A SIMPLE METHOD FOR THE QUANTITATIVE DETER-

MINATION OF PREGNANEDIOL IN HUMAN URINE

BYE. B. ASTWOOD AND G. E. SEEGAR JONES
(From the Departments of Obstetrics and Gynecology, the Johns Hopkins
University and Hospital, Baltimore)

(Received for publication, September 14, 1940)

Since the discovery by Venning and Browne (1) of sodium preg-

nanediol glucuronidate in human urine and the perfection of a
quantitative method for its determination by Venning (2) this
compound has received wide attention because of its close relation
to the metabolism of the corpus luteum hormone. This subject
has recently been reviewed by Marker and Hartman (3). It is
apparent from conflicting results in the literature that difficulties
have been encountered in measuring the excretion of the sodium
pregnanediol glucuronidate complex. During the menstrual cycle,
when only small amounts of the material are present in the urine,
the identity of the final product is sometimes questionable. Fur-
thermore, spontaneous hydrolysis of the complex to free preg-
nanediol during the period of collection may occur. The resulting
inconstant recoveries are particularly noticeable in hot weather,
when refrigeration is not available. This latter difficulty was met
by Buchcr and Geschickter (4) who, using a modification of Hart-
mann and Lecher’s (5) method as developed by Weil (6), were
able to recover the free pregnanediol from the acetone used in the
Venning procedure. The total excretion was estimated by adding
this moiety to that recovered in the combined form. However,
wide application of such methods is limited by the technical diffi-
culties involved. With these factors in mind, a procedure has
been developed which is sufficiently simple and accurate that it
may be applied to routine clinical use. It involves three steps:
(1) liberation of free pregnanediol by acid hydrolysis of the urine
in the presence of toluene, (2) the separation of most of the impuri-
ties from the toluene extract by the precipitation with alcoholic
397

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398 Determination of Pregnanediol

alkali, and (3) the final quantitative crystallization of the pure

pregnanediol from aqueous alcohol.

Method
Urine specimens are collected, with a few drops of toluene as
a preservative. The entire 24 or 48 hour volume or a suitable
aliquot thereof is used. As 1 liter is a convenient amount, the
figures given in the following steps are for this volume of urine;
proportional amounts are used with other volumes. In some non-
pregnancy urines, when very small amounts of pregnanediol are
to be determined, 2 or 3 liter volumes should be taken, while
with late pregnancy urine 50 to 500 cc. volumes are suitable.
1 liter of urine is placed in a 2 liter round bottom flask and 50 cc.
of toluene are added. A wide bore reflux condenser, fitted with
a cork stopper or ground glass connection, is attached and the
urine heated to boiling. 100 cc. of concentrated hydrochloric
acid (sp. gr. 1.19) are added slowly through the condenser and
boiling continued for 15 minutes. The temperature of this boiling
mixture is 100-105”. The flask is then cautiously removed to a
vessel of cold water (violent boiling may occur if the hot mixture
is shaken) ; the urine is brought to room temperature and trans-
ferred to a separatory funnel. The toluene layer, together with
the layer of urine-toluene emulsion, is removed and the urine
layer is reextracted twice with 25 cc. portions of toluene. The
combined toluene extracts and emulsions are filtered through a
Buchner funnel, with gentle suction, and the residue thoroughly
washed with small amounts of toluene. If the filtration is per-
formed without excessive suction, the emulsion is completely
broken down. The filtrate is poured into a small separatory
funnel, the urine layer is discarded, and the toluene extract is
washed once with 50 cc. of water. The toluene is now transferred
to a dry 400 cc. beaker, care being taken that no droplets of water
are included, and the extract boiled on an electric hot-plate until
the last traces of water are dispelled. 10 cc. of a 2 per cent solu-
tion of sodium hydroxide in absolute methyl alcohol are added
and boiling continued until all the alcohol is driven off and the
quantity of toluene is reduced to half its original volume. After
the extract is allowed to stand at room temperature until cool,
it is filtered with gentle suction into a 125 cc. Erlenmeyer flask
 E. B. Astwood and G. E. S. Jones 399
and the abundant gelatinous precipitate is thoroughly washed
with three successive small amounts of toluene. The filtrate
should be clear and free of all traces of a pink or brown color;
otherwise the previous step must be repeated. The clear, pale
greenish yellow filtrate is now taken to dryness on the hot-plate,
the last traces of toluene being carefully removed by a stream of
air to avoid overheating the residue. 10 cc. of 95 per cent ethyl
alcohol are added and the residue is completely dissolved by heat-
ing. While heating is continued, 40 cc. of hot aqueous 0.1 N
sodium hydroxide are added slowly. The stoppered flask is al-
lowed to stand at room temperature until cool and is then placed
in the refrigerator overnight.
The precipitate is collected by filtration, washed with water,
dissolved in 10 cc. of alcohol, and precipitated a second time in
the same manner, 40 cc. of distilled water being used instead of
the sodium hydroxide solution. The pregnanediol should now
be colorless and entirely crystalline; if not, one further precipita-
tion from aqueous alcohol is carried out. The purified substance
is now collected by filtration, transferred by means of ethyl alcohol
to a tared vial, dried in an oven at 90”, and weighed. A melting
point is taken on each sample recovered and, if this be below 220”,
a mixed melting point is determined with a pure sample of preg-
nanediol. When the recovered material is largely pure preg-
nanediol, its melting point is raised when it is mixed with an
authentic sample; the determination is considered to be satis-
factory if there is no depression of the mixed melting point.

