Hyphenated Techniques, Applications of in Mass Spectrometry

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Hallam HE (1973) Vibrational Spectroscopy of Trapped Species. London and New York: Wiley. Hadzi D (ed) (1997) Theoretical Treatments of Hydrogen Bonding. London and New York: Wiley. Jeffrey GA (1997) An Introduction to Hydrogen Bonding. Oxford University Press. Kamlet MJ, Solomonovici A and Taft RW (1979) Linear solvation energy relationships, 5: correlations between infrared ∆ν values and the β scale of hydrogen-bond acceptor basicities. Journal of the American Chemical Society 101: 3734–3739.

Pimentel GC and McClellan AL (1960) The Hydrogen Bond. San Francisco: Freeman. Rao CNR, Dwivedi PC, Ratajczak H and Orville-Thomas WJ (1975) Relation between O–H stretching frequency and hydrogen bond energy: re-examination of the Badger–Bauer rule. Journal of the Chemical Society, Faraday Transactions II 71: 955–966. Scheiner S (1997) Hydrogen Bonding: A Theoretical Perspective. Oxford University Press. Tomkinson J (1992) The vibrations of hydrogen bonds. Spectrochimica Acta 48A: 329–348.

Hyphenated Techniques, Applications of in Mass Spectrometry WMA Niessen, hyphen MassSpec Consultancy, Leiden, The Netherlands Copyright © 1999 Academic Press

Introduction One of the most fascinating fields of instrumental development in mass spectrometry (MS) is hyphenation: the on-line coupling of various techniques to MS. Apart from the obvious combinations of straightforward coupling of gas chromatography (GC) or liquid chromatography (LC) to MS, a wide variety of other combinations has been described. This contribution pays attention to the rationale of hyphenated techniques and briefly indicates a number of typical applications. The key idea behind hyphenation is the significant gain in signal-to-noise ratio, and thus improvement in detection limit, that can be achieved by multidimensional methods. This concept was nicely pictured by Yost and co-workers, in the early 1980s (Figure 1). While in hyphenation the response or signal achieved decreases with the increasing number of couplings or dimensions, the (chemical) noise decreases even faster due to the increased selectivity, thus resulting in an improved signal-to-noise ratio. Another important issue is the ability to avoid sample losses and sample contamination in on-line rather than off-line combinations. In principle, one can discriminate between various approaches to hyphenation, depending on the primary objective: (a) On-line separation technique coupled to MS, e.g. GC-MS and LC-MS.

MASS SPECTROMETRY Applications

(b) On-line sample pretreatment in combination with a separation technique coupled to MS. (c) On-line multidimensional separation techniques coupled to MS. (d) On-line coupling of a separation technique to multiple detection strategies, one of which is MS. Obviously, a combination of these strategies is also possible. Examples of these approaches are given below.

Tandem mass spectrometry The first hyphenated approach to be considered is the on-line combination of MS and MS, i.e. tandem mass spectrometry (MS-MS). A variety of combinations of different mass analysers have been described, including quadrupole and magnetic-sector analysers as MS1, and quadrupole, magnetic-sector, ion-trap and time-of-flight analysers as MS2. Instruments like triple-quadrupoles are widely used for MS-MS, either as stand-alone systems with sample introduction via a solids insertion probe or flow-injection analysis, or in on-line combination with GC or LC. The work of Yost and co-workers and of Hunt and colleagues exemplify these methods. In the most common mode, i.e. the product-ion mode, MS1, is used to select a precursor ion with a particular m/z from the variety of ions generated in the ion source. The mass selected ions are dissociated via collisions with an inert gas in a collision cell, and

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selective-reaction monitoring (SRM) mode, selecting a particular precursor ion in MS1 and selecting and detecting one or more product ions in MS2. The excellent selectivity, the high level of confidence of identity, and the ability to use stable isotopically labelled internal standards are important arguments for the use of SRM in this type of routine applications.

