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Coupling mass spectrometers in tandem MS/MS) can greatly increase the specificity of MS ana I ysis without significantly decreasing its unusual sensitivity and speed, particularly for trace levels of preselected compounds in complex organic mixtures. MS/MS also gives more detailed structural information for larger organic molecules in submicrogram quantities. Fred W. McLaffetty Cornell University, Ithaca, NY, U.S.A. Analytical mass spectrometry (MS) applied to organic molecules must be considered a mature scientific field.’ The combination of MS with gas chromatography (GC/MS), first reported 25 years ago, extended its range of application to the analysis of complex mixtures, combining the separating power of chromatography with the sensitivity, specificity, and The combination of MS with speed of MS. high-performance liquid chromatography (LC/MS),

MS/MS can be thought of as analogous to GC/MS or LC/MS, using another mass spectrometer as a separator, or as an MS with a second dimension of MS specificity. begun a decade ago, has again extended such applications to less volatile and more labile compoundsl. This should have suggested to the alert researcher the eventual development of other combined techniques, but I must confess that the remarkable growth of tandem mass spectrometry (MS/MS)le6 in the last few years caught me by surprise. MS/MS can be thought of as analogous to GC/MS or LC/MS, using another mass spectrometer as a separator, or as an MS with a second dimension of MS specificity. Recent progress in the field has been overwhelming; at least that is my impression on attempting to edit 25 outstanding chapters for a book6 covering the key areas of MS/MS.

Targeted compound analysis Much of the unusual growth of GC/MS and LC/MS owes itself to their unique applicability to the identification and quantitation of components in complex organic mixtures. Although MS/MS has been applied to ‘total’ unknowns of this type, itsforte is the analysis of preselected components. For such a case ionization conditions in the first mass spectrometer can

The specificity of mass spectrometry can be unusually high because of the literally hundreds of individual masses at which peaks can occur in a mass spectrum. be selected to enhance the yield of ions characteristic of the preselected molecule, as well as to minimize the formation of interfering ions from other components. For aqueous samples, for example, chemical ionization 0 165~9936/82/CCWaooO/$Ol

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can be employed using water as the ionizing reagent gas. The sensitivity for components of higher proton affinities is thus enhanced by several orders of magnitude without removing the water. The ions characteristic of the targeted component are separated by the first mass spectrometer (MS-I) and caused to fragment by interaction with a collision gas (termed collisionally activated dissociation, CAD). Separation ofthe resulting CAD mass spectrum in MS-II provides another dimension of specificity to the analysis; primary ions from the targeted compound can be distinguished from primary ions of the same mass from other components if their CAD mass spectra differ significantly. The specificity of mass spectrometry can be unusually high because of the literally hundreds of individual masses at which peaks can occur in a mass spectrum; this specificity can be used in two dimensions by selecting one or more peaks characteristic of the desired component in MS-I, fragmenting these ions by CAD, and separating the resulting product ions in MS-II. Replacing the chromatograph of GC/MS or LC/MS with a mass spectrometer has advantages for particular mixtures in which mass spectrometric separation is superior to chromatographic separation; isotopicallylabeled compounds are probably the most dramatic example. However, there are also many isomeric compounds that are much better separated by chromatography. The most easily recognized advantage of MS is probably speed. Although the time necessary for ion formation, separation, and detection is dependent on the particular mass spectrometer used, it is usually lo-“-lo-“; doubling such times by using MS/MS has little effect on analysis speed, especially when one considers the minutes usually required for efficient chromatographic separation. In MS the time for sample introduction, pump-out of residual sample, and data collection can vary widely, depending on the system and desired sensitivity, but in many cases continuous analyses are possible. Although this is especially true with gaseous or liquid samples, with direct probe insertion of solid samples through a vacuum lock into the ion source, analysis times of one sample per minute have been achieved. One of my favorite illustrations of targeted compound analyses by MS/MS is that of the direct analysis of lettuce for the insecticide parathion’. A 1 mm* sample (10 mg) is cut from the lettuce leaf and introduced by direct probe into the MS ion source. 0 1982 Elsevicr Scientific Publishing Company

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Heating the sample drives off volatiles whose negative chemical-ionization mass spectrum contains a peak at virtually every mass up to m/z 400; most of the peaks from mass 200+00 vary in abundance by less than an order of magnitude. Thus, to measure the parathion molecular ions at m/z 291, they must be distinguished from the ‘lettuce juice’ ions at this mass, whose relative abundance varies from sample to sample. When the mass 291 ions of pure parathion, separated by MS-I, undergo CAD, they produce two prominent fragment ions at masses 154 and 169 by cleavage of the P-O (with H rearrangement) and 0-Ph bonds, respectively, in (C~H~O)~-PS-O-CSH~-NO~-. The abundances of these ions are measured by computercontrolled setting of MS-II alternately on masses 154 and 169, while MS-I is set on mass 291. In spiked samples, this ratio agreed well with that of pure parathion and also gave a straight line plot of amount vs. abundance down to 10-l’ g, showing analytical linearity from 2.5 ppb-I ppm. The average standard deviation without use of internal standards was - 16%, running -50 samples per hour.