EXPERIMENTAL

Precipitation* of Pregnanediol from Aqueous Alcohol--In order

to estimate the amount of pregnanediol which might be lost by
remaining in solution, the solubility of pregnanediol in various
concentrations of aqueous alcohol was determined in the following
manner. Known amounts of pregnanediol in 95 per cent ethyl
alcohol were diluted with water while hot. The solutions were
cooled and placed in the refrigerator overnight. By preparing a
series of such solutions with decreasing amounts of pregnanediol,
it was possible to det.ermine roughly how much was needed in
order to give a precipitate. Such an experiment is shown in
Table I, from which it may be seen that within the accuracy of
 Determination of Pregnanediol

this method prcgnanediol is completely insoluble when 3 or more

volumes of water are added to 1 volume of alcohol. It would
appear, therefore, that two or three precipitations from 10 cc. of
alcohol, with 30 or more cc. of water, would result in no detectable
loss. This is apparently true, also, in the presence of the other
neutral substances present in the extract at this stage, as similar
experiments were conducted in which cholesterol and pregnanediol-
free neutral urine extracts were added to alcohol, together with
known amounts of pregnanediol. No increased solubility of preg-
nanediol was noted in the presence of these substances in the
amounts used. It appears probable, however, that small losses
could occur at1 this step in the presence of large quantities of
other lipids.

TABLE I
Approximate Solubilities of Pregnanediol in Aqueous Alcohol
95 per cent alcohol, cc.. ;;
Water added, cc..

m7. ml. m!l. WJ.

Pregnsnediol in solution
Room temperature. > 1 .O 1.0 0.25 <0.25 <0.25 <0.25
After refrigeration.. >1 .O 0.5 0.25 <0.04 <0.04 <0.04

