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Fluoxymesterone analysis by gas chromatography
CLINICA CHIMICA ACTA
FLUOXYMESTERONE
MARYON
253
ANALYSIS
BY GAS CHROMATOGRAPHY*
W. RUCHELMAN
Department of Medicine, The University of Texas, M. D. Anderson Hosfiital and Tumor Institute at Houston, Texas 77025 (U.S.A.) (Revised manuscript
received April I, 1969)
SUMMARY
A new method for analyzing fluoxymesterone by gas chromatography utilizes the large difference in polarity between it and other ketosteroids. Its retention time is selectively shifted beyond their range. This novel procedure of separation might also be expanded to evaluate other steroids and drugs of unusually high polarity in biological fluids.
A new method for analyzing fluoxymesterone (FT) (Halotestin)** by gas chromatography (GC) has been developed in this laboratory. FT is an oral anabolic agent known since 1956 (ref. I). It has a particularly favorable anabolic - androgenic ratio which makes it useful for treatment of debilitated hospital patients2. At present, there is a research project here or the effect of androgenic hormone therapy with FT on the urinary excretion of erythropoietin in normal men and women3. It was for this later study that our method of analysis was devised. It is necessary to measure the urinary excretion of FT after ingestion of various size doses. This GC method evaluates urinary FT and its isomers by utilizing the large difference in polarity (due primarily to the fluorine atom) between FT and other ketosteroids (KS). A computer search of the literature was conducted by The Upjohn Company4 in 1966. It showed no GC procedures, with only two other methods being used. A quantitative bioassay with respect to the action of FT on the seminal vesicles of castrated rats showed only slight promise in 1962 (ref. 5). Current analytical procedure consists of a tetrazolium calorimetric method, similar to that described in the U.S. Pharmacopeiaa for the assay of prednisolone ‘, for steroids with a reducing functional group of the a-ketol type. Such a calorimetric procedure may be adequate for the analysis of FT in compressed tablets which have no interfering substances, but would not be suitable for that of FT in biological fluids where many similar steroids are also * Presented in part at the Sumposium on Biomedical Applications of Gas Chromatography at the Seventh National Meeting of the Society for Applied Spectroscopy, Chicago, Illinois, May ‘3-17, 1968. ** Systematic equivalent: ga-fluoro-1 rp-hydroxy-IT-methyl-testotsterone: ga-fluoro-r7a-methyl4-androstente-r1B,17,%diol-3-one. CbZ. Chim. Ada, 26 (1969)
253-262
RUCHELMAN
254
present which might give color. We felt it necessary to devise a method by means of GC for fractionating the androgenic steroids and determining quantitatively the presence of FT and its isomers in the urine.
Fig. I. Testosterone 294.5?.
(M.W. 228.41; m.p. 155”) and Fluoxymesterone
(M.W. 339.45; m.p. zg2.5-
In observing the molecular structures of FT and testosterone (T) (which happens to be the most potent but virilizing androgen) (Fig. I), FT differs from T by the TABLE
I
RETENTION TIMES BY GAS-LIQUID CHROMATOGRAPHY Retention time (min)
Steroids *
T Epi-T FT FT-dione 17.KS-TMS (longest ret. time, for 11-OH-E *) 17-KS T T-TMS Ed-T Epi-T-TMS FT 17-KS-TMS (longest ret. time, for II-OH-E*)
9.4
8.5 36.8 16.8
3% XE-60, 3ft. x 4 mm, N, 18 lb. inlet, col. temp. 228’
4.5 13.1 51 21.9 48 17.4
4% XE-60, 6 ft. x 5 mm, N, IO lb, inlet, col. temp. 212’
33
T FT I7-KS-TMS (longest ret. time, for II-OH-E*).
151.5 315
T T-TMS Epi-T Epi-T-TMS FT FT-TMS
79.5 30.8 73.5 24.9 315 13.5
Cholesterol-TMS FT-TMS
Column conditions
3% XE-60, 12 ft. x 5 mm, N, 13 lb. inlet, col. temp. 211’.
87
3o 425,627
3% XE-60, 12 ft. x 5 mm, N, 24 lb. inlet, col. temp. 211'
4% XE-60, 12 ft. x I.5 mm, N, IO lb. inlet, col. temp. 225’
* Abbreviations in tables : FT-dione (gcr-fluoro-I7B-hydroxy-I7-methyl-4-androstene-3,11-dione). I I -OH-E (I I ,!-hydroxyetiocholanolone). Clin. Chim.
Acta,
26 (1969) 253-262
GASCHROMATOGRAPHY OF FLUOXYMESTERONE
2.55
addition of three substituents (gu-fluoro; II,&01; and 17a-methyl). They introduce a large difference in polarity. This can be shown, first, by the longer retention times for FT on various GC columns when compared to T, epitestosterone (epi-T) (where the 17-OH is a), androsterone (A), an d other r7-ketosteroids (17-KS) (Table I). This is also shown by its small RF value on thin-layer chromatography (TLC) plates in several solvent systems (Table II). On these TLC plates, the spots of FT, FT-dione, TABLE II Steroid (Main isomer)
Distance traveled (in cm)
RF* (Solv. front 29.7
FT T Epi-T A
12.4 12.6 12.5
0.42 0.42 0.42 0.39 (Solv. front 33.4 cm)
Ft T Epi-T A FT T Epi-T A
11.6
0.12
3.9 8.95 8.9
0.27 0.27 0.38 (Solv.
