Anal. Chem. 1982, 54, 2465-2470
ex0
endo
Yet under the conditions of the derivatizution, only one peak was observed on a capilllary column. This may be due either to the known, high temperature reversibilit,yof the Diels-Alder reaction yielding only the thermodynamically more stable product or to the coelution of the two isomers. In any event, the mass spectra of both endo and exo products are expected to be very similar. In addition to the possibility of generating exo-endo isomers, pairs of enantiomers may also be formed. For example: CcI
&
C& .C -I.
2 H5
CH3
This poses no problem its long as the original olefin does not have a chiral center since the enantiomers would not be separated on normal, achiral column packings. On the other hand, with an optically active olefin a mixture of diastereomers would result that may tlhen be separated and used to advantage in the determination of the absolute configuration of an optically active olefin. The derivatization technique described in this paper works quite well for the disubstituted olefins but is not satisfactory for highly hindered olefins such as @)-%methylmethylbut2-enoate which gave no adduct with either 1 or 2 even after
2485
heating for 48 h. Although some trisubstituted olefins such as 2-methyl-2-heptene do produce adducts with 2, the mass spectra of these adducts are dominated by ion A and the other fragments are not of sufficient intensity to allow the position or substitution of the original double bond to be determined. The large increment in mass upon derivatization of double bonds is somewhat of a problem. It makes the method less useful for polyenes, because the polyadduct would be difficult to elute from a gas chromatographic column. This limitation may diminish in the future with the availability of high temperature, thinly coated, capillary columns which transmit even n-CsJ3122 (15).
LITERATURE CITED (1) Wolff. R. E.; Wolff. G.; McCloskey, J. A. Tetrahedron 1988, 22, 3093. (2) Egllnton, G.;Hunneman, D. H.; McCormick, A. Org. Mass Spectrom. 1988. 7 , 593. (3) Arlga, T.; Araki, E.; Murata. T. Anal. Blochem. 1977, 8 3 , 474. (4) Dommes, V.; Wirtz-Peltz, F.; Kunau, W.-H. J. Chromatogr. S d . 1978, 74, 360. (5) Budzikiewicz, H.; Busker, E. Tetrahedron 1980, 36, 255. (6) Ferrer-Correla, A. J.; Jennlngs, K. R.; Sen Sharma, D. K. Org. Mass Spectrom. 1978, 1 7 , 867-872. (7) Ferrer-Correle, A. J.; Jennings, K. R.; Sen Sharma, D. K. Adv. Mass Spectrom. 1978, 7 , 287. (8) Chai, R.; Harrison, A. G. Anal. Chem. 1981, 53, 34. (9) Newcomer, J S.; McBee, R. T. J. Am. Chem. SOC. 1949, 7 1 , 946. ( I O ) Boyd, R. K.; Beynon, J. H. Org. Mass Spectrom. 1977, 12, 163-165. (11) Weston, A. F.; Jennings, K. R.; Evans, S.; Elliott, R. M. Int. J. Mass Spectrom. Ion Phys. 1978, 2 0 , 317-327. (12) Karni, M.; Mandelbaum, A. Org. Mass Spectrom. 1980, 75, 53-64. (13) Hoffmann, R. W.; Hauser, H. Tetrahedron 1985, 27, 289-902. (14) Norman, R. 0.C. "Principles of Organlc Synthesls", 2nd ed.; Halsted Press: New York, pp 288-295. (15) Dielmann, G.; Meler, S.;Rapp, U. HRC CC, J . High Resolut. Chromas togr. Chromatogr. Commun. 1979, 2 , 343-350.
RECEIVED for review April 30,1982. Accepted August 23,1982.
This work is supported by a grant from the National Institutes of Health (DRR 00317).
Analysis of the Chemical Ionization Reagent Plasma Produced in Liquid Chromatogr;aphy/Mass Spectrometry by Direct Liquid Introduction Mass Spectrometry/Mass Spectrometry R. D. Voyksner"' and J. Ronald Hasir Laboratory of Environmental Chemistry, National Instltute of Environmental Health Sciences, P.0. Box 12233, Research Triangle Park, North Carolina 27709
Maurice M. Bursey Department of Chemistry, IJniversity of North Carolina, Chapel Hill, North Carolina 27514
A tandem quadrupole mass spectrometer Is used to identify ion-molecule reaction products In two common reversedphase solvents, acetonitriie/water and methanoVwater. The collision-induced dissoclation spectra of ntormal and isotopic labeled solvents and high-resoiutlon datal provlde the information to deduce the emlpiricai formulas and structures for the cluster Ions. The solvent effect on samplle protonation and fragmentation Is discussed. This solvent reagent study represents the first steps In understanding the spectra obtained by direct liquid 1ntroduci:ionLWMS.
