Comparison of corticosteroid derivatives by gas chromatography-mass

Comparison of Corticosteroid Derivatives by Gas Chromatography-Mass Spectrometry T. A. Baillie, C. J. W. Brooks, and B. S. Middleditch’ Chemistry Department, University of Glasgow, Glasgow, W.2, Scotland (Great Britain)

Representative derivatives used or proposed for the gas chromatography of corticosteroids have been compared with respect to their ease of preparation, stability, retention characteristics, and mass spectra. Retention indices on OV-1 and Dexsil-300GC phases are reported, together with salient mass spectrometric data. The sensitivity with which different derivatives can be detected by monitoring single, characteristic fragment ions has been investigated for the example of Reichstein’s Substance S: the limit of detection in the most favorable case (17,21-anhydro derivative) was 400 pg.

THESUSCEPTIBILITY of free corticosteroids t o decomposition during gas chromatography has led to studies of derivatives designed for greater stability (1). Further criteria are required as a result of the increasing importance, in steroid analysis, of combined gas chromatography-mass spectrometry (GC-MS) (2). I n this paper we have attempted a collation of some features of corticosteroid derivatives in respect to their value for GC-MS. Among the types of side-chain commonly found in the corticosteroids, the dihydroxyacetone grouping is particularly prone to decomposition ( 3 ) . The first derivatives shown to possess gas chromatographic stability were the bismethylenedioxy derivatives ( 4 ) but neither these nor the 17,21-diacetates (5) have proved of general value. Following the introduction of the 0-methyloxime (MO) derivative for ketosteroids (6), it was shown by Gardiner and Horning (7) that the 20-MO 21-trimethylsilyl (TMS) ethers were effective in stabilizing steroidal dihydroxyacetones. These and the corresponding 20-MO 17,21-diTMS ethers (8) remain the most generally serviceable derivatives, especially for quantitative work. They are included in this survey together with examples of cyclic boronate esters (9, IO), dimethylsiliconides (11, 12), and oxetanones (13, 14). [The interesting enolic TMS ethers Present address, Institute for Lipid Research, Baylor College of Medicine, Houston, Texas 77025 (1) E. C. Horning, C. J. W. Brooks, and W. J. A. VandenHeuvel, Aduaa. Lipid Res., 6 , 213-392 (1968). (2) C. J. W. Brooks, in “Mass Spectrometry,” Vol. 1, The Chemical Society, London, 1970, pp 288-307. (3) W. J. A. VandenHeuvel and E. C. Horning, Biochem. Biophys. Res. Commun., 3, 356 (1960). (4) M. A. Kirschner and H. M. Fales, ANAL. CHEM.,34, 1548 (1962). (5) C. J. W. Brooks, ibid., 37,636 (1965). (6) H. M. Fales and T. Luukkainen, ibid., p 955. (7) W. L. Gardiner and E. C. Horning, Biochim. Biophys. Acta, 115, 524 (1966). (8) E. M. Chambaz and E. C. Horning, Anal. Lett., 1, 201 (1967). (9) C . J. W. Brooks and J. Watson, in “Gas Chromatography 1968.” C. L. A. Harbourn.. Ed... Institute of Petroleum, London, 1969; pp 129-141. (10) C. J. W. Brooks and D. J. Harvey, J . Chromatogr., 54, 193 (1971). (11) R. W. Kelly, Steroids, 13,507 (1969). (12) R. W. Kelly, J . chi-omatogr., 43,229 (1969). (13) Upjohn Co., Brit. Patent 869,564 [Chem. Abstr., 56, 2490 ( 1962)l. (14) T. A. Baillie and C. J. W. Brooks, observations to be reported. 30

