The quantitative estimation of oestriol using gas-liquid chromatography

473

CLINICA CHIMICA ACTA

THE

QUANTITATIVE

ESTIMATION

OF OESTRIOL

USING

GAS-LIQUID

CHROMATOGRAPHY

S. J. RICHARDSON Depavtment of Pathology, General Hospital, Middlesbrough (U.K.) (Received

May

15,

Igp)

SUMMARY

A method is described for the routine determination of oestriol in pregnancy urine using gas-liquid chromatography with flame ionisation detector. The steroids are chromatographed as their trimethylsilyl ethers on 1.5% XE-60. The results are quantitated by measuring peak heights and comparing the oestriol response to the response for oestrone incorporated as an internal reference. The influence of sugar on the recovery of oestriol is studied. The method is also compared with the manual methods of Brown and Oakey et al.

INTRODUCTION

The influence of pregnancy on the level of oestrogenic activity was noted more than forty years ago. Smith1 reported in 1927 that oestrogenic activity increased during pregnancy and decreased after delivery. In 1933, Spielman et aJ.2 found blood oestrogens markedly decreased on foetal death and this was used to diagnose the condition. In the same year Smith and Smith3 published results in which oestrogens were bio-assayed in blood and urine of pregnant women. ResultW demonstrated decreasing urinary levels of oestrogens prior to subsequent clinical evidence of placental insufficiency. In the intervening years it has been shown by many workers using chemical methods that the amount of oestrogens excreted per 24 h was related to the foetal well-being. A review of the literature up to 1964 was made by DiczfalusyG and in this he showed the relative merits of oestriol and pregnanediol determinations during the course of the pregnancy. Oestriol excretion has been shown to be associated with foetal well-being and this association probably arises from the complicated synthesis of oestriol, which involves both foetal and placental participation. In this respect it differs from pregnanediol which, so far as is known, is related to an aspect of placental function and has little to do with the activity or otherwise of the foetus itself. The use of gas-liquid chromatography for the determination of oestriol has been suggested by several workers7-10. However these did not meet the requirements which were thought necessary, namely a system which would estimate oestriol in pregnancy urine easily and specifically, and so be suitable for routine use. Clin. Chim. Acta, 29 (1970) 473-480

RICHARDSON

474 EXPERIMENTAL

Reagents

All chemicals were of AR quality obtained from British Drug Houses, Poole, England, unless otherwise stated. Absolute ethyl alcohol AR (J. Burrough, London) ; hexamethyldisilazane, trimethylchlorosilane and dimethyldichlorosilane (Hopkin & Williams, Chadwell Heath, England) ; oestrone and oestriol (Koch Light Labs., Colnbrook, England). Saturated Na,CO, solution pH 10.5 (according to Brownll) is prepared by adding 150 ml of 20% NaOH to I 1 of 8% NaHCO,. Oestrone reference solution containing IOO pg/ml is prepared by dissolving 25 mg of oestrone in 250 ml of absolute alcohol. Standard oestriol solution containing 80 pg/ml is prepared by accurately weighing out 20 mg of oestriol and dissolving in 250 ml of absolute alcohol (in a volumetric flask). Instrument

and

for gas-lipid

coditions

&omatogra$hy

A Pye 104 Gas Chromatograph (Pye Unicam, Cambridge, England) with flame ionisation detector was used with a 5 ft. x 4 mm coiled glass column. The column was packed with 1.5% w/w p-cyanoethyl-substituted methyl polysiloxane XE-60 (Applied Science Labs., State College, Pennsylvania, U.S.A.) stationary phase, on 100-120 mesh acid/alkali-washed kieselguhr “Embacel” (May & Dagenham, England) solid support. The isothermal operating temperature for the oven was 205’ with the nitrogen carrier gas flow rate adjusted to give a retention time for oestriol trimethylsilyl ether of between 6-8 min, approximately rgo ml/min at 20’ with an inlet pressure of 1.52 x 10s N/m*. The amplifier attenuator setting was 5 x 10-l~ with an injection of of a r-p1 sample. The solid support and the internal surfaces of the empty column, adaptor and glass wool plugs were all silanised prior to use, by washing with a 5% solution of dimethyldichlorosilane in toluene, followed by methanol and finally heating in a hot air oven at 105’ for I h. The kieselguhr was coated with stationary phase by adding 0.15 g XE-60, dissolved in 40 ml of acetone, to IO g of solid support. This mixture was allowed to stand for 30 min after which the solvent was removed by evaporation and the coated support heated at 105” for 16 h. This was then packed into the column under pressure. Prior to use the column was conditioned by flowing carrier gas through it for 24 h with the oven at 220’. Treatment of urine sam$le

