553 GAS CHROMATOGRAPHY

PROFILE

APPLICATION G. L. Adessi,

TO PREGNANCY

BIOMEDICALE

Manuscrip"sreceived on:

:

URINE

Tran Quang Nhuan and M. F. JayleW

D. Eichenberger, uER

OF ESTROGENS

DES STS

PERES

B/7/74 ABSTRACT

A method for the simultaneous quantitation of 7 estrogens in pregnancy urine is described. It involves enzymatic hydrolysis, extraction of free steroids, ion-exchange column chromatography and quantitation of the trimethylsilyl derivatives by gas chromatography on OV 1. Data obtained from normal and twin pregnancies and from women with anencephalic fetus or intra uterine fetal death are analysed. The sensibility of the method is about 40 pg of each estrogen by liter of urine.

INTRODUCTION The interest quantitation Numerous

of the estrogens methods

termination

of a simple

extracted

of various

of the three

“classical”

ADLERCREUTZ

and LUUKKAINEN’s

ge resin

and al.

“classical”

estrogens

ge chromatography from pregnancy

steps.

liter

Vohme

EBERLEIN

(6), KAPLAN in urine.

since they require

urine.

a minimum

such as

(5) by using an anion exchanchange in estrogen

This purification and al.

process

isola-

was used by

(7,8) to assay the three

In this paper,

We can thus describe a simultaneous

the

a large

we apply this ion exchan-

method on AG 1 X 2 to study the profile

fast method which allows requires

complex

methods.

MORREAL

estradiol

method (4) used to evaluate

: AG 1 x 2 brought about a complete

tion and purification

: estrone,

for the de-

On the other hand, the few methods

are extremely

number of purification

urine is obvious.

were proposed

estrogens

(1, 2,3).

a simultaneous

from pregnancy

complexities

1’7 6 and estriol

total estrogens

method allowing

a simple,

evaluation

of estrogens

reliable

and

of 7 estrogens

of 40 pg of each of them to be present

and

in one

of urine (14).

25,

Nwnber

4

S

‘PDEOXDI

April,

1975

MATERIALS

AND

METHODS

A) Reagents All reagents were of analytical grade. The ethyl acetate was distilled before use. Estrogens standards were purchased from Steraloids, epicoprostanol from Sigma Chemical Co. N, O-bis-(trimethylsilyl)-acetamide (BSA) and trimethylchlorosilane (TMCS) were obtained from Pierce Chemical Co. Helix pomatia juice containing 120 000 U Fishman of fi glucuronidase and 1 000 000 U Roy of sulfatases per ml was purchased from Industrie Biologique Frangaise, Gennevilliers (France).

graph cases

Mass spectra were obtained on a LKB 2091 gas chromato- mass spectrometer ; the separator was kept at 280” C ; in all the spectra were recorded at 70 eV ionization energy.

B) Hydrolysis To 50 ml of 2 M acetate buffer pH acid if necessary) and was carried out at 45”

24 hours urine collection were added 5 ml of 4. 8 (adjusting the final pH to 4. 8 with acetic 0. 5 ml of Helix pomatia juice. The incubation C for 16 hours.

C) Extraction The hydrolysate was extracted two times with 100 ml of and the combined solethyl acetate/hexane/ ethanol (v/v/v/ ; 7/2/l) vent washed with 20 ml of Na2 CO3 (9 % ; w/v) pH 10. 5. Ethyl acetate was evaporated to dryness. D) Ion - exchange chromatography The columns were prepared according to a modified method of EBERLEIN (5) and MORREAL and al (6). A glass column (1 x 12 cm) equipped with a 50 ml bowl was filled with AG 1 X 2 Cl resin suspended in distilled water. The resin was allowed to settle and the column of 4 cm height was washed with the following reagents : 25 ml H20. 2 x 5 5 ml H20, 20 ml 80 % methanol and 20 ml methanol. ml 0.5 MNaHCO The extract was 9’hen applied to the column with 3 x 1 ml of methanol followed by 15 ml of methanol and 5 ml of 80 % methanol. The estrogens were eluted with 35 ml of 80 % methanol. The methanolic estrogens fraction was evaporated in vaccuo. E) Gas liquid chromatography The dried residue was dissolved in 4 ml of ethanol and a suitable aliquot was transferred with 20 rg of epicoprostanol as internal standard, to a conic tube and evaporated under vacuum. 100 pl of N, 0-bis-(trimethylsilyl)-acetamide-trimethyl chlorosilane (4/l ; v/v) were added to the dry residue and heated at 70” C for 3 hours. 1 to 5 pl were injected, GLC was carried out with a 4 meter X 2 mm

