HOUK: GITALOXIN IN DIGITOXIN
793
Determination of Gitaloxin in Digitoxin
By ALBERT E. H. HOUK (Food and Drug Administration, Department of Health, Education, and Welfare, Washington 25, D.C.)
METHOD
Reagents
(a) Propylene glycol-hydrochloric acid solution.—Mix equal volumes propylene glycol and
concentrated HC1, and store in refrigerator.
Prior to use, warm the solution to 20°.
(b) Gitoxin standard solution.—1.00 mmg/
ml. Dissolve 10.0 mg USP Reference Standard
Gitoxin, C 41 H 64 0 14 , in MeOH-CHCl3 (1 + 2),
dilute to 100 ml, and mix. Dilute 1.00 ml of this
stock solution to 100 ml with MeOH-CHCl3
(1 + 2), mix, and store all solutions in the refrigerator. Prior to use, warm the dilute
gitoxin solution to 20°.
Apparatus
Photofluorometer.—Input filter with maximum transmission near 360 m/i, and output
filter with maximum transmission near 470
rap. Calibrate the instrument, using the reagent blank for zero setting and 2.5, 5.0, 7.5,
and 10.0 mmg gitoxin standard, treated as
directed below, for obtaining measurements
in the optimum range of the scale.
Determination
Transfer 25.0 ml benzene-CHCl3 eluate obtained from the formamide-siliceous earth
column (1) and a suitable volume of gitoxin
standard solution to separate small beakers.
Evaporate just to dryness on steam bath with
aid of air current. Cool, add 10.0 ml propylene
glycol-HCl reagent to each beaker, and place
in bath at 20° for 28 minutes. Mix frequently.
Determine fluorescence of each solution, using
reagent as blank, 30 minutes after addition of
reagent. From the gitoxin standard and calibration line, calculate the % fluorescent substances, as gitaloxin, in the digitoxin sample.
Discussion
Gitaloxin, like gitoxin, produced a color
with alkaline picrate reagent; its intensity,
however, varying wdth specific conditions,
was about % that of digitoxin. It reacted
with Keller-Kiliani reagent, but gave less
than 1/20 the color of digitoxin with mdinitrobenzene reagent (4). Highly purified
digitoxin gave no fluorescence with propylene
glycol-HCl reagent. Under the same conditions, gitaloxin and gitoxin gave similar
strong fluorescence. Gitoxin is preferable to
gitaloxin as a standard for determining
gitaloxin since it is widely available and has
been introduced as a USP Reference
Standard.
The AOAC method for USP digitoxin (1),
with the proposed modification for gitaloxin
type compounds, was applied to purified
digitoxin, a mixture of digitoxin and gitaloxin,
USP Reference Standard Digitoxin, and
five commercial USP digitoxin samples.
Analytical data in Table 1 indicate that
gitaloxin was eluted wdth digitoxin in the
AOAC method of analysis. If present in
appreciable quantity, it must be corrected
for, and the correction can be determined
by the proposed method. In agreement with
Haack and collaborators (2), USP Reference
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The AOAC method (1) for the analysis
of digitoxin preparations separates digitoxin
from other glycosides by column chromatography.
However, a new glycoside,
gitaloxin, recently isolated from the leaves
of Digitalis purpurea and identified as 16formylgitoxin, has been reported by Haack,
et al. (2) to constitute as high as 6% of
commercial digitoxin preparations. Since
gitaloxin has chromatographic and solubility
properties similar to digitoxin, an initial
study was made to ascertain whether there
was any interference with the AOAC digitoxin assay, and to determine quantities
present in USP grade digitoxin. It was found
that gitaloxin is eluted quantitatively with
digitoxin from formamide-Celite columns and
that it gives about % as much color with
alkaline picrate as digitoxin does. Also,
gitaloxin can be determined in the presence
of digitoxin by an adaptation of Murphy's
fluorometric procedure for gitoxin (3). A
maximum of 2.6% gitaloxin was found in a
preliminary survey of commercial digitoxin.
794
JOURNAL OF THE A.0.A.C (Vol. 43, No. 4, 1960)
Table 1. Assay of digitoxin samples for gitaloxin-type compounds
Digitoxin Fraction
1
2
3
4
5
6
7
8
Description
Digitoxin, purified
Digitoxin, purified-gitaloxin (9 + 1)
USP Reference Std. Digitoxin
Commercial Digitoxin A
Commercial Digitoxin B
Commercial Digitoxin C
Commercial Digitoxin D
Commercial Digitoxin E
Standard Digitoxin was found to contain
no gitaloxin. In the five commercial digitoxin
samples analyzed, gitoxin was still the chief
contaminant, although three samples, all
from the same source, contained over 2%
gitaloxin.
Acknowledgments
Gitaloxin reference sample was obtained
through the courtesy of E. Haack, C. F.
Boehringer & Soehne G.m.b.H., Mannheim,
Germany. Paper chromatography indicated
the presence of small amounts of two con-
(%)
98.0
95.5
98.0
91.0
92.0
86.3
91.3
94.3
Fluorescent
Substances
(Gitaloxin,
etc.)
Other
Glycosides
Fraction
(%)
<%)
0.2
9.6
0.0
0.7
0.6
2.3
2.6
2.2
0.9
1.1
1.9
4.6
4.8
10.2
7.8
6.3
taminants, one more polar and one less polar
than gitaloxin.
REFERENCES
(1) "Changes in Methods," This Journal,
56 (1958).
(2) Haack, E., Kaiser, F., Gube, M.,
Spingler, H., Arzneim.-Forsch., 6,
(1956).
(3) Murphy, J. E., J. Am. Pharm. Assoc,
Ed., 43, 659 (1954).
(4) "Changes in Methods," This Journal,
57 (1959).
41,
and
176
Sci.
42,
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Sample
Total
Digitoxides