- Email: [email protected]
Rapid estimation of oestriol in pregnancy urine by gas-liquid chromatography
Clinica Chimica Acta, 46 (1973) 201-203 0 Elsevier Scientific Publishing Company,
BRIEFTECHNICAL
Amsterdam
- Printed in The Netherlands
201
NOTE
CC* 5591 Rapid Estimation of Oestriol Chromatography
in Pregnancy Urine by Gas-liquid
The advantages of gas-liquid chromatography (G.L.C.) for measuring oestriol in pregnancy urine have been known for some years. Although relatively rapid chromatographic methods have been described 1,2, these often involve time consuming extraction and purification procedures prior to G.L.C. The procedure of Nelson3 involved a relatively short extraction procedure but in our opinion had the following disadvantages: (I) it was necessary to duplicate each test because oestriol was used as an internal standard; (2) the procedure for acetylation was not always reproducible in our hands, (3) relatively large volumes of solvents and reagents were required; (4) the glassware required for this procedure was somewhat cumbersome. Smith and Stitchl, described a method for urinary oestriol in which phenolphthalein replaced oestriol as an internal standard. However, the extraction procedure prior to G.L.C. was more lengthy in the latter method than in Nelson’s procedure. Because of the desireability of having a urinary oestriol assay that is both rapid and accurate, we modified Nelson’s method by scaling down the volumes of reagents used in the extraction procedure by a factor of IO. The method was also altered by eliminating one chloroform wash and two chloroform extraction steps, and an additional saving on time was obtained by using phenolphthalein instead of oestriol, as an internal standard. These modifications resulted in a more simple and accurate procedure for estimating oestriol in pregnancy urine where a single result was obtained in 75 min which is a considerable saving on time compared with previous methods. The day to day reproducibility of the present procedure was also excellent. EXPERIMENTAL All 24-h urine collections were diluted to 2 1 with distilled water prior to analysis. When the 24-h volume was greater than 2 1 it was diluted to 3 1. Acid hydrolysis and extraction were carried out in the same test tube. Five ml of the diluted pregnancy sample was added to a glass tube (20 x 2.9 cm, fitted with a 24129 ground glass socket) followed by I ml concentrated HCl. The tube was tightly capped and placed in a briskly boiling water bath for 30 min. After cooling, I ml of phenolphthalein solution (IO rng% in ethanol) was added to the tube followed by I ml saturated NaOH solution. The alkaline solution was then washed with IO ml chloroform by shaking for 10 sec. Chloroform was removed with a Pasteur pipette attached to a vacuum line and the alkaline solution was again acidified with I ml cont. HCl. Oestrogens in the aqueous phase were extracted with chloroform (2 x 15 ml) by vigorous shaking each time for I min. The chloroform (30 ml) was filtered through anhydrous sodium sulphate into a boiling tube (22 cmx3.0 cm) and evaporated at 64’ over a stream of nitrogen on a Iz-place manifold. One ml acetic anhydride and 0.1 ml cont. HCl were
202
BRIEF TECHNICAL NOTE
added to the residue; the tube was tightly capped and placed in an oven at 115’ for 15 min. Excess reagents were evaporated at 95” over a stream of nitrogen and the residue dissolved in 0.1 ml ethanol, after which 2 ~1 was injected on to the G.L.C. column of a Pye 104 gas chromatograph. Chromatography
Column, glass, 5 ft x 0.25 in; stationary phase, 3% OV-210 on Diatomite “CQ” (J. J.s Chromatography Ltd., King’s Lynn, England) ; carries gas, nitrogen, flow rate 50 ml/min; column temperature, 285”; injection temperature, 295”; detector, flame ionisation. Retention time for oestriol triacetate, 6 min; retention time for phenolphthalein, 8 min. Excellent chromatographic separation was achieved using a 5-ft column packed with 3% OV-210 on Diatomite CQ. Both the oestriol triacetate and phenolphthalein diacetate peaks were well separated from all other peaks which appeared in the 250 urine samples assayed to date. The short retention times for each substance on the 5-ft column are an advantage.
