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A simplified estriol determination in pregnancy urine
CLISICA
CHIMICA
A SIJIPLIFIED
.\I
Dqhrtnwnt Stw
Hydr
171
ACTA
ESTRIOL
I:. IIC)S~N?‘H.L\L
AND AKHTAR
of
Laboratories, Long Pavk. Sew York rroq
(IilXXJl\+Xl Sf2pteITlber
DETERMINATION
IN PREGNANCY
URINE
YIASEK?;
Island Jewish (U..S.A .)
,Ilrdical
C&w,
*j, 1970)
Estriol is determined in pregnancy urine after acid hydrolysis by extraction, silylation with bis-(trimethylsilyl)acetamide, and gas chromatography on OV-17. Recoveries of standards are quantitative. Pregnanetriol does not interfere, while epiestriol is a negligible interferent. A variety of basic and neutral drugs and metabolites also do not interfere, while only insignificant peaks are found in the analytical region of chromatograms from non-pregnant urine.
ISTRODUCTIOK
The determination of estriol in late pregnancy urine, which gives an indication of fetal viability, has recently received considerable attention. Because of their specificity and potential speed, and the variability in the amount of estriol in total Sober chromogensl, gas-chromatographic methods were investigated, with a view toward increasing their simplicity, Methods which employ enzymatic hydrolysis of estriol glucuronide and sulfate conjugates have the disadvantages that inhibitors of glucuronidase and sulfatase may be present in urine*, and that pregnanetriol( a pre-corticoid metabolite) is difficult to separate from estriol chromatographically as the trimethylsilyl ethers usually employed. Acid hydrolysis, however, at 100’ for I h gave minimal decomposition of estriol but completely destroyed pregnanetriol. Following acid hydrolysis the estriol was extracted, silylated, and chromatographed on an OV-17 column, using cholestane as an internal standard. MATERI.XS (I) 5-a-Cholestane (Sigma Chemical Co., St. Louis, MO.), IO mg/roo ml in reagent-grade ethyl acetate. (2) Stock estriol: IO mg/roo ml estriol (Mann Research Laboratories, Mountainside, N. J.) in absolute ethanol. (3) Working estriol (I mg/roo ml) : One ml stock estriol is diluted to IO ml with reagent-grade methylene chloride.
ROSENTHAL,YASEE?;
172 (4) 6 N Hydrochloric acid. (5) 5 N Sodium hydroxide. (6) bis-(Trimethylsilyl)acetamide
(BSA), Sigma Chemical
Co.
METHOD
Hydrolysis
Five ml of urine of a 24-h specimen, or, less satisfactorily, of a morning collection, is placed into a 5o-ml centrifuge tube with 4 ml of 6 N HCl. A silica boiling chip is added and the tube placed into an electric heating block at 105” for I h. Extraction
and derivatizatiola
To the cooled tube is added 5 N NaOH until the pH is 9 $- 0.2. The urine is extracted with 20 ml of ether, using a vortex mixer for 3 min. The phases are allowed to separate, or centrifuged if necessary. The aqueous phase is aspirated off. To the ether layer Drierite (about I g) is added; the mixture is filtered through Whatman I PS paper. To a 5-ml aliquot is added 0.1 ml of cholestane solution (containing rot’g cholestane) and the solution evaporated to dryness either with a Buchler Rotary Evapo--Mix, or in a stream of nitrogen. (If the former technique is used, the last traces of solvent vapor are finally removed by a stream of nitrogen for a few seconds.) In the case of urine specimens containing more than a small amount of glucose (e.g., > “light” on Labstix, Ames Co., Elkhart, Indiana), hydrolysis should not be performed directly. The following simple procedure was found to give the best results. To 5 ml of the urine was added 3.5 g ammonium sulfate and the mixture swirled on a vortex mixer until the added solid had dissolved. The mixture was centrifuged at 2000 rpm for IO min or until the supernatant was clear. The supernatant liquid was aspirated off and discarded. To the residue was added 5 ml of water, the mixture swirled to dissolve the estriol conjugates, and this solution was then treated as a glucosefree urine. To the dried residue is added 0.1 ml BSA and the mixture is heated in a block at 65” for 30 min. Gas chromatography
The gas chromatograph used was a Perkin-Elmer model 881, fitted with a x/8 in. x6 ft. stainless steel column containing 3% OV-17 on silanized acid-washed Chromosorb W. The chromatographic conditions were : column temperature, 240” ; detector and injector temperatures, 300’; helium carrier flow, 40 ml/min ; hydrogen flame ionization detector. One ~1 of the BSA solution is injected into the chromatograph; cholestane and estriol peaks appear at 10.2 and 13.4 min respectively. Except in the case of very small amounts of estriol it was found adequate to quantitate the estriol by peak height rather than peak area. Results are expressed as mg estriol/z4 h or mg estriol/g creatinine3. RESULTS
Part (A) of Fig. I shows the standard curve of estriol concentration t’s. estriol/ cholestane peak height ratio. Part (B) shows the recovery of known estriol concenClin.
