1302
Anal. Chem. 1983, 55, 1302-1305
occasions where this property is an advantage. To avoid overflow or roundoff in the recursions, the actual word length of the signal must not exceed the available computer word length. Since the initial word length after the A/D conversion grows in the recursions by an amount which depends on the filter width N , a maximal N can be evaluated. For the Savitzky-Golay filter D2this bound is approximately given by (35)
where 6 is the difference between the computer word length and the ADC resolution in bits. The corresponding (exact) formulas for A are
N1dG-1
of 24 bit (including sign bit). Together with a 12 bit ADC, the maximal N values are 10,63, and 2047. If these bounds are too low, double precision (56 bit mantissa) may be chosen. This enables a filter width N of more than lo4for each of the three algorithms. However, it must be noted that this results in a further slow down of the execution. Before starting the recursions at k = ko - N , the algorithms must be properly initialized to avoid artifacts or even instabilities. This can most effectively be done by setting all values f [ k ] ,k < ko, preceding the first signal value f [ k o ]as well as all registers g,[ko - N - K ] , n 2 1, K 2 1, equal to zero.
LITERATURE CITED Bromba, M. U. A.; Ziegler, H. Anal. Chem. 1979, 5 1 , 1760-1762. Savitzky, A.; Golay, M. J. E. Anal. Chem. 1964, 36, 1627-1638. Ziegler, H. Appl. Spectrosc. 1981, 3 5 , 88-92. Bromba, M. U. A.; Ziegler, H. Anal. Chem. 1981, 53, 1583-1586. Vasseur, C. P. A.; et al. IEEE Trans. 1979, IM-28, 259-262. Condal, L.; Royo, J. Aflnldad 1981, 3 8 , 483-490. Hogenauer, E. B. IEEE Trans. 1981, ASSP-29, 155-162. Whlttem, R. N.; Stuart, W. I.; Levy, J. H. Thermochlm. Acta 1982, 57, 235-239. Bromba, M. U. A.; Ziegler, H. Int. J. Clrcult Theory Appl. 1983, I f , 7-32. Giannelli, G.; Altamura, 0. Rev. Scl. Instrum. 1978, 47, 27-31. Bromba, M. U. A., Dissertation, Paderborn, 1981, pp 67-76. Bromba, M. U. A.; Ziegler, H. Anal. Chem. 1983, 55, 648-653. De Blasl, M.; Giannelll, G.; Papoff, P.; Rotunno, T. Ann. Chlm. (Rome) 1975, 6 5 , 183-196. Gasser, Th.; Muller, H.-G. Lect. Notes Math. 1979, No. 757, 23-68. Tretter, S. A. "Introduction to Discrete-time Signal Processlng"; Wiley: New York, 1976. Rablner, L. R.; Goid, B. "Theory and Application of Digital Signal Processing"; Prentlce-Hall: Englewood Cliffs, NJ, 1975.
(36)
(parallel form) and
N
I26-1- 1
(37) (cascade/parallel form with scaling). Restricting our discussion to FORTRAN implementations, we have the choice between integer and real format. The integer word lengths are 16 or 32 bit (including sign bit). If we assume an ADC resolution of 12 bit (including sign bit), the maximal N values for the three algorithms are 63,1022, and 524 287 in the 32-bit case. For an integer word length of 16 bit, only the scaled cascade/parallel form works (N 5 7). If a double-word integer format is not available, the recursions may also be implemented with real format numbers (which significantly increases the execution time). Here the mantissa has a length
RECEIVED for review January 7,1983. Accepted March 4,1983.
