THE DETERMINATION OF MENADIONE AND METHODS

FOR THE IDENTIFICATION OF QUINONES*

BY WILLIAM J. CANADY AND JOSEPH I-1. ROE

(From the Department of Biochemistry, School of Medicine,
George Tl’ashington University, Washington, D. C.)

(Received for publication, October 5, 1955)

Sovelli (1) described a color reaction for menadione, in which the com-
pound was reacted with 2,4-dinitrophenylhydrazine, after which sodium

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hydroxide was added to alka1init.y. The green-colored product formed
was extracted from the mixture with butyl alcohol. Attempts (24) have
been made to apply this reaction quantitatively with variable results.
Poor sensitivity, among ot)her things, was the main objection. Analytical
procedures based on this principle have been published for the determina-
tion of menadione in pure solution, in pharmaceuticals (3, 4), and in ani-
mal feeds (5).
It has been shown in this laborat’ory that the alkali salts of the 2,4-di-
nitrophenylhydrazine derivat,ive of menadione in ethanol have an intense
blue color, which can be used for the identification and determination of
the compound (6). Details of this procedure are reported in this paper.

Methods
Reagents-
1. Concentrated ammonium hydroxide.
2. 2,4-Dinitrophenylhydrazine reagent prepared by saturating 100 ml.
of absolute ethanol with 2,4-dinitrophenylhydrazine and filtering.
3. Concentrated hydrochloric acid.
4. Fisher’s adsorption alumina (80 to 200 mesh).
5. Anhydrous sodium sulfate.
6. Absolute ethanol.
Procedure for Determination of Afenadione-1 ml. of solut,ion containing
0.5 to 4.0 pmoles of menadione is diluted to 10 ml. with absolute ethanol in
a calibrated centrifuge tube, and the contents of the tube are mixed. 3
gm. of anhydrous sodium sulfate and 3 gm. of Fisher’s adsorption alumina
(80 to 200 mesh) are added; the tube is shaken vigorously and centrifuged.
1 ml. of the supernatant fluid is transferred to a test-tube cuvette. A
standard solution containing 0.2 pmole of menadione per ml. is made up in
* The data of this paper were taken from a dissertation submitted by William J.
Canady in partial fulfilment of the requirements for the degree of Doctor of Philos-
ophy, George Washington University, Washington, D. C.
563
564 DETERMINATION OF MENADIONE

90 per cent ethanol and 10 ml. are subjected to the sodium sulfate-alumina
treatment. 1 ml. of this solution is transferred to a second cuvette to
serve as a standard. 1 ml. of 90 per cent ethanol is added to a third cu-
vette to serve as a blank for the standard. 0.1 ml. of concentrated hydro-
chloric acid and 2 ml. of 2,4-dinitrophenylhydrazine reagent are added to
each of the three cuvettes. A blank for the unknown filtrate is prepared
by adding 0.1 ml. of concentrated hydrochloric acid to 1 ml. of the above
supernatant fluid in another cuvette. All tubes are incubated for 2 hours
in a water bath at 37”. At the end of this time 2 ml. of alcoholic 2,4-di-
nitrophenylhydrazine reagent are added to the unknown blank and this is
followed immediately by the addition of 1 ml. of concentrated ammonium

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hydroxide. 1 ml. of concentrated ammonium hydroxide is then added to

400 500 600 700

WAVE LENGTH IN MlLLlMlCRONS

FIG. 1. Absorption curve of t,he color obtained with menadione by the procedure
described.

each of the other three cuvettes. All tubes are diluted to 10 ml. with ab-
solute ethanol and read in a calorimeter at 635 mp.
The absorption curve for the color formed is shown in Fig. 1. The op-
tical density of this color follows Beer’s law when concentrations of mena-
dione up to 0.4 pmole per ml. are used. Fisher’s adsorption alumina is
used to remove interfering substances. Urine contains a considerable
amount of the latter.
Extraction Method for Urine-The useful sensitivity of this method,
when working with urine, may be extended by diluting 5 ml. of urine to
50 ml. with water and extracting with 100 ml. of ether. The urine is
diluted with water to prevent emulsification during extraction. After
washing the ether twice with equal volumes of water, it is carefully evapo-
rated (the vitamin is somewhat volatile) and the residue is taken up in
absolute ethanol. The final volume should be 10 ml. 5 gm. of anhydrous
alumina are added, and the mixture is shaken, then centrifuged. 1 ml. of
the supernatant fluid is analyzed by the 2,4-dinitrophenylhydrazine pro-
cedure as outlined above.
 W. J. CANADY AND J. H. ROE 565

Recoveries of added menadione from human and rat urine are shown in
Table I.