Chim.
Acta,
31 (1971)
171-174
ESTRIOL IN PREGNANCY URINE
e.Se-
I73
ESTRIOL
PEAK HEIGHT RATIO, ESTRIOL CHOLESTANE
i$isi/q 0
IO
20
30 ESTRIOL,
40
50
ng
Fig. I. (A), Estriol standard curve; (B), recovery of estriol standards added to urine. Amounts of estriol indicated are the actual amounts injected (in I ,d) into gas chromatograph, representing amount of estriol in 0.0125 ml urine. (Cholestane is roe ng in all samples). Recoveries from urine were obtained by adding estriol standards (in ethanol) to urine, and carrying through the entire procedure. trations added to urine. Comparison of the two curves shows that recovery of estriol from urine is quantitative. Estriol taken through the hydrolysis was 95% recovered. Thus, it is permissible to use the estriol standard solution in the routine procedure without hydrolysis or extraction. Pregnanetriol taken through the hydrolysis step was 100% destroyed, within the limits of experimental observation. Urine from eleven third-trimester pregnant women gave estriol excretions of 6.1-31.0 mg/24 h. In addition, single morning specimens obtained from eleven additional pregnant women were analyzed as mg estriol/g creatinine. Results are given below : Patient I 2 3 4 5 6
;: 9 IO
II
No.
Weeks pregnant
mg estriol/g
37 36 38 36 33 35 35 38 28 34 34
29.0 34.2 20.4 49.0
cveatinine
12.2
4.8 9.9 II.0
13.9 19.8 22.0 __-
_____
More than forty male and non-pregnant female urine specimens, selected randomly from hospitalized patients, have been examined by this procedure. In no case were chromatographic peaks observed in either the estriol or cholestane regions corresponding to more than 0.2 mg/l of either substance in urine. No peaks were observed in most specimens. BSA derivatives of the following basic and neutral drugs and metabolites were Clin. Chim. Acta. 31 (1971) 171-174
174
KOSENTHAL,
YASfiES
found to elute far from the estriol and cholestane regions: reserpinc, glutethimide, dextro-amphetamine, ~hlorpn)mazint~, meprobamate, rhlordiazepoxide, diazepam, metanephrine, and nornletancpllrinr~. Acidic drugs were not studied, since they would not be extracted at pf-I 0. ~~)-ketoestr~ldiol was also As an example of a ring i)-k&r& estrogen mctaholitc’, found not to interfere wit11 the cstriol peaks. The only substance Eouiid to interfere wit11 t.lre cstriol peak was epicstriol, whose tris ~trimethylsilyl ether elutrd approsimately 0.3 min slower than that of of estrioi. Epiestriol, however, has b~rz found to be it quite minor constituent pregnancy urinf?, being excreted in ~l~]~~Iltities of the order of 2 or .J’fj, that of rstriol. Moreover, the complete (~er~vati~ation of cpiestriol by ISA is con~idcrably slower than that of rstriol; we estimate tllat nr) more tllan 2o”,, epiostriol is coniplrtel~~ ethcrifled by IS,4 during the jo-min reaction period, Hydroxy-“‘ntaining partial trimethg/lsilyl ethers elute later tluni tltt* tris- ethers and thus do not interfere in the analysis. I)ISCUSSION
The metilod presented there appears to be one of the simplest yet reported. Acid hydrolysis was found to give very satisfactory results, avoiding the problem of urinary inhibitors ~o~n~tirnes found with enzymatic llydro~ysi~, Extraction of the hydrolyzed urine, ~it~~ou~ the necessity of purjfyin~ the crude extrsct4, gave ver\~ straightforward chromatagraphic rest&r; after silylation, It ww &a found that catalysis by trimethylsilyl rhloride” in the silylation reaction was unnecessary, ttms corrosive effect of this compound on the stainless steel avoiding the potentiall) components of the chromatograph. The only potential intcrferents found were pre~nanetri(~l and epiestriol. The former is destroyed by the acid hydrolysis used in the procedure, while the latter, for the reasons discussed above, appears to be a very insignificant interfering suhstance.
