Hyphenated methods in mass spectrometry

International Journal o f Mass Spectrometry and Ion Processes, 118/119 (1992) 857-873 Elsevier Science Publishers B.V., Amsterdam

857

Hyphenated methods in mass spectrometry* J.

van der Greef a'b and W.M.A. Niessen b

aTNO Btotechnology and Chemistry Institute, Department of Structure Elucidation and Instrumental Analysts, P O. Box 360, 3700 AJ Zelst (Netherlands) bDtvlston of Analytical Chemistry, Center for Bto-Pharmaceutteal Sciences, P 0 Box 9502, 2300 RA Leiden (Netherlands) (Received 26 August 1991)

ABSTRACT The on-hne couphng of analytical methods opens up the possibility of tuning the selectivity of the analysis. In this paper, attention is focused on the so-called "hyphenated" methods, where a separation technique, especially liquid chromatography or capillary electrophoresls, is coupled to mass spectrometry Recent developments in interfacing liquid-phase techmques to mass spectrometry are reviewed Other important aspects discussed in detail are related to compatlbdlty, quantitatlon, chemistry and mass analysis

INTRODUCTION

The on-line coupling of methods is of enormous potential in analytical chemistry because the selectivity of the analysis can be tuned in an optimal way, which in turn can be translated to a higher speed of analysis or an improvement of the determination limits. As a consequence of their power for both qualitative and quantitative analyses, techniques based on mass spectrometric detection have provoked great interest. The term "hyphenated methods" is, amongst others, used to describe the approaches based on the coupling of a separation method such as gas chromatography (GC), supercritical fluid chromatography (SFC), high performance liquid chromatography (LC) or electromigration methods (capillary zone electrophoresis (CE) and isotachophoresis (ITP)) with mass spectrometry in all its different forms. These types of technique are discussed in this paper, while no attention is paid to various other hyphenated approaches such as inductively coupled plasmamass spectrometry (ICP-MS) or various multidimensional MS combinations. No attention is paid to the well-established method of GC-MS. Thus, this review focuses on those hyphenated methods comprising a separation technique combined with mass spectrometry, which are presently still under * Paper presented at the 12th International Mass Spectrometry Conference, Amsterdam, The Netherlands, 26-30 August 1991. 0168-1176/92/$05.00

© 1992 Elsevier Science Publishers B.V. All rights reserved.

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considerable development, and aims at clarifying the trends in this research area in the last three years (1988-1991). In this review, only some selected aspects are highlighted since a comprehensive review has recently been published on this subject focusing on principles and strategies in development and application of liquid chromatography-mass spectrometry (LC-MS) and related techniques (SFC-MS and CE-MS) [1]. Several other general reviews in this area have been published [2-5]. Furthermore, only minor attention is paid to developments in continuous-flow fast atom bombardment (CF-FAB), as this technique is reviewed by Caprioli ([6], see also refs. 7 and 8). After a general discussion of hyphenated methods, the developments in LC-MS interfacing are briefly reviewed. Then LC-MS, CE-MS and SFC-MS are discussed in an integrated way, paying attention to aspects of compatibility, quantitation, chemistry, and mass analysis. HYPHENATED METHODS

The general concept of a hyphenated method is one that consists of different building blocks: sample pretreatment, separation, interface, detection and data handling. For an optimum result with the method, all four building blocks have to be tuned carefully: each block individually and especially their mutual relations. Often, the analytical procedures are limited by the sample pretreatment and separation processes despite the fact that high technology mass spectrometric detection and data handling are available. Therefore, more and more emphasis is placed on the interweaving of separation, interfacing and detection [9]. Hyphenated methods benefit from developments in the various fields, e.g. from developments in liquid chromatography, LC-MS interfacing, and mass spectrometry. This has given progress in recent years an exponentional character. The implementation of other LC methods, the development of new "soft" ionization methods, and the improved performance of mass analyzers have opened largely unexplored fields of application. An interesting feature of the newer LC-MS approaches, which reflects the maturity of the technique, is their strong integrated character. Separation technique, interface, and ionization technique are becoming more and more indistinguishable as separate units of the newly designed systems. Merging of interfacing and ionization principles especially can clearly be seen. In a first-generation interface such as the moving-belt interface (MBI) an attempt is made to overcome the apparent incompatibility between the introduction of a liquid into the high vacuum of a mass spectrometer by removing the liquid. In the second-generation designs existing "soft" ionization methods have been coupled with liquid introduction; CF-FAB is a clear example of this approach. In the current third-generation LC-MS systems interfacing and

