The Thermospray Interface

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 could be used for the majority of analytes did nothing to advance the acceptance of LC-MS as a routine 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. 

The thermospray interface overcame many of the problems encountered with the moving-belt and direct-liquid-introduction interfaces and with the advent of this, LC-MS became a routine analytical tool in a large number of laboratories. This was reflected in the fact that this was the first type of interface made available commercially by the majority of the manufacturers of mass spectrometers. 

It was also the first of a number of interfaces, with the others being electrospray and atmospheric-pressure chemical ionization, in which ionization is effected directly from solution within the interface itself, i.e. the mass spectrometer was not used to produce ions from the analyte simply to separate them according to their m/z ratios. 

Ionization involving these interfaces may be considered to comprise the following four stages, with the differences between the techniques being associated with the ways in which these stages are carried out  

Thermospray may be defined as the controlled, partial or complete, vaporization of a liquid as it flows through a heated capillary tube. 

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In summary, it can be said that prior to the development of the thermospray interface there were an increasing nnmber of reports of the analytical application of LC-MS [3] bnt in this present anthor s opinion, based on a nnmber of years of using a moving-belt interface, the technique could not be considered to be routine . The thermospray interface changed this and with the commercial intro-dnction of the combined APCI/electrospray systems in the 1990s the technique, for it now may be considered as a true hybrid technique, has reached maturity (although this should not be taken as a suggestion that there will be no further developments). 

The advantages and disadvantages of this type of interface, particnlarly in comparison to the moving-belt interface which was available at the same time, are listed below. This was one of the first LC-MS interfaces to be made commercially available and, although used in a number of laboratories, its development was halted premamrely by the introduction of the thermospray interface (as we shall see later). 

The introduction of the thermospray interface provided an easy-to-use LC-MS interface and was the first step in the acceptance of LC-MS as a routine analytical technique. It soon became the most widely used LC-MS interface of those available in the mid to late 1980s. 

With the thermospray interface (Figure 4.38(a)), the mobile phase, usually containing an ammonium ethanoate buffer, is passed through a heated probe (350-400°C) into an evacuated source chamber where it forms a supersonically expanding mist of electrically charged droplets. The liquid evaporates to leave charged solid particles which then release molecular ions such as MH+ and, VI by an ammonia chemical ionization (Cl) process. The analyte ions are skimmed off into the mass spectrometer whilst the vaporized solvent is pumped away. An electron beam is also employed to enhance the production of ions by Cl. 

Ion Rathe, a technician in the LC-MS laboratory at MDS Pharma Services, checks out the thermospray interface on one of the LC-MS units in this laboratory. 

Most of the direct and indirect (transport) interfaces described here use chemical ionization (c.i.) ion-sources, which are not well suited to such polar, non-volatile compounds as tri- and higher oligosaccharides. The thermospray interface, which can operate on an ion-evaporative mode, is capable of producing intact molecular ions from such nonvolatile, polar molecules and should be useful in oligosaccharide analysis. Molecules of this type, however, can also be easily analyzed by fast-atom-bombardment ionization, and use of this technique, coupled to direct liquid introduction and moving-belt interfaces, has been reported. The latter system has been applied to complex oligosaccharide analysis. ... 

LC-PB-MS has been investigated as a potential confirmatory method for the determination of malachite green in incurred catfish tissue (81) and of cephapirin, furosemide, and methylene blue in milk, kidney, and muscle tissue, respectively (82). Results showed that the mobile-phase composition, nebulization-de-solvation, and source temperature all play an important role in the sensitivity of the method. The sensitivity increases with decreasing heat capacity of the mobile phase in the order methanol acetonitrile isopropanol water and with decreasing flow rate. A comparison of the PB with the thermospray interface showed that less structural information was provided by the latter, whereas the sensitivity was generally lower with the thermospray interface. 

In the thermospray interface, aqueous mobile phases containing an electrolyte such as ammonium acetate are passed at flow rates of 1-2 ml/min through a heated capillary prior entering a heated ion source. The end of the capillary lies opposite a vacuum line. Nebulization takes place as a result of the disruption of the liquid by the expanding vapor formed at the capillary wall upon evaporation of part of the liquid in the capillary. This results in formation of a supersonic jet of vapor containing a mist of fine, electrically charged droplets. 

The next major advance in LC-MS interfacing was developed by Blakely and Vestal (55, 56). To circumvent the solvent elimination problem, Blakely et al. (55) developed the thermospray interface that operates with aqueous-organic mobile phase at typical 4.6-mm i.d. column flow rates, 1-2 mL/min. The thermospray technique works well with aqueous buffers. This feature is an advantage when the versatility of the reversed-phase mode is considered. In fact, with aqueous buffers, ions are produced when the filament is off. A recent improvement in the thermospray technique is the development of an electrically heated vaporizer that permits precise control of the vaporization (56). This... 

