ANALYTICAL CHEMISTRY, VOL. 50, NO. 8, JULY 1978
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Pyrolysis Gas-Liquid Chromatography of Structurally Related Steroids F. M. Menger,” J. J. Hopkins, G. S. Cox, M. J. Maloney, and F. L. Bayer Department of Chemistry, Emory University, Atlanta, Georgia 30322
Fifteen structurally related steroids were subjected to high temperatures (1000 “C) in the absence of O2 and the resulting breakdown products were then separated wlth a gas chromatograph. It was found that the steroids have characteristic pyrochromatographic “fingerprints” and that thermal dlsintegration patterns are sensitive to minor structural and stereochemical differences. For example, 5P-androstan-17-one and its epimer 5a-androstan-17-one each have a large peak missing in the other. Pyrochromatography can differentiate individual steroids as well as discern sets of sterolds containing various functional groups.
In pyrochromatography (PGLC), materials are subjected t o high temperatures in the absence of 02,and the resulting breakdown products are then separated with a gas chromatograph ( I ) . PGLC studies have been carried out on a wide variety of materials including hydrocarbons ( Z ) , bitumens (3), fibers (4), coatings ( 5 ) ,polymers (6),wood (7), alkaloids (81, macrolide antibiotics (9),and dinucleotides (10). A sufficient body of data has accumulated to demonstrate a key fact about pyrochromatography: reproducibility under a standard set of conditions is excellent if sufficient care is taken (11). Gough and Jones (12)have shown that even laboratory-to-laboratory reproducibility is of a high order when experimental variables (column packing, flow rate, column length, etc.) are prescribed exactly. We have shown that PGLC data are sufficiently reproducible that they may be used in computer search programs (13). Thus far the major application of pyrochromatography has been in the area of identification and differentiation. Different solids generally have different pyrochromatographic “fingerprints” which serve t o identify the solid. PGLC identification has found its way into criminology (141, extraterrestial exploration (15), and agriculture (16) among other fields. Even closely related bacteria can be differentiated by their pyrochromatograms ( I 7). Pyrochromatographic analysis of pure nonvolatile compounds has demonstrated that PGLC data vary with molecular structure (18). Thermal degradation may someday complement electron impact in structure elucidation work, but unfortunately the sensitivity of PGLC to small molecular modifications has not yet been thoroughly evaluated. To help rectify this situation, we have secured the pyrochromatograms of a series of structurally related steroids (Figure 1). We were interested to determine, for example, if PGLC can discriminate between two steroids which differ only in their stereochemistry at the A / B ring juncture. Although steroids have been subjected to the PGLC method in the past (19-23), no systematic “structure-activity’’ experiment exists in the literature. A comment is in order on why steroids were selected for our work. First of all, a wealth of steroids is available commercially or by synthesis. Thus, it is possible to obtain isomeric steroids which differ only in the position of a carbonyl, hydroxyl, or double bond; steroids with and without an angular methyl group, D-ring functionality, or A-ring 0003-2700/78/0350-1135$01 .OO/O
aromaticity. The availability of a series of related compounds facilitates a study of fragmentation as a function of structure. Moreover, steroids are nonvolatile so that complications or interferences by sublimation are minimized.
EXPERIMENTAL Pyrochromatograms were obtained with the aid of a Chemical Data Systems Pyroprobe-120 interfaced to a Hewlett-Packard 5830A processor-controlledgas chromatograph with a dual flame ionization detector. A small quartz boat containing 50-100 kg steroid (weighed with a Cahn Model 4400 electrobalance) was inserted into the platinum heating coil of the pyrolyzer probe. For optimum reproducibility, the sample pellet was always placed in the center of the coil. The coil was heated at a rate of 75 “C/ms until a maximum of 1000 “C was reached; this temperature was maintained for 5 s before cooling. The pyrolysis products were chromatographed on a 3.65 m X 6 mm 0.d. X 2 mm i.d. silanized glass column packed with 5% carbowax 2OM-TPA on 110/120 mesh Anakrom ABS. Temperature programming settings were the following: initial temperature 55 “C; hold 6.0 min; increase 6”/min until 165 “C; hold 55 min. Other settings: interface temperature 150 “C; injection port temperature 180 “C; FID temperature 250 “C; chart speed 0.50 cm/min; flow 20 mL/min N2. The nitrogen was prepurified gas passed through Linde 5A molecular sieve and an Alltech Oxytrap. The Carbowax column withstood intensive use for over a year without deterioration in the quality of the pyrochromatograms. Eventual replacement of the column with another of the same type did not affect peak