Abstract:
A method of and apparatus for analyzing a substance takes a stream of ions in said substance and supplies the ions to a collision cell including a quadrupole rod set for guiding the ions and a buffer gas. An RF voltage is applied to the quadrupole rod set to guide ions. An additional alternating current signal is applied to the quadrupole rod set at a frequency selected to cause resonance excitation of the secular frequency of a desired ion, whereby said desired ions are excited and undergo collision with the buffer gas causing fragmentation. The alternating current signal is then modulated, whereby periods in which said alternating current signal is applied alternate with periods in which said alternating signal is not applied. The ion spectrum after fragmentation is collected to generate one set of data for one spectrum, representative of the ion spectrum when the alternating current signal is applied, and another set of data for another spectrum, representative of the ion spectrum when the alternating current signal is not applied. These two spectra can then be subtracted.

Description:
FIELD OF THE INVENTION 
     This invention relates mass spectrometers, and more particularly is concerned with collision-induced dissociation (CID) in a tandem mass spectrometer. The invention is particularly intended to enable multiple stages of fragmentation, and hence mass analysis or spectroscopy, to be effected in a collision cell. 
     BACKGROUND OF THE INVENTION 
     Radio frequency (RF) only multipole spectrometers, more particularly quadrupole spectrometers, are widely applied in mass spectrometry and nuclear physics, due to their ability to transport ions with minimal losses. During such transportation of the ions, the initial ion positions and velocities change, but the total phase space volume occupied by the ion beam remains constant (see Dawson, P. H., “Quadrupole Mass Spectrometry and its Applications,” Elsevier Scientific Publishing Co., New York, 1976). However, if a buffer gas is introduced into the ion guide, a dissipative process occurs, due to ion molecule collisions, and this enables an ion beam to be focused onto the quadrupole axis after the initial velocities have been damped. 
     Collisional quadrupole or other multipole devices have been used as an ion guide providing an interface between an ion source and a mass spectrometer, or alternatively as a collision cell for collision-induced dissociation (CID) experiments. As a straightforward interface, collisional damping reduces the space and velocity distributions of the ions leaving the ion source, thus improving the beam quality. For CID experiments, primary ions having relatively large velocities enter the multipole and collide with buffer gas molecules, and so collision-induced dissociation takes place. The multipole helps to keep both primary ions and fragment ions, resulting from the collision-induced dissociation, close to the axis and to deliver them to the exit for further analysis. Collisions inside the multipole spectrometer again act to reduce the space and velocity distribution of the ion beam. 
     Ion motion in a perfect quadrupole field is governed by Mathieu&#39;s equation (See Dawson as cited above); ions oscillate around the quadrupole axis at an appropriate fundamental frequency which is determined by their m/z and quadrupole parameters, and is independent of ion position and velocity. If the frequency of any periodic forces acting on ions coincides with the ion fundamental frequency, then resonance excitation takes place. Similar resonance excitation is widely applied in quadrupole ion trap or in ion cyclotron resonance mass spectrometers (R. E. March, R. J. Hughes, “Quadrupole storage mass spectrometry,” 1989, John Wiley &amp; Sons). 
     These properties of spectrometers have been employed in many ways. Thus, in U.S. provisional patent application 60/046,926 filed May 16, 1997 (and related U.S. patent application Ser. No. 09/066,556 and Canadian patent application 2,236,199), there is disclosed a high pressure MS-MS system. This was intended to provide improvements to a conventional triple quadrupole mass spectrometer arrangement, employing two precision quadrupole mass spectrometers separated by an RF-only quadrupole which is operated as a gas collision cell. The first mass spectrometer is used to select a specific ion mass-to-charge ratio (m/z), and to transmit the selected ions into the RF-only quadrupole or collision cell. In the RF-only quadrupole collision cell, some or all of the parent ions are fragmented by collisions with the background gas, commonly argon or nitrogen, at a pressure of up to several millitorr. The fragment ions, along with any unfragmented parent ions are then transmitted into the second precision-quadrupole which is operated in a mass resolving mode. Usually, the mass resolving mode of this second spectrometer is set to scan over a specified mass range, or else to transmit selected ion fragments by peak hopping, i.e. by being rapidly adjusted to select specific ion m/z ratios in sequence. The ions transmitted through this spectrometer are detected by an ion detector. A problem with this conventional arrangement is that the two mass resolving quadrupoles are required to operate in the high vacuum region (less than 10 −5  torr), while the intermediate collision cell operates at a pressure up to several millitorr. That earlier invention was intended to simplify the apparatus and eliminate the necessity for separate RF-only and resolving spectrometers at the input to the apparatus. Instead, a single quadrupole is provided, operating in the RF-mode to act as a high pass filter. Additionally, this quadrupole is provided with an AC field, which can be identified as a “filtered noise field”, which contains a notch in the frequency range corresponding to the mass of an ion of interest. This notch can be moved, to select and separate desired ions. 
     Other older proposals can be found, for example, in U.S. Pat. No. 5,420,425 (Bier et al. and assigned to Finnigan Corporation). This relates to an ion trap mass spectrometer, for analyzing ions. It has electrodes shaped to promote an enlarged ion occupied volume. A quadrupole field is provided to trap ions within a predetermined range of mass to charge ratios. Then, the quadrupole field is changed so that trapped ions with specific masses become unstable and leave the trapping chamber in a direction orthogonal to the central axis of the chamber. The ions leaving the spectrometer are detected, to provide a signal indicative of their mass-to-charge ratios. One method that is taught in this patent is to first introduce ions within a predetermined range of mass-to-charge ratios into the chamber and subsequently to change the field to select just some ions for further manipulation. The quadrupole field is then adjusted so as to be capable of trapping product ions of the remaining ions, and the remaining ions are then dissociated or reacted with a neutral gas to form those product ions. Subsequently, the quadrupole field is changed again, to remove, for detection, ions whose mass-to-charge ratios lie within the desired range, which ions are then detected. 