The advantages of liquid chromatogralphy over gas chroGraduate student, Delpartment of Chemistry, University of North Carolina, Chapel Hill, NC 27514. 0003-2700/62/0354-2485$01.25/0
matography as R means of sample separation for classes of compounds which are thermally labile, very polar, or nonvolatile have justified the increasing interest in liquid chromatography/mass spectrometry (LC/MS) (I, 2). A variety of interfaces developed to introduce the effluent from the LC into the mass spectrometer include the moving belt (3),silicone membrane separator (4), direct liquid introduction (DLI) (5), atmospheric pressure ionization (6),and the heated wire (7). The DLI method utilizes the solvent as the reagent gas for chemical ionization. Very little has appeared in the literature on the nature of this reagent plasma. Insight into the processes that occur in the source that give rise to analyte ions, the degree of fragmentation observed for the analyte, and the identity of the many solvent cluster ions observed will provide information needed to optimize the technique. This information is important if one wants to choose the best conditions 0 1982 American Chemlcal Society
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ANALYTICAL CHEMISTRY, VOL. 54, NO. 14, DECEMBER 1982
Table I. Ions Observed for a 50/50 Mixture of Methanol/Water, Their Collision-Induced Fragments (Collision Energy, 40 V), Their Empirical Formulas, and Ion Structures Deduced
m/z 15 18 19 33 47
re1 intens
CID fragment ions
5 7 27
100 27 4
15 33, 31, 1 5 33, 19, 1 5 47, 33, 1 5
51 65
69
79 83
3
47, 33, 1 5 65, 51, 33
97 111
2 1
65, 4 7 , 33, 1 5 79, 65, 47, 33
5
not only for LC but for MS as well, and it may aid in understanding the LC/MS results obtained for solute species. Mass spectrometry/mass spectrometry (MS/MS) has been applied to the determination of ion structures through collision-induced dissociation (CID) of the mass selected ion (8-10). Since the CID spectrum is characteristic of the structure of the ion, MS/MS has been applied in fundamental studies of unimolecular and ion-molecule reactions as well as identification and quantitation of components in mixtures (11,12). The ability to study the fragmentation patterns of selected cluster ions enables the verification of ion structures which were only previously postulated. The clusters formed from the DLI process are initially mass selected by the first quadrupole. These transmitted ions are collisionally activated with a neutral gas in the collision cell. The ability of the collision cell to shunt mass discriminating fields while passing focusing fields from the quadrupoles results in a high collection efficiency of fragment ions. The second quadrupole is scanned to detect these fragments. The same type of experiment can be performed on a reverse Nier Johnson instrument. The magnet selects the nominal mass beam which undergoes high energy collisional activation in the collision cell located in the second field free region of the instrument. The electric static analyzer performs the kinetic energy analysis on the resulting fragment ions. The reverse Nier Johnson instrument has the solvent mixtures introduced through the reservoir inlet and employs different source geometry, source pressure, and collision energy from the tandem quadrupole. The cluster ions formed from the two instruments cannot be assumed to be identical. The CID spectra can verify the qualitative identity of the ion clusters formed in either source. These clusters must be identical for the high-resolution measurements to be meaningful in deducing empirical formulas. A comparison of the DLI formed clusters in the quadrupole, with the ones formed from reservoir introduction of solvent into the source of a magnetic instrument or ion cyclotron resonance spectrometer, will indicate the relevance of previously reported studies with water, methanol, and acetonitrile in postulating ion structures. In the work described here the DLI interface is used as a solvent introduction method for the tandem quadrupole (MS/MS). The ion-molecule reaction products in two common reversed-phase solvent mixtures, acetonitrile/water and methanol/water, are identified. EXPERIMENTAL SECTION The instrument used to perform the experiments consists of a Waters 6000A pump and a Waters U6K injector. Since this work did not involve separations the column and UV detector were omitted. A flow rate of 1mL/min was sent to the interface, a Hewlett-Packard direct introduction probe, where approximately 1% of this effluent entered the mass spectrometer. The droplets
empirical formula CH3 H,O H,O CH,O C,H,O CH,O, C,H,O, C3H1102 C2H1103
C3H1303 C4Hl
63'
structure
[ CH,OH-HI+ [CH,OH-CH,]' [ CH,OH-H-OH,]+ [(CH,),Q-H-OH,]' or [ CH,OH-H-OHCH 1' [(CH,),O-H-OHCH, [ CH,OH-H-OH-H-OHCH,]' or [ CH,OH-H-O( CH,)-H-OH,]' [ CH,OH-H-O( CH,)-H-OHCH,]' [( CH,),O-H-O( CH,)-H-OHCH, ]+
5.