Identification of Naturally Occurring Quaternary Compounds by Combined Laser Desorption and Tandem Mass Spectrometry D. V. Davls and R. G. Cooks" Department of Chemistry, Purdue Unlverslty, West La fayette, Indiana 47907
B. N. Meyer and J. L. McLaughlin Department of Medlcinal Chemistry and Pharmacognosy, School of Pharmacy and Pharmacal Sciences, Purdue University, West Lafayette, Indlana 47907
Laser desorption of quaternary alkaloids can be used to generate the Intact cations whlch can be mass selected and then Identifled by recording a spectrum of fragment Ions generated by coilislon-induced dlssoclatlon. The resultlng MS/MS spectra show much less chemical noise than do the corresponding laser desorptlon mass spectra. This procedure allows Identlficatlon of the quaternary alkaloid candicine chlorlde (N,N,N-trimethyltyramlne chloride, 1) In crude extracts of cactus material at the level of 1 part per thousand (dry weight). Procedures for rapldly surveying plant material for quaternary alkalolds are given and tested for candlclne In six specks of cacti. Results agree with those from a much less specific chromatographic method. Secondary Ion mass spectrometry provides similar capabllltles and two new quaternary alkaloids, O-methyicandlclne and N,N,N-trimethyl-& methoxyphenylethylamlne, were discovered with the ald of this procedure.
Tandem mass spectrometry, often called mass spectrometry/mass spectrometry (MS/MS), is an analytical method 0003-2700/83/0355-1302$0 1.50/0
which is useful in direct mixture analysis for trace compounds ( 1 , 2 ) . In the basic MS/MS experiment, a spectrum of fragment ions is used to characterize a selected ion and hence the neutral compound for which it is the surrogate. The separatory power of this procedure can be enhanced by such refinements as thermal profiling ( 3 , 4 )and its specificity by the generation and charge inversion (5, 6) of negative ions. In addition, the use of alternate scanning methods (7-11), such as the recording of parent and neutral loss spectra, allows the recognition of classes of compounds instead of individual targeted molecules. Here we illustrate the usefulness of the MS/MS experiment when combined with laser desorption (12-15), one of the newer ionization methods based on desorption from the solid state (16). The combination of laser desorption (LD) and tandem mass spectrometry has previously been demonstrated (17). In fact, tandem mass spectrometry is an appropriate adjunct to most desorption ionization procedures because it provides structural information which may otherwise be unavailable or may be obscured by interfering ions (17-19). Desorption methods of ionization are, in turn, particularly successful in ionizing precharged species including organic salts (20). There is 0 1983 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 55, NO. 8, JULY 1983
Scheme I EHn
;+
H,C'
'CH,
m/z 5 8
P
m/x 121
CANDICINE
H' m / z 60
CH,
m h 44
currently strong interest in laser desorption of organic compounds (12,21). Direct mixture analysis by laser deeorption has been attempted (22) but some form of molecular separation is desirable, and this can be achieved by tandem mass spectrometry as is shown herein. The identification of quaternary alkaloids in cacti hias been selected to test further the capabilities which desorption ionization brings to MS/MS. There are several reasons for this choice. First, tandem mass spectrometry has already proven of value for direct mixture analysis of cactus and other alkaloids susceptible to chemical ionization (3,24-26). Second, as precharged organic salts, quaternary alkaloids should be well suited to ionization by laser desorption (12-14). Third, this intrinsically interesting class of biomolecules (including, for example, the curme arrow poisons) has been relatively little studied because tedious procedures are required for their isolation. Fourth, work on cacti, as on most plant families, has not emphasized quaternary ammonium compounds and only six quaternary cactus alkaloids have been identified. Of these, three are from a single species Lophophoru williamsii (Lem.) Coult. (23) and a fourth, candicine (1,Scheme I), has been identified in just seven species. The specific goals of the work presented here are 2-fold: first to determine whether LD (or the related desorption ionization methods) add new capabilities to MS/MS by allowing direct detection and recognition of quaternary alkaloids, such as candicine; second, to utilize the speed and ease of implementation of this procedure in surveying various