1720
Anal. Chem. 1987, 59, 1720-1725
of 2.5 to 1 the detection limit for water would be about 2.5 PPm.
ACKNOWLEDGMENT Richard Solomon of our laboratory developed the headspace GC method and used it for the comparative determinations of water in formulations of DBNPA. Danial Martin of our laboratory performed the comparative GC determination of water in Telone-I1 soil fumigant. Registry No. DBNPA, 10222-01-2; H20,7732-18-5;Telone.11, 542-75-6; 2-mercaptoethanol, 60-24-2. LITERATURE CITED (1) Mitchell, J., Jr.; Smith, D. M. Aquametry, Paif 111: Wiley-Iciterscience: New York, 1980. (2) Mitchell, J., Jr.; Smith, D. M. Aquametry. P a f I ; Wiley-Interscience: New York, 1977. (3) Blasius, E.; Janzen, K. P.;Adrian, W.; Klein, W.; Klotz, H.; Luxenbur-
ger, H.: Mernke, E.; Nguyen, V. B.; Nguyen-Tien. T.: Rausch, R.; Stok-
erner, V.; Toussaint, A. Talanfa 1980, 27. 127. (4) Fehrman, V.; Schnabel, W.; Fresenius’ 2.Anal. Chem. 1974, 269(2)
116. (5) Bjorkquist, B.; Toivoner, H. J . Chromatogr. 1979, 778, 271. (6) Roof, L. B. U.S. Patent 3935097, 1976. ( 7 ) Smith, D. M.; Mitchell, J., Jr.; Aquametry, fart I I ; Why-Interscience:
New York, 1984. (8) Helfferich, F. Ion Exchange; McGraw-Hill: London, 1962; p 104. (9) Jones, H. C.; Carroll, C. G. Am. Chem. J . 1904, 32, 521-583. (10) Jones, H. C.; Lindsay, C. F. Am. Chem. J. 1902, 2 8 , 329-370. ( 1 1) Snyder, L. R.; Kirkland, J. J. Introduction To Modern Liquid Chromatography, 2nd ed.; Wlley: New York, 1979; p 812. (12) Analytical Method ML-AM-81-45. available from the Methods Editor, The Dow Chemical Company, Michigan Divlsion, Analytical Laboratories, 574 Building, Mldland, Michigan 48667. (13) Gjerde, D. T.; Schrnuckler, G.; Fritz, J. J . Chromatogr. 1980, 787,
230.
RECEIVED for review July 21, 1986. Resubmitted February 17, 1987. Accepted March 16, 1987. A United States Patent Application bas been filed covering the subject of this contribution.
Estrogen Conjugates in Late-Pregnancy Fluids: Extraction and Group Separation by a Graphitized Carbon Black Cartridge and Quantification by High-Performance Liquid Chromatography Franca Andreolini,’ Claudio Borra,l Federica Caccamo, Antonio Di Corcia,* and Roberto Samperi
Dipartimento di Chimica, Universitci “La Sapienza” di Roma, Piazza Aldo Moro, 00185 Roma, Italy
A stepwise elution system was elaborated for the fractionatbn in classes of conlugatlon of estrogens in pregnancy body fluid samples by exploiting the presence of charged actlve centers on the surface of graphitized carbon black (Carbopack). After biological samples were percolated through an experlmental Carbopack cartridge, flve classes of estrogens were eluted In the followlng order: unconjugated, A-ring glucuronides, D-ring glucoronides, monosulfates, double conjugates. After solvent elirnlnation, each class of conjugation was subfractionated and quantifled by Ion-pair high-performance liquid chromatography. The major advantages of this procedure over those based upon ion exchangers are that minimum sample manipulation is required, It permits analysis of double conjugates, and the time of analysis is drastically reduced. Recoveries of all compounds of interest ranged between 90% and 98%. The levels of estrogens and thelr conjugates in late pregnancy urine, serum, and amniotic fluid samples were measured.