‘2.55 0
5.26 5.05 7.7
* TLC plates were 20
x
cm)
0
0.16 0.15 0.23
Solvent system
Ligroin-toluenemethanol-water (5:5:7:3. 43h) Benzene-ethylacetath)
(1.1, 2
front 33.6 cm)
Benzene-ether (I:I, 2 h)
40 cm.
T and epi-T fluoresced in the UV at 2537 A, and both they and A appeared in I, vapors. Due to this increase in polarity of FT, a polar-type phase for GC, therefore, seemed to be advisable. Also because of the large polarity and high molecular weight of FT, exploratory experiments were set up to determine whether derivative formation might be of advantage in GC. Derivatives allow a way of increasing area response and of selectively shifting a compound from its background. Kirschners used the bis-methylenedioxy derivative of cortisone for this purpose, and for thermal stability. The FT molecule (Fig. I) contains one relatively unhindered hydroxy group at the 17 position which should convert to a trimethylsilyl ether (TMS). There is a sterically blocked hydroxy at C-II, which under conditions of silanization used with the 17-KS herea, would probably not convert. TMS derivatives might have shorter retention, and they often permit resolution of close isomers, too. When a standard solution of FT (IO mg/roo ml) and a standard solution of cholesterol were converted to TMS, four major peaks resulted for FT, which had a combined area which might have been expected for one peak. Even on the Iz-foot x 1.5 mm glass column (Table I), the retention times of these four FT peaks were very close to the solvent front (tetrahydrofuran) (THF) and close together (4, 5, 6 and 7 min). The cholesterol TMS showed only one peak with a retention time of 30 min, under the same conditions. The several TMS peaks which resulted from FT would have been covered by other 17-KS TMS when run from a urine extract. Perhaps the four peaks resulted from partial conversion to TMS of both hydroxy groups (17- and Clin. Chim. Acta, 26
(1969) 253-262
256
RUCHELMAN
II/?-diol), of two close isomers of FT. (On the rz-foot column of 3% XE-60 at low carrier gas pressure, although the retention time was over 5 h, the skew FT peak indicated two isomers. They only differed, perhaps, by the a- or fi-orientation of a hydrogen atom.) Therefore, conversion to FT-TMS was not advisable for quantitation. Shorter columns would reduce the unduly long retention time of FT as the free steroid; however, good resolution was necessary, too. It was decided to remove the less polar 17-KS from the vicinity of FT on the column by condensing their retention times close to that of the solvent front. Letting the GC remove these steroids from the retention time of interest, that of FT, would be as valuable as possibly several additional separation steps by wet methods. It would not be necessary to form derivatives, either, which require more time and manipulations. Starting with this new idea, we first wanted to observe FT as the free steroid, from a standard solution. Fig. 2 shows symmetrical peaks for various concentrations of FT as a free steroid on a 3% XE-60 column, 3-foot x 4 mm, column temperature 226’, injector 260°, detector 260’. N, was 18 pounds inlet pressure. These were Gaussian peaks, good for quantitation, for amounts of about 2 ,ug to 16 clg per injection of 2 ~11.
Fig. 2. FT Standards,
by GLC.
Fig. 3 shows how the free steroid was isolated on this column and clear of other, less polar 17-KS hydrolyzed from the urine by our method. (This preparation of the urine will be explained later.) Compounds with retention times close to FT are Clin. Chim.
Acta,
26 (1969)
253-262
GAS CHROMATOGRAPHY
OF FLUOXYMESTERONE
HYDROLYZED Ibrh.,
UNHYDROLYZED IAfter FT,
257
URINE
URINE
HYDROLYZED URINE ,*,,a FT,
Fig. 3. FT from urine samples, after ingestion of low-level amounts.
possible isomers due to metabolic changes in the body. This urinary FT plus the three isomers amounted to about 500 pg/24 h after ingestion of a 20 mg daily dose. This is a low level. We have often used whole sample injection for small amounts of FT with our automatic injection system for GP. Fig. 4 shows the chromatogram for a patient, a male, 6g years old, with lymphoma, who was on 200 mg/day of FT. He excreted 2 mg/24 h in the urine. PATIENT’S URINE wig+Lml FT,
Fig. 4. FT from patient’s urine, after ingestion of high-level amounts. Clin. Chim. Acta, 26 (1969)
253-262
258
RUCHELMAN
Fig. 5 shows how FT added to urine samples matched the retention time of free ET, and was unchanged by a mild hot acid hydrolysis of only 15 min. Also, FT added to tap-water showed no damage after hydrolysis. (Although other 17-KS may not be completely hydrolyzed in less than an hour Q,FT was).
Fig. 5. Recovery
studies
of hydrolysis
by mild hot-acid
for 15 min.
