Abstract:
Systems and methods for analyzing compounds in a sample. In one embodiment, the present technology is directed towards a method of analyzing a sample, comprising: emitting ions from the sample; selecting the emitted ions for a designated ion; fragmenting the designated ions; scanning for a plurality of designated ion fragments; determining a designated fragment chromatographic trace for each designated ion fragment; and generating a combined chromatographic trace corresponding to a non-linear combination of a plurality of designated fragment chromatographic traces.

Description:
PRIORITY  
       [0001]    The present application claims priority from U.S. provisional patent application No. 60/992,172, filed Dec. 4, 2007, which is incorporated herein by reference in its entirety. 
     
    
     FIELD  
       [0002]    The present application relates generally to the field of mass spectrometry. 
       BACKGROUND  
       [0003]    The analysis of a substance to determine its composition may be necessary for many applications, including toxicology, forensics and environmental testing, as well as food and drug research. Often, samples to be analyzed are analyzed for the presence of numerous different analytes of interest. Such samples may, for example, be in the form of bodily fluids taken from test subjects, which fluids often include both drug metabolites of interest, as well as irrelevant endogenous ions from the test subject. Within complex samples, correctly determining the presence or absence as well as the quantities of a large number of analytes of interest, can be difficult and time-consuming. 
         [0004]    Mass spectrometers are often used for producing a mass spectrum of a sample to find its composition. This is normally achieved by ionizing the sample and separating ions of differing masses and recording their relative abundance by measuring intensities of ion flux. For example, with time-of-flight mass spectrometers, ions are pulsed to travel a predetermined flight path. The ions are then subsequently recorded by a detector. The amount of time that the ions take to reach the detector, the “time-of-flight”, may be used to calculate the gion&#39;s mass to charge ratio, m/z. 
         [0005]    Additional information (in addition to an ion&#39;s precursor mass) can then be obtained by fragmenting the ion via CID (collision induced dissociation) in a collision cell (or other means) to generate an MSMS spectrum. In most instruments with MSMS capabilities, the process of generating a mass spectrum, selecting a precursor ion and generating an MSMS (mass spectrum/mass spectrum) spectrum can be performed in an automated mode. This mode of acquisition is frequently referred to as Information Dependant Acquisition (IDA) or Data Dependant Experiment (DDE). 
         [0006]    Chromatographic equipment such as a liquid chromatograph may be used to elute or release ions from a sample into the mass spectrometer over a period of time. Multiple reaction monitoring (MRM) or other techniques may be used to analyze the ions received by the mass spectrometer. 
         [0007]    For complex samples, LC/MS quantitiation techniques using MRM frequently involve interfering matrix components exhibiting the same Q 1  and Q 3  masses as the analytes of interest. As a result, it may be difficult to determine which peak in a chromatogram represents the particular analyte of interest. There may also be small changes in retention time that increase the difficulty of peak finding. When dealing with a small number of analytes, this problem can usually be addressed by using specific sample cleanup techniques, isotopically enriched versions of the analytes as internal standards, or even sufficient manual intervention. For large numbers of analytes, however, such solutions are impractical. 
         [0008]    The applicants have accordingly recognized a need for systems and methods for analyzing and identifying ions from samples. 
       SUMMARY  
       [0009]    In one aspect, the present technology is directed towards a system for analyzing analytes in a sample. The system includes an ion source for emitting ions from the sample, a mass spectrometer adapted to receive the ions from the ion source, a controller operatively coupled to the mass spectrometer and configured to control the first mass analyzer to analyze for a designated ion of interest and to control the second mass analyzer to analyze for a designated ion fragment of interest. The system also includes data storage for storing at least one analyte parameter set, wherein each analyte parameter set includes: a designated precursor ion, a plurality of designated ion fragments, and a retention time window. 
         [0010]    The mass spectrometer includes a first mass analyzer to select ions received from the ion source, an ion fragmenter configured to fragment ions received from the first mass analyzer, a second mass analyzer configured to select ion fragments received from the ion fragmenter, and at least one detector configured to detect ion fragments received from the second mass analyzer. 
