High-Resolution Gas Chromatography of Plasma Steroidal Hormones and Their Metabolites Milos Novotny," M. P. Maskarinec, A. T. G. Steverink, and Raleigh Farlow Deparfment of Chemistry, Indiana University, Bloomington, Ind. 4 740 1
A method for multicomponent determination of steroids In small plasma samples is described. Complex mixtures of plasma steroids are resolved by means of glass capillary gas chromatography as methoxime-trimethylsllyl derivatives. Fractionation of plasma steroids into different conjugate types Is employed prior to gas-chromatographic determination. An effective column preconcentratlon procedure combined with high resolving power of glass capillary columns allows high-sensitivity recording of steroid profiles with the flame ionization detector. In addition, a nitrogensensitive (thermionic) detector is conveniently used for both selective detection of ketonic steroids within the complex profiles and for selected high-sensitivity measurements. Profiles of three different conjugate fractions can be obtained from 1-5 mi plasma samples in addition to the isolated determination of plasma testosterone by electron capture detection.
Determination of steroid hormones and their metabolites and conjugates in blood is of great importance in both fundamental metabolic studies and routine clinical endocrinology. With a wider acceptance of gas-liquid chromatography as an analytical tool in steroid analysis, considerable attention has also been paid to the development of highsensitivity detection methods by both conventional flame ionization and electron capture detectors (1-3). These applications of gas chromatography generally aim at measurements of a limited number of steroids. Mass-fragmentographic detection of selected plasma steroids, as recently exemplified by a determination of testosterone ( 4 ) , provides impressive sensitivity and selectivity, but this analytical tool is obviously too costly for general use. A need for methods that would be highly sensitive, but also rapid and easy to automate for clinical laboratory, has stimulated considerable interest in techniques of radioimmunoassay ( 5 )and competitive protein binding (6-8). Recording of total steroid profiles from human urine by gas chromatography has been primarily facilitated by advances in derivatization methods (9, I O ) so that a great number of important metabolites can be monitored in one chromatographic run. Introduction of glass capillary columns with their great resolving power into the steroid field (11-13) has further advanced the potential of metabolic profiling. Such columns are particularly useful when dealing with complex mixtures and samples that contain hardto-separate isomers. Following the successful application of glass capillary columns to chromatography of urinary steroids carried out by one of us ( 1 1 ) , it was of great interest to investigate whether similar approaches could be used for multicomponent determination of plasma steroids from relatively small samples. Two major problems have been recognized with determination of steroids in blood samples: 1) Concentrations of hormones and their metabolites are low compared to those of a number of interfering substances (for example, lipids) so that extensive sample clean-up must be performed; and 468
ANALYTICAL CHEMISTRY, VOL. 48, NO. 3, MARCH 1976
2) total amount of sample available for analysis is usually small, and the use of high-sensitivity methods becomes mandatory. While sample purification procedures previously described by Sjovall et al. (14-17) provided good alternatives for obtaining fractions of sufficient purity, the combination of high-resolution glass capillary column, an effective sample preconcentration method ( I 8 ) ,and high-sensitivity detection has made steroid determinations in small amounts of human plasma feasible. This work demonstrates that, after a precolumn concentration, high-resolution steroid profiles from free and conjugate fractions can be effectively recorded as methoximetrimethylsilyl derivatives (9) with samples of 1-5 ml of plasma. A recently described nitrogen-sensitive detector (19, 20) which is responsive to methoxime derivatives formed from ketonic steroids, can further aid in tentative identification of some profile constituents. The nitrogensensitive and electron capture detectors can also be COUpled with highly inert glass capillary columns for high-sensitivity measurements of some selected hormones; even smaller plasma samples are sufficient for such single-component determinations.