In this chromatogram, unlike in Fig. 3, there are no other compounds with retention times close to FT. Therefore, these compounds are not changed versions of FT due to the hydrolysis procedure. They did not match the retention time of FTdione (Table I). The 3-OH derivative of FT would have had a much longer retention time than FT due to its extra absorption properties. No other similar compounds were available. It was felt that this tentative identification of the isomers, as described under TMS, would suffice at present. No synthesis or further conversion of FT was undertaken to produce compounds for absolute identification-too many, subtle changes, with exacting sterochemistry, were felt possible, Smaller amounts of added FT were recovered in comparable percentage. Quantitation was now possible by GC. This allowed us to analyze for any’ free FT that was excreted in the urine, or any FT in the conjugated form (after it was hydrolyzed). MATERIALS
ET-obtained as Halotestin, No, U-6040, from The Upjohn Co., Kalamazoo, Mich., mp (on Fisher melting block, uncorrected), zgz.5~z94.5”, without decomposition. FT-d&e-The Upjohn Co., Kalamazoo, Mich. Silica Gel HFzM (No. 7739) from B~nkman Instruments, Inc., Westbury, L.I., N.Y. T, E$i-T, A, and other 17-E?. Man Research Lab., Inc., New York, N.Y., and Ikapharm, Ramat-Gan, Israel. All solvents: from Fisher Scientific Co., Houston, Tex. Reagent grade. Stored over drying agents, redistilled when necessary. Hexamethyldisilazane and trimethylchlorosilane reagents. Applied Science Labs., Inc., State College, Penn. Ctin. Chiwz. Acta, 26 (1969) 253~262
GAS CHROMATOGRAPHY
OF FLUOXYMESTERONE
259
XE-60 (G. E. nitrile silicone gum) phase on 80/100 mesh Gas-Chrom Q, obtained as 3% and as 4%, also coated by Applied Science Labs., Inc., State College, Penn. METHOD
Our work was initiated with FT, as oral tablets of Halotestin, which also contained a carrier, a dye, and stabilizing agents. FT could be leached from these tablets by various organic solvents, including THF, acetone, and ethanol, which also extracted the other substances in different amounts. Identification was indicated by retention times on various polar GC columns and also by TLC. Quantitation became possible upon receipt of a 5-gram sample of pure FT from The Upjohn Company. The retention time of this pure FT matched that of FT leached from the oral tablets. Standard solutions of FT were made up in several strengths in THF. The most acceptable was a standard of 208 ,ug/ml, which was used for low-level amounts of FT in urine, and for high-level amounts when they were diluted. For the relationship between concentration and peak area, I, 2, 3, and 4 ml were evaporated in individual conical, r5-ml centrifuge tubes, then redissolved in IOO ~1 of THF each. An injection of exactly 2 1’1 was made of each sample. Several chromatograms of each were run in succession, and an average taken. Fig. 2 shows chromatograms of the increasing amounts of FT per injection. The areas were measured by means of a Disc Integrator (Disc Instruments, Inc.), corrected for drift where relevant. Also, by triangulation, where overlap of isomers in some urine samples. Quantitative GC by GillI’ with comparison of triangulation and Disc integration shows a standard deviation of 0.80% and 0.47% respectively. Liquid injection was with a IO-$ syringe (Glass Engineering Co., Houston, Texas.). Fig. 6 is a Concentration vs. Area graph, with a slope of I.47 X IO@ g/count. It is made up from the measurements of the chromatograms shown in Fig. 2. This
Kg.
6. Linearity
of concentration
us. peak area of FT. C&z. Chim. Acta, 26 (1969) 253-262
260
RUCHELMAN
shows the linearity of the various amounts of FT, as far as detector response and column behavior toward this steroid. Using this graph, area (in counts) of peaks after injection of various amounts of standard solutions of FT were matched with concentration on they-axis. They agreed with calculations by Sr: 1%. Each week a new set of chromato~ams is run. The slope changes slowly after the initial column conditioning, with a slightly larger area per injection as the weeks progress. The column was four weeks old when these chromatograms were run. Although several compounds of large polarity, including zr-fluoro-progesterone (progesterone, zr-~uoro-II~,I7a-dihydroxy-4~-methyl) were tested for the possibility of an internal standard, none seemed adequate. 21-Fluoro-progesterone decomposed with GC.