         [0011]    The controller is responsive to the analyte parameter set, and during the retention time window for each analyte parameter set the controller is configured to control the first mass analyzer to select for the corresponding designated precursor ion and to control the second mass analyzer to select for the corresponding designated ion fragments. The controller is configured to determine a chromatographic trace for each designated ion fragment in the analyte parameter set and wherein the controller is configured to determine a combined chromatographic trace corresponding to a non-linear combination of a plurality of designated fragment chromatographic traces for the analyte parameter set. 
         [0012]    Each chromatographic trace may comprise a plurality of data points, each data point corresponding to an intensity of ion fragments detected by the detector at a point in time, and the controller may be configured to determine the combined chromatographic trace for an analyte set by, for each point in time during the corresponding retention time window, multiplying the values of each corresponding data point in each chromatographic trace. 
         [0013]    In another aspect, the technology is directed towards a system for analyzing ions emitted from an ion source. The system includes a first mass analyzer adapted to receive and to select ions from the ion source, an ion fragmenter configured to fragment ions received from the first mass analyzer, a second mass analyzer configured to select ion fragments received from the ion fragmenter, and a detector configured to detect ion fragments received from the second mass analyzer. 
         [0014]    The system also includes a controller operatively coupled to the first and second mass analyzers, to the fragmenter and to the detector, wherein the controller is configured to control the first mass analyzer to analyze for a designated ion of interest and to control the second mass analyzer to select for a designated ion fragment of interest. The system further includes data storage for storing at least one analyte parameter set, wherein each analyte parameter set includes a designated precursor ion, a plurality of designated ion fragments, and a retention time window. 
         [0015]    The controller is responsive to the analyte parameter set, and during the retention time window for each analyte parameter set the controller is configured to control the first mass analyzer to select for the corresponding designated precursor ion and to control the second mass analyzer to select for the corresponding designated ion fragments. The controller is further configured to determine a chromatographic trace for each designated ion fragment in the analyte parameter set and the controller is configured to determine a combined chromatographic trace corresponding to a non-linear combination of a plurality of designated fragment chromatographic traces. 
         [0016]    Each chromatographic trace may comprise a plurality of data points, each data point corresponding to an intensity of ion fragments detected by the detector at a point in time, and the controller may be configured to determine the combined chromatographic trace for an analyte set by, for each point in time during the corresponding retention time window, multiplying the values of each corresponding data point in each chromatographic trace. 
         [0017]    In yet a further aspect, the present technology is directed towards a method of analyzing a sample, comprising: emitting ions from the sample; selecting the emitted ions for a designated ion; fragmenting the designated ions; scanning for a plurality of designated ion fragments; determining a designated fragment chromatographic trace for each designated ion fragment; generating a combined chromatographic trace corresponding to a non-linear combination of a plurality of designated fragment chromatographic traces. 
         [0018]    In some embodiments, the process of generating a combined chromatographic trace comprises multiplying the designated fragment chromatographic traces together to generate the combined chromatographic trace. 
         [0019]    The method may further comprise generating a report containing data corresponding to the determined retention time. 
         [0020]    In another aspect, the invention may be directed to computer readable media configured to cause a mass spectrometer having a computer controller to perform the method. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS  
         [0021]    The present invention will now be described, by way of example only, with reference to the following drawings, in which like reference numerals refer to like parts and in which: 
           [0022]      FIG. 1  is a schematic diagram of a mass spectrometer made in accordance with the present disclosure; 
           [0023]      FIG. 2  is a is a representative example of analyte parameter data as may be stored in the data storage of the mass spectrometer of  FIG. 1 ; 
           [0024]      FIG. 3A  is a representative example of a chromatographic trace corresponding to a first designated ion fragment in a parameter set of  FIG. 2 ; 
           [0025]      FIG. 3B  is a representative example of a chromatographic trace corresponding to a second designated ion fragment in a parameter set of  FIG. 2 ; 
           [0026]      FIG. 3C  is a representative example of a chromatographic trace corresponding to a third designated ion fragment in a parameter set of  FIG. 2 ; 
           [0027]      FIG. 4  is a representative example of a combined chromatographic trace in accordance with the present disclosure corresponding to a non-linear combination of the designated fragment chromatographic traces of  FIGS. 3A ,  3 B and  3 C; 
           [0028]      FIG. 5  is a flow diagram illustrating the steps of a method of analyzing a compound in accordance with the present disclosure. 