MS/MS structure elucidation: molecules which are difficult to purify A particularly promising application for MS/MS is in obtaining structural information from trace-level complex molecules which are difficult to purify by chromatography and other techniques. This should be especially important for high molecular weight compounds such as petroleum hydrocarbons, polymers, peptides, and other biologically important compounds. In these cases a small change in structure may cause no appreciable change in chromatographic behavior; however, if it changes the molecular weight, A particularly promising application for MS/MS is in obtaining structural information from trace-level complex molecules which are difficult to purify by chromatography and other techniques. the resolving power (20,000-200,000) of many mass spectrometers commercial double-focusing would be sufficient for separation. Methods for the ionization of higher molecular weight samples must be developed further, but this is currently a very active field of interest, with ionization of molecules with mol. wts of 3,000-10,000 being achieved by various methods. Most commercial double-focusing mass spectrometers have an upper mass limit of - 1,000 for 8 kV ions, which can be extended to mass 3,000 with a special 23 Kgauss magnet. However, research instruments with larger radius magnets, such as those at the University of New South Wales’ and Cornell5 extend this limit past mass 10,000. An example is preliminary structure information on the nonapeptide bradykinin, obtained independently using MS/MS by Professor Derrick and his students’ and by C. J. Proctor and I. J. Amster in our laboratory at Cornell. Bradykinin can be ionized using the fast

atom bombardment technique of Barber’, and the protonated-molecular ion of m/z 1060 separated by MS-I from those of other components. CAD of the separated ions then produces a secondary mass spectrum measured by MS-II; this shows the classical sequence peaks indicating part of the amino acid sequence of the peptide, with a substantial reduction of background peaks from other components and the glycerol matrix used in FAB. These results from our laboratory were recently obtained with inadequate data acquisition facilities, and a more comprehensive investigation is now in progress.

MS/MS structure elucidation: fragment ion structure information The mass spectrum provides information on the mass of the molecule and on the masses of pieces of the molecule; isotopic abundances and/or exact mass measurements can provide elemental compositions of these fragments. Interpretation of the mass spectrum involves postulations of one or more molecular structures consistent with these molecular fragments. The larger the molecule, the more information is necessary to elucidate its structure; MS/MS can aid in this by determining the actual structures of fragment ions and their spatial relationships to each other, which can greatly reduce the number of possible molecular structures consistent with the fragment information. Further, MS/MS information can be obtained on samples too small for NMR; computer automation could reduce the sample requirements to less than those even for the most advanced Fourier-transform IR spectrometers. In this approach the normal mass spectrum of the unknown molecule is determined by MS-I, and from each separated fragment ion the MS-II spectrum can provide information on its structure. The CAD spectrum of multikilovolt primary ions, ignoring those product ions formed by low energy processes, is quantitatively characteristic of the ion’s structure, independent of its internal energy or mode of formation. The fragmentation mechanisms governing CAD spectra appear to follow the same rules as those for electron or chemical ionization mass spectra, and so the structure of a fragment ion from an unknown molecule can often be determined from its CAD mass by interpretation or matching against spectrum reference CAD spectra. In recent studies of this type at Cornell this technique has been applied to the fentanyl narcotic phenyl-CHzCH(CHa) - N - piperidinyl - C2H5 and to a series of N (phenyl) - CO The EI mass spectra of the 3-hydroxypregnanes”. latter compounds contain a characteristic m/z 234 peak representing the original A, B, and C rings of the steroid. Four different CAD spectra were obtained for characteristic of the four possible these ions, stereoisomers 5a, 3o-, 5cr, 3p-, 5p, 3cr-, and 50, 3/3-. With normal EI or CI mass spectra it is not possible to obtain unequivocal stereochemical evidence without a reference spectrum of the unknown

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compound, or without comparison of the spectra of all possible stereoisomers; in this case MS/MS makes it possible to make absolute stereochemical assignments to such 3-hydroxysteroids containing a wide variety of substituents in the D ring.

could cost as little as $50,000 each in a few years. The tremendous growth in research, publications, and interest in MS/MS certainly indicates that there could be a sales volume to justi such a price.

