Enhancement of the Fluorescence of Progesterone (and Other Steroids) in Sulfuric Acid JOSEPH
C. TOUCHSTONE and TARAS MURAWEC
Department o f Obstetrics and Gynecology, School o f Medicine, University o f Pennsylvania, Philudelphia, Pa.
)Certain
steroids when heated with
2 N potassium hydroxide in methanol a t 60" C. for 30 minutes b e f o r e being dissolved in sulfuric acid showed greater fluorescence intensity than when dissolved in sulfuric acid alone. Prior treatment with potassium hydroxi d e caused a hundredfold increase in the fluorescence o f progesterone in sulfuric acid. The estrogens gave a lower fluorescence b y this treatment. This reaction was studied with a variety of steroids.
F
procedures offer an approach to the study of steroidal substances present in biological fluids in submicrogram amounts. There are many references to the use of fluorescence in sulfuric acid for the determination of steroids, particularly the estrogens (4) and hydrocortisone ( 5 ) . Many steroids, hon-ever, do not show sufficiently intense fluorescence mith present methods to permit their determination. ilbelson and Bondy ( 1 ) have utilized the observation of Bush ( 2 ) that A4-3-ketosteroids on paper chromatograms shon- a fluorescence n hen treated JT-ith sodium hydroxide LUORORIETRIC
400
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solution. They used potassium tertbutoxide in tert-butyl alcohol t o develop fluorescent solutions of A4-3-ketosteroids. Chen (S) has utilized coupling with salicyloyl hydrazide for fluorometry of steroids. A previous report (6) indicated that phosphoryl chloride in sulfuric acid, although enhancing the fluorescence of estrogens and certain other steroids, was not effective for fluorometry of progesterone. The heating of certain steroids in 2 5 potassium hydroxide in methanol followed b y solution in sulfuric acid offers certain advantages for fluorometric determination. This results in a tenfold increase of fluorescent intensity of several steroids over that shown when dissolved in sulfuric acid alone. Progesterone showed a hundredfold increase. Other steroids, particularly the estrogens, showed decreased fluorescence intensity in sulfuric acid because of the alkali treatment. This paper describes the use of the reagent and the results of excitation and fluorescence spectra determinations made n ith a Farrand spectrofluorometer. Data on the effect of the alkali treatment on the absorption spectra in sulfuric acid are also presented.
Figure 1. Fluorescence spectrum o f progesterone in 88% sulfuric acid after heating with 2 N methanolic potassium hydroxide
__-
Progesterone - - - Blank
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ANALYTICAL CHEMISTRY
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EXPERIMENTAL
Methanol TI as treated with potassium hydroxide pellets a t least 24 hours and finally distilled froin fresh potassium hydroxide. The reagent was made by dissolving potassium hydroxide in methanol to make a 2 5 solution, which is stable for several days. If ethanol is used in place of methanol, the solutions become colored on standing, but can be used for fluorometry if prepared daily. Baker reagent grade sulfuric acid was diluted with m t e r to give a final concentration of 88%. Diluted acid gave higher fluorescence intensities than concentrated acid. The purity of the steroids was checked by paper chromatography. The steroid solution (or aliquot of extract) n a s pipetted into a 25-ml. Erlenmeyer flask and dried in a vacuum oven or evaporated n i t h a stream of nitrogen. A flask containing the equivalent amount of solvent was used as a blank. After evaporation of the solvents 0.5 ml. of the alkali n-as added and the mixtures n ere allowed to stand for 10 minutes before being heated a t 60" C. for 30 minutes. The solutions were then cooled to room temperature, 5.0 ml. of 88% sulfuric acid nere added (less if microcuvettes nere available), and the mixture was heated a t 120" for 15 minutes or allowed to stand at room temperature for 15 minutes. The most favorable conditions in sulfuric acid after the alkali treatment varied with the steroid. For progesterone, room temperature showed maximal results, while 120" C. for 15 minutes was suitable for 1'7hydroxyprogesterone. Steroid levels of 0.01 to 1.0 y per ml. of sulfuric acid could be measured satisfactorily. For absorption determinations 10 y per ml. v, ere rrquired.
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Figure 2 . Absorption spectrum of progesterone (10 y per ml.) in sulfuric acid a f t e r alkali treatment -- Heated at 60' C. for 30 minutes with 2N methanolic KOH, then dissolved in 88% H&Oc 88% H?SOaonly at room temperature
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Table I.
Fluorometric Properties of Steroids
KOhle R TE F
+ 88% H&Oa 120"-15'
--
F.I.
E
F
F.I.
