RAPID COMMUNICATIONS IN MASS SPECTROMETRY, VOL. 5,391-394 (1991)
Charge-remote Fragmentation of Taurine-conjugated Bile Acids William James Griffiths*+ Chemistry Department, University of the West Indies, Mona Campus, Kingston 7, Jamaica, West Indies
Borje Egestad and Jan Sjovall Department of Physiological Chemistry, Karolinska Institutet, Box 6O400, S-104 01 Stockholm, Sweden
Taurine-conjugatedbile acids are an important group of biological metabolites. When investigated by negativeion fast-atom bombardment collision-induceddissociation mass spectroscopy they show charge-remotefragmentations of the [M - HI- pseudomolecular ion. These fragmentationsprovide information on the positions of ring substituents remote from the charge site. In the present work we have compared the negative-ion fast-atom bombardment collision-induced dissociation spectra of six dinerent conjugated bile acids.
The major pathway of degradation of cholesterol in animals is conversion to bile acids and conjugated bile acids. Bile acids and conjugated bile acids are formed in the liver and secreted into the small intestine where their function is to aid the absorption of lipids. Fast-atom bombardment (FAB)' has become the favoured ionization method for the study of polar and involatile biomolecules.2 Both positive- and negativeion FAB spectra show prominent pseudomolecular ions and thus allow the determination of the molecular weights of biological compounds. Little fragmentation is generally observed in either positive- or negative- ion FAB spectra, and thus little structural information becomes available. The FAB ionization technique has opened new possibilities in the studies of bile acid metabolism and excretion. Crude biological extracts can be directly analysed, and FAB ionization mass spectrometry of urine extracts permits diagnosis of inherited and acquired diseases in bile acid biosynthesis and metabolism, such as cerebrotendinous xanthomat~sis,~ Zellweger's d i s e a ~ e , ~ 3P-hydroxy-A5-C2,-steroid dehydrogenase/isomerase deficiency' and 5P-reductase of urine and plasma extracts from d e f i ~ i e n c y .Spectra ~,~ patients with cholestatic liver disease of unknown etiology show complex patterns of pseudomolecular ions, the diagnostic value of which is presently unknown. Although the molecular weights of compounds in a mixture can easily be determined from FAB spectra, structural information concerning these compounds is not usually available. To obtain increased structural information when using the FAB ionization technique, collision-induced dissociation (CID) spectra of pseudomolecular ions are recorded. The coupling of FAB and CID has been extensively used in recent years in the analysis of biological compounds.8,9 Cheng et d.," have used the FAB/CID techni ue in the study of steroids. Beynon and co-workers", and also Tomer, Gross and co-worker~'~. l4 have studied bile acids and their conjugates using FAB/CID. Beynon and co-workers", studied conjugated bile acids using a reversed-geometry, double-focusing mass spectrometer and recorded mass-analysed ion kinetic energy
9
* Author to whom correspondence should be addressed.
' Present address: Department of
Physics, Uppsala University, Box
530, S-751 21 Uppsala, Sweden.
0951-4198/91/093391-04 $05.00
01991 by John Wiley & Sons, Ltd.
(MIKE ) spectra resulting from the CID of the positive pseudomolecular ions [M(Na) (Na)]' (i.e. , the sodium adducts of sodium bile acid salts). Gross and c o - ~ o r k e r sperformed '~ complementary work on some conjugated bile acids by recording the daughter-ion spectra obtained from the CID of pseudomolecular anions [M - HI- generated by negative-ion FAB. This work was carried out on a three-sector instrument of EBE geometry, and as in the work of Beynon, daughter ions were mass analysed by a final electric sector. Tomer, Gross and co-workersi3 observed chargeremote fragmentations of the taurine-conjugated bile acids. These are fragmentations that occur remote from the charged site, and recent studies of this type of reaction have revealed a wealth of structural information on numerous biomole~ules.~~In the case of taurine conjugates this means fragmentations within the steroid ring structure. Thus positions of substituents in the ring structure can be determined from the resultant daughter-ion spectra. Clearly the combined techniques of FAB ionization and CID fragmentation should be very useful for the study of the structure of conjugated bile acids. In the present work we have compared the negativeion FAB/CID of the taurine-conjugated bile acids, taurocholate (1), taurochenodeoxycholate (2), taurodeoxycholate (3), taurolithocholate (4), taurocholanoate (5), and taurodehydrocholate (6) whose structures are shown in Fig. 1.