Acknowledgements Instrumentation Metastable ion decompositions, as well as those from collisional activation (CAD), can be studied in a wide variety of ways; the latter were called ‘Aston bands’ in honor of the early mass spectrometrist who identified their source. Most CAD studies of the late 1960s and 1970s were done with ‘reverse geometry’ doublefocusing mass spectrometers, using the magnetic field as MS-I and the electrostatic analyzer as MS-II. If the separated primary ion AB+ dissociates to give A+, the kinetic energy of A+ will represent the mass ratio A+/AB+ of the original kinetic energy, and so will be focused by the electrostatic analyzer at A+/AB+ of its original value. However, the kinetic energy released in CAD broadens this value and seriously reduces the resolution of the MS-II spectrum (or the MS-I spectrum, depending on the scanning mode used). In instruments used more recently, performance is greatly improved by employing a high-resolution doublefocusing mass spectrometer for either MS-I or MS-II (‘triple analyzer’) or even for both5.

The current cost is probably the most serious disadvantage of MS/MS instruments, with prices far beyond that of the best GC/MS systems.

As research collaborators over the last 15 years I have been fortunate in having a series of very creative and capable graduate students and postdoctorates who deserve the bulk of the credit for the research which is reported here. I am also grateful to the National Institutes of Health (Grant GM16609) and the Army Research Office, Durham (Grant DAAG29-79C -0046) for generous financial support.

References 1 Burlingame, A. L., Dell, A. and Russell, D. H. (1982) Anal. Chem. 54, 363R Kondrat, R. W. and Cooks, R. G. (1978) Anal. Chem. 50,81A Yost, R. A. and Enke, C. J. (1979) Anal. C%em. 51, 1251A McLafferty, F. W. (1980) Act. Chem. Res. 13, 33 McLafferty, F. W. (1981) Science 214, 280 McLafferty, F. W. (ed.) (1982) Tandem Mass Spectrometry, John Wiley and Sons Inc., New York 7 Slaybach, J. R. B. and Story, M. S. (1981) Znd. Res. Dev., Febr. 129 8 Neumann, G. M. and Derrick, P. J. (1982) in Tandem Mass Spectrometry (F. W. McLafferty, ed.), Ch. 10, John Wiley and Sons Inc., New York 9 Barber, M., Bordoli, R. S., Elliott, G. J., Sedgwick, D. and Tyler, A. N. (1982) Anal. Chem. 54, 645A 10 Cheng, M. P., Barbalas, M. P., Pegues, R. F. and McLafferty, F. W. (submitted for publication)

Professor McLaffe@ came to the Department of Chemistry, Cornell University, Ithaca, NY 14853, U.S.A. in I!?68 after holding positions that included Director of Dow Chemical’s Eastern Research Laboratory for basic research and Professor at Purdue University. His research interests in molecular mass spectrometry include theory, mechanisms, instrumentation, computerization, and applications to chemical and biological problems. He is a member of the U.S. National Academy of Sciences and an honorary member of the Italian Chemical Society. He has received the American Chemical Society Awards in Ana&tical Chemistry and in Chemical Instrumentation, and the Spectroscopy Society of Pittsburgh Award.

The tandem quadrupole of Enke and Yost3 not only has unit resolution for both MS-I and MS-II, but has the further advantage of facile computer control, of great importance for selected ion monitoring in targeted compound analysis, Ion energies of such instruments are relatively low (lo-100 eV); for CAD an RF-only quadrupole region is used to focus the ionic products into MS-II, so that this MS/MS instrument is often called a ‘triple quadrupole’. Recent investigations indicate interesting trade-offs in the analytical applicability of these low energy collisions vs. the multikilovolt collisions of magnetic MS/MS instruments. The current cost is probably the most serious disadvantage of MS/MS instruments, with prices far beyond that of the best GUMS systems. However, this author has predicted that routine MS/MS instruments

New advisory editor for TrAC We are pleased to announce that Dr Alain Lamotte, director of the Service Central d’Analyse (SCA) in France, has recently become an advisory editor for TrAC. The SCA is a laboratory of the Centre National de la Recherche Scientifique (CNRS), and its main function is to provide a high grade analytical service. Dr Lamotte has been its director since it was formed in 1978 and we welcome him to our advisory

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board.