The Determination of Testosterone in Hamster Prostate by Gas Chromatography Mass Spectrometry with Selected Metastable Peak Monitoring? Simon J. Gaskell, Robert W. Finney and Maureen E. Harper Tenovus Institute for Cancer Research, Welsh National School of Medicine, Heath Park, Cardiff CF4 4XX, UK

Quantitative analyses of testosterone, as the methyl oxime t-butyldimethylsilyl ether, are performed by gas chromatography mass spectrometry with selected monitoring of the metastable peak corresponding to the fragmentation [M]+’+[M- C4H9]+in the field free region preceding the electric sector of a double focusing mass spectrometer. A detection limit of e. 30 pg is observed during analyses of the standard compound. The method is applied to the quantitative determination of testosterone in extracts of prostatic tissue from the golden hamster, using epitestosterone as the internal standard. The analytical specificity is similar to that achieved during gas chromatography high resolution mass spectrometry with selected ion detection of [MI+’ ions; gas chromatography low resolution mass spectrometry is of inadequate specificity.

INTRODUCTION The complexity of extracts of biological tissues places severe demands o n the specificity of procedures employed for the quantitative determination of trace components. In this laboratory, gas chromatography high resolution mass spectrometry with selected ion monitoring (SIM) is routinely employed in the determination of CI9 steroids in various human and animal tissues.14 Recently, we described the technique of GCMS with selected metastable peak monitoring as an alternative to GC high resolution MS with SIM for highly specific quantitative analy~es.’.~ The technique was illustrated by the determination of 5a -dihydrotestosterone in human blood plasma. In the present paper we describe the extension of the method to the quantitative analysis of testosterone in prostatic tissue of the golden hamster. The specificity of the method has been compared with that achieved during conventional GC low resolution MS with SIM and GC high resolution MS with SIM. with selected metastable peak In our earlier monitoring (using a VG-Micromass 70-70 F mass spectrometer), focusing of the metastable peak was facilitated by the use of a linked magnetic.field/electric field scan d e ~ i c eHere . ~ we describe a procedure employing an older double focusing mass spectrometer (a Varian 731), not equipped with a linked scan facility, where focusing of the metastable peak is achieved by adjustment of the accelerating voltage.

Extraction of tissue Dorsal and ventral prostatic tissue from 20 golden hamsters (Mesocricetus uurutus) was dissected free of t Abbreviations: MO = methyl oxime; TBDMS = t-butyldimethylsilyl.

capsular tissue and fat, weighed (total 3.5 g) and homogenized in 100mM Tris HCl (pH 7.4). Aliquots corresponding to one-sixth of the total homogenate were removed and epitestosterone (2 ng in 100 pl ethanol) was added to each as internal standard. Each aliquot was extracted with acetone (40 volumes). Acetone was removed under vacuum and the extract was suspended in methylene chloride (10 ml). After washing with aqueous sodium hydroxide (1 M; 2 ml) and water, solvent was removed under vacuum.

Fractionation of extracts Non-polar lipid constituents of the extracts were removed by differential solubility as described by Ismail et af.’ In some cases, additional purification steps were included. Thus, an extract, after removal of non-polar lipids, was applied to a silica TLC plate (0.1mm Flexiplate: Camlab, Cambridge, UK) which was developed in chloroform+acetone (185 : 10, v/v). The appropriate fraction was recovered as previously described.* As an alternative purification procedure, the extract of a further aliquot of tissue homogenate, after removal of non-polar lipids, was dissolved in 200 p1 methanol + chloroform + wajer (9 :2 : 1, by vol., solvent A) and applied to a column of Lipidex 5000 (4 cmx 0.5 cm; Packard, Downers Grove, Illinois, USA), swollen in solvent A. A sample of eluate (0-2 ml) was collected, evaporated to dryness and redissolved in 200 pl hexane+ethanol(4 :1,v/v; solvent B). The solution was applied to a column of Sephadex LH20 (4 cm x 0.5 cm; Pharmacia, Uppsala, Sweden), swollen in solvent B, and an eluate fraction (0-4 ml) collected. Solvent was removed under nitrogen.

