Abstract:
A mass spectrometer apparatus has one mass analyzer stage for selecting and rejecting ions having a particular mass-to-charge ratio and one fragmentation stage downstream from the first mass analysis stage for causing fragmentation of ions. Another mass analysis stage downstream from the fragmentation stage selects ions of a particular mass-to-charge ratio and rejects other ions. A synchronization unit is connected between the two mass analysis stages, for causing ions excited and removed in the one mass analysis stage to have the same mass-to-charge ratio as the ions selected by the other mass analysis stage, whereby ions detected by the detector are ions selected by the other mass analysis stage and generated by fragmentation in the fragmentation stage.

Description:
FIELD OF THE INVENTION 
     This invention relates to mass spectrometers, and more particularly relates to multiple stage multipole spectrometers and a technique for removing a background spectrum. 
     BACKGROUND OF THE INVENTION 
     Presently, mass spectrometers, particularly triple quadrupole mass spectrometers, are often used to analyze fairly complex reaction or fragmentation schemes. Thus, it is often desired to select a parent ion, fragment the parent ion to generate daughter ions, and subsequently fragment the daughter ions to create further fragment or granddaughter ions. Typically, only a single stage of fragmentation can be performed in a continuous beam triple quadrupole mass spectrometer. Subsequent stages of fragmentation are performed using alternative techniques such as radial excitation, a method common in the field of ion traps. Here the secular frequency ω of the parent ion is excited using a suitable excitation field such as an auxiliary dipole or quadrupole field. The degree of excitation depends on the amplitude of the auxiliary field; at low voltages, typically less than several volts, the ion is excited, but not ejected. At high amplitudes, typically several volts, the ion is ejected from the rod array and strikes the rods. It is well known that the secular frequency ω is approximately given by the equation ω=(a+q 2 /2)Ω/2, for q≦0.4, where a and q are the stability parameters arising from the Matthieu equation and Ω is the RF drive frequency. Thus, the excitation field is tuned to a frequency for exciting the parent ion, at an amplitude that yields good fragmentation without ejecting the ion. A detector or mass spectrometer stage is then used to determine the amount of granddaughter or secondary fragmentation ions present, which in turn can be used to determine the structure and composition of the original starting material. The problem with such a scheme is that there are numerous paths by which different components might be present in the final ion sample that is measured. This in turn creates a requirement to track the background spectrum, so as to determine the true spectra created by the secondary fragmentation of a selected primary fragment or daughter ions. For example, some of the ions present in the final sample may not have been created by fragmentation of the selected daughter ion, but rather, may have resulted from the initial fragmentation. 
     Techniques have been proposed in the art for software subtraction methods, for example by simply subtracting the background from the signal on a point by point basis. These are best suited for cases where the signal of interest is high relative to a low background component, e.g. S/N&gt;&gt;1. In contrast, in the sort of scheme outlined above, i.e. continuous flow, multiple stage fragmentation, it is likely for the background signal to be as large or much larger than the signal of interest, S/N&lt;1. It is very difficult to resolve this kind of situation downstream of a mass spectrometer. 
     Further known techniques include using broadband excitation which remove all ions within a specific mass range. Use of a broadband excitation in a continuous flow device is feasible but it requires higher power and implementation is costly and difficult. 
     The use of resonant excitation to selectively remove specific m/z ions is well known in the field. In a paper entitled “A Technique for Mass Selective Ion Rejection in a Quadrupole Reaction Chamber” by J. Throck Watson et al, there is a proposal to selectively reject from the r.f.-only collision cell of a tandem quadrupole mass spectrometer certain ions, with a view to enhancing analysis of different reactional fragmentation schemes. ( International Journal of Mass Spectrometry and Ion Processes,  93 (1989) 225-235). A further paper entitled “A Notch Rejection Quadrupole Mass Filter” by Philip E Miller et al ( International Journal of Mass Spectrometry and Ion Processes,  96 (1990) 17-26) discloses a technique in which a quadrupole is tuned to permit a wide range of masses to be transmitted, and to have a notch which selectively rejects one or more masses. In both these papers, there is no suggestion that the mass selected for rejection be tuned or linked in any way with the later stage in the spectrometer. 
