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Adsorbents for detection, isolation and evaluation of ethynyl steroids
--NOTESFOR
ADSORBENTS
DETECTION, OF
Albert0
ISOLATION
ETHYNYL
Ercoli,
February
13,
17a -Ethynylated potent
oral
evidence catabolites,
2-6.
ethynyl
help.
in pharmaceutical The ability well silver
have prepared silver
Having
even
of foreign
connection,
metabolism
is mostly
from
could
also
as
furnished
in their
1 and 17a-ethynyl-lQsensitive
biological be useful
acetylenes
to give
in mind the high specificity
adsorbents
has
conserved
a specific,
steroids
therapy
and rapid
fluids for
would
evaluating
be them
7.
or of silver
small
used in hormone
17a-ethynylestradiol
Such a method
or compounded
capture
Italy
of their
chain
ethynylated
forms
acetylides,
Gardi
Laboratories
(Como),
study
of monosubstituted
known 8.
and Rinaldo
increasingly
side for
In this
of isolating
of effective
are The
as reported
norsteroids method
steroids
the
EVALUATION
1964
compounds.
that
Vitali
Research
Casatenovo
Received
AND
STEROIDS
Roman0
Vister
479
acetylide-silver
impregnated
with silver
quantities
nitrate
complexes,
amounts
’ and tested
of ethynylated
with metals
of the formation
with small
nitrate
salts
steroids
is
of we
of metallic
their
ability
from
large
to amounts
materials.
EXPERIMENTAL Preparation
of the adsorbents
Silver on silica gel in a solution of 1.5 of AgN03 in 20 ml hydroxide was then
: 10 g of silica gel g of glucose in 150 of water containing added with stirring
G according to Stahl was suspended ml of water. A solution of 160 mg 1 ml of concentrated ammonium at room temperature. After heating
480
STEROIDS
3:4
at 50°C in a water bath for 10 min. with constant stirring, the silica gel was filtered and washed thoroughly with water. The product, slurried with water, was employed for coating glass plates (0.2 mm thick film), which were dried in the usual manner for thin-layer chromatography. : 10 g of Florisil, 60-100 mesh, was treated with 160 mg -Silver on Florisil of AgN03 as reported above. The product was washed with water and dried at 80°C for 40 min.
Silver nitrate - silica gel : 160 mg of AgNO3 was dissolved in the water necessary to slurry 10 g of silica gel G according to Stahl, and the usual procedure followed for the preparation of thin-layer plates. RESULTS In Table silica
1 are reported
gel for some
steroids, obtained
were located Quantitative
obtained
nitrate
by spraying aspects
systems - silica
of ether
gel are also reported.
of the silver
The values The compounds
acid solution.
salt formation
were checked
with
1
Rf-values of ethynylated and non-ethynylated silica gel G with silver or silver nitrate.
steroids
on thin-layers
Silver ether acetone
Compound
S-methyl ether 3-cyclopentyl ether
Estriol 17-Epiestriol 19-Nortestosterone 17a-Ethynyl-19-nortestosterone 17a-Ethynyl-19-nortestosterone 3-cyclopentyl enol ether A5-Androstene-3/3,17P-diol 17a-Ethynyl-A5-androstene-3P,
of silvered
and non-ethynylated
or acetone.
with phosphomolybdic
TABLE
Estrone Estradiol 17a-Ethynylestradiol II II
on thin-layers
17a-ethynyl-17P-hydroxysteroids
with the solvent on silver
the Rf-values
Silver nitrate ether acetone
0.92 0.85 0 0 0 0.13 0.37 0.52 0
0.92
0.93
0.93
0.86 0 0 0 0.77 0.77 0.73 0
0.88 0 0 0 0.17 0. 60 0. 62 0
0.90
0 0.54 0 ~__
0 0.83 0
0 0.66 0
0 0.87 0
acetate
17P-diol _____-_
of
0 0 0 0.79
0.83 0.88 0
April
1964
STEROIDS
Given amounts
ethynylestradiol. 20 pg were applied ascending
to silvered
chromatography
centered
to another reaction procedure 97.7
carried
employing
+ 1.4%.
