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Gas chromatographic analysis of estrogens
OBSTETRICS Gas chromatographic analysis of estrogens KEI HONDA* DONALD OSTERGARD STANLEY KUSHINSKY** Torrance, California A rapid gas chromatographic method is described for the measurement of estriol in the urine of females in or near the third trimester of pregnancy.
A N u M B E R of methods have been reported for estimating the concentration of estriol in the urine of females in the second or third trimester of pregnancy. 1 - 6 The methods may be classified as relatively specific or relatively unspecific, depending on whether a chromatographic purification is employed in which estriol is separated from at least some other estrogens. Regardless of the specificity of the methods, the concentration in urine of what is alleged to be estriol invariably is reported to increase during the course of normal pregnancy, and there is general agreement that one indication of fetal distress is an abrupt decrease in the daily urinary excretion of estrioL The study reported here was undertaken in order to develop a routine method, compatible with existing facilities in our labora-
tory, for estimating the concentration of estriol in late pregnancy urine. In this paper are described the details of the method and the experiments performed to determine the validity of the method.
Method and material Reagents and solvents. All reagents and solvents were of analytical grade except for acetone which was "Nanograde" (Mallinckrodt). No additional purification was done. The sources of the following chemicals are designated in parentheses: Estriol (ParkeDavis & Company), 14C-estriol-16a-glucuronide (Dr. Mortimer Levitz, Department of Obstetrics and Gynecology, New York University School of Medicine). Heptafluorobutyric anhydride was prepared from the acid (3M Company, St. Paul, Minnesota) as described by Exley. 7 Gas chromatograph. A Packard Instruments Company (Downers Grove, Illinois) Model 7800 gas chromatograph was employed in these studies. The instrument was equipped with two columns, two electron capture detectors, and a W estronics Model DIIA dual-pen recorder. Coiled glass columns 2 mm. x 180 em. were packed with Gas Chrom Q (Applied Science) coated with
From the Department of Obstetrics and Gynecology, Division of Reproductive Biology, Harbor General Hospital, Torrance, and the UCLA Schoo~ of Medicine. *Present address: Department of Obstetrics and Gynecology, Niigata University School of Medicine, Asahi-Machi, Niigata City, Japan. **Address for rePrint requests: 1000 West Carson Street, Torrance, California 90509.
528
Volume 104 Number 4
Gas chromatography of estrogens
529
XE-tiO ( 2 per cent w/ w) . Carrier gas, "prepurified" nitrogen, was passed through a mixed molecular sieve (4A and 13X) for additional purification. The flow rate was adjusted to 60 mi. per minute through each column. The temperatures employed are as follows: Column, 190° C.; injector, 195° C.; and detector 200° C. Under these conditions the retention time for estriol heptafluorobutyrate is approximately 7 minutes. Preparation of derivative.* A known quantity of estriol or the extract of late pregnancy urine is dissolved in 0.1 mi. of acetone and 0.05 mi. of heptafluorobutyric anhydride is added. After 10 minutes at room temperature the solution is evaporated to dryness under a gentle stream of nitrogen at 40° C. Measurements of radioactivity. A NuclearChicago scintillation counter, Model 6860, operated at settings optimized for counting by the channels ratio method was employed in these studies. The scintillation solvent used was that described by Bruno and Christian9 except that the concentration of naphthalene was 7 per cent instead of 5 per cent. To insure solubility, 0.5 mi. of methanol was added to the dried sample in the counting vial prior to adding the scintillation solvent. Method in detail. A 24 hour specimen of urine is diluted to 2 liters. To two 50 mi. centrifuge