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Comparative chromogenicities and discrepant spectra obtained between a heterogeneous and homogeneous Zimmermann reaction
MICROCHEMICAL
JOURNAL
21, 466-477
(1976)
Comparative Chromogenicities and Discrepant Spectra Obtained between a Heterogeneous and Homogeneous Zimmermann Reaction* R. WATKINS
AND C. S. FELDKAMP
Departments of Pathology, Wayne State University School of Medicine and Detroit General Hospital, Detroit, Michigan
E. EPSTEIN William Beaumont Hospital, Royal Oak, Michigan
R. J. THIBERT
AND
B. ZAK
Departments of Pathology, Wayne State University School of Medicine and Detroit General Hospital, Detroit, Michigan, and Department of Chemistry, University of Windsor, Windsor, Ontario, Canada Received
July
7, 1976
Chromogenicity of 17-ketosteroids (17-KS) by Zimmermann reaction using metadinitrobenzene (MDB) in an alkaline medium has not been thoroughly evaluated for all procedures. Methods in which the medium is primarily aqueous, and in which the reactions can be carried out either heterogenously, by a biphasic precipitation reaction (4,6-8) or homogeneously, in a monophasic system (2,3,9), both using Hyamine 1622 (H1622) as a solubilizing detergent, report at least three different orders of chromogenicity for the more common 17-KS encountered (3,4,9). These conflicting reports must be somewhat disturbing to users of the H1622 techniques. The seemingly minor differences in reaction matrices between the several apparently similar procedures do not project as large enough factors to account for the discrepancies, yet they do not result in similar reported chromogenicities generated. Therefore, two methodological examples of widely divergent systems were selected for comparative evaluation. One is heterogeneous and one homogeneous in the color generating phase, with the former a manual modification (6-8) and the latter a semiautomated modification (3) of a manual procedure (4). They differ primarily in the use of methanol (heterogeneous) or ethanol 1 Supported tion.
in part
by a grant-in-aid
from
the Detroit 466
Copyright @ 1976 by Academic Press, Inc. All rights of reproduction in any form reserved.
General
Hospital
Research
Corpora-
CHROMOGENICITIES
OF ZIMMERMANN
REACTIONS
467
(homogeneous) to solubilize extract residues, in the concentration of alkali (KOH) added, in reaction temperature and in the time of reaction before absorbance measurements are obtained. Because controversy of this kind is worthy of resolution, a purpose of the present report is to determine the differences, and if possible any reasons for those differences for the varied behavior of the seven most common 17-KS, when determined by the two types of analytical techniques, since in only one procedure, the semiautomated system, was any attempt made to determine reaction characteristics for all seven compounds. The areas investigated by means of a spectrophotometric study included formation curves for reaction plateaus, the related reaction speeds, chromogenicities, spectral fine structure, peak maxima, color stabilities, and molar absorptivities, all obtained with the seven 17-KS, dehydroisoandrosterone (DHI), androsterone (A), etiocholanolone (E), 1 1-hydroxyandrosterone (HA), 11-hydroxyetiocholanolone (HE), 1 I-ketoandrosterone (KA), and 11-ketoetiocholanolone (KE). It will be established that significant differences do exist in the two analytical systems which mostly account for the reported variations. The heterogeneous system to be described here is one wherein the color reaction is isolated in a precipitated phase which is subsequently solubilized on the reduction of the alkalinity of the system by dilution, whereas the homogeneous procedure is one in which no precipitation is involved, and the reaction takes place in a single phase reaction mixture. MATERIALS
AND METHODS
Reagents The reagents used for the heterogenous procedure were identical to those previously described (6,7). Those used for the homogeneous procedure were as described by Egloff et al. (3) in their description of a semiautomated determination of urinary 17-ketosteroids. Hyamine 1622 solutions: prepare 2.5 and 5% aqueous solutions, and a mixture of 1 part ethanol and 4 parts of 2.5% Hyamine 1622 solution. Potassium hydroxide solutions: prepare 2.0 and 10 N (3,6,7) aqueous solutions. 17-Ketosteroid standards: prepare stock methanolic solution standards of 10 mg/ml of dehydroisoandrosterone (DHI), eticholanolone (E), androsterone (A), 11-hydroxyetiocholanolone (HE), 11-hydroxyandrosterone (HA), 1 lketoetiocholanolone (KE), and 11-ketoandrosterone (KA). Prepare ethanolic solutions of the same compounds at the same concentrations. Prepare a stock standard in the ethanol-Hyamine 1622 solution by dissolving 10 mg of each 17-ketosteroid in 20 ml of ethanol, and then diluting to 100 ml with 2.5% of Hyamine 1622 solution. Metadinitrobenzene solution (saturated): shake 2 g of metadinitrobenzene with 1 liter of 5.0% Hyamine 1622 solution. Clear the reagent by filtration after saturation is complete.
468
WATKINS
ET AL.
