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Fast atom bombardment mass spectrometry of estrogen glucuronides and sulfates
599
295!3
FAST ATOM BOMBARDMENT MASS SPECTROMETRY OF ESTROGEN GLUCURONIDES AND SULFATES Joachim G. Liehr, Carl F. Beckner, Annie M. Ballatore and Richard M. Caprioli Analytical Chemistry Center and Department of Biochemistry and Molecular Biology, University of Texas Health Science Center at Houston, Houston, Texas 77030 Received 4-12-82 ABSTRACT Fast atom bombardment (FAB) mass spectra of 13 intact, underivatized glucuronides and/or sulfate salts are reported. Spectra are charalterized by abundant ions formed by attachment of a proton, [M+H.] , or of an alkali ion, [M+alkali] , to the glucuronide or sulfate salt. Fragment ions were of low intensity. FAB spectra can be used to ombtain the molecular weight of a sample, to assess its purity and to identify the nature of the alkali of the glucuronide or sulfate salt. INTRODUCTION In mammals, most estrogens present in plasma, bile, urine or feces are conjugated (1) with glucuronic acid and/or sulfuric acid. Minolr quantities of other classes of conjugates, such as N-acetylglucosamines, have been isolated, but their contributions to steroid profiles are negligible.
Structural analysis of hormone metabolites
presents problems due to the small quantities isolable from biological sources and due to the polar character of glucuronides and sulfates. Therefore, steroid extracts are usually separated into conjugate fractions by chromatography and hydrolyzed enzymatically or by solvolysis before steroid profiles or structural analyses can be obtained using mass spectrometric methods (2).
Direct analysis by GC-MS or
gas chromatography of estrogen glucuronides (3-6) and sulfates (7,8) has been reported but is not widely accepted as a method for steroid profiling because of the chromatographic difficulties involved in
Vohme
39, Number 6
S
TDROIDES
June, 1982
these procedures.
Alternative ionization methods (field desorption)
in mass spectrometry for the direct analysis of estrogen conjugates have been tried (9), but the expense of the equipment prevents its widespread use. A novel ionization technique in mass spectrometry, fast atom bombardment
(FAB) (10,11,12), is promising for the analysis of estro-
gen conjugates.
Existing equipment can be modified to accept a FAB
ion source and mass spectra can be obtained with this technique from highly polar or thermally labile substances (13).
Thus, FAB mass
spectra have been reported (13) for underivatized peptides, carbohydrates, nucleotides, antibiotic substances and other polar compounds. In order to determine the suitability of this novel mass spectrometric method for the analysis of steroid hormones, a series of underivatized estrogen glucuronides and sulfates were examined by FAB mass spectrometry. MATERIALS AND METHODS Diethylstilbestrol (DES) monosulfate was synthesized following published procedures (14). DES monoglucuronide was a generous gift from Dr. W. Kruger and Dipl.-Chem. R. Mahrwald, Forschungsinstitut Manfred von Ardenne, Dresden, GDR. All other substances were purchased from Sigma Chemical Co., St. Louis, Missouri. Fast atom bombardment mass spectrometry was performed on a Finnigan MAT 3300/Incas mass spectrometer, modified as follows to accept an Ion Tech BllNF saddle field atom gun and B50 power supply (15). The Ion Tech gun was placed on the analyzer housing previously connected to the GC interface assembly. The ion focusing device is a standard Finnigan MAT 3300 electron impact ion source with the filament and trap removed. Further, the ion volume was replaced by a flat plate so as to act as a repeller while allowing for better pumpout characteristics. The FAB gun is normally operated with xenon (99.999% purity) at 0.5 cclmin at 10 psi. The accelerating voltage is approximately 8 KeV which results in 40-50 pamp of ion5current. Under these conditions, torr. the analyzer pressure is about 10
The sample is placed on a teflon coated tool.steel probe fitted with a copper tip machined at an an&La,of approximately 25' with re,spectto the atom beam. RESULTS AND D~S~SS~~~ FAB mass spectra were recorded for a series of 13 uncierlvatised estrogen glucuronides and sulfates (Table 1). Among these were conjugates of estrone, estradiol, estriol, and DES. Monosulfates and
CMW
372)
395
salt)+Na]+
[WNa
Fig.