tubes are added a known quantity of estriol-16a-glucuronide-16-C 14 ( 40 me. per milligram). Aliquots of 1.5 and 3.0 mi. of urine then are transferred to the centrifuge tubes and the volume of each tube is increased to 7 mi. with water. If the volume of the original specimen is greater than 2 liters, it is diluted to the nearest integral multiple of 1 liter and the dilution to 7 mi. is modified so that the fractions of the 24 hour urine analyzed remain constant regardless of the original volume of urine. To each centrifuge tube is added 1.2 mi. of 12N HOI and the tubes are heated in an aluminum pressure cooker at 107° C. for one hour. After cooling to room temperature the urine is adjusted to pH 11.5 with saturated NaOH. The aqueous phase is washed once with an
equal volume of toluene and the toluene is discarded. One milliliter of saturated NaHC0 3 solution is added and the aqueous phase is adjusted to pH 8 with 12N H2S04. The estrogens then are extracted from the aqueous phase with 3 x Yz volume of ethyl acetate. The combined ethyl acetate phase is washed with 2 x 7io volume of water and evaporated to dryness in vacuo. (As many as 12 specimens may be evaporated simultaneously by means of two six-port manifolds designed for this purpose.) The residue is dissolved in 10 mi. of acetone and 4 mi. is removed for measurement of the recovery of radioactivity. Another aliquot (0.1 or 0.2 mi.) is removed for reaction with heptafluorobutyric anhydride under the conditions described previously. (The smaller aliquot is taken for the 3.0 mi. specimen of urine and the larger for the 1.5 ml. specimen of urine.) After evaporation of the volatile components under a stream of nitrogen the residue is dissolved in 0.2 mi. of acetone and 2 ~-tl is applied to the column. The area under the peak corresponding to estriol heptafluorobutyrate after correction for the aliquots taken and the recovery of radioactivity is used to determine the quantity of estriol present in the 24 hour specimen of urine. The area under the peak is determined by multiplying the height of the peak by the width at half-height. Quantitation is achieved by means of a daily prepared plot of area under the peak versus known quanti-
*The procedure used was developed by M. Hiroi and S. Kushinsky. 8
Fig. 1. Gas chromatography of urinary estriol (as the heptafluorobutyrate).
a: 0
1-
uw
E3
1-
w
0
LL.
0 w
z
0
a.
w
a:
10 20 0 10 RETENTION TIME (MINUTES)
20
530 Honda, Ostergard, and Kushinsky
Am .
Table I. Replicate analysis of urinary estriol performed on the same day --------
---------·--·-~-----
Estriol (mg./24 hr.) Specimen lA IB IC
ID
lE IF Mean Range
S.D. 2A 2B 2C
:m 2E 2F Mean Range S.D. 3A 3B
3C 3D
3E 3F Mean Range S.D.
Co!umn I
Column 2
Mean
18.3 17.0 19.0 18.1 16.6 16.9 17.6 16.6-19.0
18.4 1~ ') '-~ 18.9 18.3 16.5 16.8 17.6 16.5-18.9
18.4 17.1 19.0
1.7
:.!.!
8.6 6.9 s. 7 S.5 7.0 7.8 7.9 6.9-8.6 0.9 2.6 2.6 2.5 2.4 2.4 2.6 2.5 2.4-2.6 0.3
8.:{ 6.8 ll.5 8.6 7.0
H 7.7 6.8-8.6
1.3 2.5 2.5 2.4 2.4 :!.3 2.5 2.4 2.:>-2.5 11.4
18.~
16.6 16.9 17.7 16.6-19.0 1.0 8.5 6.9 8.6 8.6 7.fl 7.6 7.8 6.9-8.6
1.3 2.6 2.6 2.5 2.4 2.4 2.6 2.5 2.4-2.6 0.3
-----
ties of estriol which are reacted with heptafiuorobutyric anhydride. Two tracings of typical chromatograms are shown in Fig. 1. Variation of replicate analyses. Three specimens of urine were analyzed in sextuplicate on the same day. Gas chromatography of each sample was done on two columns. The results, mean values, ranges, and standard deviations are summarized in Table I. In each case there are only small variations among the replicate values. Analysis on successive days. Three specimens of urine were analyzed in duplicate on 3 successive days. The values obtained the second day of analysis are somewhat lower than those of the first and third days, but the magnitude of the differences is not considered excessive for the clinical use of this assay which is contemplated. The results arc summarized in Table II.
June 15, 1969 & Gynec.
.f. Obst.