Procedures Homogeneous
procedure. Pipet 0.5 ml of the standard into a test tube and carefully evaporate off its methanol. Two alternatives are then available for solution of the residue. Dissolve the residue with 0.5 ml of the H1622-ethanol solution by vigorous vortexing, or dissolve the residue with 0.1 ml of ethanol, and then add 0.4 ml of 2.5% Hyamine. To either, add 0.6 ml of metadinitrobenzene solution and 2.6 ml of 2 N KOH. The second method was used because of difficulty encountered sometimes in dissolving the residue quantitatively in the vortexing step. The solution was warmed at 50” in a water bath for 5 min and then scanned immediately across the visible range from 700-400 nm. This procedure is a close manual approximation of the described automated procedure, an approximation which was necessary in order to carry out the comparative evaluation study. Heterogeneous procedure. This was identical to the one previously described (2,3). Briefly, 10 N KOH and MDB in H1622 are added to a methanolic solution of the residue from an evaporated ether extract of urine. The alkali precipitates the H1622 which removes the forming MDB-17-KS complex from solution. At the completion of the reaction, the precipitate is dissolved by adding more H1622 and the absorbance is measured against a reagent blank. RESULTS
AND DISCUSSION
Figs. IA-G are graphic illustrations of the formation curves for the seven 17-KS when using the homogeneous Zimmermann reaction (3). Several facts are evident from inspection of these results. The rise curves (RC) are different for the several compounds. Some of the compounds are stable after full color formation while others, the 11-ketosteroids, show evidence of fading along the time path of the RC. Full color formation occurs at different times ranging from quite slow to quite fast. This is illustrated by the fast reactions to full color formation (FCF) for the llketo compounds as compared to the partial color formation (PCF) obtained after 5 min heating at 50”. The other compounds react noticeably slower than the 11-keto compounds. Different partial color formations, and this is the method of measurement of the automated homogeneous procedure, are evident for all of the compounds. The spectral characteristics obtained with the seven compounds vary widely and dissimilarities in fine structure are evident for PCF spectra and FCF spectra for the different compounds with the poorest symmetry obtained with the more commonly investigated trio of steroids, DHI, E, and A. The plateaus of the rise curves pictured are not the same as the peak maxima at full color formation. A partial explanation for that phenomenon lies in the fact that the rise curves were graphed at the wavelength of 520
CHROMOGENICITIES
OF
ZIMMERMANN
REACTIONS
469
FIG. 1. Rise curves (RC) for Zimmermann reaction versus time followed at 520 nm. Full color formation (FCF) spectra are also shown along with partial color formation (PCF) spectra for A, 1 I-ketoandrosterone (KA); B, 1 I-ketoetiocholonolone (KE); C, etiocholanolone (E); D, dehydroisoandrosterone (DHI); E, androsterone (A); F, llhydroxyandrosterone (HA); and G, 1I-hydroxyetiocholanolone (HE),
nm which was used in the automated procedure (3), whereas the peaks of the curves ranged across 70 nm. One of the stated advantages in automation is that one need not wait for full color formation in a color reaction as long as measurements can be made at precise times on the rise curve of the reaction of an analyte. Obviously that rationalization disintegrates
470
WATKINS
ET AL.
when one measures mixtures in which rate of formation of color, stability of color, and peak maxima are variables in a common reaction for a mixture of compounds. Strikingly different molar absorptivities for the different 17-KS compounds occur in a situation involving presumptive intensive properties (5), in which so many variables appear to be out of control during the quantitative phase of the procedure. The spectra of Figs. 2A-D were obtained as representatives of the rise curves of the heterogeneous system. The spectra of the three common 17-KS, DHI, E, and A are not shown here because they were reported in a previous publication and their formation curves, peak maxima, and equivalence in chromogenicity are already available (6). Since a precipitate of HI622 is formed in the reaction medium by the action of the strong alkali to which the Zimmermann chromogen (ZC) adheres, perhaps by absorption or post-precipitation, it is not possible to make a continuous measurement for a rise curve as was described for the homogeneous technique. Therefore, increments of 5 min between scans beginning with zero time for the first spectra obtained were used to follow the rate of formation of color for the 11-hydroxy and 11-keto 17-KS in the precipitation system where solubilization of the precipitate by the addition of more detergent to dilute the alkalinity of the solutions to be measured occurred at the times specified on the peaks of the curves. Some indeterminate error is evident in the different peaks, and this can be partially attributed to the fact that the still incomplete reaction of 17-KS with MBD continues after the solubilization of the precipitated H1622 and color complex has been affected by dilution even though that reaction is somewhat slower than when the two phases are present. Several other facts are also evident from the results displayed here. All spectra have an identical peak maximum at 520 nm. None of the spectra show the asymmetrical characteristics obtained with the same steroids when using the automated procedure (3). All rates of formation are similar, so that a common reaction time is easily selected for a mixture of steroids. All of the reactions are stable within the time limits studied and no fading is evident for the four compounds shown here or for the other three compounds previously tested (6). The chromogenic characteristics of the two modifications of what may appear on the surface to be the same reaction are described in Figs. 3 and 4. In Fig. 3, the results of the heterogeneous reaction are shown for the same concentration by weight of DHI as a standard and the 1 l-keto and 11-hydroxy 17-KS, KE, HA, HE, and HA. The hydroxy compounds show less reaction in terms of absorbance on a weight basis than DHI. However, on an equivalent basis they are virtually the same, indicating that the hydroxy group at the eleven position has no apparent effect on the reaction or on the final chromogen. The 1l-keto compounds are simi-
CHROMOGENICITIES
OF
ZIMMERMANN
471
REACTIONS
,
2A “,
HA
WAVELENGTH
WAVELENGTH
= 05 L S EW 5 03 02 01 0
700 +-.ot+ +.ot+ WAVELENGTH
. +-Loo-
4 WAVElENGlH
FIG. 2. Spectra of intermittent reacttons to show rise curve for the Zimmermann reaction versus time for the heterogeneous aqueous technique for 17KS for A, HA; B, HE; C, KA; and D, KE.