1
FAB
mass
spectrum
of
sodium
estrone
(MW
Fig.2
FAB
mass
spectrum
estradiol-17@
of
d-sulfate
604)
dipotassium
3-sulfate
17_(B-D-glucuronidel
monoglucuronides and two mixed conjugates (glucuronide/sulfate) were examined to determine which classes of estrogen metabolites can be studied using this novel technique. As can be seen from the spectra shown in Fig. 1 and Fig. 2, FAB spectra of estrogen conjugates were clear and simple and were distinguished from the conventional electron impact or chemical ionization induced mass spectra by the low abundance or absence of any fragment ions.
Ions were formed by attachment of a proton or an alkali ion
to the uncharged molecule.
[M+H]+ and/or [M+Na]+ or [M+K]+ ions were
formed from salts (M representing the molecular weights of sodium or potassium glucuronides or sulfates).
The samples were dissolved in a
glycerol/water or glycerol/dimethyl sulfoxide mixture and were directly ionized from this matrix. In all cases examined, FAB spectra of estrogen sulfate salts were dominated by alkali attachment ions [M+alkali]+: ion [M+Na]+ and potassium salts by [M+K]+.
sodium salts by an
Despite the highly polar
character of alkali attachment ions, these were the most abundant ions in the spectra of estrogen sulfates. FAB spectra of estrogen glucuronide salts were characterized by two dominant ions in the upper mass region, an [M+H]+ and an [M+alkali]+ ion for each salt examined.
Fragment ions were of low
intensity or absent. With decreasing sample concentration, ions resulting from ionization of the glycerol solvent were found to increase. were:
These ions
m/z 115 [M+Na]+; m/z 131 [M+K]+; m/z 185 [2M+H]+; m/z 207
[2M+Na]; and m/z 223 [2M+K]+.
Ionized glycerol tri- and tetramers of
varying intensity were also detected.
At high sample concentrations
S
TD&OSDS
603
.,.a
.N
.
(app. 5-10 pg/@)
solvent ions were of low intensity when compared to
sample ions (8s shown in Fig. 1 and 2). An estimate was made of the sensitivity range of FAB with steroid conjugates. Using estrone sulfate solutions of varying concantration, it was possible to record a signal to noise ratio of 6.511 at m/z 395, @+Wa]
+
, with
47 nnml of oatnple (186 ng of estrone sulfate). This
sensitivity was achieved by adding sodium chloride to the glycerol/ Hz0 solvent system to increase the Na' concentration in the solvent. Without elevated Na' concentration the signal to noise at m/z 395 was lower, because formation of I[M+Na]'ions directly depends on the Nf
concentration. In summary, all spectra were characterizedby abundant molecular
species. Protonated molecular ions and/or [M+alkali]+ ions of high intensity were prominent and allowed an unambiguous deduction of the molecular weight of the sample. The nature of the alkali ion in the estrogen sulfate or glucuronide salt, usually sodium or potassium, can be determined from the mass difference between protonated and alkali attachment ions. Since most ion current formed by ionization of sample molecules is concentrated in one or two ions, the quasi-molecularions, the purity of the sample can be readily assessed. Especially polar impurities, organic or inorganic salts, which go undetected by conventional mass spectrometric techniques, may be easily identified. The low yield of fragment ions presents difficultieswith regard to the characterizationof isomers, but this information should be available using more complex techniques such as collision-induceddecomposition. The sensitivity of this novel technique at the present
S
TDEOSD-
605
experimental stage is not yet satisfactory to researchers in steroid hormone analysis. Improvements in the future may, however, make it a valuable tool in this field of research. ACKNOWLEDGMENT This work was supported in part by a grant from the National Institute of Health (Grant No. 1 ROl CA 27539). REFERENCES 1.
2.
3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15.