The ratio, peak height oestriol/peak height internal standard, was calculated, and the amount of oestriol in the 5-ml aliquot of diluted urine was determined by reference to a calibration curve constructed from ratios obtained by adding known
Fig. I. Oestriol calibration curve. Means (0) and their standard deviations (I), for ratios obtained by adding various known amounts of oestriol to s-ml aliquots of non-pregnancy urines after acid hydrolysis are shown (1~= 30).
amounts of oestriol and internal standard to 5-ml aliquots of non-pregnancy urine after hydrolysis (Fig. I). Oestriol per 5 ml diluted urine~number of 5-ml aliquots in total volume (diluted) = oestriol per 24 h. Reproducibility
Aliquots of three pregnancy urines were stored at -20’ and assays were carried out on each urine over a period of 30 days. Results are shown in Table 1.
203
BRIEFTECHNICALNOTE
Acc~acy
The accuracy of the method was tested by diluting a pregnancy urine to varying degrees with non-pregnancy urine and assaying each dilution for oestriol. When oestriol values were plotted against per cent dilution, a straight line through the origin was obtained. TABLE
I
REPRODUCIBILITY Specimen
I 2 3
Number assays 6 6 6
OFTHE
of
METHOD
Mean value
Range
(mg/24
(mgl+
II.7 6.7 30.1
hJ
h)
I2.2-1I.Z 7.1- 6.3 31.7-28.9
Standard deviation
Coejicient of variation
0.361 0.330 0.90
3.085 4.925 2.990
(Oh)
CONCLUSION
A rapid G.L.C. method for the assay of oestriol in pregnancy urine has been described. The method has been in routine use in this laboratory for almost 12 months with excellent results. ACKNOWLEDGEMENT
We are grateful to Professor E. O’Dwyer and his staff in the Department of Obstetrics and Gynecology for their help. D. W. NUGENT P. F. FOTTRELL
Department of Biochernist~y, University College, Galway (Ireland) I S. J. RICHARDSON, Clin. Chim. Acta, zg (1970) 473. 2 P. D. SMITH AND S. R. STITCH, Clin. Chim. Acta, 36 (1972) 3 G. H. NELSON, Am. J. Obstet. Gynecol., 106 (1970) 649.
Received December 18, Igp
439.
BRIEFTECHNICAL
Amsterdam
- Printed in The Netherlands
201
NOTE
CC* 5591 Rapid Estimation of Oestriol Chromatography
in Pregnancy Urine by Gas-liquid
The advantages of gas-liquid chromatography (G.L.C.) for measuring oestriol in pregnancy urine have been known for some years. Although relatively rapid chromatographic methods have been described 1,2, these often involve time consuming extraction and purification procedures prior to G.L.C. The procedure of Nelson3 involved a relatively short extraction procedure but in our opinion had the following disadvantages: (I) it was necessary to duplicate each test because oestriol was used as an internal standard; (2) the procedure for acetylation was not always reproducible in our hands, (3) relatively large volumes of solvents and reagents were required; (4) the glassware required for this procedure was somewhat cumbersome. Smith and Stitchl, described a method for urinary oestriol in which phenolphthalein replaced oestriol as an internal standard. However, the extraction procedure prior to G.L.C. was more lengthy in the latter method than in Nelson’s procedure. Because of the desireability of having a urinary oestriol assay that is both rapid and accurate, we modified Nelson’s method by scaling down the volumes of reagents used in the extraction procedure by a factor of IO. The method was also altered by eliminating one chloroform wash and two chloroform extraction steps, and an additional saving on time was obtained by using phenolphthalein instead of oestriol, as an internal standard. These modifications resulted in a more simple and accurate procedure for estimating oestriol in pregnancy urine where a single result was obtained in 75 min which is a considerable saving on time compared with previous methods. The day to day reproducibility of the present procedure was also excellent. EXPERIMENTAL All 24-h urine collections were diluted to 2 1 with distilled water prior to analysis. When the 24-h volume was greater than 2 1 it was diluted to 3 1. Acid