introduced by Chambaz and Madani (15) have not been compared directly with the aforementioned derivatives in our laboratory.] Gas chromatographic and mass spectrometric data for derivatives of other corticosteroids [e.g., acetonides of 17a,20-diols (16) and 20,21-diols (17, 18)], and for some products of side-chain degradation, are also reported. EXPERIMENTAL

Steroids and Derivatives. Steroids were obtained commercially, mainly from Ikapharm (Ramat-Gan, Israel) and Sigma London Chemical Company Ltd. (London, S.W.6, England). Derivatives were prepared by conventional methods, using 0.4 mg (ca. 1 pmole) of steroid, except in the following instances. The oxetanone from Reichstein’s Substance S, i.e., 17a,21-oxidopregn-4-ene-3,20-dione, was prepared by the general procedure reported by the Upjohn Co. (13): the reaction product contained 21-fluoro-17a-hydroxypregn4-ene-3,20-dione, which was removed by repeated thinlayer chromatography using as mobile phase ethyl acetate/ light petroleum, bp 60-80 “C, (1 :1 v,!v). Small-scale preparations of Substance S methylboronate and MO TMS ether derivatives were carried out using 4 pg of steroid in each case. [We did not effect a satisfactory preparation of the dimethylsiliconide on this scale.] The following procedure is illustrative. Substance S (4 pg) was mixed with a solution of methylboronic acid (4 molar equiv.) in ethyl acetate (6 pl), and kept in a closed tube at room temperature for 15 min. An aliquot (l/q) was used for GLC, which indicated that the boronate had been formed in 85% yield. The remainder was diluted to 100 pl, and 2 p1 (equivalent to 60 ng of Substance S) was used for “single ion monitoring.” Gas Chromatography. A Perkin-Elmer F-1 1 dual-column chromatograph was used, with 2 m x 3 mm i.d. “silanized” glass columns packed with 1 OV-1 and 1 Dexsil-300GC, respectively, on Gas Chrom Q, 100-120 mesh (Applied Science Laboratories, State College, Pa.). Flame ionization detectors were used, with nitrogen as carrier gas. Retention indices were measured with respect to n-alkanes co-injected with the steroid derivatives. In the LKB 9000 instrument, a 3 m X 3 mm i.d. column (1 OV-1) was used with helium as carrier gas. Mass Spectrometry. Mass spectra were recorded, at electron energy 70 eV, except for those of Figure 2 (22.5 eV), using a n LBK 9000 gas chromatograph-mass spectrometer. The column temperature was 245 “C, with the flash heater a t 260 “C, the molecule separator at 250 “C, and the ion source at 250 “C. The helium flow rate was 25 ml/min. The trap current was 60 p A ; accelerating voltage 3.5 kV; electron multiplier voltage 2.1 kV for recording full spectra,

ANALYTICAL CHEMISTRY, VOL. 44, NO. 1, JANUARY 1972

z

z

(15) E. M. Charnbaz and C. Madani, Excerptn Med. I/zt. Coirgr. Ser., 210,abstract No.192, p 97 (1970). (16) M . L. Lewbart and J. J. Schneider, J . Org. Chem., 34, 3505, ‘ 3513 (1969). (17) H. Adlercreutz. S. Laiho. and T. Luukkainen, Symposium “Gas Chromatographic Determination of Hormonal Steroids,” Rome, September 22, 1966: Academic Press, New York. 1967, p 69. (18) E. Bailey, Steroids. 10, 527 (1967).

