ACKNOWLEDGMENT Acknowledgment is made to K. J. Alkerton and W. D. Lord for assistance i n experimental work, and t o T h e Steel Company of Canada, Ltd., for permission to publish.
LITERATURE CITED Bhargava and W. G. Hines, Paper presented at the 50th Annual Conference of the Chemical Institute of Canada, Toronto, June 1967. (2) 0. P. Bhargava, G. F. Pitt, J. F. Donovan, and W. G. Hines, Technicon International Congress 1970, New York, N.Y.; published in the Congress Proceedings. (1) 0.P.
(3) 0. P. Bhargava, G. F. Pitt, and W. G. Hines, Talanta, 18,793 (1971). (4) 0. P. Bhargava, J. F. Donovan, and W. G. Hines, Paper presented at the 25th Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy, Cleveland,Ohio, March 1974. (5) P. E. Kilsby, Technicon International Congress 1970, New York, N.Y.; published in the Congress Proceedings. (6) 0. P. Bhargava and W. G. Hines, Anal. Cbem., 40,413 (1968).
RECEIVEDfor review April 29, 1976. Accepted J u l y 1, 1976. P a p e r presented at t h e P i t t s b u r g h Conference on Analytical Chemistry and Spectroscopy, Cleveland, Ohio, March 1976.
Determination of Total Estrogens in Urine with 3-Methyl-2benzothiazolinone Hydrazone Hugh Y. Yee* and Bobette Jackson The Deparfnient of Pathology, Hutzel Hospital, 432 East Hancock, Detroit, Mich. 4820 1
An estrogen method has been developed that utilizes a colorimetric reaction consistlng of the oxidative coupling of 3methyl-2-benzothiazolinone hydrazone (MBTH) to the phenol portion of the steroid molecule. Colorlmetric measurements are made at 530 nm manually or with an AutoAnalyzer at an analysis rate of 50 samples per hour. Comparison with a fluorometric method gave a correlation coefficient of 0.96 and with a gas chromatographic one, a coefficient of 0.94, so that the proposed method may be suitable as an alternative method for the determination of total estrogens in pregnancy urine samples.
T h e importance of assaying urinary estrogen concentrations t o assess fetal growth and well being has been established and documented ( I ) . To date, most procedures have not combined simplicity a n d specificity. Perhaps, t h e most rapid m a n u a l method for use is one that uses Amberlite XAD-2 resin t o separate estrogens from urine, reaction with a Kober reagent, and a fluorometric measurement (2). A comparable colorimetric method has n o t been m a d e available, as relatively longer times are needed ( 3 ) . We have investigated a procedure to assay urinary estrogens that uses either ethyl ether-ethanol extraction ( 4 )or ammonium sulfate precipitation (5) of t h e steroids and reagents that a r e suitable for manual or automated quantification. Larger laboratories with a greater number of estrogen assays t o perform would t e n d to use an automated procedure, and m a n y excellent procedures are available (6-9). Most of t h e published procedures are carried oat at an analysis rate of 15 t o 40 samples per hour, a n d necessitate the use of a fluorometer. W e have succeeded i n extending the manual method reported here t o a semiautomated estrogen method with an analysis rate of 50 samples per hour. T h e color reaction used i n t h i s procedure has been previously used for t h e determination of phenols in water supplies (IO, 11).
EXPERIMENTAL Apparatus. The manifold used for the automated procedure is shown in Figure 1. The AutoAnalyzer system (Technicon Corp., Tarrytown, N.Y.) consisted of sampler 11, pump 11, colorimeter, and recorder. Manual measurements were made with a Gilford Model 300-N spectrophotometer (Gilford Instruments Laboratory, Oberlin, Ohio). An International B-20 A high speed refrigerated centrifuge 1704
* ANALYTICAL CHEMISTRY. VOL.
