272
ANALYTICAL CHEMISTRY, VOL. 51, NO. 2, FEBRUARY 1979
Hydroxyl Ion Negative Chemical Ionization Mass Spectra of Steroids T. A. Roy and F. H. Field" The Rockefeller University, New York, New York 10021
Yong Yeng Lin and Leland L. Smith The University of Texas Medical Branch, Galveston, Texas 77550
The OH- negative chemical ionization spectra of 35 steroids have been investigated. Spectra are dependent upon the nature, number, and proximity of the functional groups in the several compounds. The predominant reaction observed is proton abstraction, and (M - 1)- is found for all but two compounds. Other reactions involve nucleophilic attack by OH- on the sample molecule and nucleophilic attack by (M - 1)- on N,O. These processes are frequently accompanied by loss of H, and H20, which sometimes aids in determining the nature and environment of substituents in the compounds. The amount of the fragmentation of the molecules is relatively small. The overall sensitivity of the method is somewhat greater than that for CH, positive chemical ionization with the group of steroids investigated.
In this paper we report on the mass spectral behavior of 35 steroids under OH- negative chemical ionization (NCI) conditions. Smit and Field ( I ) have described the formation of OH- by the electron bombardment of a mixture of N 2 0 and CH4 and the use of OH- as a reactant ion in NCI. Their study showed that reaction of OH- with a variety of organic compounds was quite mild, and, in most cases, abundant (M 1)-ions were formed. We refer the reader to their report ( I ) for a detailed discussion on the formation of OH- and a brief review of negative ion publications. T h e present investigation has attempted to provide information on OH- negative ion spectra which might serve as the basis of a n analytical method for an important group of biomolecules. The ammonia, methane, and isobutane positive chemical ionization (PCI) spectra of a similar group of steroids have been determined by Lin and Smith (2).
EXPERIMENTAL The steroids examined were commercial samples from Steraloids Inc., Wilton, N.H., or Research Plus Steroid Laboratories, Inc., Denville, N.J., or were prepared in our laboratories. All samples were analyzed by thin-layer and gas chromatography and, in some instances, by high pressure liquid chromatography. Mass spectra were obtained with a Biospect mass spectrometer manufactured by Scientific Research Instrument Corp., Baltimore, Md. The instrument was modified for negative ion detection using the negative ion detection system developed by Smit, Rosseto, and Field ( 3 ) . All samples (1-2 pg) were introduced by means of the direct insertion probe. The spectra were recorded on an Infotronics Corp. (Houston, Tex.) CRS 160 mass spectrometer digital readout system. Ionization of the reaction mixture was effected by electrons emitted from a rhenium filament. The electron accelerating voltage was adjusted to give maximum ion intensity, generally about 450 V in the OH- NCI experiments and about 250 V in some CHI PCI experiments. The source pressure was maintained at 3.0 Torr (2/1 mixture of CH,/N,O) in the OHNCI experiments and 1.0 Torr in the CH4 PCI experiments. The source temperature was maintained at 200 "C in all experiments. The reader is referred to the study of Smit and Field ( I ) for a 0003-2700/79/035 1-0272$0 1.OO/O
detailed description of the effects of temperature, pressure, and reactant gas ratio on the reactant ion spectrum.