EXPERIMENTAL Sample Preparation. Venous blood samples from various normal volunteer subjects and one pregnancy case were obtained at a standard clinical facility. One- to 5-ml plasma samples were obtained using EDTA as an anticoagulant. The plasma sample of a female patient with untreated Cushing syndrome was kindly supplied by R. B. Schnute, Department of Medicine, Indiana University School of Medicine, Indianapolis, Ind. A modified procedure of Sjovall et al. (14-17) was used to extract and fractionate different steroidal conjugates through Sephadex LH-20 column chromatography. Three main fractions containing (a) a mixed fraction with primarily free steroids, but possibly also some glucuronides; (b) monosulfates; and (c) disulfates, were isolated. Fraction (a) was further treated with 0.5 ml Glusulase (Endo Laboratories, Garden City, N.Y.) containing some 100,000 units of P-glucuronidase, a t pH 4.55, for a period of 24 h at 37 "C. Liberated steroids were successively extracted with 2 X 20 ml methylene chloride and 2 X 20 ml ethyl acetate, further purified with 3 X 20 ml 0.1 N NaOH and 3 X 20 ml distilled water, and dried over MgS04. A further chromatographic step, using Amberlyst A-26 as the column packing and ethanol as the mobile phase [as described by Shackleton et al. ( 1 7 ) ] ,was carried out with this fraction to remove other interfering compounds (lipids). If a single determination of testosterone is desired, a so aliquot of fraction (a) is taken for a second chromatographic step on Sephadex LH-20 using the solvent system, 99:l dichloromethaneethanol, as described by Newsome et al. (18) and the marker dyes described by Drewes and Kowalski (21). Testosterone is first converted into methoxime (9) and then to heptafluorobutyryl derivative with N-heptafluorobutyrylimidazole (22) prior to its determination by the electron capture detector. Fractions (a) through (c) were converted into methoxime-trimethylsilyl derivatives prior to gas chromatography. The overall analytical scheme for all determinations is presented in Figure 1. Capillary Gas Chromatography. Steroid profiles from fractions (a) through (c) were recorded with a model 1400 (Varian Aerograph) gas chromatograph equipped with the flame ionization detector, a modified injection system permitting the use of the pre-
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Figure 1. Total analytical scheme for determination of plasma steroids column concentration technique (18) and glass capillary columns. Another model 1400 gas chromatograph was dedicated to operate with the electron capture detector. A low-volume 63Ni detector (Varian Aerograph) was coupled with the response linearizer (Antek Instruments, Houston, Texas) and pulsed with 30 V, pulse width 4 us, and pulse interval 100 ~ s Purified . Nz was used as a carrier gas in glass capillary columns (approximately 1 ml/min) and as a make-up gas (25 ml/min) a t the column outlet to eliminate the dead-volume problems with the electron capture detector. All measurements that involved the nitrogen-sensitive detector and comparisons of its sensitivity with the standard flame ionization detector were carried out with a model 990 Perkin-Elmer gas chromatograph. The injection system and the detector base were modified to allow work with glass capillary columns. Glass capillary columns of various lengths used in this study were prepared from soft glass by gas-phase etching and silylation (23, 241, and a subsequent coating with GE SE-30 (Applied Science Laboratories) by the static procedure ( 2 5 ) .Except for the use of electron capture detector, helium carrier gas was employed throughout the experiments. From 100 u1 of the solutions of methoxime-trimethylsilyl derivatives in the silylation agent for fractions (a) through (c), 25-pl aliquots were used for analyses. After evaporation of the silylation agent, the residues were re-dissolved in 100 u1 n-heptane, and 20-rl aliquots introduced into the precolumn (18) with a subsequent solvent removal,