+
EXPERIMENTAL
Negative-ion FAB spectra were obtained on a 7070 E mass spectrometer (VG Analytical, Manchester, UK) fitted with a VG FAB source and Ion-Tech atom gun (Teddington, UK). Xenon atoms were used to bombard the sample, the ion gun conditions being typically 8 kV accelerating potential and 1-2 mA discharge current. The accelerating potential was 6 kV. Samples were prepared by dissolving a small amount of the conjugated bile-acid sodium salt in a methanollwater (70% solvent 1 yg/yL) and then placing 1 yL of solution onto the FAB target which was itself coated by a drop of glycerol. Sample lifetimes were approximately 15 min. CID spectra were generated using argon as the collision gas in the first field-free region gas cell, at a Received 12 July 1991 Accepted 31 July 1991
REMOTE-CHARGE FRAGMENTATION OF CONJUGATED BILE ACIDS
392
80 60 -
A 40
-
20 0' 200
A Figure 1. Charge-remote fragmentation of steroid ring structure in (a), taurocholate R,= R2= R3= OH (1); taurochenodeoxycholate R, = Rz= OH, R3= H (2); taurodeoxycholate R1= R3= OH, R2 = H (3); taurolithocholate R1= OH, R2= R3= H (4); taurocholanoate R1= R2= R3= H (5); and (b), taurodehydrocholate (6). Cleavage A occurs within ring A, cleavage B within ring B, and cleavage C within ring C.
pressure which gave a reading of 2 X Torr on the nearby analyser ion gauge (this pressure was sufficient to cause a 50% reduction in ion-beam intensity). Daughter ion (BIE is a constant) linked scanslawere recorded of the [M -HI- pseudomolecular ions of the conjugated bile acids, and constant neutral-mass loss (BIEis a constant) linked scans were also recorded to establish the origin of a particular neutral loss. The sodium salts of the conjugated bile acids were obtained from the Sigma Chemical Co. (St Louis, MO, USA and used without further purification. RESULTS AND DISCUSSION Negative-ion FAB spectra were recorded of all the conjugated bile acids studied. All showed prominent [M - HI- pseudomolecular ions, and very little fragmentation was evident. Shown in Figs 2-7 are the CID daughter-ion spectra of the [M -HI- pseudomolecular ions generated from the six taurine-conjugated bile acids studied. The dominant fragmentations were loss of 16u and 18u and those shown in Table 1.
L66
L26
I
I
2;o 250
, '
,
3bO
(
I
l
400
350
l
I
I1
L
4ko
I0
Figure 3. Spectrum of daughter ions produced by CID of [M - HIions of taurochenodeoxycholate.
Charge-remote fragmentation is important for all compounds studied; cleavages A and B as shown in Fig. 1 are seen for all samples, while cleavage C is seen for taurodeoxycholate, taurolithocholate and taurodehydrocholate. By studying the charge-remote fragmentations the structure of the conjugated bile acids can be obtained. For example the two isomers taurochenodeoxycholate and taurodeoxycholate can be differentiated from their CID spectra." Clearly both isomers have an OH group attached to ring A of the steroid structure as is seen from the fragmentation resulting from cleavage A, i.e., mass loss of 72u (C,H,O). But taurochenodeoxycholate has an OH group on ring B, while taurodeoxycholate does not. This is evident from cleavage B ; taurochenodeoxycholate loses a mass of 156u (C9HI6O2),while taurodeoxycholate loses a mass of 140u (C9Ht60).The presence of the OH group on ring C in taurodeoxycholate is confirmed by a mass loss of 2 1 0 ~(C13H2202). is a conConstant neutral-mass loss ( B I E stant) linked scans were recorded for each of the conjugated bile acids. They confirmed that the daughter ions listed in Table 1 were formed by a neutral-mass loss from the pseudomolecular [M - HIparent ions. Tomer, Gross and c o - ~ o r k e r salso ' ~ studied the negative ion FAB/CID spectra of taurocho-
LBZ
/I
'"I
L96
I
I
100
490
8-
6-
4-
2-
0;
1
,
I
I
I
I
L85
LL2
358 1
I
1
I
1
1
I
I
1
I
I
1
I
'1
rn/z
Figure 2. Spectrum of daughter ions produced by CID of [M - HIions of taurocholate.