A baseline urine and urine after several days of ingestion of FT were collected for 24 h each. An aliquot of IIO ml was withdrawn for routine identification of 17-KS9 and an aliquot of IIO ml for FT. The balance would then be dialyzed to a urine concentrate as described previouslylZ, and reconstituted with saline to original volume. From this latter, IIO ml would be withdrawn again for routine r7-KS in order to determine the amount remaining within the dialysis membrane, and IIO ml for FT analysis. Sometimes, the concentrate would be analyzed without reconstitution, as it was in the membrane. The balance of the reconstituted volume would be used for erythropoietin studies”. The first step was to extract the original, unhydrolyzed urine samples with ether, which was then dried and evaporated. THF solvent would be added (o.c5 to 0.10 ml, depending upon expected amount of FT) for injection into GC. This chromatogam would show an unconjugated FT or any isomers of it. (Fig. 3, middle chromatogram, showing any steroids excreted in “free” form.) The other, aqueous fraction of each urine sample would then be hydrolyzed as for the routine 17-KS by a mild hot-acid method* but only for 15 min. Enzymatic hydrolysis for three days had revealed basically the same amounts of FT and its isomers, about 95% & 5% were recovered by either method (Fig. 5). This indicates that FT and its other isomers are conjugated mainly as glucuronides. After routine purification and removal of acids and phenolic steroids, the ether extract of the hydrolyzed, neutral androgens was dried and evaporated, with THF added for injection into GC. (Fig. 3, bottom chromatogram, showing that FT and Isomer No. I are excreted as glucuronic acid salts. Apparently, isomers Nos. z and 3 are not excreted in this way but are organic-soluble.) Determinations were complete within a day. Gas chromatography This analysis was run on a Barber-Coleman Series 5ooo. The 4 mm i.d. columns were glass: g-foot U-tubes, 6-foot U-tubes, and rz-foot W-tubes. They were packed with 3% or 4% XE-60 phase on Gas Chrom Q support, coated by Applied Science Labs. Glass wool plugs were siliconized. Column conditions are given in Table I. The attenuator of the GC was set at 5, the sensitivity at 1000. The 5 mV recorder speed was 20 in/h.
CGn.Chim.
Acta, zb(1969) 253-262
GAS CHROMATOGRAPHY
OF FL~~XYMEST~RONE
261
DISCUSSION
The aim of this research was to develop an accurate, reproducible and relatively rapid procedure for analysis of FT in urine and reconstituted urine from dialysis. The amount of FT excreted ranged from 500 c/g to z mg/zq h. The amount of FT and that of the 17-KS within the dialysis membrane remained the same per ml. That is, I pugFT/ml, if IOOOml before dialysis, was I /‘g/ml if 17 ml afterwards. GC proved to be a sensitive means of analysis. Since less than 5% of ingested FT (plus its close isomers) can be accounted for in the urine, either some is excreted in the feces or in the form of other metabolites in the urine. In the routine 17-KS analysis, there were several large peaks as TMS derivatives which had retention times past the usual steroid TMS’s. Future work entails the tracing of metabolites of FT as excreted in the urine. Far less than the minimum quantity of 150 pg required to stimulate radioiron utilization in polycythemic mice has been accounted for. It is felt that the metabolites of FT will probably contain a fluorine atom, which may be traced by means of an electron capture cell on GC analysis. This will probably allow further identification of Isomers Nos. I, 2, and 3. These metabolites might be of prime importance in physiological mechanisms of erythropoietin. FT and its metabolites in plasma will also be analyzed. This CC method isolates FT and any close isomers from other 17-KS by concentrating them near the solvent front far from the retention time of FT. This is a new way of resolution without derivative formation or alteration of the molecule by other chemical means. This type of analysis might also be expanded to evaluate other steroids and drugs of unusually high polarity in biological fluids. It certainly appears promising for future experimental work, as such compounds could thus be selectively shifted beyond the range of their background interferences. ACKNOWLEDGMENT
The author wishes to acknowledge the helpful information and advice about FT from Doctor Raymond Alexanian, and the able technical assistance of Brenda Prewitt and Pamela Herrera. This study was supported by U.S.P.H.S. Research Grant No. Ca 05831. The supply of FT and FT-dione used in this study was supplied by The Upjohn Company, Kalamazoo, Michigan. REFEKENCES I M. E. HERR, J. A. HOGG AND R. H. LPVIN, J. Am. Chem. Sot., 78 (1~56) 500. 2 L. I?. FIESER AND M. FIESER, Steroids, Reinhold Publishing Corp., New York, 1959. p. 593, 3 R. ALEXANIAN, W. K. VAUGHN AND M. MI. RUCHELMAN, J. Lab. C&n. Med., 70 (1967) 777. 4 Computer tape of references sent to DR. SHIZUTOMO KATSUTO, this dept., by DR. H. H. ANGE~L of ‘The Upjohn Co,. Kalamazoo, Mich, in Feb., 1966. 5 E. J. CLEGG AND T. M. FARLEY, J. Repvod. Fertit., 4 (1962) 125. 6 The P~ayrn~ope~u ofthe U.S. of America (The U. S. Ph~rrn~a~e~a~, r7 th rev., Mack Publishing Co., Easton, Pa., x965, p. 887. 7 P. W. O’CONNEL, Biological Screening Office, ‘The Upjohn Co., Kalamazoo, Mich., personal communication, 1966. 8 M. A. KIRSCHNER AND H. M. FALES, Anal. Chem., 34 (196~) 1548. Clin. Chim. Acta, 26 (1969) 253-262
262 g M. W. RIJCHELMAN AND V. W. COLE, Clin. Chem., 12 (1966) 771. IO M. W. RUCHELMAN, J. Gas Chrom., 4 (1966) 265. II J. M. GILL AND H. M. MCNAIR, Aerograph Res. Notes, Fall Issue (1956). 12 R. ALEXANIAN, Blood, 28 (1966) 344.