lar in absorbance to DHI on a weight basis but are higher than DHI in absorbance on an equivalance basis, lending credence to the notion that the 1 l-ketone positions may be involved in the Zimmermann reaction perhaps either by changing the chromogenic nature of the final reaction compound or by reaction with MDB. This ability to potentiate chromogenicity of the 17-KS has been previously reported (II). The results agree with those reported by Vestergaard for a pyridine version of the Zimmermann reaction (IO) but they dispute his statement about the H1622 procedure, that the “chromogenicity of the 17-oxosteroids formed in the 17-ketogenic assay varies greatly if the Zak and Epstein procedure is used.” Another interesting and important aspect of the reaction is shown in the figure in that the width of the DHI spectrum is greater than those of the other four compounds. Since DHI is commonly used as a standard, this could create a problem if the spectra were subjected to the Allen correction for irrelevant absorption, a correction which is commonly applied in an effort to eliminate errors of a nondescriptive and undetermined kind (1). Some further work is in progress on the evaluation
472
WATKINS
ET AL
of the potential flaws and limitations in the correction of the Allen equation. If one compared E to HE on a weight basis, one would expect that the same calorimetric reaction would yield a lower value for the heavier compound. It has previously been reported that an 1 1-beta-hydroxy group has a suppressing effect on the 17-ketosteroid reaction (11), a fact which is not indicated by the studies described above unless the comparison is made on a weight basis. The weight basis of comparison may be a less proper approach then an equivalence basis for the comparative chromogenicities of equally reacting compounds. Figure 4 shows the paired spectra of DHI, E, and A in the precipitation technique where both methanol and ethanol were used to dissolve the residues of steroids. After a 20 min wait in the precipitated state for both procedures, each reaction was then diluted with the same volumes of HI622 solution to solubilize the precipitates. The weights of steroids used were 40, 35, and 30 E.cgfor E, A, and DHI, respectively. When methanol was used, the linear chromogenicity of the reactions of the three compounds is obvious for what is obviously an equivalency of that reaction when using the reagent makeup of the heterogeneous technique. However, when ethanol was substituted for methanol, the order of chromogenicity changed and E was more sensitive than A even though there was 40 pg of A present as compared to 35 pg of E. This is a striking loss in the equivalency of reaction and the molar absorptivity of the reaction by what appears on the surface to be a miniscule change in reaction conditions. From subsequent observations, it was learned that ethanol also decreased the rate of formation of color of the Zimmermann chromogen. When a comparison was
FIG. 3. Chromogenicitycomparison for equal weights (40 pg) of KA, KE, DHI, HA, and HE using the heterogeneous precipitation technique.
CHROMOGENICITIES
OF ZIMMERMANN
REACTIONS
473
WAVELENGTH
Sensitivity and chromogenicity comparison for DHI (30 pg), E (35 pg), and A (40 wg) for methanol versus ethanol in the heterogeneous technique with a 20 min reaction time in the precipitated state. FIG.
4.
made between methanol (M) and ethanol (E) in the heterogeneous reactions using DHI to demonstrate the spectra obtained at the plateaus of reaction when using each added solvent, the spectra of Fig. 5 were obtained. Even at full color formation, and with alkalinity kept constant for both reactions, the substitution of methanol with ethanol reduced the final color by 36% of the intensity obtained with methanol, which again points out the striking effect of what appears to be a small change in the matrix of reaction. It can be seen therefore, that the slope ratio of sensitivity between the two procedures is 1.5 in favor of the methanolic reaction. In addition, when one considers the variation in time of formation for the seven compounds when using the homogeneous reaction and the different wavelengths of peaks maxima between 450 and 520 nm, it is understandable that determination of absorbance for mixed reaction speeds and variable wavelengths such as is obtained with these seven steroids can lead to nonuniform measurement even when the procedure appears uniform. Uniformity of color for different times of color formation can only be attained at a common plateau for the time factor, or this reaction variable will be misused. One of the key differences between the two procedures, aside from the methanol to ethanol substitution, was the alkalinity of the reaction matrix. In the heterogeneous system, the alkalinity is high enough to precipitate the detergent, H1622, which causes the formed chromogen to leave the solution phase and attach itself to the detergent, or perhaps forms on the surface of the detergent, a phenomenon which appears to speed up the reaction by removing the product of reaction. In contradistinction, in
474
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ET AL.
g.0450
WAVELENGTH
5. Comparative sensitivity for methanol (M) versus ethanol (E) in the precipitation technique using plateau reaction time in the precipitated state. FIG.