Recovery of added estriol and estriol-16aglucuronide-16-C14. Estriol-16a-glucuronide16-C14 was added to four centrifuge tubes containing 10 J.Lg each of estriol, four containing 20 p.g of estriol, and four containing no estriol. Three milliliters of a pooled specimen of late pregnancy urine was added to each and the analysis of estriol was carried out in the usual manner. The recovery of radioactivity up to the step in which the heptafluorobu tyrates are prepared and the quantity of estriol found in each specimen are summarized in Table III. The average recovery of radioactivity was 76.0 per cent, while the average recovery of added estriol was 71 per cent and 73 per cent, respectively, when 10 and 20 J.Lg were added. The reCO\,.eries of added estriol have been corrected for procedural losses of radioactivity. Comment
It is not generally appreciated that the concentration of estriol found in urine after hydrolytic conditions does not necessarily reflect accurately the total concentration of estriol (free and combined) present in the intact urine. Correction for procedural losses by the addition of isotope-labeled estriol, one of the glucuronides, or a mixture of conjugates of estriol and measurement of the recovery of radioactivity at or close to the end of the procedure involve a number of questionable assumptions. The extent of the invalidity depends on many factors. Among these are whether the labeled estriol is added as the free compound or as one or more of the conjugated forms, the composition of the added mixture (if a mixture) and the endogenous mixture, differences in the stability of estriol and its conjugated forms to the conditions of the analysis, at what step the isotope is added, at what step the recovery is determined, and whether the measurement of radioactivity represents the content of labeled intact estriol rather than a decomposition product of estriol with the isotope intact. In procedures wherein no chromatography is employed, there is no way to determine whether the recovery of radioactivity reflects the recovery
Volume 104 Number 4
Gas chromatography of estrogens
531
Table II. Replicate analysis of urinary estriol performed on successive days Estriol (mg./24 hr.) Analysis 2
Analysis 1 Specimen
Column 1
4A 4B Mean
20.5 19.3
SA 5B Mean
10.2 9.5
6A 6B Mean
12.7 12.2
I
Column 2
19.7 18.5 10.3 9.7 12.3 12.5
I
Col~
Mean
umn 1
20.1 18.9 19.5
18.2 17.3
10.3 9.6 10.0
7.5 7.1
12.5 12.4 12.5
10.5 10.3
I
Col~
umn 2
I
18.4 17.3 7.5 7.6 10.0 9.6
Analysis 3
Col~
Mean
umn 1
18.3 17.3 17.8
21.2 19.3
7.5 7.4 7.5
8.8 9.1
10.3 10.0 10.2
11.5 12.1
I
CoZ.. umn 2
21.0 19.1 9.0 9.3 11.6 11.6
I
Mean
Mean
Range
S.D.
21.1 19.2 20.2
19.1
1 7.8-20.2
2.3
8.9 9.2 9.1
8.8
7.5-10.0
1.8
11.5 10.2-12.5
1.1
11.6 11.9 11.8
Table III. Recovery of estriol and estriol-16-C 14 -16a-glucuronide added to late pregnancy unne
Specimen
Estriol added (p.g)
Recovery of C14 (%)
Determined content of estriol* Column 1
I Column
2
I
Mean
Recovery of added estriol (%)*
7A 7B 7C 7D Mean
0 0 0 0
76.9 77.5 77.8 79.3
42.3 40.1 40.4 39.7
41.1 40.3 41.4 38.5
41.7 40.2 40.9 39.1 40.5
8A 8B 8C 8D Mean
10 10 10 10
73.4 76.2 74.8 76.4
50.1 46.6 46.7 46.7
49.6 47.1 46.5 47.2
49.8 46.9 46.6 47.0 47.6
70.8± 14.9 (S.D.)
9A 9B 9C 9D Mean
20 20 20 20
53.8 55.9 54.4 56.4 55.1
73.2± 5.7 (S.D.)
74.8 77.7 74.6 73.0 76.0 ± 2.1 (S.D.)
