Patent Application
20150346152
The applicants' teachings pertain to analytical chemistry including mass spectrometry methods and apparatus.
The number of and exact location of carbon-carbon double bonds (CCDBs) within a molecule (e.g., lipids such as fatty acids, triacylglycerols, etc.) can be of great importance to understanding the chemical reactivity of such molecules. In some cases, such lipids are metabolites from human or animal subjects, and the identification of CCDB number and position is essential as a diagnostic tool in health care. In other cases, lipids are present in modern biofuels, and the presence of CCDBs can affect combustion efficiency and processing parameters.
The unambiguous identification of CCDB number and location in a molecule can be performed by using mass spectrometry, specifically a technique known as ozone-induced dissociation (OzID), which uses the well-established reaction of ozone with CCDBs to cleave these functionalities in a specific, characteristic manner. However, the general use of OzID requires manual intervention and a priori knowledge regarding the presence of CCDBs in an analytical sample. Accordingly, there remains a need for improved methods and systems for identifying CCDBs in analytes, while simultaneously characterizing the remainder of the structural features of these analytes by using other techniques of mass spectrometric analysis.
The foregoing are among the objects attained by the applicants' teachings, which provide, in some aspects, methods and apparatus for mass spectrometry analysis that identify the location of carbon-carbon double bonds (CCDBs), if any, in an analyte by (1) subjecting its ions to collision-induced dissociation (CID) and (2) determining relationships between the ions and/or fragments produced by such CID. The methods and apparatus selectively subject analyte ions to ozone-induced dissociation (OzID) based on those relationships and determine the number and/or location(s) of CCDBs, if any, from reaction products of OzID.
Related aspects of the applicants' teachings provide such methods and apparatus that determine the masses of charged or neutral fragments (so called neutral losses) resulting from CID and that utilize those masses in the determination of whether to subject analyte ions to OzID.
Further aspects of the applicants' teachings are set forth in the claims attached hereto.
Methods and apparatus according to the applicants' teachings are advantageous, among other reasons, in that they make possible the mass spectrometric analysis, e.g., of lipids, petrochemicals, and polymers (among other compounds), including the determining the number and/or location(s) of CCDBs therein, on time-scales typically associated with liquid chromatography.
These and other features of the applicants' teachings are set forth herein.
The skilled person in the art will understand that the drawings, described below, are for illustration purposes only. The drawings are not intended to limit the scope of the applicant's teachings in any way:
Referring to
Although the spectrometer 12 and computer 22 are each shown, here, as a separate units housing respective constituent components, in some embodiments those components may be housed otherwise. Thus, for example, the computer 22 (or one or more components thereof) may be housed with the spectrometer 12, one or more components of the spectrometer may comprise stand-alone equipment, and so forth all by way of example. For these reasons, among others, the terms “apparatus” and “systems” are used interchangeably herein.
The ion source 14 is configured to emit ions generated from the analyte or sample (not shown) to be analyzed. The ion source 14 is constructed and operated (e.g., by a human operator, computer 22, and/or otherwise) in the conventional manner known in the art of mass spectrometry, as adapted in accord with the teachings hereof. The ion source 14 can include, but is not limited to, a continuous ion source, such as an electron impact (EI), chemical ionization (CI), or field desorption-ionization (FD/I) ion sources (which may be used in conjunction with a gas chromatography source); an electrospray (ESI) or atmospheric pressure chemical ionization (APCI) ion source (which may be used in conjunction with a liquid chromatography source); a desorption electrospray ionization (DESI); or a laser desorption ionization source such as a matrix assisted laser desorption ionization (MALDI), laser desorption-ionization (LDI) or laserspray (which typically utilizes a series of pulses to emit a pulsed beam of ions).
Ions generated by the ion source 14 are transmitted to mass filter 16, which is configured to select (or filter) a subset of ions within a chosen mass-to-charge ratio range and/or based on intensity of the analyte ions for transmission into the reaction region 18. The mass filter is constructed and operated (e.g., by a human operator, computer 22, and/or otherwise) in the conventional manner known in the art, as adapted in accord with the teachings hereof. The mass filter 16 can include, but is not limited to, a quadrupole mass filter, an ion trapping device (such as a 3D or 2D quadrupole ion trap, a C-trap, or an electrostatic ion trap), all by way of example.
Ions emitted by the mass filter 16 are admitted into the region 18 for reaction with a reagent gas or gas mixture under a prescribed pressure. The mass filter 16 is constructed and operated (e.g., by a human operator, computer 22, and/or otherwise) in the conventional manner known in the art, as adapted in accord with the teachings hereof. It can be injected from source 18a with an inert reagent gas of the type known in the art that is typically used in collision-induced dissociation (CID) reactions, e.g., helium, neon, nitrogen, argon, xenon, or air, by way of non-limiting example, and/or, from source 18b, with ozone so as to form a mixture with the inert gas. The reaction region 18 can include, but is not limited to, a quadrupole mass filter, an ion trapping device (such as a 3D or 2D quadrupole ion trap, a C-trap, or an electrostatic ion trap), all by way of example. Injection of the region 18 from sources 18a, 18b can be controlled by computer 22 and/or by an operator in the conventional manner known in the art, as adapted in accord with the teachings hereof.
Ions admitted to the reaction region 18 may pass through the region without incurring any structural fragmentation, or they may fragment as a result of collision with atoms/molecules of the gas mixture present in the region 18 and/or as a result of dissociation (e.g., under the influence of ozone). Some or all or the ions may be trapped for a period of time in the region before passing through.
