Analysis of Steroid Hormones Using High Resolution Liquid Chromatography Sidney Siggia and Robert A. Dishman’ University of Massachusetts, Amherst, M a s s . Fast, high resolution liquid chromatographic separations of structurally similar androgens, estrogens, progestins, and adrenal corticosteroids were obtained with modest instrumentation at column inlet pressures less than 600 psi. The effect of the solid support of the reversed phase liquid-liquid partition chromatography of these steroids was investigated. At a given inlet pressure, improved separations were obtained using a combination of liquid-liquid partition and liquid-solid absorption effects. A hydrophobic solid support is used which exhibits lipophilic adsorption even after coating with the stationary phase. Using this combination of absorption and partition effects, optimization of separations can be achieved.
A FAST, smsmve, easily quantitated, high efficiency method of analysis for mixtures of thermally labile steroids is desirable because of the importance of these compounds in the pharmaceutical industry and clinical studies. Paper chromatography, thin-layer chromatography, conventional column chromatography, and gas chromatography are used extensively in the separation of steroids. However, each of these chromatographic methods, as well as conventional multistep extraction techniques, suffers from one or more limitations for the fast analysis of steroid mixtures. Since many of these limitations can be eliminated by the use of high speed, high resolution liquid chromatography (LC), this technique should be the method of choice for the analysis of many steroid mixtures. Thin-layer, paper, and conventional column chromatography are methods generally applicable for steroid separations. They require no derivatization and are simple and inexpensive to carry out. However, they all have serious inherent limitations with regard to speed of separation, ease of quantitation, and separation efficiency(1). Gas chromatography has become a popular method of analysis for steroids because of its speed, relative ease of quantitation, and high resolution capability. However, gas chromatography suffers from one major limitation in the analysis of steroids in that many of the biologically important steroids either have low volatility or are too thermally labile for direct gas chromatographic analysis. For these steroids, derivatization techniques are required for satisfactory, intact separations. I n fact, many thermally stable steroids are often derivatized to obtain better chromatographic behavior at conveniently low temperatures (2,3). There are several problems inherent to the derivatization of mixtures of steroids. Some of these problems include: known yield of derivatives can be difficult to achieve without careful study ; either different conditions for derivatization or different derivatives may be necessary for the various steroids Present address, Instrument Dept., Union Carbide, White Plains, N.Y. (1) J. F. K. Huber, J. Chromatogr. Sci, 7 , 85 (1969).
(2) A. Karmen and J. L. Marsh in “Lipid Chromatographic Analysis,” Vol. 2, G. V. Marinetti, Ed., Marcel Dekker, Inc., New York, N. Y., 1969, Chapter 10. (3) H. H. Wotiz and S . J. Clark, “Gas Chromatography in the Analysis of Steroid Hormones,” Plenum Press, New York, N. Y . , 1966.
present in a mixture; multifunctional steroids can give mixtures of derivatives; prior knowledge of the composition of the mixture is needed to ensure proper derivatization; derivatization techniques are time consuming and often require a recovery or purification step prior to injection. Recent work in several laboratories (4-13) has shown that high resolution liquid chromatographic separations can be achieved routinely. By using narrow bore columns, small diameter supports, low dead volume injectors and detectors, and pressurized eluant delivery to achieve desired flows, fast separations of similar compounds can be achieved with column efficiencies per unit time, previously only associated with gas chromatography. Waters, Little, and Horgan (11) have shown that the separating power is by far the most important factor in liquid chromatography. They showed that for a representative system if one compares two packing materials, one with twice the separating power of the other, the only way to obtain similar results is to take eight times as much time, or operate at 51 times as much pressure drop. This paper deals with the development of column packings which have high separating power for various polar and moderately polar steroids. Many of these steroids are difficult to separate or quantitate by other methods. Representative members of several classes of steroid hormones were separated on a variety of columns under different conditions. The effect of the solid support is studied with regard to optimization of the separations at a given column inlet pressure. A combination of liquid-liquid partition and liquid-solid adsorption effects is used to obtain increased separation power under a given set of chromatographic conditions. EXPERIMENTAL
