LC/MS electrospray ionization

There are three major ionization sources now used for steroid LC-MS electrospray ionization MS (ESI), atmospheric pressure chemical ionization (APCI), and atmospheric pressure photoionization (APPI). Under optimal conditions, all ionization methods have similar absolute sensitivities. ESI is preferred for polar and charged (steroid conjugates) molecules, but derivatization or chemical modification of less-polar molecules may be necessary for this technique to match the sensitivity of APPI or APCI. 

Figure 4.11. Schematic diagram of an LC/MS electrospray ionization (ESI) interface showing the nebulization of the LC eluent into droplets, evaporation of the solvent, and the ionization of the analytes, which are pulled inside the MS. Diagram courtesy of Agilent Corporation.

This effect is seldom made use of in GC-MS analysis in contrast to being the prevailing reaction in ESI-LC-MS (electrospray ionization), but must be taken into account on evaluating Cl spectra. The enhanced formation of adducts is always observed with intentional protonation reactions where differences in the proton affinity of the participating species are small. High reagent gas pressure in the ion source favours the effect by stabilizing collisions. 

Among the currently available interfaces for drug residue analysis, the most powerful and promising appear to be the particle-beam (PB) interface, the thermospray (TSP) interface that works well with substances of medium polarity, and the atmospheric pressure ionization (API) interfaces that have opened up important application areas of LC to LC-MS for ionizable compounds. Among the API interfaces, electrospray (ESP) and ionspray (ISP) appear to be the most versatile as they are suitable for substances ranging from polar to ionic and from low to high molecular mass. Ionspray, in particular, is compatible with the flow rates used with conventional LC columns. In addition, both ESP and ISP appear to be valuable in terms of analyte detectability. 

Atmospheric pressure ionization (API) techniques are the most commonly used techniques in DM studies. Since the ionization occurs at the atmospheric pressure, API can be characterized as a soft ionization technique. There are three commonly used API sources [59] that can directly couple LC with MS electrospray ionization (ESI) [60], atmosphere pressure chemical ionization (APCI) [61-63], and atmospheric pressure photoionization (APPI) [64,65], The properties of the compound, such as its structure, polarity, and molecular weight, lead to the selection of one of these ionization techniques for sample analysis. 

Idborg-Bjorkman, H., P-O. Edlund, O. M. Kvalein, I. Schuppe-Koistinen, and S. P. Jacobsen. 2003. Screening of biomarkers in rat urine using LC/mass electrospray ionization-MS and two-way data analysis. Analytical Chemistry 75 4784 792. 

GC-MS, gas chromatography-mass spectrometry LC-MS, liquid chromatography-mass spectrometry FAB-MS, fast atom bombardment-mass spectrometry MS/MS, tandem mass spectrometry ESI-MS, electrospray ionization-mass spectrometry MALDI-ToF-MS, matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry. 

MS has proved to be a very powerful technique in the analysis of flavonoids and phenolic acids mainly due to its high sensitivity and the possibility of coupling with different chromatographic techniques such as gas chromatography (GC-MS), capillary electrophoresis (CE-MS), and especially LC-MS. Nowadays, techniques such as LC-DAD-MS, and particularly LC-DAD-electrospray ionization (ESI)/MS, are regarded as necessary tools for the analysis of phenolics in natural matrices. ... 

This is true for ion trap hybrids with FT-ICR-MS (see Sect. 4.4.7) and Orbi-trap (see Sect. 4.4.9) instruments, but also for the ion trap-time-of-flight hybrid system. The latter system has been pioneered by the group of Lubman [87, 88]. It has become commercial available for both MALDI and LC-MS applications [89]. All these hybrid MS system are frequently applied in combination with LC and electrospray ionization in, for instance, drag metabolite identification studies and in various proteomics-related studies. 

Although substantial progress has been made in determining acrylamide, a recent interlaboratory comparison showed that many current methods are not satisfactory for samples in complex matrices. Even with relatively simple samples, the range of results was too wide to be acceptable. Thus, more improvements in analytical methodolc are certainly in order. A fully validated improved LC/MS/ MS method was recently reported that uses isotope dilution, LC, and electrospray ionization M /MS. Excellent results were obtained on chocolate powders, cocoa, and coffee. 

Alkyl phosphonic acid degradation products provide good ionization and fragmentation with MS and MS/MS detection. Typical ionization techniques coupled with LC are electrospray ionization (ESI) and atmospheric pressure... 

For mixture.s the picture is different. Unless the mixture is to be examined by MS/MS methods, usually it will be necessary to separate it into its individual components. This separation is most often done by gas or liquid chromatography. In the latter, small quantities of emerging mixture components dissolved in elution solvent would be laborious to deal with if each component had to be first isolated by evaporation of solvent before its introduction into the mass spectrometer. In such circumstances, the direct introduction, removal of solvent, and ionization provided by electrospray is a boon and puts LC/MS on a level with GC/MS for mixture analysis. Further, GC is normally concerned with volatile, relatively low-molecular-weight compounds and is of little or no use for the many polar, water soluble, high-molecular-mass substances such as the peptides, proteins, carbohydrates, nucleotides, and similar substances found in biological systems. LC/MS with an electrospray interface is frequently used in biochemical research and medical analysis. 

