Procedures for Data Processing in Chromatography Mass Spectrometry

A data set acquired with a GC-MS device will be used as the base to describe the common procedures of data processing in chromatography-MS. In GC-MS, samples are eluted into the source of the mass spectrometer at their characteristic retention times. For a Gaussian-shaped peak, the concentration of the sample in the source starts at a low value, increases to its maximum value, and then decreases symmetrically. Part of the challenge of MS is to record a characteristic mass spectrum for this sample of constantly changing concentration, given that a certain amount of time is required to scan the mass filter across its mass range. Modern mass spectrometers (considered here to be quadrupole, sector, or ion trap mass spectrometers) scan across the usual mass range in a short time. The instrument specifications for a sector instrument may show that the scan speed of the mass analyser can be as fast as 0.1 s decade_1. In this context a decade is the mass range between 10 and 100 Da, or similarly between 100 and 1000 Da. In a sector instrument the scanned parameter is the magnetic field strength, and the ion m/z passing through the instrument varies with the square of this value. In a quadrupole or ion trap instrument, the scan parameter is linearly proportional to the mass of the ion in the analyser.

Approximately ten scans of the mass analyser are required to characterize any GC peak. This requirement stands regardless of the width of the GC peak, and, as the peaks in GC narrow with the achievement of higher separation resolutions, the requirements on the scan speed of the mass analyser become more onerous. Changing concentration of the sample in the source will distort the true intensities of the ions observed in the measured mass spectrum in each individual scan, so mass spectra recorded across the entire width of the peak are averaged together to create a mass spectrum that contains more accurate ion intensities. To ease the burden, the mass analyser can be forced to scan faster and faster, but eventually a fundamental hardware constraint limits the scan speed. Faced with these limits, the analyst can narrow the mass range across which the analyser must scan, or choose to monitor only certain significant ions from within the mass range. In either case, the analyst risks losing the ability to accurately identify unexpected compounds in a complex mixture.

The discussion that follows is software-independent. Each computer system will handle the operations described in its own unique fashion, and there may be specialized procedures beyond those that are described here. It is expected that the analyst will understand the general principles involved, and then take the time to explicitly explore these functions on the particular instrument to be used for analysis.

Background subtraction Resolution in chromato-graphy is established by the time between adjacent peaks in which no sample components are eluting into the source of the mass spectrometer. Generally, the higher the chromatographic resolution, the more 'empty' space there is in the chromatogram. The mass spectrometer is recording mass spectral data even during the times when nothing is eluting from the chromatograph. These scans become the mass spectra of the background. The background mass spectrum is not constant. During a GC-MS run the background changes because of increased bleed from the column at increased temperatures encountered during a temperature ramp, low-level, highly retained components, and the continual desorption of organic compounds and contaminants absorbed throughout the system. As background is not constant, the details of background subtraction are not constant, although the correct procedure is generally accepted.

There is no error-free method of arbitrarily reducing the contributions of background in a mass spectrum to zero. However, based on the assumption that the sample background and ionization is independent of the elution and ionization of the sample component itself, then the number of scans averaged together to create a background mass spectrum should be equal to the number of scans averaged together to provide the mass spectrum of the sample. Simplisti-cally, if ten mass spectral scans are necessary to completely characterize a peak, and these ten scans will be averaged together, then the 'background' mass spectrum should also be the average of ten scans. The scans taken for the background should be taken from as close before the peak elution, and from as close after the peak elution, as practical. There is no guarantee that the intensities of background ions in the mass spectrum will be reduced to zero, but they will be minimized relative to those ions that are derived from the sample component. Simple mathematical subtraction may lead to negative relative intensities for some ions; these are arbitrarily set to zero.

When two sample components elute very close to one another, it is not possible to follow the before/after procedure just outlined. In the simple identification of a background mass spectrum, advantage is taken of the fact that the background does not change rapidly. The portion of the chromatogram from which the background mass spectrum, is taken is simply the first encountered as one moves to a point in the chromatogram when it is assumed that there is no contribution from the eluting compound. A more enlightened use of background subtraction involves creating a high quality mass spectrum from each of two closely eluting or even overlapped components. In this case advantage is taken of the fact that peak intensities that are reduced in intensity to zero do not distort the mass spectrum, and that the probabilities that ion masses overlap are small. In the case described, one can enter as background mass spectra those scans that are clearly from the second of two components with the assurance that the ions from this component will be effectively removed from the mass spectrum of the first.

The intensity behaviours of ions that belong to the background and those that belong to an eluting peak are different. This difference can be highlighted mathematically. The first derivative of the values for ion intensities that are changing will have a nonzero value. However, the first derivative of the ion intensities in an eluting peak will be characteristic in crossing zero at the retention time. This difference in behaviour can be used to identify ions from the sample as opposed to ions from the background. The mathematical result is an 'enhanced' mass spectrum. Note that the first derivatives for different ions will cross zero at slightly different times, depending on the rate of scan of the mass analyser.