Apparatus. A schematic of the liquid chromatograph used in this study is shown in Figure 1. The pump is a Milton Roy Controlled Volume Minipump Model 196-31. This pump has a maximum discharge pressure of 1000 psi and a micrometer stroke adjustment allowing capacity adjustment from 0-160 ml/hr. Pulse damping is achieved by use of a 0-600 psi stainless steel Bourdon gauge (Linde Division, Union Carbide) and a 2-m section l/l&ch by 0.01-inch i.d. stainless steel tubing. A precolumn 12-inch by 2.8-mm i.d., was packed with 160-180 mesh Anakrom AB coated with the stationary phase (10z). All fittings and tubing used were Teflon (Du Pont), Kel-F, or stainless steel. The column (4) J . F. K. Huber and J. A. R. J. Hulsman, Anal. Chirn. Acta, 38, 305 (1967). ( 5 ) J. H. Knox and M. Saleem,J. Chromatogr. Sci., 7 , 614 (1969). (6) J. C. Giddings, ANAL.CHEM.,35, 2215 (1963). (7) L. R. Snyder and D. L. Saunders, J . Chromarogr. Sci., 7, 195
(1969). (8) L. R. Snyder, ibid., p 352. (9) Ibid., p 595. (10) R. P. W. Scott and J. G. Lawrence, ibid., p 71. (11) J. L. Waters, J. N. Little, and D. F . Horgan, ibid., p 293 (1969). (12) C. Horath and S. R. Lipsky, ibid., p 109. (13) J. J. Kirkland, ibid., p 7.
ANALYTICAL CHEMISTRY, VOL. 42, NO. 11, SEPTEMBER 1970
1223
Injection Tee
I
I
-.
n
T
o waste
Figure 1. Schematic of liquid chromatograph
fittings and injection tee were obtained from Chromatronix Inc., Berkeley, Calif. Injections were made with a 5-p1, high pressure syringe (Hamilton, HP-305) through a rubber septum 1.5 cm into the column bed. The maximum pressure used on this system was 600 psi. All columns were 500 mm X 2 mm straight borosilicate glass tubing (Chromatronix Inc.). A battery operated Beckman D U Spectrophotometer equipped with a Deuterium source lamp (Beckman part No. 96280) was used as the detector. A Varian G-2000 strip chart recorder (Varian Associates, Palo Alto, Calif.) equipped with 1-IO00 mV variable span was wired across the null meter. Using this high input impedance recorder, no further impedance matching was required. The cell was constructed after the design of Jentoft and Gouw (14) from a stainless steel block (3.3 x 7.8 x 1.0 cm) to fit into the sample positioning assembly of the spectrophotometer. The cell has a path length of 1 cm and a volume of 20 pl. With this cell, no beam condensing system was necessary. Base-line adjustment at a given wavelength was made by zeroing both the dark current and sensitivity controls to some arbitrary but constant point near the left extreme of the meter. The recorder pen was then set using the zero off-set on the recorder. Operated in this manner, the recorder signal is not directly proportional to % transmittance or concentration. However, reasonably linear calibration curves of quantity injected us. response can be constructed for concentration ranges over two orders of magnitude. The detection of 10 ng of fast eluting steroids could be obtained with a signal to noise ratio of greater than 10 to 1. Elevated temperatures could be employed on this apparatus using column jackets connected to a circulating water bath. All results reported in this study were obtained at ambient temperature23.0 f 1 "C. Reagents. The steroids were either purchased from Mann Research Laboratories, New York, N. Y., or were supplied by Anthony M. Gawienowski, Department of Biochemistry, University of Massachusetts, Amherst, Mass. Except for the estrogens, all the steroids studied have a 4-en-3-one chromophore with molar extinction coefficients of approximately 16,000 and absorption maxima between 240-250 mp. Estrogens were detected at 280 mp. The steroids were chromatographed as received. Eluants. Reversed phase chromatography was employed throughout this study since many of the more labile steroids are quite polar and elute relatively slowly using nonpolar eluants. Water and water-methanol solutions were used as