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ionization have merged into a combined technique: the interface method is simultaneously an ionization technique, as is the case of thermospray (TSP) and electrospray (ESP). Liquid chromatography mass spectrometry is evolving from a combination of two techniques (LC/MS), via an interfaced, hyphenated technique (LC-MS) to an integrated approach (LCMS). Similar developments can be seen in SFC-MS and CE-MS coupling. The liquid-based soft ionization methods have also gained a place in the common MS technology as they are widely used as sample introduction techniques, not only because of the versatility and simplicity of flow injection analysis, but also especially because of their potential for analyte ionization. This is, for instance, reflected by the number of contributions at the annual meetings of the American Society for Mass Spectrometry (ASMS) in which liquid introduction techniques, viz LC-MS interfaces, are applied. A steady increase in the number of contributions can be seen: e.g. 10% in 1984, 17% in 1989, and 27% in 1991. Obviously, these numbers reflect a trend rather than the actual situation in the mass spectrometric laboratory; new methods are largely over-emphasized in the number of ASMS contributions. CE-MS and SFC-MS applications are still a minority, i.e. 1.5% and 0.5°,/0 respectively, of the total number of contributions. DEVELOPMENTS IN LC-MS I N T E R F A C I N G

Only a few of the greater than 25 LC-MS interface designs that have been reported over the past 20 years are still used in practice. These are MBI, TSP, CF-FAB, particle-beam (PBI), ESP, ionspray or pneumatically-assisted electrospray (ISP), and the heated-nebulizer interface (HNI). The relative importance of the first five interfaces and their promise for the future can be extrapolated from Fig. 1, where for each of these five interfaces the percentage

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of ASMS contributions is given for the years 1988 to 1991. Again, an overemphasis upon new techniques can be observed. For instance, the majority of the ESP and ISP contributions are devoted to characterization of high molecular weight proteins, and not to on-line LC-MS or CE-MS application. In daily practice, TSP is presently the most widely used LC-MS interface, followed by PBI in environmental applications and CF-FAB in biological and pharmaceutical applications. ESP and HNI hold great promise for the near future, especially in biological and pharmaceutical application areas, emphasizing the enormous growth in importance of atmospheric-pressure ionization. Other approaches to interfacing LC to atmospheric-pressure ionization are also described, e.g. the so-called atmospheric-pressure spray (APS), which is a TSP-like nebulizer [10,11]. Figure 2 may serve as a means to distinguish the basic features of the various LC-MS interfaces, and also depicts the three generations of LC-MS interfacing mentioned above. Three general approaches are followed in LC-MS interfacing: (a) nebulization of the column effluent, removal of the mobile phase constituents, vaporization of the analyte and subsequent ionization, as done with the MBI and PBI; (b) direct ionization from the (miniaturized) effluent stream, as done in CF-FAB; and (c) nebulization of the column effluent into either an atmospheric-pressure or a reduced-pressure region, desolvation of the droplets, followed by either gas-phase chemical ionization or ion evaporation, as done in TSP, ESP, ISP and HNI. A paper on strategies in developing LC-MS interfaces and comparison of interfaces from a technological point of view has recently been published [12]. Various review papers on specific interfaces have also been published, e.g. on MBI [13], TSP [14], CF-FAB [6-8], atmospheric-pressure ionization [15,16], and ESP [17-20].

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Some of the recent instrumental developments of the most widely used LC-MS interfaces are briefly reviewed. The application of the MBI in LC-MS has further diminished; the MBI is still successfully used in SFC-MS [21,22]. No significant instrumental improvements of the MBI have been reported. The analysis of derivatized modified DNA bases at the low picogram-high femtogramme level using the MBI in the negative chemical ionization (CI) mode may serve as a good example of an MBI application [23]. Another interesting application of the MBI in LC-MS is its use in combination with ion-pairing chromatography in combination with a micro-membrane suppressor, as used in ion chromatography, for the analysis of pyridinium and inmidazolium salts and carboxylic and sulphonic acids [24]. In the field of TSP various interesting developments have been reported. The use of laser-drilled pinholes at the tip of the vaporizer has been demonstrated, which appears especially useful in the analysis of sulphonated azo dyes [25], peptides (up to 1500 Da) [26] and other ionic compounds. The laser-drilled pinholes also improve the reproducibility of the vaporizers and avoid the need to replace the complete vaporizer upon clogging, e.g. by silica particles dissolved from the column. The demonstration of the analysis of high molecular weight peptides and proteins by TSP, as demonstrated with TSP mass spectra of myoglobin and other proteins (up to 30 kDa) by Straub and Chan [27], is another milestone in TSP development. The many successful reported applications of TSP LC-MS in not only qualitative, but also certainly in quantitative analysis, are perhaps of more practical importance (see below). TSP interfacing to ion trap mass spectrometers (ITMS) [28] has also been reported recently. Many developments were reported with the relatively new PBI. The implementation of the PBI on magnetic sector instruments further widens the potential of the PBI in LC-MS as well as in serving as a rapid and versatile alternative to direct insertion probe analysis; high resolution measurements for accurate mass determination are possible as well [29,30]. On-line FAB mass spectra from a PBI have also been demonstrated [31]. A modified PBI, featuring TSP nebulization, a spray chamber for desolvation and removal of the larger droplets, a countercurrent gas diffusion membrane separator, and a two-stage momentum separator has also been described and is commercially available [32]. Several studies are devoted to the possibilities of the PBI in quantitative (environmental) analysis [33,34]. The observation of carrier effects with the use of mobile phase additives such as ammonium acetate or oxalate has interesting prospects for improving the determination limits and linearity obtainable with the PBI, although the mechanism of this effect is not yet understood [33,34]. Analyte derivatization to enhance vaporization of the analyte particles in the ion source and/or to introduce functional groups