Kober
Quantitative
in large
procedure
given
below
for
of this solution,
containing
of 2.5 ml of benzene
The column
according
of lipoid a sesame
from
oil
solution
nitrogen
chloride
for clinical
pg of the steroid,
Quantitative
on normal showed
employed
recovery estimations
by NH4Cl.
filtrates
was dissolved
ether (Rf = 0.6).
gave an average
One ml
was diluted to 20 with 1 g of silvered
to a beaker, treated
at 80°C
and washed with a mixture
1 : 2)
to the same procedure
silver
and heated with stirring
chromatography
3-cyclopentyl
in a
use l2.
filled
was then transferred
The collected
acetate-cyclohexane
achieved
was eluted with 50 ml of ether and then with 50 ml
and the residue
Thin-layer
steroids
of ethynylestradiol
of 10 ml of ethanol and 5 ml of benzene,
5 ml of ethanol.
area.
of ethynylated were
was
gave no
the starting
material
proposed
135.8
recovery
to the same procedure,
in a mixture
was filtered
15
to the Brown’s
The average
acid lo.
The adsorbent
Florisil
for
The Kober
of acetone.
of ammonium
gently
and
under nitrogen.
ml with ether and applied to the top of a column Florisil.
to a
evaporated
recovered
ether 1 l1 recently
3-cyclopentyl
of adsorbent
and transferred
and heated
and determination
amounts
as in an
transferred
subjected
extraction
4 to
was aspirated,
76% sulphuric
in the residue
from
The solution
out on the residue
Estradiol,
chromogen
contained
in a mixture
test tube and carefully was
A 1 x 1 cm square
5 mg of NH4Cl,
swirling.
ranging
and developed
point was scraped
It was then suspended
with occasional
gel plates
with ether.
2.5 ml of ethanol containing min.
of ethynylestradiol
silica
around the deposition
test tube.
481
The Kober
The
of 10 ml of benzene
in a measured
a single
with 20 mg
for 15 min.
were evaporated
silica
suspended
to dryness
reaction
for ethynylestradiol
under
amount of ethanol.
gel G plates spot
and
(system
ethyl-
of ethynylestradiol carried
out according
(maximum
at 540mp)
of 100 + 3%. were also made without previous
The adsorbent
was treated
removal
of
as above with the ethanol-
STEROIDS
benzene mixture was carried above,
without added NH&l.
out as follows.
The Kober reaction
The dry residue,
containing 2 to 20 ng of product,
Kober reagent
(prepared
by dissolving
in this case
obtained accordingly
was treated
with 2.5 ml of
2 g of hydroquinone
in 100 ml of
63% (v/v) sulphuric acid) and heated on a steam bath for 20 min. cooling in an ice-water mixture
for 5 min.,
After
O-7 ml of water was added and the
was heated again for 10 min. on a steam bath.
an ice-water
as
After
for 5 min. and standing at room temperature
cooling in
in the dark
for 10 min. the solution was read at 516 rnp against a blank similarly prepared.
The values were corrected
The standard calibration
Allen.
of ethynylestradiol on silvered
silica
shown in Fig. 1.
to the formula
ether prepared
gel or Florisil
by adsorbing
salt
the compound
and eluting with an ethanol-benzene
as well as that of the free acetylenic
According
of
curve was obtained with the silver
3-cyclopentyl
This curve,
mixture.
according
to this procedure
the average
steroid, recovery
are was
100.2 + 0.87%.
DISCUSSION All the 17a-ethynylsteroids silver gel G.
salt formation
tested
gave rise
after application
on thin-layers
By using ether as the developing
ethynylated
steroids
using acetone, epiestriol, acetylenic
salts from the application
of ethynylated
past presented
such as estriol
Similar
from large
quantities
difficulties,
to the lack of a free phenolic hydroxyl
By
and 17~ of the
results
reported
of foreign
of ethynylestradiol
ether in an oily solution at very high dilution, particular
line.
gel plates.
as for instance in the example
3-cyclopentyl
silica
the less polar of the non
point was noted.
- silica
steroids
of silvered
In either system no migration
removed.
nitrate
and quantitative
from the starting
even the more polar compounds,
were clearly
materials,
solvent,
could be neatly displaced
were obtained on silver Isolation
to rapid
had in the
mainly due in this particular at C3.