RESULTS G e n e r a l Reactions. We will first discuss the general characteristics of the spectra and later turn our attention to a more detailed discussion of individual spectra. Table I lists the spectra obtained for 30 of the steroids investigated. The spectra of the five cholesteryl esters studied are given in Table 11. As is well known, the nature of the tuning of a quadrupole mass spectrometer markedly affects the discrimination properties of the instrument. As best we could determine, the spectra of the compounds in Table I were obtained under non-mass discriminating conditions, which are relatively easy to achieve because the spectra do not span a wide mass range. The spectra of the compounds in Table I1 do span a large mass range, and for these the instrument was tuned so as not to discriminate against the high mass ions and perhaps to emphasize them somewhat. The predominant reaction for all these compounds is proton abstraction to form the (M - 1)- ion. We assume this abstraction involves hydrogens attached to or adjacent to groups or atoms which lend some degree of stability to the resulting anion, e.g., oxygen, carbon-carbon double bonds, etc. T h e relative intensity of the (M - 1)-ion varies from 100% in the spectrum of the saturated ketone, 5a-cholestan-3-one (4) to 0% in the spectra of cholesteryl benzoate (32) and Sa-cholestane (1). The numbers in parentheses following the names of the steroids refer to the numbers identifying the compounds in Tables I and 11. Following t h e initial reference t o a compound, we will normally refer to it by its assigned number only. An (M - 3)- ion is observed in t h e spectra of a number of the compounds, particularly those containing the hydroxyl group or the carbon-carbon double bond. We postulate that the formation of this ion involves loss of H2 following initial proton abstraction. In the case of alcohols, this can produce a resonance stabilized enolate ion. Thus, in 5a-cholestan-3P-01 (12), for example,
Obviously, loss of H2 can occur across the 2,3 C-C bond also. T h e presence of the (M - 3)-ion in alkenes, dienes, etc., also probably involves loss of H2 from an initially formed (M - 1)ion. Abstraction of an allylic hydrogen can generate a n allylic carbanion as (M - 1)-, and subsequent loss of H2 serves to lower the energy of the ion by extending the conjugated system and further delocalizing the negative change. Thus, in cholest-5-ene (a), for example,
An (M - 5)- ion is also sometimes observed with these types C 1979 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 51, NO. 2, FEBRUARY 1979
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ANALYTICAL CHEMISTRY, VOL. 51,
NO. 2 ,
FEBRUARY 1979
Table 11. OH- NCI Spectra‘ of Several Cholesteryl Esters (XOOCR)b mass and relative intensities m / e ( R I )
no. 31 32 33
34 35 (I
Same
compound mol. wt. cholesteryl 428 acetate cholesteryl 490 benzoate ch olesteryl 624 palmitate cholesteryl 650 oleate cholesteryl 652 stearate conditions as in Table I. X
(M
(RCO0)59(37)
- 1)427(53)
(RCOOH,O)-
(XOH2O)367( 5 )
(XO H,O H2
(XO H,O 2H, )-
I-
365(5)
121(100)
=
623( 3 9 )
25 5( 47 )
365(4)
649(63)
281(28)
263( 4 )
367(5)
651(53)
283(39)
265(3)
367(5)
363(3)
cholesteryl group (C2-H45).
of compounds, and the ion is almost surely formed by the same kind of H2loss process as is involved in (M - 3)- ion formation. An (M - 19)- ion is observed, to some degree, for all 22 steroids in Table I which contain a t least one hydroxyl group. We believe t h a t this ion is formed by the elimination of the elements of water following the initial proton abstraction. In those cases where the (M - 1)-is the alkoxide, a resonance stabilized allylic anion can be formed, and we suggest as an illustrative possibility
The (M + 43)- ion is found in the spectra of the saturated monoalcohols and some of the carbon-carbon unsaturated steroids. The (M + 25)- and (M + 15)- ions are only found in the spectra of the compounds containing carbon-carbon unsaturation. The (M - 1)- nucleophile is presumably the alkoxide ion in the case of the saturated monoalcohols and the allylic carbanion in the case of the carbon-carbon double bond containing compounds. The (M 43)- and (M + 25)ions have been reported by Smit and Field ( I ) , who proposed that the ions represented the species (M - H + N20)- and (M H + N 2 0 - H,O)-, respectively. A recent study ( 4 ) has indicated that reaction of N 2 0 with various organic ions in a flowing afterglow system may be characterized as addition of N 2 0 followed by loss of H20,N2,or CH20. We suggest that the (M + 15)- observed in the present study represents the species (M H + N 2 0 - N2)-. T h e mechanism proposed ( I ) to explain the loss of H 2 0 from the (M + 43)- ion was based on initial nucleophilic attack of the (M - 1)-ion a t the central atom of N 2 0 . Both of the principal resonance structures of N 2 0 assign a positive charge to the central atom, and this would seem a logical point of attachment. However, work by Dawson and Nibbering ( 5 )using labeled N 2 0 indicates that attachment actually takes place a t the terminal nitrogen, a t least when CH2=C-. is the nucleophile. Revising the mechanism for elimination of H 2 0 from ( M - H + N20)- is not complicated when this point of attachment is used. T h e (M + 43)- ion formed with a simple alkene can lose water and cyclize to form a pyrazole anion
+
~
H
U
H
(3)
An (M - 21)- ion is often observed in the spectra of the alcohols
as well, and we presume that this represents the loss of both H2 a n d H 2 0 from the (M - 1)- ion in such a way as to lower the energy of the system. T h e (M 37)- ion observed in the spectra of all the diols and two triols in Table I doubtless results from the loss of a second water molecule. The energy and intensity of this ion may be related to the position of the hydroxyl groups in the molecule. Generally, where elimination of a second molecule of water will serve to lower the energy of an ion by increasing resonance (electron delocalization) we observe relatively large (M - 37)- ions. Specific examples of these observations will be discussed later. Another general type of reaction observed between the OHion and the cholesteryl esters, specifically, appears to involve nucleophilic attack by OH-. T h e major pathway for nucleophilic attack by OH- involves cholesteryl-oxygen bond cleavage. This results in the formation of the conjugated base of the acid moiety ~
R$-o-R’+
OH-
-+
RC-O-+
~
-
-
N=N-O-
+
&
+
3
N I N - 0 - 4
HOR’
‘d
(4) This is a departure from condensed phase chemistry where nucleophilic attack by OH- occurs at the carbonyl carbon of the ester, which results in acyl-oxygen bond cleavage. 0
Assuming the same initial point of attachment of N20, we tentatively suggest the following mechanism for the elimination of N2 from the (M + 43)- ion f.--P-.