ratios but did cause small variations in retention times (which could be changed back to the original ones by adjusting the N2 flow rate). Differences in peak amplitudes in repeat runs arose from the variations in sample size (50-100 wg). This was inconsequential because peaks were always compared using “area % ” (a processor-calculated parameter referring to the area of a peak as a percentage of the total area of the chromatogram). Although i t is recognized that the extent of secondary reactions in pyrolysis can change with sample size (24,25), we noted no dependence of the relative peak areas on the sample size within the 2-fold range. The steroids were provided by Steraloids (Wilton, N.H.) and Amersham/Searle (Arlington Heights, Ill.). Melting points and specific rotations and/or thin-layer chromatograms were secured for all the compounds. Several steroids were recrystallized with no noticeable effect on the pyrochromatograms. The effects of possible minor impurities were considered unimportant for two reasons. First, we concentrated on only a few major changes in the fingerprints; peaks less than 1 area 70 were not needed to differentiate the steroids in Figure 1. Second,control experiments, in which a steroidal “impurity” was purposely added to a sample of another steroid, indicated that pyrochromatograms are not sensitive t o low levels of contaminant. A few steroids, especially monofunctional compounds with low melting points, gave a broad poorly defined peak at high retention times. This probably stems from volatilization of the steroid prior to pyrolysis. Prevolatilization prevented our securing a pyrochromatogram on androstane, a nonfunctionalized steroid. Changing the physical state of the steroid by grinding or melting did not affect the pyrochromatograms. Likewise, changing the pyrolysis surface from quartz to platinum was of no consequence. Mixtures of two steroids melted together produced pyrochromatograms which were essentially the summation of the individual 0 1978 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 50, NO. 8, JULY 1978
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Figure 4. Pyrochrornatograrn of 5a-androstan-3-one (steroid 3)
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Figure 5. Pyrochrornatograrn of 5a-androstan-3P-ol-17-one (steroid
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Figure 6. Pyrochromatogram of 5a-androstan-3a-ol-17-one (steroid 10)
Figure 3. Pyrochrornatograrn of Sa-androstan-17-one (steroid 2)
ones. This suggests that relatively few peaks arise from fragment-fragment or fragment-molecule reactions.
RESULTS AND DISCUSSION Typical steroid pyrochromatograms (Figures 2-7) were complete in 40 min (retention time, RT, of 4-octanone under identical conditions = 13 min). Over 50 peaks were obtained for each steroid of which about half could be considered “significant” (>1%of the total area or peak heights >1 cm). Small saturated and unsaturated hydrocarbons, possessing RT less than 5 min and comprising 40-60% of the total area, were not used for diagnostic purposes. Steroids listed in Figure 1have characteristic “fingerprints”, and we specify below only the most apparent differences. Pyrochromatograms have been
ANALYTICAL CHEMISTRY, VOL. 50, NO. 8, JULY 1978
Figure 7. Pyrochromatogram of estrone (steroid 15)
provided for six of the 15 steroids in Figure 1to illustrate many of the PGLC characteristics. 1. Four prominent peaks are common to all steroids (RT = 6.2, 13.9,19.3, and 24.3 min). To assess reproducibility, we focus on the 24.3 “marker” which, depending on the steroid, varies in size from 1.3 to 6.1 area %. The largest observed discrepancy in R T between two duplicate runs done within the same day is 0.10 min; the average difference is only 0.04 min. Retention times collected over 5 months with all the steroids fall within 24.25 f 0.15 min. Variations in peak area for repeat runs are less satisfactory owing in part to the dominant contribution of the low-RT peaks mentioned above. Irreproducibility in the area data also stems from baseline variations. On the average, relative areas for the 24.1 peak in repeat runs differ by somewhat more than 10%. Estrone has a 6.2 marker with an average area % for 8 runs of 3.39 (0 = 0.49) and a 13.9 marker with an average area % of 1.58 (u = 0.32). These values are typical of those found throughout our work. 2. Polyunsaturation in the A/B rings diminishes substantially the size of the 9.5/10.0 min “doublet” found in all the pyrochromatograms. Thus, estrone (steroid 15) and 1,4,6-androstatriene-3,17-dione (steroid 14) have doublets roughly 25% the area of the others (compare Figure 7 for estrone with Figures 2-6). 3. In contrast to all other steroids examined, 5a-androstan-3-one (steroid 3) has a major peak at R T = 25.8 min (2-2.5%). This peak, rising above the markers in Figure 4, may result from the fact that among the steroids in Figure 1 only steroid 3 has saturated B, C, and D rings. 