0 rn/z
Figure 4. Spectrum of daughter ions produced by CID of [M - HIions of taurodeoxycholate.
REMOTE-CHARGE FRAGMENTATION OF CONJUGATED BILE ACIDS
'O0I
8o 464
60 -
I
t
L38
60 -
-
40 -
4 50
410
20 342
0
L92
100 -
80
40
393
288 I
I
I
I
I
426 II
Lh[L
2 o l l
I,I
0200
rn/2
LbO
Figure 5. Spectrum of daughter ions produced by CID of [M - HIions of taurolithocholate.
m/z
Figure 6. Spectrum of daughter ions produced by CID of [M -HIions of taurocholanoate.
Figure 7. Spectrum of daughter ions produced by CID of [M - HIions of taurodehydrocholate.
late, taurochenodeoxycholate and taurolithocholate. They used a three-sector instrument in a tandem mass spectrometry (MS/MS) study. They desorbed their pseudomolecular ions from a triethanolamine matrix, and used xenon atoms for sample bombardment. Pseudomolecular [M - HI- ions were selected by the first two sectors (MS-I) of EB geometry and transmitted into a third field-free region gas cell containing helium collision gas. Daughter-ion spectra were acquired by scanning the third sector (MS-11) an electrostatic analyser. Tomer, Gross and co-workers found water loss to be the dominant fragmentation, and all three conjugated bile acids to give charge-remote fragmentations by cleavages A and B, with the resultant daughter ions having relative abundances of about 10%. Cleavage C was found to occur for taurocholate and taurolithocholate, with daughter-
Table 1. Charge-remote fragmentations miz
(M-H)
Compound
5b0
(Charge-remote fragmentation) cleavages-resultant miz (%RA) A B C
Others (%RA)
1
Taurocholate R, = R 2 = R,= OH
514
442( 10)
3S8( 10)
-
498(8S) 496( 100) 48S( 10)
2
Taurochenodeoxycholate Rl = R, = OH,, R,= H
498
426(20)
342(20)
-
482( 100) 480(80) 466( 20)
3
Taurodeoxycholate Rl =OH, R,= H, R3= OH
498
426(30)
358(10)
288(15)
482(100) 480(90) 466( IS)
4
Taurolithocholate R,= OH, R, = R, = H
482
410(20)
342(10)
288(5)
466( 100) 464(60) 4.50(25) 426(10)
5
Taurocholanoate R,= R, = R, = H
466
410(2S)
342(10)
6
Taurodehydrocholate
508
438(80)
356(1S)
450( 100) 448(2S) 422( 10) 288(20)
494(80) 492(100) 480(30) 464(40)
394
REMOTE-CHARGE FRAGMENTATION O F CONJUGATED BILE ACIDS
ion relative abundances of approximately 5YO. Taurochenodeoxycholate, as in the present work, was not found to give cleavage C. It is interesting to compare these results to those obtained in the present work in which the linked scan (BIE is constant) technique was used. A difference between the results of the present work and those from the MS/MS study is that cleavage C was not observed for taurocholate in the present work, but was seen in the MS/MS study. Although the relative abundances for the various daughter ions differ in this work and in the MS/MS study this variation is not unexpected as different instruments, collision gases, and scanning methods were used. CONCLUSIONS The taurine-conjugated bile acids studied show prominent charge-remote fragmentation reactions. Analysis of the FAB/CID spectra from the [M - HI- pseudomolecular ions gives information concerning the position of the ring functional groups, and in the case of taurochenodeoxycholate and taurodeoxycholate, isomers can be differentiated.” This is of major importance in the study of biological extracts. Negative-ion FAB is the favoured ionization technique for the analysis of biological extracts as the accompanying lack of fragmentation allows pseudomolecular ions to be identified. Clearly if CID studies are carried out on three pseudomolecular ions, structural information concerning the compounds in a particular extract will be forthcoming. Certain conjugated bile acids are excreted in the urine of healthy humans, while others may be excreted by diseased patient^."-'^ Clearly, urine analaysis by negative-ion FAB/CID will allow the bile-acid content to be characterized and this will be beneficial in the diagnosis of diseased patients.