(Damon/IEC Division, Needham Heights, Mass.) was used to centrifuge the ammonium sulfate precipitates. Spectra were obtained with a Coleman Model 124 spectrophotometer (Coleman Instruments, Inc., Maywood, Ill.) with Model 165 recorder. Fluorometric measurements were made with an Aminco Model SPf 125 spectrofluorometer (American Instruments Co., Silver Spring, Md.). A Varian Aerograph Model 1440 chromatograph was used for the assay of estrogens by gas chromatography. Reagents. Isolation, Hydrolysis, and Purification. Reagents used were 6 N H2SO4; 0.5 N HzS04 in ethanol; 0.5 N HzS04 in methanol; 1 N NaOH; 0.1 N NaOH; acetate buffer (2 molh.; pH 4.7); Glusulase, an enzyme mixture from Helix Pomatia containing approximately 200 000 unitdm1 of glucuronidase and 100 000 units/ml of sulfatase (Endo Laboratories, Garden City, N.Y.); 1 M K2C03; (NH&S04; ethyl ether; methanol (aldehyde free); ethanol. Manual Color Development. Use 0.2% w/v ceric ammonium sulfate (G. F. Smith, Columbus, Ohio) in 1.5%v/v HzS04, store in an amber colored bottle; 0.15% w/v aqueous solution of 3-methyl-2-benzothiazolinone hydrochloride (MBTH) (Aldrich Chemical Co., Milwaukee, Wis.), keep refrigerated when not in use and discard after 2 weeks; 0.3% w/v EDTA (disodium salt); estriol standards 20 and 40 mg/l. in 25% v/v methanol or 100%methanol. Automated Color Development. Wash water, add 0.1 ml Brij-35 (30%solution; Technicon Corp., Tarrytown, N.Y.) per liter; 0.2% w/v ceric ammonium sulfate in 2% v/v H2S04, store in an amber colored bottle; 0.05% w/v MBTH in aqueous solution, keep refrigerated when not in use and no more than a %week supply; 0.3%w/v EDTA (disodium salt), add 0.1 ml Brij-35 per liter; stock estriol standard, 1mg/ml in ethanol; dilute with 25% v/v ethanol to obtain standards of 5,10, 20, 30, and 40 mg/l. Procedure. Isolation: ( a ) A m m o n i u m Sulfate Precipitation. In a 40-ml glass-stopper centrifuge tube containing 3.5 g (NH&S04, pipet 5 ml of urine from a 24-h collection, add 0.1 m16 N HzS04, and mix the contents. Warm the contents of the tube in a 55 "C water bath for several minutes. Stopper the tube and vortex vigorously until the ,salt dissolves. Transfer the contents of the centrifuge tube to aplastic centrifuge tube. Centrifuge the tube in a high speed centrifuge a t 17 000 rpm at 0-5 "C for 30 min. Remove the tube from the centrifuge ana aspirate off the supernatant liquid, taking care not to remove any of the precipitate. Add 2 drops of 1N NaOH and 1ml of water. Vortex the contents. Add 3 ml of water and mix. Centrifuge for 5 min at 3000 rpm to pack any undissolved material. Transfer the purified urine extract to a clean 40-ml glass-stopper centrifuge tube. Hydrolysis. Add 1ml of acetate buffer and 0.5 ml of Glusulase. Mix and incubate the contents a t 55 "C for 120 min. Purification: ( a ) A m m o n i u m Sulfate Isolation. Cool the tube containing the hydrolysate. Add 25 ml of ethyl ether and shake vigorously 3 times for 10-s intervals (release pressure after each 10-s shaking). After the layers have separated, aspirate off the lower phase. Wash the ether phase by shaking with 10 ml of 1M KzC03 and aspirating off the lower phase. Wash with 10 ml of water and aspirate off
48, NO. 12, OCTOBER 1976
AUTOMATED ESTROaEN
2:1
OH
ceric reagent
Dl
colorimeter 530 nm i5mm Vc Figure 1. Manifold diagram for automated color development
