LC/MS performance

Method development is important. LC-MS performance, probably more than any other technique involving organic mass spectrometry, is dependent upon a range of experimental parameters, the relationship between which is often complex. While it is possible (but not always so) that conditions may be chosen fairly readily to allow the analysis of simple mixtures to be carried out successfully, the widely variable ionization efficiency of compounds with differing structures often makes obtaining optimum performance for the study of all components of a complex mixture difficult. In such cases, the use of experimental design should be seriously considered. 

Tang, L., Fitch, W. L., Alexander, M. S., and Dolan, J. W. (2000). Expediting the method development and quality control of reversed-phase liquid chromatography electrospray ionization mass spectrometry for pharmaceutical analysis by using an LC/MS performance test mix. Anal. Chem. 72 5211-5218. 

Liquid chromatography-mass spectrometiy (LC-MS) based on atmospheric-pressure ionization (API) was demonstrated as early as 1974 (Ch. 3.2.1). However, it took until the late 1980 s before API was starting to be widely applied. Today, it can be considered by far the most important interfacing strategy in LC-MS. More than 99% of the LC-MS performed today is based on API interfacing. In this chapter, instrumentation for API interfacing is discussed. First, vacuum system for MS and LC-MS are briefly discussed. Subsequently, attention is paid to instrumental and practical aspects of electrospray ionization (ESI), atmospheric-pressure chemical ionization (APCI), and other interfacing approaches based on API. The emphasis in the discussion is on commercially available systems and modifications thereof. Ionization phenomena and mechanisms are dealt with in a separate chapter (Ch. 6). Laser-based ionization for LC-MS is briefly reviewed (Ch. 5.9). 

The analysis of cocaine and its metabolites has been performed by means of GC-MS, but this demands extensive sample pretreatment and derivatization. LC-MS has been evaluated as an alternative. Simultaneous LC-ESI-MS and fluorescence detection of cocaine, benzoylecgonine, and cocaethylene in human hair was described by Clauwaert et al. [84]. The effect of mobile-phase composition and pH on the ESI LC-MS performance of cocaine, ecgoitine methyl ester, and benzoylecgoitine was investigated by Jeanville et al. [85]. The best sensitivity was achieved with a 1 1 mixture of 60% acetonitrile-40% acetone in 100 mmol/1 ammonium acetate. The pH has little influence on the response, suggesting that gas-phase rather than liquid-phase ionization is important. 

Table 15.1 Environmental applications in LC-MS performed by thermospray interface (TSP).

LC-MS should be considered as a hybrid technique, in which the features of both LC and MS are optimally used to get the best possible result. Optimum separation conditions in terms of mobile-phase composition and stationary phase for a ultraviolet (UV) detector may widely differ from the separation conditions ideally suiting the mass spectrometer. Unlike in the early days of LC-MS, performing LC-MS in most laboratories is no longer the task of specialized mass spectrometrists. The technique is entering the chromatography laboratory as well. And in many instances separate LC-MS laboratories are set up, where specialists from the two disciplines work together to get the best results. 

In the 1990s and 2(XX)s, new products were designed and developed exclusively for LC-MS performance. A broader scale of application occurred with the development of LC-MS-based methods for the analysis of novel pharmaceuticals. 

Voyksner et al. (15) reported the results from a systematic examination of the effects of solvent composition, source pressure and source temperature on the LC/MS performance. In order to do so, they had to develop an appropriate apparatus that would allow them to change the source pressure in a controlled manner and determine the nature of the ionization that resulted. The apparatus they used is shown in Figure 11. 

Mellon, F.A., Bennett, R.N., Holst, B. et al. (2002) Intact glucosinolate analysis in plant extracts by programmed cone voltage electrospray LC/MS performance and comparison with LC/MS/MS methods. Anal. Biochem., 306, 83-91. 

The liquid chromatography - tandem mass spectrometry (LC/MS/MS) technique was proposed for the determination of corticosteroids in plasma and cerebrospinal fluid (CSF, liquor) of children with leucosis. Preliminai y sample prepai ation included the sedimentation of proteins, spinning and solid-phase extraction. MS detection was performed by scanning selected ions, with three chai acteristic ions for every corticosteroids. The limit of detection was found 80 pg/ml of plasma. 

