2450
Anal. Chem. 1902, 5 4 , 2456-2461
P P R tRX ui,max
pressure, index 0 inlet; index L refers to outlet. average pressure. resolution, AtR/uti retention time of component x maximum obtainable response of a detector; upper index c or m refers to concentration or mass flow sensitive detector, respectively linear velocity at point z average velocity volume of a phase; index s or m refers to stationary or mobile phase specific area per unit column volume constant in eq 19 determined by the nature of the phase system and the component fraction of the column volume occupied by the gase phase pressure resistance factor capacity factor; index refers to component reduced velocity vdp/Dm;index min refers to minimum in h-v plot pressure ratio, po/pL. standard deviation of the peak, first index t (time), u (volume), or z (length), second index refers to component constant describing flow regime in injector
Lauer, H. H.; Poppe, H.; Huber, J. F. K. J. Chromatogr. 1977, 732,1. Peichang, L.; Liangmo, 2.; Chinghai, W.; Guanghua, W.; A h , X.; Fangbao, X. J. Chromatogr. 1979, 766, 25. Dandeneau, R.; Hawkes, S. Chromatographla 1080, 73,666. Lewis, D. A.; Vourous, P.; Karger, B. L. Chromatographia 1982, 75, 117. Gaspard, G.; Vidal-Madjar; Guiochon, G. Chromatographia 1982, 75, 125. Knox, J. H.; Saieem, M. J. Chromatogr. Sci. 1069, 7, 614. Giddings, J. C. Anal. Chem. 1863, 35. 1338, Knox, J. H.; Gilbert, M. T. J. Chromatogr. 1979, 186,421. Giddings, J. C. "Dynamics of Chromatography"; Marcel Dekker: New York, 1965; Part I. Purneii, H. "Gas Chromatography"; Wiley: New York, 1967. Khan, M. A. "Gas Chromatography Hamburg, 1962"; van Swaay, Ed.; Buttersworth, London, 1962; p 3. Jonker, R. J.; Poppe, H.; Huber, J. F. K. J. Chromatogr. 1079, 186, 311. de Jong, A. W. J.; Kraak, J. C.; Poppe, H.; Nooltgedacht, G. J. Chromafogr. 1080, 793,161. Haarhof, P. C.; van der Linde, H. J. Anal. Chem. 1968, 36,573. Desty, D. H.; Goldup, A.; Swanton, W. T. "Gas Chromatography 1961"; Brenner, N., et ai., Eds.; Academic Press: New York, 1962; p 105. Schettler, P. D.; Giddings, J. C. Anal. Chem. 1965, 37, 835. Struppe, H. 0. I n "Gas-Chromatographie 1961"; Schroder, M., Metzner, K., Eds.; Akademie-Veriag: Berlin, 1962; p 409. Giddings, J. C. Anal. Chem. 1867, 39, 1027. Karger, B. L.; Synder, L. R.; Eon, C. Anat. Chem. 1978, 52, 2126. Smit, H. C.; Walg, H. L. Chromatographia 1975, 8, 311. Littlewood, A. B. "Gas Chromatography"; Academic Press: New York, 1970.
LITERATURE CITED Smit, H. C. Chromatographia 1970, 3,515. Guiochon, 0. "Advances in Chromatography"; Giddhgs, J. C., Keller, R. A., Eds.; Marcel Dekker: New York, 1969; Vol. 6,p 179. Huber, J. F. K.; Lauer, H. H.; Poppe, H. J. Chromafogr. 1975, 122, 377.
RECEIVED for review May 12, 1982. Accepted September 3, 1982. Part of this work was supported by the Nederlandse Gasunie N.V.
Characterization of Glucuronides by Chemical Ionization Mass Spectrometry with Ammonia as Reagent Gas Thomas Cairns" and Emii G. Siegmund Department of Health and Human Services, Food and Drug Administration, Office of the Executive Director of Regional Operations, 7521 West Pic0 Boulevard, Los Angeles, California 90015
The ammonia chemical ionlration mass spectra of seven glucuronides have been determined by using the movlng belt LC interface for probe sample Introduction. Fragmentation proceeded vla cleavage of the glucuronic bond wlth the constituent aglycone moiety being identlfled as elther an adduct or protonated specles. Dominant In these spectra are a highly characterlstlc ion at m / r 194 corresponding to the ammonium adduct species wlth the glucuronlc portion of the molecule. Fragmentationpathways have been establlshed via the additional Informationfrom the use of ND, as reagent gas for determinatlonof exchangeable hydrogens. This technlque has proved to be convenient for introduclng lndlvldual samples to obtaln spectra resembling those reported using desorptlon chemical lonlratlon condltlons.