The silvered
Florisil
case
April 1964
STEROIDS
483
0
0*7
/
046
?
0 /
0
0.3
0
012
/
/
e
’/
0
0
011
/‘ /
0
/
0 0
l
l
20
10
,
l
30
PS
Fig. 1. Calibration curves for ethynylestradiol 3-cyclopentyl ether, free (el ), Ecorr = 2 E540 - (E500 + E5a0), and as silver salt (0 -O )* Ecorr = 2 E516 = (EqT6 + E556), in the Kober reaction.
STEROIDS
484
process
gave excellent
on the column, material.
results
here.
3:4
Prompt
and the ether and acetone
By extracting
the adsorbent
derivative
could be recovered
extraction
was carried
compound
was quantitatively
as silver
the ethynyl
salt.
when the
in the process
of NH4C1, the free acetylenic
and column steroids
the adsorbent process
here,
procedures
from foreign
A further
this reaction
It has
a lower
was confirmed
been
colour
steroidal
silver
salts,
us to develop
maximum
is quite
fluids
appears
can be made
to either the heavy metal or
technique.
concerns reported intensity
by our results
The investigations
3,ethers.
even
The applicability
acetylenes
is also
of the
quite
with 3-methyl-l-pentyn-3-01.
point of interest
phenolsteroids.
materials
that many modifications
monosubstituted
as had been verified
in isolating
in biological
with regard
or the impregnation
to non-steroidal
obvious,
allowed
it is clear
described
Alternatively,
regenerated.
of ethynylated
Of course,
all lipoid
with benzene-ethanol
evident and a new route for their estimation to be open.
occurred
elution eliminated
out in the presence
The utility of both thin-layer small amounts
salt formation
the Kober
reaction
3 that ethynylestradiol
of the Kober
a procedure
reaction
according
extinction
itself
in This
and its
on the corresponding
to the suggestions
which furnished
with high and repeatable
gives
than do the natural estrogens.
in both ethynylestradiol
performed
on ethynyl
a sharp
of Nocke 13,
adsorption
values.
REFERENCES 1. 2.
3.
Stimmel, B. F., and May, J. A. t J. CLIN. ENDOCRINOL. 11, 408 (1951). U. f and Noeke, W., ACTA ENDOCRINOL. Breuer, H, a Dardenne, AND HORMONES See also Breuer, H. f VITAMINS 33, 10 (1960). 20, 315 (1962). Brown, J. B. I and Blair, H. A. F. a PROC. ROY. SOC. MED. 53, 433 (1960).
April 1964
4. 5. 6. 7. 8.
9.
10.
11. 12. 13,
STEROIDS
485
37, 14 (1961). Langecker, H. + ACTA ENDOCRINOL. Arai, K., Golab, T., Layne, D. S. I and Pincus, G. p ENDOCRINOLOGY 71, 639 (1962). Layne, D. S., Golab, T., Arai, K., and Pincus, G. p BIOCHEM. PHARMACOL. 12, 905 (1963). Cf. Becker, A., and Ehinger, F. a 2. ANAL. CHEM. 198, 162 (1963). The formation of a complex with mercuric nitrate has been utilized by Langecker 4 to detect ethynyl steroids after paper chromatography. To visualize the Hg-complex, sodium sulfide was employed. See also ref. 6. The use of adsorbents containing silver nitrate for quite different purposes has been reported by de Vries, B. f CHEM. IND. (London) 1049 (1962) ; Barrett, C. B., Dallas, M. S. J., and Padley, F. B., ibid. 1050 (1962) ; Gupta, A. S., and Dev, S., J. CHROMATOG. 12, 189 (1963) ; Urbach, G., ibid., 12, 196 (1963), and others. 8 t 196 (1952). This procedure, Brown, J. B., J. ENDOCRINOL. developed by Brown for estriol, gives optimal results also for 17aethynylestradiol. We are indebted to Dr. F. Galletti of our laboratories for this personal communication. Ercoli, A. p and Gardi, R., CHEM. IND. (London) 1037 (1961). Symposium on “Eteri steroidali enolici e fenolici in clinica e in biologia” FOLIA E~OCRINOL. (Pisa) 14, suppl. to issue No. 6 (1961). Nocke, UT,, BIOCHEM. J. 78, 593 (1961).