considering the amount of chromogen generated, the homogeneous reaction appears to be about two-thirds complete at equilibrium. There is an alkalinity above which precipitation of the H1622 occurs and more chromogen appears to be formed, so an experiment to demonstrate this is shown as follows. In Fig. 6 are the spectra obtained when the alkali content of the reaction system was varied across a wide range for the heterogeneous procedure but the reaction time before the further addition of HI622 solution was kept constant. In the beginning, when the KOH normality was low, the chromogenicity varied, as did the shape of the spectra. A gray area in which cloudiness occurred appeared beyond 5 N, and as the normality was increased to 10 N, the spectral symmetry stayed constant and only the molar absorptivity changed. Until one uses a solution beyond 5 N, no precipitation occurred, the equilibrium lay somewhat to the left, and the spectra were structured differently when the reaction remained homogeneous than when it became heterogeneous. Beyond 10 N, the precipitate did not dissolve on the addition of the same volume of H1622 solution used for the lower normalities because the resulting normality was still too high to solubilize without further dilution, so no further studies were carried out. Under the described conditions, 10 N KOH appears best suited for the reaction described because of its action in the
CHROMOGENICITIES
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REACTIONS
475
600 500 700 WAVELENGTH FIG. 6. Effect of varying the normality of KOH from 1N to ION using the precipitation 400
technique. Precipitation
occurs between SN and 7N.
HETERO
HOMO
400 WAV: SGTH
FIG. 7. The heterogeneous versus the homogeneous technique under described reaction conditions at 3.5 ml vol. (Hetero) and 3.7 ml vol. (Homo).
476
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ET AL.
precipitation of H1622 and its ability to increase the amount of color obtained. Figure 7 shows a sensitivity comparison between the homogeneous procedure performed manually and the heterogeneous procedure at the same concentrations of DHI in the two solvents used to prepare standards. Each set of standards was scanned after using the reaction characteristics described for them. The manual version of the automated homogeneous procedure used conditions of reagents, volumes, reaction time, and reaction temperature which were as close as possible to the automated description (3). The final volumes chosen for the comparison favored the heterogeneous system carried out at 3.2 ml as compared to 3.7 ml for the homogeneous system. The differences obtained are mainly mainfested by dissimilarities in spectral structures and in molar absorptivities. In this case, considering sensitivity and previously described variations in chromogenicities, the homogeneous procedure appears to be a poorly modified version of the heterogeneous procedure. SUMMARY A spectrophotometric study is shown for two modifications of a 17-KS procedure in which nearly all aqueous reaction media were used. Although the apparent differences in matrices of reaction between the two procedures appear to be minute, a small volume of ethanol for one is substituted for the same volume of methanol in the other, the final alkalinity of the latter procedure is stronger, and the reaction temperature was increased, wide differences in spectral results are found between the two methods. The ethanolic, lower alkalinity procedure is a homogeneous, single-phase reaction while the methanolic, higher-alkalinity procedure is a heterogeneous system in which a precipitation of detergent occurs and the precipitate becomes an integral part of the color reaction. The rate of formation of the ethanolic procedure is variable for the different 17-KS but it is constant in the methanolic procedure. The chromogenicities of color formation between the two procedures differ in that the methanolic system provides uniform and predictable spectra with more equivalent molar absorptivities. Lastly, the methanolic system shows a much greater sensitivity owing, apparently, to a more complete final reaction. This inability to form full color for several compounds in the homogeneous system can be related in some way to the large variations in ability to form that color owing to matrix composition.
REFERENCES 1. Allen, W., A. Simple method for analyzing complicated absorption curves of use in the calorimetric determination of urinary steroids. J. Clin. Endocrinol, 10, 71-83 (1950). 2. Edwards, R. W. H., Makin, H. L. .I., and Barratt, T. M., The steroid ll-oxygem&~n index: A rapid method for use in the diagnosis of congenital adrenal hyperplasie. J. Endocrin. 30, 181-194 (1964). 3. Egloff, M., Degrelle, H., and Jayle, M. F., Dosage semi-automatique en flux continu des 17-Cetosteroides Urinaires. C/in. Chim. Acta 59, 147-1.54 (1975). 4. Epstein, E., An aqueous Zimmermann reagent for the determination of 17-ketosteroids. Clin Chim. Acta 7, 735-737 (1962). 5. Ewing, G. W., “Instrumental Methods of Chemical Analysis,” 3rd ed. pp. 4bl04. McGraw-Hill, New York, 1969.
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REACTIONS
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6. Feldkamp, C. S., Epstein, E., Thibert, R. J., and Zak, B., Spectrophotometric study of both the Zimmermann reaction and the application of a corrective measure for irrelevant absorption. Micro&em. J. 21, 45-59 (1976). 7. Feldkamp, C. S., Epstein, E., Thibert, R. J., and Zak, B., Spectrophotometric study of drug interferences in an aqueous 17-ketosteroid reaction. Micro&em. J. 20, 523-533 (1975). 8. Kraushaar, L. A., Epstein, E., and Zak, B., Characteristics of a 17-ketosteroid reaction. Clin. Chem. 12, 282-288 (1966). 9. Rudd, B. T., and Galal, 0. M., The Zimmermann reaction-past and present. Assoc. Clin. Biochem. 4, 175-179 (1967). JO. Vestergaard, P., and Sayegh, J. F., Semi-automated assays for urinary 17-ketogenic steroids. A comparison of bismuthate and periodate oxidation methods. C/in. Chim. Acta 14, 247-262 (1966). 11. Wilson, H., Chromogenic values of various ketosteroids in a micro modification of the Zimmermann reaction: Comparison with the macro procedure, Arch. Biochem. Biaphys. 52, 217-235 (1954).