     Accordingly, in a multiple stage mass spectrometer performing multiple stages of mass spectrometry (MS), particularly where there are multiple fragmentation stages, it is highly desirable to provide some technique for removing the background. More particularly, it is desirable to provide a technique for removing the background in real time or “on the fly” rather than relying on some later processing technique to remove the background. 
     SUMMARY OF THE INVENTION 
     In accordance with the present invention, there is provided a mass spectrometer apparatus comprising; 
     a first mass analyzer stage for selecting ions having a particular mass-to-charge ratio; 
     a fragmentation stage downstream from the first mass analysis stage for causing fragmentation of ions; 
     a second mass analysis stage downstream from the fragmentation stage for selecting ions of a particular mass-to-charge ratio and rejecting other ions; 
     and a synchronization unit connected between the first and second mass analysis stages, whereby ions excited and removed in the first mass analysis stage have the same mass-to-charge ratio as the ions selected by the second mass analysis stage, whereby ions detected by the detector are ions selected by the second mass analysis stage and generated by fragmentation in the fragmentation stage. 
     Another aspect of the present invention provides a method of analyzing an ion stream, the method comprising: 
     (1) selecting ions having a particular mass-to-charge ratio from the ion stream; 
     (2) causing fragmentation of the remaining ions to generate fragment ions; 
     (3) subsequently selecting ions having a desired mass-to-charge ratio for detection, and detecting the quantity of ions having the desired mass-to-charge ratio; and 
     (4) synchronizing the selected mass-to-charge ratio in step (1) with the desired mass-to-charge ratio in step (3), whereby detected ions having the desired mass-to-charge ratio will have been generated by fragmentation in step (2). 
     Both the apparatus and method of the present invention can be applied to a scheme where two or more fragmentation stages take place. In such a situation, there would be provided upstream of the first mass analysis stage, an initial mass analysis or spectrometer stage, for selecting a parent ion, and a further fragmentation stages, for causing fragmentation of the parent ion, to generate daughter ions. These daughter ions would then pass into the first mass analysis stage, for selection, and the fragmentation stage would then generate granddaughter ions from the daughter ions, etc. 
     Mass analysis and fragmentation can be carried out in any suitable apparatus. However, it is preferred for these steps to be carried out in a quadrupole mass spectrometer. Such a spectrometer can provide a plurality of stages, each comprising a quadrupole rod set aligned with adjacent stages, and configured to carry out the various steps in mass analysis, fragmentation, etc. 
    
    
     BRIEF DESCRIPTION OF THE DRAWING 
     For better understanding of the present invention and to show more clearly how it may be carried into effect, reference will now be made, by way of example, to the accompanying drawings, in which: 
     FIG. 1 shows a preferred embodiment of an apparatus of the present invention and which shows schematically a cross-section through an apparatus in accordance with the present invention; and 
     FIGS. 2 a,    2   b  and  2   c  are mass spectra showing the use of the apparatus of the present invention. 
    
    
     DESCRIPTION OF PREFERRED EMBODIMENT 
     An apparatus in accordance with the present invention is indicated generally by the reference  10 . The apparatus  10  includes a housing  12  provided in known manner with a pump  14 . The pump  14  can comprise two or more pumps configured in known manner. An ion source is indicated at  16 , and this can comprise any suitable ion source, such as an electrospray ion source, and it can operate at atmospheric pressure. 
     The ion source  16  injects ions into a first quadrupole Q 1 , which is operated at a low pressure, for example of 2×10 −5  torr. Downstream from the first quadrupole Q 1  is a collision cell  20  including three separate quadrupole sections indicated at Q 2   a,  Q 2   b  and Q 2   c.  As detailed below, the first and last quadrupole sections Q 2   a,  Q 2   c  in this collision cell  20  are operated in an RF only mode, to cause fragmentation. This is achieved by applying an RF signal and a desired excitation frequency or set of frequencies to excite a particular ion. The excitation for quadrupoles Q 2   a,  Q 2   c  is sufficient just to cause fragmentation and not loss of the ions. 