A 5-ml aliquot of a 24-h urine specimen together with IO ml of water and 3 ml of cont. HCI is placed in a 25 x 150 mm test tube. The tube is then heated in a boiling water bath for 45 min. After hydrolysis the tube is cooled and the contents poured into a roe-ml separating funnel. The tube is washed twice with 5 ml of water and these washings are also added to the funnel. This solution is extracted with 3 x20 ml of diethyl ether and the aqueous phase discarded. The pooled ether extract is washed with 12 ml of saturated Na,CO, solution followed by 5 ml of water and these washings are discarded. The ether is extracted with IO ml and then 5 ml of N NaOH and the combined extracts acidified with 5 ml of 3 N H,SO,. The ether phase is discarded. The C&n.Chim.Acta,

zg (1970)

473-480

CHROMATOGRAPHIC

ESTIMATION

475

OF OESTRIOL

aqueous phase is washed with IO ml of petroleum ether-toluene I :I (v/v), separated and the organic phase washed with 5 ml of water. This washing is added to the aqueous phase and the organic phase discarded. The aqueous solution is extracted with 3 x 20 ml volumes of ether which are combined, dried with anhydrous Na,SO, (approximately 5 g) and decanted into a roe-ml round-bottomed flask followed by a ro-ml ether washing of the Na,SO,. The ethereal solution is concentrated using a rotary evaporator, to a volume of 2-3 ml. This solution is then transferred quantitatively to a 25-ml stoppered tube, using a washing of 1-2 ml of ether for the flask. Derivative formation

To this tube and a second tube, which is to be used for an external standard, is added I ml of a IOO ,ug/ml solution of oestrone. To the standard tube only is added I ml of an 80 pg/ml solution of oestriol. The contents of each tube are evaporated to dryness using the rotary evaporator. To the dry residue in each tube is added 0.2 ml of hexamethyldisilazane and 0.1 ml of trimethylchlorosilane, the stopper inserted, and the tube carefully rotated to wet the wall of the tube. They are then placed in a hot air oven at 105’ for 15 min. Afterwards the tubes are removed from the oven, the excess reagents evaporated as before, and restoppered. 0.2 ml of n-hexane is added to the cooled tube which is then rotated to wet the wall and finally a vortex mixer is used to achieve complete dissolution. The tube is kept stoppered except when adding or removing materials. One+1 aliquots are then subjected to GLC and finally the peak heights measured. CALCULATION

The detector response for each substance is obtained by measuring the height of the recorder peak. Then the ratio of the response for oestriol compared with that for oestrone from the same injection is calculated, i.e. oestriol peak height oestrone peak height

= Rs, RT~, etc.,

for standard and tests. The ratio of peak heights is equal to the ratio of the concentrations of the two substances. The amount of oestrone is the same in both the standard and test, and the amount of oestriol in the standard is 80 ,ug. Therefore: RT

Quantity of oestriol in 5 ml urine = K x 80 pg.

RESULTS

AND

DISCUSSION

Hydrolysis

The hydrolysis of the oestriol conjugates using acid was investigated to find the optimum conditions for maximum yield of free oestriol, and the influence of glucose on this process. From these experiments the result of varying acid concentration and time of heating is shown in Figs. I and 2. These show that the maximum yield of oestriol, from the hydrolysis of the conjugates, is obtained when 5 ml urine and I ml HCl are Clin. Chim. Acta, 29 (1970) 473-480

RICHARDSON

476

,

1.0 15 05 ml ol cow. HCI added

I

2.0

r

0.5 Duration

I

I.0 of

L

I.5 heating

20 (h)

Fig. I. Influence of increasing amounts of HCl on the hydrolysis of oestriol conjugates contained in 5 ml of pregnancy urine and heated for 45 min. The optimum volume of HCl for maximum yield of free oestriol is 16%. -o-nurine I, -A-aurine 2. Fig. 2. Influence of duration of heating on the hydrolysis of oestriol conjugates contained in 5 ml of pregnancy urine with 16% HCI. The optimum time for maximum yield of free oestriol is 45 min. -.-*urine 3, -x-X - urine 4. -O-Ourine 5.