     The above process describes how the technique of MS/MS (or MS 2 ) is applied in an ion trap configuration. A related technique of MS/MS/MS (or MS 3 ) can be provided by isolating one of the product ions of the first MS 2  process, and eliminating all but the selected product ion from the trap. The selected product ion mass is then excited so that it fragments through collisions with the buffer gas in the trap. The range of secondary product ions formed in this two-stage process is then scanned from the trap for detection, so that a mass spectrum is recorded. The spectrum consists of fragments of a fragment from the original parent ion. The process can be extended by trapping and isolating one of the secondary product ions, and then fragmenting that ion mass, in order to form an MS/MS/MS/MS or MS 4  spectrum, and ultimately the process can be extended to an MS n  spectrum. Ion losses occur at each stage, however, so that sensitivity decreases as the number of steps increases. Nevertheless, this technique of MS n  can be a useful tool to help elucidate the structure of organic ions. 
     Another approach to obtaining an MS 3  spectrum is described in U.S. Pat. No. 6,011,259 by Craig Whitehouse, Thomas Dresch and Bruce Andrien of Analytica of Brantford, which shows how a multipole ion guide can be combined with a time-of-flight (TOF) mass spectrometer to provide MS n  analysis. They describe the method of using resonant excitation to excite one ion mass in the quadrupole ion guide in order to fragment a selected ion (without isolating or rejecting the other ion masses). By turning the excitation on and off several times per second, a background subtracted spectrum of the fragments of the desired precursor ion can be created. This works very well with TOF mass spectrometers where the TOF is pulsed at a higher frequency than the pulsing of the excitation. 
     The method described produces an MS 2  spectrum of the selected ion. In order to produce an MS 3  spectrum (designated by the inventors of that patent as MS/MS 2 ), two frequencies must be added, first exciting just the first precursor ion, then adding another frequency to excite both the first precursor ion and the selected product of that precursor ion together, and subtracting the spectra to obtain an MS 3  spectrum. A total of three spectra must be collected sequentially: a first spectrum without any excitation, then a spectrum with only one excitation frequency (first MS 2  spectrum), then a spectrum with both frequencies added simultaneously (MS 3 ). The second spectrum must be subtracted from the first spectrum in order to generate the MS 2  spectrum and identify the primary product ion of interest, and then the third spectrum must be subtracted from the second spectrum in order to generate an MS 3  spectrum (in other words, an MS 2  spectrum of the primary product ion). 
     The first technique taught above is complex, and requires a number of separate quadrupoles or the like, and the ability to move the ions sequentially through the different quadrupole sections. The technique taught in the Finnigan patent is complex and requires a number of steps. Also, it is concerned with ion traps and not a flow quadrupole. While all of the above methods can be used to obtain MS 3  spectra (or higher order), they all suffer from some limitations or drawbacks. The ion trapping methods require isolation of the first parent ions before fragmentation, and then sequential steps of fragmentation, isolation and fragmentation in order to reach MS 3 . The initial ion mass which is fragmented is not mass selected. 
     The method described in Whitehouse et al to achieve MS 3  is complex, and reduces the duty cycle for the overall process (as described by the inventors of the &#39;259 patent) to 33% for MS 3 . Also, the mass-selective specificity of the first fragmentation step, obtained by exciting the ion radially to collide and fragment is much less than that achievable by using a mass spectrometer (a quadrupole mass filter for example). Therefore, the method taught is both less sensitive and less mass-selective than that described in the present application. Finally, these inventors fail to recognize that simple subtraction alone will not always give an accurate representation of the true fragment spectra; the present inventors have realized further statistical analysis is required to eliminate uncertainties due to poorly subtracted spectra. 
     Accordingly, it is desirable to provide one technique which, in one device, readily enables ions of a selected mass-to-charge ratio to be subject to collision-induced-dissociation (CID) or fragmentation, so that the fragments can be transported further for subsequent analysis. It is desirable to provide this in a single device, since movement of ions from one device to another inevitably leads to some losses. Similarly, the techniques of the Finnigan patent works effectively with pulse ion sources, but inefficiently with continuous ion flow, for instance from an electrospray ion source. In this field, spectrometers are frequently used to analyze small samples, and often, high efficiency is required, if any reliable reading or measurement is to be obtained. 
     SUMMARY OF THE INVENTION 
     In accordance with a first aspect of the present invention, there is provided a method of analyzing a substance, the method consisting of:
         (1) creating a stream of ions in said substance;   (2) supplying the stream of ions and a collision gas to a multipole and providing an RF signal to the multipole, whereby the multipole functions as a collision cell;   (3) fragmenting said ions in the RF multipole by collisions with the gas molecules, in order to form primary fragment ions;   (4) supplying additional alternating current to the multipole at a frequency selected to cause resonance excitation of a desired primary fragment ion mass-to-charge ratio, whereby ions with said desired primary fragment ion mass to charge ratio are excited and undergo collisions with the gas molecules causing production of secondary fragment ions;   (5) modulating the alternating current signal applied in step (4) whereby periods in which said alternating current signal is applied alternate with periods in which the alternating signal is not applied;   (6) detecting the ion signal after fragmentation with a mass spectrometer and collecting one set of data for one spectrum, representative of the ion spectrum when the alternating current signal is applied and another set of data for another spectrum, representative of the ion spectrum when the alternating current signal is not applied;
 
whereby said other spectrum can be subtracted from said one spectrum, to generate a subtracted spectrum showing the secondary fragment ions without the presence of the primary fragment ions except for any said primary fragment ions which are generated by step (4), whereby to obtain MS 3  information.
       