Clin. Chim. Acta, 26 (1969) 253-262
RUCHELMAN
FLUOXYMESTERONE
MARYON
253
ANALYSIS
BY GAS CHROMATOGRAPHY*
W. RUCHELMAN
Department of Medicine, The University of Texas, M. D. Anderson Hosfiital and Tumor Institute at Houston, Texas 77025 (U.S.A.) (Revised manuscript
received April I, 1969)
SUMMARY
A new method for analyzing fluoxymesterone by gas chromatography utilizes the large difference in polarity between it and other ketosteroids. Its retention time is selectively shifted beyond their range. This novel procedure of separation might also be expanded to evaluate other steroids and drugs of unusually high polarity in biological fluids.
A new method for analyzing fluoxymesterone (FT) (Halotestin)** by gas chromatography (GC) has been developed in this laboratory. FT is an oral anabolic agent known since 1956 (ref. I). It has a particularly favorable anabolic - androgenic ratio which makes it useful for treatment of debilitated hospital patients2. At present, there is a research project here or the effect of androgenic hormone therapy with FT on the urinary excretion of erythropoietin in normal men and women3. It was for this later study that our method of analysis was devised. It is necessary to measure the urinary excretion of FT after ingestion of various size doses. This GC method evaluates urinary FT and its isomers by utilizing the large difference in polarity (due primarily to the fluorine atom) between FT and other ketosteroids (KS). A computer search of the literature was conducted by The Upjohn Company4 in 1966. It showed no GC procedures, with only two other methods being used. A quantitative bioassay with respect to the action of FT on the seminal vesicles of castrated rats showed only slight promise in 1962 (ref. 5). Current analytical procedure consists of a tetrazolium calorimetric method, similar to that described in the U.S. Pharmacopeiaa for the assay of prednisolone ‘, for steroids with a reducing functional group of the a-ketol type. Such a calorimetric procedure may be adequate for the analysis of FT in compressed tablets which have no interfering substances, but would not be suitable for that of FT in biological fluids where many similar steroids are also * Presented in part at the Sumposium on Biomedical Applications of Gas Chromatography at the Seventh National Meeting of the Society for Applied Spectroscopy, Chicago, Illinois, May ‘3-17, 1968. ** Systematic equivalent: ga-fluoro-1 rp-hydroxy-IT-methyl-testotsterone: ga-fluoro-r7a-methyl4-androstente-r1B,17,%diol-3-one. CbZ. Chim. Ada, 26 (1969)
253-262
RUCHELMAN
254
present which might give color. We felt it necessary to devise a method by means of GC for fractionating the androgenic steroids and determining quantitatively the presence of FT and its isomers in the urine.
Fig. I. Testosterone 294.5?.
(M.W. 228.41; m.p. 155”) and Fluoxymesterone
(M.W. 339.45; m.p. zg2.5-
In observing the molecular structures of FT and testosterone (T) (which happens to be the most potent but virilizing androgen) (Fig. I), FT differs from T by the TABLE
I
RETENTION TIMES BY GAS-LIQUID CHROMATOGRAPHY Retention time (min)
Steroids *
T Epi-T FT FT-dione 17.KS-TMS (longest ret. time, for 11-OH-E *) 17-KS T T-TMS Ed-T Epi-T-TMS FT 17-KS-TMS (longest ret. time, for II-OH-E*)
9.4
8.5 36.8 16.8
3% XE-60, 3ft. x 4 mm, N, 18 lb. inlet, col. temp. 228’
4.5 13.1 51 21.9 48 17.4
4% XE-60, 6 ft. x 5 mm, N, IO lb, inlet, col. temp. 212’
33
T FT I7-KS-TMS (longest ret. time, for II-OH-E*).
151.5 315
T T-TMS Epi-T Epi-T-TMS FT FT-TMS
79.5 30.8 73.5 24.9 315 13.5
Cholesterol-TMS FT-TMS
Column conditions
3% XE-60, 12 ft. x 5 mm, N, 13 lb. inlet, col. temp. 211’.
87
3o 425,627
3% XE-60, 12 ft. x 5 mm, N, 24 lb. inlet, col. temp. 211'
4% XE-60, 12 ft. x I.5 mm, N, IO lb. inlet, col. temp. 225’
* Abbreviations in tables : FT-dione (gcr-fluoro-I7B-hydroxy-I7-methyl-4-androstene-3,11-dione). I I -OH-E (I I ,!-hydroxyetiocholanolone). Clin. Chim.
Acta,
26 (1969) 253-262
GASCHROMATOGRAPHY OF FLUOXYMESTERONE
2.55
addition of three substituents (gu-fluoro; II,&01; and 17a-methyl). They introduce a large difference in polarity. This can be shown, first, by the longer retention times for FT on various GC columns when compared to T, epitestosterone (epi-T) (where the 17-OH is a), androsterone (A), an d other r7-ketosteroids (17-KS) (Table I). This is also shown by its small RF value on thin-layer chromatography (TLC) plates in several solvent systems (Table II). On these TLC plates, the spots of FT, FT-dione, TABLE II Steroid (Main isomer)
Distance traveled (in cm)
RF* (Solv. front 29.7
FT T Epi-T A
12.4 12.6 12.5
0.42 0.42 0.42 0.39 (Solv. front 33.4 cm)
Ft T Epi-T A FT T Epi-T A
11.6
0.12
3.9 8.95 8.9
0.27 0.27 0.38 (Solv.
