Analysis of Small Volatile Molecules


The majority of instruments utilize capillary columns thereby allowing relatively simple connection to the mass spectrometer. Earlier systems used packed columns that required some form of separator in order to reduce the amount of carrier gas entering the mass spectrometer ionization chamber. A 5% poly(diphenyldimethylsiloxane) stationary phase is frequently used because of its wide general applicability and stability. Samples can be introduced using an autoinjector or manually by syringe; headspace gases, solutions in volatile solvents, solid-phase microextraction (SPME) systems and thermal desorption with cryofocussing can all be used. In some cases it is necessary or advisable to derivatize the sample in order to enhance its thermal stability.

Mass spectrometers of any type can be coupled, from huge magnetic sector machines to time-of-flight systems and the small 'bench-top' quadrupole or ion trap instruments. When the mass spectrometer is op erated in positive ion mode with electron impact ionization at 70 eV spectra generated can be compared with those in the extensive databases organized by NIST and Wiley. Using computerized file-handling techniques, spectra can be compared very rapidly and a list of possible compounds can be compiled. The mass spectrometer can also be operated in negative ion mode which allows improved sensitivity with particular analytes especially with chemical ionization.


Figure 1 shows typical results from a GC-MS study using a bench-top quadrupole system. The sample was obtained by extraction of serum taken after administration of an oestradiol prodrug. The upper

Figure 1 GC-MS of serum extract following oestradiol prodrug administration: (top) total ion chromatogram; (middle) mass chromatogram for mjz = 272; (bottom) mass spectrum for peak at Rt = 28.76 min and NIST library spectrum for oestradiol.
Figure 2 Mass chromatogram for m/z = 74 identifying FAMEs.

trace shows the total ion chromatogram (TIC), the second trace shows the mass chromatogram for m/z = 272 which is diagnostic for oestradiol. The mass spectrum obtained for this component and the NIST library match are the lower traces.

Use of mass chromatograms of diagnostic ions allows facile recognition of homologous series such as fatty acid methyl esters (FAMEs). Figure 2 shows the mass chromatogram for m/z = 74 for the FAME derivatives prepared from an archeological sample; this valuable information assisted in the interpretation of the pottery artifact.

The signal at m/z"74 is due to methyl ethanoate formed in the mass spectrometer by a McLafferty rearrangement shown in Figure 3. This process can occur with any FAME having an available hydrogen atom at position 4 and thus provides a useful diagnostic ion.

Selected ion recording (SIR) measures the ion current for a restricted range of ions instead of the whole spectrum. This is of particular use in biomonitoring studies for example where the analyte is well characterized from the chromatographic and spectrometric point of view, affording improved sensitivity and precision.

Figure 4 is a graphical representation of the data from an occupational health study of urinary amines: paired samples of urine taken at the beginning and end of a working day were analysed for a particular aromatic amine. A protocol was used that freed the amine from excreted conjugates followed by ex-

Figure 3 McLafferty rearrangement in a FAME M # ' to form m/z = 74.

Figure 3 McLafferty rearrangement in a FAME M # ' to form m/z = 74.

Figure 4 Urinary amine concentrations of paired samples.

traction and derivatization. The graph emphasizes the difference between the amounts of amine in the paired samples; although the values were well below regulatory limits, those from the fifth pair led to a change in working practice for that donor.

Amines can be converted into perfluoroacyl derivatives which are more stable thermally and chemically; such derivatives give improved sensitivity in electron-capture detectors and this is also manifest in negative ion chemical ionization mass spectrometry. This approach can be adopted for the analysis of materials that form relatively stable gas phase anions in the mass spectrometer. Figure 5 shows data from another typical example: prostanoids such as PGF2a are converted into the t-butyldimethylsilyl ether/penta-fluorobenzoyl ester derivatives. Under negative ion chemical ionization conditions using either methane or ammonia as reagent gas, the ester function is lost in a fragmentation reaction to form a fragment ion at m/z = 695 corresponding to the silyl ether car-boxylate anion shown in the figure. This ion shows satellites due to silicon and carbon isotopes at 696 and 697 that are in accord with calculated distributions. Detection levels for this analyte are in the low pg ijL-1 (ppm) range for full scan data and fg (ppb) range with selected ion recording.

The final example of this first group of applications involving small volatile species crosses the boundary into analysis of large intractible materials and concerns a pyrolysis study. Occasionally there may be insufficient sample to carry out a normal extraction prior to GC-MS analysis, this is particularly so with conserved archeological material and microscopic biopsy samples. In such circumstances, microscale sealed vessel pyrolysis GC-MS can be applied: Figure 6 shows the TIC obtained from a hair sample (2 mg) taken from an Egyptian mummy. The individual components of the pyrolysate can be identified

Figure 5 Mass spectrum of derivatized PGF2tt in negative ion chemical ionization and structure of anion corresponding to m/z = 695.

by comparison with library spectra and interpreted in terms of the mummification process.

This pyrolysis method can be applied to other complex analytes including whole cells in order to investigate occluded materials or for the identification of chemical modification of biopolymers such as starch.

The quantitative analysis of halogenated dibenzo-dioxins and -furans in biological (and environmental) matrices is a good example of the combination of high resolution GC (HRGC) and MS (HRMS) technologies using the isotopes of chlorine and carbon

Figure 7 Protocol for pretreatment of dioxin/furan samples prior to HRGC-HRMS.
Figure 6 Total ion chromatogram from GC-MS of pyrolysis products from archaeological hair sample.

to facilitate identification and quantification. Prior to any instrumental analysis, the flesh, vegetation or other material has to be prepared according to a quality assured protocol that is summarized in Figure 7.

Intrinsic to the procedure is the use of standards: the first standard to be added is 13C12-2,3,7,8-tetrachlorodibenzodioxin upon which quantitation is based. Another standard, 13C12-1,2,3,4-tetra-chlorodibenzodioxin is added after the chemical manipulation of the sample is complete and immediately prior to GC-MS analysis. Comparison of the signals from the two standards then allows the efficiency of the extraction process to be assessed. Typical data sets are shown in Figures 8 and 9: both show four traces, the upper two traces are the high resolution

Figure 8 HRGC-HRMS SIR data from dioxin/furan analysis of meat extract.

SIR data for m/z = 319.8965 and 321.8936 corresponding to the molecular ions of tetrach-lorodibenzodioxins (TCDD), i.e. C12H4(35Cl4)O2 and C12H4(35Cl337Cl)O2 respectively. The lower two traces in each case correspond to the ions of m/z = 331.9368 and 333.9339 for the 13C-isotopomers, i.e.

13C12H4(35Cl4)O2 and 13C12H4(35Cl337Cl)O2 for the two standards.

The data shown in Figure 8 represents TCDD levels below regulatory limits whereas those in Figure 9 (from an environmental sample) were significantly higher.

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