     In one embodiment, the method further includes the step of processing the data sets by applying statistical analysis to reject spectra having statistically insignificant variations in the ion signal. 
     Preferably, the statistical analysis is implemented in a software program and performed automatically. 
     More preferably, the statistical analysis is performed in real time so that spectra having statistically insignificant variations in the ion signal are not displayed. 
     In one embodiment, the alternating current signal is at a frequency that excites the desired primary fragment ion. 
     Preferably, the method includes passing the stream of ions through a first mass analyzer to select a precursor ion of interest, and passing the precursor ion into the collision cell. 
     More preferably, the method includes providing a potential difference between the first mass analyzer and the collision cell, to accelerate the precursor ion into the collision cell, whereby the precursor ions gain sufficient velocity to collide with the buffer gas to cause fragmentation, and wherein step (4) comprises applying an alternating current signal to excite a fragment of the precursor ion, said fragment comprising the desired ion. 
     The method can include applying a second alternating current signal to the quadrupole rod set, to excite a fragment ion resulting from resonance excitation of said desired ion, thereby to generate secondary fragment ions and wherein step (5) comprises modulating the second alternating current signal. It will be appreciated that it may be possible to apply a number of different excitation signals to cause fragmentation of fragments from the previous step. 
     Another aspect of the present invention provides an apparatus, for analyzing a substance by resonance excitation of selected ions and selective collision-induced dissociation, the apparatus comprising:
         an ion source for generating a stream of ions;   a collision cell, including a quadrupole ion guide, for receiving a stream of precursor ions and provided with a collision gas, for collision-induced dissociation between the parent ions and the buffer gas;   a power supply connected to the quadrupole rod set for generating an RF field in the quadrupole rod set for guiding ions and for applying an additional alternating current field at a frequency selected to excite a desired ion;   a modulation means connected to the power supply, for modulating the alternating current signal, whereby periods in which said alternating current signal are applied alternate with periods in which the alternating current signal is not applied.       

     Preferably, the apparatus additionally includes a detector for detecting fragment ions exiting the collision cell, a switch connected to the detector, two data storage devices connected to the switch, and a connection between the modulation control unit and the switch, whereby the switch switches detected data for periods when the alternating current signal is applied to one data storage device and collected data for periods when the alternating current signal is not applied to the other storage device. 
     To enable a second excitation step to be effected, the apparatus can include a second power supply connected to the quadrupole rod set, a second modulation unit connected to the second power supply and also to the switch, before applying a second alternating current signal, for excitation of a second ion. 
     Preferably the apparatus includes a first mass analysis section for selecting a parent ion and a final mass analysis section, including the detector, for analyzing fragment ions from the collision cell. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWING FIGURES 
       For a 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  is a schematic of a first embodiment of an apparatus in accordance with the present invention; 
         FIG. 2  is a schematic of an apparatus in accordance with a second embodiment of the present invention; 
         FIGS. 3   a - 3   e  are mass spectra showing analysis of bosentan and fragments thereof; 
         FIGS. 4   a,    4   b  and  4   c  are spectra showing fragmentation of taxol; 
         FIGS. 5   a  and  5   b  are detailed graphical spectra of fragments obtained from fragmentation of a fragment of mass  202  of bosentan; 
         FIGS. 6   a - 6   c  and  7   a - 7   c  are mass spectra showing MS 3  and MS 4  fragmentation schemes for reserpine; and 
         FIGS. 8   a - 8   d  are subtracted MS 3  mass spectra of Reserpine at various excitation amplitudes. 
     
    
    
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 
     A description is first given of the apparatus in  FIGS. 1 and 2 . The two apparatus are largely similar, except for the final mass analysis stage.  FIG. 1  shows a variant with a quadrupole rod set and detector as the final mass analysis stage, while this is effected by a time-of-flight section in FIG.  2 . 
     Referring first to  FIG. 1 , the first variant of the apparatus is indicated at  10 . In known manner, the apparatus  10  includes a first quadrupole rod set generally indicated as Q 0 . Q 0  is intended to collimate and reduce the energy of ions received from an electrospray source or the like. In known manner, upstream of Q 0 , there would be an ion inlet, skimmers, intermediate pressure stages and the like, all intended to remove gas and reduce pressure down to that required for mass analysis (these elements and associated pumps are not shown). Q 0  collimates the ion beam and further serves to reduce gas pressure. 
     Ions from Q 0  pass through an interquad aperture  12  into a quadrupole rod set Q 1 , which functions as a first mass analysis section. In known manner, Q 1  is supplied with resolving RF and DC voltages. These can be conventional and the power supplies are not shown. 
     From Q 1 , the ions pass through into a collision cell housed in a chamber generally indicated  14 . The collision cell includes a quadrupole rod set Q 2 . The chamber  14  includes, at either end, an inlet interquad aperture  16  and an exit interquad aperture  18 . 
     The ions then pass into a final quadrupole Q 3 . Q 3  again would be provided with resolving RF and DC voltages, and the power supply for these is not shown. Finally, the ions pass through to a detector  20 . 
     In known manner, appropriate DC potentials would be provided between the different quadrupole sections Q 0 , Q 1 , Q 2  and Q 3  and also appropriate potentials on the interquad apertures  12 ,  16 ,  18 , together with an appropriate potential drop to the detector  20 . These various potentials ensure movement of ions axially, from left to right in  FIG. 1 , in known manner. 
     Quadrupoles Q 1 , Q 3  would be maintained at a low pressure of 10 −5  torr, as is known for mass resolving quadrupoles. Chamber  14  is operated as a collision cell and would be provided with a suitable collision gas (source not shown). Typically, it is operated at a pressure in the range 0.5-20 mTorr. A suitable collision gas is nitrogen. 
     In accordance with the present invention, a first MS step is effected in Q 1 . This selects a parent or precursor ion, which then passes into the rod set Q 2  of the collision cell. To effect a second MS step, MS 2 , ions are accelerated into a quadrupole collision cell (Q 2 ), effecting fragmentation through collision with a low pressure gas in Q 2  to generate primary fragment ions. A radio frequency (RF) source  22  for rod set Q 2  is indicated, for example, 1,000 volts at 2 MHz. An auxiliary RF voltage would be provided in a quadrupolar, dipolar or any other suitable manner, i.e. with the cos ωt provided to one opposite pair of rods in the quadrupole rod set Q 2 , and −cos ωt provided to the other, diagonally opposite pair of rods of the rod set Q 2 . 
     It will be appreciated that while each fragmentation step is designated MS 2 , MS 3 , etc., the final MS step is effected in Q 2  (or other downstream mass analyzer). Also, the number of fragmented ion steps is 1 less than the total number of MS steps, i.e. MS 2  has one fragmentation step, MS 3  has two fragmentation steps, and MS n  has n−1 fragmentation steps. 
     Another way of expressing this is to note that a mass analysis step as such is not effected in the collision cell or quadrupole Q 2 . Rather fragment ions are generated in Q 2  for mass-analysis in a downstream mass analyzer. Thus a reference, for example, to MS 2  occurring in Q 2  means that fragment ions are generated in Q 2 , and that these are then mass analyzed downstream in Q 3 , to provide the second mass analysis step. 
     Note also, while this description of the preferred embodiment assumes in all cases a first mass analysis step in Q 1 , this may not be essential. Thus, it may be desirable to analyze ions from a pure, single component sample, if only to record the characteristic fragmentation characteristics of the chosen component. In such a case, it may be possible to omit the first mass selection step in Q 1 . In this case, the total number of fragmentation steps will equal the number of mass-analysis steps. 
     Now, in accordance with the present invention, the rod set Q 2  is further excited to effect either one or a multiple steps of excitation. 
     Firstly, a further excitation step MS 3  is effected by an excitation source  24  provided with a modulation control unit  26 , whose function is explained below which causes secondary fragment ions to be generated from the primary fragment ions. To effect a third or tertiary fragmentation step, a second power supply  28  is provided, connected to a second modulation control unit  30 . Each of the power supplies  24 ,  28  can provide a similar signal to the rod set Q 2 , the signal as being selected to excite different fragments, as detailed below, and the basic scheme is described in relation to the third mass selection step MS 3 , involving two fragmentation steps, with the control unit  24 . 
     Each ion has a secular frequency v. which is related to the drive frequency Ω/2π, and the following Mathieu parameter β, as follows: 
             v   =           β   ⁢           ⁢   Ω     2     ⁢           ⁢   for   ⁢           ⁢   n     =   0             (   1   )             
 