Perhaps the most severe restriction experienced by mass spectrometric techniques is the inability to deal consistently and effectivelywith thermally labile and polar molecules. The basic operational framework that the sample must be vaporized prior to ionization usually results in thermal degradation since the energy required for such evaporization is excessive. Modern mass spectroscopists, therefore, have been grappling with the problem of how to routinely analyze sig-
nificant biological molecules such as the class of drug conjugates known as glucuronides. Characterization for such glucuronides has been established by a wide variety of techniques such as negative ion desorption chemical ionization ( I ) , derivatization followed by E1 or CIMS (2), "in beam" CI (3))field desorption (4) and the use of pyridine as reagent gas (5). More recently, the application of fast atom bombardment (FAB) mass spectrometry has been reported (6) in the successful analyses of nonvolatile and/or thermally labile organic molecules such as polypeptides, antibiotics, and glucosinalates. All these attempts have indeed advanced the characterization process of glucuronides by observing molecular weight information, but such diverse techniques are time-consuming and highly technical in nature and are not always commonly available to the majority of biochemists and analytical chemists. The recent availability of the liquid chromatography moving belt interface as a superior sample introduction probe especially with regards to thermal degradation (7) has made the use of chemical ionization with ammonia as reagent gas a less complex technique for potential characterization of underivatized glucuronides (8). The recent reported success achieved by this experimental approach (9) in the characterization of an important glycoside, amygdalin, encouraged study extrapolation to certain model glucuronides as well as several key estrogenic glucuronides.
This article not subject to US. Copyrlght. Published 1962 by the American Chemical Soclety
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ANALYTICAL CHEMISTRY, VOL. 54, NO. 14, DECEMBER 1982
This paper describes the application of this experimental technique to the charact,erization of glucuronic acid (I),p nitrophenol glucuronide (II), phenolphthalein glucuronide (III),estriol 17@-glucuromide[E3-17G] (IV), estriol 16a-glucuronide [E3-16G](V), estrone glucuronide [E,-G] (VI), and estradiol 17P-glucuronide [E2-17G](VII). Additionally, the employment of deuterated ammonia (ND,) in parallel studies as reagent gas has permitted more detailed fragmentation pathways to be postulated. Such studies have provided a unique and predictive insight into the general mode of fragmentation of such molecules under these preselected conditions. The importance of substitution by NHS has played a key role in clearly delineating the glucuronic character of these molecules. HO~OH
H@H
~
150
i
~
1
(NH31 168
~
COOH
0
2
mi=
OH
H
COOH
(111) QH
Figure 1. Chemical ionization mass spectra of glucuronic acid (I): (A) NHJas reagent gas at a flash vaporization temperature of 200 OC, and (8) ND, as reagent gas at a flash vaporization temperature of 220 OC.
tsrface unit. Samples (100-500 ng dissolved in water/methanol) were directly deposited onto the moving belt interface (2.25 cm/s). Kapton belts were used with a flash vaporization temperature of between 200 O C and 220 O C , a CI source temperature of 180 " C , and a source pressure of 0.8 torr (adjusted to maximize the intensity of the [NH4]+ion at m / z 18). Samples. Glucuronides(I, II,and 111)were commercial samples from Sigma Chemical Co. and were used without further purification. The estrogen glucuronides (IV, V, VI, and VII) were kindly provided by William Slikker of the National Center for Toxicological Research (10). RESULTS AND DISCUSSION Glucuronic Acid (I). Figure 1illustrates the mass spectra obtained for glucuronic acid using both NH3 and ND, as reagent gases. In the case of NHS (Figure 1A) the appearance of an ion at m / z 194 corresponded empirically to [M NH4 - HzO]+. The observed mass shift of 7 daltons of this ion to m / z 201 when ND3 was used as reagent gas (Figure 1B) indicated that the total number of acidic hydrogens exchanged for deuterium together with those present from the reagent gas was seven. If the ion m / z 194 did correspond to [M + NH4 - H20]+,then the anticipated number of deuterium atoms in that dehydrated adduct ion would be seven. Maquestiau et al. (11) have recently studied the structure of ammonium adduct ions using mass analyzed ion kinetic energy spectroscopy and concluded that for ketones, the [M + 18]+ is a carbinolamine ion and not a shared proton ion. Subsequent loss of water from this carbinolamine would then yield the ion [M + NH4 - HzO]+. In the chemical ionization