~~~

Table I. Retention Index Values and Salient Mass Spectrometric Data for Corticosteroid Derivatives m/e values and relative abundances of characteristic ions Retention indices Base 1260" Iano Peak, Compound mie > 80 Fragment ions ov-1 Dexsil-a00GC Substance S (17~~,21-Dihydroxypregn-4ene- 3,20-dione) 3,20-diMO 21-TMS 3165

3290

386

3,20-diMO 21-d9-TMS

3155

3270

386

3,20-diMO 17,21-diTMS

3070

3135

517

17,21-dimethylsiliconide

2890

3170

91

17,21-methylboronate

2820

3110

244

17,21-anhydro-derivative (oxetanone) DOC (21-Hydroxypregn-4-ene3,20-dione) 21-TMS

2775

3120

286

3005

3320

299

3,20-diMO 21-TMS

3030

3130

460


2935

3075

110

20P,21-Dihydroxypregn4-en-3-one 20,21-diTMS

3095

3285

283


2850

3135

124

20,21-acetonide

2890

3165

357

2890

2985

117

3,17,2&triTMS

2765

2775

255

3-(acetoxydimethy1)silyl17,20-dimethylsiliconide

2885

2980

157


2585

2725

342

17,20-acetonide

2665

2815

86

3220

3425

116

3-MO 17,20,21-triTMS

3160

3205

388

methylboronate

2970

3245

372

acetonide

3015

3310

287

2960

3110

357

2910

3010

429

2505 (230") 2615 (230")

2900

286

2800

344

2730

3000

124

2745

2935

125

SP-Pregnane-3a,l7a,20@ triol 3,20-diTMS

17a,20P,21-Trihydroxypregn-4-en-3-one diTMS

17a-Hydroxyprogesterone ( 17a-Hydroxypregn-4ene-3,20-dione) 3,20-diMO 3,20-diMO 17-TMS

$:? 229

148

136

Androst-4-ene-3,17-dione

free 3,17-diMO Methyl 3-oxoandrost-4-ene17P-carboxylate free 3-MO


31

~

Retention indices ps00

Compound 3-Oxoandrost-4-ene-17pcarboxaldehyde free 3,2@diMO

1215"

OV-I

Dexail-300GC

2660

2990

2750

2900

Methyl 17a-hydroxy-3-oxoandro st-4-ene-17pcarboxylate free

Table I. Continued m/e values and relative abundances of characteristic ions Base peak, M+. m/e > 80 Fragment ions 300 (66) 358 (100)

124 358

285 (8) 343 (7)

282 (7) 327 (37)

258 (62) 273 (23)

244 (41) 153 (49)

229 (14) 151 (39)

215 (30) 137 (54)

125 (80)

3-MO

1.7-3.7 kV for single ion monitoring; exit slit, 0.2 m m ; entrance slit, 0.12 mm. Spectra were obtained using a recording oscillograph. Single ion monitoring was carried out at 22.5 eV with a Rikadenki potentiometric recorder as previously described (19). Much of the electrical "noise" originating in the electron multiplier was filtered uiu a 25,000 p F capacitor (20). RESULTS AND DISCUSSION

cultly-separable byproducts. It was, nevertheless, decided to include oxetanones in the survey, as a more practical preparative method may eventually emerge. ACETONIDES OF DIOLS. The method of Bailey (18) was found satisfactory for the small-scale preparation of both 17,20- and 20,21-diols: the use of perchloric acid as a catalyst (16) would doubtless be superior for the formation of 17,20-diol acetonides in larger quantity. The acetonide obtained from the 17a,20@,21-triol is assumed to be the 20,21-derivative (16-18). Gas Chromatographic Properties. Gas chromatographic retention indices for the various derivatives are listed in Table I. Satisfactory peaks were obtained in all instances. It should be noted that although the Dexsil-300GC column has been found suitable (20) for certain steroidal tertiary alcohols, it would not be generally applicable to compounds containing free hydroxyl groups. While the data do not suffice for detailed correlations, the following useful regularities are discernible.