Derivatization Standard steroids (0.5-25 ng) and tissue extracts were converted to methyl oxime derivatives by dissolving in a

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solution of methoxyamine hydrochloride in pyridine (30 p.1; 15 mg ml-l) and allowing to stand overnight at room temperature. Pyridine was removed under vacuum and t-butyldimethylsilyl (TBDMS) ethers prepared by addition of 30 pl t-butyldimethylchlorosilane + imidazole + dimethylformamide (1: 1:6 , by wt; Applied Science Laboratories, State College, Pennsylvania, USA) and standing at room temperature overnight. Excess reagent was removed by chromatography on Lipidex 5000, as described previously.' Gas chromatography mass spectrometry

GCMS analyses employed a Varian 2700 gas chromatograph interfaced via a two-stage Watson-Biemann separator to a Varian MAT 731 double focusing mass spectrometer. Separations were achieved on a glass column (3 m X 3 mm, i.d.) of 1% OV-1 on Gas Chrom Q (100/120 mesh) at 280 "C. For SIM at high mass spectrometric resolution ( m / A m 8500, 10% valley), ions of m/z 431.3219 were focused, using the peak matching unit, by reference to the ion of m / z 430.9729 derived from perfluorokerosene which was independently introduced into the ion source. Selected ion monitoring at low mass spectrometric resolution (m/Am 1000) was subsequently performed after removal of perfluorokerosene. For selected metastable peak monitoring experiments, the appropriate metastable peak corresponding to the fragmentation m/z 431+m/z 374, was located by the following procedure. A sample (c. 0.5 Fg) of testosterone methyl oxime, TBDMS ether was introduced by direct insertion probe. The daughter ion of m / z 374 was located by adjustment of magnet current at a nominal accelerating voltage of 8 kV (recorded value 8.96 kV). The accelerating voltage was increased to 10.34 kV to focus the metastable peak. After withdrawal of the direct insertion probe, fine adjustment of the accelerating voltage was made during GCMS analysis of testosterone methyl oxime, TBDMS ether.

RESULTS The mass spectra (70eV) of the methyl oxime (MO), t- butyldimethylsilyl (TBDMS) ether derivatives of testosterone (1) and epitestosterone (2)each include

molecular ions and [M-C4H9]+ fragment ions at high abundance." The intense molecular ions are unusual for TBDMS derivatives and are probably attributable to charge stabilization by the 3-methyl oxime, 4-ene system. The very low intensity of the molecular ion in the spectrum of the MO, TBDMS derivative of the isomeric compound, dehydroe iandrosterone (3),is consistent with this suggestion. 18 During analysis of testosterone MO, TBDMS, the metastable peak corresponding to the fragmentation, [M]"+[M-C4H9]' ( m / z 431-+m/z 374), in the field free region preceding the electric sector was located by initial focusing of the 'normal' daughter ion and adjustment of the accelerating voltage (as described in the Experimental section). Earlier ex eriments with selected metastable peak monitoring5*!employed an instrument equipped with a linked magnetic field @)/electric field ( E )scanning device for the focusing of metastable peaks. Thus, the parent ion was initially focused and the appropriate metastable peak located by adjustment of magnetic and electric fields such that the ratio of B / E remained constant. By this procedure, only those daughter ions, formed in the field free region preceding the electric sector, which possess the same velocity as the parent ion are focused. The resultant energy discrimination affords a high effective r e s ~ l u t i o n . ~ When '" metastable peaks are located (as in the present work) by adjustment of the accelerating voltage after initial focusing of the 'normal' daughter ion, imperfect energy focusing is achieved by the electric sector. The extent of the loss of effective resolution in switching from conventional selected ion monitoring to metastable peak monitoring was assessed, in the present work, by comparison of the shapes of the 'normal' daughter ion peak (m/z 374) and the metastable peak corresponding to the transition, mf z 431-+mlz 374. Figure 1shows the profiles obtained by slow scanning of the voltage applied across deflection plates located after the magnetic sector. While the metastable peak is clearly the broader, the loss of effective resolution appears to be minor in this example. The results shown in Fig. 1 also indicate the relative intensities of peaks corresponding to ions of m / z 374 formed in the ion source and in the field free region preceding the electric sector (ion source focusing conditions were in each case adjusted for maximum sensitivity). The intensity of the metastable peak [Fig. l(b)]is approximately 8% of that of the peak corresponding to daughter ions formed in the ion source [Fig. 1(a)]. The

OSi(CH,),C,H,

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Figure 1. Detection of ions of mlz 374 derived.from testosterone MO, TBDMS ether by fragmentation of mlz431 in (a) the ion source, and (b) the field free region preceding the electric sector. Peak profiles were obtained by slow scanning of the voltage applied across deflection plates located after the magnetic sector.