JOURNAL
21, 466-477
(1976)
Comparative Chromogenicities and Discrepant Spectra Obtained between a Heterogeneous and Homogeneous Zimmermann Reaction* R. WATKINS
AND C. S. FELDKAMP
Departments of Pathology, Wayne State University School of Medicine and Detroit General Hospital, Detroit, Michigan
E. EPSTEIN William Beaumont Hospital, Royal Oak, Michigan
R. J. THIBERT
AND
B. ZAK
Departments of Pathology, Wayne State University School of Medicine and Detroit General Hospital, Detroit, Michigan, and Department of Chemistry, University of Windsor, Windsor, Ontario, Canada Received
July
7, 1976
Chromogenicity of 17-ketosteroids (17-KS) by Zimmermann reaction using metadinitrobenzene (MDB) in an alkaline medium has not been thoroughly evaluated for all procedures. Methods in which the medium is primarily aqueous, and in which the reactions can be carried out either heterogenously, by a biphasic precipitation reaction (4,6-8) or homogeneously, in a monophasic system (2,3,9), both using Hyamine 1622 (H1622) as a solubilizing detergent, report at least three different orders of chromogenicity for the more common 17-KS encountered (3,4,9). These conflicting reports must be somewhat disturbing to users of the H1622 techniques. The seemingly minor differences in reaction matrices between the several apparently similar procedures do not project as large enough factors to account for the discrepancies, yet they do not result in similar reported chromogenicities generated. Therefore, two methodological examples of widely divergent systems were selected for comparative evaluation. One is heterogeneous and one homogeneous in the color generating phase, with the former a manual modification (6-8) and the latter a semiautomated modification (3) of a manual procedure (4). They differ primarily in the use of methanol (heterogeneous) or ethanol 1 Supported tion.
in part
by a grant-in-aid
from
the Detroit 466
Copyright @ 1976 by Academic Press, Inc. All rights of reproduction in any form reserved.
General
Hospital
Research
Corpora-
CHROMOGENICITIES
OF ZIMMERMANN
REACTIONS
467
(homogeneous) to solubilize extract residues, in the concentration of alkali (KOH) added, in reaction temperature and in the time of reaction before absorbance measurements are obtained. Because controversy of this kind is worthy of resolution, a purpose of the present report is to determine the differences, and if possible any reasons for those differences for the varied behavior of the seven most common 17-KS, when determined by the two types of analytical techniques, since in only one procedure, the semiautomated system, was any attempt made to determine reaction characteristics for all seven compounds. The areas investigated by means of a spectrophotometric study included formation curves for reaction plateaus, the related reaction speeds, chromogenicities, spectral fine structure, peak maxima, color stabilities, and molar absorptivities, all obtained with the seven 17-KS, dehydroisoandrosterone (DHI), androsterone (A), etiocholanolone (E), 1 1-hydroxyandrosterone (HA), 11-hydroxyetiocholanolone (HE), 1 I-ketoandrosterone (KA), and 11-ketoetiocholanolone (KE). It will be established that significant differences do exist in the two analytical systems which mostly account for the reported variations. The heterogeneous system to be described here is one wherein the color reaction is isolated in a precipitated phase which is subsequently solubilized on the reduction of the alkalinity of the system by dilution, whereas the homogeneous procedure is one in which no precipitation is involved, and the reaction takes place in a single phase reaction mixture. MATERIALS
AND METHODS
Reagents The reagents used for the heterogenous procedure were identical to those previously described (6,7). Those used for the homogeneous procedure were as described by Egloff et al. (3) in their description of a semiautomated determination of urinary 17-ketosteroids. Hyamine 1622 solutions: prepare 2.5 and 5% aqueous solutions, and a mixture of 1 part ethanol and 4 parts of 2.5% Hyamine 1622 solution. Potassium hydroxide solutions: prepare 2.0 and 10 N (3,6,7) aqueous solutions. 17-Ketosteroid standards: prepare stock methanolic solution standards of 10 mg/ml of dehydroisoandrosterone (DHI), eticholanolone (E), androsterone (A), 11-hydroxyetiocholanolone (HE), 11-hydroxyandrosterone (HA), 1 lketoetiocholanolone (KE), and 11-ketoandrosterone (KA). Prepare ethanolic solutions of the same compounds at the same concentrations. Prepare a stock standard in the ethanol-Hyamine 1622 solution by dissolving 10 mg of each 17-ketosteroid in 20 ml of ethanol, and then diluting to 100 ml with 2.5% of Hyamine 1622 solution. Metadinitrobenzene solution (saturated): shake 2 g of metadinitrobenzene with 1 liter of 5.0% Hyamine 1622 solution. Clear the reagent by filtration after saturation is complete.
468
WATKINS
ET AL.