Acknowledgements W.J.G. wishes to thank the University of the West Indies for providing him with a ‘study and travel’ grant which partially financed
his visit to the Karolinska Institutet. This work was supported by the Swedish Medical Research Council (grant no. 03X-219).
REFERENCES 1. M. Barber, R. S. Bordoli, R. D. Sedgwick and A. N. Tyler, J. Chem. SOC. Chem. Commun. 325 (1981). 2. K. L. Rinehart Jr., Trends Anal. Chem. 2, 10 (1983). 3. B. Egestad, P. Pettersson, S. Skrede and J. Sjovall, Scand. J . Clin. Lab. Invest. 45, 443 (1985). 4. A. M. Lawson, M. J. Madigan, D. Shortland and P. Clayton, Clin. Chim. Acta, 161,221 (1986). 5. P. T. Clayton, J. V. Leonard, A. M. Lawson, K. D . R. Setchell, S. Andersson, B. Egestad and J. Sjovall, J. Clin. Invest. 79, 1031 (1987). 6. P. T. Clayton, E. Patel, A. M. Lawson, R . A. Carruthers, M. S. Tanner, B. Strandvik, B. Egestad and J. Sjovall, Lancet, 1283 (1988). 7. K. D . R. Setchell, F. J. Suchy, M. B. Welsh, L. Zimmer-Nechemias, J. Heubi and W. F. Baltistreri, J. Clin. Invest. 82, 2148 (1988). 8. A. J. Alexander, P. Thibault, R. K. Boyd, J. M. Curtis and K. L. Reinhart, In!. I . Mass Spectrom. Ion Processes. 98, 107 (1990). 9. J. A. Zirrolle, E . Bettazoli, M. Gross and R. C. Murphy, J. A m . SOC. Mass Spectrom. I , 325 (1990). 10. M. T. Cheng, M. P. Barbalos, R. F. Peguers and F. W. McLafferty, J. A m . Chem. SOC. 105, 1510 (1983). 11. J. G. Liehr, E. E. Kingston and J. H. Beynon, Biomed. Mass Spectrom. 12, 95 (1985). 12. E. E. Kingston, J. H. Beynon, R . P. Newton and J. G. Liehr, Biomed. Mass Spectrom. 12, 525 (1985). 13. K. B. Tomer, N. J. Jensen, M. L. Gross and J. Whitney, Biomed. Enuiron. Mass Spectrom. 13,265 (1986). 14. K. B. Tomer and M. L. Gross, Biomed. Enuiron. Mass Spectrom. 15,89 (1988). 15. N. J. Jensen, K.B. Tomer, M. L. Gross, J. A m . Chem. SOC. 107, 1863 (1985). 16. M. L. Gross, Ado. Mass Spectrorn. llA,792 (1989). 17. J. Adams, Mass Spectrom. Rev. 9, (1990). 18. K. R. Jennings and R. S. Mason, in Tandem Mass Spectrometry, ed. by F. W. McLafferty, p. 197, Wiley-Interscience (1983). 19. W. J. Griffiths, B. Egestad and J. Sjovall, Rapid Cornmun. Mass Spectrom. 5, 196 (1991). 20. P. Black, Clin. Chim. Acta, 44, 199 (1973). 21. P. Black, K. Spaczynski and W. Gerok, Hoppe-Seyler’s Z. Physiol. Chem. 355,749 (1974). 22. A. Stiehl, in ‘Clinics in Gastroenterology’, ed. by G. Paumgartner, p. 45, Saunders, London, Philadelphia, Toronto (1977). 23. K. D. R. Setchell and J. M. Street, Semin. Liver Dir. 7 , 85 (1987).