Choline and acetylcholine were extracted from mouse brain homogenates, in the presence of 2H9-choline and 2H4-acetylcholine, and the extract was analyzed directly by HPLC-MS on a column (15 cm x 4.6 mm) of Ultrasphere-I.P. ODS. The mobile phase was 0.1 M ammonium acetate-2 mM pyridine containing octanesulfonic acid (50mg/L, pH 5), and was eluted at a flow rate of 1.25mL/min. The column was coupled to a quadrupole mass spectrometer by the thermospray interface, and selected ions were monitored at mjz 104, 113, 146 and 150 for choline, [2H9] choline, acetylcholine and [2H4] acetylcholine, respectively. 

MS has been used for a number of years as a powerful tool for the study of drug biodeposition and metabolism [83-87]. These types of studies have traditionally been carried out by gas chromatography-MS techniques. However, the use of LC-MS has been growing, especially since the introduction of the thermospray interface. With the advent of liquid-phase ionization techniques, such as electrospray, it has become possible to use LC-MS for the structural characterization of highly polar molecules at very low concentrations. 

Thermospray. The thermospray interface was introduced and developed by Blakley and Vestal [14], In their approach, a liquid flow from HPLC was directed through a resistively heated capillary connecting to the MS ion source. The heat and vacuum would evaporate the solvent from a supersonic beam of mobile phase produced in the spray, creating charged small microdroplets. These small liquid droplets were further vaporized in the heated ion source. Ions present in the ion source were then transferred to the mass analyzer, and residual vapors were pumped away. 

The interfaces that effectively replaced the transport system were the thermospray and electrospray sample introduction systems. The thermospray interface, a diagram of which is shown in figure 23, is a development from the direct inlet system of McLafferty. The successful use of the thermospray interface was first reported by... 

The DLI interface was widely used in LC-MS applications between 1982 and 1985. The DLI interface did not survive the introduction of the thermospray interface, which removed some of the drawbacks of the DLI interface, i.e., the flowrate limitation of 50 pl/min and the problems with clogging of the diaphragms. Furthermore, thermospray added new ionization modes next to the solvent-mediated Cl used in DLL... 

The thermospray interface (Vestec Inc., Houston, TX) was operated at a vaporizer temperature of 180-195°C and a source temperature of 250 C. The vaporizer temperature was adjusted to optimize the solvent signal which correlated closely to optimal conditions for the analyte [14]. Most of the compounds were analyzed using ion evaporation ionization, although a few proved more sensitive under discharge ionization conditions (discharge needle at 1000 V). 

Figure 1 shows the LC/MS thermospray total ion chromatogram of 0.25 p.g standard mixture of the six sulfonylurea herbicides. Gradient HPLC conditions were used to separate the six compounds in less than 25 minutes total run time. The mobile phase composition was kept isocratic at 30% acetonitrile/.05M formic acid for the first 15 minutes to separate the four herbicides HARMONY, ALLY, OUST and GLEAN. A gradient from 30% acetonitrile to 60% in 10 minutes was then used to elute EXPRESS and CLASSIC. An acidified mobile phase is used with sulfonylureas to keep them in the undissociated form which is retained on the HPLC column (3). Organic acids are recommended for use with LC/MS to prevent the formation of deposits in the mass spectrometer source and to prevent clogging of the thermospray interface probe tip. In this work we used formic acid. 

Combined liquid chromatography/mass spectrometry (LC/MS)can play an important role in both qualitative and quantitative bioanalysis. LC/MS can be performed with a number of interfaces. Three interfaces are presently available in our laboratories i.e., the thermospray interface (TSP), the moving-belt interface (MBI), and continuous-flow fast atom bombardment (CF-FAB). These interfaces are supplementary with respect to their applicability and the type of information that can be obtained. 

The introduction of the thermospray interface in the mid to late 1980s provided the first efficient LC-MS connecting technique. With the relatively new interface techniques of electrospray interface and the complementary atmospheric pressure chemical ionization interface (APCI), the full potential of the LC-MS system can now be achieved. 

Table (11) gives an overview on the LC-MS methods described in the literature. The vast majority of studies has been performed with the thermospray interface in the positive ionization mode. Thermospray detection is not very sensitive [279], For procyanidins a detection limit of 1 ig has been reported [269], No data are available on the detection limits of procyanidins using other types of interfaces. So far, LC-MS has not yet been applied to the quantitative analysis of procyanidins. 

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