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favorable in the ionization, e.g., by incorporation of pentafluorobenzyl groups, further broadens the application area and improves the determination limits of the PBI [35,36]. Despite the progress in the development of the PBI, it cannot (yet) compete with the MBI in terms of determination limits. A wider field of application is the most important development in CF-FAB, i.e. not only biochemical applications with peptides and proteins, but also applications in the areas of pharmaceuticals, bioanalysis, phospholipids, pesticides and natural products [6-9]. Modification and characterization of the CF-FAB target is another topic of interest [37-39]. The field undoubtedly developing the fastest is that of ESP and ISP. The capability of obtaining mass spectra containing a series of peaks owing to multiply-charged ions of high molecular weight peptides and proteins has rapidly moved mass spectrometry into the field of biochemistry. Accurate molecular-weight determination (___0.1%) of biomacromolecules with high sensitivity opens possibilities for rapid characterization of recombinant proteins, hemoglobin variants, and so on [17-20,40-42]. Various hardware modifications of the ESP interface, originally described by Whitehouse et al. [43], have been reported [44,45]. ESP interfaces and ion sources have been described for high-resolution magnetic sector [46,47] and Fourier transform ion cyclotron resonance and (FT-ICR) [48] instruments, allowing the determination of the charge state of a particular peak in the MS or MS-MS spectrum, and for ITMS [49,50] (see below). In most on-line LC-MS applications ISP is used, which allows somewhat higher flow rates than ESP (see e.g. refs. 51 and 52). The HNI appears to be a highly versatile, robust and sensitive approach to LC-MS. Recent examples of its application are the determination of methandrostenolone [53] and a renin inhibitor [54]. Unfortunately, it is available from only one instrument manufacturer. An alternative approach, the APS system [10,11], is successfully applied in qualitative and quantitative analysis as well. COMPATIBILITY OF I N T E R F A C E S IN L C - M S A N D C E - M S

In evaluating the compatibility of an LC separation with a particular LC-MS interface and MS ionization technique, several aspects have to be taken into consideration. The type of analytes, and more specifically their polarity, primarily determines the choice of the LC-MS interface used in the analysis, provided of course that a choice of LC-MS interfaces is available within the laboratory. The choice of the LC-MS interface in turn determines the available ionization techniques and puts restrictions on the flow rate that can be used in the chromatography. Furthermore, the analyte polarity also

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Fig. 3. Apphcation areas of various LC-MS interfaceswith respect to analyte polarity, allowable flow rate and LC method. determines to a large extent the LC phase system, i.e. combination of mobile and stationary phases, that must be used in the separation. The interrelations between the analyte polarity, interface choice, flow rate allowed, and LC method of choice are schematically depicted in Fig. 3. Typically, the PBI in electron impact (El) or chemical ionisation (CI) operation can be applied for the analysis of the non-polar to medium-polar analytes, but developments with FAB ionization [31] might widen this range. TSP and HNI cover a rather broad polarity range, which is one of the reasons for their popularity. CF-FAB operates well for highly polar and ionic compounds; its popularity is probably due to the relatively minor modifications that are needed in the mass spectrometer in order to perform CF-FAB. ESP also provides excellent performance for the highly polar and ionic analytes, but its applicability extends to the analysis of polar and ionic high molecular weight compounds (up to 150 kDa). With increasing polarity of the analytes the applied LC phase system changes from normal-phase separations, using hexane modified with small amounts of polar solvents as mobile phase, via reversed-phase separations, using mixtures of water and an organic modifier such as methanol or acetonitrile, to ion-pair, ion-exchange or ion chromatography. In the latter cases, the mobile phase contains relatively high concentrations of non-volatile additives which are not compatible with routine LC-MS operation. Moreover, compatibility problems also arise as a result of the generally narrow flow rate range in which each LC-MS interface can be used. Since the MS acts as a mass-flow sensitive detector rather than as a concentration sensitive detector, e.g. a UV detector, solvent splitting inevitably results in a loss in signal by a factor equal to the splitting ratio. Basically, the LC-MS