‘2.55 0
5.26 5.05 7.7
* TLC plates were 20
x
cm)
0
0.16 0.15 0.23
Solvent system
Ligroin-toluenemethanol-water (5:5:7:3. 43h) Benzene-ethylacetath)
(1.1, 2
front 33.6 cm)
Benzene-ether (I:I, 2 h)
40 cm.
T and epi-T fluoresced in the UV at 2537 A, and both they and A appeared in I, vapors. Due to this increase in polarity of FT, a polar-type phase for GC, therefore, seemed to be advisable. Also because of the large polarity and high molecular weight of FT, exploratory experiments were set up to determine whether derivative formation might be of advantage in GC. Derivatives allow a way of increasing area response and of selectively shifting a compound from its background. Kirschners used the bis-methylenedioxy derivative of cortisone for this purpose, and for thermal stability. The FT molecule (Fig. I) contains one relatively unhindered hydroxy group at the 17 position which should convert to a trimethylsilyl ether (TMS). There is a sterically blocked hydroxy at C-II, which under conditions of silanization used with the 17-KS herea, would probably not convert. TMS derivatives might have shorter retention, and they often permit resolution of close isomers, too. When a standard solution of FT (IO mg/roo ml) and a standard solution of cholesterol were converted to TMS, four major peaks resulted for FT, which had a combined area which might have been expected for one peak. Even on the Iz-foot x 1.5 mm glass column (Table I), the retention times of these four FT peaks were very close to the solvent front (tetrahydrofuran) (THF) and close together (4, 5, 6 and 7 min). The cholesterol TMS showed only one peak with a retention time of 30 min, under the same conditions. The several TMS peaks which resulted from FT would have been covered by other 17-KS TMS when run from a urine extract. Perhaps the four peaks resulted from partial conversion to TMS of both hydroxy groups (17- and Clin. Chim. Acta, 26
(1969) 253-262
256
RUCHELMAN
II/?-diol), of two close isomers of FT. (On the rz-foot column of 3% XE-60 at low carrier gas pressure, although the retention time was over 5 h, the skew FT peak indicated two isomers. They only differed, perhaps, by the a- or fi-orientation of a hydrogen atom.) Therefore, conversion to FT-TMS was not advisable for quantitation. Shorter columns would reduce the unduly long retention time of FT as the free steroid; however, good resolution was necessary, too. It was decided to remove the less polar 17-KS from the vicinity of FT on the column by condensing their retention times close to that of the solvent front. Letting the GC remove these steroids from the retention time of interest, that of FT, would be as valuable as possibly several additional separation steps by wet methods. It would not be necessary to form derivatives, either, which require more time and manipulations. Starting with this new idea, we first wanted to observe FT as the free steroid, from a standard solution. Fig. 2 shows symmetrical peaks for various concentrations of FT as a free steroid on a 3% XE-60 column, 3-foot x 4 mm, column temperature 226’, injector 260°, detector 260’. N, was 18 pounds inlet pressure. These were Gaussian peaks, good for quantitation, for amounts of about 2 ,ug to 16 clg per injection of 2 ~11.
Fig. 2. FT Standards,
by GLC.
Fig. 3 shows how the free steroid was isolated on this column and clear of other, less polar 17-KS hydrolyzed from the urine by our method. (This preparation of the urine will be explained later.) Compounds with retention times close to FT are Clin. Chim.
Acta,
26 (1969)
253-262
GAS CHROMATOGRAPHY
OF FLUOXYMESTERONE
HYDROLYZED Ibrh.,
UNHYDROLYZED IAfter FT,
257
URINE
URINE
HYDROLYZED URINE ,*,,a FT,
Fig. 3. FT from urine samples, after ingestion of low-level amounts.
possible isomers due to metabolic changes in the body. This urinary FT plus the three isomers amounted to about 500 pg/24 h after ingestion of a 20 mg daily dose. This is a low level. We have often used whole sample injection for small amounts of FT with our automatic injection system for GP. Fig. 4 shows the chromatogram for a patient, a male, 6g years old, with lymphoma, who was on 200 mg/day of FT. He excreted 2 mg/24 h in the urine. PATIENT’S URINE wig+Lml FT,
Fig. 4. FT from patient’s urine, after ingestion of high-level amounts. Clin. Chim. Acta, 26 (1969)
253-262
258
RUCHELMAN
Fig. 5 shows how FT added to urine samples matched the retention time of free ET, and was unchanged by a mild hot acid hydrolysis of only 15 min. Also, FT added to tap-water showed no damage after hydrolysis. (Although other 17-KS may not be completely hydrolyzed in less than an hour Q,FT was).
Fig. 5. Recovery
studies
of hydrolysis
by mild hot-acid
for 15 min.