On-line chromatography–mass spectrometry

Figure 1 Hyphenation of analytical techniques. Dependence of the signal S, the noise N, and the signal-to-noise (S/N ) ratio on the number of analytical stages.

the product-ions are subsequently mass-analysed by MS2. In this setup, MS1 can be considered as a separation technique, while MS2 is operated as a conventional mass analyser and detector. Analogies between MS-MS and GC-MS have frequently been drawn (Figure 2). It has been argued whether a chromatographic separation is actually still required in a combination with MS-MS, given the excellent selectivity that can be achieved. However, it is currently generally agreed that some separation is required, at least in the analysis of samples of biological or environmental origin, in order to avoid rapid contamination of the ion source and in order to reduce and/ or avoid analyte ion suppression effects, i.e. in electrospray ionization. MS-MS is currently very widely used in combination with chromatographic separation methods, especially LC. The obvious reason for this is the frequent use of soft ionization techniques in LC-MS interfacing, i.e. electrospray and atmospheric-pressure chemical ionization. MS-MS allows additional structural information as well as the molecular mass information to be obtained. On-line LC-MS-MS is currently the method-of-choice in quantitative bioanalysis in (pre-)clinical pharmacological studies during drug development in pharmaceutical industries. In these studies, the instrument is operated in

The on-line combination of a chromatographic separation technique (GC, LC, but also thin-layer chromatography (TLC), capillary zone electrophoresis (CZE) and supercritical fluid chromatography (SFC)) with MS enables the mass spectrometric characterization of components in complex mixtures after separation with minimal or no sample loss. It is especially useful in the identification of minor or trace components that are difficult to collect by fractionation of the column effluent or would be easily lost. Furthermore, as already indicated above, on-line GC-MS and LC-MS-MS are important tools in quantitative bioanalysis as well. Obviously, fractionation in large series of samples for routine quantitative applications would be extremely time-consuming and ineffective. GC-MS plays an important role in many application areas, including the characterization of components in petroleum and derived products and in essential oils, the identification and quantitation of compounds of environmental interest such as polychlorodibenzodioxins and related compounds, polycyclic aromatic hydrocarbons, pesticides and herbicides, and a variety of other microcontaminants. In addition, GC-MS is important in the analysis of compounds of pharmacological, forensic and/ or toxicological interest, including drugs, anaesthetics, steroids, growth hormones and drugs of abuse.

Figure 2

Schematic comparison of MS-MS and GC-MS.

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LC-MS is applied in complementary fields, where the analytes are not amenable to GC-MS. LC-MS is for instance applied in the identification and quantitation of pesticides, herbicides, surfactants and (sulfonated) azo dyes in environmental samples, in the identification of drugs during drug development, their degradation products and metabolites, in the quantitative bioanalysis of drugs and related compounds in biological tissues and fluids, in the characterization of natural products, such as alkaloids, taxoids, toxins, as well as endogenous compounds like acylcarnitines, prostaglandins, bile acids. Furthermore, LC-MS plays an important role in biochemical and biotechnological applications of MS, via the electrospray MS and electrospray LC-MS analysis of biomacromolecules like peptides, proteins and DNA fragments. Methods and some applications of GC-MS and LC-MS are discussed in a separate contribution. The on-line combination of TLC and MS via FAB, liquid SIMS, or MALDI has also been frequently described. The TLC-MS combination, recently reviewed by Somsen and co-workers, is applied for a wide variety of compounds, including drugs and their metabolites, antibiotics, steroids, alkaloids, lipids, bile acids, porphyrins, dyes and peptides.