species of cacti for the occurrence of these compounds. EXPERIMENTAL SECTION Instrumental Section. Experiments were performed on a mass-analyzed ion kinetic energy (MIKE) spectrometer, a tandem mass spectrometer which has been described elsewhere (27).A new source (28) has been fitted to this instrument to allow laser desorption as well as conventional chemical ionization (CI) experiments The source is fitted with two 0.32 in. diameter X 0.040 in. thick fused silica windows, located on opposite sides of the ion block to allow optical access to the ionization region. Two additional fused silica windows, on either side of the source vacuum housing, form high vacuum seals while allowing the laser beam to transit the instrument. If the laser beam pasaies completely through the source, alignment is facilitated. Samples were dissolved in either methanol or water and deposited on a silver or platinum foil; excess solvent was removed by gentle heating. The foil supporting the dry sample was attached as an extension to the conventional solids probe of the mass spectrometer. This probe enters the source chamber from the back and is intercepted from below at a 90° angle by the laser beam, with the ions being extracted collinearly with the probe axis. The sample was irradiated by the 1064-nm line of a Quanta Ray DCR-1A pulsed Q-switched NdYAG laser. The puliae width was nominally 10 ns and the pulse energy approximately 0.1 J/pulse (55-60 J/pulse in terms of 1 A energy). The laser repetition rate was approximately 15 Hz and the irradiated spot size
1303
on the foil approximately 2 mm2,yielding power densities on the order of 108-10e W/cm2. The detection system (29) consisted of a standard Galileo Channeltron continuous dynode electron multiplier (Model No. 4830), the output of which is fed to an Analog Technology Corp. current to frequency (I/F) converter (Model No. 170). This converter serves two purposes; first, it integrates the signal from each laser shot avoiding pulse pile-up due to high ion flux, as sometimes seen in pulse counting methods previously used, and second, it converts data to the digital domain. The TTL pulse train from the I/F converter is sent to a digital counter (PAR Model No. 1109) which is synchronized with the laser such that the laser shot initiates the counter which counts for a specified time, The output of the counter is then dumped directly to a Hewlett-Packard minicomputer (Model No. 2100). The data acquisition software of this system (29) allows for signal averaging, background subtraction, and Gaussian smoothing. While it was possible to observe mass spectra by using the laser as the only means of ionization, these signals were not intense enough to generate satisfactory MS/MS data for the compounds of interest. However, when laser desorbing the sample into a CI plasma (30), the signal intensity increased by an order of magnitude and MS/MS spectra of high quality could be acquired. This procedure, however, does lead to increased background in both the mass and the MS/MS spectra. All of the data shown here were obtained with both the laser beam and the CI plasma contributing to the ionization process; however, there is no evidence that CI serves m y role other than to create a plasma which assists in efficient extraction of the ions formed by LD. (For example, no ions due to the quaternary compounds could be detected unless the laser was on while the converse is not true.) The CI reagent gas in these experiments was isobutane at a pressure of 0.15-0.2!5 torr as measured on an MKS baratron capacitance manometer connected directly to the source chamber. All MS/MS spectra were obtained by collision-induced dissociation in the second field-free region of the instrument. Air was used as the collision gas at a nominal pressure of (2-4) X loT6 torr measured by a I3ayard-Alpert type ion gauge. The accelerating potential in all experiments was 7 kV. Reproducibility of spectra was 10-20% due to variability in relative intensities caused by the nonreproducibility of individual laser shots. A typical MS/MS spectrum consists of approximately 700 data points, each point being generated by a single laser shot. The spectra shown here, however, consist of several summed individual MS/MS spectra, and, hence, shot-to-shotvariability is reduced. Secondary ion mass spectra (SIMS)were obtained with a Riber SQ 156L instrument, with an argon primary beam and current densities of 10-8-10-9 A/cm2. Samples were supported on silver foil. Experimental procedures for SIMS are detailed elsewhere (31).