W.R. Slaunwhite, Jr., R.Y. Kirdani, and A.A. Sandberg, Handbook of Physiology, Section 7: Endocrinology,Vol. II, Part 1, R.O. Greep, E.B. Astwood, and S.R. Geiger, eds., American Physiological Society, Washington, D.C., 1973, p. 485 and references cited therein. C.J.W. Brooks and S.J. Gaskell, Biochemical Applications of Mass Spectrometry,First SupplementaryVolume, G.R. Waller and O.C. Dermer, eds., John Wiley & Sons, New York, 1980, p. 611 and references cited therein. B. Spiegelhalder,G. R8hle, L. Siekmann, and H. Breuer, J. Steroid Biochem., I, 749 (1976). B. Spiegelhalder and H. Breuer, 2. Klin. Chem. Klin. Biochem., 2, 254 (1974). R.M.‘Thompson, J. Steroid Biochem., 7_, 845 (1976). H. Miyazaki, M. Ishibashi, M. Itoh, N. Morishita, M. Sudo, and T. Nambara, Biomed. Mass Spectrom.,3_,55 (1976). J.C. Touchstone and M.F. Dobbins, J. Steroid Biochem., 2, 1389 (1975). T. Cronholm, Steroids, 2, 285 (1969). H. Adlercreuts, B. Soltmann, and M.J. Tikkanen, J. Steroid Biochem., 5_, 163 (1974). M. Barber, R.S. Bordoli, R.D. Sedgwick, and A.N. Tyler, .I.Chem. Sot. Chem. Commun., 325 (1981). M. Barber, R.S. Bordoli, R.D. Sedgwick, and A.N. Tyler, Biomed. Mass Spectrom., 2, 492 (1981). M. Barber, R.S. Bordoli, R.D. Sedgwick, A.N. Tyler, and E.T. Whalley, Biomed. Mass Spectrom.,8_, 337 (1981). Section on FAB-MS, Abstracts, 29th Annual Conf. on Mass Spectrom. and Allied Topics, May 24-29, 1981, Minneapolis, Minn., p.351. P.A. Barford, A.H. Olavesen, C.G. Curtis, and G.M. Powell, Biochem. J., 9, 423 (1977). R.M. Caprioli, C.F. Beckner, and L.A. Smith, Biomed. Mass Spectrom., submitted.
295!3
FAST ATOM BOMBARDMENT MASS SPECTROMETRY OF ESTROGEN GLUCURONIDES AND SULFATES Joachim G. Liehr, Carl F. Beckner, Annie M. Ballatore and Richard M. Caprioli Analytical Chemistry Center and Department of Biochemistry and Molecular Biology, University of Texas Health Science Center at Houston, Houston, Texas 77030 Received 4-12-82 ABSTRACT Fast atom bombardment (FAB) mass spectra of 13 intact, underivatized glucuronides and/or sulfate salts are reported. Spectra are charalterized by abundant ions formed by attachment of a proton, [M+H.] , or of an alkali ion, [M+alkali] , to the glucuronide or sulfate salt. Fragment ions were of low intensity. FAB spectra can be used to ombtain the molecular weight of a sample, to assess its purity and to identify the nature of the alkali of the glucuronide or sulfate salt. INTRODUCTION In mammals, most estrogens present in plasma, bile, urine or feces are conjugated (1) with glucuronic acid and/or sulfuric acid. Minolr quantities of other classes of conjugates, such as N-acetylglucosamines, have been isolated, but their contributions to steroid profiles are negligible.
Structural analysis of hormone metabolites
presents problems due to the small quantities isolable from biological sources and due to the polar character of glucuronides and sulfates. Therefore, steroid extracts are usually separated into conjugate fractions by chromatography and hydrolyzed enzymatically or by solvolysis before steroid profiles or structural analyses can be obtained using mass spectrometric methods (2).
Direct analysis by GC-MS or
gas chromatography of estrogen glucuronides (3-6) and sulfates (7,8) has been reported but is not widely accepted as a method for steroid profiling because of the chromatographic difficulties involved in
Vohme
39, Number 6
S
TDROIDES
June, 1982
these procedures.
Alternative ionization methods (field desorption)
in mass spectrometry for the direct analysis of estrogen conjugates have been tried (9), but the expense of the equipment prevents its widespread use. A novel ionization technique in mass spectrometry, fast atom bombardment
(FAB) (10,11,12), is promising for the analysis of estro-
gen conjugates.