The authors wish to thank Dr. Harry for making available additional unpublished
Goldenberg of Kio-Science details of their analytical
L.abnrataries procedure,
CHIMICA
A SIJIPLIFIED
.\I
Dqhrtnwnt Stw
Hydr
171
ACTA
ESTRIOL
I:. IIC)S~N?‘H.L\L
AND AKHTAR
of
Laboratories, Long Pavk. Sew York rroq
(IilXXJl\+Xl Sf2pteITlber
DETERMINATION
IN PREGNANCY
URINE
YIASEK?;
Island Jewish (U..S.A .)
,Ilrdical
C&w,
*j, 1970)
Estriol is determined in pregnancy urine after acid hydrolysis by extraction, silylation with bis-(trimethylsilyl)acetamide, and gas chromatography on OV-17. Recoveries of standards are quantitative. Pregnanetriol does not interfere, while epiestriol is a negligible interferent. A variety of basic and neutral drugs and metabolites also do not interfere, while only insignificant peaks are found in the analytical region of chromatograms from non-pregnant urine.
ISTRODUCTIOK
The determination of estriol in late pregnancy urine, which gives an indication of fetal viability, has recently received considerable attention. Because of their specificity and potential speed, and the variability in the amount of estriol in total Sober chromogensl, gas-chromatographic methods were investigated, with a view toward increasing their simplicity, Methods which employ enzymatic hydrolysis of estriol glucuronide and sulfate conjugates have the disadvantages that inhibitors of glucuronidase and sulfatase may be present in urine*, and that pregnanetriol( a pre-corticoid metabolite) is difficult to separate from estriol chromatographically as the trimethylsilyl ethers usually employed. Acid hydrolysis, however, at 100’ for I h gave minimal decomposition of estriol but completely destroyed pregnanetriol. Following acid hydrolysis the estriol was extracted, silylated, and chromatographed on an OV-17 column, using cholestane as an internal standard. MATERI.XS (I) 5-a-Cholestane (Sigma Chemical Co., St. Louis, MO.), IO mg/roo ml in reagent-grade ethyl acetate. (2) Stock estriol: IO mg/roo ml estriol (Mann Research Laboratories, Mountainside, N. J.) in absolute ethanol. (3) Working estriol (I mg/roo ml) : One ml stock estriol is diluted to IO ml with reagent-grade methylene chloride.
ROSENTHAL,YASEE?;
172 (4) 6 N Hydrochloric acid. (5) 5 N Sodium hydroxide. (6) bis-(Trimethylsilyl)acetamide
(BSA), Sigma Chemical
Co.
METHOD
Hydrolysis
Five ml of urine of a 24-h specimen, or, less satisfactorily, of a morning collection, is placed into a 5o-ml centrifuge tube with 4 ml of 6 N HCl. A silica boiling chip is added and the tube placed into an electric heating block at 105” for I h. Extraction
and derivatizatiola
To the cooled tube is added 5 N NaOH until the pH is 9 $- 0.2. The urine is extracted with 20 ml of ether, using a vortex mixer for 3 min. The phases are allowed to separate, or centrifuged if necessary. The aqueous phase is aspirated off. To the ether layer Drierite (about I g) is added; the mixture is filtered through Whatman I PS paper. To a 5-ml aliquot is added 0.1 ml of cholestane solution (containing rot’g cholestane) and the solution evaporated to dryness either with a Buchler Rotary Evapo--Mix, or in a stream of nitrogen. (If the former technique is used, the last traces of solvent vapor are finally removed by a stream of nitrogen for a few seconds.) In the case of urine specimens containing more than a small amount of glucose (e.g., > “light” on Labstix, Ames Co., Elkhart, Indiana), hydrolysis should not be performed directly. The following simple procedure was found to give the best results. To 5 ml of the urine was added 3.5 g ammonium sulfate and the mixture swirled on a vortex mixer until the added solid had dissolved. The mixture was centrifuged at 2000 rpm for IO min or until the supernatant was clear. The supernatant liquid was aspirated off and discarded. To the residue was added 5 ml of water, the mixture swirled to dissolve the estriol conjugates, and this solution was then treated as a glucosefree urine. To the dried residue is added 0.1 ml BSA and the mixture is heated in a block at 65” for 30 min. Gas chromatography
The gas chromatograph used was a Perkin-Elmer model 881, fitted with a x/8 in. x6 ft. stainless steel column containing 3% OV-17 on silanized acid-washed Chromosorb W. The chromatographic conditions were : column temperature, 240” ; detector and injector temperatures, 300’; helium carrier flow, 40 ml/min ; hydrogen flame ionization detector. One ~1 of the BSA solution is injected into the chromatograph; cholestane and estriol peaks appear at 10.2 and 13.4 min respectively. Except in the case of very small amounts of estriol it was found adequate to quantitate the estriol by peak height rather than peak area. Results are expressed as mg estriol/z4 h or mg estriol/g creatinine3. RESULTS
Part (A) of Fig. I shows the standard curve of estriol concentration t’s. estriol/ cholestane peak height ratio. Part (B) shows the recovery of known estriol concenClin.