Samples. Candicine (N,N,N-trimethyltyramine)chloride was synthesized by methylation of hordenine (Nfl-dimethyltyramine) (32). Extract samples were prepared with dried, pulverized, plant materials. Suppliers of the plants were Abbey Gardens, Reseda, CA (Trichocereus puchanoi Br. and R., T. werdermannianus Bckbg.), Desert Botanical Gardens, Kerrville, TX [Lophophora williamsii (Lem.) Coult., T. spachianus (Lem.) Ricc.], and Grigsby Cactus Gardens, Vista, CA [Coryphanthagreenwoodii H. Bravo, T. pasacana (Web.) Hr. and R., T. fuluilanus Ritt.]. All of these cactus species have been previously investigated for alkaloids (33). Preparation of Samples for LDMS/MS. Three grams of plant material was extracted with ethanol, and the residue was partitioned between 20 mL each of CHCl, and H20at pH 10. The water layer was freeze-dried, and the residue reconstituted in CHBOHor H20 for mass spectral analysis. (The condensed ethanol extract could also be used without further processing and was found to yield similar results.) Samples were usually admixed with excess (ca. 101) sodium or ammonium chloride prior to LD because this improves ion yields and spectrum persistence (34). Quantitative TLC. Samples were prepared for thin-layer chromatography (TLC) in analogous fashion, except that after partitioning between CHCl, and water, an additional washing of the CHC1, layer with ll2O was performed. The H20fractions were then combined and reduced in volume. The remaining solution was then diluted to a final total volume of 10 mL with H20. A standard solution of candicine chloride, 1 mg/mL H20,was also
ANALYTICAL CHEMISTRY, VOL. 55, NO. 8, JULY 1983
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LO-CI MS/MS SPECTRUM OF CANOICINE CHLORIOE m/.
1 .
1 0
140
Table I. TLC and LD MS/MS Determination of Candicine in Cactus Extracts
IS0
I 0
1 0
0
60
0
20
1
SPECTRUM OF TRICHOCEREUS PASACANA
LO-CI MS/MS
m/z 180
plant species Trichocereus pasacana Trichocereus werdermannianus Trichocereus pachanoi Trichocereus spachianus Trichocereus fuluianus Lophophora williamsii
LD MS/ MS +
+
-
TLC +(4PPt)U -
-
t (8 PptY
-
a Value in parts per thousand for ethanolic extract examined, Concentration of candicine per dry weight of plant material is calculated to be 0.075%for 2'. pasacana and 0.093% for T. spachianus.
TRICHOCEREUS SPACHIANUS m/z
180
J
Flgure 1. Laser desorption MS/MS spectrum of m / z 180 from candiclne chloride (top), T.pasacana (center), and T. spachlanus (bottom).
prepared, and known volumes of both the standard and samples were spotted on TLC plates (aluminum oxide, Bakerflex). The plates were developed in MeOH-CC14-glacial acetic acid, 2812:1, and visualized with iodoplatinatespray reagent (35). Quantitative evaluation was made by comparison of spot sizes for a series of standard samples. RESULTS AND DISCUSSION The MS/MS spectrum of candicine chloride (l),Figure la, illustrates the quality of data obtainable from quaternary alkaloid salts using the combination of laser desorptionchemical ionization (LD-CI) and tandem mass spectrometry. The methodology provides molecular weight information and a structurally diagnostic set of fragment ions. The major ions, their probable origins, and strudures are illustrated in Scheme I. The reactions involved are suggested on the basis of known unimolecular gas phase chemibtry. The dominant peak in the spectrum (m/z 60) corresponds to formation of protonated trimethylamine (36). The next most abundant peak (m/z 121) is generated by scission of the C-N bond, probably with formation of a stabilized phenonium ion (37). Also occurring a t low mass are ions characteristic of protonated amines. Having established the suitability of LD-CI MS/MS for characterizing candicine itself, the alkaloid was sought in crude cactus extracts. Trichocereus pasacana, a species which had been reported (32)to contain candicine, was first investigated. The plant extract, coated onto a silver foil was irradiated with the magnet set to pass ions of m/z 180. The electric sector was scanned, and data were acquired synchronously with the firing of the laser. The results shown in Figure 1confirm the presence of the quaternary alkaloid in Trichocereus pasacana. Ion intensities are not markedly attenuated in the course of this experiment (some minutes), providing adequate opportunity for signal averaging. Note also that the similarity of the two spectra confirms that the fragmentation taking place are not affected by the presence of the matrix in the plant extract. Other cactus species were also examined. T. spachianus, also reported to contain candicine (38), was used to confirm the applicability of the methodology. Results are shown in Figure IC,and they confirm the presence of the alkaloid in this plant. A group of cactus species, in the same genus but not previously examined for quaternary alkaloids, was also surveyed for the presence of candicine. In none of the three species of cacti chosen, Trichocereus pachanoi, Trichocereus