Procedures Homogeneous
procedure. Pipet 0.5 ml of the standard into a test tube and carefully evaporate off its methanol. Two alternatives are then available for solution of the residue. Dissolve the residue with 0.5 ml of the H1622-ethanol solution by vigorous vortexing, or dissolve the residue with 0.1 ml of ethanol, and then add 0.4 ml of 2.5% Hyamine. To either, add 0.6 ml of metadinitrobenzene solution and 2.6 ml of 2 N KOH. The second method was used because of difficulty encountered sometimes in dissolving the residue quantitatively in the vortexing step. The solution was warmed at 50” in a water bath for 5 min and then scanned immediately across the visible range from 700-400 nm. This procedure is a close manual approximation of the described automated procedure, an approximation which was necessary in order to carry out the comparative evaluation study. Heterogeneous procedure. This was identical to the one previously described (2,3). Briefly, 10 N KOH and MDB in H1622 are added to a methanolic solution of the residue from an evaporated ether extract of urine. The alkali precipitates the H1622 which removes the forming MDB-17-KS complex from solution. At the completion of the reaction, the precipitate is dissolved by adding more H1622 and the absorbance is measured against a reagent blank. RESULTS
AND DISCUSSION
Figs. IA-G are graphic illustrations of the formation curves for the seven 17-KS when using the homogeneous Zimmermann reaction (3). Several facts are evident from inspection of these results. The rise curves (RC) are different for the several compounds. Some of the compounds are stable after full color formation while others, the 11-ketosteroids, show evidence of fading along the time path of the RC. Full color formation occurs at different times ranging from quite slow to quite fast. This is illustrated by the fast reactions to full color formation (FCF) for the llketo compounds as compared to the partial color formation (PCF) obtained after 5 min heating at 50”. The other compounds react noticeably slower than the 11-keto compounds. Different partial color formations, and this is the method of measurement of the automated homogeneous procedure, are evident for all of the compounds. The spectral characteristics obtained with the seven compounds vary widely and dissimilarities in fine structure are evident for PCF spectra and FCF spectra for the different compounds with the poorest symmetry obtained with the more commonly investigated trio of steroids, DHI, E, and A. The plateaus of the rise curves pictured are not the same as the peak maxima at full color formation. A partial explanation for that phenomenon lies in the fact that the rise curves were graphed at the wavelength of 520
CHROMOGENICITIES
OF
ZIMMERMANN
REACTIONS
469
FIG. 1. Rise curves (RC) for Zimmermann reaction versus time followed at 520 nm. Full color formation (FCF) spectra are also shown along with partial color formation (PCF) spectra for A, 1 I-ketoandrosterone (KA); B, 1 I-ketoetiocholonolone (KE); C, etiocholanolone (E); D, dehydroisoandrosterone (DHI); E, androsterone (A); F, llhydroxyandrosterone (HA); and G, 1I-hydroxyetiocholanolone (HE),
nm which was used in the automated procedure (3), whereas the peaks of the curves ranged across 70 nm. One of the stated advantages in automation is that one need not wait for full color formation in a color reaction as long as measurements can be made at precise times on the rise curve of the reaction of an analyte. Obviously that rationalization disintegrates
470
WATKINS
ET AL.
when one measures mixtures in which rate of formation of color, stability of color, and peak maxima are variables in a common reaction for a mixture of compounds. Strikingly different molar absorptivities for the different 17-KS compounds occur in a situation involving presumptive intensive properties (5), in which so many variables appear to be out of control during the quantitative phase of the procedure. The spectra of Figs. 2A-D were obtained as representatives of the rise curves of the heterogeneous system. The spectra of the three common 17-KS, DHI, E, and A are not shown here because they were reported in a previous publication and their formation curves, peak maxima, and equivalence in chromogenicity are already available (6). Since a precipitate of HI622 is formed in the reaction medium by the action of the strong alkali to which the Zimmermann chromogen (ZC) adheres, perhaps by absorption or post-precipitation, it is not possible to make a continuous measurement for a rise curve as was described for the homogeneous technique. Therefore, increments of 5 min between scans beginning with zero time for the first spectra obtained were used to follow the rate of formation of color for the 11-hydroxy and 11-keto 17-KS in the precipitation system where solubilization of the precipitate by the addition of more detergent to dilute the alkalinity of the solutions to be measured occurred at the times specified on the peaks of the curves. Some indeterminate error is evident in the different peaks, and this can be partially attributed to the fact that the still incomplete reaction of 17-KS with MBD continues after the solubilization of the precipitated H1622 and color complex has been affected by dilution even though that reaction is somewhat slower than when the two phases are present. Several other facts are also evident from the results displayed here. All spectra have an identical peak maximum at 520 nm. None of the spectra show the asymmetrical characteristics obtained with the same steroids when using the automated procedure (3). All rates of formation are similar, so that a common reaction time is easily selected for a mixture of steroids. All of the reactions are stable within the time limits studied and no fading is evident for the four compounds shown here or for the other three compounds previously tested (6). The chromogenic characteristics of the two modifications of what may appear on the surface to be the same reaction are described in Figs. 3 and 4. In Fig. 3, the results of the heterogeneous reaction are shown for the same concentration by weight of DHI as a standard and the 1 l-keto and 11-hydroxy 17-KS, KE, HA, HE, and HA. The hydroxy compounds show less reaction in terms of absorbance on a weight basis than DHI. However, on an equivalent basis they are virtually the same, indicating that the hydroxy group at the eleven position has no apparent effect on the reaction or on the final chromogen. The 1l-keto compounds are simi-
CHROMOGENICITIES
OF
ZIMMERMANN
471
REACTIONS
,
2A “,
HA
WAVELENGTH
WAVELENGTH
= 05 L S EW 5 03 02 01 0
700 +-.ot+ +.ot+ WAVELENGTH
. +-Loo-
4 WAVElENGlH
FIG. 2. Spectra of intermittent reacttons to show rise curve for the Zimmermann reaction versus time for the heterogeneous aqueous technique for 17KS for A, HA; B, HE; C, KA; and D, KE.