hydrolysis and extraction were carried out in the same test tube. Five ml of the diluted pregnancy sample was added to a glass tube (20 x 2.9 cm, fitted with a 24129 ground glass socket) followed by I ml concentrated HCl. The tube was tightly capped and placed in a briskly boiling water bath for 30 min. After cooling, I ml of phenolphthalein solution (IO rng% in ethanol) was added to the tube followed by I ml saturated NaOH solution. The alkaline solution was then washed with IO ml chloroform by shaking for 10 sec. Chloroform was removed with a Pasteur pipette attached to a vacuum line and the alkaline solution was again acidified with I ml cont. HCl. Oestrogens in the aqueous phase were extracted with chloroform (2 x 15 ml) by vigorous shaking each time for I min. The chloroform (30 ml) was filtered through anhydrous sodium sulphate into a boiling tube (22 cmx3.0 cm) and evaporated at 64’ over a stream of nitrogen on a Iz-place manifold. One ml acetic anhydride and 0.1 ml cont. HCl were
202
BRIEF TECHNICAL NOTE
added to the residue; the tube was tightly capped and placed in an oven at 115’ for 15 min. Excess reagents were evaporated at 95” over a stream of nitrogen and the residue dissolved in 0.1 ml ethanol, after which 2 ~1 was injected on to the G.L.C. column of a Pye 104 gas chromatograph. Chromatography
Column, glass, 5 ft x 0.25 in; stationary phase, 3% OV-210 on Diatomite “CQ” (J. J.s Chromatography Ltd., King’s Lynn, England) ; carries gas, nitrogen, flow rate 50 ml/min; column temperature, 285”; injection temperature, 295”; detector, flame ionisation. Retention time for oestriol triacetate, 6 min; retention time for phenolphthalein, 8 min. Excellent chromatographic separation was achieved using a 5-ft column packed with 3% OV-210 on Diatomite CQ. Both the oestriol triacetate and phenolphthalein diacetate peaks were well separated from all other peaks which appeared in the 250 urine samples assayed to date. The short retention times for each substance on the 5-ft column are an advantage.
The ratio, peak height oestriol/peak height internal standard, was calculated, and the amount of oestriol in the 5-ml aliquot of diluted urine was determined by reference to a calibration curve constructed from ratios obtained by adding known
Fig. I. Oestriol calibration curve. Means (0) and their standard deviations (I), for ratios obtained by adding various known amounts of oestriol to s-ml aliquots of non-pregnancy urines after acid hydrolysis are shown (1~= 30).
amounts of oestriol and internal standard to 5-ml aliquots of non-pregnancy urine after hydrolysis (Fig. I). Oestriol per 5 ml diluted urine~number of 5-ml aliquots in total volume (diluted) = oestriol per 24 h. Reproducibility
Aliquots of three pregnancy urines were stored at -20’ and assays were carried out on each urine over a period of 30 days. Results are shown in Table 1.
203
BRIEFTECHNICALNOTE
Acc~acy
The accuracy of the method was tested by diluting a pregnancy urine to varying degrees with non-pregnancy urine and assaying each dilution for oestriol. When oestriol values were plotted against per cent dilution, a straight line through the origin was obtained. TABLE
I
REPRODUCIBILITY Specimen
I 2 3
Number assays 6 6 6
OFTHE
of
METHOD
Mean value
Range
(mg/24
(mgl+
II.7 6.7 30.1
hJ
h)
I2.2-1I.Z 7.1- 6.3 31.7-28.9
Standard deviation
Coejicient of variation
0.361 0.330 0.90
3.085 4.925 2.990
(Oh)
CONCLUSION
A rapid G.L.C. method for the assay of oestriol in pregnancy urine has been described. The method has been in routine use in this laboratory for almost 12 months with excellent results. ACKNOWLEDGEMENT
We are grateful to Professor E. O’Dwyer and his staff in the Department of Obstetrics and Gynecology for their help. D. W. NUGENT P. F. FOTTRELL
Department of Biochernist~y, University College, Galway (Ireland) I S. J. RICHARDSON, Clin. Chim. Acta, zg (1970) 473. 2 P. D. SMITH AND S. R. STITCH, Clin. Chim. Acta, 36 (1972) 3 G. H. NELSON, Am. J. Obstet. Gynecol., 106 (1970) 649.
Received December 18, Igp
439.