Preparation of Derivatives. MO TMS ETHERS. The results confirm the general efficacy of the two-stage procedure (7), whereby reactive ketonic groups are converted t o methyloximes and these products are trimethylsilylated, with or without reaction of 17a-hydroxyl groups (8, 21-24). This study was limited t o 11-deoxysteroids, but conditions have already been well established for the trimethylsilylation of 1lp-hydroxycorticosteroids (21, 24). The conversion of 11-ketones to their enol-TMS ethers occurs only slowly even under vigorous conditions (22,24). DIMETHYLSILICONIDES. In our hands, these derivatives were satisfactorily obtained only from a dihydroxyacetone Methoximation of carbonyl groups increases the retention and from a 17a,20-diol: conditions suitable for the formaon OV-1, but decreases it on Dexsil. tion of a 20,21-dimethylsiliconide (12) were not found. Both Among the derivatives formed without side-chain degradathe reagent (diacetoxydirnethylsilane) and the derivatives tion, the methylboronates have the lowest retention were extremely susceptible to hydrolysis. indices, with the exception of the oxetanone from SubMETHYLBORONATES. As already described ( I O ) , the prepastance S (on OV-1) and the M O triTMS ether of 17a,ration of these derivatives was effected (for dihydroxyacetones 20P,21-trihydroxypregn-4-en-3-one(on Dexsil). The and diols) by mixing the steroid with methylboronic acid in latter exception arises because of the promotive effect a n organic solvent. This extreme simplicity allowed the of methoximation o n elution from the polar phase. formation of methylboronates from as little as 4 pg of steroid Trimethylsilylation of the 17a-hydroxyl group is advandiol. [The presence of additional hydroxyl groups complitageous in reducing the retention times to values that are cates the reaction ( I O ) . ] comparable (on Dexsil) with those of methylboronates. OXETANONESFROM STEROIDAL DIHYDROXYACETONES. It is evident that the only practical derivatives for the These derivatives were prepared from large amounts (100 mg) analytical separation of all six corticosteroid types are of steroid by a published procedure (13). The yields were the M O TMS ethers. For the four types containing unsatisfactory, and 21-fluorosteroids were formed as difia- or ,&diol groupings, the methylboronates provide a possible alternative with the advantage of lower reten(19) C. J. W. Brooks, A. R. Thawley, P. Rocher, B. S. Middletion times: thus, the pregn-4-en-3-ones possessing the ditch, G. M. Anthony, and W. G. Stillwell, J . Chromatogr. Sci., dihydroxyacetone and 17a,20P,21-triol side-chains yield 9, 35 (1971). M O (full-) TMS ethers with Zov-l 3070 and 3160, respec(20) C. J. W. Brooks and B. S. Middleditch, Cliii. Chim. Acta, tively, while the corresponding methylboronates have 34, 145 (1971). I o v - ~2820 and 2970. (21) E. M. Chambaz and E. C. Homing, Aml. Biochem., 30, 7 (1969). (22) N. Sakauchi and E. C. Homing, A i d . Lett., 4, 41 (1971). No marked differences were noted a t the 1-pg level in the (23) L. L. Engel, A . M. Neville, J. C. Orr, and P. R. Raggatt, response of the flame ionization detector to the various Steroids, 16, 377 (1970). derivatives. G a s chromatograms of the five representative (24) L. Aringer, P. Eneroth, and J.-A. Gustafsson, ibid., 17, 377 derivatives of Substance S, recorded consecutively under (1971). 32


io 1

z

0

5

10

15

20

time ( m i n u t e s )

time ( m i n u t e s )

Figure 1. Gas chromatograms of derivatives of Substance S (a) 17,21-anhydro derivative (b) 17,21-methylboronate ( c ) 17,21-dimethylsiliconide ( d ) 3,20-diMO 17,21-diTMS ( e ) 3,20-diMO 21-TMS 6 ft. 1 OV-1 at 220 “C

constant conditions, are shown in Figure 1. The important question of the lower limits of detection is discussed below in relation to mass spectrometric data. Mass Spectrometric Characteristics. Salient features of the mass spectra of the compounds studied are cited in Table I. Ions below m/e 80 have been disregarded in assignment of base peaks. Molecular ions were generally prominent: only the 20P,21-diol diTMS ether failed to give one, because of the supervention of a-cleavage of the TMS ether. Similar dominance of ether fragmentations accounted for the low abundance of the characteristic “ring A” ion (25) of m/e 124 in the TMS ethers of the 20,21-diol and 20,21-ketol, and in the 20,21-di TMS ether of the 17a,20/3,21-triol. The fragmen(25) R. H. Shapiro and C . Djerassi,J. Amer. Chem. SOC.,86, 2825 (1964).