S

555

-A?B&UIDb

glass column packed with 1 % OV I on 100-120 mesh Chromosorb WBP with 50 ml NZ/min. ~ on a 900 Perkin-Elmer with a hydrogen flame ionization detector. Inlet temperature was 250” C, detector temperature was 300” C and the column temperature was programmed at 1. 5” C/min. starting at 180 or 200” C. Estrogens were identified by their relative retention time to epicoprostanol and/or by their methyl unit (MU).

lGD..JABfLITY

unit values

DATA

Characterization of estrogens was performed or relative retention time to epicoprostanol

through methyl (Table I). Using I.6

epi E3

1.068

TABLE I - Methyl unit values (MU) and relative retention time (RRT) to epicoprostanol for trimethylsilyl (TMSi) derivatives of estrogens (1 % OVI, column temperature programmed at 1. 5” C/min. , starting at 190” C ; silylation reagent : BSA/TMCS (v/v ; 4/l), 3 hours at 70” C).

this compound as internal standard, a quantitative analysis med by peak height with a good reproductibility (Table II).

was perfor-

556

s

TMSi

E1

E2

TPlEOXDl

16 a-OH

16 0x0

E1

E2

E3

16 epi E

E4 3

RX

. 834

1.034

.688

1.022

1. 18

1. 09

1.19

s

.021

.017

.017

.016

.012

. 16

.021

2. 52

1. 64

2.47

1. 60

1.04

1.47

1. 76

cv

%

TABLE II - Quantitative determination of estrogens by peak height using epicoprostanol as internal standard (mean of 5 determinations) (Rx = peak height CV = coefficient

ratio

epic~~‘~~teanoI

, s = standard

deviation

(2) ,

of variation).

Precision of the method was determined by calculating the standard --_---deviation in a replicate assay of pregnant women urine pool. The standard deviation for 10 replicate determinations is given in Table III. The mean of the coefficient of variation is 4. 95 %.

TMSi

mean mg/l

S

mg/l

cv

%

El

E2

16 a-OH

16 0x0

E1

E2

E3

16 epi

E4

E3

5. 24

.98

3.79

1.93

39.38

. 83

1.21

.2887

. 0663

.0198

.074

1. 302

.0354

.0695

3.83

3.30

5.51

16.76

15.22

1

1 4.25

( 5.74

TABLE III - Values of 10 replicate determinations for estrogens in a pool of pregnant women urine (s = standard deviation (+), CV = coefficient of variation).

Accuracy - of the method was tested by adding pure estrogens standards _---in ethanol to 5 males urines before enzymatic hydrolysis. The added amounts corresponded to 5 to 40 mg/l in order to agree with the relative concentration of each estrogen in pregnancy urines (Table IV). The recovery was corrected for endogenous material.