It is known that only a fraction of the steroid hormones are present in biological materials as unconjugated forms, the major part of them being conjugated with sulfuric and glucuronic acids. Although steroid conjugates do not possess a direct biological activity, they can act as precursor hormone reservoirs able to be converted to either uncorijugated precursor steroids or physiologically active steroids (1. 2 ) . Moreover, the level of each conjugate in biological materials is controlled by complex metabolic and transport mechanisms, which may be altered by pathological disturbances. For these Present address: Department of Chemistry, Indiana University,
Bloornington, IN 47406.
0003-2700/87/0359-1720$01.50/0
reasons, a more complete understanding of the relationship of steroids with diseases may be achieved from the knowledge of the levels of these conjugates in normal and abnormal status. The recent trend in clinical chemistry is that of devising analytical systems for measuring one selected intact steroid conjugate (3-5) or all the conjugated forms of a single steroid (6-9) or the conjugation profile of one class of steroids. Group separation of urinary steroid conjugates has been performed by anion exchangers (10-12). After fractionation, steroids are deconjugated, derivatized, and analyzed by capillary gas chromatography. With this procedure and the use of the chromatograph coupled to a mass spectrometer, impressive results have been obtained as to the determination in pregnancy urine of the conjugate profile not only of the three main estrogens but also of their metabolites (13). Although various modifications have been tried in order to simplify and increase the speed of all steps, this methodology is time-consuming, prone to errors due to excessive manipulation of the sample, and thus unsuitable for routine analysis. In addition, the procedure cited above does not permit determination of double conjugates and it has not been employed for body fluids other than urine. The elution and separation of synthetic mixtures of intact estrogen conjugates have been successfully performed by ion-pair “high-performance” liquid chromatography (IP-HPLC) (14, 15). By the use of this technique, we succeeded recently in determining the conjugation profile of estriol in various body fluids of pregnancy (9). The purification of the biological specimens was accomplished by solid-phase extraction with Vulcan (Carbopack B), a well-known example of graphitized carbon black (GCB),which has been extensively used for isolating analytes of clinical interest from biological materials (16-18). Interestingly, during the experiments we made in order to find the most effective eluant phase for simultaneous de1987 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 59, NO. 13, JULY 1, 1987
sorption of estriol and its ionogenic, conjugated forms, we observed that these latter compounds were so strongly adsorbed that no simple solvent system was able to desorb them. Although Vulcan is known to behave as a natural reversed phase, this material was supposed by us to contain on its surface particular chemical groups able t o bind anions via electrostatic forces. The purpose of this work has been that of evaluating the ability of Vulcan in fractionating steroids according t o their classes of conjugation in order to develop more convenient HPLC methods for the measurement of the three main estrogens and their major conjugates in fluids of the late human pregnancy, such as urine, serum, and amniotic fluid. EXPERIMENTAL SECTION Chemicals and Solvents. Chloroform was of analytical grade and was used after redistillation in a glass system. The other solvents were of HPLC grade and were used as supplied. Tetrapropylammonium bromide (TPABr)and tetramethylammonium hydroxide (TMAOH) 2.2 mol/L in methanol were from Fluka AG (Buchs, Switzerland). When necessary (see later), TPABr was converted to its chloride form by percolating water containing TPABr 20 mmol/L through a column filled with a strong anionic exchanger in the chloride form. The solvent mixtures utilized for the purification and