490 500
40 0 8 0
390 450
500 500
20 0 12 0
450 450 460 460 390 460
500 500 500 500 450 500
6 9 21 22 7
0 0 0 0 0 15 0
450 450 460 460 390 460
500 500 500 500 450 500
10 19 20 25 13 22
2 0 8 0
390 450
480 510
4 0 2 0
450 460 460 390 450 450 390 450
510 520 510 460 500 510 450 500
20.0 :3 0 :3 5 3 0 10 0 19 0 23 0 18 0
510 i 0 460 510 2 0 460 510 6.0 460 460 Digitoxin Room temperature. * Relative fluorescence intensity above blank. Galvanometer deflection ( 1-1 00) X sensitivity (0.01-1000). c E. hlnximum of excitation spectrum. F. Maximum of emission spectrum.
500
18 0
Steroid Progesterone
F.1.b
Fc
Ec
F.I.
u
390 450
0 0 0 0 0 0
390 450
4-10 500
0.2 0.4
390 460
440 510
3 0 3 0
Pregnanolone Allopregnanedio1-3~,20a Pregnanediol-3aj20a Pregnanetriol
17cu-Hydro~).progesterone
450 450 460 460 390 450
500 500 500 500 460 500
0.4 0.9 10.0 9.0 5.0 15.0
460 460 460 460 390 460
510 510 510 510 460 510
Estrone
390 450
460 470
20 0 400.0
300 440
470 480
67 0 670 0
390 450
480 500
Estradiol-1 70 Estriol Corticosteronr Cortisone Hydrocortisone Testosterone Androstane-3,lT-dione
450 460 460 390 460 460 390 460
490 530 500 460 520 520 460 510
500 0 150.0 36 0 1. 0 43.0 2.5 0.2 0.2
450 460 460 390 460 460 390 460
480 520 510 450 520 510 460 510
500 0 150 0 6 0 3 0 30 0 10 0 0 7 0 2
450 460 460 390 450 450 390 450
500 520 520 470 500 510 450 500
4 22 30 37 23 50
30 2 1 1 3 7 3 2
0 0 4 4 7 0 8 2
0 0 0 0 0 0
0
RESULTS
The fluorescence produced by various steroids after treatment with 2 S pot,assium hydroxide in methanol follon-ed by solution in sulfuric acid may be varied, depending upon the heating prior to addition of sulfuric acid and the heating of the final sulfuric acid solution. Because the methanol was largely evaporated in the initial heating step, t h e conccntrntion of the potassium hydroxitic apparently n-as not critical. A 10% solution of potassium hydroxide in methanol \vas a k o used with satisfactory results. So major day-to-da!variations ocwirred in the intensity of the fluorescenw of progesterone and 17liydros!-progesteronc:. I n Table I are listed the maxima of the excitat,ion and fluorescence spectra and the fluorescence intensities of a number of steroids treated as follolvs: 1. Dissolved in 887, sulfuric acid a t room tcniperature
2. As in 1, then heated a t 120" C. for 15 minutes 3. Heated in 211- potassium hydro.;ide in methanol a t 60" C. for 30 minutes, then dissolved in 88% sulfuric acid a t room temperature 4. As in 3, but heated in 88% sulfuric acid a t 120" C. for 15 minutes These determinations were made on a Farrand n h d e l 1754-17 spectrophotofluorometer using quartz cuvettes (10 x 10 x TO mni.). KO attempt was made to correct for the spectral rcsponse of the multiplier phototube of the instrument. Table I indicates that A4-3-ketosteroids did not all show a greater fluorescence intensity with the reagent than TT hen sulfuric acid alone was present. The fluorescence of the estrogens in sulfuric acid n as greatly diminished by the primary treatment with 2 3 potassium hydroxide in methanol. Testosterone and androstane-3,li-dione shoned increases in the intensity of the /
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T
t 300
:
1--~7--/-rii 1 1 1 / 400 500 WAVE LENGTH, Mr
Figure 3. Absorption spectrum of estrone (10 y per ml.) in sulfuric acid after alkali treatment -H e a t e d at 60' C. for 30 minutes with 2N methanolic KOH, t h e n dissolved in 8870 H~SOI 88% HlS04 only at room t e m p e r a t u r e
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emission. Other steroids shoned decreases in fluorescence intensity due t o prior treatment, with the modified procedure. Figure 1 shows the fluorescence spectrum of progesterone after treatment with the methanolic potassium hydroside and dissolving in 887, sulfuric acid, as described, and the blank result'ing from the same conditions of development. The various other steroids shon-ed only slight differences in the maxima of the emission spectra after treatment iyith potassium hydroxide in methanol plus sulfuric acid when compared x i t h sulfuric acid alone. second maximum for excitation and emission IT-as indicat,ed in some instances. Sulfuric acid alone with or without heating gave more fluorescence for the estrogens and certain other steroids than the procedure using 2 5 potassium hydroside in methanol. Figure 2 sho1v-s the result of prior treatment with potassium hydroxide in methanol on the absorption spectrum of progesterone dissolved in sulfuric acid, as compared with that obtained by dissolving in 887, sulfuric acid alone. In 8891, sulfuric acid progest,erone has a niaximuni at 292 mp only. After the modified procedure a second maximum a t 370 mp appeared. This is probably responsible for the fluorescence seen a t 490 mp when excitation is carried out a t 390 mp. (The effect of alkali on the steroid molecule under these conditions is under investigation.) Figure 3 presents the absorption spectra of estrone dissolved in 88% sulfuric acid and after treatment b y the present procedure, which produced a A \
VOL. 32, NO.
