Thermospray interface HPLC

A group of techniques employing differential selection of solute ions relies on nebulisation and ionisation of the eluent, with some discrimination of ion selection in favour of the solute. Main representatives are APCI [544] and thermospray [545]. In a thermospray interface a supersonic jet of vapour and small droplets is generated out of a heated vaporiser tube. Controlled, partial vaporisation of the HPLC solvent occurs before it enters the ion source. Ionisation of nonvolatile analytes takes place by means of solvent-mediated Cl reactions and ion evaporation processes. Most thermospray sources are fitted with a discharge electrode. When this is used, the technique is called plasmaspray (PSP) or discharge-assisted thermospray. In practice, many... 

Kiehne and Engelhardt (283) developed HPLC-MS with thermospray interface (ammonium acetate solution for buffer ionization) to determine flavonols, flavonol 0-glycoside, flavone C-glycosides, CF, TB, theogalline, and theanine from green tea. 

In thermospray interfaces, the column effluent is rapidly heated in a narrow bore capillary to allow partial evaporation of the solvent. Ionisation occurs by ion-evaporation or solvent-mediated chemical ionisation initiated by electrons from a heated filament or discharge electrode. In the particle beam interface the column effluent is pneumatically nebulised in an atmospheric pressure desolvation chamber this is connected to a momentum separator where the analyte is transferred to the MS ion source and solvent molecules are pumped away. Magi and Ianni (1998) used LC-MS with a particle beam interface for the determination of tributyl tin in the marine environment. Florencio et al. (1997) compared a wide range of mass spectrometry techniques including ICP-MS for the identification of arsenic species in estuarine waters. Applications of HPLC-MS for speciation studies are given in Table 4.3. 

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. 

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. 

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. 

One of the initial accounts of the use of RP-HPLC with fluorescence detection centered on the determination of LAS in river water without any preconcentration. In this approach, a CIS column was used with a methanol-water eluent containing 0.1 M NaC104 and provided a partial separation of LAS isomers from clean aqueous samples with a concentration of 0.1 % LAS. Kikuchi et modified the method to make use of gradient elution with acetonitrile and water using UV detection. Mottaleb et al. demonstrated the use of HPLC-UV-FTIR using the modified thermospray interface for the analysis of LAS. 

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]. 

Figure 7.9 HPLC-MS thermospray system used in the analysis of a mixture of drugs, (a) Thermospray interface, (b) total ion chromatogram, and (c) mass spectra of peaks E, F, Q and S. (Reproduced by permission of VG Instruments.)...

Originally the electrospray interface, like the thermospray interface, was limited to use with very low flow rates of mobile phase from capillary or microbore HPLC columns or capillary electrophoretic separations. The acceleration of droplet evaporation by... 

Schroder [88,89] analyzed fluorinated surfactants in water and wastewater using HPLC coupled by a thermospray interface to a tandem mass spectrometer (MS/MS). Alternatively, the chromatographic column was bypassed and the analyte was injected into the mass spectrometer (FIA, flow injection analysis). 

Schroder [20b] used high-performance liquid chromatography (HPLC)/ mass spectrometric (MS)/MS with a thermospray interface [20c] to detect, identify, and quantify metabolites of Fluowet OTN, a nonionic fluorinated surfactant with the structure R F2,i+iCH2CH2(OCH2CH2)wOH. The biodegradation was limited to the poly(oxyethylene) hydrophile [20b]. The absence of fluoride ions indicated that the perfluorocarbon chain was not degraded. 

Continuous flow FAB has been demonstrated for HPLC detection of the components of ditallowdimethylammonium chloride fabric softener. In this technique, the matrix substance is added to the HPLC colunrn effluent prior to introduction to the MS. MS-MS was demonstrated for confirmation of identity. While continuous flow FAB is typically performed with aqueous HPLC systems, the solubility of dialkyl quats is such that normal phase HPLC with nonpolar solvents is preferred. Lawrence was able to adapt a FAB system to this analysis, using a matrix solution of 75 25 glycerol/methanol (118). The use of FAB for analysis of surfactants has dropped off sharply with the availability of electrospray and thermospray interfaces for HPLC-MS. 

The nebulization and evaporation processes used for the particle-beam interface have closely similar parallels with atmospheric-pressure ionization (API), thermospray (TS), plasmaspray (PS), and electrospray (ES) combined inlet/ionization systems (see Chapters 8, 9, and 11). In all of these systems, a stream of liquid, usually but not necessarily from an HPLC column, is first nebulized... 

Principles and Characteristics Thermospray ionisation (TSP) involves introduction of a relatively high flow (0.2-2mLmin ) of solvent into the ion source of a mass spectrometer, and is therefore suitable as an interface for HPLC-MS, using standard bore columns. A vaporiser probe (essentially a resistively heated capillary tube of about 100 xm i.d.) acts as a transfer line for taking solvent and solute into the source. The source is heated to prevent condensation of the solvent, and the temperature of the capillary is chosen so as to ensure vaporisation of the solvent. In this way, a vapour jet is generated, which contains small, electrically charged droplets if the solvent is at least partially aqueous and... 

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