It was of interest to determine the fate of other substances

present in the neutral fraction when this precipitation was carried
out, for one might expect that other urinary constituents may
behave in a fashion sufficiently similar to pregnanediol to be pres-
ent in the final product. The three most abundant neutral lipids
known to be present in human urine were studied. These were
cholesterol, androsterone, and a hydrocarbon designated by Hart
and Northrup (7) and by Marker (8) as heptacosane. Cholesterol
in amounts up to 8 mg. and the hydrocarbon in amounts up to
100 mg. were found to form stable colloidal solutions when precipi-
tated in the above manner from 50 cc. of 19 per cent alcohol. On
filtration these colloidal suspensionsleave no residue. When pres-
ent in larger amounts, some residue is retained on the filter. An-
drosterone separates as plate-like crystals from 50 cc. of the 19 per
cent alcohol when present in amounts exceeding 4 mg.; amounts
smaller than this remain in solution. Therefore, an excess of
 E. B. Astwood and G. E. S. Jones 401
16 mg. of cholesterol, 200 mg. of the hydrocarbon, or 8 mg. of
androsterone per liter of urine would be necessary in order to
interfere with the final purity of the pregnanediol, if the two pre-
cipitations from aqueous alcohol are carried out. These amounts
are well outside the range of values thus far reported for these
neutral substances in normal human urine.
Precipitation of Impurities from Toluene Extract-It is of impor-
tance that excess water be avoided in handling the toluene extract.
Water in excess of the small amount resulting from chemical reac-
tion may introduce impurities which are not removed by the sub-
sequent step. The presence of water also results in a large gelati-
nous precipitat,e which is difficult to wash. Excess hydrochloric
acid is removed by washing the original toluene extract, thus
minimizing the water of reaction. Dissolved water is then largely
dispelled by boiling.’ The 2 per cent sodium hydroxide solution
in methyl alcohol is prepared from clean dry sodium hydroxide
pellets and absolute methyl alcohol. An alternative to the alco-
holic sodium hydroxide solution is a 2 per cent methyl alcohol
solution of sodium methylate, prepared by adding sodium to abso-
lute methyl alcohol. Such a solution has been used with satis-
factory results.
It seemed possible that some loss at this stage, through adsorp-
tion of the pregnanediol on the abundant precipitate, might be
incurred. Therefore, precipitates from several batches of urine
extract were combined and thoroughly extracted with boiling
ethyl alcohol. After the filtered alcohol extract was evaporated
off, the residue was taken up in toluene and the toluene-soluble
material purified in the usual manner. No pregnanediol was
recovered by this method from fourteen combined precipitates.
Apparently, therefore, the loss of pregnanediol in this stage of
purification is negligible.
Extraction of Pregnanediol from Urine by Toluene-1 liter
volumes of urine from which 5 to 80 mg. of pregnanediol had been
recovered by the extraction method described were exhaustively
reextracted with toluene. In no case was any further pregnanediol
recovered. In five instances the three toluene extracts described
1 An occasional difficulty in avoiding excess water may be met by the
addition of 0.5 to 1 gm. of powdered calcium oxide to the boiling extract
before cooling.
402 Determination of Pregnanediol
in the original procedure were worked up separately. The re-
coveries indicate that approximately 85 per cent of the free
pregnanediol is taken up by the toluene during hydrolysis; the re-
mainder usually appears in the second extraction, while only
occasionally does the third extract contain a further trace. The
volumes of toluene used were therefore considered sufficiently
large to remove all available pregnanediol. Inasmuch as the solu-
bility of pure pregnanediol in toluene at room temperature does
not exceed 2 mg. per cc., no attempts were made to use smaller
volumes, for fear of losses in subsequent steps.
Hydrolysis of Combined Pregnanediol by Acid-The rate of hy-
drolysis of the sodium pregnanediol glucuronidate complex was
studied by adding measured amounts of an aqueous sodium preg-
nanediol glucuronidate solution to male urine and boiling for
various periods of time in the presence of different concentrations
of hydrochloric acid. The amount of free pregnanediol liberated
was then determined by the method outlined above.
The first series of hydrolyses was made on 100 cc. samples of
male urine containing 10 mg. of sodium pregnanediol glucuroni-
date. To this volume of urine amounts of concentrated hydro-
chloric acid from 5 to 15 volumes per cent were added; the mixtures
were boiled for periods of 23 to 60 minutes. The amounts of
free pregnanediol recovered are plotted in Fig. 1, from which it
can be seen that with 5 volumes per cent of acid the hydrolysis is
slow and that a maximal yield of 66 per cent was obtained after
1 hour’s boiling. When 10 or 15 volumes per cent were used, a
maximal yield of 70 per cent was obtained in 2$ and 10 minutes
respectively, but with longer boiling smaller amounts were ob-
tained. It appeared from this that 10 volumes per cent of acid
would give satisfactory results if destruction of the compound
could be avoided. Consequently, a second series of hydrolyses
was made on 1000 cc. volumes of urine to which 0.05 volume of
toluene was added. In Fig. 2 are shown the results of such an
experiment. The 10 volumes per cent of acid gave maximal yields
within 10 minutes and prolonged boiling for 4 hours resulted in
little destruction of the compound.
A third series of hydrolyses was made on 50 mg. amounts of
combined pregnanediol in 1000 cc. of distilled water to which were
added 50 cc. of toluene and 100 cc. of concentrated hydrochloric
 E. 13. Astwood and G. E. S. Jones 403

acid. A maximal yield of 80 per cent was obtained after 15

minutes of boiling and continued boiling for 4 hours resulted in
only slight destruction. In Fig. 2 these results are compared
with similar experiments in which no toluene was used. It is
apparent that the presence of toluene serves to prevent destruc-
tion of pregnanediol by the acid.
% %
EiO- 80-
70- 70.
60- 60-
50- 50-
40- 40.
30. 30-
,’ \
20- ,’
20- \
a
!O- IO-

2.5 5 IO 20 30 -60 i I'0 15 30 6'0 126 2bo

FIG. 1 FIG. 2
FIG. 1. Liberation of free pregnanediol from 10 mg. of sodium preg-
nanediol glucuronidate on boiling with acid in 100 cc. of normal male urine.
The dots represent 5, the crosses 10, and the triangles 15 volumes per cent
of concentrated hydrochloric acid. The ordinate represents per cent of
pregnanediol recovered; the abscissa, time of boiling in minutes (logarith-
mic scale).
FIG. 2. Hydrolysis, in 1000 cc. of an aqueous solution, of 50 mg. amounts
of sodium pregnanediol glucuronidate with 10 volumes per cent of con-
centrated hydrochloric acid. The dots represent results with, the triangles
without 50 cc. of toluene. The crosses show the rate of hydrolysis in 1000
cc. of male urine with 100 cc. of concentrated hydrochloric acid and 50
cc. of toluene.