7,JUNE 1960
823
new peak in the spectrum. There was a decrease in the fluorescence intensity of estrone in sulfuric acid due to this treatment, but this vas not associated with an equivalent decreaqe in absorption a t 450 mp. Preliminary studies concerning the action of the alcoholic potassium hydroxide on the progesterone molecule have indicated that a substance more polar than progesterone was formed. Paper chromatography in the methylcyclohexane-propylene glycol system showed a substance of mobility between that of desoxycorticosterone and progesterone. The new substance resulted from treatment with niethanolic po-
tassium hydroxide and n as isolated after neutralization of the alkali and extraction with ether. I n contrast, progesterone itself can be recovered unchanged after standing at room temperature in concentrated sulfuric acid solution. The nen- fluorescence method for progesterone is sensitive to 0.05 y and has been used successfully for the determination of this substance in peripheral blood plasma. The results of these determinations mill be published later. LITERATURE CITED
(1) Abelson, D., Bondy, P. I
Routine phosphate analyses can b e simplified by the use of a unique instrument which automates a variety of colorimetric procedures. Two classical colorimetric methods have been adapted to the programming facilities of the apparatus with the result that orthophosphate or total inorganic phosphate determinations can be performed at sampling rates of 20 per hour on solutions containing condensed phosphates. The standard deviation of a single measurement by either Within these analysis is about 1%. precision limits the accuracy of the determinations is equal to that of classical methods. Untreated aqueous solutions of heavy-duty synthetic detergent products are sampled a t 1 or 0.1 0% concentrations. Continuous nonequilibrium dialysis within the systems described is responsible for relatively interference-free operation with such samples. Considerable time is saved, and the methods used appear to b e generally applicable to other problems in phosphate analysis.
E
for automating complete colorimetric procedures becomes economically desirable where large numbers of samples must be processed by a routine consisting of essentially repetitive operations. The Technicon AutoAnalyzer (Technicon Instruments Corp., Chauncey, N. Y.) automates a vaQUIPMEKT
824
ANALYTICAL CHEMISTRY
Co., Edgewafer, N. 1.
riety of colorimetric manipulations including sampling solutions, diluting, adding reagent solutions sequentially, mixing, filtering, separating interferences, heating or cooling, and measuring and recording transmittance values relative to the blank for subsequent calculation b y the analyst Typical applications of this equipment have included the determination of urea nitrogen ( 7 ) and the determination of streptomycin and penicillin in industrial fermentation media (3). The selective determination of orthophosphate in the presence of condensed phosphates was selected for study with the instrument. This investigation led logically to the determination of t o t a l inorganic phosphate in mixtures. TKO classical colorimetric methods were adapted to the programming facilities of the instrument. For the orthophosphate analysis, reduction of phosphomolybdic acid with 1-amino-2-naphthol-4-sulfonic acid a t room teniperature and moderate acidity was chosen. These conditions were established by Fiske and Subbarow (4) and are sufficiently mild to prevent extensive degradation of condensed phosphates. Boltz and Mellon’s (1) method of reducing phosphomolybdic acid n-ith hydrazine a t high temperature and high acidity was chosen for the colorimetry of total phosphate determinations. This Colorimetric system possesses the re-
quired tolerance for the high concentration of mineral acid which must be introduced a t an earlier stage for rapid quantitative degradation of condensed phosphates; furthermore, the system is extremely sensitive, stable, and relatively interference-free.
I
EXPERIMENTAL
Description of Equipment. T h e Technicon Aut0~4nalyzer n-as conceived by Skeggs ( 1 1 ) and has been described by Muller (9). Addition of a second heating bath, an extra set of dialyzer plates, and matched pairs of 660- and 805-mp interference filters for the colorimeter provides all the equipment needed for the phosphate applications. The mechanics and principles of operation of the lutohnalyzer system have been discussed by Ferrari, Russo-Alesi, and Kelly (3) ; substantially identical instrument components are used for phosphate analysis. Flow Schemes. Both phosphate flow schemes have s i r poly(viny1 chloride) tubes in the proportioning p u m p for t h e continuous delivery of streams of constant composition. T h e tubes used have internal diameters between 0.035 and 0.081 inch and are color-coded b y t h e manufacturer for nominal deliveries in milliliters per minute. Four tubes deliver reagent solutions or diluents; the other two deliver air. The seventh tube in each manifold is the only tube d i i c h is not introducing a stream of constant composition; during one interval this