53.5 56.6 55.0 56.0
54.0 55.2 53.8 56.8
*Corrected for recovery of C 14 •
of radioactive structurally intact estriol. Where no chromatography other than vapor phase is employed and the recovery of radioactivity is determined prior to chromatography rather than by means of a stream splitter* in the effluent the validity of the correction for procedural losses is equally ambiguous. Even when recovery of radioactivity is determined after a chromatographic procedure, prior to quantitative measurement, such as by means of a *Some preliminary experiments of this type have been carried out in our laboratory but a satisfactory solution is as yet unavailable (J. Coyotupa and S. Kushinsky, unpublished observations).
color test, there is the possibility that decomposition occurs between the time the resolution is achieved and the time that the measurement is made. Preliminary experiments with sequential chromatography may eliminate most of such uncertainty. But, only rarely is sequential chromatography even considered. 11 The recovery of estriol in the experiments described herein may be considered in any of several perspectives. Clearly, the one with the fewest. assumptions leads to the conclusion that the average recovery of estriol was only 55 per cent. This is based on the gas
532 Honda, Ostergard, and Kushinsky
chromatographically determined recovery without correction for loss of radioactivity. The apparent inconsistency between the recovery of radioactivity and of gas chromatographically identified estriol is likely to be a result of a stabilizing effect of the glucuronide during the treatment with hot acid. Any hypothetical decomposition product of estriol must be presumed to be extractable by ethyl acetate, yet gas chromatographically different from estriol to be compatible with the observed results. It is likely that the estriol is excreted in urine principally as the 16a-glucuronide2 • 12 although in some subjects the 3-glucuronide and possibly some other more complex conjugates may be more abundant. Consequently, it is more reasonable to use the 16a-glucuronide of radioactive estriol to check for procedural losses than to use the free compound. The specificity achieved with the method reported here is considered by us to be adequate in light of the purpose for which the method was developed. Among the factors considered before deciding that the specificity is adequate were the following: 1. Estrone and estradiol and probably many other estrogens are readily separated from estriol gas chromatographically as the heptafluorobu tyrates. 2. The pen of the recorder was close to the baseline except for the solvent peak and the peak associated with estriol. 3. Estriol is known to be the most abundant estrogen present in late pregnancy urine and potentially interfering substances are
June 15, 1969 Am. J. Obst. & Gynec.
likely to be separated during the preliminary partitioning procedures or on the gas chromatographic column. 4. Additional purification by thin-layer or other chromatographic procedures was expected to add to the time required for analysis without significant improvement in the specificity. The additional time also would have precluded completion of a reasonable number of assays in a single working day and thereby have defeated a major purpose of the assay; namely, a rapid, clinically useful procedure. The reproducibility of the assay was shown to be very satisfactory both with replicate measurements on the same day and on successive days. The latter situation is particularly relevant since it is generally considered that a precipitous decrease in the concentration of estriol in urine on 2 successive days is indicative of fetal distress. 2 The absolute accuracy of the method described here is not subject to clear definition or verification for reasons discussed earlier. We consider it likely that the values obtained reflect reasonably well the concentration of estriol present in late pregnancy urine. The authors are grateful to the 3M Company (St. Paul, Minnesota) for providing heptafluorobutyric acid and to Mrs. Frances Nakamura for assistance in one phase of this work. The investigation was supported in part by a NIH-GRS grant from the Attending Staff Association, Harbor General Hospital, NIH Grants AM-09908 and RCDA 5K3-AM-31,321 (SK), an ACS Institutional Research Grant (UCLA), and a grant from The Ford Foundation.
REFERENCES
1. Brown, J. B., and Coyle, M. G.: J. Obst. & Gynaec. Brit. Emp. 60: 219, 1963. 2. Beling, C. G.: Acta endocrinol., Suppl. 79, 1963. 3. Yousem, H. L., and Strummer, D.: AM. J. 0BST. & GYNEC. 88: 375, 1964. 4. Schindler, A. E., and Herrmann, W. L.: Gynaecologia 161: 446, 1960. 5. Jaffe, S. H., and Levitz, M.: AM. J. OBsT. & GYNEC. 98: 992, 1967. 6. Frandsen, V. A.: The Excretion of Oestriol in Normal Human Pregnancy, Copenhagen, 1963, Ejnar Munksgaard Forlag.
7. Exley, D.: Memoirs Soc. Endocrinol. 16: 117, 1966. 8. Hiroi, M., and Kushinsky, S.: Submitted for publication. 9. Bruno, G. A., and Christian, J. E.: Anal. Chern. 33: 1216, 1961. 10. Honda, K., and Kushinsky, S.: Submitted for publication. II. Idler, D. R., Kimball, N. R., and Truscott, B.: Steroids 8: 865, 1966. 12. Hahne), R. J.: Endocrinology 38: 417, 1967.