RESULTS AND DISCUSSION The development of a precolumn procedure (18) for concentration (solvent removal) and direct sample introduction into the capillary column was of fundamental importance to reproducible high-sensitivity detection of plasma steroids in this work. Whereas superior resolutions of complex steroid mixtures with glass capillary columns had already been demonstrated with urinary samples (11-131, it is primarily the combination of effective sample preconcentration, high resolving power of such columns and highsensitivity detection that have made feasible the recording of plasma steroid profiles reported in this work. The use of flame ionization detector for monitoring concentrations of plasma hormones is highly desirable, in spite of a presently more popular selective detection. I t is demonstrated here that its sensitivity is adequate even for relatively small blood samples, provided that efficient sample clean-up and preconcentration are utilized. Efficient peak resolution by capillary gas chromatography contributes further to the specificity of such determinations by possible separation of the compounds of interest from contaminants. ANALYTICAL CHEMISTRY, VOL. 48, NO. 3, MARCH 1976
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Figure 2. Profiles of plasma steroids of a normal male detected by
the flame ionization detector ( A ) Free-glucuronide fraction; (8)monosulfate fraction: and (C)disulfate fraction. Chromatographic conditions: 20 m X 0.26-mm i.d., glass capillary column coated with GE SE-30; injector, 270 O C : detector, 270 O C
Although the procedures of Sjovall et al. (14-17) may be considered too laborious for routine clinical work, they provide a very effective fractionation scheme and the basis for obtaining information on the degree of conjugation of individual plasma steroids. In spite of many steps involved in the sample preparation prior to gas-chromatographic determination, we find the adapted procedure quite reproducible. Reproducibility of profile determinations was checked with large samples of plasma obtained from the blood bank. Overall variations in peak heights among successively run identical specimens are estimated to be f10% or less. Blood samples of several normal male and female subjects were analyzed and individual differences of pri470
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Flgure 3. Profiles of plasma steroids of a female patient with Cushing syndrome (A), (B),and (C)have the same meaning as in Figure 2. Chromatographic conditions, same as Figure 2
marily quantitative nature concerning the degree of conjugation were noticed within the groups of same sex. Typical profiles obtained from the plasma of a normal male are shown in Figure 2. The free-glucuronide fraction shows a continual spectrum of steroids beginning with 17-ketosteroids (androsterone elutes on the capillary column used at 208 "C) and their oxygenated metabolites, pregnane derivatives, up to corticoids and their metabolites. This elution pattern can be roughly compared with the chromatogram of standard derivatized steroids that will be later shown in Figure 4.
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by ( A ) the flame ionization detector, and (B) nitrogen-sensitive (thermionic detector) Chromatographic conditions: 21 m X 0.26-mm i.d. glass capillary column coated with GE SE-30; injector, 275 ‘C;detector, 275 OC. Key: 1. androsterone; 2, etiocholanolone; 3, dehydroepiandrosterone; 4, androstenedione; 5, androstanediol; 6, allopregnanedioi; 7, pregnanediol; 8, pregnenolone; 9, pregnanetriol; 10, progesterone; 1 1, estriol, 12, tetrahydrocortisol; 13, allotetrahydrocortisoi; 14, cholesterol. Amounts injected: 20-30 ng of each compound, except 5-10 ng of Compounds 4, 5,and 10
I t should be emphasized that the purpose of this study was primarily the development of analytical techniques and demonstration of the scope of such methods for detailed biomedical studies. Consequently, blood hormone levels of the studied subjects were not followed for a longer period of time, and the consideration of diurnal variations, time of sampling vs. the secretion by endocrine glands, etc., were outside the scope of this work. In addition to utility of high-sensitivity, high-resolution gas chromatography in fundamental studies of hormonal secretion in both humans and experimental animals, these or similar methods can prove beneficial in endocrinological