Colum,n C%ronaatogmphy of 2 ,I$-Dinitrophenylhydrazine Derivatives

o.j Quinones
A 2,4-dinitrophenylhydrazinc reagent, suitable for identification, is
made up by saturating 100 ml. of approximately 6 N hydrochloric acid
with 2,4-dinitrophenylhydrazine and filtering.

TABLE I
Recoveries of Menu&one Added to Urine

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Urine source mount added, Y per ml Recovered Per cent recovered

Y
Human. ................. 10 10.6 106
“ .................. 10 10.8 108
I‘ .................. 10 9.9 99
“ .................. 10 9 90
“ .................. 10 10 100
“ .................. 20 20 100
“ .................. 20 20 100
“ .................. 20 19.6 98
“ .................. 20 19.5 97.5
“ .................. 20 18.4 92
‘I .................. 20 21 105
“ .................. 40 40 100
Rat. ..................... 10 11 110
“ ..................... 20 19.9 99.5
I‘ ...................... 40 40 100
I‘ ...................... 40 40 100
“ ...................... 40 38 95

To 1 ml. of the unknown solution containing 0.1 to 10 y of the compound

made up in 90 per cent ethanol is added 0.2 ml. of the 2,4-dinitrophenyl-
hydrazine reagent. The quinone is allowed to react as completely as pos-
sible. This may take as long as 1 or 2 days at room temperature with
some of the less reactive compounds. After a suitable time, the reaction
mixture is made alkaline by the addition of 1 ml. of concentrated am-
monium hydroxide and gently evaporated to dryness. If desired, the
solution read in the tube in the quantitative procedure for menadione, de-
scribed above, may be used. It can be evaporated to dryness and subse-
quently treated in the same manner. Either mixture is then purified by
column chromatography in preparation for paper chromatography. How-
ever, in some cases it might be desirable to avoid heating the compound
with alkali in order to lessen the possibility of alteration of a substituent.
566 DETERMINATION OF MENADIONE

This is true for the derivative of vitamin K1, which will give rise to a new
compound with a greater Rr if exposed to such treatment. In such a case
an extra&on of the acid reaction mixture with ligroin or ether will generally
accomplish the results desired. Water is added and the ligroin or ether
layer is collected and evaporated. The residue is taken up in the acetone-
ligroin mixture as described below.
A small chromatographic tube is packed with Fisher’s adsorption alumina
(80 to 200 mesh) and washed with acetone-ligroin mixture. The same
solvent mixture containing the 2,4-dinitrophenylhydrazine derivative of
the quinone is then placed on the column and washed with more acetone-
ligroin mixture. It will be noted that during this time the excess 2,4-di-
nitrophenylhydrazine continues down the column, while the quinone

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derivative, easily recognizable as a violet or blue band, is held quite firmly.
The p-benzoquinones generally appear red-violet, while naphthoquinones
and anthraquinones form blue bands. The band is then eluted by means
of an acetone-water mixture. The proportions may be altered to give best
results wit,h the particular compound under investigation. 100 parts of
acetone to 2 parts of water, by volume, will be satisfactory for elution in
most cases. This mixture was the eluting solvent used in these investiga-
tions, except in the case of the anthraquinone-sulfonic acid derivative in
which a 5: 1 mixture of acetone to water was used. The latter derivative
is difficult to elute, a broad band being obtained. @-Naphthoquinone-4-
sulfonic acid does not give a blue color on the column, although it can be
chromatographed on paper. The other compounds investigated gave good
results. Although mixtures can be separated on the column, they are
usually eluted together and separated on paper. The fact that acetone is
allowed to contact these derivatives for a short period of time does not
seem to do any harm. The melting point of the 2,4-dinitrophenylhydra-
zine derivative of 1,4-naphthoquinone (278.5’) is the same whether the
derivative is recrystallized five times from ethanol or purified by one
passage over the column. This melting point is in agreement with that
given by Shriner and Fuson (7).
Identijication by Paper Chromatography-Whatman No. 2 filter paper is
used. The solution collected from the alumina column is placed on t,he
paper with a fine capillary pipette so that a small spot is obtained. The
paper is then placed in a sealed jar containing c.p. concentrated ammonium
hydroxide as a solvent. The solvent is allowed to run up the paper to
produce an ascending chromatogram. For most purposes the solvent need
travel only 4 to 5 inches. The compounds are placed 1.5 inches from the
starting point of the solvent. No equilibration is necessary. Other
solvent systems such as acetone-ammonium hydroxide or alcohol-
ammonium hydroxide have advantages for specific cases. Generally, the
more a quinone is substituted with aliphatic side chains, the slower it will
 IV. J. CANADY AND J. H. ROE 567