       
    
    
     DETAILED DESCRIPTION  
       [0029]    Referring to  FIG. 1 , illustrated therein is an analysis system referred to generally as  10 , made in accordance with the present disclosure. The system  10  is configured to be capable of performing scheduled MRM in accordance with the present disclosure, as will be understood. 
         [0030]    The analysis system  10  includes a mass spectrometer  11  (which may be an MS/MS system such as a hybrid quadrupole time-of-flight or triple quadrupole system. The spectrometer  11  comprises a suitably programmed controller or central processing unit (CPU)  12  having a suitably programmed analysis engine  14  stored in RAM or other suitable computer-readable media. Alternatively, the engine  14  may reside on a CPU remote from the CPU  12 , for remote processing of the data. An input/output (I/O) device  16  (typically including an input component  16   A  such as a keyboard or control buttons, and an output component such as a display  16   B ) is also operatively coupled to the CPU  12 . Data storage  17  is also provided. 
         [0031]    The system  10  also includes an ion source  20 , configured to emit ions, generated from the sample  21  to be analyzed. The ion source  20  may be a continuous ion source, for example, such as an electron impact or chemical ionization source (which may be used in conjunction with a gas chromatography source), or an electrospray or atmospheric pressure chemical ionization ion source (which may be used in conjunction with or operatively coupled to a liquid chromatography source), or a desorption electrospray ionization (DESI), or a laser desorption ionization source, as will be understood. 
         [0032]    The ion source  20  can also be provided with an ion transmission ion guide, such as a multipole ion guide, ring guide, or an ion mass filter, such as a quadrupole mass filter, or an ion trapping device, as generally known in the art (not shown). For brevity, the term ion source  20  has been used to describe the components which generate ions from the sample  21 , and emit analyte ions of interest for detection. Other types of ion sources  20  may also be used, such as a system having a tandem mass filter and ion trap. Preferred ion sources are those which emit the ions from the sample  21  over a range of times, to enable mass analysis by the mass spectrometer  11  using MRM or other suitable techniques. 
         [0033]    As will be understood, liquid chromatography may be used to separate compounds dissolved in solvent from other substances in the sample  21 , and release or emit such compounds for MS analysis. As a result of the different timings for the chemical interactions that take place during the LC phase, the analytes of interest are released over time. The release times for specific analytes can be estimated, based on the expected chemical interactions. 
         [0034]    As noted above, the spectrometer  11  may comprise a triple quadrupole mass spectrometer, having triple rod sets Q 1 , Q 2  and Q 3 . The rod sets Q 1  and Q 3  may be controlled by the processor  12  (via the trigger engine  14 ) to select or filter for ions having a particular m/z. In contrast, the Q 2  rod set is provided with a chamber and configured to operate as a collision cell or fragmenter for fragmenting the ions received from Q 1 . The resulting ion fragments may be passed through to, and selectively filtered by, rod set Q 3 , before being detected or recorded by the detector  22 . 
         [0035]    Optics  24  or other focusing elements, such as an electrostatic lens can also be disposed in the path of the emitted ions, typically between the Q 3  rod set and the detector  22 , for focusing the ions onto the detector  22 . 