practice where even a 2-day analysis time may be acceptable in view of detailed information on the concentrations of numerous steroids. However, correlations of plasma steroid profiles with the clinical status of various patients are needed. The analysis of pathological sample included in this study (a female patient with untreated Cushing syndrome) is demonstrated in Figure 3. While the elevations (Figure 3, A ) observed in the corticosteroid region (225 to 250 O C ) could be considered typical for this disorder, much biochemical information could possibly be obtained in such and similar cases by relating plasma profiles to clinical symptoms, following the profiles during disease treatment, etc. The chromatograms obtained in this work suggest that even with the high resolving power of glass capillary col-
umns, many profile constituents still overlap. This is, perhaps, not surprising in view of the possible existence of many steroid isomers. Several barely resolved peaks in the chromatograms shown in this work (that could not have been noticed with conventional packed columns) clearly show the magnitude of this problem. While identification of profile constituents mainly relies on retention data and GC-MS information, additional techniques, such as selective derivatization, enzymatic treatment (26), and peak shift methods, may be required. A simple method that can provide more information on the nature of certain profile constituents is the use of a nitrogen-sensitive detector (19) described in this work. Following the preparation of volatile steroid derivatives according to the method of Gardiner and Horning (91, steroidal ketones are conveniently converted to methoxime derivatives, whereas remaining hydroxy groups can be silylated. Now, because of the presence of nitrogen in their molecules, derivatized ketones can be selectively detected by the nitrogen detector in the midst of interfering peaks. In addition, a simple comparison of chromatograms obtained with this detector and those recorded with the conventional flame ionization detector clearly reveals the presence of ketonic steroids. Figure 4 shows a comparison of chromatograms obtained from the standard mixture with the two detectors. Although the sensitivity of the nitrogen detector for most nitrogen-containing compounds is usually 50 times higher than that of the flame ionization detector (20) for a comparable noise level, the presence of silicon in derivatized steroids appears to cause a decrease in response. Some advantages of this detector for steroid determinations can be pointed out: 1) Significant enhancement of response for steroids containing two ketonic groups, such as androstenedione and progesterone, is demonstrated. “Shoulders” on both peaks can be most likely attributed to a partial rpsoluANALYTICAL CHEMISTRY, VOL. 48,
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tion is directly proportional to the area under a detected peak, efficient capillary columns that yield sharp chromatographic peaks are highly desirable for such trace analyses. The detection conditions for androstenedione and progesterone are particularly favorable, as two nitrogen-containing groups are introduced into these molecules; minimum detectable amounts (two times the noise levels) are about 0.1 ng. The detector was less sensitive for methoxime-trimethylsilyl testosterone (detection limit of 1 ng), because of the mentioned negative effect of silicon on the detector response. However, considerably lower levels of testosterone can be measured by the electron capture detector coupled with the glass capillary column. Figure 6 shows testosterone detection from the extract of male plasma compared with elution of the standard compound. Reliable detection with even low-level samples (e.g., in normal women; typically 0.5 ng/ml) is feasible with this method.
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ACKNOWLEDGMENT We thank Richard B. Schnute, Department of Medicine, Indiana University School of Medicine, for supplying the pathological plasma sample used in this research.
LITERATURE CITED TlMEIMIN.1