Samples containing mixtures of peptides can be analyzed directly by electrospray. Alternatively, the peptides can be separated and analyzed by LC/MS coupling techniques such as electrospray or atmospheric pressure chemical ionization (APCI). 

In the following chapters, the basic principles of HPLC and MS, in as far as they relate to the LC-MS combination, will be discussed and seven of the most important types of interface which have been made available commercially will be considered. Particular attention will be paid to the electrospray and atmospheric-pressure chemical ionization interfaces as these are the ones most widely used today. The use of LC-MS for identification and quantitation will be described and appropriate applications will be discussed. 

The pump must provide stable flow rates from between 10 ttlmin and 2 mlmin with the LC-MS requirement dependent upon the interface being used and the diameter of the HPLC column. For example, the electrospray interface, when used with a microbore HPLC column, operates at the bottom end of this range, while with a conventional 4.6 mm column such an interface usually operates towards the top end of the range, as does the atmospheric-pressure chemical ionization (APCI) interface. The flow rate requirements of the different interfaces are discussed in the appropriate section of Chapter 4. 

Under many experimental conditions, the mass spectrometer functions as a mass-sensitive detector, while in others, with LC-MS using electrospray ionization being a good example, it can behave as a concentration-sensitive detector. The reasons for this behaviour are beyond the scope of this present book (interested readers should consult the text by Cole [8]) but reinforce the need to ensure that adequate calibration and standardization procedures are incorporated into any quantitative methodology to ensure the validity of any results obtained. 

Ionization methods that may be utihzed in LC-MS include electron ionization (El), chemical ionization (Cl), fast-atom bombardment (FAB), thermospray (TSP), electrospray (ESI) and atmospheric-pressure chemical ionization (APCI). 

Matrix-assisted laser desorption ionization (MALDI) is not yet a technique that has been used extensively for LC-MS applications. It is included here because it often provides analytical information complementary to that obtained from LC-MS with electrospray ionization, as illustrated later in Chapter 5. 

The fundamental piece of information on which the subsequent spectral analysis is based is the total-ion-current (TIC) trace. Such a trace, obtained from the LC-MS analysis of a pesticide mixture, is shown in Figure 3.13, together with the UV trace recorded simultaneously. For the purposes of this discussion, the HPLC and MS conditions used to generate the data, other than the fact that electrospray ionization was used, are irrelevant. 

Electrospray ionization occurs by the same four steps as listed above for thermospray (see Section 4.6). In contrast to thermospray, and most other ionization methods nsed in mass spectrometry, it shonld be noted that electrospray ionization nnnsnally takes place at atmospheric pressure. A similar process carried out under vacuum is known as electrohydrodynamic ionization and gives rise to qnite different analytical results. This technique has not been developed into a commercial LC-MS interface and will not be considered further. 

Factors may be classified as quantitative when they take particular values, e.g. concentration or temperature, or qualitative when their presence or absence is of interest. As mentioned previously, for an LC-MS experiment the factors could include the composition of the mobile phase employed, its pH and flow rate [3], the nature and concentration of any mobile-phase additive, e.g. buffer or ion-pair reagent, the make-up of the solution in which the sample is injected [4], the ionization technique, spray voltage for electrospray, nebulizer temperature for APCI, nebulizing gas pressure, mass spectrometer source temperature, cone voltage in the mass spectrometer source, and the nature and pressure of gas in the collision cell if MS-MS is employed. For quantification, the assessment of results is likely to be on the basis of the selectivity and sensitivity of the analysis, i.e. the chromatographic separation and the maximum production of molecular species or product ions if MS-MS is employed. 

The vast majority of LC-MS analyses cnrrently in use employ either electrospray ionization or APCI. In the previons example, electrospray ionization was employed because of the highly polar nature of the analytes bnt, as discussed above in Sections 4.7 and 4.8, this and APCI are, to a large extent, complementary, with APCI being used for low- to medium-polarity analytes and electrospray for medinm- to high-polarity analytes. There are many compounds, therefore, for which the best ionization technique is not immediately obvious and their relative merits must be investigated. 

In this study, the effect of mobile-phase flow rate, or more accurately, the rate of flow of liquid into the LC-MS interface, was not considered but as has been pointed out earlier in Sections 4.7 and 4.8, this is of great importance. In particular, it determines whether electrospray ionization functions as a concentration-or mass-flow-sensitive detector and may have a significant effect on the overall sensitivity obtained. Both of these are of great importance when considering the development of a quantitative analytical method. 

Although the use of MALDI per se is not covered in this present text, the data cited here clearly show that it is complementary to LC-MS employing electrospray ionization. 

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