lar in absorbance to DHI on a weight basis but are higher than DHI in absorbance on an equivalance basis, lending credence to the notion that the 1 l-ketone positions may be involved in the Zimmermann reaction perhaps either by changing the chromogenic nature of the final reaction compound or by reaction with MDB. This ability to potentiate chromogenicity of the 17-KS has been previously reported (II). The results agree with those reported by Vestergaard for a pyridine version of the Zimmermann reaction (IO) but they dispute his statement about the H1622 procedure, that the “chromogenicity of the 17-oxosteroids formed in the 17-ketogenic assay varies greatly if the Zak and Epstein procedure is used.” Another interesting and important aspect of the reaction is shown in the figure in that the width of the DHI spectrum is greater than those of the other four compounds. Since DHI is commonly used as a standard, this could create a problem if the spectra were subjected to the Allen correction for irrelevant absorption, a correction which is commonly applied in an effort to eliminate errors of a nondescriptive and undetermined kind (1). Some further work is in progress on the evaluation
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of the potential flaws and limitations in the correction of the Allen equation. If one compared E to HE on a weight basis, one would expect that the same calorimetric reaction would yield a lower value for the heavier compound. It has previously been reported that an 1 1-beta-hydroxy group has a suppressing effect on the 17-ketosteroid reaction (11), a fact which is not indicated by the studies described above unless the comparison is made on a weight basis. The weight basis of comparison may be a less proper approach then an equivalence basis for the comparative chromogenicities of equally reacting compounds. Figure 4 shows the paired spectra of DHI, E, and A in the precipitation technique where both methanol and ethanol were used to dissolve the residues of steroids. After a 20 min wait in the precipitated state for both procedures, each reaction was then diluted with the same volumes of HI622 solution to solubilize the precipitates. The weights of steroids used were 40, 35, and 30 E.cgfor E, A, and DHI, respectively. When methanol was used, the linear chromogenicity of the reactions of the three compounds is obvious for what is obviously an equivalency of that reaction when using the reagent makeup of the heterogeneous technique. However, when ethanol was substituted for methanol, the order of chromogenicity changed and E was more sensitive than A even though there was 40 pg of A present as compared to 35 pg of E. This is a striking loss in the equivalency of reaction and the molar absorptivity of the reaction by what appears on the surface to be a miniscule change in reaction conditions. From subsequent observations, it was learned that ethanol also decreased the rate of formation of color of the Zimmermann chromogen. When a comparison was
FIG. 3. Chromogenicitycomparison for equal weights (40 pg) of KA, KE, DHI, HA, and HE using the heterogeneous precipitation technique.
CHROMOGENICITIES
OF ZIMMERMANN
REACTIONS
473
WAVELENGTH
Sensitivity and chromogenicity comparison for DHI (30 pg), E (35 pg), and A (40 wg) for methanol versus ethanol in the heterogeneous technique with a 20 min reaction time in the precipitated state. FIG.
4.
made between methanol (M) and ethanol (E) in the heterogeneous reactions using DHI to demonstrate the spectra obtained at the plateaus of reaction when using each added solvent, the spectra of Fig. 5 were obtained. Even at full color formation, and with alkalinity kept constant for both reactions, the substitution of methanol with ethanol reduced the final color by 36% of the intensity obtained with methanol, which again points out the striking effect of what appears to be a small change in the matrix of reaction. It can be seen therefore, that the slope ratio of sensitivity between the two procedures is 1.5 in favor of the methanolic reaction. In addition, when one considers the variation in time of formation for the seven compounds when using the homogeneous reaction and the different wavelengths of peaks maxima between 450 and 520 nm, it is understandable that determination of absorbance for mixed reaction speeds and variable wavelengths such as is obtained with these seven steroids can lead to nonuniform measurement even when the procedure appears uniform. Uniformity of color for different times of color formation can only be attained at a common plateau for the time factor, or this reaction variable will be misused. One of the key differences between the two procedures, aside from the methanol to ethanol substitution, was the alkalinity of the reaction matrix. In the heterogeneous system, the alkalinity is high enough to precipitate the detergent, H1622, which causes the formed chromogen to leave the solution phase and attach itself to the detergent, or perhaps forms on the surface of the detergent, a phenomenon which appears to speed up the reaction by removing the product of reaction. In contradistinction, in
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g.0450
WAVELENGTH
5. Comparative sensitivity for methanol (M) versus ethanol (E) in the precipitation technique using plateau reaction time in the precipitated state. FIG.