Existing equipment can be modified to accept a FAB
ion source and mass spectra can be obtained with this technique from highly polar or thermally labile substances (13).
Thus, FAB mass
spectra have been reported (13) for underivatized peptides, carbohydrates, nucleotides, antibiotic substances and other polar compounds. In order to determine the suitability of this novel mass spectrometric method for the analysis of steroid hormones, a series of underivatized estrogen glucuronides and sulfates were examined by FAB mass spectrometry. MATERIALS AND METHODS Diethylstilbestrol (DES) monosulfate was synthesized following published procedures (14). DES monoglucuronide was a generous gift from Dr. W. Kruger and Dipl.-Chem. R. Mahrwald, Forschungsinstitut Manfred von Ardenne, Dresden, GDR. All other substances were purchased from Sigma Chemical Co., St. Louis, Missouri. Fast atom bombardment mass spectrometry was performed on a Finnigan MAT 3300/Incas mass spectrometer, modified as follows to accept an Ion Tech BllNF saddle field atom gun and B50 power supply (15). The Ion Tech gun was placed on the analyzer housing previously connected to the GC interface assembly. The ion focusing device is a standard Finnigan MAT 3300 electron impact ion source with the filament and trap removed. Further, the ion volume was replaced by a flat plate so as to act as a repeller while allowing for better pumpout characteristics. The FAB gun is normally operated with xenon (99.999% purity) at 0.5 cclmin at 10 psi. The accelerating voltage is approximately 8 KeV which results in 40-50 pamp of ion5current. Under these conditions, torr. the analyzer pressure is about 10
The sample is placed on a teflon coated tool.steel probe fitted with a copper tip machined at an an&La,of approximately 25' with re,spectto the atom beam. RESULTS AND D~S~SS~~~ FAB mass spectra were recorded for a series of 13 uncierlvatised estrogen glucuronides and sulfates (Table 1). Among these were conjugates of estrone, estradiol, estriol, and DES. Monosulfates and
CMW
372)
395
salt)+Na]+
[WNa
Fig.
1
FAB
mass
spectrum
of
sodium
estrone
(MW
Fig.2
FAB
mass
spectrum
estradiol-17@
of
d-sulfate
604)
dipotassium
3-sulfate
17_(B-D-glucuronidel
monoglucuronides and two mixed conjugates (glucuronide/sulfate) were examined to determine which classes of estrogen metabolites can be studied using this novel technique. As can be seen from the spectra shown in Fig. 1 and Fig. 2, FAB spectra of estrogen conjugates were clear and simple and were distinguished from the conventional electron impact or chemical ionization induced mass spectra by the low abundance or absence of any fragment ions.
Ions were formed by attachment of a proton or an alkali ion
to the uncharged molecule.
[M+H]+ and/or [M+Na]+ or [M+K]+ ions were
formed from salts (M representing the molecular weights of sodium or potassium glucuronides or sulfates).
The samples were dissolved in a
glycerol/water or glycerol/dimethyl sulfoxide mixture and were directly ionized from this matrix. In all cases examined, FAB spectra of estrogen sulfate salts were dominated by alkali attachment ions [M+alkali]+: ion [M+Na]+ and potassium salts by [M+K]+.
sodium salts by an
Despite the highly polar
character of alkali attachment ions, these were the most abundant ions in the spectra of estrogen sulfates. FAB spectra of estrogen glucuronide salts were characterized by two dominant ions in the upper mass region, an [M+H]+ and an [M+alkali]+ ion for each salt examined.
Fragment ions were of low
intensity or absent. With decreasing sample concentration, ions resulting from ionization of the glycerol solvent were found to increase. were:
These ions
m/z 115 [M+Na]+; m/z 131 [M+K]+; m/z 185 [2M+H]+; m/z 207
[2M+Na]; and m/z 223 [2M+K]+.
Ionized glycerol tri- and tetramers of
varying intensity were also detected.
At high sample concentrations
S
TD&OSDS
603
.,.a
.N
.