of cyclohexanone with [NH,]+ a protonated imine was shown to be a major product of the ion/molecule reaction via an intermediate carbinolamine (12). In a study of factors affecting reactivity in ammonia CI reactions by Keough and DeStefano (13),experimentalwork was interpreted to support a hydroxyl group replacement mechanism involving a transition state in the formation of the [M + NH4 - HzO]+ions. These authors observed that the production of [M NH4]+ ions was more dependent on NHBpressure than the [M NH4 - HzO]+ ions and hence concluded that a transition state involving collision with NH4+was mainly responsible for the resultant substitution ion. While the variation in relative abundances of [M NH4]+to [M + NH4 - HzO]+using benzyl alcohol as model compound did demonstrate a linear relation as a function of ammonia pressure, the interpretation of the data in verifying that a single collision with NH4+ was responsible for the substitution ion cannot be viewed as conclusive proof. The observed decrease in the relative abundance of [M^+ NH4 - HzO]+ as the source pressure increased is
+
'
OH
(VI) (VII) The main objective of this study protocol was not to determine lower detection lev& but rather to establish structural information from samples within the concentration range of 100 to 500 ng. Such data frlom a detailed structural elucidation study could then provide a predictive insight into the selection of suitable ions for single ion monitoring experiments when low level detection was mandated for exploratory work on biological extracts. EXPERIMENTAL SECTION Apparatus. All spectra were obtained on a Finnigan Model 3300 quadrupole mass spectrometerequipped with a CI source, Model 6000 data system and Finnigan LCMS moving belt in-
+
+
+
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ANALYTICAL CHEMISTRY, VOL. 54, NO. 14, DECEMBER 1982
puzzling if indeed a single collision transition state mechanism is operative. The argument advanced that the increased relative abundance of [M + NH4]+is mainly due to collisional stabilization should be viewed with suspicion. However, such a marked decrease in relative abundance of the [M NH, - HzO]+ion is an indication that a stepwise mechanism via the collision of a neutral NH3 and the [MH - H20]+ion cannot be supported. Lin and Smith (14) have also studied the [M + NH4 - OH]+ ions produced in the ammonia CI spectra of a group of steroidal alcohols and concluded that nucleophilic substitution was responsible. It would seem reasonable that these mechanisms could explain the present experimental observations. Which actual route is involved is not yet certain and cannot be derived from the present experimental evidence. However, another reaction route might involve the formation of an [MH - HzO]+ion to give an intermediate carbonium ion (the elimination ion) which can undergo nucleophilic attack by NH3. Keough and DeStefano (13) had already discounted a similar reaction mechanism from data collected at various source pressures. However, the model compounds in their studies did not belong to the hydroxy acid class and the real possibility of a different reaction mechanism could exist. In support of such a different reaction mechanism, some published results have already provided added support to the bimolecular collision mechanism as being responsible for substitution ions in NH, CIMS studies. Since the appearance of the ion at m / z 194 is prevalent in the spectra of all seven glucuronides studied, initial fragmentation at the glucuronic bond would produce a carbonium ion similar to that via protonation and loss of water. An analogous ion of this general structural type, Le., [MH - HzO]+,has recently been shown by Suzuki et al. (15)to occur in the ammonia CI spectra of certain aminoglyoside antibiotics via cleavage of the glycosidic bonds. Resonance stabilization to the corresponding oxonium ions was suggested. Tabet et al. (16)have concluded that an SN2substitution involving a Walden inversion is primarily responsible for the formation of the ion [M NH, - HzO]+ in two pairs of diastereoisomeric unsaturated tertiary alcohols. The importance of neigboring group participation via hydrogen bonding in stabilizing the quasi-molecular ion was found to be highly dependent on the spatial arrangement of the surrounding electronegative centers. Establishment of this S N 2 mechanism has added weight to the possible prevalence of nucleophilic substitution particularly with polyhydroxy compounds. More recently, the report by Djerassi et al. ( 1 7 ) on the NH3 DCI spectra of several epimeric 3hydroxy steroids (and their ether and carboxylic acid ester derivatives) has been rationalized as a demonstration of an S N 1 type reaction in the formation of the substitution ion [M - OR NH3]+ via an intermediate elimination ion species [MH - HOR]'. We believe the mechanism to be operating in the case of glucuronic acid to be similar. Formation of the ion at m / z 194 could be formed via the [MH - H20]+ion to yield an intermediate carbonium ion which could then undergo attack by NH3 (Scheme I). The alternate and more probable route of formation is via the carbinolamine. Although this proposed reaction scheme fits the empirical data, critical additional experiments using ion kinetic energy spectrometry should be performed to validate this speculation. Therefore, for future discussion purposes in this paper the structure of the ion at m / z 194 has been drawn as illustrated in Scheme I and could be derived via the carbinolamine route or by the postulated substitution mechanism. The intermediate carbonium ion produced by loss of OH could resonance stabilize via formationof the corresponding oxonium ion using the ether oxygen of the ring. A similar reaction has already been observed (9) for a disaccharideglycoside under similar conditions where primary fragmentation at the central glycosidic bonds
+
+
+
Scheme I. Fragmentation Pathway for Glucuronic Acid (I) [MH
OH
- H20]+
[M
COOH
coon
OH
OH
Ho+2
* NHq -
H20]+
COOH
- H20
formed a carbonium ion which was then subjected to substitution by NH3. In order to explain the spectra recorded with ND3as reagent gas the other ions observed [mlz 168,150,132 and 1151 must represent open chain structures (Scheme I) to account for the deuterium exchange patterns experimentally observed. Indeed the retention of the primary adduct ion species is worthy of comment. The adduct ion has been observed to lose both water and carbon dioxide rather than NH3. From these data it could be assumed that the site of adduct formation is not any single selected atom or bond but the "pseudo" chelating influence exercised by the multiple hydroxyl functions in the molecule despite decarboxylation and successive dehydrations. The detailed attention paid to glucuronic acid was thought to be paramount in fully understanding the spectra to be obtained from glucuronides. The spectra obtained for glucuronic acid were found to be extremely temperature sensitive. Nucleophilic collision with ammonia was favored at the higher temperature of 220 OC (Figure 1B) relative to 200 "C (Figure 1A). In fact the higher temperature of 220 "C also caused the predictable marked increase in dehydration, Le., base peak observed was m/z 119, corresponding to m / z 115 when NH3 was employed as reagent gas. Similar trends have been reported by Cotter and Fenselau (18)by observance of the protonated molecular ion abundance of p-nitrophenol glucuronide at various source temperatures using direct exposure CIMS. Furthermore, the effect of varying the flash vaporization temperature did not produce a competition between protonation and adduct formation. p-Nitrophenol Glucuronide (11). The choice of p nitrophenol glucuronide as a model compound for study was made on the basis of the attention it had already received in the literature (1,18). This molecule represents a low molecular weight member of the glucuronide class and thus the results might permit a general preview of the expected fragmentation pathway to be encountered in much larger molecules. Figure 2 illustrates the mass spectra obtained for p-nitrophenol glucuronide using both NH3 and ND3 as reagent gases. The presence of a low abundance ion at m / z 315 (Figure 2A) corresponded to the ion [M + NH, - H,O]+. Unfortunately, the exact character of this ion could not be formulated since no counterpart ion was observed when ND3 was employed as reagent gas. Two main ions were observed, mlz 157 and m l z 194. The assignment of mlz 157 to an adduct ion with p nitrophenol (Scheme 11) was established by observing a 5 dalton shift to m/z 162 when ND3 was used (Figure 2B): four deuteriums present as ND4and the remaining as an exchanged hydroxyl hydrogen. The possible nature of the structure corresponding to mlz 194 ion (Figure 2A) has already been outlined in the discussion for glucuronic acid. To explain the spectra observed for p-nitrophenol glucuronide, the characteristic feature of the primary fragmentation of the glucuronic
ANALYTICAL CHEMISTRY, VOL. 54, NO. 14, DECEMBER 1982
2459
coon
I L.-
m/z
icb
Flgure 2. Chemical ionization mass spectra (of p -nitrophenol glucuronide (11): (A) NH, as magent gas at a flash vaporization temperature of 200 O C , and (B) ND3 as reagent gas et a flash vaporization temperature of 220 O C .
Scheme 11. Fragmentation Pathway for p-Nitrophenol Glucuronide (11)
'
An
'
An
______I_5c
r-
zoo
2%
mlz
Figure 3. Chemlcal ionization mass spectra of phenolphthalein glucuronide (111): (A) NH, as reagent gas at a flash vaporization temperature of 200 O C , and (B) ND, as reagent gas at a flash vaporization
temperature of 200 O C .