Charge-remote Fragmentation of Taurine-conjugated Bile Acids William James Griffiths*+ Chemistry Department, University of the West Indies, Mona Campus, Kingston 7, Jamaica, West Indies
Borje Egestad and Jan Sjovall Department of Physiological Chemistry, Karolinska Institutet, Box 6O400, S-104 01 Stockholm, Sweden
Taurine-conjugatedbile acids are an important group of biological metabolites. When investigated by negativeion fast-atom bombardment collision-induceddissociation mass spectroscopy they show charge-remotefragmentations of the [M - HI- pseudomolecular ion. These fragmentationsprovide information on the positions of ring substituents remote from the charge site. In the present work we have compared the negative-ion fast-atom bombardment collision-induced dissociation spectra of six dinerent conjugated bile acids.
The major pathway of degradation of cholesterol in animals is conversion to bile acids and conjugated bile acids. Bile acids and conjugated bile acids are formed in the liver and secreted into the small intestine where their function is to aid the absorption of lipids. Fast-atom bombardment (FAB)' has become the favoured ionization method for the study of polar and involatile biomolecules.2 Both positive- and negativeion FAB spectra show prominent pseudomolecular ions and thus allow the determination of the molecular weights of biological compounds. Little fragmentation is generally observed in either positive- or negative- ion FAB spectra, and thus little structural information becomes available. The FAB ionization technique has opened new possibilities in the studies of bile acid metabolism and excretion. Crude biological extracts can be directly analysed, and FAB ionization mass spectrometry of urine extracts permits diagnosis of inherited and acquired diseases in bile acid biosynthesis and metabolism, such as cerebrotendinous xanthomat~sis,~ Zellweger's d i s e a ~ e , ~ 3P-hydroxy-A5-C2,-steroid dehydrogenase/isomerase deficiency' and 5P-reductase of urine and plasma extracts from d e f i ~ i e n c y .Spectra ~,~ patients with cholestatic liver disease of unknown etiology show complex patterns of pseudomolecular ions, the diagnostic value of which is presently unknown. Although the molecular weights of compounds in a mixture can easily be determined from FAB spectra, structural information concerning these compounds is not usually available. To obtain increased structural information when using the FAB ionization technique, collision-induced dissociation (CID) spectra of pseudomolecular ions are recorded. The coupling of FAB and CID has been extensively used in recent years in the analysis of biological compounds.8,9 Cheng et d.," have used the FAB/CID techni ue in the study of steroids. Beynon and co-workers", and also Tomer, Gross and co-worker~'~. l4 have studied bile acids and their conjugates using FAB/CID. Beynon and co-workers", studied conjugated bile acids using a reversed-geometry, double-focusing mass spectrometer and recorded mass-analysed ion kinetic energy
9
* Author to whom correspondence should be addressed.
' Present address: Department of
Physics, Uppsala University, Box
530, S-751 21 Uppsala, Sweden.
0951-4198/91/093391-04 $05.00
01991 by John Wiley & Sons, Ltd.
(MIKE ) spectra resulting from the CID of the positive pseudomolecular ions [M(Na) (Na)]' (i.e. , the sodium adducts of sodium bile acid salts). Gross and c o - ~ o r k e r sperformed '~ complementary work on some conjugated bile acids by recording the daughter-ion spectra obtained from the CID of pseudomolecular anions [M - HI- generated by negative-ion FAB. This work was carried out on a three-sector instrument of EBE geometry, and as in the work of Beynon, daughter ions were mass analysed by a final electric sector. Tomer, Gross and co-workersi3 observed chargeremote fragmentations of the taurine-conjugated bile acids. These are fragmentations that occur remote from the charged site, and recent studies of this type of reaction have revealed a wealth of structural information on numerous biomole~ules.~~In the case of taurine conjugates this means fragmentations within the steroid ring structure. Thus positions of substituents in the ring structure can be determined from the resultant daughter-ion spectra. Clearly the combined techniques of FAB ionization and CID fragmentation should be very useful for the study of the structure of conjugated bile acids. In the present work we have compared the negativeion FAB/CID of the taurine-conjugated bile acids, taurocholate (1), taurochenodeoxycholate (2), taurodeoxycholate (3), taurolithocholate (4), taurocholanoate (5), and taurodehydrocholate (6) whose structures are shown in Fig. 1.