tations of other types of side-chain derivative competed more evenly with those characteristic of the nucleus: compounds retaining the 4-en-3-one group yielded ions of mje 124, and the corresponding 0-methyloximes gave ions of mje 125 (26). The mass differences between the various derivatives, and their individual fragmentation modes, together provide a wide range of mass spectrometric ions suitable for analytical characterization. DIHYDROXYACETONE. Mass spectra of the five principal types of derivative of Substance S are shown in Figure 2. [Line diagrams of the diMO diTMS ether (23) and methylboronate (27) have been published before, but are included for (26) F. Dray and I. Weliky, Anal. Biochem., 34, 387 (1970). (27) C . J. W. Brooks, B. S. Middleditch, and D. J. Harvey, Org. Mass Spectrom., in press.


33

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convenient comparison.] The diMO monoTMS ether and its &analog (Table I) gave closely similar fragmentation patterns (with the expected mass shifts): the base peak in each case was at m/e 386, representing loss of the trimethylsilanol moiety. The diMO diTMS ether, in contrast, gave no significant peak at [M - 90]+' : the dominant fragmentations proceeded cia loss of methoxyl radicals. The characteristic (23) ion at mle 273 doubtless comprises rings A, B, and C with C-14 and C-18, and corresponds t o the well-known fragment of this type observed for cyclic boronates (9, 10) 34

-

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,111

1/11

Ill

ill, li

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W' N

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and dimethylsiliconides (11) with the added 29 mass units due to the M O group. The dimethylsiliconide gave a particularly intense molecular ion: the base peak at m/e 91 is structurally insignificant. A notable feature of this spectrum is the peak at [M - 281f' : a similar ion was recorded by Kelly ( Z J ) for cortisol 11-dimethylsilyl ether 17,21-dimethylsiliconide. The nature of this elimination has not yet been firmly established, but it probably represents loss of C O . No corresponding peak occurs in the spectrum of the methylboronate, which in other respects parallels the dimethylsiliconide. The oxeta-


silanol, affording the base peak, [M - 193]+. A more balanced fragmentation occurred with the methylboronate, leading to the “ring A” fragment (m/e 124) as the base peak, and to prominent ions M+ ‘ , [M - 421+’, and m/e 85 [“boronThe acetonide (17) exhibited ate” fragment C ~ H ~ B O Z(27)]. +’ yet another mode of fragmentation, giving the expected [M - 15]+ as base peak: the intense peak at m/e 101 is doubtless due t o the dimethyldioxolenium ion C6H9OZ+, paralleling the ion of m / e 85 from the boronate. The ion a t m / e 101 is prominent in the spectrum of 2Oa-dihydroprednisolone acetonide recorded by Bailey (18). 17a,20/3-D10~. No sample of 17a,20-dihydroxypregn-4en-3-one was available at the time of this work, and the steroid type used was thus not uniform with the others. The spectra of the TMS ethers are dominated by peaks arising by cleavage of C(20)/C(21), with subsequent eliminations of trimethylsilanol from nuclear fragments. The three cyclic derivatives yield individual fragmentation patterns. Each affords an ion comprising the side-chain moiety, at mle (99 X I :

none undergoes distinctive fragmentations, notably the loss of ketene which affords the base peak. This probably arises primarily from the oxetanone moiety, because similar ions are prominent (14) in the spectra of oxetanones derived from 4,5-dihydro-derivatives of Substance S and cortisone. Two other interesting ions, [M - 58]+’ and [M - 59]+, apparently result from extrusion of the side-chain:

[M-581 Previously reported mass spectra related to those discussed above include those of tetrahydro-S M O diTMS ether (28), cortisol dimethylsiliconide ( I I ) , and the diMO (full-) TMS ethers of cortisol and cortisone (24). Spectra of cortisol 11,17,2l-triTMS ether, 3,20-di-enol-TMS ether, and cortisone 17,21-diTMS ether, 3,11,20-tri-enol-TMS ether (24) contained minor peaks at mle 332. These are the base peaks in the 20 eV spectra of the 3,17,21-triTMS ether 20-enol-TMS ethers of tetrahydrocortisol and tetrahydrocortisone, recorded in our laboratory on behalf of Dr. E. M. Chambaz (IS) and representing the complete side-chain with C (15) to C (17), less one hydrogen atom. ~ ~ , ~ ~ - K E TThe o L 21-TMS . ether of deoxycorticosterone undergoes the expected major a-cleavage of C(20)/C(21), with charge retention on either of the fragments. The abundant ion a t m]e 143 is noteworthy: an ion of this mass was observed in about 3 0 x relative abundance in the spectrum of 11~,2l-dihydroxy-5a-pregnane-3,20-dione diTMS ether (29). The following mode of fragmentation may be envisaged:

