421

PROTECTION OF ESTROGENIC HORMONES BY ASCORBIC AC I 'D DURING CHROMATOGRAPHY Charles E. Morreal, Ph. D. and Thomas L.

Dao,

M.

D,

Department of Breast Surgery and Endocrine Research Laboratory Roswell Park Memorial Institute Buffalo, New York 14263 Received: 1122175 Oxidative decomposition of phenolic hormones during thin-layer chromatography can be avoided by the incorporation of ascorbic acid into the plates. The application of this technique to the purification of urinary extracts is described.

Previous work in this laboratory has dealt with the determination by gas chromatography (GC) of low levels of estrogens in the urine of men and in pre- and post-menopausal women with breast cancer. [la21 The known susceptibility of phenolic compounds to air oxidation 131 prompted us to develop procedures which would eliminate those losses due to oxidative rupture of trace amounts of these hormones. Thus, we developed a method of accomplishing our purification by thin-layer chromatography (TLC) on plates treated with ascorbic acid in a fashion similar to that demonstrated for corticosteroids [41 and for catechol estrogens,

151 except

that the present method offers a more uniform

distribution of the anti-oxidant. METHODS AND MATERIALS The labeled compounds estrone-6,7-3H, estradiol-6,7-3H,and estriol-6,7-3H (specific activities of 40-50 CifmM) were obtained from the New England Nuclear Corporation and the radioactivity was counted on a Packard Tri-Carb scintillation counter Model 3375 by standard techniques. All reagents were fractionally distilled before use. The ethyl acetate was treated with phosphorous pentoxide before distillation. Estrone (El), estradiol-176 (E2) and estriol (E3) were all obtained from Mann Research Laboratories. Silica gel H was obtained from Brinkmann Instruments, Inc., while the ascorbic acid was purchased from Nutritional Biochemicals Corp.

VoZwne 25, Nwnber 3

S

TIIROLD~

March, 1975

Preparation of TLC Plates: A solution of 500 mg ascorbic acid in 80 ml glass distilled water was extracted with 2 x 50 ml ether to remove nonpolor organic impurities. The aqueous solution was then used to form a Blurry with silica gel H (35 g) which was used to prepare glass TLC plates (5 x 20 cm) of thickness 250 urn. These plates were activated by heating at 110' for 30 minutes. Upon each plate was applied under a stream of nitrogen between 20 and 200 ng of either estrone, estradiol, or estriol and 40,000 cpm of corresponding radioactive tracer to facilitate recovery calculations. This procedure was followed both for those plates which contained ascorbic acid and those which contained silica gel alone. The plates were developed in 5% ethanol in benzene for estrone and estradiol and in ethyl acetate for estriol. The plates to which estrogens had been applied were placed in a holding tank flushed with nitrogen while the plates on which standard material was applied for reference were sprayed with a solution of 30% phosphoric acid in ethanol and heated at 1OOO. The appropriate zones were scraped into 0.5 ml water and extracted with 3 x 3 ml ether. Gas Chromatography: Aliquots of the purified estrogenic fractions were counted and the remainders, to which were added 100 ng 2MeOE3 as an internal standard were evaporated and converted into the heptafluorobutyrate esters by the addition of 0.1 ml of 50% heptaflurobutyric anhydride in acetone. The solutions were kept at room temperature for one hour and then evaporated in a vacuum dessicator and left standing overnight. GLC was carried out with an 8 foot x 2 mm silanized glass column packed with 3% OV17 on 100-200 mesh Gas Chrom Q (Applied Science Laboratories) with 50 cc/minute N2 on a Packard 7400 Series gas chromatograph with a Nickel-63 e ctron capture detector and a Model 878 electrometer set at 4 x lo-$8 amperes. Inlet and detector temperatures were 2600. Estradiol and estriol were run at a column temperature of 220° while estrone was run at 245O C. DISCUSSION AND RESULTS In the course of our work on the quantitative evaluation of daily urinary excretion of estrogenic hormones in patients with breast cancer, we began to notice that as our techniques improved, the ability to detect low levels of these hormones declined. An extensive search for the cause of this problem involved a detailed study of the numerous steps through which the urinary extracts are carried - selection of urinary aliquot, enzymic hydrolysis, acid hydrolysis, ion exchange chromatography, selective extraction, TLC and finally gas chromatography. No irregularity was noticed by this search except that the loss appeared in the TLC step.