(app. 5-10 pg/@)
solvent ions were of low intensity when compared to
sample ions (8s shown in Fig. 1 and 2). An estimate was made of the sensitivity range of FAB with steroid conjugates. Using estrone sulfate solutions of varying concantration, it was possible to record a signal to noise ratio of 6.511 at m/z 395, @+Wa]
+
, with
47 nnml of oatnple (186 ng of estrone sulfate). This
sensitivity was achieved by adding sodium chloride to the glycerol/ Hz0 solvent system to increase the Na' concentration in the solvent. Without elevated Na' concentration the signal to noise at m/z 395 was lower, because formation of I[M+Na]'ions directly depends on the Nf
concentration. In summary, all spectra were characterizedby abundant molecular
species. Protonated molecular ions and/or [M+alkali]+ ions of high intensity were prominent and allowed an unambiguous deduction of the molecular weight of the sample. The nature of the alkali ion in the estrogen sulfate or glucuronide salt, usually sodium or potassium, can be determined from the mass difference between protonated and alkali attachment ions. Since most ion current formed by ionization of sample molecules is concentrated in one or two ions, the quasi-molecularions, the purity of the sample can be readily assessed. Especially polar impurities, organic or inorganic salts, which go undetected by conventional mass spectrometric techniques, may be easily identified. The low yield of fragment ions presents difficultieswith regard to the characterizationof isomers, but this information should be available using more complex techniques such as collision-induceddecomposition. The sensitivity of this novel technique at the present
S
TDEOSD-
605
experimental stage is not yet satisfactory to researchers in steroid hormone analysis. Improvements in the future may, however, make it a valuable tool in this field of research. ACKNOWLEDGMENT This work was supported in part by a grant from the National Institute of Health (Grant No. 1 ROl CA 27539). REFERENCES 1.
2.
3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15.
W.R. Slaunwhite, Jr., R.Y. Kirdani, and A.A. Sandberg, Handbook of Physiology, Section 7: Endocrinology,Vol. II, Part 1, R.O. Greep, E.B. Astwood, and S.R. Geiger, eds., American Physiological Society, Washington, D.C., 1973, p. 485 and references cited therein. C.J.W. Brooks and S.J. Gaskell, Biochemical Applications of Mass Spectrometry,First SupplementaryVolume, G.R. Waller and O.C. Dermer, eds., John Wiley & Sons, New York, 1980, p. 611 and references cited therein. B. Spiegelhalder,G. R8hle, L. Siekmann, and H. Breuer, J. Steroid Biochem., I, 749 (1976). B. Spiegelhalder and H. Breuer, 2. Klin. Chem. Klin. Biochem., 2, 254 (1974). R.M.‘Thompson, J. Steroid Biochem., 7_, 845 (1976). H. Miyazaki, M. Ishibashi, M. Itoh, N. Morishita, M. Sudo, and T. Nambara, Biomed. Mass Spectrom.,3_,55 (1976). J.C. Touchstone and M.F. Dobbins, J. Steroid Biochem., 2, 1389 (1975). T. Cronholm, Steroids, 2, 285 (1969). H. Adlercreuts, B. Soltmann, and M.J. Tikkanen, J. Steroid Biochem., 5_, 163 (1974). M. Barber, R.S. Bordoli, R.D. Sedgwick, and A.N. Tyler, .I.Chem. Sot. Chem. Commun., 325 (1981). M. Barber, R.S. Bordoli, R.D. Sedgwick, and A.N. Tyler, Biomed. Mass Spectrom., 2, 492 (1981). M. Barber, R.S. Bordoli, R.D. Sedgwick, A.N. Tyler, and E.T. Whalley, Biomed. Mass Spectrom.,8_, 337 (1981). Section on FAB-MS, Abstracts, 29th Annual Conf. on Mass Spectrom. and Allied Topics, May 24-29, 1981, Minneapolis, Minn., p.351. P.A. Barford, A.H. Olavesen, C.G. Curtis, and G.M. Powell, Biochem. J., 9, 423 (1977). R.M. Caprioli, C.F. Beckner, and L.A. Smith, Biomed. Mass Spectrom., submitted.