Table I. Comparison of Adsorption on Uncoated Supports fat, min Anakrom No. Steroid CTFE Zipax AB 1 6P-Hydroxycortisone 1.85 .39 0 2 Aldosterone 9.00 1.77 0 4 Cortisone 18.5 1.07 0 15 4-Androstentrione 19.1 2.28 0 8 Corticosterone 34.7 2.38 0 11 11-Deoxycortisol 45.5 2.81 0.08 17 A1,CAndrostadiene170-01-3-one 39.8 1.31 0.07 14 Deoxycorticosterone 87.5 1.81 0.10 Columns: 485 mm X 2 mm i.d. Eluant : CTFE-30 (v/v) methanol in water Zipax and Anakrom AB-water Flow: 0.25 ml/min Packing: CT'FE, sieved to pass 325 mesh Zipax, 20-37 p Anakrom AB, 140-325 mesh
eluants depending upon the steroids being separated. Eluants containing up to 33% methanol (v/v) could be used without excessive bleeding. Stationary Phase. Amberlite LA-1 [n-dodecenal (trialkylmethyl) amine] obtained from Rohm and Haas, Philadelphia, Pa., was used as the stationary phase throughout this study. Columns prepared using LA-1 as the stationary phase exhibited good separation power for the steroids studied, gave symmetrical peaks with no signs of decomposition for even the most labile steroids, and yielded good column life. Columns have been used over three months without significant deterioration of performance. Support. Various solid supports were studied. The use of three supports is reported here. Anakrom AB (Analabs, Inc., Hamden, Conn.) was obtained as 100-110 mesh. This diatomaceous earth support was then ground, sieved (140-325 mesh), and washed in concentrated "0,. The slurry was then filtered, neutralized with NaOH, and washed with water. The dried support was then used without further screening. Plaskon CTFE-2300 was obtained from Allied Chemical Co. This terpolymer, consisting mainly of trifluoro ethylene, was obtained as a fine powder and sieved. The fraction which passed 325 mesh was used in this study. Unlike fine powders of Teflon, CTFE powder is easy to work with. No low temperature packing techniques were used as have been recommended for Teflon packings (15). The CTFE was easily coated with LA-1 and exhibited a rather high capacity for the amine before serious packing problems were observed. This hydrophobic support showed considerable lipophilic adsorption properties in water and water-methanol systems. The effect of this absorption was noticed even at the highest stationary phase loadings studied and was used to considerable advantage in many separations. Zipax chromatographic support was obtained from D u Pont Instruments. This support, first reported by Kirkland (13, 16, 17), consists of spherical silica beads (20-37 p ) with controlled surface porosity (CSP). The support was used as obtained. Column Preparation. In each case the stationary phase was coated by evaporating, with stirring, a slurry of the support in a solution of the stationary phase in dichloromethane. Each support was coated with the stationary phase at a level which gave close to optimal results. The Zipax support was coated with 1.0% LA-1 (13). The Anakrom AB support was coated with 15 % LA-1. This loading gave good results (15) D. M. Ottenstein,J. Gas Chromatogr., 1, 11 (1963).
(14) R. E. Jentoft and T. H. Gouw, ANAL.CHEM., 40,923 (1968). 1224
41, 218 (1969). (16) J. J. Kirkland, ANAL.CHEM., (17) J. J. Kirkland, J. Chrornafogr. Sci., 7, 361 (1969).
ANALYTICAL CHEMISTRY, VOL. 42, NO. 11, SEPTEMBER 1970
~~
Table 11. Effect of Solid Support on Steroid Separations Using Coated Columns
No. 2 4 15 8
t R , min Anakromb CTFE= AB 4.58 4.26
Steroid Aldosterone
k
Rij
Zipaxe 3.68
CTFE 0.152
AB 0.036
Zipax 0.287
Cortisone
5.08
4.80
4.11
0.373
0.0862
0.437
4-Androstentrione
6.53
5.67
5.07
0.765
0.284
0.772
Corticosterone
8.10
6.30
7.66
1.19
0.426
1.68
11
11-Deoxycortisol
11.86
7.21
6.64
2.57
0.652
1.34
17
Al,.?-Androstadiene17P-ol-3-one
22.7
12.59
9.29
5.13
1.85
2.24
14
Deoxycorticosterone
47.6
23.7
20.12
11.86
4.30
5.98
CTFE
AB
Zipax
1.28
0.27
0.66
1.84
0.95
1.18
1.63
0.63
1.46*
3.01
0.78
0.81
4.88
3.14
0.88
5.4
3.5
3.6
Columns: 485 mm X 2 mm i.d. Eluant: water Flow: 0.25 ml/min Packing: a 24% LA-1 on CTFE. b 14% LA-1 on Anakrom AB. c 1% LA-1 on Zipax. * Elution order reversed in the case of Zipax;
and is easily packed. The CTFE support was loaded at various levels. All columns were packed by adding small amounts of the dry packing to a funnel attached to the top of the column. Then the column was alternately tapped on the end and on the sides until no further compression of the packing bed was observed. This process was then repeated until the column was full. Low density packings such as CTFE often required additional packing to be added after the column was operated for a time at maximum pressure. However, this further packing procedure had no apparent detrimental effects on column efficiency. No further changes in the bed volume were observed.