Scheme 111. Fragmentation Pathway for Phenolphthalein Glucuronide (111)
I
acid bond results in the production of the neutral aglycone moiety, p-nitrophenol, and an intermediate carbonium ion, a glucuronic acid entity. The aglycone moiety is then ionized via adduct formation to give an [aglycone + NH4]+ion, Le., mlz 157, while the postulated glucuronic acid intermediate carbonium ion collides with a neutral ammonia to give m / z 194 (Scheme 11). Games (8) had already observed similar ions in the characterization of 2-naphthyl-P-~-glucuronide by LCMS-CI-NH3. This iclnization process has provided ions which have structural elucidation significance. In spite of the absence of molecular weight evidence, the chemical character of the molecule can be deduced by the presence of the ion at m/z 194, Le., glucuronide. The nature of the aglycone portion can then be extrapolateld by turning attention to mlz 157 which represents the molecular weight of the aglycone plus NH4. Parallel studies using ND3 can further establish the number of acidic hydrogens in the aglycone by deuterium exchange. Under negative ion CI (1)the characterization of p-nitrophenol glucuronide was definitely accomplished by the appearance of a molecular anion [MI-. at m / z 315 and a fragment ion at mlz 139 corresponding to [p-nitrophenol]--. Clearly both NICI and ammonia CI cause the same bond cleavage between the glucuronic acid and the aglycone. However, under ammonia CI conditions this aglycone moiety has the opportunity to pick up a proton to yield the neutral aglycone which can then tiiibsequently undergo ionization by NH4+to form an adduct species. The structural advantages gained by using ammonia CI is the appearance of the ion a t m / z 194. The marked appearance of this ion clearly establishes the compound under investigation as a glucuronide. It would seem that sample introduction via the moving belt interface which minimizes thermal decomposition (19) followed by ionization with NH4+can provide an alternate ap-
proach to characterization. To put this theory to the test another model glucuronide of higher molecular weight was examined. Phenolphthalein Glucuronide (111). The spectra recorded for phenolphthalein glucuronide are illustrated in Figure 3. If the primary fragmentation pathway observed for p-nitrophenol glucuronide was to be operative, then the presence of mlz 194 (Figure 3A) with a 7 dalton shift to m / z 210 (Figure 3B)must be evident. Such is the case. The second half of the argument advanced for p-nitrophenol glucuronide remained to be satisfied. The appearance of an ion at mlz 319 should correspond to an adduct species between the aglycone moiety and NH4+. However, the total number of deuterium atoms in the corresponding ion (mlz 322) when N D 3 was used was only three. To explain this ion, the aglycone moiety must undergo protonation (Scheme 111)and not adduct formation with NH,+. The remaining two deuteriums can be accommodated by exchange phenomenon with the two hydroxyl hydrogens. Once again the main fragmentation pathway using ammonia as reagent gas has been cleavage of the glucuronic bond with subsequent release of the neutral aglycone for collision with NH4+. For determination of the molecular weight of the aglycone, however, the additional parallel experiment with ND3 is essential to indicate either adduct formation or simple protonation. From the data obtained from these two model glucuronides it was now thought that extrapolationto several key estrogenic glucuronides would provide further insight into the postulated
2480
ANALYTICAL CHEMISTRY, VOL. 54, NO. 14, DECEMBER 1982 i
1106
I
*
. a
Scheme V. Fragmentation Pathway for Estrone Glucuronide (VI)
194
I
11s
I1
.E
a C
mi.
Figure 4. Ammonia chemical ionization mass spectra of estrogen glucuronides (flash vaporization temperature, 200 "C): (A) estriol 17/3-glucuronlde (IV); (6) estriol 16a-glucuronide (V); (C) estrone glucuronide (VI): and (D)estradiol 17/3-gIucuronlde (VI I).