heated for 45 min. The volume of HCl must be increased to 3 ml when the urine is diluted with IO ml of water, to obtain the same yield (see below). The conditions for acid hydrolysis must be rigidly standardised to achieve reproducible results. For this reason a boiling water bath was used in preference to heating under reflux over a gas or electric heater. It was found that urine sugar (identified by paper chromatography as glucose) reduced the yield of free oestriol during the hydrolysis of the conjugates by an amount proportional to the glucose concentration, as is shown in Fig. 3. These findings are in keeping with the results published by several workers18-14 who suggest making a dilution of the urine in water of I in IO prior to hydrolysis. Fig. 3 shows that a urine glucose concentration of I g% suppresses the recovery of free oestriol from an undiluted urine by about 15%. However, by diluting I part urine (I g% glucose content) with z parts water the suppression is of the order of only 2%. Therefore by making this dilution, urines with a glucose content of up to I g% can be processed without further treatment. This will accommodate most of the urines encountered. Those with a glucose content greater than I g”/o should be fermented with brewer’s yeast until the glucose is reduced to this level, prior to hydrolysis. This can be carried out in less than 2 h. Extraction The ethereal extract of the hydrolysed urine is washed with saturated NaCO, solution, as suggested by Brown 11, to remove the acidic fraction and the traces of carbonate are removed by a water wash, The NaOH wash extracts the phenolic fraction containing the oestrogens. This is then acidified and washed with petroleum ether-toluene mixture to extract the less polar entities which include oestrone. This hydrocarbon wash is included in the scheme to remove any endogenous oestrone SO that a known amount can be added later to act as an internal reference standard. C&L Chim

Acta, zg (1970) 4733480

CHROMATOGRAPHIC

ESTIMATION

iii,,,

,

[L

Concenkation

2 of

3 glucose

4 (g%)

477

OF OESTRIOL

:: 8

40 yg

80 Oestriol

120

lb

added

Fig. 3. The effect of increasing concentrations of glucose on the recovery of oestriol from urine. A similar result is obtained whether the oestriol is initially present in either the conjugated (endogenous) or free state (added). For this experiment the recovery of oestriol is assumed to be IOO~/~ when there is no glucose present. Fig. 4. Relationship between processed and unprocessed oestriol standards. a. -o-onon-extracted standard, b. -x-xextracted standard. a. Shows the quantitative formation of the TMSE derivatives. b. Shows the linear recovery of oestriol after being added in the unconjugated form to a male urine and processed by the complete method.

Unfortunately this wash also removes a significant amount of oestriol, but by washing the hydrocarbon phase with water and then combining this wash with the original aqueous phase, the removal of oestrone is better than 95% while the loss of oestriol is reduced to less than 2%. These levels are then within acceptable limits. Derivative fomation and quantitation When the dry oestrogen extract is heated with a mixture of hexamethyldisilazane and trimethylchlorosilane at 105’ for 15 min in a tightly stoppered tube the formation of the trimethylsilyl ethers of oestrone and oestriol is found to be quantitative and reproducible. There appears to be no advantage in incorporating a solvent in this reaction mixture. The excess reagents are removed using a rotary evaporator, with the tube immersed in a boiling water bath. It was found that the introduction of silylation reagents into the column resulted in permanent detector contamination and loss of sensitivity. The complete removal of these reagents is therefore essential. A solution of the silyl ethers in n-hexane is stable for a minimum of 48 h when contained in a tightly stoppered tube, to exclude moisture, at room temperature. Oestrone is used as an internal reference standard because it has a suitable retention time, is stable under the conditions used and its endogenous concentration in the test solution is known to be very small after the preparative treatment described. Recovery studies Oestriol standard was added to .=,-ml aliquots of a male urine which were then processed as for a pregnancy urine. The results (see Fig. 4) show that the recovery is consistent over the range of the experiment and is approximately 80%.

Clin. Chz’nz. Acta,

29 (1970) 473-480

RICHARDSON

478

Re$roducibility and @ecisio?z A late pregnancy urine was analysed in a series of batches and individually, on a number of separate occasions to a total of 22 analyses. The results are shown in Table I. Table II gives the standard deviation and coefficient of variation for both the pregnancy urine described above and a male urine with oestriol added. The latter one shows an average recovery

TABLE I ANALYSES OF A LATE PREGXANCY NumbevlBatch 1a

2a

3” 4a 5a 62. 7b

Sb gb lob I-lb

TABLE RECOVERY

of 79%.

URINE

MIS d

Number/Batch

51 54 52 52 52 53 52 53 53 55 50

Izb

54 57 57 53 57 53 54 54 56 57 54

I3C I4C l5C 16~ 17d

18d Igd zod 218 22f

II OF OESTRIOL

FROM

MALE

5 ml male urine+ 80 pg oestriol

5 ml pregnancy

urine

AND

PREGNANCY

URINES

Range,w

M&Z%

Standard deviation

Coejkient vaviation

59-66 50-57

63 54

2.2

3.65 3.7

2.0

of (%)

Corm$arison This GLC method was then compared with two other procedures : the methods of BrownIl and Oakey et al.15. The Steroid Laboratory, Royal Victoria Infirmary, Newcastle, kindly undertook the analyses by these two techniques. Figs. 5 and 6

Brown

Oakey et. al. (~19124 h)

(mg124h)

Fig. 5, Correlation between the GLC method and the Brown method. Fig. 6. Correlation between the GLC method and the Oakey method. c&.