the lower phase. Pipet a 10-ml aliquot of ether into a 40-ml conical test tube and evaporate the ether to dryness in a 55 "C bath. For automated color development, dissolve the sample residue in 0.5 ml of ethanol, making certain the sides of the tube are rinsed. Add 1.5 ml of water and mix well. For manual color development, substitute 2.0 ml of methanol. The solution is equivalent to 2 ml of urine. Isolation: ( b )Ether-Ethanol Extraction. In a 50-ml glass-stopper centrifuge tube containing 2.5 g (NH&S04, pipet 5 ml of urine, add 0.1 m16 N HzS04,and mix the contents. Add 10 ml of ether-ethanol 3:l v/v, and shake vigorously 3 times for 20-9 intervals (release pressure after each 20-9 shaking by carefully opening the stopper). Aspirate off the urine layer. Transfer the solvent to a clean 40-ml centrifuge tube and evaporate to dryness. Add 4 ml of water and mix. Hydrolysis. Add 1ml of acetate buffer and 0.5 ml of Glusulase. Mix and incubate the contents a t 55 "C for 120 min. Purification: ( b ) Ether-Ethanol Isolation. Extract the cooled hydrolysate with 25 ml of ether and shake vigorously 3 times for 10 s each (release pressure after shaking). Aspirate off and discard the aqueous phase. Wash the ether phase with 10 ml of 1M KzC03 followed by 10 ml of water. Pipet a 10-ml aliquot into a 15-mlconical test tube and evaporate the ether to dryness in a 55 O C bath. Redissolve the residue in 3 ml of ethyl ether. Add 1.6 ml of 0.1 NaOH and vortex. Aspirate off the ether phase. Transfer the alkaline phase to a clean test tube. Add 0.4 ml of 0.5 N HzS04 in ethanol for automated color development or 0.5 N HzS04 in methanol for manual development. Mix well. Color Development and Measurement: Manual. Pipet 1ml of the sample solution into a clean test tube. Pipet 1 ml of each standard, 20 and 40 mg/l., into separate tubes. For a reagent blank, use 1ml of 25% v/v methanol or 100%methanol. Add 1 ml of 0.15% MBTH to all tubes and mix. Let stand 5 min. Add 1ml of 0.2% ceric reagent, mix, and let stand 5 min. Measure the absorbance a t 530 nm vs. the blank. Calculate the concentration of the sample from the standards. Multiply the concentration in mg/l. of the urine sample by the number of liters of the 24-h collection to obtain the mg/24 h. If the absorbance from a urine control is greater than anticipated, it may be necessary to prepare the estriol standards in a urine matrix. Color DeuelaAment, Measurement, and Calculations: Automated. Place the manif6ld on the pump (Figure l), attach reagent lines, and pump reagents for a t least 10 min to obtain a stable baseline. Place the 530-nm filters and the No. 1 aperture in position. Set baselines. If the 100%T baseline drift is pronounced, replace the sample line. Load the standards on the tray followed by three water wash cups, controls, water wash cup, and groups of six samples. Separate each
Table I. Recovery of Added Estriola Added
Calcd value
Found
Recovery, %
Recovered
5.8 7.8 8 .O 2.2 110.0 4.0 9.8 9.1 3.9 91.5 16.4 10.0 15.8 10.6 106.0 20.0 25.8 24.3 18.5 92.6 a Estriol was added to a urine containing 5.8 mg/l. of estrogen. All specimens were assayed in triplicate.
0.0 2.0
~
~~~~
~
group of samples with a water wash cup. Begin sampling a t a rate of 50/h (2:l sample wash; 48-s sample/24-s wash). After the highest concentration standard is recorded and the pen returns to or near 100% T, re-adjust the baseline if necessary. After all samples have been recorded, remove reagent lines and place them into water and pump water through the system for a t least 20 min. Plot % T values of the standards vs. their respective concentrations using semilogarithmic graph papw. Read the concentrations of the samples directly from the plot. Multiply the concentration in mgh. by the number of liters of the 24-h urine collection to obtain mg/24 h.