RESULTS AND DISCUSSION All steroids chromatographed are numbered consecutively by retention volume within each class. For the androgens and adrenal corticosteroids, retention data are reported under identical conditions for easy comparison. All injections were made from methanol solutions of the steroids. Injections of 1 to 10 pl of solution were made containing between 0.05 and 1.0-pg of each steroid. Effect of the Support on Separations. The separations achieved in liquid-liquid partition chromatography are greatly affected by the adsorbent properties of the solid support. Table I compares the adsorbent properties of the three supports studied in a reversed phase system (HzOeluant). The adjusted retention times ( t p ) for eight steroids are given for columns (485 mm X 2 mm i.d.) prepared from the uncoated supports where: tR'
E
tR
-
tm
is the retention time measured from injection, and tm is the elution time for an unretained solute (KNOa). In each case the flow was 0.25 ml/min. Comparison of the adjusted retention volumes shows that the Anakrom AB is essentially inert in a reversed phase system while the untreated Zipax shows some adsorption. The CTFE exhibits strong adsorption even with a less polar eluant (30% methanol v/v). (Retention times of these steroids were inconveniently long on this column using a water eluant.) tR
The adsorption isotherms for the steroids studied were linear on CTFE over the concentration range studied. This was evidenced by the symmetrical peak shapes obtained upon elution. However, very fast eluting steroids were observed to tail slightly at high flows indicating a kinetic contribution. Anakrom AB showed no significant tailing. Untreated Zipax gave serious tailing for several steroids; however, this is reduced by coating (1 %) with stationary phase. Silanizing the Zipax support is recommended by Kirkland (18) for reversed phase chromatography and should eliminate much of this tailing. Work in this laboratory has shown that silanization of porous glass supports promotes lipophilic adsorption on the resulting hydrophobic surface. This adsorption has been used to advantage in reversed phase steroid separations. The effect of these adsorption properties on the retentions and resolutions obtained using coated column packings can be seen from Table 11. The retention times ( t R ) and capacity ratios ( k ) of several steroids on LA-1 coated columns using these three supports are compared using HzO eluants. The capacity ratios were calculated from :
k =
wt of compound in stationary phase wt of compound in mobile phase
_ t R_' -
rm
For direct comparison of the separation power of these columns, the resolutions (R,) for each pair of adjacent steroids are calculated from :
tRf and rRi are the retention times of compounds i and j ; Y t , and Y t t are their respective peak widths (min). It is readily seen that the resolution achieved using the CTFE support is far superior to the less active supports. Note, however, the elution order of androstentrione and corticosterone are reversed on the Zipax column. (Resolutions were calculated on
(18) J. J. Kirkland, DuPont, Wilmington, Del., private communication, 1970.