fractionation of the estrogen and their conjugates with the Carbopack cartridge were as follows: (A) 100 mmol/L of formic acid in methanol; (B) chloroform/methanol (60/40, by volume); (C) 250 mmol/L of formic acid in chloroform/methanol (27/73, by volume); (D) 250 mmol/L of formic acid in chloroform/methanol (60/40, by volume); (E) 5 mmol/L of TMAOH in chloroform/ methanol (10/90, by volume); (F)0.5 mmol/L of TMAOH in chloroform/methanol (80/20, by volume); (G) 5 mmol/L of TMAOH in chloroform/methanol (80/20, by volume). The following authenthic steroids were purchased from Sigma Chemical Co. (St. Louis, MO): androsterone (A), androsterone sulfate (A-S), estriol (E3), estradiol (E2), estrone (El), estriol 3-glucuronide (E3-3-G),estradiol 3-glucuronide (E2-3-G),estrone 3-glucuronide (E,-G), estriol 16-a-glucuronide(E3-16-G),estradiol 17-P-glucuronide (Ez-lT-G), estriol 3-sulfate (E3-3-S),estradiol 3-sulfate (Ez-3-S),estrone 3-sulfate (E,-S), estradiol 3-sulfate 17-@glucuronide(E2-3-S-17-G).Estriol 3-sulfate 16-a-glucuronide (E3-3-S-16-G),commercially unavailable, was prepared (19)and purified (9) as reported elsewhere. Preparation of the Carbopack Cartridge. The Carbopack cartridge is an experimental kit developed and kindly supplied by Supelco (Bellefonte, PA). The cartridge consists of a 6-cm x 1-cm-id cylindrical polypropylene tube, which is one-sixth full of 250 mg of GCB with a particle size range between 125 and 20 pm. Polyethylene frits are located above and below the adsorbent bed to hold the minute particles in place and keep the chromatographic column intact. The cartridge fits directly into the vacuum manifold. Vacuum was obtained with a water pump, taking no care to ensure a low and costant flow-rate of the samples percolating through the cartridge. Before use, the cartridge was washed by passing through it, sequentially: 3 mL of chloroform, 2 mL of methanol, and 2 mL of water. Procedure with Serum and Amniotic Fluid Samples. One milliliter of pregnancy serum or amniotic fluid was deproteinized with 5 mL of methanol at 0 OC followed by centrifugation at 2000g for 3 min. For amniotic fluid samples, the supernate was applied directly to the Vulcan column. On the contrary, to remove phospholipids, which interfere with the analysis, the methanolic extract of serum samples was diluted with 0.5 mL of water and passed through a CIB chemically bonded silica column (Sep-PAK, Waters Associates) followed by 4 mL of methanol/water (80/20, by volume). The entire effluent was collected and percolated through the Vulcan column. After this, we passed sequentially through the column: (1)10 mL of solvent A, discarded; (2) 5 mL of methanol, discarded; (3) 3 mL of solvent B, collected (It contains unconjugated estrogens.); (4) 3 mL of methanol, discarded; ( 5 ) 5 mL of solvent C, collected (It contains estrogens A-ring glucuronides.); (6) 5 mL of solvent D, collected (It contains estrogen D-ring glucuronides.); (7) 2 mL of methanol, discarded; (8) 5 mL of solvent E, discarded; (9) 2 mL of methanol, discarded; (10) 5
1721
mL of solvent F, collected (It contains estrogen sulfates.) (11)5 mL of solvent G, collected (It contains double conjugated estrogens.). With Urine Samples. One milliliter of pregnancy urine was diluted with 9 mL of water and passed through the Carbopack cartridge. After it was washed with 5 mL of water followed by 10 mL of solvent A, the scheme described above was followed starting from step 5, as unconjugated estrogens are virtually absent in urine (7). Whatever the body fluid analysed and the fraction of estrogens collected, solvents were removed by evaporation in a water bath at 60 ”C under a stream of nitrogen. Before heating, the solution containing double conjugates was suitably neutralized, in order to avoid partial decomposition. The residue was reconstituted in 50 pL of the HPLC mobile phase (starting composition) and injected into the HPLC apparatus. HPLC. Liquid chromatography was carried out with Varian, Model 5000 having a Rheodyne Model 7125 injector with a 40-pL loop and equipped with both UV (Varian, Model 2050) and fluorometric (Perkin