On-line sample pretreatment With the advent of LC-MS technologies, the on-line combination with various sample pretreatment strategies received considerable attention. The rationale for on-line sample pretreatment is to avoid sample losses and sample contamination during the off-line transfer from one step of the analytical procedure to another. In addition, an on-line procedure greatly facilitates automation of the complete procedure, thereby speeding up the analysis. The most successful and most widely applied approach is on-line solid-phase extraction (SPE) in combination with LC-MS. A number of instruments have been developed and commercialized for this combination, e.g. the Varian AASP, the Gilson ASPEC, the Spark Holland Prospekt, and the Merck OSP-2. In all these automated systems, the sample constituents within a certain polarity range are trapped onto a short cartridge column (or Empore disk) containing reversedphase LC packing material. The cartridge or disk may be washed with water to remove hydrophilic sample constituents. Subsequently, the analytes of interest are desorbed and transferred to an LC column for separation. These on-line SPE strategies serve for both sample pretreatment and analyte preconcentration. While on-line SPE-LC-MS was initially mainly

developed for quantitative bioanalysis, the most important current application is in environmental analysis, i.e. in the selective sample pretreatment and analyte preconcentration of pesticides, their degradation products as well as other microcontaminants from surface water as an on-line part of the SPE-LCMS or SPE-LC-MS-MS analysis. The results of the group of Brinkman show that analyte preconcentration by factors up to 103 or 104 are feasible in pesticide analysis from surface water, enabling concentration detection limits well below 0.1 µgL–1. Consecutively, the same group demonstrated that SPE can also be applied in an on-line combination with GC-MS. An example is the SPE-GC-MS analysis of 10 mL of river Rhine water spiked at the 0.5 µgL–1 level with 80 microcontaminants, such as chlorobenzenes, aromatic compounds, anilines, phenols and organonitrogen and organophosphorus pesticides. In addition to SPE, other sample pretreatment methods have been combined with GC-MS (see Goosens and co-workers), LC-MS or to MS(-MS) directly, e.g. on-line membrane sample introduction, solid-phase micro-extraction (SPME), supercritical fluid extraction and membrane dialysis. A variety of on-line sample pretreatment procedures have been described for the coupling to on-line capillary zone electrophoresis (CZE)-MS, including capillary isotachophoresis, electrodialysis, liquid– liquid electroextraction and SPE on Empore disks. The latter system was applied to the analysis of the neuroleptic drug haloperidol in patients’ urine by on-line Empore-disk SPE-CZE-MS.

Multidimensional separation techniques Multidimensional separation techniques have been developed with a number of objectives such as increased peak capacity and/or improved resolution for the separation of highly complex samples, shortened analysis time via heartcut and partial analysis of fractions from complex samples, and enhanced detection of trace components. Both GC-GC, LC-GC, and LC-LC methods as well as various other multidimensional combinations involving CZE or SFC have been described and coupled to mass spectrometry. In most cases, the multidimensional approach comprises a combination of two chromatographic columns, either containing two different stationary phases (both GC and LC), or developed with two different mobile phases (LC only). The sample is injected onto the first column. Part of the chromatogram is heart-

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cut, either sampled onto a retention gap or short trapping column or transferred directly, and subsequently analysed on the second column, which is then interfaced to an MS. A typical setup for LC-LCMS is shown in Figure 3. Several examples of on-line multidimensional GC-MS were reviewed by Ragunathan and coworkers including the characterization of essential oils in order to determine the composition, to identify or quantify enantiomers, or to study the chemotaxonomy of the oil, and the determination of coplanar polychlorobiphenyls congeners. The on-line combination of LC-GC-MS is applied in a variety of analytical problems, such as the analysis of (trace amounts of) aromatic compounds in complex fossil fuel fractions or in vegetable oil, the identification of microcontaminants in surface water, and the identification of impurities in pharmaceutical products. The on-line combination of LC-LC-MS has been investigated for a number of reasons. In addition to the general prospects of multidimensional separation techniques, especially enhanced selectivity, there was special interest in the ability to perform LC-LC with two different mobile-phase compositions. In this way, it should be possible to avoid problems with mobile-phase incompatibility due to the use of nonvolatile mobile-phase constituents. A good example of this approach is the determination of enantiomers of β-blockers in plasma samples, described by Edholm and co-workers. Racemic mixtures of a β-blocker like metoprolol can be separated on a α1-acid glycoprotein column. However, the chromatography requires the use of a 20 mM phosphate buffer (pH 7) in the mobile phase, which is not compatible with on-line LC-MS. Therefore, the chiral column was coupled via a set of two trapping columns to a common reversed-phase LC column. After separation, the two enantiomers were sepa-

Figure 3 system.