A N u M B E R of methods have been reported for estimating the concentration of estriol in the urine of females in the second or third trimester of pregnancy. 1 - 6 The methods may be classified as relatively specific or relatively unspecific, depending on whether a chromatographic purification is employed in which estriol is separated from at least some other estrogens. Regardless of the specificity of the methods, the concentration in urine of what is alleged to be estriol invariably is reported to increase during the course of normal pregnancy, and there is general agreement that one indication of fetal distress is an abrupt decrease in the daily urinary excretion of estrioL The study reported here was undertaken in order to develop a routine method, compatible with existing facilities in our labora-
tory, for estimating the concentration of estriol in late pregnancy urine. In this paper are described the details of the method and the experiments performed to determine the validity of the method.
Method and material Reagents and solvents. All reagents and solvents were of analytical grade except for acetone which was "Nanograde" (Mallinckrodt). No additional purification was done. The sources of the following chemicals are designated in parentheses: Estriol (ParkeDavis & Company), 14C-estriol-16a-glucuronide (Dr. Mortimer Levitz, Department of Obstetrics and Gynecology, New York University School of Medicine). Heptafluorobutyric anhydride was prepared from the acid (3M Company, St. Paul, Minnesota) as described by Exley. 7 Gas chromatograph. A Packard Instruments Company (Downers Grove, Illinois) Model 7800 gas chromatograph was employed in these studies. The instrument was equipped with two columns, two electron capture detectors, and a W estronics Model DIIA dual-pen recorder. Coiled glass columns 2 mm. x 180 em. were packed with Gas Chrom Q (Applied Science) coated with
From the Department of Obstetrics and Gynecology, Division of Reproductive Biology, Harbor General Hospital, Torrance, and the UCLA Schoo~ of Medicine. *Present address: Department of Obstetrics and Gynecology, Niigata University School of Medicine, Asahi-Machi, Niigata City, Japan. **Address for rePrint requests: 1000 West Carson Street, Torrance, California 90509.
528
Volume 104 Number 4
Gas chromatography of estrogens
529
XE-tiO ( 2 per cent w/ w) . Carrier gas, "prepurified" nitrogen, was passed through a mixed molecular sieve (4A and 13X) for additional purification. The flow rate was adjusted to 60 mi. per minute through each column. The temperatures employed are as follows: Column, 190° C.; injector, 195° C.; and detector 200° C. Under these conditions the retention time for estriol heptafluorobutyrate is approximately 7 minutes. Preparation of derivative.* A known quantity of estriol or the extract of late pregnancy urine is dissolved in 0.1 mi. of acetone and 0.05 mi. of heptafluorobutyric anhydride is added. After 10 minutes at room temperature the solution is evaporated to dryness under a gentle stream of nitrogen at 40° C. Measurements of radioactivity. A NuclearChicago scintillation counter, Model 6860, operated at settings optimized for counting by the channels ratio method was employed in these studies. The scintillation solvent used was that described by Bruno and Christian9 except that the concentration of naphthalene was 7 per cent instead of 5 per cent. To insure solubility, 0.5 mi. of methanol was added to the dried sample in the counting vial prior to adding the scintillation solvent. Method in detail. A 24 hour specimen of urine is diluted to 2 liters. To two 50 mi. centrifuge tubes are added a known quantity of estriol-16a-glucuronide-16-C 14 ( 40 me. per milligram). Aliquots of 1.5 and 3.0 mi. of urine then are transferred to the centrifuge tubes and the