Elmer, Model 650-10s) detectors, in series. A 4.6-mm x 25-cm column filled with 5-ym particle size, C18 reversed-phase packing and a guard column containing “Pelliguard”, both from Supelco, were used. Solvent A was a phosphate buffer (5 mM, pH 3.2) containing TPABr, 20 mmol/L; solvent B was acetonitrile/methanol (60/40, by volume). The flow rate was 1.5 mL/min. Chromatographic conditions are reported in detail in Results and Discussion. The concentrations of estrogens and their conjugates in standard and patient samples were calculated by comparing peak heights produced by the analytes in the sample with those of reference standards. These were prepared for chromatography by mixing appropriate volumes of working standard solutions and, after solvent removal, by reconstituting the residue with 50 pL of the HPLC mobile phase. RESULTS AND DISCUSSION As mentioned above, attempts to desorb anionic compounds, such as estrogen conjugates, from the Vulcan surface by washing it extensively with various, neutral solvent mixtures a t high elution strength failed. This effect can be explained by assuming that the GCB surface is contaminated by certain, positively charged chemical impurities which may be a burnt-off residue left over from the heating at 2700-3000 “C of carbon black. Recently, we had experimental evidence (20) for the existence of chromene-like structures included in the surface framework of GCB. This oxygen complex in the presence of oxygen and water can be rearranged to form a structure similar to benzpyran and benzpyrylium salts according to:
The surface density of this oxygen complex was assessed by determining the maximum amount of an ionogenic compound, such as E3-3-S,that can be chemically adsorbed on a given amount of Vulcan. After slow percolation through 250 mg of the GCB column of aqueous solutions containing different concentrations of E3-3-S,chloroform/methanol(60/40, by volume) was passed through to remove the steroid physically adsorbed. Then, the fraction of E,-3-S still remaining adsorbed via electrostatic forces was recovered by using chloroform/methanol basified with TMAOH and quantified by HPLC. From the data obtained, which are shown in Figure 1, by assigning to Vulcan a surface area of 100 m2/g (22),we calculated a surface density of chromene groups equal to 54 nmol/m2. It is interesting to note that this value is in fair agreement with those obtained by measuring both the amount of HzOz (20) and OH- (21) liberated on suspending Vulcan in water, thus substantiating the hypothesis made on the nature of the oxygen complexes present on the GCB surface. The peculiarity of GCB of acting as an anion exchanger as well as a nonspecific adsorbent is well illustrated in Figure
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ANALYTICAL CHEMISTRY, VOL. 59, NO. 13, JULY 1, 1987
E" D
P
.4
U
E 0 L
a c
p
.2
D
1
3
2 amount in water,
4
mg
Flgure 1. Plot of the amount of E 3 3 S chemisorbed on 250 mg of GCB vs. the amount of estrogen dissolved in water.
2, where the separation of some, selected steroids representative of each type of steroid both unconjugated and conjugated is shown. In this experiment, steroids were adsorbed onto the GCB surface from water. Moreover, the initial sequence of percolation of the eluants was modified with respect to that followed in practice in order to avoid the coelution of androsterone and its glucuronide. In accordance with the behavior shown by a commonly used anion exchanger (22),the acidification of a suitable, organic solvent mixture caused complete desorption of the steroid glucuronides, while the more acidic sulfates and sulfoglucuronides still remained adsorbed onto the GCB surface. These compounds were effectively removed from the adsorbent by adding a basic agent to a chloroform/methanol mixture. Moreover, by properly adjusting the base concentration, the complete separation of sulfates from double conjugated steroids was achieved. Because of the double nature of GCB, this material offers the advantage over conventional ion exchangers in that extraction of both charged and uncharged analytes from a complex matrix, their fractionation, and purification can be made with a single analytical step, provided that a careful choice of washings and eluants is done. As an example, complete separation of androsterone and its conjugates from