Schematic diagram for a coupled-column LC-LC-MS

rately trapped onto two short columns and subsequently transferred to the second LC column for analysis with a mobile phase containing ammonium acetate, which is well compatible with the thermospray LC-MS interfacing applied in this study. Subsequently, it was demonstrated that by the use of MS-MS the second separation step could be avoided: the effluent from the trapping column can be directly introduced into the MS-MS system, operated in SRM mode. This phase-system switching approach is well suited for solving this type of mobile-phase incompatibility problem.

Multiple detection strategies The increasingly more difficult analytical problems to be solved is an important impetus in all developments in hyphenated techniques, but especially in multidimensional detection strategies. Development of adequate separation techniques for the analysis of complex samples is often a difficult task, independent of the question whether one is interested in only one compound present at trace level, or in characterization and structure elucidation of most sample components. Multidimensional separation techniques were developed in order to achieve the separation of the components of very complex samples. MS is an extremely powerful technique for the identification and structure elucidation of unknowns. The sensitivity of MS as a fairly universal technique in this area is unsurpassed. However, in many instances the information obtained from MS is not sufficient to enable unambiguous identification of the unknown compounds. Furthermore, in certain application areas it is obligatory to identify the components with such a high level of confidence that independent results from a second technique in addition to MS are required, as for instance for impurity screening of pharmaceutical products. Improved levels of confidence in identification and structure elucidation can be achieved by the use of multiple detection strategies. Although detection with a variety of detectors can be achieved via multiple analysis of the sample on systems equipped with different detectors, there has been a growing interest and need for systems which enable the use of multiple detectors coupled to a single analytical separation system. This is especially important in two areas, i.e. in the analysis of very complex samples where multiple detection strategies coupled to one single data-acquisition and processing system can greatly facilitate the interpretation of the data, and in the high throughput analysis where minimization of the analysis time is of utmost importance.

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Several dual or multiple detection systems involving MS have been described. In this respect, one should not forget that the mass spectrometer is a destructive detector. Therefore, the mass spectrometer should be the final detector in a series. Combination with another destructive detector, e.g. a flame ionization detector, is possible only after splitting the column effluent, while in-line coupling with a nondestructive detector, e.g. a UV absorbance detector, is only possible when this detector does not result in too much chromatographic peak broadening. In several cases, the second nondestructive detector is used in parallel rather than in series, e.g. because the sensitivities of both detectors are widely different, or in order to avoid the loss of chromatographic resolution due to excessive peak broadening by the first detector in the series. The most widely applied dual detector strategies are those where MS is combined with another spectrometric technique, in order to obtain complementary structural information. Two types of spectrometric detectors have been widely used in an in-line combination with GC-MS, i.e. Fourier-transform infrared (FT-IR) and atomic emission spectrometry (AED). Both FT-IR and AED are used in parallel with the MS, i.e. after a split. GC-FT-IR-MS systems are commercially available. When the measured IR spectrum of an unknown is not present in the IR spectral library, the FT-IR part of the system can help in the identification of an unknown by providing information on functional groups and the structural backbone. While homologues cannot readily be identified from IR spectra but easily discriminated by MS, the FT-IR part can be used as an important tool in discriminating between structural isomers where MS generally fails. Typical applications of GC-FT-IR-MS can be found in the analysis of essential oils, flavours and fragrances, and in the identification of isomeric reaction products or other types of isomeric compounds. The potential of a dual detection GC-FT-IR-MS system was also explored for the identification of illicit drugs, e.g. in tracing banned stimulants used by athletes. The combination of GC-AED-MS to our knowledge is not (yet) commercially available. The element specificity of the AED can be of great help in identifying particular classes of compounds in complex samples, e.g. in environmental screening. The AED provides information on the presence of certain elements, e.g. halogens, nitrogen, or phosphorus, in the chromatographic peaks detected. This facilitates the search for relevant peaks as well as the interpretation of the mass spectra of particular compounds in a complex chromatogram. In addition, the AED can