for n=0
 
     For q&lt;0.6, this reduces to 
                 For   ⁢           ⁢   q     &lt;   0.6     ,       this   ⁢           ⁢   reduces   ⁢           ⁢   to   ⁢           ⁢   v     =         (     a   +       q   2     2       )       1   2       ⁢     Ω   2                 (   2   )             
 
where a and q are standard Mathieu parameters given by: 
             a   =       8   ⁢   cU         mr   2     ⁢   Ω               (   3   )               q   =       4   ⁢   eV         mr   2     ⁢     Ω   2                 (   4   )             
 
     Thus, for a=0, the relationship reduces to: 
             v   =       q   ⁢           ⁢   Ω       2   ⁢     2                 (   5   )             
 
     In accordance with the present invention, an excitation voltage is applied to the rod set Q 2  at a frequency which is twice the secular frequency, i.e. with a frequency of ω=2v ion. This would be at a potential v, in the range of 0.5 to 20 volts. This potential will be added to each of the potentials supplied to each pair of rods of the rod set Q 2 . Thus, the potential supplied to the pairs of rods would be as follows:
 
V cos Ωt+v cos (ωt+φ)  (6)
 
−V cos Ωt−v cos (ωt+φ)
 
where φ is simply a factor to allow for the fact that the two signals need not necessarily be in phase.
 