I
%
CHgOH
90 A
I
I
'.Solvent A I Sdvent B 'Solvent c I I I I I
E2
the corresponding estrogen forms was achieved by properly selecting the composition of the eluants. This effect is due to the particular affinity that the flat surface of GCB has for the aromatic ring of estrogens, which results in lower mobility of these compounds with respect to androsterone. It is noteworthy that this selectivity is not remarkably decreased even when glucuronide or sulfate groups are attached to the steroid moiety. The retardation of D-ring glucuronides with respect to A-ring glucuronides, which results in complete separation of E2-3-G from E2-16-G, is probably due to the presence of the free phenolic group in the latter compound, which is able to interact with certain active centers of the GCB surface. This hypothesis is supported by the anomalously high breakthrough volume measured for phenol when adsorbed from water on Vulcan (23).However, the subfractionation of estrogen glucuronides was not complete as El-G and E3-16-G were partially coeluted. Increasing the column efficiency by doubling the weight of the adsorbent remarkably reduced this carryover from 15% to 4% (Figure 3). This modification should be adopted if the Carbopack cartridge is introduced in an analytical scheme involving a hydrolysis step of estrogens after group separation. In this case, especially when pregnancy urine samples are analyzed, the noncomplete subfractionation of glucuronides results in a double error, that is understimation of E3-16-G and a fairly large overstimation of E3-3-G,as the concentration of the former compound in pregnancy urine is much higher than that of the latter one. A correct assessment of the level of urinary E3-3-Gcan be of aid in the management of some pathological disturbances of pregnancy, such as preeclampsia (7). On the other hand, the increase of the column size of the adsorbent decreases the speed of analysis, as for each class of conjugation a proportionally larger volume of eluant has to be collected and eliminated by evaporation. Since the analytical scheme elaborated by us is based upon direct determination of intact conjugates, we chosed to maintain the column size of Vulcan as small as possible in order to develop a method that could be readily applied for routine use. The effect of the flow rate a t which the biological samples and the solvent systems were percolated through the Vulcan column on the recovery and class separation of steroids was evaluated by varying it from 0.5 mL/min, which is the flow rate usually employed with conventional ion exchangers, to 6 mL/min, which is the maximum flow rate possible with the
I
I
I
Solvent D I Solvent E I Solvent I I E2-17G 1 A-S I E2-3S
F
I I
I Solvent G 1 I
IE2-3S-17G I I
70
50.
30.
10.
a 1 0 1 2 14 1 6 18 2 0 2 2 2 4 2 6 2 8 30 3 2 3 4 36 38 40 42 44 4 6 4 8 5 0 5 2 rnL Figure 2. The group separation of some steroids and their conjugates by the Carbopack cartridge. See the Experimental Section for the abbreviations 2
used.
4
6
ANALYTICAL CHEMISTRY, VOL. 59, NO. 13, JULY 1, 1987
Table I. Optimized Chromatographic Conditions for Subfractionation and Quantification of Estrogens
I Solvent C
I Solvent
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D
class of conjugation
solvent B,
wavelength, nm
%
fluorometer UV
start end time, min
unconjugated
U" Sb A-ring glucuronides U S D-ring glucuronides U S sulfates U
S
sulfoglucuronides
I
I
Solvent
50
30
10
2
4
6
8
10 1 2
14 16
38 40e 38 60 38 50 38 60 38
40
282' 308d 215 275 304 280 215 282 308
30
275
304
40
275
304
35
280 215
" U = urine. b S = serum. 'Excitation wavelength. dEmission wavelength. eTo purge the column, the percentage was then increased to 60% in 5 min.
Solvont D
I 70
U S
38 20 38 20 38 20 38 20 38
mL
Flgure 3. The rate of coelutlon of E,-G and E3-16G by varying the amount of Vulcan filling the cartridge: upper, 250 mg; lower, 500 mg.