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interfaces can be divided into two groups: interfaces limited to flow rates in the/~1 min- J range such as CF-FAB and ESP; and interfaces restricted to flow rates in the ml min -~ range such as TSP, PBI and HNI. Compatibility problems with respect to flow rate and mobile phase composition can be solved in various ways. Considerable progress has been made in this respect in the last few years. This progress is highlighted here with some examples. A multi-dimensional approach for target compound analysis, referred to as the phase-system switching (PSS) approach [55,56], has been developed to solve compatibility problems with mobile phase composition as well as with flow rate. A schematic set-up of the PSS approach, which is based on valveswitching techniques and coupled-column chromatography approaches, is shown in Fig. 4. The set-up for PSS consists of an analytical LC system followed by a trapping column (with small internal diameter for applications in combination with CF-FAB and ESP), the interface of choice and the mass spectrometer. The procedure is as follows. In step 1 the chromatography is performed with a favorable, but incompatible, mobile phase at the optimum flow rate. In step 2 the valves are switched just before and after the elution of the analyte(s) of interest, thereby heartcutting the eluting fraction(s) and collecting and enriching these fractions on the trapping column(s). In step 3 the hydrophilic non-volatile mobile phase additives, e.g. buffers or ion-pairing agents, are washed away with water. Finally, in step 4 the analytes are eluted into the interface/MS system using a flow rate and solvent composition which

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are favorable for the interface chosen. The versatility of the PSS approach is demonstrated by the variety of combinations of LC phase system, LC-MS interface and flow rates that have been reported in the past few years (see Table 1 [55-65]). Another important feature of the PSS approach is that the determination limits can be improved on one hand as a result of the analyte focusing that is achieved on the trapping column, and on the other hand because no splitting is needed in the coupling of conventional columns (3-4.6 mm i.d.) to a low flow rate interface. The latter case is an example of on-line miniaturization, while the high loadability of the system is maintained. Another approach in flow rate matching is the use of miniaturized separation systems. It must be emphasized that the miniaturization as such, basically to avoid splitting, does not improve the concentration detection limits, because the decrease in maximum injection volume is balancing the reduced splitting. Analyte focusing by injection in a solvent mixture of low elution power, followed by gradient elution, is an elegant way to increase the loadability as shown in several cases for packed fused-silica columns working in the flow rate region of 3-5 #1 min- t and directly coupled to CF-FAB [66,67] or ESP. The current progress in micro-LC, especially with respect to instrumental aspects and the ability to reliably and reproducibly produce microbore and fused-silica-packed microcapillary columns, will activate the micro-LC applications. Recently, nanoscale packed columns (50-75#m) have been applied in analyte focusing mode, which increased the loadability from 10 nl to 10#1 [68]. The loadability problem is a major limitation in CE-MS as well. So far, the potential of CE-MS has been illustrated [69-73], but papers related to applications at low determination limits are very scarce. The reason for this is that the typical injection volumes are in the range of 10 nl. In order to improve the concentration detection limits achievable in CE-MS, the use of a PATRIC array detector in the mass spectrometer instead of a conventional electron multiplier has been demonstrated [73]. More recently, the potential of multidimensional electromigration methods as a solution to this problem has been investigated. At present, the on-line coupling of isotachophoresis and capillary electrophoresis (ITP-CE) is performed [74]. The schematic diagram of the experimental set-up of ITP-CE is given in Fig. 5. The ITP itself has been coupled with mass specrometry before and was shown to be attractive in MS-MS studies [75]. In the multi-dimensional ITP-CE set-up the ITP process is used for its selectivity but, more importantly, for its capability to concentrate the zones of analytes present in low concentrations. By careful selection of the leading and terminating buffers, concentration factors of 500-1000 have been demonstrated in ITP-CE analysis of FITC-labeled peptides using laser-induced fluorescence detection and of several nucleotides

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using UV detection. The coupling of ITP-CE to MS is currently under investigation. A third example of compatibility matching is the coupling of ion chromatography to MS via a micromembrane suppressor system. This approach has been demonstrated by Simpson et al. [76] in the LC-MS analysis of amino sugars using TSP and by Conboy et al. [77] in the LC-MS analysis of quaternary ammonium and sulphate compounds using ISP. The micromembrane suppressor has also been applied to couple ion-pair chromatography to an MBI [24]. Despite the necessary splitting with a factor of 50-100 for ISP, a detection limit for a quaternary ammonium salt of 40pg with ISP has been reported. QUANTITATION