fuluilanus, and Trichocereus werdermannianus, could candicine be detected in spite of the fact that each is known to accumulate the less methylated alkaloids (33). A fourth species, Lophophora williamsii, has been reported (39) to contain candicine (TLC analysis), but it was not evident in later work (23) or in this assay. It is estimated (see below) that the detection limit for candicine in these extracts is 0.1 fig/mg dry weight. Since the survey methodology applied here is new, an independent procedure was used to confirm its reliability. The method chosen, thin-layer chromatography (TLC), has good sensitivity but low specificity. Comparison of the TLC and MS/MS results for the six species of interest is provided in Table I. Agreement between the two techniques serves to establish the usefulness of the faster and more selective MS/MS methodology. Both methods yield semiquantitative results, the former established the concentration of candicine in the extracts with positive responses at approximately 5 ppt. Given an average extract size of 1mg this leads to an absolute sensitivity to candicine of 5 fig in the presence of the matrix. The excellent signal-to-noise ratio exhibited by the spectra of candicine at these levels leads one to estimate an ultimate detection limit at least an order of magnitude better than this, even without further sample workup. This study of quaternary amine salts in cacti by direct mixture analysis shows the additional capabilities which desorption ionization techniques bring to MS/MS. Specifically, the methodology provides a reliable means of surveying for quaternary alkaloids and makes their identification easier and faster than when using classical techniques. Further enhancement in sensitivity, at some loss in specificity, is available by monitoring only the major fragment ions instead of recording full MS/MS spectra. In other experiments, this multiple reaction monitoring technique has been observed to decrease detection limits by approximately 2 orders of magnitude (40). Related ionization methods can be expected to have comparable value in the type of investigation discussed here. For example, the secondary ion mass spectrum (SIMS) of candicine has been obtained, and its resembles the LD MS/MS spectrum, although there are intriguing differences in immonium ( m / z 58) and ammonium (m/z 60) fragment ion abundances. Matrices such as ammonium chloride enhance ion intensities in SIMS (41) as well as LD spectra, further illustrating the similarities between these ionization methods. The SIMS spectra of two quaternary alkaloids, present in an extract from Coryphantha greenwoodii, showed prominent ions at m/z 194 corresponding to methylated analogues of candicine. A close structural relationship to candicine is further indicated by the presence in both spectra of abundant ions at m/z 135 (loss of trimethylamine) and by ions at m / z 60 and 58. The two compounds are distinguishable by the 135+/194+ ratio which differs by a factor of 2. Laser de-
ANALYTICAL CHEMISTRY, VOL. 55, NO. 8, JULY 1983
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is ineffective in creating ions. Whatever the mechanism of ionization, a pulsed laser is an effective means of producing intact cations of quaternary alkaloids.
1
160
140
la0
100
a0
80
40
20
1
LO-Cl MS/MS SPECTRUM OF m/r 180’
ACKNOWLEDGMENT B. N. Meyer acknowledges support as the Charles J. Lynn Memorial Fellow of the American Foundation for Pharmaceutical Education. Registry No. 1, 6656-13-9; 143, 3761-58-8; 2, 777-74-2; 3, 85553-38-4. LITERATURE CITED
LD-CI
MS/MS SPECTRUM OF m/r BO+
Figure 2. Illustration of chemical noise rejection by the second stage of analysis In the LD MS/MS experiment. Inserts show mass spectra. Top is background; bottom is sample.