Chim.
Acta,
31 (1971)
171-174
ESTRIOL IN PREGNANCY URINE
e.Se-
I73
ESTRIOL
PEAK HEIGHT RATIO, ESTRIOL CHOLESTANE
i$isi/q 0
IO
20
30 ESTRIOL,
40
50
ng
Fig. I. (A), Estriol standard curve; (B), recovery of estriol standards added to urine. Amounts of estriol indicated are the actual amounts injected (in I ,d) into gas chromatograph, representing amount of estriol in 0.0125 ml urine. (Cholestane is roe ng in all samples). Recoveries from urine were obtained by adding estriol standards (in ethanol) to urine, and carrying through the entire procedure. trations added to urine. Comparison of the two curves shows that recovery of estriol from urine is quantitative. Estriol taken through the hydrolysis was 95% recovered. Thus, it is permissible to use the estriol standard solution in the routine procedure without hydrolysis or extraction. Pregnanetriol taken through the hydrolysis step was 100% destroyed, within the limits of experimental observation. Urine from eleven third-trimester pregnant women gave estriol excretions of 6.1-31.0 mg/24 h. In addition, single morning specimens obtained from eleven additional pregnant women were analyzed as mg estriol/g creatinine. Results are given below : Patient I 2 3 4 5 6
;: 9 IO
II
No.
Weeks pregnant
mg estriol/g
37 36 38 36 33 35 35 38 28 34 34
29.0 34.2 20.4 49.0
cveatinine
12.2
4.8 9.9 II.0
13.9 19.8 22.0 __-
_____
More than forty male and non-pregnant female urine specimens, selected randomly from hospitalized patients, have been examined by this procedure. In no case were chromatographic peaks observed in either the estriol or cholestane regions corresponding to more than 0.2 mg/l of either substance in urine. No peaks were observed in most specimens. BSA derivatives of the following basic and neutral drugs and metabolites were Clin. Chim. Acta. 31 (1971) 171-174
174
KOSENTHAL,
YASfiES
found to elute far from the estriol and cholestane regions: reserpinc, glutethimide, dextro-amphetamine, ~hlorpn)mazint~, meprobamate, rhlordiazepoxide, diazepam, metanephrine, and nornletancpllrinr~. Acidic drugs were not studied, since they would not be extracted at pf-I 0. ~~)-ketoestr~ldiol was also As an example of a ring i)-k&r& estrogen mctaholitc’, found not to interfere wit11 the cstriol peaks. The only substance Eouiid to interfere wit11 t.lre cstriol peak was epicstriol, whose tris ~trimethylsilyl ether elutrd approsimately 0.3 min slower than that of of estrioi. Epiestriol, however, has b~rz found to be it quite minor constituent pregnancy urinf?, being excreted in ~l~]~~Iltities of the order of 2 or .J’fj, that of rstriol. Moreover, the complete (~er~vati~ation of cpiestriol by ISA is con~idcrably slower than that of rstriol; we estimate tllat nr) more tllan 2o”,, epiostriol is coniplrtel~~ ethcrifled by IS,4 during the jo-min reaction period, Hydroxy-“‘ntaining partial trimethg/lsilyl ethers elute later tluni tltt* tris- ethers and thus do not interfere in the analysis. I)ISCUSSION
The metilod presented there appears to be one of the simplest yet reported. Acid hydrolysis was found to give very satisfactory results, avoiding the problem of urinary inhibitors ~o~n~tirnes found with enzymatic llydro~ysi~, Extraction of the hydrolyzed urine, ~it~~ou~ the necessity of purjfyin~ the crude extrsct4, gave ver\~ straightforward chromatagraphic rest&r; after silylation, It ww &a found that catalysis by trimethylsilyl rhloride” in the silylation reaction was unnecessary, ttms corrosive effect of this compound on the stainless steel avoiding the potentiall) components of the chromatograph. The only potential intcrferents found were pre~nanetri(~l and epiestriol. The former is destroyed by the acid hydrolysis used in the procedure, while the latter, for the reasons discussed above, appears to be a very insignificant interfering suhstance.
The authors wish to thank Dr. Harry for making available additional unpublished
Goldenberg of Kio-Science details of their analytical
L.abnrataries procedure,