     Thus, to effect the different steps of MS 3  and MS 4 , it is a matter of selecting different frequencies of ω, corresponding to ions of interest, as explained in greater detail in relation to the examples below. Alternatively, however, from equation (5) it is evident that one could select a different RF voltage, or select a different q for a constant excitation frequency. 
     Additionally, an important aspect of the invention is to modulate the additional excitation provided by the power supplies  24 ,  28 . For this purpose, each power supply  24 ,  28  is shown with a respective modulation control unit  26 ,  30 . For some purposes, it may be suitable or possible to provide a single modulation control unit and a single power supply, which together are switchable between the different characteristics required for each fragmentation step. 
     Modulation control units  26 ,  30  effectively turn on and off the power supplies  24 ,  28 , with a square wave signal at a frequency of, for example 2 Hz. In other words, the power supply  24 ,  28  as the case may be, would be turned on for 0.25 seconds, turned off for 0.25 seconds, etc. The reason for this is to provide data with and without excitation, to enable subtraction of the different signals obtained. Comparing results with excitation on and excitation off for any lengthy time period is impractical, since any analyzer or detector tends to show drift for a variety of reasons. That is, a signal measured will drift by the order of a few percent over time. In many cases, as detailed below, comparison of two signals, with excitation on and excitation off, amounts to obtaining a small difference between two relatively large signals. If either one of these has drifted significantly, then this can lead to a major error in the small, calculated difference. 
       FIG. 1  also shows a modification to a conventional mass spectrometer apparatus, required by the present invention. Thus, the detector  20  is connected to a switch  32 . The switch  32  is connected to and controlled by either one of the modulation control units  26 ,  30 . The switch  32  has two outputs connected to separate data storage devices  34 ,  36 . Thus, the data storage device  34  is for when there is no excitation and the data storage device  36  is for when excitation is provided. 
     Then, in use, when modulation is effected by either of the units  26 ,  30 , and note that this is irrespective of any voltage set by the power supply  24 ,  28 , the output from the detector  20  is switched by the unit  32  alternately between the two data storage devices  34 ,  36 , in synchronism with the modulation. This enables collection of two sets of data, one when excitation is effected and one when excitation is not effected. As detailed below, this gives different spectra, which can be subtracted from one another. 
     Still referring to  FIG. 1 , significantly, the use of a pre-selecting mass filter in the present application allows the first fragmentation to be non-selective, via a potential gradient. This is preferable because typically much more energy can be deposited into the initial ions, which may include hard to break bonds and massive molecules. The present inventors have appreciated that there would be sufficient cooling of the primary fragment ions and any residual precursor ions in the second multipole to permit efficient radial excitation for subsequent MS steps. 
     Reference will now be made to FIG.  2 . This shows an apparatus indicated generally by the reference  40 . The apparatus  40  is similar to the apparatus  10 , and for simplicity and brevity, like components are given the same reference numeral and the description of these components is not repeated. In brief, the apparatus  40  includes the first three quadrupole rod sets Q 0 , Q 1  and Q 2 , and associated control and power supply elements. 
     However, here, to replace the final quadrupole Q 3  and detector  20 , there is provided a time-of-flight (TOF) mass analyzer  42 . In known manner, the TOF analyzer of section  42  includes a gating region  44  and a detector  46 . Thus, in use, ions pass into the gating region  44  and are gated or pulsed out to travel down the main body of the TOF  42 , following a drift tube, until detected at a detector  46 . 
     It will be appreciated that any suitable form of TOF could be provided. Thus, the TOF could comprise a reflectron or the like. 
     Reference will now be made to  FIGS. 3-6  and also to Tables 1 and 2, which show mass spectra data collected in accordance with the present invention. All this data was collected on an apparatus using a TOF section, as in FIG.  2 . 
     Referring first to  FIG. 3   a,  there is shown a mass spectrum resulting from carrying out the first two MS steps, MS 1  and MS 2 , on bosentan, a low mass chemical or drug, with a mass of  580 . Thus, in Q 1 , the voltages are set to select m/z  580  from bosentan, which is then accelerated into Q 2  to fragment it, to generate the spectrum shown in  FIG. 3   a;  it will again be appreciated that the second mass analyzing step is in fact effected in TOF mass analyzer  42 . As shown, this includes some residual amount of the original bosentan at mass  580  and other significant peaks of fragments at  508  fragments close to mass  200  and others. 
       FIGS. 3   b - 3   d  then show subtracted spectra obtained by applying the third MS step, MS 3 , with a frequency set to excite an ion with an m/z  508 ,  202  and  280 , respectively. For example, fragmentation of m/z  508  is achieved by applying a 4.5 volt excitation signal at a frequency of 220 kHz. As indicated on  FIG. 3   b,  this effects MS/MS/MS (or MS 3 ). 
       FIG. 3   b  shows a subtracted spectrum. Thus,  FIG. 3   b  shows the spectrum obtained by effecting the triple MS technique, with the spectrum of  FIG. 3   a  subtracted. Here, any negative quantities are shown as zero. For example, the peak for mass  508  will, clearly, be much less in  FIG. 3   b,  so the subtraction of the spectrum of  FIG. 3   a  would give a negative value; in  FIG. 3   b,  this is graphed. This technique has the effect of subtracting any fragments that were present as a result of the MS 2  ion fragmentation. However, as explained below, further analysis of  FIGS. 3   b - 3   d  is required to determine which peaks are true MS 3  and which peaks result from incompletely subtracted spectra, due to signal fluctuations alone. 
       FIG. 3   e  shows a scan obtained by effecting modulation with modulation control unit  26 , to provide the received signal into the two separate data streams, to collect two sets of data. However, the voltage supplied by the unit  24  is set to zero. In effect,  FIG. 3   e  shows the subtraction of what in theory should be two identical outputs. As can be seen, the spectra does show some measurable peaks. Note that these peaks result from, in effect, the subtraction of two relatively large quantities, to give a small difference. The vertical scale in  FIG. 3   e  is different from that in the other figures. What this shows is that there will, in practice, be some fluctuation of the signal, and this can be some measure of the fluctuation for individual fragments, and it can be noted that the fragment  202  shows a significant fluctuation. Thus, a statistical analysis of the significance level of the subtracted ion signal is used, as explained below. Processing of data sets collected for the statistical analysis allows identification and possible elimination of the non-coherent variations in the ion signal. 
     While the statistical analysis is presented here by way of equations and tables, it will be appreciated that the analysis may be automated by implementing it in a software program running on a data processor, so as to process the data as it is recorded. This will permit rapid “real time” determination of whether the value of the subtracted data is significant. Optionally, only data which is determined to be significant after such statistical analysis can be selected for presentation. 
     Turning to  FIGS. 4   a  and  4   b,  these show test results and spectra obtained for the drug taxol.  FIG. 4   a  shows a basic two-step MS 2  process. That is, taxol was selected in Q 1 , for transmission into Q 2 ; the taxol is then accelerated into Q 2  with a suitable potential difference, to cause CID or fragmentation of the taxol in Q 2 . The spectra in  FIG. 4   a  was then obtained. 
       FIG. 4   b  then shows the spectrum obtained by further excitation, i.e. MS 3 .  FIG. 4   b  is a subtracted spectrum. This shows a significant range of fragments for approximately 100 m/z to 400 m/z. Notably, even though there are significant peaks in this range in  FIG. 4   a,  the same ions are also generated by the subsequent fragmentation. 
       FIG. 4   c  again shows a subtraction spectrum obtained without any excitation. In other words, with modulation unit  26  actuated, to cause the data to be divided into two sets of data, but with the power supply  24 , set to give zero excitation. Surprisingly, for taxol, this shows a significant residual background. 
     Referring now to  FIGS. 5   a  and  5   b  these show, in greater detail, a graphical representation of the signal obtained around the peak  124  and  122 , as a result of exciting the fragment  202 ; thus these figures show details of the scan of  FIG. 3   c.    
       FIG. 5   a  shows two peaks  50  and  51 . Peak  50  is the signal obtained with the additional excitation provided by the unit  24  turned off, and this also shows error bars indicating the variance in the signal obtained. Peak  51  shows the signal obtained with power supply  24  actuated, to provide excitation of fragment  202 , generating an additional quantity of the ion around mass  124 . A subtracted spectrum would effectively show peak  51  minus peak  50 . This demonstrates that a fragmentation of ion  202  does add significantly to a fragment at mass  124 . 
       FIG. 5   b  shows similar peaks  52  and  53  at mass  122 . Again, error bars for the peak  52  are shown. Peak  52  shows the spectra with no excitation of ion  202 , while peak  53  shows the spectra with  202  excited. This shows where the two peaks are effectively identical, allowing for a margin of error. In other words, fragmentation of ion  202  does not add significantly to the signal at mass  122 . 
     Thus, in order to ascertain which fragment signals are significant, the present invention incorporates statistical analysis for determining when fragmentation of a particular ion has added to the signal for a smaller fragment, and when no such effect is present. This is based on two basic principles, namely: firstly, simply subtracting the two peaks, as indicated for the peaks in  FIGS. 5   a,    5   b  and determining that there is a significant additional added signal, when there is a significant and measurable difference between the two peaks; and comparing two peaks to determine if there is significant fluctuation in values. This latter feature is explained in greater detail in relation to Tables 1 and 2. 
     Referring first to Table 1, this shows four sets of data, for different peaks at, approximately 124, 98, 106 and 79, where it is determined that fragmentation of the  202  ion did add significantly to a peak. These peaks were chosen, representative of, respectively, “medium”, “little”, “big” and very little peaks, the adjectives indicating relative peak size. For each ion, there are two columns, indicating the count made, with excitation on and excitation off respectively. 
     Thus, for ion  124 , counts are obtained at masses ranging from 124.0131 to 124.0735. The final column calculates a significance factor |T| or “Sig” using a statistical method called the “T” test. This test permits comparison of two parent populations to determine the degree to which they are different. This method is derived from probability statistics assuming a Gaussian distribution of ions in time; other probability functions may be used. While this statistical method considers the magnitude of the significance factor, other methods may take polarity information into consideration. The value of |T| is calculated by the following equation: 
                  T        =     Sig   =                    detected   ⁢           ⁢   ion   ⁢           ⁢   signal     ,       alternating   ⁢           ⁢   current   ⁢           ⁢   on     -                   detected   ⁢           ⁢   ion   ⁢           ⁢   signal     ,     alternating   ⁢           ⁢   current   ⁢           ⁢   off                           σ   2     ⁢           ⁢   alternating   ⁢           ⁢   current   ⁢           ⁢   on     +                 σ   2     ⁢           ⁢   alternating   ⁢           ⁢   current   ⁢           ⁢   off                              (   7   )             
 