In this book, I have tried to show the way in which high performance liquid chromatography-mass spectrometry (LC-MS) has developed, somewhat slowly it has to be said, into a powerful hybrid analytical technique. 

Q-ToF-LC-MS-MS quadrupole time-of-flight mass analyser in combination with (high performance) liquid chromatography and tandem mass spectrometry... 

To understand those aspects of high performance liquid chromatography which are essential to the application of LC-MS. 

There are a number of specialist texts in which high performance liquid chromatography (HPLC) is described in varying amounts of detail (Lindsay [2] Robards et al. [3] Meyer [4]). It is not, therefore, the intention of this author to provide a comprehensive description of the technique but merely to discuss those aspects which are essential to the successful apphcation of the LC-MS combination. 

One of the functions of an LC-MS interface is to remove the mobile phase and this results in buffer molecules being deposited in the interface and/or the source of the mass spectrometer with a consequent reduction in detector performance. Methods involving the use of volatile buffers, such as ammonium acetate, are therefore preferred. 

This can potentially be overcome by the use of microbore HPLC columns with flow rates which are directly compatible with mass spectrometer operation, although the necessary decrease in injection volume results in little overall gain in the concentration of sample reaching the mass spectrometer. In addition, at the time that the DLI was available, the use of microbore HPLC, which introduces another set of potential problems related to chromatographic performance, was probably as widespread as the use of LC-MS It has been assessed [2] that in around 25% of the reported applications of DLI, microbore HPLC has been utilized. 

Although each of the previously described interfaces has advantages for particular types of analyte, there are also clear limitations to their overall performance. Their lack of reliability and the absence of a single interface that conld be used for the majority of analytes did nothing to advance the acceptance of LC-MS as a rontine technique. Their application, even with limitations, did, however, show very clearly the advantages that were to be gained by linking HPLC to MS and the efforts of many to find the ideal LC-MS interface were intensified. 

In this chapter, seven types of LC-MS interfaces have been described and their performance characteristics compared. Any modifications to the HPLC conditions that are required to allow the interface to operate effectively have been highlighted. 

In the vast majority of GC-MS applications, the chromatographic conditions employed have little or no effect on the operation of the mass spectrometer. This means that the spectrometer may be tuned for optimum performance and a number of samples containing different analytes can be analysed without operator intervention. This is not the case with LC-MS where the chromatographic conditions will invariably have a significant, compound-dependent, effect on the mass spectrometry conditions required to obtain useful analytical data. 

Table 5.22 Comparison of method performance for LC-ToF-MS and LC-MS-MS determination of Idoxifene...

Studies on carotenoid autoxidation have been performed with metals. Gao and Kispert proposed a mechanism by which P-carotene is transformed into 5,8-per-oxide-P Carotene, identified by LC-MS and H NMR, when it is in presence of ferric iron (0.2 eq) and air in methylene chloride. The P-carotene disappeared after 10 min of reaction and the mechanism implies oxidation of the carotenoid with ferric iron to produce the carotenoid radical cation and ferrous iron followed by the reaction of molecular oxygen on the carotenoid radical cation. Radical-initiated autoxidations of carotenoids have also been studied using either radical generators like or NBS.35... 

Another successful adaptation of the fully extended DFG S19 approach is the determination of, e.g., fenpyroximate in all type of berries by LC/MS/MS with APCI monitoring of positive ions directly in the S19 raw extract, and further the determination of trifluralin by LC/MS/MS with APCI monitoring of negative ions after performing a short SPE cleanup on an ion-exchange material. Similar approaches have used CC/MS/MS for, e.g., fenpropimorph and kresoxim methyl in St. John s Wort and peppermint. 

Reversed-phase Cig chromatography column. Keystone Scientific Betasil, 100 x 2.0-mm i.d., 5-pm particle size, 100 A, Part No. 105-701-2-CPF TSQ 7000 LC/MS/MS system with electrospray ionization (ESI) or atmospheric pressure chemical ionization (APCI) interface and gradient high-performance liquid chromatography (HPLC) unit, or equivalent Vacuum manifold for use with SPE cartridges (Varian Vac Elut 10 or equivalent)... 

---