Chinz. Acta,

29 (1970)

473-480

CHROMATOGRAPHIC

ESTIMATION

OF OESTRIOL

479

show the results obtained. The arithmetic means of the results are related by the ratios: Brown:GLC:Oakey = 0.8:1:1.7*.

. . .

75 ,‘OE

*..

30

‘..

31

32

33

. .-* *. . *. J.. . :. . . .* . . . . **

34

Week Fig.

7.

thod.

35 of

36

37

. .* . :

38

39

40

Pregnancy

The excretion of oestriol in urine during the lrtter weeks of pregnancy by this GLC me-

Pregnancy urines were obtained from ante-natal clinics and analysed with a view to constructing a normal excretion pattern for this method, results are shown in Fig. 7. The 24-h urines were collected without preservative and analysed within 36 h

of completing the collection.

0 Tome (min)

Fig. 8. GLC trace of a urine extract and standards. El = oestrone; E, = oestniol. The standard E, peak represents 0.4 ,ug and the urine E, peak represents 0.058 ,ug which is equivalent to 2.3 mg/rooo ml 24-hour urine specimen. * It is suggested that the discrepancy between the Oakey method and the other two methods is due to the former being less specific and also because it compensates for losses incurred during the extraction. The Brown and GLC methods do not attempt to take the losses into consideration when calculating the result. C&z. Chim.

Acta,

zg (1970)

473-480

480

RICHARDSON

CONCLUSION

With the conditions described it is possible to measure down to between I-Z mg/Iooo ml z4-hour urine specimen, and up to about 40 mg for a similar volume of urine. Values greater than this can be determined by re-injecting a smaller volume of the derivative solution. A GLC trace obtained from a urine and standard is shown in Fig. 8. It is quite easy, using this procedure, to obtain a result for I or 2 urines within 23 h of receiving the specimens, while a batch of 8 urines can be analysed comfortably by one operator in one working day. ACKNOWLEDGEMENTS

This work constituted part of a MSc. thesis submitted to the University of Newcastle upon Tyne. The author wishes to thank: Professor A. L. Latner and Dr. P. Smith of that University for their interest and also for making available the results quoted in the text; Dr. S. Wray and Mr. F. E. Harper for their interest and encouragement and F. E. H. for critically reading this manuscript; also Dr. P. N. Coleman, Mr. D. W. Briggs and Dr. H. K. B. Geiser who kindly made available the ante-natal specimens. REFERENCES J. S~VIITH, Bull.Johns Hopkins Hosp., 41 (1927) 62. SPIELMAN, M. A. GOLDBERGER AND R. T. FRANK, J. Amer. Med. Assoc., IOI (1933) 266. V. S. SMITH AND 0. W. SMITH, Proc. Sot. Exptl. Biol. Med., 30 (1933) 918. V. S. SMITH AND 0. W. SMITH, J.Clin. Endocrinol.,I (1941)470. 5 G. V. S. SMITH AND 0. W. SMITH, Physiol. Rev., 28 (1948) I. 6 E. DICZFALUSP, Federation Proc., 23 (1964) 791. 7 T. LUUKKAINEN, W. J. A. VANDEN HEUVEL AND E. C. HORNING, Biochim. Biophys. Acta, 62 I 2 3 4

M. F. G. G.

(1962) 153. 8 H. H. WOTIZ AND S. C. CHATTORAJ, Anal. Chew., 36 (1964) 1466. g H. L. YOUSEM AND D. STRUMMER, Amer. J. Obstet. Gynaecol., 88 (1964) 375. IO H. ADLERCREUTZ AND T.LUUKKAINEN,~~ M. B. LIPSETT(E~.),G~~ Chromatography ofsteroids i?zBiologicalFluids, Plenum Press,New York, 1965, pp. 215-228. II J. B. BROWN, Biochem J., 60 (1955) 185. I2 R. HOBKIRK, A. ALFHEIM AND S. BUGGE, J. Clin. Endocrinol. Metab., 19 (1959) 1352. 13 E.S. TAYLOR, A.HAMNER,P.D.BRUNS AND V. E.DROSE, Amer.J.Obstet. Gynaecol.,85 (1963)

IO. I4 H.L.YousEM, 15 R.E.OAKEY,R. 15 (1967) Clin. Chim.

D. SOLOMAN AND D. STRUMMER, Amer.J. A.BRADSHAW,S.S.ECCLES,S.R.STITCHAND

35. Acta,

1-g (1970)

473-480

Obstet. Gynaecol.,g5 (1966) 595. R.F.HEYs, Clin.Chirn.Acta,