Radiant energy – Ionic separation or analysis – Methods
Reexamination Certificate
2003-03-31
2004-06-08
Lee, John R. (Department: 2881)
Radiant energy
Ionic separation or analysis
Methods
C436S161000, C435S007100
Reexamination Certificate
active
06747272
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a method and apparatus for analyzing spectra obtained by a mass spectrometer and, more particularly, to a method and apparatus for analyzing spectra obtained by mass-analyzing ions to which molecules of a mobile phase solvent or impurities contained in the solvent are attached.
2. Description of Related Art
Where the masses of molecules (hereinafter referred to as sample molecules indicated by M) contained in a sample are analyzed using a mass spectrometer, the sample is ionized. Various methods are available for the ionization. In one of these methods, ions of the sample molecules with mobile phase solvent or impurities contained in the solvent attached to the sample molecules are generated by ionization.
A typical example of such ionization method is atmospheric pressure ionization (API) used as an interface between a liquid chromatograph (LC) and a mass spectrometer (MS).
Two kinds of API methods are available: electrospray ionization (ESI) and atmospheric-pressure chemical ionization (APCI). In either method, ionization is performed by movement of protons between sample molecules and molecules of a mobile phase solvent, such as methanol, acetonitrile, or acetic acid.
Where positive ions are detected by a mass spectrometer, protonated ions [M+H]
+
consisting of sample molecules M to which protons H
+
are attached are detected. Where negative ions are detected by a mass spectrometer, deprotonated ions [M−H]
−
consisting of sample molecules M from which protons H
+
have been abstracted are detected.
If ions produced by API are only either protonated ions [M+H]
+
or deprotonated ions [M−H]
−
, no serious problems take place. It is known, however, that adduct ions are generated in addition to protonated or deprotonated ions where molecules of mobile phase solvent or impurities contained in it are attached to sample molecules and that such adduct ions are detected by mass spectrometry and often appear in spectra.
Ions where molecules of a mobile phase solvent or impurities contained in it are attached to sample molecules as mentioned above are hereinafter referred to as impurity adduct ions. Impurity adduct ions, protonated ions, and deprotonated ions are collectively referred to as adduct ions. For example, in protonated ions, protons are adducts. In impurity adduct ions [M+NH
4
]
+
, ammonium ions are adducts as described later. Also, with respect to deprotonated ions, protons are conveniently referred to as adducts.
For example, where ionization is done by ESI using methanol as a mobile phase solvent and positive ions are detected by a mass spectrometer, it is empirically known that positive impurity adduct ions [M+NH
4
]
+
and/or [M+Na]
+
are sometimes detected in addition to protonated ions [M+H]
+
. In the ions [M+NH
4
]
+
, ammonium ions are attached to sample molecules M. In the ions [M+Na]
+
, sodium ions are attached to sample molecules M.
It is also empirically known that where methanol is used as a mobile phase solvent, ionization is performed by APCI, and positive ions are detected by mass spectrometry, positive impurity adduct ions [M+H+CH
3
OH]
+
where protons H
+
and methanol molecules are attached to sample molecules M are sometimes detected in addition to protonated ions [M+H]
+
.
Furthermore, it is experimentally known that where a sample is ionized by ESI using formic acid as a mobile phase solvent and negative ions are detected by mass spectrometry, negative impurity adduct ions [M+HCOO]
−
where formic acid ions are attached to sample molecules M are sometimes detected in addition to deprotonated ions [M−H]
−
.
Where samples are ionized by API and mass analyzed in this way, peaks of impurity adduct ions appear in the resulting spectrum, in addition to peaks of protonated or deprotonated ions. This often makes it difficult to judge the molecular weight of the sample molecules based on the spectrum or to analyze the spectrum.
Accordingly, it is quite difficult to analyze spectra obtained by ionizing samples by API and mass analyzing them. Therefore, a rich amount of experience is necessary to determine the molecular weight of sample molecules based on the spectrum or to analyze the spectrum. In addition, there is even the problem that the results of spectral analysis differ according to the degree of experience of each analyst.
The case where ionization is performed by API has been described thus far. Impurity adduct ions may also be generated where ionization is performed by methods other than API, e.g., chemical ionization (CI), fast atom bombardment (FAB), matrix assisted laser desorption (MALDI), and field desorption (FD). Where mass spectra obtained by these ionization methods are analyzed, similar problems take place. This is a first problem that the present application tackles.
Spectra obtained by ionizing samples by the aforementioned various ionization methods and mass analyzing them are compared with a very large number of mass spectra registered in a commercially available library, or database, of mass spectral data to identify the chemical formulas of observed ions. However, such registered mass spectra have all been derived by electron impact (EI) ionization that is a hard ionization method. Its feature is that the peaks of each individual molecular ion [M]
+
charged positively by release of one electron from each sample molecule are distributed in the highest mass-charge ratio (m/z) region of the resulting spectrum while peaks of fragment ions produced by fragmentation of molecular ions [M]
+
are distributed in a lower m/z region than the molecular ions. One example is a mass spectrum of toluene as shown in FIG.
1
. This spectrum is a bar-type spectrum in which the peaks of a measured mass spectrum are data processed and represented as a bar graph. That is, each peak is represented in terms of a bar.
Such a library, or database, of mass spectra owing to EI is generally applied to mass spectra obtained by a GC/MS instrument that is a combination of a gas chromatograph and a mass spectrometer. In a GC/MS measuring system, peaks of adduct ions are not contained at all in mass spectra. Consequently, where samples ionized by soft ionization methods, such as API, CI, FAB, MALDI, and FD are compared with mass spectra which are obtained by a mass spectrometer and contain many peaks of adduct ions, pattern mismatch occurs frequently, even if they are mass spectra of the same compound.
Where samples are ionized by El, molecular ions [M]
+
that are charged positively by release of one electron from each sample molecule are observed routinely. However, mass spectra obtained by ionizing samples by a soft ionization method such as API, CI, FAB, MALDI, or FD and detecting the resulting ions by a mass spectrometer contain almost no such molecular ions [M]
+
.
Where API, CI, FAB, MALDI, or FD is used, fragment ions are not readily produced because of a soft ionization method. Yet, fragment ions can be produced by ESI or APCI by using in-source CID or in-source fragmentation that produces fragment ions by applying a voltage of tens of volts to the ion introduction port (orifice) of the vacuum region from the atmospheric-pressure ionization region to momentarily accelerate ions for collision with atmospheric gas.
FAB is a somewhat harder ionization method than ESI and APCI and, therefore, fragment ions are often produced depending on the nature of the measured compound.
Where mass spectra are obtained by a soft ionization method as described, if the measurement is performed by in-source CID, spectra having many fragment ions can be derived. Yet, there is the problem that the hit (match) rate is low when a search is do
Gill Erin-Michael
JEOL Ltd.
Lee John R.
Webb Ziesenheim & Logsdon Orkin & Hanson, P.C.
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