TMSi

E

amonts added

5

1

F2 5

16 0x0

16 a-OH

E3 40

10

10

16 epi

E4

5

5

(91699)

(7840-85:

mg/l recovery % 94 (extremes) (90-99)

(9ZT97) (785os4) (9?99)

,(9:!98)

TABLE IV - Percent recovery of estrogens standards added to male urines prior to hydrolysis (average of 5 determinations). The of a procedure using GLC to analyse substances present --_ ecificitx ---_ in biological fluids depends of how pure the sample is prior to GLC. The present method, using ion-exchange chromatography to separate estrogens from neutral steroids and more acidic phenols is specific as judged with a gas chromatograph - mass spectrometer : the peaks of estrogens are homogeneous and the spectra are identical with those of reference standards (Fig. 1). We completed this study with a comparison between the result obtained by summing up individual estrogens assayed by the proposed method and those obtained for the “total estrogens” by a fluorimetric method (9). For 50 comparisons, the coefficient of correlation r is 0, 99 and the regression line calculated is : Y = 0, 98 X f 0,47 (Y : sum of the estrogens evaluated by the above described method X : “total estrogens”). The sensibilig of the method was evaluated by 35 determinations on --_-----an urine pool with low estrogens content. The mean concentration obtained is 81,8 pg/liter with a value of 38,4 pg/liter for two standard deviations. It is thus possible to conclude that the sensibility of the method is around 40 pg/liter. DISCUSSION Estrogens gates. of Helix

The hydrolysis

AND RESULTS

in urine are either of conjugates

pomatia juice is complete

free,

by sulfatases

sulfo-or

glucuro-conju-

and 6 glucuronidase

with the method described

by one

author of our group (10). the extraction

of estrogens.

sion led us to prefer nol which allows purification

Ether is the solvent

X 2 involving of estrogens

The risk of inflammability

the following

an almost

and isolation a minimal

complete

extraction

loss

(5, 6) explains

Gas chromatography

with its high resolving of 7 estrogens.

temperature

program

11 oxo-estradiol.

In fact,

1 - Mass spectra

in pregnancy

urine.

A : estrone C : estradiol

energy

Our preliminary

derivatives

with a

such as 16-0~0 and

of interference

of the TIMSI derivative

Electron

power is essen-

phase such as OV 1. We did

with compounds

this absence

of recovery

IV).

prompt us to use trimethylsilylate

any interference

resin AG 1

the high degree

(Table

and a weak polar

etha-

The method of

added to the medium

separation

hexane,

by the ion exchange

experiments

Fig.

(11).

used for

and of explo-

blend : ethyl acetate,

of estrogens

tial for the simultaneous

not observe

still commonly

(see Table

of estrogens

70 eV

; B : estetrol 17 6 ; D : 16 keto-estradiol

17 6

I)

detected

S is understandable interference

since,

substances

-lFEIEOXDrn

in most cases,

is 50 to 100 times lower

tion of the compound to be assayed. more

polar

derivatives

estrogens Table

was also a failure.

A typical

from the 36th to the 42nd weeks of pregnancy The results

gnancies

(48 analyses)

and ring-D interval.

CLketols

in maternal

for normal,

in two time intervals

Estriol

averaged

8, 5 % of the total estrogens

The average

2.

rate of estriol

pre-

: 36-40

7% %, estetrol measured

urine

twin and

obtained for 9 uncomplicated

are subdivided

and 41-42 weeks of pregnancy.

The

chromatogram

is shown on Fig.

V shows the amounts of 7 estrogens

pregnancies.

when using

OV 225 or NPGS.

of a 39 weeks pregnancy

abnormal

of such

than the concentra-

We had no success

phases like OV 17 and particularly

use of acetyl of urinary

the concentrations

4 %

during each

we found between the 36th and

E

Fig.

2 - Gas chromatogram

of estrogens

obtained from

the urine of a 39 weeks pregnancy.

IS : internal

standard

(epicoprostanol)

the 40th weeks is higher LUUKKAINEN

than the rate given by ADLERCREUTZ

(12) and by HOBKIRK

found for the ring-D by these authors.

CLketols

The rates

and al.

(13) while the rates

compounds are lower of estrone

and estradiol

than the rates

and we found

17 6 we found

S are much rates

closer

to HOBKIRK’s

variations number

are due to the different of pregnancies

reviewed

Our results

(13) than to ADLERCREUTZ’s

at the same time xy-estrone,

results

fetus,

is lower

of estradiol

that the rate of 16 a hydroare

rate represents of estradiol

values.

