate is increased considerably by means of a positive air stream.

By trial and error the thickness of the capillary is made such as to permit delivery of a spot of appropriate size. If the fluid runs too slowly, a rubber-stopper may be forced into the open end of the applicator to give an increased pressure.

After the sample has been applied to the paper, the residue from the applicator is removed by washing twice with chloroform (25 pl) and collected on the same spot, The unknown samples, together with two standards of dicoumarol, should be run on the same paper.

The chromatographic procedure and the quantitative determinations. The chromato- gram is developed overnight by one-dimensional technique with n-butanol - 3 M- aqueous ammonia, 1 : I (v/v).

After drying, the chromatogram is sprayed with diazotized sulphanilic acid and then dried again, the spots containing dicoumarol are eluted, and the optical densities of the eluates are determined at 415 mp, all as described in the previous paper (CHRIS- TENSEN 1964 a). Standards of dicoumarol that have been put through the whole proce- dure, as well as blank samples obtained from suitable places on the chromatogram, are also measured.

The optical density of the unknown (ODA), and the mean values of the optical densities of the standards (ODST) and the blanks (ODBL) are used for calculating the amount of dicoumarol in the unknown sample by means of the equation

ODA -ODBL ODST - ODBL

pg dicoumarol in the sample = x (pg dicoumarol in standard).

Modified method for human plasma. For determining dicoumarol in human plasma, a slight modification is recommended. The extraction with ethylene chloride at pH 4-5 is replaced by one with n-heptane at pH 1-2; otherwise the procedure is the same.

DETERMINATION OF DICOUMAROL IN TISSUES 301

< 2 . . . . . . . . . . . . . . . . . . . . I 67 (3 j 4-5 95 ( 5 ) 6-7 92 ( 6 )

....................

.................... 7-8 . . . . . . . . . . . . . . . . . . . . 1 85 (6)

80 (9) 98 ( 5 ) 94 (6) -

302 FLEM M ING CH RISTENSEN

4 . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 ........................... m .......................... s . . . . . ......................

Table 2.

98 98

100.0 1 2 . 0

Recovery of dicoumarol added to various biological materials and determined by paper chromatography. The drug (50 pg) was added to each sample, consisting of 4 ml of urine, 1 ml of plasma or 4 ml of homogenate (containing 0.8 g of tissue). The determinations of dicoumarol were based on aqueous standards of the drug carried through the whole

procedure, as described in the text.

Recovery rate per cent

Liver tissue

I Muscle Experiment No.

tissue Urine 1 Plasma

1 . . . . . . . . . . . . . . . . . . . . . . . . . . . I 102 . . . . . . . . . . . . . . . . . . . . . . . . . . . I 18: 2

3 ...........................

I02 103 104 I04 I04 103.4

0.9

82 102 101 91 95 95.4 i 8.0

106 91 99 91 92 98.2

1 5 . 1

also obtained if the extraction was performed at pH 1-2 with n-heptane as the solvent.

By the procedure described the drug is extracted from one phase into another in 3 stages. From the measured portions of these extracts used, it can be calculated that 52.5% of the dicoumarol in the original sample should be expected in the final chloroform extract. Usually three quarters of this amount is applied to the paper chromatogram, thus reducing the percentage to 39.4 Analysing watery standards of dicoumarol on diffe- rent occassions gave a mean value of 37.1 % (n = 14); thus 94.3% of the amount of dicoumarol expected was recovered on the final chroma- togram. Further, a certain day-to-day variation in this recovery rate was recorded. These systematic errors of the method are, however, removed by the use of dicoumarol standards put through the whole procedure.

From table 2 it appears that the recovery rate obtained with dicoumarol added to various biological materials was nearly lOO%, but that the recoveries depended somewhat on the materials examined.

Table 3 shows the total recoveries of dicoumarol administered intra- venously into two rats. The results indicate almost complete recovery. In these experiments the chromatograms of the gastro-intestinal tracts revealed a substance not completely separated from dicoumarol.