apparatus used. No significant variation was observed in terms of recovery and group fractionation of estrogens in the range of flow rate investigated. From a practical point of view, this means that what can be done in about 6 h by an anion exchanger (12) is accomplished in about 20 min by a Carbopack cartridge. At present, extremely large analysis times for measuring the conjugation profile of steroids is one of the major obstacles to the convertibility of research methodologies to routine clinical assays. The influence of the nature and amount of the biological fluid applied to the GCB column on the recovery and group separation of steroids was assessed by processing increasing amounts of pooled pregnancy urine, serum, and amniotic fluid, respectively. Increasing the urine volume had the effect of progressively increasing the mobility of conjugates with the result that E3-3-G and E2-3-G, which are the earliest eluted estrogen conjugates, are partially lost in the methanol effluent. Recoveries and group separation of the other conjugates were unaffected by passing from 1to 4 mL of urine. Probably, the partial loss of the two glucuronides in the methanol washing can be due to partial formation of ion pairs with organic cations present in the urine, which increases their mobility. For the sake of rapidity and simplicity, no attempt was made to eliminate this effect by adopting a preliminary passage of the urine samples through a cation exchanger, as the limit of detection of the method was sufficiently low to determine estrogen conjugates present in only 1mL of late pregnancy urine. In experiments with serum, when the methanolic extract was directly passed through the Vulcan column, a severe carryover between A-ring and D-ring glucuronides occurred.
This may be explained by the fact that phospholipids, which are abundant in serum, are coextracted with steroids by methanol and partially saturate both specific and nonspecific adsorptive sites of the GCB surface. By use of a method elaborated by Andemon and Sjovall(24) and slightly modified by us, the isolation of steroids from phospholipids was achieved by passing the methanolic extract suitably diluted with some water through a C18-substituted silica column. Under the experimental conditions used, phospholipids were almost completely blocked by the modified silica cartridge while steroids passed unretained through it and were collected directly on the Carbopack cartridge. For amniotic fluid samples, the step mentioned above could be omitted since the relatively low amount of phospholipids usually present in amniotic fluid did not affect the group separation of steroids. In Table I are reported chromatographic conditions that, depending upon the particular fraction injected, were optimized in terms of detectability, accuracy, and elution time. On analyzing amniotic fluid, we followed the same chromatographic conditions as those for serum. Contrary to E3 and E2,El and its two derivatives are about 30 times less fluorescent. To detect them, we coupled a UV detector to a fluorometer. With respect to 280 nm, estrogens possess a much higher UV adsorption at 215 nm. This effect was exploited for determining small amounts of estrone and its derivatives, as those present in pregnancy serum. A t 215 nm, however, the detector was saturated because of the presence of TPABr added to the mobile phase for ion-pair HPLC. This difficulty was eliminated by replacing the bromide ion, as described in the Experiment Section, with a less W absorbing ion, such as chloride. Nevertheless, gradient elution at 215 nm was precluded as it caused too large a base-line drift. Figure 4 shows some typical chromatograms. For each body fluid considered, analytical recoveries were evaluated by supplementing pooled pregnancy biological specimens with known amounts of the estrogens and their conjugates and reassaying. In all cases, mean recoveries ranged between 90% and 98%. Under the experimental conditions used, the limits of sensitivity (signal to noise ratio = 3) for all the compounds of interest were calculated and are reported in Table 11. The precision of the method and a rough assessment of the normal levels of estrogens and their conjugates present in late pregnancy body fluids were performed by pooling biological samples, respectively, from 9, 6, and 4 urines, sera and amniotic fluids of apparently healthy women in their third trimester of pregnancy. Each one of the three resulting pools
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ANALYTICAL CHEMISTRY, VOL. 59, NO. 13, JULY 1, 1987
, I
I
I
35
30
I
time 4 0 (min)
b
E3
e
time (min)
5
15
20
10
5
I
E2-3
Ii
E3-16G -3 S E3-3G
d
1
X
1
I
I
t l m o 30 (mid
I
I
I
I
I
25
20
15
10
5
I
I
time (min)
I
20
I
_
I
I
I
15
10
5
y
1
Figure 4. Some, selected chromatograms of purified specimens from pregnant women: a, unconjugated estrogens in serum; b, estrogen sulfates in amniotic fluid: c, estrogen A-ring glucuronides in urine: d, mixed conjugates in urine. Dotted line = UV signal; full line = fluorometry signal.
