Case 83 -- Insecticide Poisoning


GAS CHROMATOGRAPHY - MASS SPECTROSCOPY

Gas chromatography coupled to mass spectroscopy (GC-MS) provides a particularly powerful method for identifying drugs or toxins in serum, urine, and other body fluids, and is used in our laboratories as part of a comprehensive drug screen. To be measured by GC-MS technique, the drugs or toxins and their metabolites must be capable of being volatalized at temperatures of less than about 265-300C, depending upon the particular GC-MS instrument used. Compounds with higher boiling points cannot be analyzed with this technique.

The preferred patient sample for GC-MS analysis is urine, although serum and gastric contents can also be analyzed. Specimen preparation begins with the addition of activated charcoal to the sample. The activated charcoal provides a relatively non-selective, but extensive, surface area to which many different types of organic molecules will bind. The patient sample is then decanted off the charcoal, and the organic substances bound to the charcoal are extracted with the solvent methylene chloride. The extracted material is decanted from the activated charcoal, and then evaporated using a stream of dry nitrogen gas. The resulting residue, which contains the drugs or toxins, is re-dissolved in a small amount of methanol. Barbital is added to provide a known internal standard.

A small aliquot (1-2 L) of the preparation is then injected into the gas chromatograph port of the GC-MS instrument. The principle of gas chromatography is that a gas passed over a solid or liquid surface to which it has some tendency to bind will be "slowed" compared to a gas which passes over the same surface, but has no tendency to bind. The time that it takes for a gas to pass through the column is called its retention time; gases which tend to bind to the column have longer retention times than do gases which do not.

A typical gas chromatography column consists of a glass column packed with beads composed of silica gel, activated charcoal, or molecular sieve particles. The column used in the GC-MS procedure for detection of drugs and metabolites has a slightly different design. Instead of using a relatively short column packed with beads, this system uses a long (approximately 15 meter), narrow (0.5 mm), coiled, fused silica capillary tube which is internally coated with cross-linked methyl-silicone. This type of very long column tends to have a higher resolution, with better separation of gases, than does a conventional column. Gases with more polarity tend to bind with greater avidity to this coating, thereby slowing them, than do less polar gases. A further refinement present in the GC-MS is that the coiled capillary column is placed in an oven whose temperature is slowly raised during the approximately 1/2 hour gas chromatography run. This slow rise in temperature helps to elute compounds with higher boiling points that may not be in gaseous phase in the beginning of the run.

The elution characteristics of a gas through a column of this type are quite complex and are affected by the flow of the inert gas, the chemical characteristics of the organic compound, the changing temperature, and the characteristics of the surface to which the gases bind. In practice, this is not a problem, because the elution characteristics of a particular type of column are determined and validated by the manufacturer, who tests a wide variety of substances and then empirically determines their retention times by the column. The retention times are tabulated and are available on-board the computer which controls the GC-MS instrument. The internal barbital standard which is added to the sample provides a reference for the performance of the column and also for the technique of sample preparation.

The gas chromatography step provides considerable information about the identification of a compound, since only a small number of compounds will have retention times of a particular value. Tabulated lists of retention times for thousands of compounds are available both in book form and in computer libraries. However, the gas chromatography cannot completely characterize a compound, since more than one substance may have the same retention time.

A more definitive answer is obtained by coupling the gas chromatograph technique with mass spectroscopy, which is capable of providing a great deal of additional information about the eluted substances. In practice, while there are many pairs of compounds with similar retention times, and many pairs of compounds with similar mass spectra, the combination of retention time and mass spectra usually provides a definitive identification of a compound.

The principle of mass spectroscopy is that a gaseous compound can be ionized and partially fragmented by an electron beam, and the mass/charge ratios of the resulting charged fragments can be measured. The simplest way to conceptualize a mass spectroscope is by imagining that a gas enters a small hole in the machine; its molecules are fragmented and charged by being hit by the electron beam; the resulting fragments are deflected to a different extent by an electric field depending upon their mass/charge ratios; and then the fragments are "splattered" against different portions of the far wall. The mass spectra can then be obtained by measuring where on the wall different fragments hit.

Some early mass spectrometers actually used the strategy described in the preceding paragraph, which required a large number of detectors corresponding to different mass/charge ratios. Modern mass spectrographs typically use only one detector, and instead use a complex analyzer (the quadrupole analyzer) to change the electric field experienced by the beam of molecular fragments so that only fragments with a particular mass/charge ratio will hit the detector at a particular time. Since the quadrupole mass analyzer is continuously, systematically, varying the field experienced by the fragments, mass spectra can still be obtained.

The placing of a mass spectrograph in tandem with a gas chromatograph instrument tends to produce an enormous amount of data, since a mass spectrograph is obtained for each different retention time. GC-MS machines are linked to a powerful computer to store and analyze the data that are generated. Historically, mass spectral data were identified by hand by comparison to tabulated values; now this task is usually automatically provided by the computer which uses large libraries of on-board spectral data. A laboratory may still need to consult one of the book formats spectral libraries, if the computer fails to identify a compound.


FINAL DIAGNOSIS


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