Tables 4 and 5 show the comparative determinations of dicoumarol by the paper chromatographic and ultraviolet spectrophotometric methods. The results obtained with human plasma (table 4) were similar, and the paper chromatograms did not reveal spots other than those of dicoumarol. For rat livers (table 5 ) the results differed rather markedly in two experi-

DETERMINATION OF DICOUMAROL I N TISSUES

I . . . . . . . . . . . . . . . . 2 . . . . . . . . . . . . . . . . 3 . . . . . . . . . . . . . . . . 4 . . . . . . . . . . . . . . . .

303

7.6 8.1 17.4 17.3 21.5 26.5 16.8 17.4

Table 3.

1 . . . . . . . . . . . . . . . . 2 . . . . . . . . . . . . . . . . 3 . . . . . . . . . . . . . . . . 4 . . . . . . . . . . . . . . . .

Recovery of dicoumarol from two rats. The animals each received 5.20 mg of the drug intravenously 5 min. before they were killed. Blood, skin, gastro-intestinal tract and carcass were separately analysed for their content of dicoumarol by paper chromatography. The

weights of the animals 1 and 2 were 169 and 195 g respectively. Cast.-intest. = gastro-intestinal tract.

38.0 49.4 34.2 51.0 33.2 31.2 19.6 18.5

I I Recovery mg Total recovery

Animal No. 1 I Cast.- i Blood ~ Skin 1 intest. 1 Carcass 1

I I I I I I

mg 1 per cent

1 . . . . . . . . . . . . . . 0.99 0.51 0.33 3.37 5.20 100 2 . . . . . . . . . . . . . . 1 0.83 1 0.38 1 0.26 1 3.91 1 5.38 ! 103

I

Table 4. Comparison between dicoumarol determinations per- formed by paper chromatography and ultraviolet spec- trophotometry (AXELROD er a/. 1949). Plasma samples were obtained from patients receiving anti-coagulant therapy with dicoumarol. Concentrations in pg/ml.

Ultraviolet Plasma sample spectropho- No.

Table 5. Comparison between dicoumarol determinations per-

formed by paper chromatography and ultraviolet spec- trophotometry (WEINER et a/. 1950). Rat livers were ob- tained from animals injected intravenously with dicou- marol. Concentrations in pg/g of wet tissue weight.

I I

Rat liver ~ Paper ~ UItraviolet No. chromatography s p e c ~ ~ [ ~ o -

3 04 FLEMMING CHRISTENSEN

ments (no. 1 and 2), and the paper chromatograms revealed the presence in the livers of dicoumarol-treated rats of several unknown substances. One of these substances, with an Ri-value of 0.55, was always the pre- vailing one and was especially abundant present in the rat livers from experiments 1 and 2. It reacted with diazotized sulphanilic acid to give a coloured dye-stuff indistinguishable from that obtained with dicoumarol. As all of the unknown substances mentioned were apparently not present in the livers of rats untreated with the drug, they were presumed to be metabolites of dicoumarol.

Discussion According to most of the methods hitherto described, dicoumarol is

extracted, at an acid reaction, from the biological material before its assay, and it is therefore of importance for the drug to be extracted at a reproducible rate. With human plasma, to which most methods have been devoted, high extraction rates have generally been reported. Thus, a mean recovery rate of 97.9% of dicoumarol added to human plasma was obtained by the ultraviolet spectrophotometric method of AXELROD et al., (CHRISTENSEN 1964 b). However, as far as is known, few figures are avail- able for recovery of dicoumarol from other biological materials. With the ultraviolet spectrophotometric method of WEINER, which is the method commonly used for tissue determinations, GREEN et al. (1956) reported recoveries from 84 to 103 % of dicoumarol added to liver homogenates. We have also used this method in a series of recovery experiments and obtained recovery rates of 85-103 % with kidney homogenates and 78-85 % with muscle tissue homogenates.

As indicated by these results, the recovery rates are somewhat variable if dicoumarol is extracted from tissues at a strongly acid pH; our study indicates that an almost complete recovery of the drug can be achieved by extraction at pH 4-5.