[&

+

p

.+

m/e (99 + X) For the dimethylsiliconide, this ion ( m / e 157) and the molecular ion were the only strong peaks, but the methylboronate also gave abundant ions representing the complementary nuclear fragment (with the elimination of HzO). The base peak of the acetonide spectrum at m/e 86 (C6HloO+’)evidently arises from the acetonide group. A possible mode of formation would be :

+

L:=siMez

-{B-

m/e 86

~ ~ c Y , ~ O P , ~ ~ - T The R I O LdiTMS . ether yielded the base peak at m/e 116, characteristic of such derivatives (30), and an intense peak [M - 133]+ (cf,ref. 24) ascribable to loss of a rearranged side-chain:

to

0=SiMez

m/e 143

The spectrum of the diMO TMS ether, in agreement with data previously reported (24, 29), included strong peaks at [M - 174]+‘ and [M - 1871f and quasi-complementary peaks a t m/e 175 and 188-the latter representing the side-chain with one and two ring D carbon atoms, respectively, as proposed by Gustafsson and Sjovall (29). The 20,21methylboronate, for which evidence favors a A17(20)-structure, yielded a spectrum dominated by the “boronate” fragment at m / e 110 ( 9 , 2 7 ) . The three derivatives compared here illustrate well the manner in which the base peak can be directed to various regions of the spectrum. 20,21-D10~. The diTMS ether of 20P,21-dihydroxypregn4-en-3-one gave no discernible molecular ion because of the predominance of a-cleavage and elimination of trimethyl(28) P. Koepp, J. A . Vollmin, M. Zachmann, and H. C. Curtius, Acta Endocrinol. (Copenhagen), 66, 756 (1971). (29) J.-A. Gustafsson and J. Sjovall, Eur. J . Biochem., 6 , 236

(1968).

rOTMS

OTMS

[M-133]+ In the 3-MO 17,20,21-triTMS ether the base peak was due to the corresponding ion from direct cleavage of C(17)/C(20). As in previous examples, the cyclic derivatives displayed modes of fragmentation markedly different from those of TMS ethers. The methylboronate gave a n intense molecular ion together with the “ring A” ion ( m / e 124), a nuclear (30)C . J. W. Brooks, E. M. Chambaz, W. L. Gardiner, and E. C . Homing, Excerpta Med. Int. Coag. Ser., 132, 366 (1967).


35

fragment (m/e 287) resulting from loss of the (rearranged) boronate side-chain (27) and further ions due t o additional losses of H 2 0or ketene. I n the acetonide, the ion of m/e 287 dominated the spectrum. ~ O , ~ ~ ~ Y - K E No T O stable L. cyclic derivatives were obtained from 17a-hydroxyprogesterone. The spectra of the diMO and diMO TMS ether both gave as base peaks [M - 31]+. The latter spectrum also contained a series of even-mass ions mainly comprising the side-chain M O group rather than the ring A M O group (26, 31): detailed assignments would require mass measurements combined with a study of labelled derivatives. The characteristic ion at m/e 273 (23) has been mentioned above with respect to the dihydroxyacetone analogs. SIDECHAIN DEGRADATION PRODUCTS. The mass spectra of these compounds require little comment, as their main features arise from the 4-en-3-one group and its M O derivative, and are well-defined (25, 26). Abundant molecular ions were observed. It is noteworthy that the principal nitrogencontaining ions from the 3,20-diMO of the 17P-aldehyde appear to arise from the ring A moiety. A reversal of this situation might be expected for the corresponding 17a-trimethylsilyloxy-3,20-diMO, by analogy with the results observed for the 17-hydroxyprogesterone derivative (see above). Detection of Corticosteroids by “Single Ion Monitoring.” The detection of particular compounds by means of a small set of characteristic ions in their mass spectra (32-34) is now a well-established technique which has recently been elegantly refined (35). In suitable instances, the very simple technique of focusing at a single m/e value may afford a satisfactory means of detecting and estimating steroids a t low concentrations (20, 36-39). The success of such procedures depends critically upon a number of factors, the relative importance of which must be assessed experimentally. The principal considerations in respect of corticosteroids (for which derivative formation is a prerequisite) are as follows: The ease, reliability, selectivity and completeness of derivative formation. The stability of the derivatives. Their retention times, and the quality of the chromatographic peaks. The degree t o which the derivatives may be lost by adsorption or destruction during gas chromatography. The character of the ions to be selected for monitoring. [The choice of fragment will depend on the structural features that it is desired to detect.] The nature of probable interference by ions from other compounds in the sample, and its avoidance-as far as possible-by optimal selection of the sample preparation method, type of derivative, stationary phase, and chromatographic conditions.