provide a fairly accurate determination of the elemental composition, thereby often excluding the need for accurate mass determination by expensive magnetic sector instruments. Furthermore, GC-AEDMS can be applied in the analysis of organometallic compounds, e.g. organomercury and organotin compounds. The availability of nondestructive flow-through detectors for LC, e.g. UV absorbance and fluorescence detectors, readily leads to the application of dual detection systems in LC-MS. While the additional information of single-wavelength UV detection is generally limited, the availability of efficient and sufficiently sensitive UV-photodiode array detectors enables the on-line acquisition of UV spectra as well as mass spectra (Figure 4). Although the structural information available from a UV spectrum is generally limited, especially in a relatively undefined solvent mixture during gradient elution, the on-line combination can successfully be used in facilitating identification when appropriate UV spectral libraries are available. The combination of retention time in a well defined LC system, the match of a UV spectrum, and the molecular mass (and possible structural information) from MS are especially useful for confirmation of identity, e.g. in multiresidue screening for pesticides in environmental samples or antibiotics in food products. Furthermore, an excellent feature of UV-PDA systems in combination with appropriate software is peak purity assessment, based on changes of the UV spectra over the chromatographic peak. This feature may be helpful in impurity profiling by LC-UV-PDA-MS. Although the use of FT-IR in combination with LC is of growing importance, the on-line or in-line combination of FT-IR and MS is not yet frequently applied. However, the on-line combination of LC-MS and NMR is currently introduced as an important tool in identification of impurities, degradation products, and/or metabolites of pharmaceutical products. A recent example of on-line LC-NMR-MS is the characterization of ibuprofen metabolites in human urine. Simultaneous acquisition of electrospray MS and NMR spectra of synthetic drug products in an ‘open-access’ setting has also been reported.

Final considerations and perspectives Hyphenation in relation to MS is an important research topic because in many instances the optimum tuning of the two or more parts of the setup requires

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Figure 4

An example of typical results from an LC-UV-PDA-MS setup for dual detection of separated compounds.

considerable attention. Good knowledge of both parts is required in order to avoid the often inevitable compatibility problems and to obtain best possible results. However, hyphenated MS techniques have already widely proved their value in solving real analytical problems. In principle, the use of advanced data-processing software could be considered as a hyphenated approach as well, but in general is not. However, one can question whether the use of principal component analysis and similar data-processing techniques does not have a similar impact on the analytical procedure and its result as the hardware hyphenation discussed above. Software for both controlling the hyphenated combination of techniques and efficient processing of the data is of utmost importance to the breakthrough and general use of a hyphenated technique. In this respect, the development of automatic tuning and calibration software for GC-MS and more recently LC-MS, of computer spectral library searching after electron ionization, and of efficient quantitation software packages after both GC-MS and LCMS can be considered as important steps in the development of hyphenated techniques. Control of the complete (often multivendor) instrumentation,

including the GC or LC chromatograph, the sample processor or autosampler, on-line or off-line second (scanning) detectors from within a single software platform, often the MS control and acquisition software, is also essential and obligatory for the success of the technique. Some instruments enable high levels of automation and control via the use of macro scripts or instrument control languages. Such approaches may be used for optimization and/or control of the various steps in the hyphenated technique, but also for artificial-intelligence type of applications where the software makes decisions concerning the analytical strategy to follow or the type of experiments to perform on the basis of the data acquired. The latter is, for instance, applied in data-dependent product-ion scanning during GCMS-MS and LC-MS-MS. Progress is also made in software for mass spectral interpretation, especially for the so-called deconvolution of (mixed) ion envelopes of multiply charged protein ions generated by electrospray ionization, and for the peptide sequencing by MS-MS techniques. In that respect, the development of tools to search extensive protein and DNA databases using peptide maps, peptide molecular masses, LC retention time data, and sequence tags is an enormous