Scheme IV. Fragmentation Pathway for Estriol 170-Glucuronide (IV)
OH
/+-i
a p+ [E3
+
+
NHll
predictiveness of the fragmentationpathway already exhibited. Estrogen Glucuronides (IV, V, VI, and VII). The ammonia CI spectra of four steroidal glucuronides are compiled in Figure 4. In all four compounds examined the presence of an ion at m / z 194 was evident, although somewhat weak in the spectra of [El-GI (Figure 4C) and [E2-17G](Figure 4D). In parallel studies with ND3 as reagent gas, a 7 dalton shift was observed correspondingto the assignment already placed on this ion with the glucuronic acid discussed above. However, the ions produced by the aglycone moieties deserve individual attention. There was no evidence of molecular weight information in the spectrum of this compound (Figure 4A). However, the presence of an intense ion at m/z 306 corresponded to the NH4 adduct ion with the constituent aglycone, estriol [E3] (Scheme IV). Confirmation of this fact was provided by a 7 dalton shift (Le., shift from m / z 306 to m/z 313) when ND3 was used as reagent gas. The assignments of the remaining ions to [E3H]+at m / z 289, [E3 - H]+ at m/z 287, and [E3H - H20]+at m / z 271 (Scheme IV) were confirmed by observing correct mass shifts when ND3 was employed (mass shifts of 4,2, and 2 daltons, respectively). This fragmentation pattern is consistent with that observed with p-nitrophenol glucuronide. For characterization purposes, the additional ions produced increase the effectiveness of the
mechanism of identification for this particular glucuronide. It is interesting to note, however, that the ion intensity corresponding to the protonated aglycone [E3H]+at m / z 289 is small compared to the adduct species, [E3 + NH4]+at m / z 306. This situation must reflect the competition between adduct, protonation, proton abstraction, and dehydration processes for the neutral aglycone, [E3]. The presence of the base peak at m / z 306 clearly proves that adduct formation with NH4+is highly favored relative to the other processes. Perhaps the presence of the diol grouping at C-16,17 assists in the preferred adduct formation by providing a larger cross-sectional area for attachment for NH4+ (16). E3-16G. The same arguments advanced for [E3-17G]apply to the observed spectrum for [E3-16G](Figure 4B) and the resultant fragmentation pathway. However, the presence of a protonated molecular ion at m / z 465 (confirmed by a 7 dalton shift when ND3was used) deserves special commentary. Both [E3-17G]and [E3-16G]have the same molecular weight and differ only in the site of the glucuronic acid grouping. From the biochemical standpoint it would be advantageous to be able to distinguish these isomers. The only spectral difference observed was the presence of a protonated molecular ion in the case of [E3-16Gl. This difference could be employed to suggest that the 16a-glucuronide is present. A plausible explanation for the observance of a protonated molecular ion in the case of [E3-16G] may be the stereochemical arrangement at C-16,17. In the case of [E,-17G] the glucuronide is in the plane above the steroid nucleus while in [E3-16G] the reverse is true. It could be postulated that when the glucuronide is a to the steroid ring system this stereochemical arrangement permits protonation. With the availability of other steroidal glucuronides of similar structural significance this observation could be tested. Another minor difference in the fragmentation pathway of [E3-16G]relative to [E3-17G]is the lack of an ion corresponding to [E3- H]+ at m / z 287. Once again the stereochemical arrangement at C-16,17 could influence this situation. This characterization of both estriol conjugates is a convincing display of the power of ammonia CI in dealing with polyhydroxy compounds. However, the ability to differentiate these two isomers is not yet fully established. Until the trend for a-substituted glucuronides can be observed with other compounds, the postulated differences should be cautiously viewed. El-G. The mass spectrum of this particular glucuronide is illustrated in Figure 4C. In this case the neutral aglycone produced by fission of the glucuronic link is estrone [E,] (Scheme V). Adduct formation with NH4+is clearly evident by the appearance of the ion at m / z 288 (5 dalton shift when ND3 was used). This behavior is consistent with that already
ANALYTICAL CHEMISTRY, VOL. 54, NO. 14, DECEMBER 1982
Scheme VI. Fragmentation Pathway far Estradiol 17p-Glucuronide (VII)
COOH
observed for the model glucuronides and two estriol conjugates examined above. However, the appearance of an ion at m / z 271 corresponding to the protonated aglycone (2 dalton shift observed when ND3 used) indicates that the competition between protonation and adduct formation is about even. Molecular weight evidence is available by the appearance of a protonated molecular ion at m/z 447 (5 dalton shift when ND3 used). The overall spectral profile displayed by [E,-G] is highly characteristic of its structural identity and differs sufficiently that a distinction can be made from estriol conjugates. Ez-17G. The mass spectrum for this particular glucuronide is displayed in Figure 41).The low relative abundances of the ions required for characterization are disappointing. Admittedly the presence of (anion at m/z 273 (3 dalton shift when using ND3 as reagent gan) that corresponds to the protonated aglycone, estradiol [E,], is in keeping with the trend already established above for its sister compounds. The ions at m / z 255 (1dalton shift observed when ND3 was used) (Scheme VI) correspond to the loss of water from the protonated aglycone [E,H]+.