Data averaging In the perfect chromatographic separation, the peak has a Gaussian shape. In the perfect spectroscopic detection system, the signal can thus be plotted as a convoluted function of mass analyser scan time and the number of scans across the peak profile, and the assumed constant level of noise recorded in the mass spectrum, from either chemical or electronic sources. None of these ideal assumptions is true. To increase the signal-to-noise (S/N) ratio of the mass spectrum recorded for a chromatographic peak, it is common practice, as described, to sum or average all of the mass spectra gathered as the peak elutes.

The need for data averaging is most apparent when the scan time is a significant fraction of the peak width. Given that ten scans across a peak are needed to establish the elution profile, the example described here is for just such a situation. Further, we will make the assumption that (as is commonly the case) the analyser scan is from high mass to low mass. The first scans across a peak are recorded as the concentration of the sample in the source is increasing. There is a bias in ion intensities towards the low-mass end of the mass spectrum. If a steady state sample concentration is reached, a mass spectrum with accurate and true ion intensities is recorded. However, such a situation is unlikely. After the instantaneous maximum in sample concentration is reached, the amount of sample in the source starts to decrease, and the remaining scans are recorded as the concentration of the sample is decreasing. The trailing scans will also be biased in that the ion intensities at the higher mass end of the spectrum will be too high relative to their low-mass counterparts. If the peak is symmetrical, and there are enough scans on either side of the peak maximum, the bias can be muted by simply averaging all the spectra together. This processed mass spectrum should be approximately the same as if the sample concentration in the source was constant, and it can therefore be searched against a mass spectral library.

Intuitively, the analyst wants to average all the mass spectra that are recorded for an eluting peak, even at the leading and trailing edges of the peak where the signal is first and last discernible against background. Even though a simple summation is the most common practice, it is not optimal in terms of providing the maximum S/N ratio in the processed mass spectrum. Recent work by Chang shows that only the mass spectra for which the ions are at least 38% of the maximum recorded ion abundance should be included in the summation. Furthermore, the use of a matched filter (such as used in NMR experiments) provides an additional increment in S/N ratio, provided that the shape of the matched filter parallels that of the chromatographic peak itself. The data processing applies to any combination of chromatography and detection, but is demonstrated specifically with the combination of LC and MS. The widespread use of processed mass spectrometric data to provide enhanced chromatographic resolution, based on the independence of the mass spectrometric data, makes this study particularly worthwhile. It is revealing that background subtraction has been used in mass spectral data processing for decades, and essential elements of its character are still being deduced.

Reconstructed ion chromatograms A peak eluting from a chromatographic column exhibits a characteristic retention time. When using MS as a detection technique, that retention time is determined by the point at which all the ion intensities in the mass spectrum reach their maximum value. As described previously, the first derivative of the ion intensities crosses zero. That statement was carefully crafted to differ from the statement that the retention time is the point at which the total ion current (TIC) trace reaches a maximum. The TIC summed intensity is derived from both sample-related and background ions. The two statements are usually, but not always, identical in meaning. Using the more accurate description also provides the underlying basis for introduction of the reconstructed ion chromatogram (RIC) procedure.

A data file contains mass spectra recorded as a function of time; the mass spectrum is a table of m/z values and intensities. Therefore the data file is a collection of intensities of all of the m/z channels recorded as a function of time. Any m/z value can be specified, and the intensity data can be extracted from the data set and plotted as a function of time. In the elution of a sample peak from a column into the mass spectrometer, all of the sample-related ions should follow a similar time profile as the concentration rises. It is assumed that if an ion properly 'belongs' in the mass spectrum, then its intensity profile should track in time all of the other sample-related ions. Therefore, the converse should hold. If an ion intensity trace follows the same profile as ions that are known to be in the mass spectrum, then it 'belongs' in the mass spectrum. More powerfully, if it does not follow that trace exactly, then it does not belong. The RIC is nothing but an independent series of intensity versus time traces that graphically establish spectral propriety, and provide hints when something is amiss. The plots are independently calculated, and the absolute intensity of the ion is normalized. The graphical appearance of correctness is striking in the alignment of peak maxima and in the duplication of peak shape on both leading and trailing edges. When there is an unresolved peak component, ions that belong in that mass spectra show a strikingly different trace. A peak that belongs in the background will show a slowly changing trace with multiple maxima.

If the sample analysed by GC-MS is a mixture of closely related compounds, the mass spectra of each member of that compound class will generally contain characteristic ions of the same mass. As the analyst recognizes that a correspondence exists between the ion mass and the compound class, the entire data set can be interrogated for those characteristic ions. The RIC trace should exhibit multiple maxima that correspond to the retention times of each of the individual compounds in that class. The relative areas or peak heights for each trace do not directly represent the quantitative distribution of those class members, since they reflect the relative intensity of that mass-specific ion in the mass spectrum. However, the power of the RIC in highlighting compounds within a homologous series is evident.

