Frit-FAB

Two forms of interface have been commercially developed [7] which allow analytes in a flowing liquid stream - it has to be pointed out, not necessarily from an HPLC system (see below) - to be ionized by using FAB. These are essentially identical except for that part where the HPLC eluate is bombarded with the heavy-atom/ion beam. Both of these interfaces consist of a probe in the centre of which is a capillary which takes the flowing HPLC eluate. In the continuous-flow FAB interface (Figure 4.3), the column eluate emerges from the end of the capillary and spreads over the probe tip, while in the frit-FAB interface the capillary terminates in a porous frit onto which the atom/ion beam is directed. 

Principles and Characteristics Continuous-flow (or dynamic) FAB/FTB [102] and frit FAB/F1B [103] offer a means of introducing samples in solution into a continuous flow of solvent which terminates at the modified FAB/FIB probe tip, and they extend the applicability of FAB. Samples are injected through a conventional HPLC injection valve, or solutions are simply drawn in by the high vacuum in the ionisation source of the mass spectrometer. These very similar techniques are particularly amenable to coupling with HPLC columns, and ionisation of the sample is unchanged with respect to conventional FAB and FIB/LSIMS. 

I. Pastorova, C.G. de Koster, J.J. Boon, Analytical study of free and ester bound benzoic and cinnamic acids of gum benzoin resins by GC MS and HPLC frit FAB MS, Phytochemical... 

In standard FAB, the surface of the matrix solution is depleted of analyte and suffers from radiational damage during elongated measurements. Refreshment of the surface proceeds by diffusion (limited by the viscosity of the matrix) or evaporation. Continuous-flow fast atom bombardment (CF-FAB) continuously refreshes the surface exposed to the atom beam. [107,108] The same effect is obtained in slightly different way by the frit-fast atom bombardment (frit-FAB) technique. [109,110] In addition, both CF-FAB and frit-FAB can be used for online-coupling of liquid chromatography (LC, Chap. 12) [111] or capillary electrophoresis (CE) to a FAB ion source. [112]... 

Fig. 9.15. Experimental setups of CF-FAB (a) and frit-FAB (b). Reproduced from Ref. [108] by permission (a). Academic Press, 1986. Reproduced from Ref. [110] by permission (b). Elsevier Science, 1988.

Aubagnac, J.-L. Gilles, I. Lazaro, R. Claramunt, R.-M. Gosselin, G. Martinez, J. Reduction Phenomenon in Frit FAB-MS. Rapid Commun. Mass Spectrom. 1995,9, 509-511. 

H Oka, Y Ikai, J Hayakawa, K Harada, H Asukabe, M Suzuki, R Himei, M Horie, H Nakazawa, JD MacNeil. Improvement of chemical analysis of antibiotics. 22. Identification of residual tetracyclines in honey by frit FAB/LC/MS using a volatile mobile phase. J Agr Food Chem 42 2215 -2219, 1994. 

Two approaches based on fast-atom bombardment (FAB) and introduced almost simultaneously were soon after their first description in 1985 and 1986 commercialized, i.e., the frit-FAB [80] and the continuous-flow Cf-FAB [81]. Both systems are used to introduce part of the column effluent (typically 1-10 pl/min) into a FAB somce. In the frit-FAB system, a capillary transfers the effluent to a stainless-steel or PFTE frit used as a FAB target, while in Cf-FAB system the effluent flows in a thin uniform film over the FAB target. A suitable FAB matrix, e.g., glycerol, should be added to the mobile phase. Analyte molecules are directly desorbed and ionized from the hqtrid film by FAB. These approaches are discussed in Ch. 4.6. 

Figure 4.8 Frit-FAB interface for LC-MS (Reprinted with permission from [40], 1988, Elsevier Science).

The fiit-FAB interface consists of a 40-pm-lD fused-silica capillary, which ends at a porous stainless-steel frit with 2-pm porosity. A schematic diagram of the interface tip is shown in Figure 4.8. The frit-FAB interface is commercially available from Jeol. A frit-FAB interface, based on the use of a 8 pm thick stainless-steel screen (2 pm mesh) as FAB target, was described Hogge et al. [43], and subsequently commercialized by Micromass (currently Waters) as the screen-wick interface with a thin screen with 2 pm pores at the tip of the interface probe [44]. 

In all these application areas, Cf-FAB has almost completely lost territory to electrospray interfaces. The two most important disadvantages of Cf-FAB are the limitations in the maximum allowable flow-rate and the difficulty of achieving stable conditions by balancing the solvent flow-rate, viscosity and surface tension, and the temperature, wettability, and liquid-film properties of the target. Because of its easy implementation on sector instmment, some application of Cf-FAB is still reported, especially frit-FAB. 

D.G. Watson, A.G. Davidson, B.I. Knight, The analysis of some corticosteroids and corticosteroid esters by LC-frit-FAB-AdS, Rapid Commun. Mass Spectrom., 11 (1997)415. 