was divided in five aliquots and assayed. Results are shown in Table 111. As to the levels of the urinary glucuronides and sulfates, our results agree fairly well with those found by combining ion-exchange columns and capillary gas chromatography (12), except for El-G and El-S. Contrary to the previous finding, we observed that estrone is excreted in pregnancy urines preferentially as sulfate instead of as glucuronide. The excreted amount of E3-3-S-16-Gas measured by us is in accordance with that determined by an indirect RIA procedure (7). For E2-3-S-17-G,no comparison was possible because no
data are reported in the literature. With respect to the estrogen concentrations in pregnancy serum, good agreement was found between our results and those obtained by various RIA procedures concerning E3 and its four conjugates (251, El and El-S (26),and unconjugated E2 (27). On the contfary, we measured serum concentrations of E2-3-Sand E3-3-S-16-G, which were much lower than those monitored by two different, direct RIA procedures (5, 28). Overestimations by these methods may be due to the fact that antisera insufficiently specific were used in assays that did not involve any purification step. Only very few attempts were made in the past
ANALYTICAL CHEMISTRY, VOL. 59, NO. 13, JULY 1, 1987
Table 11. Limit of Sensitivity of the Method for Estrogens in Various Body Fluids estrogen
urine, ng/24
E3 E2 El E3-3-G E,-3-G El-G E3-16-G Ez-17-G E3-3-S E2-3-S
h
serum,"
0.5 1.5 3.5 2.0 1.5 1.0 1.0 1.5 2.0 6.0 4.0 4.0 7.0
6.0 4.0 12.0 4.0 3.5 4.0 9.5 25.0 5.5 10.0
El-S E,-S-16-G Ez-3-S-17-G " T h e l i m i t i n g values serum.
ng/mL
for amniotic fluid are equal t o those for
Table 111. Precision of the Method and the Mean Content of Estrogens in Pregnancy Urine (n = 9), Serum (n = 6), and Amniotic Fluid (n = 4) serum, meanb f compound
urine, mean" f
E3
E2 El E3-3-G E*-3-G E,-G E,-16-G E2-17-G E3-3-S E2-3-S El-S E3-3-S-16-G E,-3-S-17-G
SD
SD
4.61 f 0.157 0.166 f 0.0109 14.8 f 0.38 0.101 i 0.0070 1.03 f 0.037 0.055 f 0.0042 1.21 f 0.046 0.019 * 0.002
Omg/24 h of t h e free estrogen. N o t detected. dTraces.
11.5 f 0.49 12.0 = 0.53 4.4 f 0.38 47.1 f 1.55 d 1.0 f 0.12 43.7 f 1.44
e 19.8 f 0.77 d 63.0 f 1.97 41.0 f 2.27 c bng/mL
amniotic fluid, meanb f SD
15.7 f 0.64 e d 10.1 f 0.48 c d 145.0 f 3.92 c 18.7 f 0.71 c 11.1 f 0.50 70.7 f 3.65
e
of t h e free estrogen.
to determine the pattern of conjugation of estrogens in amniotic liquid and all of these were limited to profiling E3and its metabolites. For these compounds, our data compared well with those reported elsewhere (6).
ACKNOWLEDGMENT We thank E. Raponi for the synthesis of E3-3-S-16-G,A. Pachi for useful discussions and for supplying us with amniotic fluid specimens, and G. Carfagnini for skillful assistance.
1725
Registry No. A, 53-41-8; El, 53-16-7;E*, 50-28-2;E3, 50-27-1; A-G, 1852-43-3;A-S, 2479-86-9;El-G, 2479-90-5;El-S, 481-97-0; E3-3-G, 2479-91-6;E2-3-S,481-96-9; Ez-S-G,15270-30-1;Ez-lv-G, 1806-98-0; E3-3-S, 481-95-8; E,-16-G, 1852-50-2; E2-3-S-17-G, 26923-03-5; E,-3-S-16-G, 4661-65-8.
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RECEIVED for review November 10, 1986. Accepted February 11, 1987. We are grateful to the Minister0 della Pubblica Istruzione for economic support of this work.