Selected ion monitoring Selected ion monitoring (SIM) is a procedure used in data acquisition and processing in which the mass analyser is not scanned over a mass range, but instead hops rapidly between several preselected m/z values (this is called peak hopping). For example, instead of scanning the mass range from 35 to 1000 Da in 0.2 s, the analyser will spend some time at m/z 77, some time at m/z 91, some time on the ion at m/z 135, and finally some time at m/z 180. Usually, all of these ions are those that belong in the mass spectrum of the targeted sample component. In the scanning experiment the analyser will spend 0.2 s/965 = 2.07 x 10~4 s recording the signal in each nominal mass channel. In the SIM experiment the same 0.2 s (ignoring the short time that it takes to hop between peaks) is spent monitoring the ion signal in four ion channels. The detector is integrating a signal for a period that is 242 times as long in the SIM experiment. If this is indeed where the signal is to be found, then an increase in the sensitivity of the mass spectrometric analysis can be attained simply by virtue of the fact that a longer time is spent recording the signal. S/N ratios for each individual ion trace are appropriately increased as well, since this value scales with the number of independent measurements taken. Various values for the increase in sensitivity attained with the use of SIM are found in the literature. These values range from 10 to 100 fold, and depend on the width of the ion mass window monitored, and the intensity of the ions to be found within that window. Unfortunately, ions chosen for SIM are often only those that are found in the mass spectrum of the targeted sample component. It is wise to include in the selected ions a m/z value that represents a nonsample ion, so that the true S/N ratio for the experiment can be determined.

The analyst should be clear about what is gained and what is lost in the SIM experiment. Clearly there is a gain in sensitivity. The resolution of the mass analysis is not changed, so there is no tradeoff here. What is lost is the generality of the mass spectro-metric detection. In short, the analyst must already know the identity of the target compound, for example, and the masses of the ions to be monitored. These are established in separate experiments that precede the selection of the SIM experiment. If a large amount of an unexpected sample is eluted from the GC during the SIM experiment, and this unexpected adulterant does not produce ions at the monitored masses, and there is no matrix effect as a result of its presence, it will simply go undetected, even if it elutes at exactly the same time as the targeted component. Loss of such 'insurance' capability should always be carefully considered when setting up a SIM protocol.

The output of a RIC looks identical to the output of a SIM experiment. However, in this case the RIC graphical output is the result of a data processing routine. During the data acquisition process, the mass analyser is scanned across the full mass range, and each scan is a complete mass spectrum in the stored computer file. A full data set can be interrogated repeatedly with different selected ions. A SIM experiment contains ion intensities only for those ions that were selected. The RIC provides an increase in confidence of spectral propriety, but no increase in sensitivity.

Advanced computer processing The combination of chromatography with MS would not exist today were it not for the capabilities of computers in instrument control, data acquisition, data processing, and spectral manipulation and display. Advances in computer capabilities have provided more precise control, faster and more accurate data acquisition, faster and more sophisticated data processing, and higher content and more striking visual displays of chromato-graphic and mass spectral data. Computational power has always been applied to the interpretation of mass spectral data, and computer-assisted interpretation of mass spectral data, specifically in the area of structure/spectral relationships, continues. Computer-aided interpretation, orginally applied exclusively to EI mass spectra, is now used with success in the interpretation of CI, ESI, and MS-MS data. It is analytically compelling to support this expansion, as it is unlikely that libraries of these types of mass spectral data will grow to the size of the current libraries of EI mass spectral data. The precepts behind computer applications in the interpretation of mass spectral data have been described (Karjalainen). More recent applications have increased the speed and expanded the scope of applications, but no matter what, the progress the fundamental principles continue to apply. Pattern recognition programs can be used to recognize similarities in groups of mass spectra data. Calibration, especially in isotope ratio measurements, often involves sophisticated computer-performed mathematical algorithms. Pyrolysis MS often involves searches for similarities and differences in complex mass spectra through computer algorithms. Correlation analysis is used in many different areas of MS.

The growth in computer-assisted evaluation of chromatography-mass spectrometry data has been slower. This is surprising given the sophistication of computer hardware and software, and the proliferation of chromatography-mass spectrometry instruments. The quantitative information content of GC-MS has been described using latent variables in the context of multivariate analysis. Regression and least squares methods have been used to specifically model quantitative results obtained for GC-MS of closely eluting compounds. Procrustes analyses have been used to determine the number of significant masses in GC-MS, where significant masses represent the ions in the mass spectrum that differentiate one compound from the other. Each of these recent studies suggests that there is more information to be obtained from GC-MS than we have yet mined. The promise is that the general informational methods described will be adopted seamlessly into LC-MS and CE-MS as well.

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