RESULTS AND DISCUSSION Recoveries and Correlation. Aqueous solutions containing 10, 15, 30, and 50 mg/l. of estriol-3-glucuronide and estriol-16-glucuronide (Sigma Chemical Co., St. Louis, Mo.) were assayed in replicate, and recoveries of 77-96% consistently obtained with an average recovery of 86%. T h e highest concentration of estrogen conjugate gave the lowest recovery indicating that samples of greater than 40 mgh. concentrations be diluted and repeated. Estriol added t o urine was recovered as shown i n T a b l e I. The data indicates excellent recoveries. A series of urine specimens were assayed with t h e proposed method and compared with fluorometric (12) and gas chromatographic ( 1 3 ) procedures. The data a r e shown i n Table 11. Quite satisfactory correlations a r e obtained when the proposed procedure is compared with either of the o t h e r two
methods.
ANALYTICAL CHEMISTRY, VOL. 48, NO. 12, OCTOBER 1976
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Table 11. Comparison of Proposed Method with Other Estrogen Methods0 Sample No.
1
2
3 4 5 6 7 8 9
10 11
12 13 14 15 16 17
18
MBTH
Fluorometric ( 1 2 )
GC ( 1 3 )
14.6 21.3 19.5 34.8 7.2 25.8 32.3 12.4 26.9 20.0 24 .O 20.8 16.7 7.3 8.3 23.0 21.6 13.0 18.9 13.7 21.2 11.3 19.1
16.9 20.5 17.6 33.3 6.0 27 .O 32.6 12.1 22.0 21.7 23.2 19.3 16.7 5.5 8.8 26.0 18.7 11.0 15.5 14.1 19.3 12.6 14.5
14.1 25.4 15.4 39.0 7.9 25.2 30.8 12.4 23.4 23.2 22.8 17.9 20.6 5.8 6.2 24.2 20.2 14.2 17.6 14.9 24.2 16.1 17.9
I
t1
.' *--.
Mo
\.
450
500
550
600
Wrvrlmgtk (nml
Figure 2. Spectrum of chromogen of estriol, MBTH, and ceric ion re
action
Hunig and Fritsch (14).The coupling was initially carried out under alkaline conditions with ferricyanide, but modification to use ceric ammonium sulfate in acid medium was successful with an increased sensitivity (10).The reaction is depicted in Figure 3. Specificity and Interferences. The specificity of the color Average 18.9 18.0 19.1 reaction with other representative steroids was investigated, 0 All values listed are mg/24 h. Y(MBTH) = 1.86 + 0.89 X ( G c i ; r = 0.94;SXy= 2.40. Y(MBTH)= 1.62 + 0.96 X(fluor); and these data are given in Table 111. Since the color reaction occurs with the phenol portion of the molecule, steroids not r = 0. 6; ,S , = 1.98. having this structure will not interfere. The most probable interferences in urine will be aromatic amines, phenolic acids, and neutral phenols. To eliminate interference from aromatic amine compounds, the isolation of the estrogens is carried out Table 111, MBTH Reaction with Various Steroidsa at an acid pH. Amines will be in the form of their acid salts Estriol which are relatively more water-soluble and not separated Estradiol with the steroids. Phenolic acids are removed by washing the Estrone ether extract with potassium carbonate. Androsterone Epiandrosterone The most difficult compounds to remove are the neutral Dehy droepiandrosterone phenols of which p-cresol and catechol appear to be the preCortisol dominant ones ( 1 5 ) .Fortunately, two favorable factors minPregnangdiol imize this interference: (a) phenols with a pK 7 are generally Pregnanetriol excreted as sulfate conjugates (16) (p-cresol pK = 10.3; cat0 Concentrations of all steroid solutions were 10 mg/liter echol pK = 9.5), so that these salts would most likely remain in methanol. in solution during the isolation of the steroids as well as being removed by the potassium carbonate wash; (b) substantial losses of both of these phenols would occur during the ether evaporation at 