volume of each tube is increased to 7 mi. with water. If the volume of the original specimen is greater than 2 liters, it is diluted to the nearest integral multiple of 1 liter and the dilution to 7 mi. is modified so that the fractions of the 24 hour urine analyzed remain constant regardless of the original volume of urine. To each centrifuge tube is added 1.2 mi. of 12N HOI and the tubes are heated in an aluminum pressure cooker at 107° C. for one hour. After cooling to room temperature the urine is adjusted to pH 11.5 with saturated NaOH. The aqueous phase is washed once with an
equal volume of toluene and the toluene is discarded. One milliliter of saturated NaHC0 3 solution is added and the aqueous phase is adjusted to pH 8 with 12N H2S04. The estrogens then are extracted from the aqueous phase with 3 x Yz volume of ethyl acetate. The combined ethyl acetate phase is washed with 2 x 7io volume of water and evaporated to dryness in vacuo. (As many as 12 specimens may be evaporated simultaneously by means of two six-port manifolds designed for this purpose.) The residue is dissolved in 10 mi. of acetone and 4 mi. is removed for measurement of the recovery of radioactivity. Another aliquot (0.1 or 0.2 mi.) is removed for reaction with heptafluorobutyric anhydride under the conditions described previously. (The smaller aliquot is taken for the 3.0 mi. specimen of urine and the larger for the 1.5 ml. specimen of urine.) After evaporation of the volatile components under a stream of nitrogen the residue is dissolved in 0.2 mi. of acetone and 2 ~-tl is applied to the column. The area under the peak corresponding to estriol heptafluorobutyrate after correction for the aliquots taken and the recovery of radioactivity is used to determine the quantity of estriol present in the 24 hour specimen of urine. The area under the peak is determined by multiplying the height of the peak by the width at half-height. Quantitation is achieved by means of a daily prepared plot of area under the peak versus known quanti-
*The procedure used was developed by M. Hiroi and S. Kushinsky. 8
Fig. 1. Gas chromatography of urinary estriol (as the heptafluorobutyrate).
a: 0
1-
uw
E3
1-
w
0
LL.
0 w
z
0
a.
w
a:
10 20 0 10 RETENTION TIME (MINUTES)
20
530 Honda, Ostergard, and Kushinsky
Am .
Table I. Replicate analysis of urinary estriol performed on the same day --------
---------·--·-~-----
Estriol (mg./24 hr.) Specimen lA IB IC
ID
lE IF Mean Range
S.D. 2A 2B 2C
:m 2E 2F Mean Range S.D. 3A 3B
3C 3D
3E 3F Mean Range S.D.
Co!umn I
Column 2
Mean
18.3 17.0 19.0 18.1 16.6 16.9 17.6 16.6-19.0
18.4 1~ ') '-~ 18.9 18.3 16.5 16.8 17.6 16.5-18.9
18.4 17.1 19.0
1.7
:.!.!
8.6 6.9 s. 7 S.5 7.0 7.8 7.9 6.9-8.6 0.9 2.6 2.6 2.5 2.4 2.4 2.6 2.5 2.4-2.6 0.3
8.:{ 6.8 ll.5 8.6 7.0
H 7.7 6.8-8.6
1.3 2.5 2.5 2.4 2.4 :!.3 2.5 2.4 2.:>-2.5 11.4
18.~
16.6 16.9 17.7 16.6-19.0 1.0 8.5 6.9 8.6 8.6 7.fl 7.6 7.8 6.9-8.6
1.3 2.6 2.6 2.5 2.4 2.4 2.6 2.5 2.4-2.6 0.3
-----
ties of estriol which are reacted with heptafiuorobutyric anhydride. Two tracings of typical chromatograms are shown in Fig. 1. Variation of replicate analyses. Three specimens of urine were analyzed in sextuplicate on the same day. Gas chromatography of each sample was done on two columns. The results, mean values, ranges, and standard deviations are summarized in Table I. In each case there are only small variations among the replicate values. Analysis on successive days. Three specimens of urine were analyzed in duplicate on 3 successive days. The values obtained the second day of analysis are somewhat lower than those of the first and third days, but the magnitude of the differences is not considered excessive for the clinical use of this assay which is contemplated. The results arc summarized in Table II.
June 15, 1969 & Gynec.
.f. Obst.