     The central quadrupole Q 2   b  is connected to a drive unit  26 . It is intended to remove ions with a selected mass, as detailed below. 
     A third quadrupole Q 3  is provided downstream of the collision cell  20 . A synchronization unit  24  is connected to a drive unit  30  for the quadrupole Q 3  and the drive unit  26  for the quadrupole Q 2   b  (as indicated at  27 , the quadrupoles Q 2   a,  Q 2   c  would have respective drive units in known manner). 
     A detector  32  is provided downstream from the quadrupole Q 3 , which again can be conventional. 
     In use, the quadrupole Q 1  is operated in a mass selection mode, i.e. with both RF and DC fields applied. The quadrupole Q 1  selects a parent ion having a desired mass, for example reserpine 609. 
     This is passed to the collision cell  20 . The first quadrupole in the cell  20 , quadrupole Q 2   a  is operated in an RF only mode, with a suitable radial excitation field, (typically several kHz to Mhz, 1 mV to several volts) to cause excitation of the parent ion. It then collides with the gas in the collision cell  20 . For this purpose, the collision cell is maintained under a pressure of, for example, 7-8 millitorr, with gas supplied through the gas inlet  28 . For example reserpine 609 could be fragmented to form fragment or daughter ions with masses 397, 195 and other masses. It is to be appreciated that fragmentation could also be achieved by axial acceleration. 
     The fragment ions and any remaining parent ions are then passed to the second quadrupole Q 2   b,  in the collision cell  20 . These ions would comprise any remaining parent ions, and all the daughter or fragment ions. It is desired, ultimately, to detect a secondary fragment or granddaughter ion in the quadrupole Q 3 . 
     To prevent interference from the fragments generated from the first fragmentation, the quadrupole Q 2   b  is operated to remove selectively ions with this mass. This is achieved by applying a suitable excitation signal of several mV to volts, several kHz to Mhz, in order to effectively notch the background ion. Since the frequency or frequencies of the notch and RF voltage on Q 2   b  determine which mass (or masses) is notched, these values are set to correspond to the RF/DC value of quadrupole Q 3 , using the synchronization unit  24 . For example, the quadrupole Q 2   b  could be operated to remove ions with a mass of 195, while Q 3  is set to transmit 195. (It should be noted that this notching could be performed in Q 2   a  so that Q 2   b  could be omitted, or put another way Q 2   a  and Q 2   b  could be combined as mentioned below). It should also be noted that the cells Q 2   a,  Q 2   b  and Q 2   c  could consist of individual collision cells separated by electrostatic lenses and/or differential apertures. 
     Then, all the fully enclosed remaining ions are passed to the third quadrupole Q 2   c  within the collision cell  20 . This is operated in the RF only mode with a suitable excitation signal (several kHz to MHz, several mV to volts), to excite a particular daughter or fragment ion, to generate secondary fragments or granddaughter ions. For example, an ion of mass 397 could be excited, which would generate ions of a variety of masses, including ions of a mass of 195. 
     The ion stream is then passed to the final quadrupole Q 3 . This is operated at low pressure, and with RF and DC voltages applied, as a mass resolving quadrupole. This is set to pass only ions of a desired mass. Following the example above, it would be set to pass ions having only a mass of 195. These would be detected by the detector  32 . 
     Now, since ions of mass 195 had been eliminated in the quadrupole Q 2   b,  one has effectively achieved a total subtraction of a background or interference signal in this quadrupole arrangement. Then, any ions of mass 195 detected at detector  32  can only be as the result of the fragmentation of the ion 397 in the third quadrupole Q 2   c.    