considering the amount of chromogen generated, the homogeneous reaction appears to be about two-thirds complete at equilibrium. There is an alkalinity above which precipitation of the H1622 occurs and more chromogen appears to be formed, so an experiment to demonstrate this is shown as follows. In Fig. 6 are the spectra obtained when the alkali content of the reaction system was varied across a wide range for the heterogeneous procedure but the reaction time before the further addition of HI622 solution was kept constant. In the beginning, when the KOH normality was low, the chromogenicity varied, as did the shape of the spectra. A gray area in which cloudiness occurred appeared beyond 5 N, and as the normality was increased to 10 N, the spectral symmetry stayed constant and only the molar absorptivity changed. Until one uses a solution beyond 5 N, no precipitation occurred, the equilibrium lay somewhat to the left, and the spectra were structured differently when the reaction remained homogeneous than when it became heterogeneous. Beyond 10 N, the precipitate did not dissolve on the addition of the same volume of H1622 solution used for the lower normalities because the resulting normality was still too high to solubilize without further dilution, so no further studies were carried out. Under the described conditions, 10 N KOH appears best suited for the reaction described because of its action in the
CHROMOGENICITIES
OF
ZIMMERMANN
REACTIONS
475
600 500 700 WAVELENGTH FIG. 6. Effect of varying the normality of KOH from 1N to ION using the precipitation 400
technique. Precipitation
occurs between SN and 7N.
HETERO
HOMO
400 WAV: SGTH
FIG. 7. The heterogeneous versus the homogeneous technique under described reaction conditions at 3.5 ml vol. (Hetero) and 3.7 ml vol. (Homo).
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precipitation of H1622 and its ability to increase the amount of color obtained. Figure 7 shows a sensitivity comparison between the homogeneous procedure performed manually and the heterogeneous procedure at the same concentrations of DHI in the two solvents used to prepare standards. Each set of standards was scanned after using the reaction characteristics described for them. The manual version of the automated homogeneous procedure used conditions of reagents, volumes, reaction time, and reaction temperature which were as close as possible to the automated description (3). The final volumes chosen for the comparison favored the heterogeneous system carried out at 3.2 ml as compared to 3.7 ml for the homogeneous system. The differences obtained are mainly mainfested by dissimilarities in spectral structures and in molar absorptivities. In this case, considering sensitivity and previously described variations in chromogenicities, the homogeneous procedure appears to be a poorly modified version of the heterogeneous procedure. SUMMARY A spectrophotometric study is shown for two modifications of a 17-KS procedure in which nearly all aqueous reaction media were used. Although the apparent differences in matrices of reaction between the two procedures appear to be minute, a small volume of ethanol for one is substituted for the same volume of methanol in the other, the final alkalinity of the latter procedure is stronger, and the reaction temperature was increased, wide differences in spectral results are found between the two methods. The ethanolic, lower alkalinity procedure is a homogeneous, single-phase reaction while the methanolic, higher-alkalinity procedure is a heterogeneous system in which a precipitation of detergent occurs and the precipitate becomes an integral part of the color reaction. The rate of formation of the ethanolic procedure is variable for the different 17-KS but it is constant in the methanolic procedure. The chromogenicities of color formation between the two procedures differ in that the methanolic system provides uniform and predictable spectra with more equivalent molar absorptivities. Lastly, the methanolic system shows a much greater sensitivity owing, apparently, to a more complete final reaction. This inability to form full color for several compounds in the homogeneous system can be related in some way to the large variations in ability to form that color owing to matrix composition.
REFERENCES 1. Allen, W., A. Simple method for analyzing complicated absorption curves of use in the calorimetric determination of urinary steroids. J. Clin. Endocrinol, 10, 71-83 (1950). 2. Edwards, R. W. H., Makin, H. L. .I., and Barratt, T. M., The steroid ll-oxygem&~n index: A rapid method for use in the diagnosis of congenital adrenal hyperplasie. J. Endocrin. 30, 181-194 (1964). 3. Egloff, M., Degrelle, H., and Jayle, M. F., Dosage semi-automatique en flux continu des 17-Cetosteroides Urinaires. C/in. Chim. Acta 59, 147-1.54 (1975). 4. Epstein, E., An aqueous Zimmermann reagent for the determination of 17-ketosteroids. Clin Chim. Acta 7, 735-737 (1962). 5. Ewing, G. W., “Instrumental Methods of Chemical Analysis,” 3rd ed. pp. 4bl04. McGraw-Hill, New York, 1969.
CHROMOGENICITIES
OF
ZIMMERMANN
REACTIONS
477
6. Feldkamp, C. S., Epstein, E., Thibert, R. J., and Zak, B., Spectrophotometric study of both the Zimmermann reaction and the application of a corrective measure for irrelevant absorption. Micro&em. J. 21, 45-59 (1976). 7. Feldkamp, C. S., Epstein, E., Thibert, R. J., and Zak, B., Spectrophotometric study of drug interferences in an aqueous 17-ketosteroid reaction. Micro&em. J. 20, 523-533 (1975). 8. Kraushaar, L. A., Epstein, E., and Zak, B., Characteristics of a 17-ketosteroid reaction. Clin. Chem. 12, 282-288 (1966). 9. Rudd, B. T., and Galal, 0. M., The Zimmermann reaction-past and present. Assoc. Clin. Biochem. 4, 175-179 (1967). JO. Vestergaard, P., and Sayegh, J. F., Semi-automated assays for urinary 17-ketogenic steroids. A comparison of bismuthate and periodate oxidation methods. C/in. Chim. Acta 14, 247-262 (1966). 11. Wilson, H., Chromogenic values of various ketosteroids in a micro modification of the Zimmermann reaction: Comparison with the macro procedure, Arch. Biochem. Biaphys. 52, 217-235 (1954).