The maturity of an analytical method is often best illustrated by its ability to perform automated quantitative analysis. In the case of LC-MS, quantitative work has been published for most interfaces. For TSP LC-MS even a fully automated system is described, as shown in Fig. 6 [78]. This schematic shows a real modern hyphenated system, where the mass spectrometer is just one of the building blocks of the overall system. The performance of this automated system was investigated using an extensive series of automated repeated injections of anti-asthmatic drugs at the low picomole level. For terbutaline, a coefficient of variation (CV) was measured of 9.7% at the 1 pmol level and of 3.6% at the 32pmol level in a 19h precision measurement with the automated system [78]. The HNI, which is not so widely used, also exhibits excellent performance in the quantitation of compounds of medium polarity. Fouda et al. [54] described the quantitation of a renin inhibitor in serum in the concentration range 50 pg ml- L to 10 ng ml- ~ using the HNI interface. Over 4000 clinical samples were analyzed by this method. An interesting phenomena reported

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was the observation of a carrier effect by the addition of a structural analog of the compound of interest. Both the recovery and precision of the assay improved significantly [54]. CHEMISTRY IN LC-MS AND SFC-MS

Chemistry plays a very important role in most of the "soft" ionization methods used in LC-MS. As a consequence, derivatization of analytes can be an attractive approach in LC-MS for the improvement of the detection limits. Several examples, often directed at the enhancement of the proton affinity in TSP, have recently been summarized [9]. A more recent example is the improvement of the detection limits in the TSP LC-MS analysis of cortisol as a result of a derivatization to the 21-acetate [79]. Some other examples of derivatization, directed at volatility enhancement and/or improvement of ionization properties, have been given above [23,35,36]. The strategy of analyte derivatization is also followed for the SFC-MS analysis of oligosaccharides by Reinhold and co-workers [80-83]; it is outlined in general in an excellent SFC-MS review paper [80]. An enhancement of the detection characteristics as well as an improvement in the analyte solubility in the supercritical mobile phase is achieved as a result of the derivatization. This is of special importance in SFC-MS because the solubility range and thus the applicable analyte polarity range is relatively narrow in SFC, especially when compared with LC.

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IMPACT OF MASS ANALYZERS

Considerable developments in the improvement of mass analyzers have been made in the last decade [84]. The potential of these new mass analyzers have also been tested with hyphenated methods. Some examples of the combination of mass analyzers other than quadrupole systems to LC-MS interfaces have been mentioned above, although some of the more recent developments are still in their infancy. Some aspects of the analyzer developments which are considered to be of major importance to the field of hyphenated methods are briefly discussed here. Various other aspects are dealt with by Cooks [84]. Time-of-flight (TOF) mass spectrometry has become very popular in recent years [85], and has been activated by the development of soft ionization methods such as plasma desorption and matrix-assisted laser desorption, enabling the detection of compounds with molecular weights up to 300 kDa. The options of a high mass range and fast spectrum recording are of interest in the field of hyphenated methods as well. In GC-MS, time array detection with a TOF instrument was successfully employed to realize a high speed analysis (20 scans s -~) without sacrificing chromatographic performance or detection limits [86]. In LC-MS, a liquid secondary ion mass spectrometry (LSIMS)-TOF design has been reported for initial studies in this field [87]. This system was subsequently modified to a system based on high speed transient recording, while realizing a high duty cycle [88]. These systems are especially attractive in CE-MS, where both the high scan speed and the high mass range are of interest. Optimal use of the latter advantage will become available via coupling of laser desorption in on-line LC and CE-TOF mass spectrometry. The use of external ion sources in FT-ICR has provided the possibility of on-line coupling with separation methods without losing the advantages of FTMS, i.e. high mass resolution and multi-stage tandem mass spectrometry (MS"). The potential of LC FT-ICR coupling has been demonstrated for ESP [48] and CF-FAB [89]. In the latter case, a resolution of 60.000 (full width half height) was demonstrated for Gramicidin S. Even more impressive are the developments in ion trap mass spectrometry (ITMS). In the combination of ESP and ITMS the high mass capabilities and excellent sensitivities of such systems have been demonstrated [49]. An on-line coupling of LC-MS with ITMS based on ISP has recently been realized [50]. On-line detection and molecular weight determination of cytochrome c (MW = 12200) and myoglobin (MW = 16951) was achieved (see Fig. 7). Human serum albumin (MW = 66000) was detected but molecular weight calculations were hampered by limited resolution, possibly arising from space charging. Furthermore, M S - M S - M S experiments were performed on