Small ions (re1 int E 5%) are observed in the spectra of the cholesteryl esters at m l e 367 and 365 and are presumably derived via water loss from the cholest-5-enoxide ion which would be the result of conventional OH- attack at the carbonyl carbon of the ester. These observations with the cholesteryl esters are in accord with the findings on esters in Smit and Field’s study ( 1 ) . Still another reaction type observed is that involved in the formation of the ions (M 43)-, (M + 25)-, and (M + 15)-. This reaction appears t o be a nucleophilic attack of an (M 1)- ion, produced from the sample, on the N 2 0 reactant gas.
+
+
+
NEN-O
j
(7)
The relative intensity of this group of ions, which we shall generally refer to as the (M + n)- ions, is believed ( I ) to be related to the energy of the (M - 1)- ions. We observe t h a t the introduction of additional functional groups or elements into compounds containing carbon-carbon unsaturation greatly reduces the intensity of the (M + n)- ions. We attribute this to a lowering of the energies of the (M 1)- ions ~
ANALYTICAL CHEMISTRY, VOL. 51, NO. 2, FEBRUARY 1979
by the presence of the substituents. OH- NCI Spectra of Individual Steroids. 50-Cholestane (1)is the only saturated hydrocarbon steroid included in this study, and it was not expected to show much reactivity since it was previously found ( I ) t h a t alkanes did not react with OH-. A comparison of the total ion current attributed to the sample for 50-cholestane and for several monofunctional compounds (the monoketone (4), the monoalcohol (13) and the alkene (2)) revealed that 50-cholestane was some ' / a o - ' / j o as reactive as the functionalized compounds. The ions in the spectrum of (1)present a pattern similar to that observed in the spectrum of cholest-5-ene (2), and they may result from thermal dehydrogenation of (1) in the source followed by ionization as described below for (2). Cholest-5-ene (2) and cholesta-3,5-diene (3) are the only unsaturated hydrocarbons included in Table I. The (M - 1)in the monoolefin (2) is quite weak; most of the ionization observed involves loss of hydrogen from (M- 1)- or reaction of (M - 1)-with N20. As discussed in the previous section, we infer t h a t the initial (M - 1)-in monoolefins like (2) is of high energy and reacts further to form a lower energy ion. By contrast, t h e introduction of a second double bond in conjugation with the first as in the diene (3) apparently allows for the formation of a low energy (M - 1)-ion, which does not appreciably react further (re1 int = 70%). A significant amount of hydrogen loss is observed in both (2) and (3), but the relative intensity of the (M n ) - ions in (3) is just 3% as compared to a value of nearly 50% in (2). The higher total reactivity in (2) reflects the higher energy of the (M 1)- ion in (2). (M n)- ions are also found in the spectrum of 5acholest-7-en-3-one(6) but not in the spectrum of a positional isomer, cholest-4-en-3-one (5). The two functional groups in the enone (6) are not in conjugation, and the spectrum contains ions that can be associated with each of the groups. T h u s the spectrum displays a n intense (M - 1)-ion (re1 int 71%) typical of a saturated monoketone (e.g., sa-cholestan-3-one (4)) and (M + n)- ions (re1 int 1270) such as those observed with the alkene (2). On the other hand, the carbonyl and carbon-carbon double bond in the enone ( 5 ) are conjugated and the spectrum is dominated by the (M- 1)ion (re1 int E 90%) and completely devoid of (M+ n)-ions. Here again we suggest t h a t the lack of (M + n)- ions in (5) is related to the relatively low energy of the (M- 1)-in that the removal of an allylic hydrogen produces a highly conjugated anion. Thus,
+
-
+