sorption, followed by collision-induceddissociation of m / z 194, gave similar spectra to those obtained by SIMS. Again, the facility of trimethylamine loss serves to distinguish the isomers. Based on these results and the spectral analogies with candicine itself, structures 2 and 3 were postulated. The SIMS spectrum of 2 shows m / z 194 (100%))m / z 135 (78%))m/z 105 (6%),and m / z 58 (4%). The SIMS spectrum of 3 shows m / z 194 (loo%),mlz 135 (26%))m / z 105 (7%), m / z 103 ( l l % ) , m / z 91 (5%))m / z 77 (3%))m / z 60 (3%))and mlz 58 (13%). SIMS/MS data have also been obtained on these two isomers (43))and they confirm the mass spectral results. Compound 2 is expected to lose trimethylamine more readily than 3 because of assistance by the p-methoxy substituent. Confirmation of these assignments by synthesis is described elsewhere, as are the spectroscopic data and isolation procedures (42).
One of the advantages of MS/MS is the great reduction in chemical noise (interfering components) resulting from the high specificity of the method (27). This advantage applies in laser desorption ionization as illustrated in Figure 2, which shows (inserts) a background mass spectrum compared to that of an extract of T.pasacana. These mass spectra show that any increase in signal due to the occurrence of candicine in the extract is masked by the overall increase in signa3 due to other ions. The MS/MS data (shown in the same figure) on the other hand are clearly diagnostic for candicine. Note that the spectrum illustrated (a single 60 s scan) is not an averaged spectrum, and peak intensities are subject to the shot-to-shot variation of the laser. It is also evident from the results presented here that the high powered laser does not lead to appreciable thermal decomposition, a feature which has also been noted recently with regard to continuous wave (CW) lasers (13,44).Such decomposition would have been indicated by a significant signal in the mass spectra at m / z 166 which corresponds to the protonated tertiary amine. On the other hand it is not argued that thermal desorption does not contribute to the ionization process. One merely notes that intense signals are observed only when the laser operates in the CI plasma. Resistive probe heating in the absence of the laser
(1) McLafferty, F. W. Science 1982,214, 280. (2) Busch, K. L.; Cooks, R. G. J . Chem. €doc. 1882,5 9 , 926. (3) Unger, S.E.; Cooks, R. G.; Mata, R.; McLaughlin, J. L. J. Nat. Prod. 1880,43, 288. (4) Davls, D. V.; Cooks, R. G. J. Agric. Food Chem. 1982, 30, 495. (5) Cooks, R. G. NBS Spec. Publ. (US.)1979,No. 519, 609. (6) Zakett, D.; Clupek, J. D.; Cooks, R. G. Anal. Chem. 1981,5 3 , 723. (7) Perchalski, R. J.; Yost, R. A.; Wilder, B. J. Anal. Chem. 1982, 54, 1486. (8) Haddon, W. F. Org. Mass Spectrom. 1980, 15, 539. (9) Zakett, D.; Schoen, A. E.; Kondrat, R. W.; Cooks, R. G. J . Am. Chem. SOC. 1978, 101, 6781. (10) Schuetzle, D.; Riley, R. L.; Prater, T. J.; Harvey, T. M.; Hunt, D. F. Anal. Chern. 