ANALYTICAL CHEMISTRY, VOL. 42, NO. 11, SEPTEMBER 1970
1225
Table 111. Effect of Stationary Phase Loading on Separations Using CTFE Support t R ’ (min) Loading No. Steroid 18z 24% 28z 35z 0.05 1 6@-Hydroxycortisone 0.40 0.17 0.10 2 Aldosterone 1.43 0.56 0.28 0.24 4 Cortisone 3.02 1.38 0.96 0.53 15 4-Androstentrione 3.76 2.83 2.84 3.28 2.55 8 Corticosterone 8.83 4.40 3.02 11 1I-Deoxycortisol 12.66 7.59 6.80 7.10 17 AI ,4-Androstadiene17P-ol-3-one 22.46 19.0 22.5 24.2 14 Deoxycorticosterone 52.2 43.9 51.2 57.0 Columns: 485 mm X 2 mm i.d. Eluant: water Flow: 0.25 ml/min Packing: indicated loadings of LA-1 on CTFE Table IV. Column Efficiencies TIME [MIN 1
Figure 2. Effect of solid support on separations Column: 485 mm X 2 mm i.d. Eluant: Water Flow: 0.49ml/min Packings : A . 14 LA-1 on Anakrom Ars B. 1% LA-1 on Zipax C. 23%LA-1 on CTFE
the observed elution order in each case.) Figure 2 compares the elution behavior of a mixture of four different steroids on these three columns. Chromatogram A (15% LA-1 on Anakrom AB) shows only two resolved peaks. Steroids numbered 1 and 6 as well as 9 and 10 (Table V) are unresolved. I n chromatogram B (1 % LA-1 on Zipax) steroids 1 and 6 are resolved but steroids 9 and 10 remain unresolved. In chromatogram C (24% LA-1 on CTFE) all four components are resolved. I n each of these columns, the pressure drops were approximately the same (450 psi at 0.49 ml/min). Thus direct comparison of resolution/unit time at a given pressure drop can be made. Effect of Stationary Phase Loading on Separation. In Table I11 the effect of the stationary phase loading on CTFE columns is illustrated. Adjusted retention times of several steroids are given VS. the stationary phase (LA-1) loading (wt stationary phase)/(wt coated packing). Two things are noteworthy. First, the retentions of the fast eluting, more polar adrenal corticosteroids (No 1, 2, 4, and 8) decrease with increased loading. Second, the remaining less polar steroids exhibit a minimum retention at a given loading and then increased retention at higher loadings. This behavior can be explained by considering the competition of the two separation mechanisms, Le., liquid-liquid partition and liquid-solid adsorption. As the loading is increased the contribution of the liquid-solid adsorption is decreased and that of the liquid-liquid partition is increased. Thus as the adsorption decreases, the retention decreases. Then when liquid-liquid partition becomes predominant, the retention increases with loading as expected. For the more polar steroids the adsorption is predominant over the loading range studied. The loading range from 18-28z was optimum for the steroid separations studied. At higher loadings the packing is
Packing 3 5 z LA-I on CTFE 28% LA-1 on CTFE 2 4 z LA-1 on CTFE 18% LA-1 on CTFE 14% LA-I on Anakrom AB 1 LA-I on Zipax
z
H(mm) KN03 v = 0.04 cm/sec 0.44 0.17
0.16 0.30 0.23 0.29
H(mm) cortisone v = 0.22 cm/sec 1.9 0.55
0.52 0.69 0.80 0.83
quite “wet,” and high efficiency columns are increasingly difficult t o prepare. At lower loadings the adsorption effects become quite strong and elution times for relatively nonpolar steroids can become inconveniently long. Furthermore the column efficiencies, as measured by the number of theoretical plates [ N = 16 ( t R / Y J 2 observed ], for the steroids studied decreased with loading below 18 %. Preliminary studies comparing column efficiencies for a n unretained solute with those for moderately retained steroids indicate that the strong adsorption of lower loaded and uncoated CTFE columns significantly increases the band broadening contribution of mass transfer in the stationary phase, C,. The effect of this can be seen from the simplified van Deemter equation:
(tRl)
1226
Vis the average linear flow velocity. H , the height equivalent to a theoretical plate, depends upon the sum of three terms. A , the eddy diffusion or multiple path term, is a measure of how well the column is packed. B, the molecular diffusion term, is negligible in liquid chromatography (19, 20). The magnitude of the last term, depends on the contribution t o band spreading due t o resistance t o mass transfer in the stationary phase, C,,and the resistance to mass transfer in the mobile phase, C,, as well as on V. For a n unsorbed solute, band spreading is dependent on the A term. Therefore, the difference in H measured for a sorbed and unsorbed solute in the same eluant (same C,) at the same flow gives a measure of C,. The increase in C, observed on uncoated and lightly coated CTFE columns increased H and thus decreased the number of theoretical plates obtained. In general the greatest number of effective plates (8) were obtained with the CTFE (19) I. Hal& and P. Walking, J. Chromatogr. Sci., 7, 129 (1969). (20) L. R. Snyder, ANAL.CHEM., 39,698 (1967).