Cholest-3,5-dien-7-one( 7 ) and cholest-4-en-3,6-dione (8) are both highly conjugated, and the spectra display intense (M - 1)-ions and small or zero intensities of (M + n)- ions. The spectra are analogous t o those of the a,@-unsaturatedketone (5). T h e spectra of several of the steroids are of interest since they may provide information on the relative acidity of the hydrogens associated with the carbonyl and the hydroxyl group. We noted above t h a t the spectrum of the saturated ketone (4) displays only one ion, the (M - 1)-. On the other hand, spectra of the saturated alcohols, 5a-cholestan-3B-01 (12) and 5/3-cho!estan-3a-ol (13) show (M - 1)-ions of only about 50% relative intensity, with ions representing loss of H2 and H 2 0and association with N 2 0 accounting for the remainder of the ionization. These results suggest that the (M - 1)-ion associated with the ketone is of lower energy than the (M 1)-ion in the alcohols. 3/3-Hydroxy-5a-cholestan-6-one (10) is a compound containing both the carbonyl and hydroxyl
275
group. The two groups are sufficiently isolated so as to minimize interaction between the two. Thus, we might expect the spectrum of (10) to reflect the influence of both groups on the ionization of the molecule. Here, the (M- 1)-ion is dominant (re1 int 90%); the (M - 19)- ion accounts for the remaining ionization observed. The complete absence of H2 loss or N 2 0 association and the small amount of H 2 0 loss implies that only a small fraction of the (M - 1)-ion can be attributed to proton abstraction from oxygen. Such results suggest that, in the gas phase, the hydrogens CY to the carbonyl group are more acidic than the hydroxyl hydrogen in alcohols. This would represent a departure from condensed phase chemistry where the acidity of secondary alcohols such as (12) and (13) is generally found to be 10-100 times greater than the acidity of aliphatic ketones (6). We note here that the general features of the spectra of alcohols (12) and (13) are characteristic of the alcohols in Table I. These two particular alcohols are configurational isomers, and the differences in their spectra are too subtle to allow a clear distinction between the two. This is also the case for two other pairs of configurational isomers ((17 and 18) and (21 and 22)) in Table I. A fourth pair of configurational isomers, the epoxides ( 2 6 ) and (27), display significantly different spectra and will be discussed later. Cholest-5-en-3/3-01(14) (cholesterol) and cholest-4-en-3/3-01 (15) are positional isomers. The spectrum of (14) displays a sizable (M - 1)-ion (re1 int E 70%) and also ions associated with loss of H 2 0 and reaction with N 2 0 . T h e isomer (15) displays very little (M - 1)-,an intense (M - 3)- (re1 int E S O % ) , and ions associated with the loss of H 2 0 amounting to 35% of the total ionization. No measurable (M n)- intensity is observed in the spectrum of (15). The (M - 1)-ion in (14) is somewhat greater in intensity than the (M - 1)-(re1 int E 50%) ion in the saturated alcohols (12 and 13). This difference may be experimentally significant, and we make the tentative hypothesis that a substantial amount of the (M - 1)-ion in the spectrum of (14) may in fact be an allylic anion and not the alkoxide ion, Le.,
+
-
-
(9) HO H
The hydrogens a t the C4 position will be activated by both functional groups, and the anion resulting from the removal of a proton from this position may well be of lower energy than the (M - 1)-ion in saturated alcohols. The intense (M - 3)ion in the spectrum of the alcohol (15) may represent loss of H2 from the alkoxide ion to produce a resonance stabilized enolate ion analogous to that in reaction 1. Of course, the H2 loss will not occur across the 3-4 bond in (15), but loss across the 2-3 bond is possible and would serve the function of lowering the energy of the ion. However, it is difficult to envision loss of H 2 0 from this species to give the observed (M 21)- ion (re1 int 22%). Neither is it easy to envision the production of (M- 21)- ion by loss of H 2 0 followed by loss of H2 or for these entities to be lost simultaneously. Here also we suggest that a substantial amount of the (M - 1)-ion formed may be the allylic carbanion and not the alkoxide ion. With this hypothesis we can easily account for the observed ions, e.g., -
ti0
&
&-% (M-I)
(M-19)-
In both the (M - 19)- and (M - 21)- ions, extensive resonance can occur.