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Fig. 7. LC-MS total 1on chromatogram for a synthetic protein mixture consisting of 5.5 pmol cytochrome c, 1.3 pmol human serum albumin and 3.8 pmol myoglobin. LC coupled to ion-trap MS via an ionspray interface (reprinted from ref. 50 with permission). components in a tryptic digest of the b-chain of h u m a n haemoglobin [49,50]. The performance of the ion trap system in an on-line coupling with LC separation methods is clearly demonstrated. This approach is on its way to become an exciting new tool in the field. CONCLUSIONS The examples selected from the literature of the past three years show the fast developments in the field of hyphenated methods. Both qualitative and routine quantitative analysis have been achieved with L C - M S , which makes the method an important analytical tool in addition to G C - M S . S F C - M S is more restricted owing to the smaller range o f sample polarity to be analyzed. Although having a much wider applicability range, the use of C E - M S is primarily restricted owing to the need for developments in optimizing detection and especially in enhancement of the loadability. A breakthrough in this area can be expected in the years to come. Finally, new developments in coupling mass analyzers such as TOF, F T - I C R and ITMS open a large area of new research, which will result in an even wider application field. However, balancing the separation method of choice with the mass spectrometric detection is the most important issue in the field of hyphenated methods. Developments in LC, in an interface, in MS, or in any instrument will give the major breakthrough needed. But only "hybrid vision" will do, so the hyphenated techniques must be considered as real hybrid techniques, where simultaneous, unidirectional efforts from various fields are needed in order to realize the optimum systems. REFERENCES 1 W.M A Niessen and J. van der Greef, Liquid Chromatography-Mass Spectrometry, Marcel Dekker, New York, 1992. 2 A.L Yergey, C.G. Edmonds, I A.S Lewis and M.L Vestal, Liqmd Chromatography/ Mass Spectrometry: Techniques and Applications, Plenum Press, New York, 1990

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3 M.A. Brown (Ed.), Liquid Chromatography/Mass Spectrometry: Applications in Agricultural, Pharmaceutical, and Enwronmental Chemistry, ACS Symp. Ser. 420, American Chemical Society, Washington, DC, 1990. 4 K. Vekey, D. Edwards and L.F. Zerilli, J. Chromatogr., 488 (1989) 73. 5 K.B. Tomer and C.E. Parker, J. Chromatogr., 492 (1989) 189. 6 R.M. Caprioll, Int. J. Mass Spectrom. Ion Processes, 116 (1992). 7 R.M. Capnoh (Ed.), Continuous-Flow Fast Atom Bombardment Mass Spectrometry, Wiley, New York, 1990. 8 R.M. Caprioh, Anal Chem., 62 (1990) 477A. 9 J. van der Greef, W.M.A. Nlessen and U.R. Tjaden, J. Chromatogr., 474 (1989) 5. 10 M Sakairi and H. Kambara, Anal. Chem., 60 (1988) 774. 11 M. Sakalri and H. Kambara, Anal. Chem., 61 (1989) 1159. 12 W.M.A. Nlessen, U.R. Tjaden and J. van der Greef, J. Chromatogr., 554 (1991) 3. 13 P.J. Arpmo, Mass Spectrom. Rev., 8 (1989) 35. 14 PJ. Arpino, Mass Spectrom. Rev., 9 (1990) 631. 15 E.C. Huang, T. Wachs, J.J. Conboy and J.D, Henion, Anal. Chem., 62 (1990) 713A. 16 A.P. Brutus, Mass Spectrom. Rev., 10 (1991) 53. 17 J.B. Fenn, M Mann, C.K. Meng, S.F. Wong and C.M. Whitehouse, Scaence, 246 (1989) 64. 18 J.B. Fenn, M. Mann, C.K. Meng, S.F. Wong and C.M. Whitehouse, Mass Spectrom. Rev., 9 (1990) 37. 19 R.D Smith, J.A. Loo, C.G. Edmonds, C.J. Barinaga and H.R. Udseth, Anal. Chem, 62 (1990) 882. 20 M Mann, Org. Mass Spectrom., 25 (1990) 575. 21 J.R. Perkins, D.E. Games, J.R. Startin and J. Gdbert, J Chromatogr., 540 (1991) 239. 22 J R. Perkins, D.E. Games, J.R. Startm and J. Gilbert, J. Chromatogr., 540 (1991) 257. 23 R.S. Annan, G.M. Kresback, R.W. Glese and P. Vouros, J. Chromatogr., 465 (1989) 285. 24 R.E.A. Escott, P.G. McDowell and N.P. Porter, J. Chromatogr., 554 (1991) 281. 25 M.A. McLean and R.B. Freas, Anal. Chem., 61 (1989) 2054. 26 S W. Flnk and R.B. Freas, Anal. Chem., 61 (1989) 2050. 27 K. Straub and K. Chan, Rapid Commun. Mass Spectrom., 4 (1990) 267. 28 R.E. Kaiser, Jr., J.D. Wdliams, S.A. Lammert, R.G. Cooks and D. Zackett, 562 (1991) 3. 29 L. Baczynskyj, Rapid Commun. Mass Spectrom., 4 (1990) 198 30 W.V Llgon, Jr. and S.B. Dorn, Anal. Chem., 62 (1990) 2573. 31 J.D. Kirk and R.F Browner, Biomed. Environ. Mass Spectrom., 18 (1989) 355. 32 M.L. Vestal, D. Winn, C.H Vestal and J.G. Wilkes, m M.A. Brown (Ed.), Liquid Chromatography/Mass Spectrometry: Applications in Agricultural, Pharmaceutical, and Environmental Chemistry, ACS Symp. Ser. 420, American Chemical Society, Washington, DC, 1990, p. 215. 33 T.A Bellar, T.D. Behymer and W.L Budde, J. Am. Soc. Mass Spectrom., I (1990) 92. 34 A. Apffel and M.L. Perry, J Chromatogr., 554 (1991) 103. 35 A.P Tinke, R.A M. van der Hoeven, W.M A Niessen, U.R. Tjaden and J. van der Greef, J. Chromatogr., 554 (1991) 119. 36 V. Raverdmo, J. Chromatogr., 554 (1991) 125. 37 P.S Kokkonen, W.M.A Nlessen, U.R Tjaden and J. van der Greef, J. Chromatogr., 511 (1990) 35. 38 Dynamic FAB/LSIMS setup and troubleshooting, Flsons VG Technical Note, VG Analytical Ltd, Manchester, 1990.