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parent ion, of m / z 431, has an abundance of c. 60% of that of the daughter ion formed in the ion source. In quantitative analyses of complex mixtures of biological origin, however, the limit of detection is generally dictated by the specificity of analysis, as discussed below. Figure 2 illustrates the analysis'of a mixture of the MO, TBDMS derivatives of epitestosterone (300 pg) and testosterone (375 pg) by GCMS with selected metastable peak monitoring of the fragmentation m / z 431+m/z 374. A limit of detection in the analysis of testosterone MO, TBDMS of c. 3 0 p g is indicated. The limit of detection of this procedure for dehydroepiandrosterone MO, TBDMS was, as expected, much higher-approximately 300 pg. Determination of testosterone in biological samples employed epitestosterone as an internal standard. (Epitestosterone has not been observed as an endogenous component of the samples studied). A standard curve, prepared by GCMS with metastable peak monitoring using standard mixtures of testosterone and epitestosterone MO, TBDMS, is shown in Fig. 3. Instrumental error associated with metastable peak monitoring was assessed by analyses of aliquots of a mixture of the MO, TBDMS derivatives of testosterone and epitestosterone (2.5 : 1, by wt). Injections corresponding to approximately 250pg testosterone afforded signal-to-noise ratios of about 20 : 1. The peak height ratio was determined with a coefficient of variation of 7.1% ( n = 6). Non-polar lipid constituents of crude extracts of aliquots of prostatic tissue homogenate were removed

10

5

15 Tesiosterone Ing)

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Figure 3. Calibration line, prepared by GCMS with selected metastable peak monitoring using aliquots of standard mixtures of testosterone and epitestosterone (internal standard, 2 ng), as the MO, TBDMS ether derivatives. The calibration line is that calculated by a least-squares linear regression analysis. Each experimental point is indicated as the mean of three determinations +SD, where each determination is a mean value obtained by duplicate injections of a single sample.

by differential solubility. The remaining fraction was converted by a sequential derivatization procedure to the methyl oxime, TBDMS ether. Figure 4(a) shows the analysis of the derivatized fraction by GCMS, with selected metastable peak monitoring of the fragmentation m / z 431+m/z 374. Two peaks only were observed at significant intensity, corresponding to the

Ic 1

I I

bvy 0

0 \

2 4 Time (min)

Figure 2. GCMS of a mixture of the MO, TBDMS ether derivatives of testosterone (1; 375 pg) and epitestosterone (2; 300 pg) with selected metastable peak monitoring of the reaction m/z 431 + m/z 374. (For conditions of analysis, see Experimental section.)

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Figure 4. Analyses of an extract of hamster prostatic tissue, as the MO, TBDMS ether derivative. (a) GCMS with selected metastable peak monitoring of the reaction m/z 431 + m / z 374. (b) GC high resolution MS with SIM of m/z431.3220. (c) GC low resolution MS with SIM of m/z 431. For conditions of analyses, see Experimental section. 1: testosterone MO, TBDMS; 2: epitestosterone MO, TBDMS.

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derivatives of testosterone (1)and epitestosterone (2). Determination of the peak height ratio indicated, by reference to the standard curve, a total of 3.61 ng of testosterone in the tissue extract. Analysis of an aliquot of the same sample by G C high resolution MS ( m / A m 8500, 10% valley) with SIM of m / z 431.3220 is shown in Fig. 4(b). Two peaks were again observed; by reference to a standard curve determined by G C high resolution MS and SIM, a total of 3.18ng of testosterone in the tissue extract was indicated by this procedure. In contrast to selected metastable peak monitoring and SIM at high mass spectrometric resolution, the specificity of SIM at low mass spectrometric resolution was inadequate for quantitative determination of testosterone in these tissue extracts. Figure 4(c) shows the GCMS and SIM analysis at low resolution. An intense peak of retention time intermediate between those corresponding to the derivatives of epitestosterone and testosterone was observed; the identity of this component is unknown. The relative intensity of the peak varied widely between extracts of the same tissue homogenate (in certain instances completely obscuring the peaks corresponding to the derivatives of epitestosterone and testosterone) and, indeed, between replicate analyses of the same extract. The interfering component was not removed by TLC or by a combination of reversed- and straight-phase gel chromatography procedures (see Experimental).