276
ANALYTICAL CHEMISTRY, VOL. 51, NO. 2, FEBRUARY 1979
Cholest-4,6-dien-3@-01(16) is similar to (151, having an additional carbon-carbon double bond in conjugation with the 4-ene-3-01 system. T h e spectrum of (16) displays an intense (M - 19)- (re1 int 7 7 % ) . As was seen for (ls),it is difficult to envision loss of H 2 0 from the alkoxide ion or enolate ion (alkoxide-Hz) produced from this type of allylic alcohol. On the other hand, the (M - 19) ion can be formed directly from the allylic carbanion by loss of HzO. ~
The ene-diols, (20S)-cholest-5-ene-3~,20-diol (23), (245)cholest-5-ene-3$,24-diol (24), and cholest-5-ene-3@,25-diol (25), have the two hydroxyl groups in the molecule far removed from one another. Loss of a second molecule of water from these compounds would not be expected to lower the energy of the ion particularly, and thus we observe relatively weak (M - 37) ions in their spectra (-10% re1 int). The two epoxides, 5,6t~-epoxy-5u-cholestan-3/3-ol (26) and 5,6B-epoxy-5~-cholestan-3P-ol (27) are the only pair of configurational isomers studied whose spectra reveal any significant difference. The trans-fused epoxide (26) displays an (M 1)- ion which is of significantly greater intensity than the (M - 1) ion observed in the cis-fused isomer (27). We offer no rationale for this apparent difference, which was consistent over many replicate measurements. One might expect that OH- would attack the epoxide ring as a nucleophile and open the ring; however, no (M 17)- ion, which would result from such an attack, is observed in the spectrum of either epoxide. However, the spectra of the epoxides show significantly higher (M - 19)- intensities than those of the alcohols (12) and (13), which differ in structure from the epoxides only by the lack of the epoxide group, and we think that the epoxide ring is involved, to some extent, in the ionization of the molecule. We suggest the following mechanism whereby an (M 1)- ion could be formed by a bimolecular elimination process involving OH- attack on the C6 hydrogen -
I t is interesting to note that this ion is identical to the (M 3) ion in the diene ( 3 ) discussed earlier. The (M 3)- ion in the diene (3) is presumably formed by loss of H2 from (M - 1)-,whereas the (M - 19)- ion in the diene-ol (16) apparently results from loss of H 2 0 from the (M - 1)-ion. A comparison of their relative intensities, 27% and 7 7 % , respectively, may be indicative of the relative ease of removal of H,and H,O under these experimental conditions. T h e spectra of 5c~-cholestane-3/3,5-diol(17), Wcholestane-3/3,5-diol (18), and 5u-cholestane-3P,GP-diol (19) all display prominent (M - 1)- ions (re1 int 60%). Essentially all the remaining ionization can be attributed to the (M 19)and ( M -- 37)- ions, which are of about equal intensity. It was suggested earlier that the intensity of the (M - 37)- ion may be related to the position of the hydroxyl groups in diols such as (17)-(19). These three are saturated diols, and it may be assumed that the (M 1)- ion is the alkoxide. It can be seen t h a t elimination of the second molecule of water will serve to increase the resonance in the ion and further delocalize the charge -
-
+
-
-
?$J
HO
ti0
0-
IM-191-
%!I
,73M - :&
& 2%
L$
T.+ HO 0-
) , 3 .M;&
IM-19)-
(12) We observe fairly intense (M - 37)- ions with these three alcohols, which we attribute to the stability of the conjugated systems in the ions. This view is reinforced when one observes the spectra of t h e six ene-diols (20)-(25). Cholest-5-ene-3fi,4/3-diol(20) is a vicinal diol and its spectrum displays an intense (M - 19)(re1 int 73%) ion and no measurable (M - 37)- ion. We suggest t h e following scheme for the formation of (M 19)- ions
$ f
-HZO_
0
0
H
H
T h e position of the hydroxyl groups in (20) make it difficult to envision a mechanism for the loss of a second H 2 0 molecule. I t is gratifying, therefore, that no (M - 37)- ions are observed. On t h e other hand, t h e configuration isomers, cholest-5ene-3@,7u-diol(21) and cholest-5-ene-3@,7/3-diol(22) display intense (M - 37)- ions, but in these molecules loss of the second water molecule yields a resonance stabilized system
OHHO
0.