where σ is the standard deviation. Here, a value of |T| of ˜two or less, indicates that there is a greater than ˜95% probability that the excitation on and off signals are the same. On the other hand, for this mass  124 , one can see that the values of |T|, at the peak, are in excess of 10, clearly indicative of a substantial difference, and this is borne out by the visual representation in  FIG. 4   a.  
 
     Similar results, although not quite so strong, were obtained for the peak and mass  98 . This again shows that, for nearly all values around the peak  98 , the on signal gave a higher signal than the off signal. Again, value of |T| was quite high around the peak. 
     In general, it would be noted that it is more difficult to make a clear determination for smaller peaks. 
     For a large or big peak, as shown for the mass  106 , the difference between the on and off signals was significant, and it is noted that the value of |T| reached a value of in excess of 57 close to the peak. This is clearly indicative of a substantial difference between the on and off signals, thereby indicating that the fragmentation of ion  202  did contribute significantly to the fragment and mass  106 . 
     Finally, for the ion at mass  79 , this represents another, smaller peak. This again gives a clear indication that there was a difference between the two signals. 
     
       
         
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                   
                 TABLE 1 
               
               
                   
                   
               
               
                   
                 M/Z 
                 ON 
                 OFF 
                 [T] 
               
               
                   
                   
               
             
             
               
                   
               
             
          
           
               
                   
                   
                   
                 MED#, YES 
                   
               
               
                   
                 124.0131 
                 7 
                 11 
                 0.943 
               
               
                   
                 124.0186 
                 20 
                 11 
                 1.62 
               
               
                   
                 124.0241 
                 40 
                 36 
                 0.459 
               
               
                   
                 124.0296 
                 162 
                 149 
                 0.737 
               
               
                   
                 124.0351 
                 1117 
                 874 
                 5.44 
               
               
                   
                 124.0406 
                 3854 
                 3036 
                 9.85 
               
               
                   
                 124.0461 
                 6377 
                 4865 
                 14.3 
               
               
                   
                 124.0516 
                 5321 
                 4073 
                 12.9 
               
               
                   
                 124.0571 
                 2596 
                 2164 
                 6.26 
               
               
                   
                 124.0626 
                 1420 
                 1163 
                 5.06 
               
               
                   
                 124.0681 
                 1016 
                 829 
                 4.35 
               
               
                   
                 124.0735 
                 663 
                 566 
                 2.77 
               
               
                   
                   
                   
                 LITTLE, YES 
                   
               
               
                   
                 98.0192 
                 1 
                 1 
                 0 
               
               
                   
                 98.0241 
                 4 
                 2 
                 0.816 
               
               
                   
                 98.0289 
                 13 
                 13 
                 0 
               
               
                   
                 98.0338 
                 61 
                 28 
                 3.50 
               
               
                   
                 98.0387 
                 91 
                 66 
                 1.99 
               
               
                   
                 98.0436 
                 103 
                 51 
                 4.19 
               
               
                   
                 98.0485 
                 43 
                 33 
                 1.15 
               
               
                   
                 98.0534 
                 26 
                 15 
                 1.72 
               
               
                   
                 98.0583 
                 6 
                 13 
                 1.61 
               
               
                   
                 98.0632 
                 7 
                 5 
                 0.577 
               
               
                   
                 98.068 
                 1 
                 6 
                 1.89 
               
               
                   
                 98.0729 
                 3 
                 2 
                 0.447 
               
               
                   
                 98.0778 
                 3 
                 5 
                 0.707 
               
               
                   
                 98.0827 
                 3 
                 6 
                 1.00 
               
               
                   
                   
                   
                 BIG, YES 
                   
               
               
                   
                 105.9971 
                 18 
                 11 
                 1.30 
               
               
                   
                 106.0021 
                 10 
                 7 
                 0.728 
               
               
                   
                 106.0072 
                 29 
                 11 
                 2.85 
               
               
                   
                 106.0123 
                 46 
                 19 
                 3.35 
               
               
                   
                 106.0174 
                 120 
                 58 
                 4.65 
               
               
                   