migrate in the aqueous solvent. In the case of the 2,4-dinitrophenyl-

hydrazine derivative of vitamin Ki, the 20-carbon side chain does not
allow it to move in the ammonium hydroxide system unless a quantity of
fat solvent, such as acetone, is added. A compound containing an acid
group, such as sulfonic acid, generally travels faster in the alkaline aqueous
system than the corresponding unsubstituted quinone and is slowed by the
addition of an organic solvent like acetone. However, concentrated am-
monium hydroxide is satisfactory for the separation of the majority of the
compounds tested. Since there is alkali present, the compounds are ob-
served on paper as purple or blue spots. Usually p-benzoquinones give
rise to a purple color, while naphthoquinone and anthraquinone derivatives

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are blue. The spots must be marked as soon as the paper is removed from
the jar, since they rapidly disappear as the ammonia concentration de-

TABLE II
Paper Chromatography of d,4-Dinitrophenylhydrazine Derivatives of Some Quinones
The solvent used was concentrated ammonium hydroxide at 25”.

Original compound RF

1,4-Naphthoquinone. . 0.46
Menadione (2.methyl-1,4-naphthoquinone) .. 0.34
2.Methyl-3.bromo-1,4-naphthoquinone.. 0.10 + 0.35
1,2-Naphthoquinone. .. 0.85 + streaks
1,2-Naphthoquinone-3-sulfonic acid. 0.85
p-Toluquinone.......................................... 0.73

creases. Such a system containing ammonia presenm some difficulty in

standardization, but it has been found that with care good reproducibility
can be obtained, provided the same batch of ammonium hydroxide, paper,
and jar are used each time. A sealed jar is essential.
Table II illustrates the results obtained with paper chromatography.
All experiments were carried out at 25’ with Baker’s c.p. concentrated
ammonium hydroxide.
It will be noted in Table II that two components were obtained from
2-methyl-3-bromo-1,4-naphthoquinone and from 1,2-naphthoquinone.
The method is so sensitive that very small quantities of impurities can be
picked up. In the former case, the 2-methyl-3-bromo-1,4-naphthoquinone
prepared from menadione by the method of Adams et al. (8) was recrys-
tallized repeatedly from ethanol and the melting point was in agreement
with that obtained by the authors. Two components were still produced.
It might be argued that, since the R, of one component is very close to that
of the derivative of menadione, some of the starting material may have
been present. This would seem to be ruled out by the fact that a good
568 DETERMINATION OF MENADIONE

melting point was achieved. Judging roughly from the intensities of the
spots, it does not appear that one is an impurity. The possibility of iso-
merism must be considered, since 2,4-dinitrophenylhydrazine could couple
adjacent to the bromine atom or the methyl group. In the case of 1,2-
naphthoquinone, the same situation may exist,, since the quinoid oxygen
atoms are not equivalent as in 1,4-naphthoquinone. This phenomenon has
not been observed with quinones substit.uted only with alkyl groups.

Recovery from Urine of Parenterally Administered Menadione

1 ml. of corn oil containing 1 mg. of menadione was injected into each of
five female rats weighing 220 to 255 gm. The animals had been fasted
for 24 hours previously and received only water throughout the experiment.

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TABLE III
Recovery from Rat Urine of Injected Menadione
The amount injected was 1 mg. in each rat.

Amount recovered from 24 hr. sample after hydrolysis

Rat No.
With acetic acid +
With HCI With HCl + SnClz ceric sulfate

w. nzg. mg.