ANALYTICAL CHEMISTRY, VOL. 42, NO. 11, SEPTEMBER 1970
Table V. Retention Data for Some Adrenal Corticosteroids Relative Steroid 4-Pregnene-6&17a,21-triol-3,11,20-trione (6~-hydroxycortisone) 4-Pregnene-11&21-diol-3,20-dione-18-a1 (aldosterone) 4-Pregnene-17a,20~,21-triol-3,1l-dione (Reichstein’s U) 4-Pregnene-17a,21-di01-3,11,20-trione (cortisone) 5 4-Pregnene-1lp,17a,20P,21-tetrol-3-one (Reichstein’s E) 6 4-Pregnene-11~,17a,21-triol-3,2O-dione (cortisol) 7 4-Pregnene-21-01-3,11,20-trione (11-dehydrocorticosterone) 8 4-Pregnene-11/3,21-diol-3,20-dione(corticosterone) 9 4-Pregnene-1lo, 17a-diol-3,20-dione 10 4-Pregnene-17a-01-3,11,20-trione 11 4-Pregnene-17a,21-diol-3,20-dione (11-deoxycortisol) 12 4-Pregnene-17a,21-diol-3,11,20-trione-21-acetate (cortisone 21-acetate) 13 4-Pregnene-20o,21-diol-3-one 14 4-Pregnene-21-01-3,20-dione (deoxycorticosterone) Column: 485 mm X 2 mm i.d. Eluant: water Flow: 0.25 ml/min Packing: 24% LA-1 011 CTFE No. 1 2 3 4
t ~min ,
tR‘
3.87 4.26 4.75 5.08 5.27 5.71 6.05 8.10 9.24 11.29 11.86 14.31
0.123 0.406 0.761 1.Ooo 1.14 1.46 1.70 3.19 4.01 5.50 5.91 7.69
36.6 47.6
23.8 31.8
~
z,
support loaded (LA-1) between 20-28 and the fastest steroid separations were achieved on these columns. The differences in retention behavior of these steroids at different stationary phase loadings can be used t o considerable advantage in optimizing a separation. For example, it can be seen from Table I11 that, using columns with the same number of theoretical plates, the separation of androstentrione and cortisone is significantly better at high loadings while the separation of 6-P-hydroxycortisone and aldosterone is better at lower loadings. I n general, the steroids studied showed this type of retention behavior and separations can often be optimized by varying the loading. Column efficiencies (H) for the various columns used in this study are given in Table IV. Values of H a r e given for a n unretained solute (KNOJ at a n average flow velocity of 0.04 cmi sec and for cortisone at 0.22 cmjsec (0.25 mlimin). High column efficiencies were obtained using the CTFE support. Columns exhibiting more than 6000 plates/meter for unretained solutes could be prepared routinely. No effort was made t o improve column efficiencies by further size grading the CTFE, although this should lead t o significantly higher plate numbers. Separations of Steroids by Class. The steroids studied were chosen on the basis of biological importance, structure, and availability. Whenever applicable, the steroids were chromatographed o n the same column under the same conditions t o facilitate comparison of retentions between classes. Relative retentions of the adrenal corticosteroids and the androgens are given relative to cortisone. The column lengths used are a compromise to allow representative chromatograms of steroids with capacity ratios ranging over a factor of 400 in a reasonable time. A column length of 750 mm is sufficient to resolve nearly all of the steroids tried. ADRENAL CORTICOSTEROIDS. Corticosteroids and structurally related steroids exhibiting commonly occurring combinations of functionality were chromatographed. Table V contains the retention times ( t R ) ,relative retentions (rR,), and capacity ratios ( k ) of the steroids o n a column packing of 24 LA-1 on CTFE and water mobile phase. While any pair of these steroids could be completely resolved o n this packing, many separations could be further optimized by changing the per cent loading or eluant composition. Columns with lower
k 0.046 0.151 0.284 0.373 0.424 0.543 0.638 1.19 1.50 2.05 2.21 2.87 8.89 11.86
~~
I
i
r
TIME [MINI