In this chromatogram, unlike in Fig. 3, there are no other compounds with retention times close to FT. Therefore, these compounds are not changed versions of FT due to the hydrolysis procedure. They did not match the retention time of FTdione (Table I). The 3-OH derivative of FT would have had a much longer retention time than FT due to its extra absorption properties. No other similar compounds were available. It was felt that this tentative identification of the isomers, as described under TMS, would suffice at present. No synthesis or further conversion of FT was undertaken to produce compounds for absolute identification-too many, subtle changes, with exacting sterochemistry, were felt possible, Smaller amounts of added FT were recovered in comparable percentage. Quantitation was now possible by GC. This allowed us to analyze for any’ free FT that was excreted in the urine, or any FT in the conjugated form (after it was hydrolyzed). MATERIALS
ET-obtained as Halotestin, No, U-6040, from The Upjohn Co., Kalamazoo, Mich., mp (on Fisher melting block, uncorrected), zgz.5~z94.5”, without decomposition. FT-d&e-The Upjohn Co., Kalamazoo, Mich. Silica Gel HFzM (No. 7739) from B~nkman Instruments, Inc., Westbury, L.I., N.Y. T, E$i-T, A, and other 17-E?. Man Research Lab., Inc., New York, N.Y., and Ikapharm, Ramat-Gan, Israel. All solvents: from Fisher Scientific Co., Houston, Tex. Reagent grade. Stored over drying agents, redistilled when necessary. Hexamethyldisilazane and trimethylchlorosilane reagents. Applied Science Labs., Inc., State College, Penn. Ctin. Chiwz. Acta, 26 (1969) 253~262
GAS CHROMATOGRAPHY
OF FLUOXYMESTERONE
259
XE-60 (G. E. nitrile silicone gum) phase on 80/100 mesh Gas-Chrom Q, obtained as 3% and as 4%, also coated by Applied Science Labs., Inc., State College, Penn. METHOD
Our work was initiated with FT, as oral tablets of Halotestin, which also contained a carrier, a dye, and stabilizing agents. FT could be leached from these tablets by various organic solvents, including THF, acetone, and ethanol, which also extracted the other substances in different amounts. Identification was indicated by retention times on various polar GC columns and also by TLC. Quantitation became possible upon receipt of a 5-gram sample of pure FT from The Upjohn Company. The retention time of this pure FT matched that of FT leached from the oral tablets. Standard solutions of FT were made up in several strengths in THF. The most acceptable was a standard of 208 ,ug/ml, which was used for low-level amounts of FT in urine, and for high-level amounts when they were diluted. For the relationship between concentration and peak area, I, 2, 3, and 4 ml were evaporated in individual conical, r5-ml centrifuge tubes, then redissolved in IOO ~1 of THF each. An injection of exactly 2 1’1 was made of each sample. Several chromatograms of each were run in succession, and an average taken. Fig. 2 shows chromatograms of the increasing amounts of FT per injection. The areas were measured by means of a Disc Integrator (Disc Instruments, Inc.), corrected for drift where relevant. Also, by triangulation, where overlap of isomers in some urine samples. Quantitative GC by GillI’ with comparison of triangulation and Disc integration shows a standard deviation of 0.80% and 0.47% respectively. Liquid injection was with a IO-$ syringe (Glass Engineering Co., Houston, Texas.). Fig. 6 is a Concentration vs. Area graph, with a slope of I.47 X IO@ g/count. It is made up from the measurements of the chromatograms shown in Fig. 2. This
Kg.
6. Linearity
of concentration
us. peak area of FT. C&z. Chim. Acta, 26 (1969) 253-262
260
RUCHELMAN
shows the linearity of the various amounts of FT, as far as detector response and column behavior toward this steroid. Using this graph, area (in counts) of peaks after injection of various amounts of standard solutions of FT were matched with concentration on they-axis. They agreed with calculations by Sr: 1%. Each week a new set of chromato~ams is run. The slope changes slowly after the initial column conditioning, with a slightly larger area per injection as the weeks progress. The column was four weeks old when these chromatograms were run. Although several compounds of large polarity, including zr-fluoro-progesterone (progesterone, zr-~uoro-II~,I7a-dihydroxy-4~-methyl) were tested for the possibility of an internal standard, none seemed adequate. 21-Fluoro-progesterone decomposed with GC.