ADSORBENTS
DETECTION, OF
Albert0
ISOLATION
ETHYNYL
Ercoli,
February
13,
17a -Ethynylated potent
oral
evidence catabolites,
2-6.
ethynyl
help.
in pharmaceutical The ability well silver
have prepared silver
Having
even
of foreign
connection,
metabolism
is mostly
from
could
also
as
furnished
in their
1 and 17a-ethynyl-lQsensitive
biological be useful
acetylenes
to give
in mind the high specificity
adsorbents
has
conserved
a specific,
steroids
therapy
and rapid
fluids for
would
evaluating
be them
7.
or of silver
small
used in hormone
17a-ethynylestradiol
Such a method
or compounded
capture
Italy
of their
chain
ethynylated
forms
acetylides,
Gardi
Laboratories
(Como),
study
of monosubstituted
known 8.
and Rinaldo
increasingly
side for
In this
of isolating
of effective
are The
as reported
norsteroids method
steroids
the
EVALUATION
1964
compounds.
that
Vitali
Research
Casatenovo
Received
AND
STEROIDS
Roman0
Vister
479
acetylide-silver
impregnated
with silver
quantities
nitrate
complexes,
amounts
’ and tested
of ethynylated
with metals
of the formation
with small
nitrate
salts
steroids
is
of we
of metallic
their
ability
from
large
to amounts
materials.
EXPERIMENTAL Preparation
of the adsorbents
Silver on silica gel in a solution of 1.5 of AgN03 in 20 ml hydroxide was then
: 10 g of silica gel g of glucose in 150 of water containing added with stirring
G according to Stahl was suspended ml of water. A solution of 160 mg 1 ml of concentrated ammonium at room temperature. After heating
480
STEROIDS
3:4
at 50°C in a water bath for 10 min. with constant stirring, the silica gel was filtered and washed thoroughly with water. The product, slurried with water, was employed for coating glass plates (0.2 mm thick film), which were dried in the usual manner for thin-layer chromatography. : 10 g of Florisil, 60-100 mesh, was treated with 160 mg -Silver on Florisil of AgN03 as reported above. The product was washed with water and dried at 80°C for 40 min.
Silver nitrate - silica gel : 160 mg of AgNO3 was dissolved in the water necessary to slurry 10 g of silica gel G according to Stahl, and the usual procedure followed for the preparation of thin-layer plates. RESULTS In Table silica
1 are reported
gel for some
steroids, obtained
were located Quantitative
obtained
nitrate
by spraying aspects
systems - silica
of ether
gel are also reported.
of the silver
The values The compounds
acid solution.
salt formation
were checked
with
1
Rf-values of ethynylated and non-ethynylated silica gel G with silver or silver nitrate.
steroids
on thin-layers
Silver ether acetone
Compound
S-methyl ether 3-cyclopentyl ether
Estriol 17-Epiestriol 19-Nortestosterone 17a-Ethynyl-19-nortestosterone 17a-Ethynyl-19-nortestosterone 3-cyclopentyl enol ether A5-Androstene-3/3,17P-diol 17a-Ethynyl-A5-androstene-3P,
of silvered
and non-ethynylated
or acetone.
with phosphomolybdic
TABLE
Estrone Estradiol 17a-Ethynylestradiol II II
on thin-layers
17a-ethynyl-17P-hydroxysteroids
with the solvent on silver
the Rf-values
Silver nitrate ether acetone
0.92 0.85 0 0 0 0.13 0.37 0.52 0
0.92
0.93
0.93
0.86 0 0 0 0.77 0.77 0.73 0
0.88 0 0 0 0.17 0. 60 0. 62 0
0.90
0 0.54 0 ~__
0 0.83 0
0 0.66 0
0 0.87 0
acetate
17P-diol _____-_
of
0 0 0 0.79
0.83 0.88 0
April
1964
STEROIDS
Given amounts
ethynylestradiol. 20 pg were applied ascending
to silvered
chromatography
centered
to another reaction procedure 97.7
carried
employing
+ 1.4%.