The ion analyzer 20 is positioned downstream of the ion source 14 and the reaction region 18 in the path of the ions emitted from reaction region 18. Analyzer 20, which may include a detector (not shown) separates the emitted ions and fragments as a function of mass-to-charge ratio (m/z) and generates an output representative of the number of ions at each m/z value. The ion analyzer 20 (and constituent detector) is constructed and operated (e.g., by a human operator, computer 22, and/or otherwise) in the conventional manner known in the art, as adapted in accord with the teachings hereof. The ion analyzer 20 can include, but is not limited to, a quadrupole mass filter, an ion trapping device (such as a 3D or 2D quadrupole ion trap, a C-trap, or an electrostatic ion trap), an ion cyclotron resonance trap, an Orbitrap, or a time-of-flight mass spectrometer, all by way of example.
Components 14-20 of the mass spectrometer 12 are coupled by tubing, valves and other apparatus of the type conventionally used in the art to form an flow path suitable for passage and analysis of ions generated by source 14 in accord with the teachings hereof.
Computer 22 comprises a general- or special-purpose digital data processor (stand-alone, embedded or otherwise) of the type known in the art suitable for controlling and/or providing an interface to the mass spectrometer 12, all in the conventional manner known in the art, as adapted in accord with the teachings hereof. Thus, for example, software 24 executes on computer 22 in order to facilitate and/or effect operation of the mass spectrometer 12 using information or information-dependent acquisition consistent with the teachings hereof, and data storage 26 retains mass-to-charge data output by analyzer 20 and/or data (e.g., tables of specific neutral losses from lipid ions that are known to contain CCDBs) utilized for identification of samples appropriate for OzID-based analysis.
To this end, the computer 22 and/or the operator effect operation of the mass spectrometer 12 (and, more generally, of the system 10) in accord with the workflow shown in
Referring to the drawing, the ion source 14 is used to generate ions from an analyte. Step 30.
Mass filter 16 can then be used to isolate a subset of those ions to simplify the analysis. This subset can contain a single analyte ion (one m/z value—e.g., m/z 100+/−0.5) or, if the mass filter is configured to permit passage of the full range of ions (or, alternatively, the mass filter is not applied), can contain a window of ions (e.g., m/z 100+/−20). Step 32.
Those ions are transmitted through the mass spectrometer, including the reaction region 18, and are detected by the ion analyzer without any modification, reactions, or fragmentation. Step 34. This yields information on the intact molecular masses of the chosen analyte ions.
In a subsequent analysis, the reaction region 18, which is filled with the inert target gas (e.g., nitrogen, argon) from a suitable source (see element 18a,
The software 24 then compares the mass spectrum of the intact analyte ions (from steps 30-34) with the mass spectrum of the CID fragments of those ions (from steps 36-38) and determines whether there is a relationship between intact and fragmented ions that would indicate presence of one or more CCDBs. The relationship may be based on a specific mass difference between any of the fragment ions and the intact analyte ions or on a specific CID fragment ion. The software determines the presence of CCDB based either on lookup tables or using internal fragment and/or neutral loss prediction algorithms. The software 24 can also predict ab initio the presence of CCDBs in a charged or neutral loss fragment using exact mass calculations. See step 40. (In such cases, the fragmentation step can be of value, for example, in collecting complementary CID information for the species.)
If one or more of these CCDBs is identified when the first and second mass spectra are compared, a third consecutive analysis on the same subset of analyte ions is initiated—an OzID experiment. The purpose of the OzID experiment is to identify unequivocally the position of the CCDB(s) in an analyte ion.
Here, ozone is injected into the reaction region 18 from a suitable source (see element 18b,
Post-acquisition, the software 24 compares the mass spectrum of the OzID fragments (from steps 42-44) with that of the intact analyte ions (from steps 30-34) to determine the exact position(s) of any CCDBs. See step 46. The software can utilize the mass spectrum of the CID fragments of those ions (from steps 36-38) for general structure elucidation, for example, identification of the lipid class by the headgroup fragments present.
In some embodiments, steps 30-46 are performed in real-time, i.e., in a rapid succession within the operational bounds of the spectrometer 12. This compares favorably with conventional techniques for CCDB localization and, as such, represents a unique research tool not equaled in the art.
In some embodiments, the OzID ion/molecule reactions, e.g., conducted in a q2 region of a QTRAP® mass spectrometer maintained at a high pressure (e.g., about 1 mTorr), can generate intact adduct ions [M+O3]+/−, where M denotes an analyte ion. By way of example, in some embodiments, such intact adduct ion can include the intact adduct of a lipid ion with a neutral ozone molecule. In some embodiments, a supplemental activation energy can be provided to such intact adduct ions so as to cause them to fragment into ozonolysis products, thereby increasing the yield of the OzID reaction. This can in turn increase the speed and sensitivity of the analysis. For example, in some embodiment in which such supplemental activation is employed, shorter ion/molecule reaction times are required to produce equivalent levels of diagnostic OzID product ions, and these products ions can have greater intensities given the dissociation of the intact residual adduct ions.
Supplemental activation of the intact adduct ions can be achieved in a variety of different ways. For example, the intact adduct ions can be subjected to an acceleration potential (typically a small acceleration potential, e.g., 15 volts). By way of example, in some embodiments, such an acceleration potential can be applied to the intact adduct ions between the q2 and Q3 regions of a QTRAP® mass spectrometer. In some other embodiments, the intact adduct ions can be subjected to resonant dipolar excitation, e.g., in a Q3 region of a QTRAP® mass spectrometer.
Described above are systems and methods meeting the objects set forth earlier, among others. It will be appreciated that the embodiments shown in the drawing and discussed above are merely examples and that other embodiments incorporating changes thereto fall within the scope of the applicants' teachings, of which we claim.
This application claims priority to U.S. provisional application No. 61/616,755 filed Mar. 28, 2012, which is incorporated herein by reference in its entirety.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/IB2013/000563 | 3/28/2013 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 61616755 | Mar 2012 | US |