+
EXPERIMENTAL
Negative-ion FAB spectra were obtained on a 7070 E mass spectrometer (VG Analytical, Manchester, UK) fitted with a VG FAB source and Ion-Tech atom gun (Teddington, UK). Xenon atoms were used to bombard the sample, the ion gun conditions being typically 8 kV accelerating potential and 1-2 mA discharge current. The accelerating potential was 6 kV. Samples were prepared by dissolving a small amount of the conjugated bile-acid sodium salt in a methanollwater (70% solvent 1 yg/yL) and then placing 1 yL of solution onto the FAB target which was itself coated by a drop of glycerol. Sample lifetimes were approximately 15 min. CID spectra were generated using argon as the collision gas in the first field-free region gas cell, at a Received 12 July 1991 Accepted 31 July 1991
REMOTE-CHARGE FRAGMENTATION OF CONJUGATED BILE ACIDS
392
80 60 -
A 40
-
20 0' 200
A Figure 1. Charge-remote fragmentation of steroid ring structure in (a), taurocholate R,= R2= R3= OH (1); taurochenodeoxycholate R, = Rz= OH, R3= H (2); taurodeoxycholate R1= R3= OH, R2 = H (3); taurolithocholate R1= OH, R2= R3= H (4); taurocholanoate R1= R2= R3= H (5); and (b), taurodehydrocholate (6). Cleavage A occurs within ring A, cleavage B within ring B, and cleavage C within ring C.
pressure which gave a reading of 2 X Torr on the nearby analyser ion gauge (this pressure was sufficient to cause a 50% reduction in ion-beam intensity). Daughter ion (BIE is a constant) linked scanslawere recorded of the [M -HI- pseudomolecular ions of the conjugated bile acids, and constant neutral-mass loss (BIEis a constant) linked scans were also recorded to establish the origin of a particular neutral loss. The sodium salts of the conjugated bile acids were obtained from the Sigma Chemical Co. (St Louis, MO, USA and used without further purification. RESULTS AND DISCUSSION Negative-ion FAB spectra were recorded of all the conjugated bile acids studied. All showed prominent [M - HI- pseudomolecular ions, and very little fragmentation was evident. Shown in Figs 2-7 are the CID daughter-ion spectra of the [M -HI- pseudomolecular ions generated from the six taurine-conjugated bile acids studied. The dominant fragmentations were loss of 16u and 18u and those shown in Table 1.
L66
L26
I
I
2;o 250
, '
,
3bO
(
I
l
400
350
l
I
I1
L
4ko
I0
Figure 3. Spectrum of daughter ions produced by CID of [M - HIions of taurochenodeoxycholate.
Charge-remote fragmentation is important for all compounds studied; cleavages A and B as shown in Fig. 1 are seen for all samples, while cleavage C is seen for taurodeoxycholate, taurolithocholate and taurodehydrocholate. By studying the charge-remote fragmentations the structure of the conjugated bile acids can be obtained. For example the two isomers taurochenodeoxycholate and taurodeoxycholate can be differentiated from their CID spectra." Clearly both isomers have an OH group attached to ring A of the steroid structure as is seen from the fragmentation resulting from cleavage A, i.e., mass loss of 72u (C,H,O). But taurochenodeoxycholate has an OH group on ring B, while taurodeoxycholate does not. This is evident from cleavage B ; taurochenodeoxycholate loses a mass of 156u (C9HI6O2),while taurodeoxycholate loses a mass of 140u (C9Ht60).The presence of the OH group on ring C in taurodeoxycholate is confirmed by a mass loss of 2 1 0 ~(C13H2202). is a conConstant neutral-mass loss ( B I E stant) linked scans were recorded for each of the conjugated bile acids. They confirmed that the daughter ions listed in Table 1 were formed by a neutral-mass loss from the pseudomolecular [M - HIparent ions. Tomer, Gross and c o - ~ o r k e r salso ' ~ studied the negative ion FAB/CID spectra of taurocho-
LBZ
/I
'"I
L96
I
I
100
490
8-
6-
4-
2-
0;
1
,
I
I
I
I
L85
LL2
358 1
I
1
I
1
1
I
I
1
I
I
1
I
'1
rn/z
Figure 2. Spectrum of daughter ions produced by CID of [M - HIions of taurocholate.
0 rn/z
Figure 4. Spectrum of daughter ions produced by CID of [M - HIions of taurodeoxycholate.