(31) C. J. W. Brooks and D. J. Harvey, Steroids, 15, 283 (1970). (32) D. Henneberg, Z.A/7a/. Cliem., 183, 12 (1961). (33) C. C. Sweeley, W. H. Elliott, I. Fries, and R. Ryhage, ANAL. CHEM.,38, 1549 (1966). (34) R. A. Hites and K. Biemann, ibid., 40, 1217 (1968). (35) C.-G. Hammar and R. Hessling, ibid.,43, 298 (1971). (36) H. Adlercreutz, Ab. Deut. Akad. Wiss. BerIii7, KI. Med., 1968 !21. (37) J. Sjovall and H. Reimendal, Excerpta Med. Znt. C o ~ g rSer., . 210, abstract No. 18, p 8 (1970). (38) L. Siekmann, H.-0. Hoppen, and H. Breuer, 2. A d . Cliem., 252, 294 (1 970). (39) R. W. Kelly, J . Chromutogr., 54, 345 (1971).

36

ANALYTICAL CHEMISTRY, VOL. 44,

NO. 1, JANUARY 1972

The m/e values at which similar interference may arise from impurities constantly or frequently present in the “background” of the GC-MS system. [Accidental coincidence of a selected ion with a prominent “background” ion may, in itself, compel the choice of a different derivative yielding a n ion of more suitable m/e value. Interference by ions from particular stationary phases may be avoidable, but that arising from O-rings, septa, and solvent impurities is almost inescapable.] The relationship of the actual mje values selected for monitoring to the degree of mass resolution, as well as to the sensitivity of the electron multiplier system in the region of the spectrum concerned. In this paper we have given special attention to five representative derivatives of Reichstein’s Substance S. In each instance, ions of even mass have been selected for monitoring: the majority of strong “background” peaks are of odd mass. For the diMO TMS ether, the base peak [M - 90]-’ was chosen; for the diMO diTMS ether, dimethylsiliconide, and methylboronate, the molecular ions were used; and for the oxetanone, the base peak [M - 42]+’ was employed. In each instance, the lower limit of detection was explored, with the results summarized in Figure 3. All the derivatives were satisfactorily applicable for detection and estimation of quantities above 100 ng. Below this level, marked differences in behavior were noted, and only the oxetanone remained clearly detectable at the 400-pg level. It appeared probable that the loss of response was due to adsorption of the derivatives on the chromatographic column. It may be observed that the derivatives that were most easily prepared (methylboronate; diMO 21-TMS) were also those most susceptible to loss at low sample sizes, whereas the more difficultly accessible oxetanone and dimethylsiliconide yielded the best results under these conditions. Th? derivative of choice with respect to convenience of preparation and range of detectable concentration would appear to be the diMO diTMS. This result is in harmony with the trend toward the use of fully-silylated derivatives for the analytical characterization of steroids (21, 22, 24). The recent introduction of 0-benzyloximes (BO) and BO TMS ethers (40, 41) ranks as an important complementary development in this area. The data in Table 1 suggest that derivatives suitable for detection by single ion monitoring should be obtainable from all types of corticosteroid. In certain instances, apparently suitable peaks might be inappropriate: for example, m/e values of 124, 125, and 157 represent ions that are relatively abundant in the “background” spectra of our instrument a t the temperatures (for OV-1 and Dexsil) required for steroid derivatives (20). Untoward coincidences of this kind can be circumvented either by selecting a n entirely different derivative, or by using simple homologs to shift the masses of the desired ions without any undue alteration of the fragmentation mode. Examples include d9-TMS ethers, chloromethyl(dimethy1)silyl ethers, 0-ethyloximes, 0-trimethylsilyloximes, and 0-benzyloximes. The extent to which mass spectrometric detection is likely to prove useful in the analysis of urinary corticosteroids is by no means fully explored. In our experience, single ion monitoring has been found suitable for the study of certain steroidal drug metabolites. The technique is particularly (40) P. G. Devaux, M . G. Homing, R. M. Hill, and E. C. Homing, A m / . Biochem., 41, 70 (1971). (41) P. G. Devaux, M. G. Homing. and E. C. Horning. Aiial. Lett., 4, 151 (1971).