Loss of H 2 0 from this ion would extend the conjugation and account for the prominent (M - 19)- ions observed for both epoxides. Two triols were included in the study, and their spectra show quite different patterns. (2OR)-Cholest-5-ene-3@,20,21-triol (29) is structurally analogous to cholesterol (14) differing only by the addition of vicinal hydroxyl groups in the CI7 side-chain. I t is apparent from a comparison of the spectra of these two compounds that these exocyclic hydroxyls have a substantial effect on the ionization of the triol. Compound (29) shows a lower (M 1) intensity and a higher iM - 19)- intensity than (14). It seems clear that a significant fraction of the (M - 1)- ions in (29) are exocyclic alkoxide ions, and some of these lose water to produce allylic alkoxide ions. We point out that the intense (M 19)- ion (re1 int >50%) in the spectrum of' (29) is reminiscent of the (M - 19)- ion (re1 int > Y O % ) in the vicinal diol (20) (see reaction 14). 5,Cholestane-3d.5,GB-triol (30) is structurally analogous to Sa-cholestan-3&ol (12) and 5e-cholestan-3P,5-diol (17), and its spectrum contains ions found in both these analogues. A sizable (M - 3)- ion (re1 int E 20%) is observed in the spectrum of ( 3 0 ) ,which is comparable to that observed for the monoalcohol (12). This ion presumably represents the loss of H, from an alkoxide (see reaction 1). In addition, the spectrum of (30) contains ions a t (M - 19)- and (M - 37)comparable to those seen in the spectrum of the 3,5-diol (17). In both instances, loss of water could be expected to stabilize the ion by resonance. The loss of a third hydroxyl from the triol (30) could produce a highly conjugated system, and we observe a small (M - 55)- ion (re1 int E 10'70) in the spectrum. This ion is not found in the spectrum of the triol (29); formation of such an ion in (29) would require an allene structure and would offer no stabilization due to resonance. The spectrum of 3P-hydroxy-5a-cholest-6-ene-5-hydroperoxide (28) is rather complex and not readily interpreted. Although an (M - 1)-ion is observed, its intensity is quite weak. T h e ion may originate from proton abstraction a t the
zc-Oa -
HO
+ ti0
-
-
ANALYTICAL CHEMISTRY, VOL. 51, NO. 2, FEBRUARY 1979
alcohol or the hydroperoxide function, the latter being the more acidic in the condensed phase. Ions a t (M - 19)- and (M - 37)- are the most intense in the spectrum, and these may represent loss of H 2 0 , and H 2 and H 2 0 2 ,respectively, from a n alkoxide (M 1)-. Cholesta-4,6-dien-3-one,which can be formed by elimination of H,02 and H2 from (28) is a known thermal degradation product from the hydroperoxide (7). Thus, -
T h e presence of a very weak ion a t ( M 17)- suggests that some free radical mechanisms may be occurring here. The source temperature of 200 "C might well produce some degree of thermal homolysis of the peroxide bond since D(R0-OH) = 43-44 kcal/mol for R = Me, Et, i-Pr and t-Bu (8). T h e resultant radical could then be converted to a molecular anion by resonance capture of a low energy electron in the source. Thus, -
The ions resulting from nucleophilic attack by OH- on the esters were discussed in the section on general reactions. The other major ion observed for these compounds is (M - 1)-, which accounts for a significant amount of the ionization in the spectra of cholesteryl acetate (31) and the three fatty acid esters, cholesteryl palmitate (33), cholesteryl oleate (34), and cholesteryl stearate (35). This ion may result from abstraction of one of the allylic protons adjacent to the C-5 double bond in the steroid nucleus; however, the complete absence of (M + n ) - ions in these spectra would tend to discount this possibility. A more appealing mechanism ( I ) involves abstraction of a proton o to the carbonyl group of the ester. Thus, for cholesteryl stearate
We note that there is no (M - 1)-ion in the spectrum of the benzoate ester (32), which has no hydrogens in the a-position. The relative intensities of the major ions in the spectra of the four esters studied may be dependent on the availabilities of these hydrogens. In addition, these ion intensities may also be dependent upon the relative stabilities of the carboxylate and alkoxide ions. Sensitivity. A few experiments were carried out to obtain information on the relative sensitivities of OH- NCI and CHI PCI for these steroidal compounds. The compounds chosen for investigation were 50-cholestan-3-one (4),cholestan-3-01s (12 and 13), and cholesteryl stearate (35). These are monofunctional steroids containing the important functional groups (carbonyl, hydroxyl, and ester) found in the total body of steroids here investigated. The experimental technique used involved completely evaporating from the mass spectrometer probe equal amounts of a compound (in the range 1-3 pg) using first t h e positive and then the negative mode of ionization. The intensities of the ions produced were measured continuously during the course of the evaporation,
Table 111. Relative Sensitivities XI( N C I ) / r l ( P C I ) relative compound sensitivities 5a-cholestan-3-one(4 ) 4.7 cholestan-3-ols(12.3~13) 3.8 cholesteryl stearate( 34) 7.1
277
-