DISCUSSION The inherent specificity of metastable peak analysis makes the technique an attractive possibility for quantitative determinations of trace components in complex mixtures. The use of mass analysed ion kinetic energy spectroscopy has indeed been suggested as an alternative to GCMS for mixture analyses.’* The combination of GCMS and selected metastable peak monitoring, however, is of special interest in that identification of trace components is based on both gas chromatographic and mass spectrometric parameters. In our previous

studiessv6and in the present work, GCMS with selected metastable peak monitoring has been shown to afford adequate sensitivity for the analyses of steroids in complex mixtures at the sub-nanogram level. The correct choice of derivative is clearly of crucial importance; TBDMS ethers and related derivatives appear to offer particular advantages in this respect by virtue of the intensity of high mass ions and the simplicity of the fragmentation patterns. For the example of the determination of testosterone in extracts of hamster prostatic tissue, the analytical specificity achieved by G C low resolution MS of the MO, TBDMS derivatives with selected metastable peak monitoring has been shown to be comparable with that achieved by GCMS with conventional SIM when high mass spectrometric resolution is employed. G C low resolution MS and SIM is of inadequate specificity, even when additional purification steps are incorporated in the work-up procedure. GCMS with selected metastable peak monitoring has several experimental advantages over G C high resolution MS and SIM. Thus, while a double focusing instrument is clearly required, the capability of achieving high mass spectrometric resolution is not. ‘Medium’ resolution double focusing instruments may therefore be employed. Furthermore, instrumental stability (in terms of mass drift) is less critical for GCMS with selected metastable peak monitoring than for analyses by G C high resolution MS and SIM. A comparison of the analytical precision achieved by the two techniques is in progress and will be reported elsewhere. In those instances (such as the analysis reported here) where both methods are applicable, the two sets of data are of complementary value in substantiating both the identification and the quantification of components of a natural mixture present at the nanogram level.

Acknowledgements We gratefully acknowledge the generous financial support of the Tenovus Organization. Accessories for the Varian M A T 731 were provided by Medical Research Council grant G974/125/C. We thank Professor K.Griffiths for constructive criticism and advice.

REFERENCES 1. D. S. Millington, D. A. Jenner, T. Jones and K. Griffiths, Biochem. J. 139, 473 (1974). 2. D. S.Millington, M. E. Buoy, G. Brooks, M. E. Harper and K. Griffiths, Biomed. Mass Spectrom. 2, 219 (1975). 3. P. V. Maynard, A. W. Pike, A. Weston and K. Griffiths, Eur. J. Cancer 13, 971 (1977). 4. R. W. Finney, M . E. Harper, S. J. Gaskell and K. Griffiths, J. Endocrinol. 79, 53P (1978). 5. S. J. Gaskell and D.S. Millington, Biomed. MassSpectrom. 5, 557 (1978). 6. S. J. Gaskell and D. S.Millington, in Proceedings of the 2nd lnternational Symposium on Quantirarive Mass Spectrometry in Life Sciences, ed. by A. P. De Leenheer, R. Roncucci and C. Van Peteghem, Elsevier, Amsterdam, in press. 7. D. S. Millington and J. A. Smith, Org. Mass Spectrom. 12,264 (1977).

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689 (1972). 9. S. J. Gaskell and C. J. W. Brooks, Biochem. SOC.Trans. 4,111 (1976). 10. S. J. Gaskell and A. W. Pike, in Proceedings of the 2nd International Symposium on Quantitative Mass Spectromefry in Life Sciences, ed. by A. P. De Leenheer, R. Roncucci and C. Van Peteghem, Elsevier, Amsterdam, in press. 11. R. K. Boyd and J. H. Beynon, Org. Mass Spectrom. 12, 163 (1977). 12. R. W. Kondrat and R. G. Cooks, Anal. Chem. 50, 81 A (1977).

Received 23 September 1978 @ Heyden & Son Ltd, 1979

@ Heyden & Son Ltd, 1979