Recovery of added estriol and estriol-16aglucuronide-16-C14. Estriol-16a-glucuronide16-C14 was added to four centrifuge tubes containing 10 J.Lg each of estriol, four containing 20 p.g of estriol, and four containing no estriol. Three milliliters of a pooled specimen of late pregnancy urine was added to each and the analysis of estriol was carried out in the usual manner. The recovery of radioactivity up to the step in which the heptafluorobu tyrates are prepared and the quantity of estriol found in each specimen are summarized in Table III. The average recovery of radioactivity was 76.0 per cent, while the average recovery of added estriol was 71 per cent and 73 per cent, respectively, when 10 and 20 J.Lg were added. The reCO\,.eries of added estriol have been corrected for procedural losses of radioactivity. Comment
It is not generally appreciated that the concentration of estriol found in urine after hydrolytic conditions does not necessarily reflect accurately the total concentration of estriol (free and combined) present in the intact urine. Correction for procedural losses by the addition of isotope-labeled estriol, one of the glucuronides, or a mixture of conjugates of estriol and measurement of the recovery of radioactivity at or close to the end of the procedure involve a number of questionable assumptions. The extent of the invalidity depends on many factors. Among these are whether the labeled estriol is added as the free compound or as one or more of the conjugated forms, the composition of the added mixture (if a mixture) and the endogenous mixture, differences in the stability of estriol and its conjugated forms to the conditions of the analysis, at what step the isotope is added, at what step the recovery is determined, and whether the measurement of radioactivity represents the content of labeled intact estriol rather than a decomposition product of estriol with the isotope intact. In procedures wherein no chromatography is employed, there is no way to determine whether the recovery of radioactivity reflects the recovery
Volume 104 Number 4
Gas chromatography of estrogens
531
Table II. Replicate analysis of urinary estriol performed on successive days Estriol (mg./24 hr.) Analysis 2
Analysis 1 Specimen
Column 1
4A 4B Mean
20.5 19.3
SA 5B Mean
10.2 9.5
6A 6B Mean
12.7 12.2
I
Column 2
19.7 18.5 10.3 9.7 12.3 12.5
I
Col~
Mean
umn 1
20.1 18.9 19.5
18.2 17.3
10.3 9.6 10.0
7.5 7.1
12.5 12.4 12.5
10.5 10.3
I
Col~
umn 2
I
18.4 17.3 7.5 7.6 10.0 9.6
Analysis 3
Col~
Mean
umn 1
18.3 17.3 17.8
21.2 19.3
7.5 7.4 7.5
8.8 9.1
10.3 10.0 10.2
11.5 12.1
I
CoZ.. umn 2
21.0 19.1 9.0 9.3 11.6 11.6
I
Mean
Mean
Range
S.D.
21.1 19.2 20.2
19.1
1 7.8-20.2
2.3
8.9 9.2 9.1
8.8
7.5-10.0
1.8
11.5 10.2-12.5
1.1
11.6 11.9 11.8
Table III. Recovery of estriol and estriol-16-C 14 -16a-glucuronide added to late pregnancy unne
Specimen
Estriol added (p.g)
Recovery of C14 (%)
Determined content of estriol* Column 1
I Column
2
I
Mean
Recovery of added estriol (%)*
7A 7B 7C 7D Mean
0 0 0 0
76.9 77.5 77.8 79.3
42.3 40.1 40.4 39.7
41.1 40.3 41.4 38.5
41.7 40.2 40.9 39.1 40.5
8A 8B 8C 8D Mean
10 10 10 10
73.4 76.2 74.8 76.4
50.1 46.6 46.7 46.7
49.6 47.1 46.5 47.2
49.8 46.9 46.6 47.0 47.6
70.8± 14.9 (S.D.)
9A 9B 9C 9D Mean
20 20 20 20
53.8 55.9 54.4 56.4 55.1
73.2± 5.7 (S.D.)
74.8 77.7 74.6 73.0 76.0 ± 2.1 (S.D.)
53.5 56.6 55.0 56.0
54.0 55.2 53.8 56.8
*Corrected for recovery of C 14 •
of radioactive structurally intact estriol. Where no chromatography other than vapor phase is employed and the recovery of radioactivity is determined prior to chromatography rather than by means of a stream splitter* in the effluent the validity of the correction for procedural losses is equally ambiguous. Even when recovery of radioactivity is determined after a chromatographic procedure, prior to quantitative measurement, such as by means of a *Some preliminary experiments of this type have been carried out in our laboratory but a satisfactory solution is as yet unavailable (J. Coyotupa and S. Kushinsky, unpublished observations).