Recoveries of Pregnanediol by Method Proposed-Amounts of

sodium pregnanediol glucuronidate from 2 to 200 mg. were added
to samples of urine from normal males. The volumes of urine
used were 1000, 2000, and 4000 cc. ; these were treated by the
above method and the yields calculated. The results are given
in Table II, which shows the calculated equivalent of free preg-
nanediol contained in the amount of the complex added, the
amount recovered, and the per cent recovery.
Table II shows that yields from 1 liter of urine containing 3 mg.
 TABLE II
Recovery of Pregnanodiol
-7
Sodium Calculated Deviation
pregnanediol equivalent from mean
Free pregnanediol recovered
gluww~~date of free P?c”“*ry of
Ipregnanediol* 68.02 per cent

CC. mg. ml. mg. per cent per cent

1000 0 0 0
0 0 0.8
2 1.236 0.0
2 1.236 0.0
2 1.236 0.5 41.5
2 1.236 0.6 50.6
2 1.236 1.2 100.0
5 3.10 2.3 74.2 +6.18
5 3.10 2.3 74.2 +6.18
5 3.10 2.4 77.4 +9.38
5 3.10 2.5 80.6 +12.58
10 6.18 3.9 63.1 -4.92
10 6.18 4.0 64.7 -3.32
10 6.18 4.1 66.4 -1.62
16 9.89 5.9 60.0 -8.02
20 12.36 8.2 66.4 -1.62
20 12.36 8.4 68.0 -0.02
20 12.36 8.7 70.4 $2.58
25 15.45 9.5 61.5 -6.52
30 18.54 12.0 64.8 -3.22
30 18.54 12.4 67.0 -1.02
30 18.54 13.3 71.8 +3.7s
40 24.72 16.5 66.8 -1.22
40 24.72 16.5 66.8 -1.22
50 30.90 19.9 64.4 -1.62
50 30.90 20.0 64.7 -3.62
50 30.90 21.7 70.3 +2.2s
200 123.60 80.6 65.2 -2.82
2000 0 0 0.5
2 1.236 0.0
2 1.236 0.0
2 1.236 0.1 8.33
2 1.236 1.1 91.66
2 1.236 1.4 116.66
5 3.10 1.1 35.48
5 3.10 1.6 51.61
5 3.10 1.7 54.83
4OQO 0 0 0.0
0 0 0.1
2 1.236 1.7 141.66
5 3.10 0.6 19.35
5 3.10 1.0 32.66
5 3.10 2.2 70.96
10 6.18 3.2 53.2
moo 0 0 0.0

* Calculated with the conversion factor of 0.618.

404
 .I{;. 13. A&wood and G. .E. S. Jones 405
or more of pregnanediol are consistent and average 68 per cent.
The standard deviation from the mean was 5.15 per cent and the
standard error of the mean was 1.12 per cent. Recoveries of smaller
amounts of pregnanediol were less uniform, but some quantitative
significance could be attached to determinations of 1 to 3 mg. per
liter, when 2 or 4 liter volumes were used.
Similar volumes of male urine to which no sodium pregnanediol
glucuronidate was added were hydrolyzed and extracted in the
same manner. Two of these samples yielded no precipitate at
the final step, while three gave readings of 0.1 to 0.8 mg. per liter.
The melting points of the recovered material in the determina-
tions shown in Table II ranged from 218-228“, and on mixing with
pure pregnanediol (m.p. 238’) no depression occurred.
Pregnanediol determinations on human urine during the luteal
phase of the menstrual cycle and during pregnancy have yielded
material with melting points ranging from 205-228”. These melt-
ing points were not depressed and usually were elevated when the
material was mixed with the pure substance. The lower values
are apparently due to a small amount of unidentified material not
present in hydrolysates of the combined form. The possibility
that this impurity is allopregnanediol has not been eliminated.
DISCUSSION