872

J. van der Greef et al./Int. J. Mass Spectrom. Ion Processes 118/119 (1992) 857-873

39 P.S. Kokkonen, W.M A. Niessen, U.R Tjaden and J. van der Greef, Org. Mass Spectrom., 25 (1990) 566. 40 P. Roepstrorff, Int. J. Mass. Spectrom. Ion Processes, 116 (1992). 41 A. van Dorsselaer, F. Bltsch, B.N. Green, S Jarvis, P. Lepage, R Blschoff, H.Y.J. Kolbe and C Roitsch, Blomed. Environ. Mass Spectrom, 19 (1990) 692. 42 C.H.L. Shackleton, A.M Fahck, B N. Green and H Wltkowska, J. Chromatogr., 562 (1991) 175 43 C.M. Whltehouse, R N Dreyer, M. Yamashita and J.B. Fenn, Anal. Chem, 57 (1985) 675. 44 S.K. Chowdhury, V. Katta and B T. Chalt, Rapid Commun. Mass Spectrom, 4 (1990) 81. 45 K. Hlraoka and I. Kudaka, Rapid Commun. Mass Spectrom, 4 (1990) 519. 46 M.H. Allen and I A.S. Lewis, Rapid Commun. Mass Spectrom., 3 (1989) 255. 47 C.-K Meng, C.N. McEwen and B.S Larsen, Rapid Commun. Mass Spectrom., 4 (1990) 147, 151. 48 K.D. Henry and F.W McLafferty, Org Mass Spectrom., 25 (1990) 490. 49 G.J. van Berkel, G.L. Glish and S.A. McLuckey, Anal. Chem., 62 (1990) 1284. 50 S.A. McLuckey, G J. van Berkel, G.L. Ghsh, E.C. Huang and J.D Hereon, Anal. Chem., 63 (1991) 375. 51 E.D. Lee, J.D Hereon and T.R Covey, J. Mlcrocol. Sep., 1 (1989) 14 52 S. Pleasance, M.A. Quilham, A.S.W. de Freltas, J C. Marr and A.D. Cerebella, Rapid Commun. Mass Spectrom, 4 (1990) 206. 53 P.O. Edlund, L. Bowers and J.D. Henion, J. Chromatogr., 487 (1989) 341. 54 H. Fouda, M. Nocerim, R. Schneider and C. Gedutls, J. Am Soc. Mass Spectrom., 2 (1991) 164 55 J. van der Greef, W.M A Niessen and U.R. Tjaden, J. Pharm Blomed Anal., 6 (1988) 565. 56 E.R. Verhelj, H.J.E.M Reeuwijk, G.F. LaVos, W.M.A. Niessen, U.R Tjaden and J. van der Greef, Biomed. Environ. Mass Spectrom., 16 (1988) 393. 57 A Walhagen, L.E. Edholm, C.E M. Heeremens, R.A.M. van der Hoeven, W.M.A. Niessen, U.R. Tjaden and J. van der Greef, J. Chromatogr., 474 (1989) 257. 58 W M.A. Nlessen, U.R. Tjaden and J. van der Greef, in M.A. Brown (Ed.), Liquid Chromatography/Mass Spectrometry' Apphcatlons in Agricultural, Pharmaceutical, and Environmental Chemistry, ACS Symp. Ser. 420, American Chemical Society, Washington, DC, 1990, pp. 179. 59 P.S. Kokkonen, W.M.A. Niessen, U.R. Tjaden and J. van der Greef, Rapid Commun Mass Specrom., 5 (1991) 19. 60 P.S. Kokkonen, W.M.A. Niessen, U.R. Tjaden and J van der Greef, J Chromatogr., 565 (1991) 265. 61 J. Guthne and G. Radolovlch, Proc. 39th ASMS Conf. Mass Spectrometry and Allied Topics, 1991, Nashville, TN, 1991. 62 R.J. Seymour, Proc. 39th ASMS Conf. Mass Spectrometry and Alhed Topics, Nashvdle, TN, 1991. 63 R.J Vreeken, G. Bakker, J. Brakenhoff, G.J. de Jong, R W. Frel and U.A.Th. Brmkman, paper presented at the 18th Int. Symp. Chromatogr., Amsterdam, 23-28 September, 1990 64 R.J. Vreeken, W D. van Dongen, R. Ghijssen, G J. de Jong, H. Lmgeman, U A.Th Brinkman, R.G.J. van Leuken, G.T C. Kwakkenbos and R S. Deelder, Biol. Mass Spectrom., m press. 65 R.G.J. van Leuken, G.T.C. Kwakkenbos and W.D. van Dongen, Abstracts 12th International Mass Spectrometry Conference, 26-30 August 1991, Amsterdam, p 225