color test, there is the possibility that decomposition occurs between the time the resolution is achieved and the time that the measurement is made. Preliminary experiments with sequential chromatography may eliminate most of such uncertainty. But, only rarely is sequential chromatography even considered. 11 The recovery of estriol in the experiments described herein may be considered in any of several perspectives. Clearly, the one with the fewest. assumptions leads to the conclusion that the average recovery of estriol was only 55 per cent. This is based on the gas
532 Honda, Ostergard, and Kushinsky
chromatographically determined recovery without correction for loss of radioactivity. The apparent inconsistency between the recovery of radioactivity and of gas chromatographically identified estriol is likely to be a result of a stabilizing effect of the glucuronide during the treatment with hot acid. Any hypothetical decomposition product of estriol must be presumed to be extractable by ethyl acetate, yet gas chromatographically different from estriol to be compatible with the observed results. It is likely that the estriol is excreted in urine principally as the 16a-glucuronide2 • 12 although in some subjects the 3-glucuronide and possibly some other more complex conjugates may be more abundant. Consequently, it is more reasonable to use the 16a-glucuronide of radioactive estriol to check for procedural losses than to use the free compound. The specificity achieved with the method reported here is considered by us to be adequate in light of the purpose for which the method was developed. Among the factors considered before deciding that the specificity is adequate were the following: 1. Estrone and estradiol and probably many other estrogens are readily separated from estriol gas chromatographically as the heptafluorobu tyrates. 2. The pen of the recorder was close to the baseline except for the solvent peak and the peak associated with estriol. 3. Estriol is known to be the most abundant estrogen present in late pregnancy urine and potentially interfering substances are
June 15, 1969 Am. J. Obst. & Gynec.
likely to be separated during the preliminary partitioning procedures or on the gas chromatographic column. 4. Additional purification by thin-layer or other chromatographic procedures was expected to add to the time required for analysis without significant improvement in the specificity. The additional time also would have precluded completion of a reasonable number of assays in a single working day and thereby have defeated a major purpose of the assay; namely, a rapid, clinically useful procedure. The reproducibility of the assay was shown to be very satisfactory both with replicate measurements on the same day and on successive days. The latter situation is particularly relevant since it is generally considered that a precipitous decrease in the concentration of estriol in urine on 2 successive days is indicative of fetal distress. 2 The absolute accuracy of the method described here is not subject to clear definition or verification for reasons discussed earlier. We consider it likely that the values obtained reflect reasonably well the concentration of estriol present in late pregnancy urine. The authors are grateful to the 3M Company (St. Paul, Minnesota) for providing heptafluorobutyric acid and to Mrs. Frances Nakamura for assistance in one phase of this work. The investigation was supported in part by a NIH-GRS grant from the Attending Staff Association, Harbor General Hospital, NIH Grants AM-09908 and RCDA 5K3-AM-31,321 (SK), an ACS Institutional Research Grant (UCLA), and a grant from The Ford Foundation.
REFERENCES
1. Brown, J. B., and Coyle, M. G.: J. Obst. & Gynaec. Brit. Emp. 60: 219, 1963. 2. Beling, C. G.: Acta endocrinol., Suppl. 79, 1963. 3. Yousem, H. L., and Strummer, D.: AM. J. 0BST. & GYNEC. 88: 375, 1964. 4. Schindler, A. E., and Herrmann, W. L.: Gynaecologia 161: 446, 1960. 5. Jaffe, S. H., and Levitz, M.: AM. J. OBsT. & GYNEC. 98: 992, 1967. 6. Frandsen, V. A.: The Excretion of Oestriol in Normal Human Pregnancy, Copenhagen, 1963, Ejnar Munksgaard Forlag.
7. Exley, D.: Memoirs Soc. Endocrinol. 16: 117, 1966. 8. Hiroi, M., and Kushinsky, S.: Submitted for publication. 9. Bruno, G. A., and Christian, J. E.: Anal. Chern. 33: 1216, 1961. 10. Honda, K., and Kushinsky, S.: Submitted for publication. II. Idler, D. R., Kimball, N. R., and Truscott, B.: Steroids 8: 865, 1966. 12. Hahne), R. J.: Endocrinology 38: 417, 1967.