The calculation used in determining the free pregnanediol

equivalent of sodium pregnanediol glucuronidate may be subject
to error. Venning and Browne showed that sodium pregnanediol
glucuronidate contains 1 molecule of water of crystallization and
has a molecular weight of 536. As the molecular weight o,f free
pregnanediol is 320, these authors used a factor of 0.597 for the
conversion of the complex to free pregnanediol. Bachman, Leekley,
and Hirschmann (9) found that the combined form is hygroscopic,
but that all water is removed when the material is dried in a
vacuum over phosphorus pentoxide at room temperature. It has
been assumed in the above calculations that no solvent of crystal-
lization was present in the sodium pregnanediol glucuronidate
used, and that 1 gm. of the complex contained 0.618 gm. of free
pregnanediol. The material used was repeatedly recrystallized
from 95 per cent ethyl alcohol and dried in air at 90”.
It appears unlikely that, the failure to obtain 100 per cent re-
406 Determination of Pregnanediol

covery on hydrolysis of the complex in aqueous solution can be

attributed to an error in this calculation. However, the con-
sistent loss of 20 per cent under varied conditions cannot be traced
to incomplete extraction and, as destruction is largely eliminated
by the use of toluene, it remains unexplained. The further loss
in determinations on urine is believed to be incurred in purification.
The recoveries given in Table II show that determinations on
urine containing 3 mg. or more of pregnanediol per liter may be
made with considerable accuracy. When 1 to 3 mg. of preg-
nanediol per liter is present, the error involved is large, but this
error is somewhat reduced by increasing the volume of urine
extracted.
On two occasions no pregnanediol was detected in 1 liter samples
of urine containing 1.2 mg. of pregnanediol. Some samples of
normal male urine yielded zero values, while others gave 0.1 to
0.8 mg. of some unidentified substance per liter of urine. It is
assumed that the recovered material in these cases is not preg-
nanediol, but is an impurity which may lead to error in deter-
minations on urine containing comparable amounts of pregnane-
diol. Although Engel, Thorn, and Lewis (10) isolated 64 mg. of
pregnanediol from 1000 liters of normal male urine, the zero values
shown in Table II indicate that amounts of pregnanediol of the
order found by Engel, Thorn, and Lewis are not uniformly detect-
able by the above method.
Subject to these limitations the method proposed is satisfactory.
It requires no special apparatus, it is free of technical difficulties,
and no abnormal urinary constituents such as albumin or blood
have been found to interfere. As pregnanediol is a stable sub-
stance, no special precautions are required in the collection of
specimens and the loss through hydrolysis which is so inconstant
in determination of the combined form is obviated.
To estimate the 24 hour excretion of pregnanediol the following
approximate calculation is made
24 hour urine volume
Daily pregnanediol output, mg. = volume urine used

X pregnanediol found, mg. X 1.47

which takes into consideration the average loss entailed in the
method. Although the correction factor seems large, it is over-
 E. B. Astwood and G. E. S. Jones

balanced by the uniformity of results, by the stability of free

pregnanediol, and by the simplicity of the method.

The authors are indebted to Dr. L. L. Engel for valuable advice

and criticism and to Dr. P. G. Weil for many helpful suggestions.
SUMMARY

The difficulties encountered in the determination of sodium preg-

nanediol glucuronidate are cited and a method based on the deter-
mination of the free form of pregnanediol is described. The ad-
vantages and limitations of the method are discussed.
It is recommended as a satisfactory procedure for routine clini-
cal use or for the large scale preparation of pregnanediol from
human urine.
BIBLIOGRAPHY

1. Venning, E. H., and Browne, J. S. L., Proc. Sot. Exp. Biol. and Med.,
34,792 (1936).
2. Venning, E. H., J. Biol. Chem., 119, 473 (1937); 126, 595 (1938).
3. Marker, R. E., and Hartman, C. G., J. Biol. Chem., 133, 529 (1940).
4. Bucher, N. L. R., and Geschickter, C. F., Endocrinology, 27,727 (1940).
5. Hartmann, M., and Lecher, F., Helv. chim. acta, 18,160 (1935).
6. Weil, P. G., Proc. Sot. Exp. Biol. and Med., 38, 503 (1938).
7. Hart, W. F., and Northrup, M. A., J. Am. Chem. Sot., 67,2726 (1935).
8. Marker, R. E., J. Am. Chem. Sot., 60, 2442 (1938).
9. Bachman, C., Leekley, D., and Hirschmann, H., J. Clin. Znv., 19,801
(1940).
10. Engel, L. L., Thorn, G. W., and Lewis, R. A., Am. J. Physiol., 129,
P352 (1940).