Figure 3. Separation of some adrenal corticosteroids Column: 485 mm X 2 mm i.d. Eluant: Water Flow rates: initial 0.10 ml/min A . Increased to 0.13 ml/min B. Increased to 0.21 ml/min C. Increased to 0.26 ml/min D. Increased to 0.44 ml/min Packing: 23% LA-1 on CTFE liquid phase loading are useful for the separation of the faster eluting, more polar corticosteroids. Eluants containing up t o 33% (v/v) methanol are useful for decreasing the elution volume of later eluting compounds. Figure 3 shows a chromatogram of a mixture of nine corticosteroids. Step-wise flow programming was used as noted. ANDROGENS.Retention times of some androgens and related steroids are given in Table VI. Relative retentions are given with respect to cortisone for comparison since the same column and conditions were employed as with the corticosteroids. Figure 4 shows the separation of these steroids obtained using a 23% LA-1 on a CTFE column. In general
ANALYTICAL CHEMISTRY, VOL. 42, NO. 11, SEPTEMBER 1970
1227
Table VI. Retention Data for Some Androgens No. Steroid f R , min Relative tR' 15 4-Androstene-3,11,17-trione 6.53 2.05 16 4-Androstene-llfl-ol-3,17-dione 7.98 3.10 17 A1,4-Androstadiene-l7fl-ol-3-one 22.7 13.8 18 19-nor-4-Androstene-3,17-dione 26.9 16.8 19 19-nor-Testosterone 31.9 20.4 20 4-Androsten-3,17-dione 57.2 38.8 21 Testosterone 68.6 47.0 Column: 485 mm X 2 mm i.d. Eluant : water Flow: 0.25 mlfmin Packing: 24% LA-1 on CTFE (325 m) Table VII. Retention Data for Some Progestins No. Steroid t ~min , k 4.43 1.74 21 Testosterone 22 17a-Hydroxyprogesterone 6.15 2.80 23 Progesterone 30.0 17.5 24 4-Pregnene-20@-01-3-0ne 30.6 17.9 Column: 485 mm X 2 mm i.d. Eluant: 33 methanol in water (v/v) Flow: 0.56 ml/min Packing: 2 8 z LA-1 on CI'FE
k
0.765 1.16 5.14 6.27 7.62 14.5 17.5
Ii ) ) li? w
m
Z
Bm w
LT
0
10
20
30
40
50
60
TIME [MIN.]
Figure 5. Separation of estrogens Column: 485 mm X 2 mm i.d. Eluant: Water adjusted to pH 11.5 (NaOH) Flow rates: initial 0.12 ml/min A . Increased to 0.145 ml/min B. Increased to 0.19 ml/min C. Increased to 0.49 ml/min Packing: 28% LA-1 on CTFE 17
A-
1
0
Figure 4.
irr U"(
I
I
I
10
20 TIME (MINI
30
I
40
Separation of some androgens
Column: 485 nun X 2 mm i.d. Water Eluant: Water Flow rates: initial 0.17 ml/min A . Increased to 0.49 ml/min Packing: 23% LA-1 on CTFE these steroids showed somewhat less change in retention with stationary phase loading (between 20-28x) than the corticosteroids. Therefore, in a mixture of corticosteroids and androgens, rather large differences in resolution can be obtained by varying the stationary phase loading. PROGESTJNS. Under the same conditions, the progestins eluted after all the corticosteroids and androgens studied. Therefore, these steroids were chromatographed using an 1228
aqueous eluant 33% (v/v) methanol. The retention data obtained under these conditions are given in Table VI1 for a 2 8 x LA-1 on CTFE column. Testosterone is included to facilitate comparison of the progestins retentions with those of the adrenal corticosteroids and androgens. It is noteworthy that under the conditions employed, progesterone is not satisfactorily separated from 4-pregnene-20P-ol-3-one. Therefore a different stationary phase loading or eluant would be necessary to resolve these steroids. Normal phase chromatography might give faster separations of these relatively nonpolar steroids. ESTROGENS.Estrogens were chromatographed using water or water-methanol adjusted to pH 11.5 with NaOH as mobile phases. The phenolic estrogens (pK = 9.3) are more soluble in basic than neutral eluants. Retention data for the estrogens studied are presented in Table VIII. Elution behavior was sometimes erratic using the amine stationary phase at pH's less than 10 unless columns were conditioned overnight with basic eluant. While all retentions reported here are for aqueous (pH 11.5) eluants, increased resolution can often be obtained by varying the pH or methanol content of the eluant. Figure 5 shows a separation of several of these estrogens on a 28 LA-1 on CTFE column using an aqueous eluant. Solvent programming (gradient elution) could be used to considerable advantage for these separations.
