Coupling of LC with

A very exciting development in multidimensional separation involves the coupling of LC to GC or other techniques, such as capillary electrophoresis (CE). Online coupling of LC with multidimensional GC has allowed efficient determination of the stilbene hormones in corned beef (3), whereas LC-GC coupling permitted determination of levamisole residues in milk (4). With these hyphenated techniques, the potential of selective separation is becoming increasingly apparent. 

Coupling of LC with either ISP-MS or ISP-MS-MS has been also investigated as an attractive alternative for the determination of erythromycin A and its metabolites in salmon tissue (122). The combination of these methods permitted identification of a number of degradation products and metabolites of erythromycin, including anhydroerythromycin and N-demethyerythromycin at the level of 10-50 ppb. 

Many other interactions have been used to probe the eluent from a HPLC column, and only brief mention of a few of these can be included in this volume. The coupling of LC with mass-spectrometry (LC-MS) in particular seems to have great potential and its use will no doubt increase rapidly over the next few years. 

The possibility for unambiguous identification of the analytes is offered by liquid chromatography-mass spectrometry (LC-MS). Mass spectrometry detection systems use the difference in mass-to-charge ratio (m/z) of ionized atoms or molecules to separate them from each other. Molecules have distinctive fragmentation patterns that provide structural information to identify structural components. The on-line coupling of LC with MS for the determination of drug residues in food has been under investigation for almost two decades. 

Liquid chromatography-ionspray-mass spectrometry has been shown to be an attractive approach for the determination of semduramicin in chicken liver. Tandem MS using the CID of the molecular ions further enhanced the specificity providing strucmre elucidation and selective detection down to 30 ppb. Liquid chromatography-ionspray-mass spectrometry has also been successfully applied for the assay of 21 sulfonamides in salmon flesh. Coupling of LC with either ISP-MS or ISP-MS-MS has also been investigated as an attractive alternative for the determination of erythromycin A and its metabolites in salmon tissue. The combination of these methods permitted the identification of a number of degradation products and metabolites of erythromycin at the 10-50 ppb level. Tandem MS with CID has also been... 

Coupling of LC with tandem MS may be a solution for improving detection limits by reducing the background noise, but this combination is two or three times more expensive than its LC-MS analogue. Mass spectrometry has become a standard tool in every modern laboratory, but there will be a growing need for even more sophisticated couplings. 

Coupling of LC with mass-spectrometry has been reported for the analysis of Amaryllida-29... 

There is also a demand for a universal detector that will allow quantification of drug metabolites without the need for radioisotopes or authentic reference standards. Online coupling of LC with inductively coupled plasma mass spectrometry (LC/ICPMS) offers the ability to quantify metabolites. ICPMS is an element-specific detector with almost uniform response independent of molecular structure. The response is only related to the molar content of the detected element (Axelsson et al., 2001). Unfortunately, the use of ICPMS for metabolite profiling and identification is limited to compounds containing specific elements such as P, I, F, Br, S, and selected metals. We predict that in not too distant future technologies will be available to reliably detect, identify, and quantitate major human metabolites routinely from first-in-human studies. 

The coupling of LC with mass spectrometry is not as straightforward as a similar combination of GC with MS. There are several fundamental differences in the operating environment of HPLC and mass spectrometry. The first mismatch is the solvent flow rate. The separation in conventional wide-bore analytical columns is accomplished at liqnid flow rates of 0.5 to 1.5 mL/min. Unlike GC/MS, this liqnid produces a gas flow too large for safe operation of the mass spectrometry vacnnm system (10 to 10 torr). For example, 1.0 mL of water will pro-dnce about 1.0 x 10 m of gas load when introduced in a mass spectrometer at 10 torr pressure (see Example 5.3). Liquid flow rates below 10 xL/min can be accepted safely by a mass spectrometry system. Another problem is the... 

With the development of an atmospheric-pressure ionization (API) source, coupling of LC with MS has become a routine matter. The ESI format of API is the most appropriate interface for the LCYMS combination because (1) of its potential for the analysis of a variety of nonvolatile and thermally labile molecules of low to very high molecular mass at unprecedented low detection sensitivity, (2) ionization occurs at atmospheric pressure, (3) of its compatibility with RP-LC solvents, and (4) a range of solvent flow can be accepted. As a consequence, the LC/ESI-MS combination has gained prominence in several areas of research, such as to sequence proteins to identify mixtures of compounds, tryptic maps, and posttranslational modifications in proteins to elucidate structure of metabolic products to analyze drugs, pesticides, and toxins and to screen combinatorial libraries. The development of LC/ESI-MS has also greatly advanced the science of quantification. Several reviews of LC/ESI-MS technology have appeared in the literature [37-40]. 

In the introductory Section 2.1.3, it was discussed that an important aspect of optimization can be to improve a method for its applicability in trace analysis. The nature of the mode of detection is very relevant in this case whether the applied detector is concentration proportional like the very common UV detector or mass proportional hke nebulizer-based detectors, for example, evaporative light scattering detector (ELSD) or charged aerosol detector (CAD). This textbook contains dedicated chapters on nebulizer-based or aerosol detectors (Chapter 10 on trends in detection), as well as for the coupling of LC with mass spectrometry (Chapter 1). Here, the focus is on concentration proportional detectors UV detectors (VWD, DAD), fluorescence detectors (FLD), electrochemical detectors (ECD), and refractive index (RI) detectors. 

The coupling of LC with MS requires an appropriate interface due to the incompatibilities of the two methods. In order to introduce a conventional LC flow (0.5-1.5 ml/ min) into the mass spectrometer, one should evaporate it, producing a vapor with a volume far beyond the capacity of the mass spectrometer pump systems. The selection of the interface is the key decision and a limiting factor in the utilization of the instrument. Some years ago, LC/MS systems were state-of-the-art, space-occupying machines. 

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