Kober
Quantitative
in large
procedure
given
below
for
of this solution,
containing
of 2.5 ml of benzene
The column
according
of lipoid a sesame
from
oil
solution
nitrogen
chloride
for clinical
pg of the steroid,
Quantitative
on normal showed
employed
recovery estimations
by NH4Cl.
filtrates
was dissolved
ether (Rf = 0.6).
gave an average
One ml
was diluted to 20 with 1 g of silvered
to a beaker, treated
at 80°C
and washed with a mixture
1 : 2)
to the same procedure
silver
and heated with stirring
chromatography
3-cyclopentyl
in a
use l2.
filled
was then transferred
The collected
acetate-cyclohexane
achieved
was eluted with 50 ml of ether and then with 50 ml
and the residue
Thin-layer
steroids
of ethynylestradiol
of 10 ml of ethanol and 5 ml of benzene,
5 ml of ethanol.
area.
of ethynylated were
was
gave no
the starting
material
proposed
135.8
recovery
to the same procedure,
in a mixture
was filtered
15
to the Brown’s
The average
acid lo.
The adsorbent
Florisil
for
The Kober
of acetone.
of ammonium
gently
and
under nitrogen.
ml with ether and applied to the top of a column Florisil.
to a
evaporated
recovered
ether 1 l1 recently
3-cyclopentyl
of adsorbent
and transferred
and heated
and determination
amounts
as in an
transferred
subjected
extraction
4 to
was aspirated,
76% sulphuric
in the residue
from
The solution
out on the residue
Estradiol,
chromogen
contained
in a mixture
test tube and carefully was
A 1 x 1 cm square
5 mg of NH4Cl,
swirling.
ranging
and developed
point was scraped
It was then suspended
with occasional
gel plates
with ether.
2.5 ml of ethanol containing min.
of ethynylestradiol
silica
around the deposition
test tube.
481
The Kober
The
of 10 ml of benzene
in a measured
a single
with 20 mg
for 15 min.
were evaporated
silica
suspended
to dryness
reaction
for ethynylestradiol
under
amount of ethanol.
gel G plates spot
and
(system
ethyl-
of ethynylestradiol carried
out according
(maximum
at 540mp)
of 100 + 3%. were also made without previous
The adsorbent
was treated
removal
of
as above with the ethanol-
STEROIDS
benzene mixture was carried above,
without added NH&l.
out as follows.
The Kober reaction
The dry residue,
containing 2 to 20 ng of product,
Kober reagent
(prepared
by dissolving
in this case
obtained accordingly
was treated
with 2.5 ml of
2 g of hydroquinone
in 100 ml of
63% (v/v) sulphuric acid) and heated on a steam bath for 20 min. cooling in an ice-water mixture
for 5 min.,
After
O-7 ml of water was added and the
was heated again for 10 min. on a steam bath.
an ice-water
as
After
for 5 min. and standing at room temperature
cooling in
in the dark
for 10 min. the solution was read at 516 rnp against a blank similarly prepared.
The values were corrected
The standard calibration
Allen.
of ethynylestradiol on silvered
silica
shown in Fig. 1.
to the formula
ether prepared
gel or Florisil
by adsorbing
salt
the compound
and eluting with an ethanol-benzene
as well as that of the free acetylenic
According
of
curve was obtained with the silver
3-cyclopentyl
This curve,
mixture.
according
to this procedure
the average
steroid, recovery
are was
100.2 + 0.87%.
DISCUSSION All the 17a-ethynylsteroids silver gel G.
salt formation
tested
gave rise
after application
on thin-layers
By using ether as the developing
ethynylated
steroids
using acetone, epiestriol, acetylenic
salts from the application
of ethynylated
past presented
such as estriol
Similar
from large
quantities
difficulties,
to the lack of a free phenolic hydroxyl
By
and 17~ of the
results
reported
of foreign
of ethynylestradiol
ether in an oily solution at very high dilution, particular
line.
gel plates.
as for instance in the example
3-cyclopentyl
silica
the less polar of the non
point was noted.
- silica
steroids
of silvered
In either system no migration
removed.
nitrate
and quantitative
from the starting
even the more polar compounds,
were clearly
materials,
solvent,
could be neatly displaced
were obtained on silver Isolation
to rapid
had in the
mainly due in this particular at C3.
The silvered
Florisil
case
April 1964
STEROIDS
483
0
0*7
/
046
?