Two groups

estriol,

estriol

These

conclusions

FRANDSEN

The first

on urine

and estetrol

with maternal

fetus from

The results or more. from

: 62,2

of estrogens

estrone,

can thus

estradiol

215 of the precursors

includes

17 6 and are of

16 keto-estradiol,

precursors

16 epi-

are principally

with ADLERCREUTZ

of the fetus,

origin

fetal.

(12) and

the profile

of estro-

without relative

for both cases

on the day of disappearance

an anencephalic

rate

respectively

by a fall of all estrogens

of estrogens

are signi-

(15) conclusions.

of death in utero

in presence

pregnancy

whose

are in agreement

is thus possible

weeks

group

and STAKEMAN

collected

includes

For this group

gens is characterized servation

group

The second

In case

while

17 6 and of 16 a hydroxy-estro-

43, 8 and 47, 6 % of normal

origin.

higher

4, 3 % of the normal

they represent

maternal

slightly

17 /3 and of estetrol

since

16 a hydroxy-estrone.

that in these

than the rate found

preserved

be individualized.

differ

215 and 156 %. In one woman with anence-

of estrone,

ne are relatively

These

studied

It was ascertained

and estriol

respectively

the estriol

while the rates

twin pregnancies

(12).

of 16 keto-estradiol

higher,

rate (14).

used and also to the small

pregnancies,

on the other hand the rates ficantly

estetrol

in each paper.

the rate of estrone for normal

We found a rate of estetrol

methods

for the three

ADLERCREUTZS

3 twin pregnancies

phalic

rates

(12) which are significantly lower. .. than HEIKKILA and LUUKKAINEN

lower

from

v?PIIEOXDrn

pre-

observed

of fetus heart beat.

of a low rate of estrogens

It

to differentiate

a fetus dead in utero.

we presented

The sensibility its beginning.

here

include

of the method An example

pregnancies

allows

of 36

the control

of a

out of a study being now in

V - Urine estrogens

2

[ntra uterin fetal death 2

TABLE

1

Anencephalic fetus 1

3

Twin 3

I

+

+

.I3

‘79

1.03

e 81 .81

1. 27 . 58

E1

69

.04

.14

l

.24 + .19

.32 2 .07

E2

-+ 1. 73

1.73 .86

1.42 -282 +

Bl

16 OH

in (MC/24 hours ) different (2 standard deviation)

36

36

36 - 37

41 - 42

36 - 40

36

12

Weeks of pregnancy

No of determin.

9

Normal

Pregnancies number

1. 79

1. 69 .86

pregnancies

-f

1.34 .15

E2

16 OX0

2

_+

33.20

28.20 9.18

29.14 5.94

E3

f

+

.12

N. D.

1. 84

.49

t 73

1.83 * 33

E3

.6 EPI

+

+

.I0

N. D.

2. 24

s. 49 .35

1.43 .18

E4

progress

(Fig.

3) concerns

a pregnancy

the 40th week (32 determinations). 16 epi-estriol

and estetrol

30th or 31st weeks lastly

a new plateau

estrone,

estradiol

have an almost with however followed

by a fast increase

elimination

a fast increase

by a plateau.

are parallel

curve from

of estriol,

the 26th to the

On the other

hand,

and 16 keto-estradiol

from

the 26th to the 40th week

of 17 6 estradiol

The curves

the 26th to

till the 37th week and

16 a hydroxy-estrone

continuous

keto-estradiol

: a plateau

till the time of parturition. 1’7 6,

from

The elimination

is identical

followed

controlled

around

the 35th week

of 16 a hydroxy-estrone

with wide variations

from

and of 16

one week to the

other. The confirmatory assets

corroborating

laboratories seven

abnormal whether

estrogens.

or rapidly

origin

progress

in this aim.

routinely

individual

simple

involves

determinations

or of fetal origin.

this technique estrogens

in one of the

technique

When the rate of the “total

decreasing,

the abnormality

maternal

of the data presented

the value of this very

to perform

principal

nature

Our investigations

of the

estrogens”

permits

whose

allowing

is

to decide

precursors

are of

are still in

S

26

563

I?DEOXDI

26

30

32

34

36

38

40

28

30

32

34

38

38

40

WEEKS *

26

Fig.