and the sum of the intensities of all the ions for all of the scans during a given evaporation was used as a measure of the ionization sensitivity. Prior to data collection, the instrumental parameters were adjusted to achieve unit resolution using perfluorotributyl amine for CH4 PCI and pwfluoromethyloctanoate for OH NCI. Instrumental adjustments were kept a t an absolute minimum in changing from the positive to negative mode of operation and were generally limited to slight changes in lens voltages. The relative sensitivities may be defined as the ratios of the total ionizations obtained as described above for OH NCI to those obtained for CH, PCI. The sensitivities obtained (averages of 10-15 reasonably concordant replicates) for the three compounds investigated are given in Table 111. The sensitivities in the negative mode are somewhat greater than those in the positive mode, but not enough to constitute a marked analytical advantage. In our opinion the approximate equivalence of the sensitivities is to be expected for processes involving ion-molecule reactions which are rather similar in terms of the masses of the reacting ions, the exothermicities of the reactions, and the compositions and properties of the reaction mixtures. T h e differences which d o exist between the sensitivities are probably the consequence of small effects which we do not presently understand. We have compared the OH NCI sensitivities with CH4 PCT sensitivities because the latter is among the highest to be found for positive chemical ionization systems. We have long recognized from qualitative observations that a relationship exists between the sensitivity of chemical ionization reactions and the exothermicities of the reactions, and we have recently make quantitative measurements of this phenomenon (9). Milder reagent gases such as isobutane and ammonia are in widespread use in positive chemical ionization, and the exothermicities of the reactions of t-C4H9+and NH4+from these gases with the steroids of this study will be appreciably lower than the exothermicities of the reactions of CH,+ and C2H5+ from methane with the steroids. Thus it is quite possible that the OH- NCI sensitivity with these steroids will be significantly higher than the isobutane and, especially, ammonia sensitivities. Divers Comments. The ammonia. methane, and isobutane positive chemical ionization spectra of many of these steroids have been measured by Lin and Smith (2). It is not feasible nor particularly profitable here to make a compound by compound comparison of the four kinds of chemical ionization spectra. Speaking therefore only in general terms, all four methods are soft ionization methods which produce no fragmentation of the steroid carbon skeleton. T h e amount of fragmentation involving carbon-oxygen bonds is least for NH3 PCI, OH- NCI g i-C4HloPCI, and most for CHI PCI. No markedly different structural properties are delineated by the positive and negative methods. We have found t h a t the sensitivity for OH NCI is somewhat higher than that for CH, PCI, and we pointed out that it may be significantly higher than those for i-C4Hloand NH3 PCI. We are of the opinion that the combination of low fragmentation and high sensitivity (high reaction exothermicity) is more favorable in OH- NCI than in the three PCI methods. In all of this it must be kept in mind that the PCI measurements were made a t an ion source temperature of 100 "C and a source pressure of about 0.4 Torr. The OH NCI measurements were made
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a t a source temperature of 200 "C and a source pressure of 3 Torr. More fragmentation and less ionization (sensitivity) will be encountered in the PCI methods at a higher source temperature. T h e OH- NCI spectra of virtually all of the compounds studied here could be put to good analytical use. T h e (M 1)- ion is the most intense ion in the spectra of 22 of the steroids, and it is of sufficient intensity to allow facile identification of molecular weights for all save two of them. While some association ions are observed (referred to above as (M n)- ions), the amount is small in total. and absent for most of the compounds. By contrast, the (M + NHJ+ ion is found extensively in the NH, PCI spectra, and this ion will be markedly lower in abundance or perhaps absent a t higher source temperature. T h e OH- NCI results with OH containing steroids are particularly valuable, for such compounds generally show an (M - 1)-ion of useful intensity and ions produced by the loss of 18 additional mass unit for each OH in the molecule. In some cases the relative intensities of these ions give information about the positions of the OH groups relative to each other and to other groups in the molecule. As an example, such information allows one to distinguish between the diols (20) and (21), which are positional isomers. T h e OH- NCI spectra of cholesteryl esters of fatty acids such as cholesteryl stearate are particularly attractive, for they
contain peaks giving information about the molecular weight of the component ( ( M - 1)-),the identity of the acid present (RCOO-), and the presence of the cholesterol carbon skeleton ((XO-H,O)-). In CH4 and i-C4HIoPCI the fragmentation of these molecules is so extensive that no molecular weight information is available.