     The synchronization unit  24  enables the selected masses for the quadrupoles Q 2   b,  Q 3  to be kept in complete synchronization. Thus, as the mass selected in Q 3  is stepped in time or scanned, the excitation frequency is stepped in Q 2   b.  For each step, the excitation frequency of Q 2   b  ejects a mass corresponding to mass transmitted in Q 3 . In this way, the only ion to be transmitted is a molecular ion generated by MS/MS/MS in the quadrupole Q 2   c.    
     While the invention has been described with separate quadrupole rod sets Q 2   a,  Q 2   b,  these two rod sets could be combined. The combined rod set would then provide the dual function of effecting a first fragmentation step and mass selecting an ion from ions supplied from the source  16 . This dual function quadrupole or other device, e.g. an ion trap would be located within the collision cell  20 . The following example in FIG. 2 was carried out in an instrument with Q 2   a,  Q 2   b  combined. 
     Reference will now be made to FIG. 2, which shows graphs showing characteristics of the apparatus of the present invention. FIG. 2 a  shows a mass spectrum obtained after carrying out two MS steps with an intermediate fragmentation on reserpine. The second MS step was selected to show low mass fragments. 
     Thus, as shown in FIG. 2 a,  there are significant peaks  40  around mass 130,  42  around mass 173 and  44  around mass 194. If one were to fragment the higher order fragments to generate smaller fragments around mass 130, then the existing peak at  40  would interfere. 
     Accordingly, in accordance with the present invention, as a separate experiment, during fragmentation in the combined Q 2   a,  Q 2   b  section of the instrument, a notch was imposed to remove the peak around 130/131. This is shown in FIG. 2 b.  Thus, FIG. 2 b  still shows the peaks  42  and  44 , but as indicated at  46 , the notching has removed the peak that was previously present and substantially eliminated the background around m/z 130/131. Then, the fragments can be subjected to a further fragmentation step, e.g. as in quadrupole Q 2   c  in which excitation of m/z 174 is affected. The results are shown in FIG. 2 c.    
     As shown in FIG. 2 c,  the peak  44  is still present, but as indicated at  48 , the peak formerly present around mass 173/174 has been eliminated by the fragmentation step. This fragmentation generates smaller fragments, including a substantial new peak  50  around mass 130. 
     Since the former peak  40  was removed by notching as shown in FIG. 2 b,  it is certain that the new peak  50  is the result solely of fragmentation of the mass 174 ions, and no allowance needs to be made for any background effect. 
     A further aspect of the present invention is to improve the mass resolution of the fragmentation step in quadrupole Q 2   c.  Thus, the intention of this quadrupole is to excite an ion of a particular mass or mass-to-charge ratio. However, the excitation provided may, in fact, excite ions in the range of, for example, 10 AMU. This is undesirable. 
     To mitigate or reduce this effect, a further aspect of the present invention is to additionally provide for the quadrupole Q 2   b  to remove, in addition to the ions synchronized with the quadrupole Q 3 , ions on either side of the ion to be fragmented in Q 2   c.    
     Thus, in effect, “notches” could be provided on either side of the ion to be removed in Q 2   c.  These notches would have precise edges, so the notches could be set so as to leave a narrow band width, for example 5 AMU, around the desired ion for fragmentation in Q 2   c.    
     Unlike standard ion traps, notches only are required, rather than broadband excitation presequence, since the background removal and synchronization method ensures that the ion measured is indeed a fragment ion. Since the resolution into Q 2   c  is not that poor, it is only necessary to eliminate ions immediately on either side of the ion of interest to be fragmented in Q 2   c.  As such, it is anticipated that the wave forms required would be a combination of sinusoids of different frequencies, but in view of the narrow width required for these notches, no great power requirements would be required. 
     As noted above, in comparison, software subtraction methods are best suited for cases where the signal of interest is very high. For continuous flow, multiple stage fragmentation, it is likely that the background signal is as large or much larger than the signal of interest, and this situation is very difficult to resolving using software subtraction. 
     It is also to be realized that while quadrupole mass analyzers have been shown, any suitable mass analysis technique could be used. For example, the final quadrupole Q 3  and detector  32  could be replaced by a time-of-flight section, where the arrival time bins would be synchronized with the notch.