3 - Urine

This

ACKNOWLEDGEMENT study has been supported by a CNRS grant (LA 87).

estrogens

in pregnancy

(birth weight

: 2. 900 Kg)

REFERENCES 1.

Jayle, M. F. ANALYSE DES STEROIDES HORMONAUX, vol. 2, Editor Masson (Paris), 1962, p. 396.

2.

Paulsen, C. A. ESTROGEN ASSAYS IN CLINICAL MEDICINE, Editor Paulsen, C. A. (University of Washington Press), 1965, p. 30.

3.

Adlercreutz,

4.

Adlercreutz, Suppl. 125,

H. CLIN.

CHIM.

H. and Luukkainen, 101 (1967).

ACTA.

34, 231 (1971).

T. ACTA

ENDOCRINOL.

s

564 W.R.

T&YmEOXDI

5.

Eberlein,

STEROIDS

14, 553 (1969).

6.

Morreal, C. E., 20, 383 (1972).

7.

Kaplan, H. G. and Hreshchyshyn, OBST. GYNEC. 111, 386 (1971).

M. M.,

8.

Kaplan, H. G. and Hreshchyshyn, 763 (1972).

M. M. S’I’EROIDS

9.

Adessi, G. and Jayle, 127 (1972).

10.

Jayle, M. F. ANALYSE vol. 2, Editor Masson.

11.

Adessi, G., ANN. BIOL.

12.

Adlercreutz, H. and Luukkainen, 2, 365 (1970).

13.

Y., Nilsen, Hobkirk, R. , Anuman-Rajadhum, Blaney, P. P. , CLIN. CHEM. 16, 235 (1970).

14.

Abbreviations,

Dao T. L. and Lonergan,

M. F.,

ANN.

AM.

BIOL.

DES STEROIDES

Goutte, Ch. , Christeff, CLIN. 31, 495 (1973).

P. A.,

STEROIDS

JOURNAL

CLIN.

19,

30,

HORMONAUX,

N. I. and Jayle,

T. ANN.

CLIN.

M. F. ,

RES.

M. and

trivial and systematic names : El, estrone, 3, 5 (lo)-estratrien-17-one ; E 2, estradiol 17 6 -dial ; 2 met E 1, 2178 > 1,3, 5 (lo)-estratrien-3, methoxy estrone, 3-Hydroxy, 2-methoxy-1, 3, 5 (lo)-estra3,16 atrien-17-one ; 16 HO E 1, 16 a-hydroxyestrone, Dihydroxy-1,3, 5 (lo)-estratrien-17-one ; 16 0x0 E 2, 163-Hydroxy-1,

oxo-estradiol, 3, 17 6 -Dihydroxy-1, 3, 5 (lo)-estratrien-16one ; 16, 17-epi E 3, 16, 17-epiestriol, 1,3, 5 (lO)-estratrien17 a-trio1 ; 17 epi E 3, 17 epiestriol, 1, 3, 5 (lO)3,166, estratrien-3, 16 cz, 17 a-trio1 ; E 3, estriol, 1, 3, 5 (lO)estratrien-3, 16 a, lwtriol ; 16 epi E 3, 16-epiestriol 1, 3, 5 (lo)-estratrien-3, 16 6 -trio1 ; E 4, estetrol 1,3, 5 (lO)-estratrien-3, 15 a, 16 a, 17 6 -tetrol ; Epi, epicoprostanol, 5 6 -cholestane-3cx 01. x Present

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