0.510 0.540 0.463

0.510 0.510 0.451
0.551 0.500 0.510
0.560 0.531 0.482
0.503 0.546 0.499

Each animal was placed in an individual metabolism cage and the urine
was collected at 24,48, and 72 hour intervals. At the end of each interval
the urine samples were treated in four different ways as follows: (1) No
hydrolytic, reductive, or oxidative pretreatment of urine. The result
would be expected to yield the free menadione excreted. (2) Hydrolysis
with hydrochloric acid according to Richert (9). (3) Hydrolysis with
hydrochloric acid containing stannous chloride. (4) Hydrolysis of urine
with oxidative reagents present. Ceric sulfate and glacial acetic acid were
used. The procedure followed was essentially that of Richert (10).
The results of the assays for menadione in the urine are shown in Table
III. Free menadione was not found in any of the urines in appreciable
quantities.

The methods described offer simplicity and great sensitivity. The prac-
t.ical range is from 0.05 to 0.4 pmole of menadione per ml. of solution. The
 W. J. CANADY AND J. H. ROE 569

procedures also offer the real convenience of identification by paper chro-

matography in which one makes use of the colored substance first read
in the calorimeter. Thus, in one series of operations, a cmantitative de-
termination can be made and the identity of the compound giving rise to
the color can be ascertained with accuracy and reliability. No spray re-
agents or color development of the spots is necessary. The blue color
formed is continuously under observation during the chromatography.
The results of the excretion studies (Table III) agree with those of pre-
vious workers in so far as the magnitudes of the amounts recovered are
concerned. Li et al. (11) reported that, when menadione was administered
parenterally to rats, 13 to 20 per cent was excreted in the urine, of which a
part appeared to be a /3-glycuronide. Richert (10) obtained a 31 to 34

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per cent recovery of parenterally administered menadione from rabbit urine
and stated that 30 per cent more could be recovered with eerie sulfate
hydrolysis. In the experiments described in this paper, greater recovery
was not obtained by means of ceric sulfate hydrolysis of the urine. The
fact that Richert used rabbits, rather than rats, for his experiments might
well explain the greater recovery obtained.
The observation that very little administered menadione is excreted in
the second and third 24 hour periods after administration is in agreement
with the results obtained by Solvonuk et al. (12). The latter authors re-
covered about 30 per cent of the menadione given, the major part of which
was excreted within 3 hours after administration. No free menadione was
found. This is in agreement with Richert’s finding that previous hydroly-
sis of the urine is necessary before assay of menadione (10). In their
experiments, Solvonuk et al. (12) used menadione labeled in the methyl
group with CY4.

SUMMARY

1. A method for the determination of menadione in biological materials

has been developed. The color used in this procedure has been made the
basis of methods for the identification of quinones by column and paper
chromatography.
2. The reproducibility, specificity, and precision of the described methods
are excellent.
3. Recovery experiments with menadione injected intraperitoneally into
rats have been conducted. A little over 50 per cent was recovered from the
urine during the first 24 hour period. After 24 hours very little was found
in the urine.

Appreciation is expressed to Merck and Company for contributions of

vitamin K,.
570 DETERMINATION OF MENADIONE

BIBLIOGRAPIlY

1. Novelli, A., science, 93, 358 (1941).

2. Menotti, A. R., Ind. and Eng. Chem., Anal. Ed., 14, 418 (1942).
3. Vonesch, E. E., An. jarm. 1~ bioquim., Buenos Aires, 12, 109 (1941).
4. Vonesch, E. E., Rev. farm., 84, 115 (1942).
5. Van Koetsveld, E. I<:., Rec. trav. chim. Pays-Bas, 69, 1217 (1950).
6. Canady, W. J., and Roe, J. H., Federation Proc., 13, 453 (1954).
7. Shriner, R. L., and Fuson, R. C., Identification of organic compounds, New York,
3rd edition, 265 (1948).
8. Adams, R., Geissman, T. A., Baker, B. R., and Teeter, H. M., J. dm. Chem.
Sot., 63, 528 (1941).
9. Richert, D. A., J. Biol. Chem., 164, 1 (1944).
10. Richert, D. A., J. Biol. Chem., 189, 763 (1951).

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Il. Li, L., Elliott, W. H., and Doisy, E. A., Abstracts, American Chemical Society,
116th meeting, Atlantic City, 66C (1949).
12. Solvonuk, P. F., Jaques, L. B., Leddy, J. E., Trevoy, L. W., and Spinks, J. W. T.,
Proc. Sot. Exp. Biol. and Med., 79, 597 (1952).
 THE DETERMINATION OF
MENADIONE AND METHODS FOR THE
IDENTIFICATION OF QUINONES
William J. Canady and Joseph H. Roe
J. Biol. Chem. 1956, 220:563-570.

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