1. Field of the Invention
The present invention relates to a method for sample identification by mass spectrometry and more particularly the invention is concerned with a method for sample identification based on isotope abundance.
2. Background of the Invention
Gas chromatography (GC) and liquid chromatography (LC) are important analytical techniques used today for the separation, identification and quantification of a broad range of samples and mixture of compounds. While elution time can serve for crude sample identification, mass spectrometry is by far the best and most established technology for such identification, including at trace levels. For gas chromatography mass spectrometry (GC-MS), sample identification is predominantly based on the use of extensive available 70 eV electron ionization (EI) mass spectral libraries. Library based sample identification is performed via a comparison of the experimental mass spectrum to all the library mass spectra and than the provision of a hit list (such as of 100 compounds) of candidates for the sample identity with reducing order of fitting or of a matching parameter. Accordingly, sample identification with MS libraries is predominantly based on fragment ions that provide a compound specific “finger print”. These libraries are both powerful and easy to use, however, sample identification with MS libraries is confronted with three major limitations: a) While the current libraries include a few hundred thousand compounds with the majority of all environmentally important compounds, a few millions of possible compounds are not included in the libraries, and in particular, novel synthetic organic compounds and drugs are (by definition) absent from the MS libraries; b) Occasionally, the library fails in sample identification either since the sample is not included in the library or due to coelution of two or more compounds or due to statistical errors; and c) About 30% of the sample compounds do not show a significant molecular ion in their 70 eV electron ionization MS. For these compounds sample identification through libraries alone cannot be trusted due to the possibility of false identification of a homologous compound or a degradation product. Thus, there is a need for additional supplementary and complementary means of preferably automated sample identification. An alternative approach for mass spectral sample identification is the measurement of accurate mass, typically with mass measurement precision of a few parts per million, followed by computer based conversion of that accurate mass into a list of possible elemental formulas which are arranged in order of increased deviation from the measured mass. For such inversion of experimental data into elemental formula the user must provide as an initial input parameter a short list of possible elements, otherwise the generated hit list will be too large and the calculation time could be too long even with the most powerful computers. The use of accurate mass for the provision of elemental formulas is based on the elemental specific distribution of isotopic masses. The method of accurate mass for the provision of elemental formulas is powerful but requires the use of costly mass spectrometer instrumentation such as time of flight, ion cyclotron or magnetic sectors. In addition, this method fails to provide any information if the molecular ion does not appear in the mass spectrum and can even give false identification on a fragment or impurity ion. Furthermore, in contrast to libraries, accurate mass does not provide any isomer identification information. Finally, for relatively large compounds and when the list of possible elements is not limited to very few elements, accurate mass can provide a too long list of candidates without real sample identification.
A closer look at the molecular ion in any typical mass spectrum reveals that it is actually a group of peaks spaced at 1 amu apart, emerging from the natural abundance of two or a few isotopes for most of the elements. It is well known and established that the relative height of the various molecular ion peaks that belong to the same molecule but with different isotopes (isotopomers) emerges from the relative abundances of the various isotopes and several programs are available for the calculation of the isotope abundance patterns from a given input of elemental formulas and natural isotope abundances of the various elements in that elemental formula. However, the opposite method of inversion of experimental mass spectral isotope abundance patterns into elemental formula (which is referred to as isotope abundance analysis (IAA)) is a much harder challenge. The challenges in the successful inversion of MS isotope abundance data into elemental formulas seems daunting for a few well established practical reasons: a) Isotope abundance analysis requires that the molecular ion will be available while it is missing from ordinary 70 ev EI mass spectra of more than 30% of the sample compounds; b) IAA requires that the relative heights of the various isotopomers can be accurately measured, including with low sample amounts during their short elution time from a GC or LC; c) IAA requires the absence of matrix and or sample induced self chemical ionization that distorts the experimentally measured isotope abundances due to uncontrolled degree of protonation; d) IAA requires the absence of vacuum background that distorts the measures isotope abundances, especially at low sample levels. e) IAA requires a useful method for the inversion of isotope abundance MS data into a short list of most probable elemental formulas that can provide a reliable method of sample identification. These obstacles and the seemingly limited possibility of success resulted in lack of motivation. Thus, isotope abundance analysis was generally neglected in view of the combination of lack of motivation, absence of automated effective inversion method and scarcity of useful experimental isotope abundances data.
In recent years a new type of electron ionization mass spectrometry with supersonic molecular beams (SMB) was developed, and applied with GC-MS and LC-MS. The use of SMB for analytical mass spectrometry is based on the introduction of sample compounds into an electron ionization ion source as vibrationally cold molecules in a seeded supersonic molecular beam. The electron ionization (EI) is performed in a unique fly-through EI ion source, adopted for the ionization of sample compounds while they are traveling along the ion source axis as vibrationally cold molecules, due to their cooling by the seeding gas in the supersonic expansion. The most important attribute of electron ionization of vibrationally cold sample molecules in SMB is that the molecular ion is significantly enhanced and it is practically always observed. In addition, the use of SMB with a light carrier (seeding) gas such as helium (or even vaporized solvent in LC-EI-MS of large molecules) enables the sample compounds to acquire directional hyperthermal kinetic energy. As a result, a unique mass spectral vacuum background filtration was achieved and the experimentally obtained mass spectra are clean, without vacuum background distortion. Furthermore, the collision free conditions prevailing in the EI of sample compounds in SMB ensure the full elimination of the adverse effects of self and matrix induced chemical ionization (CI). Consequently, electron ionization mass spectra of samples in SMB in both GC-MS and LC-EI-MS with SMB seems ideal for IAA, if an appropriate and preferably automated method will be developed for the inversion of its useful mass spectral isotope abundance data into elemental formula information.