J. van der Greef et aL/Int. J. Mass Spectrom. Ion Processes 118/119 (1992) 857-873

66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89

873

W.J. Henzel, J.H. Bourell and J.T. Stults, Anal. Biochem., 187 (1990) 228. J.E. Coutant, T.-M. Chen and B.L. Ackermann, J. Chromatogr., 529 (1990) 265. M.A. Moseley, L.J. Deterding, K.B. Tomer and J.W Jorgenson, 63 (1991) 1467. R D. Smith, J.A. Loo, C.J. Barinaga, C.G. Edmonds and H.R Udseth, J. Chromatogr, 480 (1989) 211. I.M. Johansson, E C. Huang, J.D. Hereon and J. Zwelgenbaum, J Chromatogr., 554 (1991) 311. M.A. Moseley, L.J. Deterding, K.B Tomer and J.W. Jorgenson, Anal. Chem., 63 (1991) 109. R M. Caprioli, W.T. Moore, M. Martin, B.B Dague, K. Wdson and S. Morning, J. Chromatogr., 480 (1989) 247. N.J. Reinhoud, E. Schr6der, U.R. Tjaden, W M.A Niessen, M.C. ten Noever de Brauw and J. van der Greef, J. Chromatogr., 516 (1990) 147. D.S. Stegehuls, H. Irth, U.R Tjaden and J. van der Greef, J. Chromatogr., 565 (1991) 265. H R. Udseth, J.A. Loo and R.D. Smith, Anal. Chem., 61 (1989) 228 R C. Simpson, C.C. Fenselau, M.R. Hardy, R.R. Townsend, Y.C. Lee and R J. Cotter, Anal. Chem., 62 (1990) 248. J.J. Conboy, J D. Henion, M.W Martin and J.A Zwelgenbaum, Anal. Chem., 62 (1990) 800. C. Llndberg, J. Paulson and A. Blomqvlst, J. Chromatogr., 554 (1991) 215. J Paulson and C. Lindberg, J. Chromatogr., 554 (1991) 149. D.M Sheeley and V N. Reinhold, J. Chromatogr, 474 (1989) 83. J.P. Caesar, Jr., D.M. Sheeley and V.N. Reinhold, Anal. Biochem., 191 (1990) 247 M.V Merntt, D.M. Sheeley and V.N. Reinhold, Anal. Biochem., 193 (1991) 24. D.M. Sheeley and V.N. Reinhold, Anal. Biochem., 193 (1991) 240. R.G. Cooks, Int. J. Mass Spectrom. Ion Processes, 116 (1992). D. Price and G.J. Mdnes, Int. J. Mass Spectrom. Ion Processes, 99 (1990) 1. E.D. Erickson, C.G. Enke, J.F. Holland and J.T. Watson, Anal. Chem., 62 (1990) 1079 K -H. Chen and R.J. Cotter, Rapid Commun. Mass Spectrom., 2 (1988) 237. W.B. Emary, I Lys, R.J. Cotter, R. Simpson and A. Hoffman, Anal. Chem., 62 (1990) 1319. C.H. Watson, G Kruppa, J Wronka and F.H. Lauklen, Rapid Commun. Mass Spectrom., 5 (1991) 249.