0 /
0
0.3
0
012
/
/
e
’/
0
0
011
/‘ /
0
/
0 0
l
l
20
10
,
l
30
PS
Fig. 1. Calibration curves for ethynylestradiol 3-cyclopentyl ether, free (el ), Ecorr = 2 E540 - (E500 + E5a0), and as silver salt (0 -O )* Ecorr = 2 E516 = (EqT6 + E556), in the Kober reaction.
STEROIDS
484
process
gave excellent
on the column, material.
results
here.
3:4
Prompt
and the ether and acetone
By extracting
the adsorbent
derivative
could be recovered
extraction
was carried
compound
was quantitatively
as silver
the ethynyl
salt.
when the
in the process
of NH4C1, the free acetylenic
and column steroids
the adsorbent process
here,
procedures
from foreign
A further
this reaction
It has
a lower
was confirmed
been
colour
steroidal
silver
salts,
us to develop
maximum
is quite
fluids
appears
can be made
to either the heavy metal or
technique.
concerns reported intensity
by our results
The investigations
3,ethers.
even
The applicability
acetylenes
is also
of the
quite
with 3-methyl-l-pentyn-3-01.
point of interest
phenolsteroids.
materials
that many modifications
monosubstituted
as had been verified
in isolating
in biological
with regard
or the impregnation
to non-steroidal
obvious,
allowed
it is clear
described
Alternatively,
regenerated.
of ethynylated
Of course,
all lipoid
with benzene-ethanol
evident and a new route for their estimation to be open.
occurred
elution eliminated
out in the presence
The utility of both thin-layer small amounts
salt formation
the Kober
reaction
3 that ethynylestradiol
of the Kober
a procedure
reaction
according
extinction
itself
in This
and its
on the corresponding
to the suggestions
which furnished
with high and repeatable
gives
than do the natural estrogens.
in both ethynylestradiol
performed
on ethynyl
a sharp
of Nocke 13,
adsorption
values.
REFERENCES 1. 2.
3.
Stimmel, B. F., and May, J. A. t J. CLIN. ENDOCRINOL. 11, 408 (1951). U. f and Noeke, W., ACTA ENDOCRINOL. Breuer, H, a Dardenne, AND HORMONES See also Breuer, H. f VITAMINS 33, 10 (1960). 20, 315 (1962). Brown, J. B. I and Blair, H. A. F. a PROC. ROY. SOC. MED. 53, 433 (1960).
April 1964
4. 5. 6. 7. 8.
9.
10.
11. 12. 13,
STEROIDS
485
37, 14 (1961). Langecker, H. + ACTA ENDOCRINOL. Arai, K., Golab, T., Layne, D. S. I and Pincus, G. p ENDOCRINOLOGY 71, 639 (1962). Layne, D. S., Golab, T., Arai, K., and Pincus, G. p BIOCHEM. PHARMACOL. 12, 905 (1963). Cf. Becker, A., and Ehinger, F. a 2. ANAL. CHEM. 198, 162 (1963). The formation of a complex with mercuric nitrate has been utilized by Langecker 4 to detect ethynyl steroids after paper chromatography. To visualize the Hg-complex, sodium sulfide was employed. See also ref. 6. The use of adsorbents containing silver nitrate for quite different purposes has been reported by de Vries, B. f CHEM. IND. (London) 1049 (1962) ; Barrett, C. B., Dallas, M. S. J., and Padley, F. B., ibid. 1050 (1962) ; Gupta, A. S., and Dev, S., J. CHROMATOG. 12, 189 (1963) ; Urbach, G., ibid., 12, 196 (1963), and others. 8 t 196 (1952). This procedure, Brown, J. B., J. ENDOCRINOL. developed by Brown for estriol, gives optimal results also for 17aethynylestradiol. We are indebted to Dr. F. Galletti of our laboratories for this personal communication. Ercoli, A. p and Gardi, R., CHEM. IND. (London) 1037 (1961). Symposium on “Eteri steroidali enolici e fenolici in clinica e in biologia” FOLIA E~OCRINOL. (Pisa) 14, suppl. to issue No. 6 (1961). Nocke, UT,, BIOCHEM. J. 78, 593 (1961).