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Figure 6. Determination of plasma testosterone from aliquot of the free-glucuronide fraction of a normal male (6) by the electron capture detector. In part ( A ) , T = testosterone standard (methoximeheptafluorobutyryl derivative), 50 pg Chromatographic conditions: 15 m X 0.25-mm i.d. glass capillary column; solution of methoxime-heptafluorobutyryl testosterone in Kheptafluorobutyryiimidazole (0.2 ml) was extracted with 5 mi n-hexane, washed with 2 X 5 ml 5 % HCI, 10% NaCI, and 2 X 5 ml 5% NaHC03, 10% NaCI, and dried with MgSOd. The hexane layer was evaporated, and the residue redissolved in 100 fil of n-hexane, from which a 1.O-fi1 aliquot was injected at 70 "C, and the column then programmed from 180 to 230 OC at 1 'Ctmin
tion of syn and anti forms of derivated steroids; 2) Determination of selected steroidal ketones can be performed even with insufficiently resolved fractions. For example, pregnenolone co-chromatographs with pregnanediol, but it can be selectively traced with the nitrogen-sensitive detector (compare 7 and 8, Figure 4). A combination of retention data with the information on ketonic steroids (obtained with the nitrogen-sensitive detector) can be of importance in routine evaluations of plasma profiles. Figure 5 shows a comparison of chromatograms obtained with the two detectors from a fraction (a) of pregnancy plasma (major peak eluted a t 220 OC was tentatively identified as progesterone.) Because of its high sensitivity, selectivity, and reliability in routine analytical work (20), the nitrogen-sensitive detector can also be used to monitor selected steroids extracted from plasma. Major purifications usually required for the flame ionization or electron capture detectors, are now hardly necessary. Furthermore, since the solute concentra-
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(1) F. Polvani, M. Surace, and M. Luisi, Ed., "Gas Chromatographic Determination of Hormonal Steroids", Academic Press, New York and London, 1967. (2) K . B. Eik-Nes and E. C. Horning, Ed., "Gas-Phase Chromatography of Steroids", Springer Veriag, New York. 1968. (3) S. J. Clark and H. H. Wotiz, in "Modern Methods of Steroid Analysis". E. Heftman, Ed.. Academic Press, New York and London, 1973. (4) I. Bjdrkhem, 0. Lantto. and L. Svensson, Clin. Chim. Acta, 60, 59 (1975). (5) G. E. Abraham, in "Modern Methods of Steroid Analysis", E. Heftmann, Ed., Academic Press, New York and London, 1973. (6) B. E. P. Murphy, Nature (London), 201, 679 (1964). (7) W. R . Slaunwhite, Jr., in "Modern Methods of Steroid Analysis", E. Heftmann, Ed.. Academic Press, New York and London, 1973. (8) H. H. Newsome, Jr., A. S. Ciements, and E. H. Borum, J. Clin. Endocrin., 34, 473 (1974). (9) W. L. Gardiner and E. C. Horning, Biochim. Biophys. Acta, 115, 524 (1966). (10) E. C. Horning and M. G. Horning, Methods Med. Res., 12, Section 5 (1970). (11) M. Novotny and A. Zlatkis, J. Chromatogr. Sci., 8, 346 (1970). (12) J. A. Vollmin, Chromatographia, 3, 233 (1970). (13) A. L. German, C. D. Pfaffenberger, J.-P. Thenot, M. G. Horning. and E. C. Horning, Anal. Chem., 45, 930 (1973). (14) J. Sjovall and R. Vihko, Steroids, 6,721 (1965). (15) R. Vihko, Acta Endocrinoi. Suppl., 109, 5 (1966). (16) J. Sjovail and R. Vihko, Acta Endocrinol., 57, 247 (1968). (17) C. H. L. Shackleton, J. Sjovall. and 0. Wisen, Clin. Chim. Acta, 27, 354 (1970). (18) M. Novotny and R. Farlow, J. Chromatogr., 103, 1 (1975). (19) B. Kolb and J. Bischoff, J. Chromatogr. Sci., 12, 625 (1974). (20) M . J. Hartigan. J. E. Purceli, M. Novotny, M. L. McConnell, and M. L.Lee, J. Chromatogr., 09, 339 (1974). (21) P. A. Drewes and A. J. Kowalski, Clin. Chem., 20, 1451 (1974). (22) M. G. Horning, A. M. Moss, E. A. Boucher, and E. C. Horning, Anal. Lett., 1, 311 (1968). (23) K. Tesarik and M. Novotny, in "Gas Chromatographie 1968". H. G. Struppe, Ed., Akademie Verlag Gmbh, Berlin, 1968, p 575. (24) M. Novotny and K. Tesarik, Chromatographia, 1, 223 (1968). (25) J. Bouche and M. Verzele, J. Gas Chromatogr., 6,501 (1968). (26) A. G. Smith and C. J. W. Brooks, J. Chromatogr., 101, 373 (1974).
RECEIVEDfor review October 16, 1975. Accepted December 15, 1975. This work was supported by the Grant No. Gm-19232, from the National Institute of General Medical Sciences, U.S. Public Health Service.