                 106.0225 
                 803 
                 437 
                 10.4 
               
               
                   
                 106.0275 
                 5560 
                 2858 
                 29.5 
               
               
                   
                 106.0326 
                 16232 
                 8273 
                 50.8 
               
               
                   
                 106.0377 
                 20957 
                 10723 
                 57.5 
               
               
                   
                 106.0428 
                 13267 
                 6652 
                 46.9 
               
               
                   
                 106.0479 
                 5185 
                 2784 
                 26.9 
               
               
                   
                 106.053 
                 2119 
                 1174 
                 16.5 
               
               
                   
                 106.058 
                 1362 
                 766 
                 12.9 
               
               
                   
                   
                   
                 V. LITTLE, YES 
                   
               
               
                   
                 79.0072 
                 0 
                 0 
                 0 
               
               
                   
                 79.0116 
                 1 
                 1 
                 0 
               
               
                   
                 79.016 
                 8 
                 2 
                 1.90 
               
               
                   
                 79.0204 
                 27 
                 9 
                 3.00 
               
               
                   
                 79.0248 
                 38 
                 12 
                 3.68 
               
               
                   
                 79.0291 
                 58 
                 9 
                 5.99 
               
               
                   
                 79.0335 
                 36 
                 5 
                 4.84 
               
               
                   
                 79.0379 
                 15 
                 5 
                 2.24 
               
               
                   
                 79.0423 
                 7 
                 4 
                 0.905 
               
               
                   
                 79.0467 
                 6 
                 5 
                 0.302 
               
               
                   
                 79.0511 
                 11 
                 5 
                 1.50 
               
               
                   
                 79.0555 
                 11 
                 2 
                 2.50 
               
               
                   
                 79.0598 
                 0 
                 3 
                 1.73 
               
               
                   
                 79.0642 
                 2 
                 1 
                 0.577 
               
               
                   
                 79.0686 
                 4 
                 0 
                 2.00 
               
               
                   
                 79.073 
                 3 
                 0 
                 1.73 
               
               
                   
                   
               
             
          
         
       
     
     Turning to Table 2, this shows sets of data indicating a situation where fragmentation of ion  202  showed little variation in the on and off signals, indicating that the peaks were essentially the same, and for which the additional third MS step added nothing to the peak. Table 2 again shows, in the same order, data for a medium, little, big and very little peaks, at masses  122 ,  131 ,  123  and  103  respectively. 
     The column for the factor |T| shows that for the mass  122 , |T| often has a value of much less than 1, and only exceeds 1 for a couple of the data points. This is clearly indicative of two peaks that are the same and have no statistically different magnitude. This data corresponds to  FIG. 4   b.    
     There is a similar effect for a small or little peak for the mass  131 . Here, the values of |T| are even smaller, and it can be seen that many of the values for the difference figure are negative or very small. 
     For a big peak at mass  123 , due to the larger size of the peaks, values for the difference and significance parameter |T| are larger Here, a review of the various values of the parameter |T| again clearly shows that these two peaks are substantially the same. 
     Finally, for mass  103 , it can be noted that the values for the difference in |T| data are all extremely small. Again, a clear indication that there is no statistically significant difference between the two peaks. 
     
       
         
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                   
                 TABLE 2 
               
               
                   
                   
               
               
                   
                 M/Z 
                 ON 
                 OFF 
                 [T] 
               
               
                   
                   
               
             
             
               
                   
               
             
          
           
               
                   
                   
                 MED#, NO 
                   
                   
               
               
                   
                 122.0154 
                 12 
                 9 
                 0.655 
               
               
                   
                 122.0208 
                 27 
                 31 
                 0.525 
               
               
                   
                 122.0263 
                 76 
                 92 
                 1.23 
               
               
                   
                 122.0318 
                 170 
                 162 
                 0.439 
               
               
                   
                 122.0372 
                 153 
                 159 
                 0.340 
               
               
                   
                 122.0427 
                 364 
                 411 
                 1.699 
               
               
                   
                 122.0481 
                 1192 
                 1289 
                 1.95 
               
               
                   
                 122.0536 
                 2480 
                 2365 
                 1.65 
               
               
                   
                 122.059 
                 2381 
                 2496 
                 1.65 
               
               
                   
                 122.0645 
                 1325 
                 1401 
                 1.46 
               
               
                   
                 122.0699 
                 622 
                 596 
                 0.745 
               
               
                   
                 122.0754 
                 285 
                 257 
                 1.20 
               
               
                   
                 122.0808 
                 159 
                 170 
                 0.606 
               
               
                   
                   
                 LITTLE, NO 
                   
               
               
                   
                 131.017 
                 1 
                 2 
                 0.577 
               
               
                   
                 131.0226 
                 12 
                 12 
                 0.000 
               
               
                   
                 131.0282 
                 18 
                 20 
                 0.324 
               
               
                   
                 131.0339 
                 26 
                 22 
                 0.577 
               
               
                   
                 131.0395 
                 32 
                 49 
                 1.89 
               
               
                   
                 131.0452 
                 132 
                 133 
                 0.061 
               
               
                   
                 131.0508 
                 324 
                 313 
                 0.516 
               
               
                   
                 131.0565 
                 463 
                 507 
                 1.41 
               
               
                   
                 131.0621 
                 335 
                 333 
                 0.077 
               
               
                   
                 131.0678 
                 172 
                 186 
                 0.740 
               
               
                   
                 131.0734 
                 212 
                 226 
                 0.699 
               
               
                   
                 131.0791 
                 385 
                 386 
                 0.036 
               
               
                   
                 131.0847 
                 405 
                 391 
                 0.496 
               
               
                   
                 131.0904 
                 204 
                 203 
                 0.050 
               
               
                   
                 131.096 
                 81 
                 86 
                 0.387 
               
               
                   
                   
                   
                 BIG, NO 
                   
               
               
                   
                 123.0259 
                 220 
                 263 
                 1.957 
               
               
                   
                 123.0314 
                 1108 
                 1098 
                 0.213 
               
               
                   
