Thermospray HPLC/MS analysis

Abian, J. and G.D. Barceld (1993). Analysis of chloro-triazines and their degradation products in environmental samples by selecting various operating modes in thermospray HPLC/MS/MS. J. Agri. Food Chem., 41 1264-1273. 

Tandem MS. The thermospray HPLC/MS/MS was performed on a Finnigan MAT TSQ-46C triple quadrupole mass spectrometer interfaced to an INCOS Data System (Finnigan MAT, San Jose, CA). The triple quadrupole was operated with the first and second quadrupoles in the RF mode during HPLC/MS operation. For HPLC/MS/MS analysis, the first quadrupole selected the [M+H] ion of the compound, while the third quadrupole was scanned over the mass range of 12-300 daltons. The second quadrupole serves as a collision chamber. Argon collision gas was added to the enclosed chamber of this quadrupole to give a pressure of 2 mtorr for collisional activation of the sample ions. 

The diversity of compounds that can be analyzed by HPLC currently preclude the use of any one HPLC/MS technique to specifically detect trace quantities. Thermospray is one of the most popular HPLC/MS techniques due to its ability to ionize nonvolatile and thermally labile compounds with minimal compromises on a HPLC separation or MS operation. Ion evaporation ionization (no filament or discharge) is suitable for numerous compounds, but often results in spectra with insufficient structural information (i.e. one ion spectra), with sensitivities varying drastically between compound classes. In cases where thermospray specificity or sensitivity is not sufficient, complementary HPLC/MS approaches need to be employed. The use of thermospray HPLC/MS and complementary techniques (i.e. immobilized enzyme bioreactors, chemical degradation and particle beam HPLC/MS) have been evaluated for the specific analysis of three major classes of compounds--peptides, pharmaceuticals, and pesticides. 

The choice of a HPLC/MS analysis method depends greatly on the characteristics of the sample (i.e. proton affinity, polarity, thermal stability and volatility) as well as the structural information and sensitivity required. The use of various techniques including enzymatic hydrolysis and physical-chemical reactions can assist in achieving the analysis goals for certain compounds. Alternatively, the use of complimentary HPLC/MS techniques such as thermospray and particle beam can be useful for the analysis of a variety of compounds, as demonstrated in this paper. Employing less commonly available instrumentation, such as tandem MS, with thermospray or particle beam can prove valuable in determining structure when other methods are unsuccessful. The further development of existing HPLC/MS techniques and the implantation of new HPLC/MS methods will continue to increase the variety of compound classes that can be routinely monitored with adequate sensitivity and specificity. 

Voyksner, R. D. Thermospray HPLC/MS for Monitoring the Environment A Chemical Analysis Series on Application of New Mass Spectrometry Techniques in Pesticide Chemistry Rosen, J. J. Wiley and Sons, NV, 1987 Vol. 91, Chapter 11, p 146. 

A method that uses high performance liquid chromatography/ mass spectrometry (HPLC/MS) for the analysis of chlorinated phenoxyacid herbicides is described. During method development different techniques were used to increase both the sensitivity and the specificity of thermospray HPLC/MS for chlorinated acid herbicides. These included the operation of the instrument in the negative chemical ionization (NCI) mode initiated by discharge and the use of a wire-repeller in the ion source for efficient extraction of positive ions. Single quadrupole repeller-induced and multiple quadrupole collision activated dissociation (CAD) experiments were also performed to increase the structural information of the mass spectra. 

M5 was shown to be parent SK F 96148 by coelution in the HPLC with authentic standard and by its LC/MS analysis (Fig. 6a). Thermospray LC/MS analysis gave an intense ammonium adduct at m/z 371, 373 and a weak protonated... 

Thernospray HPLC/MS. Urine extracts were analyzed by thermospray/HPLC/MS to Identify the metabolites present. A Finnigan 4500 series mass spectrometer Interfaced with a Vestec Thermospray unit was used to perform the analysis. The aerosol thermocouple of the Interface was replaced with a repeller which was connected to an external power supply. The samples were analyzed under thermospray/fllament chemical Ionization with pulsed +/- Ion detection. A mobile phase consisting of acetonitrlle/0.1 M ammonium acetate with a linear gradient of 9X to 65% over 30 minutes was used In the anlaysls. 