It was difficult to accept this step as the

S troublesome

one because

was excellent revealed

but scanning

recovery,

standards

deterioration developed.

the layer

there

after

counter

such an effect

of

that the

the plate

had been

is that when an extract

are numerous components which compete for

of oxygen in the atmosphere surrounding

at the origin.

on a strip

peaks with the location

of the sample was occurring

to a plate,

that there

It became evident

run concurrently.

for

indicate

of the plates

of radioactive

One explanation

is applied

plate

not only did the tracers

good correlation

authentic

423

WD=OXDI

As chromatography

proceeds,

that area of the these

components are

spread over a much wider area and the possibility

of deterioration

increases.

to add to the plate

To avoid this

material for

which would effectively

the oxygen blanket

considered

the best

availability,

its

interference sis

problem,

it was decided

compete with the urinary

over the developed

anti-oxidant

reasonable

for

cost,

with the subsequent

plate.

this

its

estrogens

Ascorbic

purpose because

water solubility

gas chromatography

acid was of its

and its

ready non-

step in the analy-

of the estrogens. The advantage

fication

of using an anti-oxidant

of estrogenic

chromatographic

hormones is shown in Figures

scans for

approximately

estriol

are compared,

a total

of 200 ng were applied

~1 of the derivatized stantial thin-layer

losses

0.4 ng estrone,

These graphs are derived

where the gas estradiol,

from plates

when ascorbic

into

of 500

the instrument.

acid was omitted

and

upon which

and from which 1 l.11of a total

sample was injected

were evident

l-3

puri-

Sub-

from the

plates,

By counting recoveries

in the chromatographic

the radioactivity

of 72.8% for

estrone,

in aliquots 67.9% for

of the TLC eluates,

estradiol

and 82.0% for

estriol

were noted.

These values

were used to adjust

and showed that when 100 ng of the estrogens treated

plates,

estrone,

109 ng estradiol,

were also applied

the analysis

obtained

to the

showed 109 ng

Excellent

when only 20 ng of each of the estrogens

results were

GC tracing of l/SOOth of the sample eluted from a TLC , regular silica gel. plate containing 200 ng El silica gel to which ascorbic acid had been -------t added.

and plates

with ascorbic were often

with breast

1.5 ug estradiol

TLC plates.

methods utilizing

treated

wide variations

a male patient estrone,

by gas chromatography and 108 ng estriol.

When the chromatographic

samples,

were applied

to the TLC plates.

FIGURE1.

plates

the GC readings

But ascorbic

2.8 pg estradiol,

cancer

regular

acid were applied

noted.

As an example,

showed a daily

and 2.6 ug estriol acid treated

and 21.9 ng estriol.

silica

plates

excretion

gel to urinary the urine of 0.4 ug

when done with regular showed 1.8 ug estrone,

of

S

TPBEOXDI

425

E2

FIGURE 2. GC tracingof 11500thof the sampleeluted from a TLC plate regularsilicagel. -------a containing200 ng E2. acid had been added. silicagel to which as=:

FIGURE 3. GC tracingof 1/500thof the sampleeluted from a TLC plate regularsilicagel.------, containing200 ng E3. silicagel to which asa; acid had been added.

S

426

TEEOXD8B

Similarly, the urine of a 7S-year old post-menopausal woman with Primary breast cancer showed no detectable amounts of any of the three major estrogens by utilizing regular TLC plates. However, plates prepared with ascorbic acid gave daily excretion levels of 1.9 ug ~1, 0.6 ug E2, and S.l ug E3. The present method of preparing glass plates with anti-oxidant protection insures a uniform concentration of ascorbic acid over the entire plate and is an improvement over the earlier methods which apply the anti-oxidant by spraying techniques over finished plates [41 or by immersion, ['I ACKNOWLEDGMENTS This work was supported by the Mary Flagler CarY Charitable Trust assistance of Miss Patricia Lonefgan and of Fund. The technical Miss Victoria Hall are greatly appreciated. REFERENCES 1.

2,

3.

4.

5.

Morreal, C. E., Dao, T. L. and Lonergan, P. A,, An Improved Method for the Detection of Estrone, Estradiol, and Estriol in Low Titer Urine. STERIODS 20, 383 (1972). Dao, T. L., Morreal, C. E. and Nemoto, T., Urinary Estrogen Excretion in Men with Breast Cancer. NEW ENG. J. MED., 289, 138 (1973). Noller, C. R., Chemistry of Organic Compounds, W. B. Saunders Co., Philadelphia, 1958, p. 505. Frgacic, S. and Kniewald, Z., Ascorbic Acid As An Antioxidant in Thin-layer Chromatrography of Corticosteriods. J. Chromatog., 3, 291 (1974). Gelbke, H. P. and Knuppen, R., A New Method for Preventing Oxidative Decomposition of Catechol Estrogens during Chromato71, 465 (1972). graphy. J. Chromatog., -