x
ANALYTICAL CHEMISTRY, VOL. 42, NO. 11, SEPTEMBER 1970
Table VIII. Retention Data for Some Estrogens Relative No. 25
min 3.60
0.008
3.98 4.02 4.20 4.87 7.30 25.8 39.8 40.4 43.0 56.4
0.305 0.336 0.477 1.000 2.90 17.3 28.3 28.8 30.8 41.3
tR,
1,3,5(l0)-estratrien-3,17~-diol-l7a-Glucosiduronic acid (Estradiol-17a-Glucosiduronic acid) 26 1,3,5(10)-estratrien-3,16a,17~-triol (estriol) 27 1,3,5(1O)-estratrien-3,16a,l7a-triol (17-epiestriol) 28 1,3,5(lO)-estratrien-3-01-16,17-dione(16-ketoestrone) 29 1,3,5(10)-estratrien-3,17~-diol-16-one (16-ketoestradiol) (16-epiestriol) 30 1,3,5(lO)-estratrien-3,16~,17~-triol 31 1,3,5(10),6,8-estrapentaen-3-01-17-0ne (equilinen) 32 1,3,5(lO)-estratrien-3,17/3-diol(estradiol) 33 1,3,5(1O)-estratrien-2-methoxy-3-ol-l7-one (Zmethoxyestrone) 1,3,5(10),7-estratetraen-3-01-17-one(equilin) 34 35 1,3,5(10)-estratrien-3-01-17-0ne(estrone) 36 1,3,5(10)-estratrien-3,16a,l7a-triol-triacetate (17-epiestriol triacetate) 37 1,3,5(lO)-estratrien-3,17P-diol17-acetate (17P-estradiol-17acetate) No elution observed under these conditions after 2 hours. Column: 485 mm X 2 mm i.d. Eluant: water, pH 11.5 (NaOH) Flow: 0.25 ml/min Packing: 28% LA-1 on CTFE
...
...
tR'
...
k 0.003 0.109 0.120 0.170 0.357 1.03
6.18 10.1
10.3 11.o 14.7
(1
. . .a
5
Table IX. Results of Trials Obtained for Known Mixtures of Steroids Relative Corticosteroids w3, used pg," found error, % 4-Pregnene-17a,20P,2l-triol-3,1l-dione 0.226 0.237 f4.9 3 a 9 4-Pregnene-11~,17a-diol-3,20-dione 0.616 0.625 +1.5 10 4-Pregnene-17a-ol-3,11,20-trione 1.008 1.003 -0.5 Androgens 4-Androstene-3,11,17-trione 0.573 0.567 -1 .o 15 b 17 A1 ,4-Androstadiene-l7P-ol-3-one 0.878 0.867 -1.3 Determined by comparison of the peak areas obtained from mixtures with calibration curves of peak area us. 1.18injected,
Mixture
a
No.
Quantitation. Although base-line drift in this instrument resulted in a change in sensitivity, it was determined that the reproducibility of peak area us. sample size injected was within 2-5z relative as long as stable base lines were observed. Table IX shows the results of trials obtained for known mixtures of steroids. Peak areas were compared to calibration curves previously prepared for each steroid. Injections were of standard mixtures of the steroids in methanol. The fast eluting steroid (No. 3) exhibited the largest deviation. This is because of the finite mixing time resulting when injecting a sample in a solvent other than the eluant. I n the case of injections of methanol solutions into water eluants, the methanol plug can tend to float up the column or
injector and spread the band entering the column. This can lead to irreproducible injection and increased error in quantitation. A different injector design should minimize this problem. ACKNOWLEDGMENT
The authors express their appreciation t o R. Hagstrom, R. Finch, and W. Thompson at Olin Corporation for their technical assistance.
RECEIVED for review May 7,1970. Accepted June 26,1970.
ANALYTICAL CHEMISTRY, VOL. 42, NO. 11, SEPTEMBER 1970
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