Almost always GC-MS has been the chosen tool for identification of alkanes from geological or living sources, and such methods in general are outlined in preceding chapters. More recently, the tandem mass-spectrometric (MS-MS) technique has been applied to the effluent from GC columns and adaptions of the standard GC-MS methods have been applied for the analysis of triterpanes and steranes (C30 compounds and the like) that often occur in petroleum and are valuable geological markers . Supercritical fluid chromatography linked to MS will undoubtedly also become a widely used technique for separation and analysis of petroleum and natural waxes, as will thermospray HPLC-MS methods. 

The ion source creates analyte ions from the neutral species in the vapor phase. Several designs of ion sources have been used for CWA degradation product analysis in recent years including thermospray ionization (TSP), atmospheric pressure chemical ionization (APCI), and electrospray ionization (ESI). These soft ionization techniques generally produce [M-H] or [M+H]" " fragments for the alkyl phosphonic acids and some other CWA degradation products. The inductively coupled plasma (ICP) is a hard ionization source and has been described in the literature. HPLC-ICP-MS has been reported for the detection of alkyl phosphonic acids.In recent years, ESI has become the most common ion source in HPLC-MS analysis in general with the APCI source a close second. The thermospray source has fallen out of favor in HPLC-MS systems since the introduction of ESI. 

System (1) has been described for the analysis of corticosteroids in urine, and uses HPLC and thermospray LC-MS technology. The method can be... 

Combination of two immobilized enzyme columns with HPLC/thermospray MS can be useful for amino acid sequencing and identification. The use of an endopeptidase bioreactor followed by HPLC separation then an exopeptidase column and MS detection can enable sequencing of 3-5 amino acids of each endopeptidase hydrolysis product. The trypsin, hydrolysis/HPLC/ carboxypeptidase A, B, and Y (1 1 1) hydrolysis/ thermospray MS analysis assist in the sequencing of Y-endorphin (Figure 2C,C ). 

Figure 2. A) Trypsin hydrolysis/HPLC/thermospray MS analysis of Y-endorphin. The two tryptic fragments (T1 and T ) were separated by HPLC (10% isopropanol in aqueous ammonium acetate (0.1 M) pH 7.1, 1 mL/min) and detected by thermospray MS. B,B ) Thermospray mass spectra of tryptic fragments and Tg separated by HPLC after hydrolysis. C,Cj) Thermospray spectra from trypsin hydrolysis followed by HPLC then carboxypeptidase A, B, and Y hydrolysis for the partial amino acid sequencing of Y-endorphin.

Figure 3. HPLC/trypsin hydrolysis/thermospray MS analysis of a mixture of two endorphins in a plasma extract. The separation was performed using 25% isopropanol in aqueous ammonium acetate (0.1 H) solution at a pH of 7.5 at a flow of 1.0 mL/min.

Pesticides. There are numerous references to the use of HPLC/MS for the analysis of pesticides and herbicides [14, 16-20]. Some major classes of pesticides and herbicides including carbamate, triazines, organophosphorus, and phenolic acid have been analyzed by HPLC/MS using Cl or ion evaporation ionization. While these ionization techniques often resulted in excellent sensitivity (thermospray/MS full scan detection limits of 1-10 ng), usually only [M+H] and/or [M+NH4] ions were formed. This limitation can be overcome using tandem4MS [20], moving belt [17], and most recently through the use of particle beam HPLC/MS. 

Kwakman and co-workers (1992) used disk extraction to isolate organophosphorus pesticides on-line with gas chromatography for rapid analysis. The method is on-line for sorption and elution using ethyl acetate as a desorbing solvent and a retention time gap approach for the GC analysis. The detection limit was 10 to 30 ng/L in tapwater and 50 to 100 ng/L in river water. Barcelo and co-workers (1993) and Molina and co-workers (1994) also found that organophosphorus pesticides and triazines could be effectively isolated on disks for further analysis by HPLC/MS using thermospray and electrospray. 

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