55 "C, since they are quite volatile ( 1 5 ) .T o Two commercially available urine control materials were determine the effects of p-cresol, samples containing 25 to 200 assayed over a period of several months to yield a mean value mg/l. [50 mg/l. is the normal amount found in urine (15)]were of 5.3 mg/l. f 1.4 (2a), n = 42 (Lederle lot 2920-690 H6; values taken through the entire procedure. A concentration of 0.2-0.4 given: 4.0 f 1.6 gas chromatography and 4.5 f 1.6 Stanbio) mg/l. of material was found at all concentrations of p-cresol. and 7.3 f 1.1 (2 a), n = 28 (Hyland lot 0401 LOO1 AA; value The concentration of residual phenolic substances of 25 given 6.6 f 1.8Brown colorimetric). A bias of approximately nonpregnancy urine specimens was determined by the pro+0.8 mg/l. was found between the proposed method and the posed method. An average concentration of 2.2 mg/l. was methods used for the commercial materials. found with a range of 0.2-3.8 mg/l. Since the estrogen conThe recoveries and correlations suggest that the proposed centrations in the majority of urines analyzed by our laboramethod is suitable as an alternative method for the estimation tory are between 5-10 mg/l. and studies with two other esof total estrogens in pregnancy urine. tablished estrogen methods indicate good correlation, the Spectral Characteristics of t h e Chromogen. The redresidual phenolic substance concentration does not appear dish violet chromogen has two absorption maxima: one a t to interfere to any significant extent. 420-440 nm and the other at 530-560 nm. The 420-440 nm Because false positive results could occur, urine collections peak of the chromogen is slightly greater (Figure 2). The apmust be from pregnant subjects. Furthermore, this procedure parent molar absorptivity a t 530 nm for estriol was 11200 f is not suitable for estimating low concentrations of estrogens 600. The absorptivities for estradiol and estrone were similar, in urine from normal subjects. so that estriol was selected as the standard. The color followed Isolation and Purification. The first procedure for the Beer's law up to a concentration of 40 mg/l. estriol. Meaisolation and purification of estrogens from urine using amsurements were made at 530 nm for either automated or monium sulfate precipitation with high speed centrifugation manual color development. was described by Cohen in a series of papers (5,17,18). OpColor Reaction. The oxidative coupling of 3-methyl-2timum conditions have been confirmed and defined (19). benzothiazolinone hydrazone (MBTH) with phenols, aromatic The alternative procedure using an ether-ethanol extracamines, and active methylene compounds was reported by 19 20 21 22 23
1706
ANALYTICAL CHEMISTRY, VOL. 48, NO. 12, OCTOBER 1976
OH
3-methyl -2 - benzothiazolinone
hydrazone
1
I
CH3
ceric
ammonium
sulfate
1 I
k
Flgurs 3. Oxidative coupling of MBTH with estrlol in the presence of
ceric Ion
tion of urine containing 50% (w/v) ammonium sulfate was reported earlier ( 4 ) . A duplicate run (n = 20) using both procedures gave results for all samples that were within f 3 mgh. with a correlation coefficient of 0.96. This evidence indicates that either means of separation is suitable for routine use. Because of greater variations in shaking with solvent in the initial extraction, the procedure employing ammonium sulfate and high speed centrifugation is preferred. Amberlite XAD-2 separation, in our hands, was not satisfactory, especially with urine samples containing drugs of abuse. Colorimetric and