REMOTE-CHARGE FRAGMENTATION OF CONJUGATED BILE ACIDS
'O0I
8o 464
60 -
I
t
L38
60 -
-
40 -
4 50
410
20 342
0
L92
100 -
80
40
393
288 I
I
I
I
I
426 II
Lh[L
2 o l l
I,I
0200
rn/2
LbO
Figure 5. Spectrum of daughter ions produced by CID of [M - HIions of taurolithocholate.
m/z
Figure 6. Spectrum of daughter ions produced by CID of [M -HIions of taurocholanoate.
Figure 7. Spectrum of daughter ions produced by CID of [M - HIions of taurodehydrocholate.
late, taurochenodeoxycholate and taurolithocholate. They used a three-sector instrument in a tandem mass spectrometry (MS/MS) study. They desorbed their pseudomolecular ions from a triethanolamine matrix, and used xenon atoms for sample bombardment. Pseudomolecular [M - HI- ions were selected by the first two sectors (MS-I) of EB geometry and transmitted into a third field-free region gas cell containing helium collision gas. Daughter-ion spectra were acquired by scanning the third sector (MS-11) an electrostatic analyser. Tomer, Gross and co-workers found water loss to be the dominant fragmentation, and all three conjugated bile acids to give charge-remote fragmentations by cleavages A and B, with the resultant daughter ions having relative abundances of about 10%. Cleavage C was found to occur for taurocholate and taurolithocholate, with daughter-
Table 1. Charge-remote fragmentations miz
(M-H)
Compound
5b0
(Charge-remote fragmentation) cleavages-resultant miz (%RA) A B C
Others (%RA)
1
Taurocholate R, = R 2 = R,= OH
514
442( 10)
3S8( 10)
-
498(8S) 496( 100) 48S( 10)
2
Taurochenodeoxycholate Rl = R, = OH,, R,= H
498
426(20)
342(20)
-
482( 100) 480(80) 466( 20)
3
Taurodeoxycholate Rl =OH, R,= H, R3= OH
498
426(30)
358(10)
288(15)
482(100) 480(90) 466( IS)
4
Taurolithocholate R,= OH, R, = R, = H
482
410(20)
342(10)
288(5)
466( 100) 464(60) 4.50(25) 426(10)
5
Taurocholanoate R,= R, = R, = H
466
410(2S)
342(10)
6
Taurodehydrocholate
508
438(80)
356(1S)
450( 100) 448(2S) 422( 10) 288(20)
494(80) 492(100) 480(30) 464(40)
394
REMOTE-CHARGE FRAGMENTATION O F CONJUGATED BILE ACIDS
ion relative abundances of approximately 5YO. Taurochenodeoxycholate, as in the present work, was not found to give cleavage C. It is interesting to compare these results to those obtained in the present work in which the linked scan (BIE is constant) technique was used. A difference between the results of the present work and those from the MS/MS study is that cleavage C was not observed for taurocholate in the present work, but was seen in the MS/MS study. Although the relative abundances for the various daughter ions differ in this work and in the MS/MS study this variation is not unexpected as different instruments, collision gases, and scanning methods were used. CONCLUSIONS The taurine-conjugated bile acids studied show prominent charge-remote fragmentation reactions. Analysis of the FAB/CID spectra from the [M - HI- pseudomolecular ions gives information concerning the position of the ring functional groups, and in the case of taurochenodeoxycholate and taurodeoxycholate, isomers can be differentiated.” This is of major importance in the study of biological extracts. Negative-ion FAB is the favoured ionization technique for the analysis of biological extracts as the accompanying lack of fragmentation allows pseudomolecular ions to be identified. Clearly if CID studies are carried out on three pseudomolecular ions, structural information concerning the compounds in a particular extract will be forthcoming. Certain conjugated bile acids are excreted in the urine of healthy humans, while others may be excreted by diseased patient^."-'^ Clearly, urine analaysis by negative-ion FAB/CID will allow the bile-acid content to be characterized and this will be beneficial in the diagnosis of diseased patients.
Acknowledgements W.J.G. wishes to thank the University of the West Indies for providing him with a ‘study and travel’ grant which partially financed
his visit to the Karolinska Institutet. This work was supported by the Swedish Medical Research Council (grant no. 03X-219).
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