forming the jet developed through a 5 wm diameter orifice and were vaporized in a desolvation chamber, on a modified Finnigan CI source as previously described (13). The Extranuclear Laboratories tandem quadrupole mass spectrometer performed the mass analysis. The ions that were transmitted through both quadrupoles were detected by an off-axis multiplier and recorded on a UV oscillograph. The acetonitrile and methanol were Burdick and Jackson, distilled in glass quality; water was J. T. Baker analyzed HPLC grade. Deuterium oxide was Aldrich Chemical gold label quality; deuterated acetonitrile from Fisher Scientific was 99% pure; and the l80water, a gift from the Weizmann Institute of Science, Israel, was 97% pure. These solvents were filtered through a 0.45 wm Millipore filter to prevent the orifice from becoming blocked. Typical operating procedure involved optimization of the jet length and probe position to give the maximum cluster intensity; this usually occurred between 0.2 and 0.4 torr as measured with a MKS Baratron capacitance manometer, type 220B. The ion current stability was monitored continuously since fluctuations can occur if the orifice becomes plugged or when the HPLC pumps do not operate properly. After source tuning the normal operating parameters of the tandem quadrupole were as follows: electron energy, 100 V; emission current, 1 mA; extractor, 2 V; source temperature, 160-180 "C. The collision gas used was nitrogen and its pressure in the collision cell was adjusted to give maximum fragment ion intensity for the proton bound dimer solvent ion. This usually resulted in a 40-60% main beam attenuation. The data for the exact mass measurements, B-E plane map (14),and fragmentation vs. reagent gas composition were obtained on a VG Micromass ZAB-2F instrument with a Finnigan-Incos 2300 data system. The instrument was operated in CI mode 0.5 mA emission, 0 V repeller, 180 "C source temperature, and 1000 resolution, except for the exact mass measurements, which were carried out at 10000 resolution. The methanol, acetonitrile, and water reagent gases were introduced into the source through a reservoir kept at 160 "C. The source housing pressure was measured to be (1.C-2.0) X lo4 torr with an ionization gauge. This corresponds to a pressure between 0.35 and 0.45 torr in the source, as measured with the Baratron capacitance manometer, which is comparable to the source pressure measured for the tandem quadrupole. The B-E plane map was acquired by scanning the magnet ( B ) at 1 s/decade over the mass range 10-170 while the electrostatic analyzer (ESA) was swept from 100 to 730 eV at 10 s/eV; helium was used as collision gas at an analyzer pressure torr. of 1 x RESULTS AND DISCUSSION Methanol/Water. The LC/MS/MS data are summarized on Table I, which contains the mass and intensity of the parent ion, the mass of the CID fragments obtained from each parent, the empirical formula, and the probable structure. Most of the ions reported for pure methanol have been previously observed (15-1 7) and the structures suggested previously correspond with the fragmentation patterns observed in Table I. The cluster at m / z 111has not been previously reported.
ANALYTICAL CHEMISTRY, VOL. 54. NO. 14, DECEMBER 1982
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Table 11. Comparison of the Ratio of Fragment Ion Intensity to the Sum of Fragment and Protonated Molecular Ion Intensity, for Different Methods of Reagent Gas Introduction
PA,
reagent gas
kcal/mol
CH4 HZ0 CH,OH C,HM
126 167 182 190
benzoic acid 1105/(1105 + 1123) .. DLI-LC/MS reservoir interface (ZAB-2F) 0.42 0.35 0.11 0.13
0.18
0.35
This ion showed losses of CH30H, CH,OH + C H , 2(CH30H), and 2(CH,OH) + CH,. This fragmentation pattern would he appropriate for the assigned structure and is consistent with the other structures. In the methanollwater solvent system fragments arise a t m / z 18,19,51, and 83, due to ion molecule reactions. As the percentages of methanol in the solvent decrease, all fragments except m / z 18 and 19 decrease in intensity. Obviously the peaks at m/z 18 and 19 are the HzO+ and H30+ ions from water. The peak at m/z 51 was assigned the structure of (CH,OH-H-OHd because of the CID fragments, m / z 33,19, and 15 by los4 of H,O, CH30H, and 2(H,O), respectively. The cluster at m/z 83 shows a similar fragment pattern with l o s s of H,O, CH,OH, and CH,OH-H,O to yield m / z 65,51, and 33, respectively. These fragments indicate that either of the assigned structures is possible. For pressures below 0.1 torr for 50150 methanol/water mixture, the main fragments recorded belong to CH,OH+ and H,O+. There was little clustering observed at the low pressures. At higher pressures (0.34.7 torr) CH,OH,+ and cluster ion intensity increases while the CH,OH+ decreased. At pressures greater than 0.7 torr, transmission decreases hecaw of the reduced mean free path of the ions. The B-E plane map (Figure 1) contains most of the mass spectral information on the methanol/water solvent mixture introduced through the reservoir. This map provides an overall view of the ion/molecule chemistry and the fragment intensity. They axis displays the CI spectrum, whose intensity is a factor of 100 lower than the rest of the map. From the data all the daughters from a selected parent ion (mass analyzed ion kinetic energy (MIKE) spectra) are shown. A representative scan line for a particular MIKE spectrum of the parent ion at m/z 65 is illustrated. Although the map was not acquired under LC/MS conditions, the MIKE spectra extracted qualitative agreed with the low-energy (40 eV) CID spectra obtained from the tandem quadrupole. The exact peak heights of the cluster ions do not match the tandem quadrupole because of different source conditions and inlet methods employed. The competition for protons should not be considered solely in terms of proton affmities of isolated solvent molecules. The enthalpy of reaction is a function of what groups have already bound to the proton to form the observed ion. Studies of the change in AH: for adding water to (H,O),H+, methanol to (H,O),(CH,OH),H+ and (CH,OH).H+, and acetonitrile to (CH3CN).HC have been carried out (18-21). Decreases in AH: up to 15-20 kcal/mol have been reported as the numher of groups attached to the core ion increases. No information The is available, to our knowledge, for (H,O),(CH,CN),H+. differences between spectra obtained under different source conditions of the ZAB and tandem quadrupole should reflect the dissimilar honding abilities of neutrals to the dominant cluster species in the two cases. A qualitative estimate of the relative proton affinities (PA) of water and methanol can he obtained from the collision products of the proton hound dimer at m / z 51 (22). At a collision energy of 15 V the primary ion gives only two frag-
diethyl 3,4-furandicarhoxylate 1167/(1167 + 1213) DLI-LC/MS reservoir interface (ZAB-2F)
0 100
0.44 0.36 0.04 0.02
0.52 0.77
1000
300
2000
500
3000
SconNumbsr
700-EESAVolloge-
Flgure 1. B-E plane map for 50150 methanollwater. obtained with the ZAE2F: C I source conditions. helium collision gas.
ments, m / z 33 and 19. Since the CH,OH,+ peak is more intense than the H30+ peak, methanol must have a greater PA than water. The reported PA of methanol in 182 kcal/mol and that of water is 167 kcal/mol(23). Both of these PAS are low compared to thme of most organics; therefore, protonation of organics by the solvent system can easily occur. From thermodynamic considerations one would expect the extent of fragmentation for an analyte ionized by CH30H/H,0 to he between that caused by methane CI and isohutane CI. In typical examples (ZAB-ZF)where the reagent is gaseous (Table 11) fragmentation decreases as the difference between PA of sample and PA of reagent gas decreases. However, in DLILC/MS the amount of fragmentation does not follow PA differences, the water solvent gave less fragmentation than methanol. This phenomenon might he explained by the high heat of vaporization and surface cohesion of water, resulting in larger nebulized droplets and thus longer drop lifetimes (24). The longer lifetimes allow the drop to condense on the desolvation chamber and then vaporize. The desolvation chamber is at a lower temperature than the source and there is a direct relationship between temperature and fragmentation (24-26).The differences in the extent of fragmentation observed in the two cases can also he explained in terms of AH: for the dominant reagent ion and the sample. If different reagent ions are responsible for sample protonation in each case, then AHrowould be different. This should influence the internal energy of the sample, resulting in varying degrees of fragmentation ohserved. Acetonitrile/Water. The other system, acetonitrile/water, was much more complex. Figure 2 shows the B-E plane map for acetonitrile/water. As with the methanol/water map, the qualitatively similar MIKE spectra can he extracted. A representative MIKE spectrum for the parent ion at m/z 83 is shown. The analysis of acetonitrile by LC/MS/MS (Table 111) indicates the complexity of this solvent system. For acetonitrile, clusters up to m/z 96 have been observed previously (27-29). The main characteristic of the CID spectra
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ANALYTICAL CHEMISTRY, VOL. 54, NO. 14, DECEMBER 1982
Table 111. Clusters Observed in CH,CN and CD,CN, Their CID Fragment Ions (40V Collision Energy), Empirical Formulas and Possible Structures CH,CN m/z intens CID fragmts 14 15 26 27 28 29 40 42
16
51 52 54 56
1 1