fluorometric measurements gave extremely high results for these extracts. Direct analysis was also unsuitable, so that separation and purification must be carried out to ensure valid results. The purification by solvent extraction or ammonium sulfate precipitation removed enzyme inhibitors, so that the rate of hydrolysis by the glucuronidase and sulfatase was increased (17).A large excess of enzyme was employed to make certain that specimens having high concentrations of estrogen conjugates were hydrolyzed within the 2-h incubation period. Automated System. An alcoholic solution of the purified and hydrolyzed estrogens is diluted with 0.3% EDTA and 0.05% MBTH added. After mixing, 0.2% ceric ammonium sulfate is added. This final solution is mixed and pumped through the flow cell and measurements made a t 530 nm. Carryover was 2% for a 5 mgh. sample after a 20 mg/l. one and 2.5% after a 40 mg/l. one. Steady state reached was 94%. Sampling rate was 50/h with a sample wash ratio of 2:l. Slightly improved washout characteristics of 1.9% could be obtained at a rate of 5Oh, 1:1,with a lower percent steady state reached and a diminishing of the peak heights. The peak heights could be restored by using a larger sample line to compensate for the shorter sampling time a t the 1:l ratio. The ceric to MBTH ratio for the automated procedure is 1mlO.2% ceric:0.23 mlO.O5%MBTH and is critical. Increased
amounts of MBTH without compensating increases in ceric ions will result in baseline drift. This drift is caused by the continuing formation of the greenish cerous ions. The drift can be controlled by maintaining the proper reagent ratio and by rapid passage of the reaction stream through the colorimeter before any substantial reduction of ceric ions takes place. Note that in the construction of the manifold, we have specified that the tubing from the last mixing coil be kept as short as possible in length.
LITERATURE CITED (1) J. W. Green, Jr., and J. C. Touchstone, Am. J. Obstet. Gynecol., 85, 1 (1963). (2) L. Lee and R. Hahnel, Clln. Chem. ( Wlnston-Salem, N.C.), 17, 1194 (1971). (3) E. S. C. Quek, J. E. Buttery, and G. F. deWltt, Clin. Chem. ( Wlnston-Salem, N.C.),19, 1204 (1973). (4) R. W. Edwards and A. E. Kellle, Acta Endocrlnol., 27, 262 (1958). (5)S. L. Cohen, J. Clln. Endocrlnol. Metab., 26, 994 (1966). (6)W. P. Barnard and R. W. Logan, Clin. Chlm. Acta, 29, 401 (1970). (7) D. G. Campbell and G. Gardner, Clln. Chlm. Acta, 32, 153 (1971). (8) I. R. Hainsworth and P. E. Hall, Clln. Chlm. Acta, 35, 201 (1971). (9) M. Lever, J. C. Powell, and S. M. Peace, Biochem. Med., 8, 188 (1973). (10) H. 0. Frlestad, D. E. Ott, and F. A. Gunther, Anal. Chem., 41, 1750 (1989). (11) P. D. Goulden, P. Brooksbank, and M. B. Day, Anal. Chem., 45, 2430 (1973). (12) A. T. Howarth and D. B. Robertshaw, Clln. Chem. ( Winston-Salem,N.C.), 17,316 (1971). (13) J. Van de Calceyde, N. Scholtls, N. Schmldt, and A. Kuypero, Clin. Chim. Acta, 25, 345 (1969). (14) S. Hbnlg and K. H. Frltsch, Justus Lleblgs Ann. Chem., 609, 143 (1957). (15) S. L. Tompsett, Clln. Chlm. Acta, 3, 149 (1958). (16) "lsolatlon and Identification of Drugs", E. G. C. Clarke, Ed., The Pharmaceutlcal Press, London, England, 196g, p 153. (17) S. L. Cohen, Can. J. Blochem., 48, 563 (1968). (18) S. L. Cohen, J. Clln. Endocrlnol. Metab., 29, 47 (1969). (19) G. S. Plnkus and J. L. Plnkus, Clin. Chem. ( Winston-Salem,N.C.), 16, 824 (1970).
RECEIVEDfor review January 20, 1976. Accepted June 25, 1976.
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