3. GAS CHROMATOGRAPHY–MASS SPECTROMETRY IN
3.2. Gas Chromatography–Mass Spectrometry–Mass
Other than n-alkanes, pristane, phytane, and other isoprenoid alkanes that are present in abundance in unaltered oils and easily recognizable by GC and GC–
MS analyses, most of the biomarker components are present in trace amounts.
Among these, naphthenic hydrocarbons, including tricyclic terpanes, pentacyclic triterpanes, and steranes, have been studied most extensively because they carry more specific information on source and depositional environment than normal and isoprenoid alkanes [21].
Figure 3 is a GC–MS reconstructed ion chromatogram (RIC) that is equiva- lent to GC flame-ionization trace of a typical crude oil. It illustrates that triterpanes and steranes cannot be detected by GC alone due to low abundance and interference of coeluting components. Through the use of mass chromato- grams of characteristic fragment ions at 191 Da for triterpanes and 217 Da for steranes from a full-scan GC–MS analysis, triterpanes and steranes can be found to elute betweenn-C22andn-C36on a nonpolar methyl silicone column. However, in order to enhance the GC–MS detection of these components, selective ion monitoring (SIM) technique for only the ions of interest can be used. Figure 4 shows the increase of the signal-to-noise ratio when only the 191- and 217-Da ions are monitored for triterpanes and steranes, respectively.
Since triterpanes (hopanes) are prevalent in the swamp/peat environment due to bacterial decay of high plant material and steranes are mostly generated by algae, the relative amounts of triterpanes and steranes have been used for estimating the organic matter type and depositional environment [22]. Figure 4 also illustrates the difficulties of measuring distributions of steranes by GC–MS.
While most triterpanes are well resolved in the 191-Da mass chromatogram,
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Figure 3 Gas chromatography–mass spectrometric reconstructed ion chromatogram (RIC) of a typical crude oil. A RIC is equivalent to a GC flame ionization detection trace.
The presence of triterpanes and steranes eluting betweenn-C20andn-C35are not evident due to low abundance and interference of coeluting compounds. Conditions: Finnigan (San Jose, CA), 9610 gas chromatograph, Supelco (Bellefonte, PA), 30 m⫻0.25 mm ID (0.1-àm film thickness) DB-1 fused silica capillary column, temperature programmed from 120 to 310°C at 8°C/min, 1-àl injection at 50 : 1 split. Mass spectrometer: Finnigan TSQ- 46B, 70-eV electron ionization, full-scan mode from 35 to 500 Da in 1 sec cycle time.
many of the isomeric steranes coelute and interfere with each other. This necessi- tates compromise when steranes are measured by GC–MS.
These coelution problems can be largely resolved by coupling MS–MS with GC, that is, GC–MS–MS. The MS–MS can be in a form of two mass analyzers in tandem that monitor precursor ions and fragment ions in respective analyzers. Other forms of MS–MS include metastable monitoring performed on a double-focusing sector mass spectrometer using specific scanning techniques [23,24]. Most of the GC–MS–MS applications for biomarkers utilize precursor (or parent) scans where the precursor ions of selected characteristic fragment ions are monitored. Tandem triple-stage quadrupole (TSQ) mass spectrometers are most suitable for this task. In a TSQ mass spectrometer, the first quadrupole is
64 Hsu and Drinkwater
Figure 4 The 191- and 217-Da mass chromatograms from SIM GC–MS analysis. Gas chromatography–mass spectrometry conditions are the same as Figure 3 except the acqui- sition being in a SIM mode rather than a full-scan mode; residence time per mass, 100 msec.
used as the first stage of the mass analyzer (MS-1) to scan or focus precursor (parent) ions, while the third quadrupole is used as the second stage of the mass analyzer (MS-2) to scan or focus product (daughter) ions. Only radio-frequency (RF) voltage is applied on the second-stage quadrupole for collimating ions. The second-stage quadrupole is often used as a collision region to enhance fragmenta- tion of precursor ions. A TSQ mass spectrometer can perform precursor (parent), product (daughter), and neutral scans [25]. Ion trap and FT-ICR mass spectrome- ters, on the other hand, can only be used for product (daughter) scans.
Although daughter scans are most commonly used in the MS–MS applica- tions, parent scans yield the most interference-free biomarker distributions. In a parent scan mode, second-stage MS is used as a filter to allow selected ions characteristic to biomarkers to fly through while first-stage MS is scanned to record the molecular ions that produce the selected ions. Only biomarker com- pounds that can yield both the molecular and characteristic fragment ions are
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detected while other coeluting nonbiomarker compounds are filtered out. Thus, GC–MS–MS provides interference-free measurement of biomarker compounds, resulting in baseline resolution of the chromatographic peaks.
The advantage of GC–MS–MS lies in the ability to distinguish among homologous series of steranes by linking their molecular ions with their character- istic fragment ion at 217 Da. Figure 5 shows typical GC–MS–MS chromato- grams for steranes. The top four traces (A through D) are selected GC–MS–MS measurements between the molecular ions and the 217-Da ion for the C27through C30 steranes, respectively. The bottom trace (E) is the integration of traces A through D, which would be similar to the 217-Da mass chromatogram from the GC–MS analysis, as shown in Figure 5. Thus, the GC–MS–MS technique can determine distributions of diasteranes, isosteranes, and regular steranes for each carbon homologue without any interference due to coelution. For example, Figure 5 shows that the two C27αββsteranes ((20R)-5α(H),14β(H),17β(H)-cholestane and (20S)-5α(H),14β(H),17β(H)-cholestane) actually coelute with the (20R)-
Figure 5 Gas chromatography–mass spectrometry–mass spectrometric chromatograms of steranes in a mature oil. Conditions are the same as Figure 3 except the acquisition being in parent scan mode rather than a full-scan mode; residence time per parent mass, 100 msec.
66 Hsu and Drinkwater
24-methyl-13α(H),17β(H)-diacholestane (C28αβR) and (20S)-24-ethyl-13β(H), 17α(H)-diacholestane (C29βαS). For the (20S)-24-ethyl-5α(H),14α(H),17α(H)- cholestane (C29ααα) and (20S)-24-ethyl-5α(H),14β(H),17β(H)-cholestane (C29
αββ), the only interference is the coelution of the C30 αβ steranes, (20S)-24- propyl-13α(H),17β(H)-diacholestane and (20R)-24-propyl-13α(H),17β(H)-dia- cholestane, that are usually present in trace amounts [28].
Steranes are routinely employed in a variety of geochemical applications.
They are used to evaluate thermal maturity of oils and source rocks from the abundance ofαααS relative toαααR, to estimate organic matter inputs (marine versus terrigenous) from the ratio of C27/C29steranes, and to assess the geological age of the source rock from the ratio of C28/C29 steranes in the oil generated [14,26,27]. The C30steranes, shown in the 414⫹→217⫹GC–MS–MS chromato- grams of Figure 5, are unambiguous indicators of contributions from marine- derived organic matter [29]. Their presence in oils and source rocks cannot be unambiguously determined by GC–MS due to severe interference from C28and C29steranes. Hence, GC–MS–MS is the only means to accurately measure this valuable sterane marker.