4. The two 5-androsten-3@-olsin Figure 1 (steroids 12 and 13) possess exceptionally large 24.3 markers (5-6%) allowing easy differentiation of these compounds from the others. Similarly, all a,@-unsaturatedketones (steroids 6, 7,and 8) display 19.3 markers 2-fold larger than those of the remaining non-aromatic steroids in Figure 1. 5. As is seen in Figures 5 and 6, epimers 5a-androstan3P-ol-17-one and 5a-androstan-3a-ol-17-one (steroids 9 and 10) differ in the region between the 19.3 and 24.3 markers. These two steroids are identical except for the stereochemistry at carbon-3. On the other hand, epimers 9 and 11, having trans and cis A/B ring junctures, respectively, give pyrochromatograms with no gross distinguishing features. 6. Epimeric steroids 4 and 5 have virtually superimposable pyrochromatograms except for an extra peak in the latter. Whereas the trans compound has a “doublet” at 26.3 and 27.1 min, the cis has a “triplet” at 26.3, 27.1, and 27.8 min. Steroids 4 and 5 are distinguishable from all the others by their sharp peak a t 18.2 rnin (note that in Figures 2-7 the 18-min region
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to the left of the 19.3 marker is devoid of a large peak). 7. 5a-Androstan-17-one (steroid 1) and its epimer 5aandrostan-17-one (steroid 2) each have a large peak missing in the other (see the 29.1 and 29.9 min peaks in Figures 2 and 3, respectively). This is an outstanding example of the sensitivity of PGLC to small structural modifications. 8. All steroids other than 5-androsten-3@-01-17-one(steroid 13) lack sharp peaks at R T >40 min. Steroid 13 has a large ( > 2 % ) peak at 45.2 min. Estrone is defined by a broad peak near 54 rnin (Figure 7). 9. The position of the carbonyl in the D-ring seems to affect the nature of the pyrochromatogram. Thus, 4-androsten3,17-dione (steroid 6) and 19-nor-4-androsten-3,17-dione (steroid 8) but not 4-androstene-3,16-dione (steroid 7)possess a conspicuous doublet in the 16.5-16.8 min region. 10. The family of a,P-unsaturated ketones (steroids 6, 7, and 8) is characterized by a medium-intensity peak (1-2%) at 34.7 min which, it will be noted, is absent from the other steroids (Figures 2-7). Computer matching of the pyrochromatograms, now in progress, will probably reveal additional and more subtle differences. But even without a computer we have shown that thermal disintegration patterns are far more sensitive to minor structural and stereochemical properties than has been realized in the past. Pyrochromatography can differentiate individual steroids, discern sets of steroids containing particular types of functional groups, and even distinguish one epimer from another. Although a much larger number of steroids must be tested, our preliminary results suffice to illustrate the potential of PGLC in structure elucidation and mechanistic studies. Identifying the steroid fragments by mass spectrometry will add another dimension to the method. Perhaps someday thermal degradation in combination with mass spectrometry will complement mass spectrometry by itself as a common tool in structure analysis.
ACKNOWLEDGMENT The equipment used in this work was purchased with funds from an NIH Biomedical Research Support Grant. We thank E. Reiner for valuable advice in the use of this equipment. LITERATURE CITED W. Simon, P. Kriemler, J. A. Voeilmin, and H. Stelner, J. Gas Chromatcgr., 5, 53 (1967). R. A. Brown, Anal. Chem., 43, 900 (1971). J. Romovaced and J. Kubat, Anal. Chem., 40, 1119 (1968). J. P. Dundon, Textile Res. J . , 34, 340 (1964). G. G. Esposito and M. H. Swann, J . Gas Chromatogr., 3, 282 (1965). E. W. Neumann and H. G. Hadeau, Anal. Chem., 35, 1454 (1963). R. F. Schwenker and L. R. Beck, J . Polym. Sci., No. 2, 331 (1963). G. Van Binst, L. Dewaersegger, and R. H. Martin, J . Chromatogr., 25, 15 (1966). D. H. Calam, J . Chromafogr. Sci., 12, 613 (1974). L. P. Turner and W. R. Barr, J . Chromafogr. Sci., 9, 176 (1962). H. L. C. Meuzelaar and R. A. in’t Veld, J . Chromatogr. Sci., 10, 213 11972). T. A.’Gough and C. E. R. Jones, Chromatographia, 8, 696 (1975). F. M. Menaer. G. A. Eostein. D. A. Goldbera, and E. Reiner. Anal. Chem., 44, 423 (i972). ’ D. W. Hill, J . Forenslc Sci. Soc., 2, 32 (1961). V. I. Oyama and G. C. Carle, J . Gas Chromatogr., 5, 151 (1967). L. K. Gaston and F. A. Gunther, Bull. Environ. Confam. Toxicol., 3 , 1 11968). E. R e h r and W. H. Ewing, Nature(London),217, 191 (1968). J. Janak, Nature (London), 185, 684 (1960). M. Gasslot-Matas and E. Juila-Danes, Chromatographia, 5, 493 (1972). M. Gassiot-Matas and E. Julia-Danes, Chromatographia, 9, 151 (1976). N. E. Vanderborgh, R. L. Hanson, and C. Brower, Anal. Chem., 47, 2277 (1975). E. Relner, “Analytical Pyrolysis”, Elsevier Scientific Publishing Co., Amsterdam, 1977 p 49. F. L. Eayer, J. J. Hopkins, and F. M. Menger, “Analytical Pyrolysis”, Elsevier Scientific Publishing Co., Amsterdam, 1977, p 217. R. L. Levy, J . Gas Chromatogr., 5, 107 (1967). C. E. R. Jones and G. E. J. Reynolds, J . Gas Chromafogr., 5, 25 (1967).
RECEIVED for review August 8, 1977. Accepted April 7, 1978.