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step forward in the routine application of hyphenated MS-MS. Excellent examples of the hybrid of hyphenated hardware, automated sample processing via computer control of the instrumentation, and automated data processing can be found in approaches recently developed in relation to combinatorial chemistry. In order to screen a particular combinatorial library in a 96-well plate, high throughput electrospray LCMS is applied. The data are postprocessed by means of a browser, which compares the molecular mass measured with expected values, taking into account the various cationized and anionized species that may be generated during electrospray ionization. By means of green and red colours, the software indicates on a screen representation of the 96-well plate which samples were found to be present and which were not. In addition to this, and using similar software strategies, the use of automated fraction collection after preparative LC under control of the data acquired by MS has been demonstrated. This hyphenated approach allows the purification of particular products from extensive combinatorial libraries based on the results of a first biological screening and prior to a more advanced biological screening, using the purified products. See also: Atmospheric Pressure ionization in Mass Spectrometry; Biochemical Applications of Mass Spectrometry; Chemical Structure Information from Mass Spectrometry; Chromatography-MS, Methods; Isotopic Labelling in Mass Spectrometry; Medical Applications of Mass Spectrometry; MS-MS and MSn.

Further reading Brotherton HO and Yost RA (1984) Rapid screening and confirmation for drugs and metabolites in racing animals by tandem mass spectrometry. American Journal of Veterinary Research 45: 2436. Edholm L-E, Lindberg C, Paulson J and Walhagen A (1998) Determination of drug enantiomers in biological samples by coupled column liquid chromatography and

liquid chromatography–mass spectrometry. Journal of Chromatography 424: 61. Hankemeier Th, Van Leeuwen SPJ, Vreuls RJJ and Brinkman UATh (1998) Use of a presolvent to include volatile organic analytes in the application range of on-line solid-phase extraction–gas chromatography–mass spectrometry. Journal of Chromatography A 811: 117. Hogenboom AC, Speksnijder P, Vreeken RJ, Niessen WMA and Brinkman UATh (1997) Rapid target analysis of microcontaminants in water by on-line singleshort-column liquid chromatography combined with atmospheric-pressure chemical ionization tandem mass spectrometry. Journal of Chromatography A 777: 81. Hunt DF, Shabanowitz J, Harvey TM and Coates ML (1983) Analysis of organics in the environment by functional group using a triple quadrupole mass spectrometer. Journal of Chromatography 271: 93. Kitson FG, Larsen BS and McEwen CN (1996) Gas Chromatography and Mass Spectrometry. A Practical Guide. London: Academic Press. Niessen WMA (1998) Liquid Chromatography–Mass Spectrometry, 2nd edn. New York: Marcel Dekker. Ragunathan N, Krock KA, Klawun C, Sasaki TA and Wilkins CL (1995) Multispectral detection for gas chromatography. Journal of Chromatography A 703: 335. Slobodnik J, Hogenboom AC, Vreuls JJ et al (1996) Tracelevel determination of pesticide residues using on-line solid-phase extraction–column liquid chromatography with atmospheric pressure ionization mass spectrometric and tandem mass spectrometric detection. Journal of Chromatography A 741: 59. Somsen GW, Morden W and Wilson ID (1995) Planar chromatography coupled with spectroscopic techniques. Journal of Chromatography A 703: 613. Vreuls JJ, Bulterman A-J, Ghijsen RT and Brinkman UATh (1992) On-line preconcentration of aqueous samples for gas chromatographic–mass spectrometric analysis. Analyst 117: 1701. Walhagen A, Edholm L-E, Heeremans CEM et al (1989) Coupled-column chromatography–mass spectrometry. Thermospray liquid chromatographic–mass spectrometric and liquid chromatographic–tandem mass spectrometric analysis of metoprolol enantiomers in plasma using phase-system switching. Journal of Chromatography 474: 257.