                 123.0368 
                 2737 
                 2943 
                 2.73 
               
               
                   
                 123.0423 
                 3539 
                 3554 
                 0.178 
               
               
                   
                 123.0478 
                 2622 
                 2738 
                 1.58 
               
               
                   
                 123.0533 
                 3409 
                 3343 
                 0.803 
               
               
                   
                 123.0587 
                 7021 
                 7081 
                 0.505 
               
               
                   
                 123.0642 
                 8916 
                 8623 
                 2.21 
               
               
                   
                 123.0697 
                 5861 
                 5698 
                 1.52 
               
               
                   
                 123.0752 
                 2345 
                 2247 
                 1.45 
               
               
                   
                 123.0806 
                 957 
                 945 
                 0.229 
               
               
                   
                 123.0861 
                 585 
                 587 
                 0.058 
               
               
                   
                   
                 V. LITTLE, NO 
                   
                   
               
               
                   
                 103.0308 
                 4 
                 9 
                 1.39 
               
               
                   
                 103.0358 
                 10 
                 10 
                 0 
               
               
                   
                 103.0408 
                 38 
                 37 
                 0.115 
               
               
                   
                 103.0458 
                 79 
                 85 
                 0.469 
               
               
                   
                 103.0508 
                 140 
                 146 
                 0.355 
               
               
                   
                 103.0558 
                 103 
                 112 
                 0.614 
               
               
                   
                 103.0608 
                 46 
                 47 
                 0.104 
               
               
                   
                 103.0658 
                 8 
                 22 
                 1.96 
               
               
                   
                 103.0708 
                 14 
                 15 
                 0.186 
               
               
                   
                 103.0758 
                 6 
                 3 
                 1.0 
               
               
                   
                 103.0809 
                 2 
                 2 
                 0 
               
               
                   
                 103.0859 
                 4 
                 2 
                 0.816 
               
               
                   
                 103.0909 
                 5 
                 4 
                 0.333 
               
               
                   
                 103.0959 
                 2 
                 3 
                 0.447 
               
               
                   
                 103.1009 
                 2 
                 4 
                 0.816 
               
               
                   
                 103.1059 
                 0 
                 0 
                 0 
               
               
                   
                   
               
             
          
         
       
     
     Referring now to  FIGS. 6   a,    6   b  and  6   c,  these show further spectra obtained for reserpine.  FIG. 6   a  again shows just the first two MS steps, where reserpine is selected in Q 1 , accelerated and fragmented in Q 2 . Additionally, here  FIG. 6   a  just shows the low mass end of the fragment spectrum up to approximately mass  200 . This shows that reserpine with an m/z of  609  generates significant fragments at 174.1 and 195.1. 
       FIG. 6   b  then shows the spectrum obtain by a third MS step, where the fragment at  174  was excited. As might be expected, this shows a much reduced peak for the mass  174 , and an increase in the number and intensity of fragments below mass  174 , notably peaks at 130.1 and 131.1 Unlike earlier figures,  FIG. 6   b  is an unsubtracted spectrum. 
     If the spectrum of  FIG. 6   a  is subtracted from  FIG. 6   b,  the spectra of  FIG. 6   c  is obtained. Note that this is on a different scale. This clearly shows a significant reduction in the peak at 195.1, as this was present in the original spectrum of  FIG. 6   a.  This spectrum also emphasizes the contribution made to the various other fragments by the third MS step, the major peaks being identified in  FIG. 6   c.    
     Reference will now be made to  FIGS. 7   a,    7   b  and  7   c.    FIG. 7   a  shows part of the spectrum of  FIG. 6   a  but only up to a mass of approximately 190. This enables a different scale to be used, to emphasize the size of the different peaks. 
       FIG. 7   b  then shows a spectrum obtained for a four-step excitation scheme. Here, the fourth MS step, MS 4  was effected utilizing the power supply  28  and modulation unit  30 . For this scheme, the excitation as a third MS step, by the power supply  24 , is continuous, without any modulation by the unit  26 . The spectrum obtained is then subject to further excitation of the mass at  130 / 131 ; these two masses are so close together, that it is impossible to obtain excitation of just one mass. Again,  FIG. 7   b  is an unsubtracted spectrum. 
       FIG. 7   c  then shows the spectrum of  FIG. 7   b,  with that of  FIG. 7   a  subtracted. This again, shows elimination of peaks due to previous fragmentation and hence solely the peaks resulting from ions generated by fragmentation of the ions of mass  130 ,  131 . It should be noted that for the fourth step MS 4  procedure, excitation from the two power supplies  24 ,  28  is provided simultaneously. As noted, the power supply  24  is unmodulated, i.e. continuous, while the excitation from power supply  28  is modulated at a modulation of, for example, 2 Hz. 
     Reference will now be made to  FIGS. 8   a - 8   d,  which show a series of spectra, indicating the effects of varying the excitation voltage.  FIG. 8   a  again corresponds to  FIG. 6   a,  and shows the fragment spectrum obtained from the initial fragmentation of the Reserpine, again showing significant peaks at 174.1 and 195.1. In this case, the larger peak at 195.1 was selected for further excitation. This was excited at a frequency of 575 kHz and at different voltages of 1.5, 2.5 and 3.5, to obtain the spectra of  FIGS. 8   b,    8   c  and  8   d.  Each of these spectra  8   b - 8   d  are subtracted spectra, that is the spectra obtained with the excitation and subsequent subtraction of the spectrum of  FIG. 8   a.  They are also unfiltered. 
     As might be expected, the peak at  195  is largely eliminated as a result of the excitation. It can be noted that at low excitation potentials, a peak is shown with an ion close to mass  190 , and this peak reduces significantly, as the excitation voltage is increased. Correspondingly, peaks with smaller fragment ions increase. This is to be expected. 
     It will be appreciated that, while the invention has been described as effected with a quadrupole, it can be carried out in any suitable collision cell, and in particular any collision cell where quadrupolar fields can be applied. Also, while using a quadrupole is preferable, it will be appreciated that other multipolar guides may be used.