A baseline urine and urine after several days of ingestion of FT were collected for 24 h each. An aliquot of IIO ml was withdrawn for routine identification of 17-KS9 and an aliquot of IIO ml for FT. The balance would then be dialyzed to a urine concentrate as described previouslylZ, and reconstituted with saline to original volume. From this latter, IIO ml would be withdrawn again for routine r7-KS in order to determine the amount remaining within the dialysis membrane, and IIO ml for FT analysis. Sometimes, the concentrate would be analyzed without reconstitution, as it was in the membrane. The balance of the reconstituted volume would be used for erythropoietin studies”. The first step was to extract the original, unhydrolyzed urine samples with ether, which was then dried and evaporated. THF solvent would be added (o.c5 to 0.10 ml, depending upon expected amount of FT) for injection into GC. This chromatogam would show an unconjugated FT or any isomers of it. (Fig. 3, middle chromatogram, showing any steroids excreted in “free” form.) The other, aqueous fraction of each urine sample would then be hydrolyzed as for the routine 17-KS by a mild hot-acid method* but only for 15 min. Enzymatic hydrolysis for three days had revealed basically the same amounts of FT and its isomers, about 95% & 5% were recovered by either method (Fig. 5). This indicates that FT and its other isomers are conjugated mainly as glucuronides. After routine purification and removal of acids and phenolic steroids, the ether extract of the hydrolyzed, neutral androgens was dried and evaporated, with THF added for injection into GC. (Fig. 3, bottom chromatogram, showing that FT and Isomer No. I are excreted as glucuronic acid salts. Apparently, isomers Nos. z and 3 are not excreted in this way but are organic-soluble.) Determinations were complete within a day. Gas chromatography This analysis was run on a Barber-Coleman Series 5ooo. The 4 mm i.d. columns were glass: g-foot U-tubes, 6-foot U-tubes, and rz-foot W-tubes. They were packed with 3% or 4% XE-60 phase on Gas Chrom Q support, coated by Applied Science Labs. Glass wool plugs were siliconized. Column conditions are given in Table I. The attenuator of the GC was set at 5, the sensitivity at 1000. The 5 mV recorder speed was 20 in/h.
CGn.Chim.
Acta, zb(1969) 253-262
GAS CHROMATOGRAPHY
OF FL~~XYMEST~RONE
261
DISCUSSION
The aim of this research was to develop an accurate, reproducible and relatively rapid procedure for analysis of FT in urine and reconstituted urine from dialysis. The amount of FT excreted ranged from 500 c/g to z mg/zq h. The amount of FT and that of the 17-KS within the dialysis membrane remained the same per ml. That is, I pugFT/ml, if IOOOml before dialysis, was I /‘g/ml if 17 ml afterwards. GC proved to be a sensitive means of analysis. Since less than 5% of ingested FT (plus its close isomers) can be accounted for in the urine, either some is excreted in the feces or in the form of other metabolites in the urine. In the routine 17-KS analysis, there were several large peaks as TMS derivatives which had retention times past the usual steroid TMS’s. Future work entails the tracing of metabolites of FT as excreted in the urine. Far less than the minimum quantity of 150 pg required to stimulate radioiron utilization in polycythemic mice has been accounted for. It is felt that the metabolites of FT will probably contain a fluorine atom, which may be traced by means of an electron capture cell on GC analysis. This will probably allow further identification of Isomers Nos. I, 2, and 3. These metabolites might be of prime importance in physiological mechanisms of erythropoietin. FT and its metabolites in plasma will also be analyzed. This CC method isolates FT and any close isomers from other 17-KS by concentrating them near the solvent front far from the retention time of FT. This is a new way of resolution without derivative formation or alteration of the molecule by other chemical means. This type of analysis might also be expanded to evaluate other steroids and drugs of unusually high polarity in biological fluids. It certainly appears promising for future experimental work, as such compounds could thus be selectively shifted beyond the range of their background interferences. ACKNOWLEDGMENT
The author wishes to acknowledge the helpful information and advice about FT from Doctor Raymond Alexanian, and the able technical assistance of Brenda Prewitt and Pamela Herrera. This study was supported by U.S.P.H.S. Research Grant No. Ca 05831. The supply of FT and FT-dione used in this study was supplied by The Upjohn Company, Kalamazoo, Michigan. REFEKENCES I M. E. HERR, J. A. HOGG AND R. H. LPVIN, J. Am. Chem. Sot., 78 (1~56) 500. 2 L. I?. FIESER AND M. FIESER, Steroids, Reinhold Publishing Corp., New York, 1959. p. 593, 3 R. ALEXANIAN, W. K. VAUGHN AND M. MI. RUCHELMAN, J. Lab. C&n. Med., 70 (1967) 777. 4 Computer tape of references sent to DR. SHIZUTOMO KATSUTO, this dept., by DR. H. H. ANGE~L of ‘The Upjohn Co,. Kalamazoo, Mich, in Feb., 1966. 5 E. J. CLEGG AND T. M. FARLEY, J. Repvod. Fertit., 4 (1962) 125. 6 The P~ayrn~ope~u ofthe U.S. of America (The U. S. Ph~rrn~a~e~a~, r7 th rev., Mack Publishing Co., Easton, Pa., x965, p. 887. 7 P. W. O’CONNEL, Biological Screening Office, ‘The Upjohn Co., Kalamazoo, Mich., personal communication, 1966. 8 M. A. KIRSCHNER AND H. M. FALES, Anal. Chem., 34 (196~) 1548. Clin. Chim. Acta, 26 (1969) 253-262
262 g M. W. RIJCHELMAN AND V. W. COLE, Clin. Chem., 12 (1966) 771. IO M. W. RUCHELMAN, J. Gas Chrom., 4 (1966) 265. II J. M. GILL AND H. M. MCNAIR, Aerograph Res. Notes, Fall Issue (1956). 12 R. ALEXANIAN, Blood, 28 (1966) 344.
Clin. Chim. Acta, 26 (1969) 253-262
RUCHELMAN