1982,5 4 , 265. (11) Shabanowltz, J.; Hunt, D. F. Anal. Chem. 1982,5 4 , 574. (12) van der Peyl, G. J. Q.; Haverkamp, J. Q.; Kistemaker, P. G. Int. J. Mass Spectrom. Ion Phys. 1982,42, 125. (13) Stoll, R.; Rollgen, F. W. Ow.Mass Spectrom. 1979, 14, 642. (14) Cotter, R. J. Anal. Chem. 1981,5 3 , 719. (15) Conzemlus, R. J.; Capellen, J. M. Int. J . Mass Spectrom. Ion Phys. 1880, 3 4 , 197. (16) Busch, K. L.; Cooks, R. G. Science 1982,278, 247. (17) Zakett, D.; Schoen, A. E.; Cooks, R. G.; Hemberger, P. H. J . Am. Chem. SOC. 1981, 103, 1295. (18) Rollgen, F. W.; Giessmann, U.; Bochers, F.; Levsen, K. Org. Mass Spectrom. 1978, 73,459. (19) Hunt, D. F.; Bone, W. M.; Shabanowitz, J.; Rhodes, J.; Ballard, J. M. Anal. Chem. 198‘1,5 3 , 1704. (20) Busch, K. L.; Unger, S. E.; Vlncze, A,; Cooks, R. 0.; Keough, T J . Am. Chem. SOC. lg82, 100, 5615. (21) Balasanmugam, K.; Dang, T. A.; Day, R. J.; Hercules, D. M. Anal. Chem. 1981,5 3 , 2296. (22) Vanderborgh, N. E.; Roland Jones, C. E. Anal. Chem. 1983, 55, 527. (23) Kapadla, G. J.; Shah, N. J.; Zalucky, T. B. J. Pharm. Sci. 1968,5 7 , 9.54.. --
(24) Pardanani, J. H.; McLaughlln, J. L.; Kondrat, R. W.; Cooks, R. G. Lloydia 1977,40, 585. (25) Pummangura, S.; McLaughlin, J. L.,; Davls, D. V.; Cooks, R. G. J . Mat. Prod. 1982. 45. 277. (26) Kruger, T.-L.; Cooks, R. 0.; McLaughlln, J. L.; Ranlerl, R. L. J. Org. Chem. 1977. 42. 4161. (27) Kondrat, R. W.; Cooks. R. G. Anal. Chem. 1978,5 0 , 81A. (28) Zakett, D. Ph.D. Thesis. Purdue Universlty, 1981. (29) Schoen, A. E. Ph.D. Thesls, Purdue University, 1981. (30) Cotter, R. J. Anal. Chem. 1980,5 2 , 1767. (31) Liu, L. K.; Unger, S. E.; Cooks, R. G. Tetrahedron 1981, 3 7 , 1067. (32) Meyer, B. N.; McLaughlln, J. L. Planta Msd. 1880, 38, 91. (33) Mata, R.: McLaughlin, J. L. Rev. Latinoma. Qulm. 1982, 12, 95. (34) Unger, S. E. Ph.D. Thesls, Purdue Unlverslty, 1981. (35) Smith, I. “Chromatographlc and Electrophoretic Techniques”; Interscience: New York, 1960; Vol. I. p 396. (36) Unger, S. E.; Ryan, T. M.; Cooks, R. G. S I A , Surf. Interface Anal. W81,3,12. (37) Shapiro, R. H.; and Jenkins, T. F. Org. Mass Spectrom. 1969,2 , 771. (38) Willaman, J. J.; Schubert, B. 0. “ Alkalold-Bearing Plants and Thelr Contalned Alkaloids”; U S . Government Printing Office: Washington, DC, 1961; p 247. (39) McLaughlln, J. L.; Paul, A. G. Lloydla 1966,2 9 , 315. (40) Kruger, T. L.; Kondrat, R. W.; Joseph, K. T.; Cooks, R. G. Anal. Biochem. 1879,9 6 , 104. (41) Liu, L. K.; Busch K. L.; Cooks, R. G. Anal. Chem. W81, 5 3 , 109. (42) Meyer, B. N.; Helfrlch, J. S.;Nlchols, D. E.; McLaughlln, J. L.; Davis, D. V.; Cooks, R. G. Y. Nat. Prod., in press. (43) Busch, K. L.; Cooks, R. G.; Gilsh, G. L.; Todd, P. J., paper presented at Pittsburgh Conference on Analytlcal Chemistry and Spectroscopy, March 1983. (44) Klstemaker, P. 0.; Lens, M. M. J.; van der Peyl, G. J. Q.; Boerbmn, A. J. H. Adv. Mass Spectrom. 1980,8A, 928.
RECEIVED for review January 3,1983. Accepted April 5,1983. This work was supported by NIH BRSG RRO-5586 (J.L.M.), by a grant from the Cactus and Succulent Society of American (J.L.M.), and by the National Science Foundation CHE 8011425 (RGC). The laser was acquired with funds provided by CONOCO.