LITERATURE CITED A. L. C. Smit and F. H. Field, J . A m . Chem. Soc., 99, 6471 (1977). Y . Y. Lin and L. L. Smith, Biomed. Mass Spectrom., in press. A. L. C. Smit, M. A . J. Rosseto, and F. H. Field, Anal. Chem., 48, 2042 (1976). V . M. Bierbaum, C. H. DePuv, and R . H. ShaDiro, J . A m . Chem. Soc., 99, 5800 (1977) J H J Dawson and N M M Nibbering, J Am Chem SOC, 100, 1929 11978) - -, D. J. Cram, "Fundamentals of Carbanion Chemistry", Academic Press, New York, 1965. L. L. Smith, M. J. Kulig, and J. I. Teng, Steroids, 22, 627 (1973). R. Hiatt, "Hydroperoxides", in "Organic Peroxides", D. Swern, Ed.. Wiley Interscience, New York, 1971, p 46. 100, 1356 M. Meot-Ner (Mautner) and F. H. Field, J . Am. Chem. SOC., (1978).
+
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RECEIVED for review August 4, 1978. Accepted November 6, 1978. The Rockefeller University portion of this work was supported in part by a grant from the Division of Research Resources, National Institutes of Health, and that of t h e University of Texas Medical Branch of National Institutes of Health Grant =HL-10160.
High Sensitivity, Continuous Flow Thermochemical Analyzer Richard S. Schifreen,' Carolyn Sue Miller,2 and Peter W. Carr" Department of Chemistry, University of Minnesota, Minneapolis, Minnesota 55455
A flow enthalpimeter for use with two fluid streams has been designed. It is based on a related device for use with one fluid stream and a bed of catalyst. Satisfactory results were obtained only when the two thermistors, which comprise part of a differential temperature measurement system, are subjected to the same net flow. I n addition, any volume located between the point of reagent mixing and the adiabatic column must be minimized. Assays for mineral acid, calcium ion, and nitrite based upon their reaction with tris(hydroxymethy1)aminomethane, ethylene bis(oxyethylenenitri1o)tetraaceticacid, and sulfamic acid, respectively, have been developed. Detection limits ranged from 10 pM to l mM, depending upon the heat of reaction and achievable base-line stability. Sample volumes of 120 pL and a flow rate of 2.5 mL/min allow for a throughput of nearly 60 sampleslh. I n general, sample volume, concentration, and flow rate affected the signal height and width in a fashion similar to that observed with an immobilized enzyme analyzer based on a related flow system. The chlef differences in behavior occur at low sample concentration.
In principle, thermochemical methods of analysis are applicable to any reaction which either generates or absorbs Present address, Clinical Chemistry Laboratory, H a r t f o r d Hospital, H a r t f o r d , C o n n . 06115. 2Present address, Technical Services, Schilling Division, M c C o r m i c k a n d Co., Inc., Salinas, Calif. 93901. 0003-2700/79/0351-0278$01 .OO/O
heat. These techniques, however, require a controlled environment and are often more tedious than other methods which provide the same information. Consequently, thermochemical analysis is often not considered for routine work when some alternative technique is available. Various attempts have been made to circumvent these limitations. In 1965, Priestly et al. introduced continuous flow enthalpimetry ( I ) . This approach was based upon mixing a reagent and sample stream in a plastic cylinder. Three thermistors monitored the temperatures of the sample, reagent, and product streams providing a differential measurement system. They recognized the need for a reaction chamber possessing high mixing efficiency and low thermal capacity and conductivity. This was accomplished by fitting the inlets to the small reaction cylinder with narrow inlets to give two opposed mixing jets. An isothermal instrument has been developed by Christensen et al. ( 2 ) . Their reaction vessel is immersed in a controlled temperature bath and maintained a t a constant temperature by a Peltier cooler and digitally controlled heater. Reaction heats are measured a t steady-state by t h e rate of heat addition required to maintain the pre-set temperature. This instrument is capable of measuring heats of reaction a t various pressures and quantitating large volume samples. Its primary advantage lies in t h a t it is independent of the heat capacity of the solution and of the characteristics of the calorimetric vessel as a result of the isothermal mode of operation. A third device has been developed by Peuschel and Hagedorn (3-5) and is being manufactured by Technicon C 1979 American Chemical Society