The accuracy of assaying dicoumarol by paper chromatography de- pends, among other things, on the material to be analysed. In an experiment with human plasma containing on the average 16.9 pg/ml of dicoumarol the accuracy (as the standard deviation) was found to be f2 .2 % (n = 8). With other materials it was somewhat less.

The lower limit for obtaining a reasonably accurate estimate of dicou- marol by paper chromatography is of the magnitude 5-7 pg in the original sample, but amounts as little as 1-2 pg can be detected qualitatively.

The nearly identical results obtained by assaying dicoumarol in human plasma by ultraviolet spectrophotometry or by paper chromatography

DETERMINATION OF DICOUMAROL IN TISSUES 305

indicate that both of these methods determine the drug accurately. Inter- ference by metabolites presumably does not take place, as was also found by AXELROD et al. (1949), who were unable to demonstrate such inter- ference when assaying dicoumarol in human plasma by their ultraviolet spectrophotometric method. This fairly simple method is thus in most instances satisfactory for determining dicoumarol in human plasma, but paper chromatography may help to give further information about that material that, in the ultraviolet spectrophotometric assay, absorbs light at 314 mp,

For the assay of dicoumarol in rat livers, the rather divergent results between those obtained by ultraviolet spectrophotometry and paper chromatography can be explained by the presence of metabolites inter- fering with the assay of the drug by the first-mentioned method. This explanation is supported by the presence, in the livers of dicoumarol- treated rats, of the metabolite, B-055, which strongly absorbs light at 314 mp if dissolved in aqueous sodium hydroxide, as in the ultraviolet spectrophotometric assay of dicoumarol. Incomplete extraction of di- coumarol from the tissues at a strongly acid pH may also contribute to the divergent results obtained. Paper chromatographic assay of dicou- marol in tissues therefore seems preferable to ultraviolet spectrohpoto- metry, at least for certain purposes, and may be useful in studying the distribution and metabolic fate of the drug in animals. The method is, however, more complicated and laborious than the ultraviolet spectro- photometric method.

Several principles, of which paper chromatography is the most impor- tant, have been used in our method to separate dicoumarol from related substances. As shown in a previous paper (CHRISTENSEN 1964 a) separation of several 4-hydroxycoumarin derivatives may be obtained by paper chromatography, and the present study indicates that dicoumarol also can be separated from its metabolites in this way. These facts, and the failure to demonstrate any spots on chromatograms of tissues from untreated rats, indicate the specificity of the method described.

summary A method is described for determining dicoumarol in biological

materials by means of paper chromatography. The mean recovery rates observed with dicoumarol added to urine, plasma, muscle, and liver tissues were 100, 103, 95, and 98% respectively. The lower limit of the method is about 6 pg of dicoumarol and the accuracy is estimated to be &3 to f10 per cent depending on the material to be analysed. The new method has been compared with ultraviolet spectrophotometric methods for assaying dicoumarol in plasma and other biological materials.

306 FLEMMING CHRISTENSEN

R E F E R E N C E S Axelrod, J., J . R. Cooper & B. B. Brodie: Estimation of Dicumarol, 3,3’-

Methylenebis (4-Hydroxycoumarin) in Biological Fluids. Proc. SOC. Exp. Biol. & Med. 1949, 70, 693-695.

Christensen, F. : Paper Chromatography of Dicoumarol and some Related Substances. Acra pharmacol. et toxicol. 1964 a, 21, 23-35.

Christensen, F.: Uricosuric Effect of Dicoumarol. Acta med. Scan. 1964b, 175,

Green, J. P., E. Ssndergaard & H. Dam: Studies on Distribution of Dicu- marol. Proc. SOC. Exp. Biol. & Med. 1956, 92, 449451.

Weiner, M., S. Shapiro. J. Axelrod, J. R. Cooper & B. B. Brodie: The Physiological Disposition of Dicumarol in Man. J . Pharm. Exp. Therap.

461-468.

1950, 99, 409-420.