100

I

’O

Q)

e0

8 I 1

sample size Ing)

m

Figure 3. “Single ion monitor” response to derivatives of Substance S

+

17,21-anhydro derivative (m/e 286) 0 17,21-methylboronate( m / e 370) 0 17,21-dimethylsiliconide ( m / e 402) A 3,20-diMO 17,21-diTMS ( m / e 548) 0 3,U)-diMO 21-TMS (m/e 386) Electron multiplier voltage 1.7-3.7 kV. Response measured as peak height, with adjustment for different amplifications(Logarithmicscales)

effective for steroids that occur in unconjugated form, such as Dianabol (17~-hydroxy-17a-methylandrosta-1,4-dien-3one) and its 6P-hydroxylated metabolite (42). Its possible application to the measurement of free urinary cortisol and 6P-hydroxycortisol(43) is under investigation. CONCLUSIONS

The results of this preliminary survey indicate that the majority of derivatives that have been developed for the gas chromatography of corticosteroids also have qualities useful for GC-MS. The range of compounds examined is, of course, very limited, and some derivatives of proved utility have not received attention, while others await evaluation. Examples of known derivatives of steroidal dihydroxyacetones which have not, to our knowledge, been applied t o gas-phase studies are the 17,21-formals (44), acetonides (45), other 17,21-acetals (46), and orthoesters (47). (42) A . M. Lawson and C. J. W. Brooks, Biochem. J., 123, 25P (1971). (43) K. Thrasher, E. E. Werk, Jr., Young Choi, L. J. Sholiton, W. Meyer, and C. Olinger, Steroids, 14, 455 (1969). (44) W. S . Allen and M. J. Weiss, J . Org. Chem., 26,4153 (1961). (45) M. Tanabe and B. Bigley, J. Amer. Chem. SOC.,83,756 (1961). (46) R. Gardi, R. Vitali, and A . Ercoli, J. Org. Chem., 27, 668 (1962). (47) R. Gardi, R. Vitali, and A . Ercoli, Gazz. 93,413 (1963).

The variety of fragmentation modes displayed by the different classes of derivative is of obvious value in the identification of corticosteroids. At the same time, it furnishes a wide choice of characteristic ions that may serve for detection of individual compounds (or common structural features) by monitoring the ion current at a single mje value or a few such values. The practicability of the mass spectrometric estimation of corticosteroids in the sub-nanogram range is clearly foreshadowed by the data presented here. ACKNOWLEDGMENT

The authors thank Miss Juliet Johnston for recording mass spectra, and Dr. J. A. Wilson for the computer program by which these were drawn. Dexsil-300GC stationary phase was kindly provided by Dr. E. C. Horning (Baylor College of Medicine, Houston).

RECEIVED for review July 9, 1971. Accepted August 23, 1971. This work was aided by a grant from the Medical Research Council to C. J. W. Brooks and by a n SRC Studentship held by T. A. Baillie. The LKB 9000 gas chromatograph-mass spectrometer was provided by SRC Grant No. B/SR/2398.


37

Comparison of corticosteroid derivatives by gas chromatography-mass

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