Suzuki et al. [57] systematically compared the responses of some oligosaccharides derivatized with PA, ABEE, PMP, 4-methoxyphenyl-3-methyl-5-pyrazolone, 2-aminoethanethiol, and 2-aminobenzenethiol in continuous-infusion ESl-MS and frit-FAB-MS. In ESl-MS, the PMP derivatives gave the best results. Reductively aminated oligosaccharides can be analysed as [M+H]" or [M+Na]" in positive-ion ESl-MS and as [M-H] in negative-ion ESl-MS. 

Cotistins 0.55-mm PTFE tube 6 4000 n-Butanol-0.04M TEA (1 1) Lower phase 0.16 34.3 Frit-FAB 9... 

Fig. 5 Frit-FAB LC/MS analysis of a cytosolic extract from mouse liver at 3 hr postinjection of microcystin-RR. Shown are (a) simultaneously monitored UV chromatogram (238 nm), (b) total ion and mass chromatograms, (c) Frit-FAB LC/MS mass spectra of microcystin-RR, (d) longer retention time area of peak X (indicated by X-L), and (e) shorter retention time area of peak X (indicated by X-S).

The immunoaffinity purification method, followed by LC/MS analysis, has also been used in other toxicological studies. When aged mice (32 weeks) were orally administered microcystin-LR at 500 pg/kg, 62% of the aged mice showed hepatic injury, whereas such changes in the liver were not found in young mice (5 weeks).Upon uptake of orally administered microcystin-LR at 500 pg/ kg, the toxin into the liver was confirmed by Frit-FAB LC/MS after the immunoaffinity purification. When microcystin-LR was intraperitoneally injected 100 times at 20 pg/kg into male ICR mice (5 weeks old, Charles River Japan, Atsugi, Japan) for 28 weeks, multiple hyperplastic nodules up to 5 mm in diameter were observed in every liver. Microcystin-LR and its cysteine conjugate were identified from the isolated mouse livers. 

Okkura, T., Takechi, T., Deguehi, S., Ishimaru, T., Maki, T., and Inouye, H., Simultaneous determination of herbicides in water hy FRIT-FAB LC-MS, J. Toxicol. Environ. Health, 40, 266-273, 1994. 

The most recent significant advance in plant hormone analysis has been the use of combined HPLC-MS for the analysis of GA conjugates, lAA conjugates and cytokinins. A number of interfaces have been developed for HPLC-MS, including thermospray, atmospheric pressure chemical ionisation, electrospray, particle beam, continuous flow fast atom bombardment (FAB) and frit-FAB (see reference [94]). GA standards have been analysed by HPLC-MS with a thermospray interface [95], an atmospheric pressure chemical ionisation interface has been used with GA conjugates [96] and cytokinins [97] while ion spray and plasma spray have been used to analyse ABA and its metabolites [98]. There are, however, many more reports on the use of frit-FAB HPLC-MS for the analysis of not only standards, but also endogenous hormones and their isotopically-labelled metabolites [18-23,99-101]. 

Direct inlet probe FAB-MS is an important tool in the analysis of compounds that are thermolabile and/or lack volatility [102]. Lack of sensitivity was initially a limiting factor but detection limits have been enhanced 10-100 fold, because of reduced suppression effects [103], with the use of a dynamic system in which the HPLC effluent is passed continuously into the ion source of the MS [104,105]. In the case of a frit-FAB HPLC interface, which is available commercially, reverse phase HPLC mobile phase, containing 1% glycerol as a matrix, is introduced into the ion source via a steel frit. The sample and matrix are then ionised on the inner surface of the frit with a beam of accelerated xenon atoms (Fig. 9). The optimum rate at which the HPLC mobile phase can be introduced into the ion source is 5 p.1 min and this necessitates the use of a reliable splitter when a conventional 2-5 mm bore HPLC column is used. Although a commercial post-column splitter is available, it is of limited value in the analysis of traee quantities of compounds. 

Tabulated positive ion frit-FAB mass spectra of lAA, lAA amino acid and sugar conjugates and related compounds [lOO], Ions without relative intensity indicated have... 

Tabulated negative ion frit-FAB mass spectra of GA glucosyl esters [99]... 

In order to carry out GC-MS analysis of cytokinins, which are mainly N -substituted derivatives of adenine that occur in plants as free bases, ribosides, ribotides and glucosides, it is necessary to convert these polar compounds into volatile derivatives. There are, however, technical problems as the more commonly used t-BuDMS and TMS derivatives are partially and completely hydrolysed, respectively, in aqueous solvents and therefore cannot be purified by reverse phase HPLC [110]. Multiple derivative formation is also known to occur during trimethylsilylation [111]. Permethyl derivatives are stable in aqueous solutions but the preparation of reagents and the derivatization procedures are both time consuming and complex and, in addition, formation of multiple derivatives can also occur [6]. Frit-FAB HPLC-MS of cytokinins is therefore particularly useful as standards and plant extracts can be analysed without recourse to derivatization [101]. Tabulated positive ion FAB mass spectra of a range of cytokinins are presented in Table... 

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