CONCLUSIONS With the limited data acquired by this study of glucuronic acid (I),two model glucuronides (I1 and 111),and four estrogen glucuronides (IV, V, VI, and VII) a number of characteristic trends can be predicted for future structural elucidation. (1) The appearance of an ion at m / z 1941is strong evidence that the compound under investigation is a glucuronide. (2) When dealing with glucuronides the presence of a strong ion corresponding to the protonated or adducted neutral constituent aglycone is usually present.
2461
(3) In steroidal glucuronides, molecular weight evidence can sometimes be observed, particularly if the stereochemical situation is optimal (Le., 16a-glucuronide). In other cases where the glucuronide grouping is 17p no molecular species will be observed. (4)Loss of water from the protonated neutral constituent aglycone can often be observed. Without the extensive use of ND3 as an additional reagent gas many of the fragmentation pathways could not have been enunciated or postulated. However, this preliminary characterization process of estrogen glucuronides should permit intelligent monitoring of biochemical matrices via LCMS. The combination of the moving belt interface for sample introduction (as a probe) with ammonia CI has proved an invaluable tool for structural characterization of such glucuronides. This technique offers a viable alternative for identification of such compounds. The ability of ammonia gas to perform nucleophilic substitution on the key carbonium ion formed from cleavage of the glucuronic acid linkage adds a new dimension to the characterization process and has been found to be beneficial in the primary assignment of a compound belonging to the glucuronide class.
LITERATURE CITED (1) Brulns, A. P. Blomed. Mass Spectrom. 1981, 8 , 31. (2) Lyle, M. A.; Pallante, S.;Head, K.; Fenselau, C. Homed. Mass Soectrom. 1979, 4 , 190. (3) Cotter, R. J. Anal. Chem. 1979, 57,317. (4) Fenselau, C.: Cotter, R. J., Johnson, L. Adv. Mass Specfrom. 1980, 8. 1159. (5) Johnson, L. P.; Subba Rao, S. C.; Fenselau, C. Anal. Chem. 1978, 50, 2022. (6) Barber, M.; Bordoll, R. S.; Sedgwick, R. D.; Tyler, A. N.; Whalley, E. T. Blomed. Mass Specfrom. 1981, 8 , 337. (7) Calrns, T.; Slegmund, E. G.; Doose, G. M. Anal. Chem. 1982, 5 4 , 953. (8) Games, D. E., Lewis, E. Homed. Mass Spectrom. 1980, 7 , 433. (9) Cairns, T.; Slegmund, E. G. Blomed. Mass Specfrom. 1982, 9 , 307. (IO) Meyers, M.; Slikker, W.; Pascoe, G.; Vore, M. J . Pharmacol. Exp. Ther. 1980, 214, 87. (11) Maquestlau, A.; Flammang, R.; Nlelsen, L. Org. Mass Spectrom. 1980, 15, 376. (12) Tabet, J. C.;Fralsse, D. Org. Mass Spectrom. 1981, 76,45. (13) Keough, T.; DeStefano, A. J. Org. Mass Spectrom. 1981, 76,527. (14) CLn, Y. Y.; Smith, L. L. Homed. Mass Spectrom. 1978, 5 , 604. (15) Takeda, N.; Harada, K.; Suzuki, M.; Tatemattsu, A.; Kubodera, T. Org. Mass Specfrom. 1982, 77, 247. (16) Bastard, J.; Do Khac Manh, 0.; Fetizon, M.; Tabet, J. C.; Fraisse, D. J . Chem. SOC.,Perkln Trans. 2 1981, 1591. (17) Tecon, P.; Hirano, Y.; Djerassi, C. Org. Mass Specfrom. 1982, 77, 277. (18) Cotter, R. J.; Fenselau, C. Blomed. Mass Spectrom. 1979, 6 , 287. (19) Cairns, T.; Siegmund, E. G.; Doose, G. M. Homed. Mass Spectrom., in press.
RECEIVED for review November 24,1981. Resubmitted June 18, 1982. Accepted September 10, 1982.