         [0036]    Referring now to  FIG. 2 , illustrated therein is a representative example of analyte parameter data  200  as may be stored in the data storage  17 . The analyte parameter data  200  includes at least one analyte parameter set  202 , and each analyte parameter set  202  includes: a m/z value corresponding to a designated precursor ion  204 , a plurality of m/z values corresponding to designated ion fragments  206 , and timing data corresponding to a retention time window  208 . While the example data is illustrated with having three ion fragments  206  per parameter set  202 , it should be understood that different numbers of fragments  206  may be determined for each set  202 . As will also be understood, the retention time window  208  corresponds to a predetermined period of time when the corresponding precursor ion  204  is expected to be emitted by the ion source  20  from the sample  21 . It should also be understood that the retention time or scanning window data  208  is not a requirement, since for certain simplified applications, the “windows” may be treated as running for the entire analysis period. 
         [0037]    Referring now to  FIGS. 3A to 3C , illustrated therein are example chromatographic traces comprising a plurality of data points, each data point corresponding to an intensity of ion fragments detected by the detector at a point in time, as may be generated by the processing of the sample  21  for analysis by the mass spectrometer  11 , as will be discussed in greater detail, below. It should be understood that charts such as those illustrated in  FIGS. 3A to 3C  corresponding to the designated fragment chromatographic traces may, but need not be, generated—rather, the designated fragment chromatographic trace data may simply be stored in data storage  17  and processed by the analyzer engine  14  in accordance with the present disclosure. 
         [0038]    Illustrated in  FIG. 3A  is a representative example of a chromatographic trace  300 A (which may be stored in data storage  17 ) corresponding to a first designated ion fragment  206 A in the parameter set  202 ′ discussed in relation to  FIG. 2 . The vertical axis  302  of the chart  300 A represents the intensity of the designated ion fragments  206 A detected by the detector  22 . The horizontal axis  304  of the chart  300 A corresponds to time and in the example the unit of measurement is minutes. In the example data, a dominant peak  310 A appears at approximately 23.03 minutes. 
         [0039]    Similarly,  FIG. 3B  is a representative example of a chromatographic trace  300 B (which may be stored in data storage  17 ) corresponding to a second designated ion fragment  206 B in the parameter set  202 ′ discussed in relation to  FIG. 2 . The vertical axis  302  of the chart  300 B represents the intensity of the designated ion fragments  206 B detected by the detector  22 . The horizontal axis  304  of the chart  300 B corresponds to time and in the example the unit of measurement is minutes. In the example data, a dominant peak  310 B appears at approximately 21.81 minutes. 
         [0040]    Also similarly,  FIG. 3C  is a representative example of a chromatographic trace  300 C (which may be stored in data storage  17 ) corresponding to a second designated ion fragment  206 C in the parameter set  202 ′ discussed in relation to  FIG. 2 . The vertical axis  302  of the chart  300 C represents the intensity of the designated ion fragments  206 C detected by the detector  22 . The horizontal axis  304  of the chart  300 C corresponds to time and in the example the unit of measurement is minutes. In the example data, a dominant peak  310 C appears at approximately 21.88 minutes. 
         [0041]    Turning now to  FIG. 4 , illustrated therein is a representative example of a combined chromatographic trace  400  (as may be stored in the data storage  17 ) generated by the analysis engine  14  and corresponding to a non-linear combination of the designated fragment chromatographic traces  300 A,  300 B and  300 C. The vertical axis  402  of the chart  400  represents the multiplied intensity values from the designated fragment chromatographic traces  300 A,  300 B and  300 C. The horizontal axis  404  of the chart  400  corresponds to time and in the example the unit of measurement is minutes. 
         [0042]    As will be understood, in operation, the CPU  12 /analysis engine  14  is responsive to the analyte parameter data  200  and specifically to the analyte parameter sets  202  (including for example,  202 ′). As will be discussed in greater detail below, the engine  14  is configured to regulate the operation of the mass analyzers Q 1  and Q 3 , to filter for the corresponding precursor ions  204  and confirmatory ion fragments  206 , during the corresponding retention time windows  208  for each analyte parameter set  202 . Once the designated fragment chromatographic traces (eg.  300 A,  300 B,  300 C) have been determined for a parameter set (eg.  202 ′), the engine  14  is further configured to generate a combined chromatographic trace (eg.  400 ) which corresponds to a non-linear combination of the designated fragment chromatographic traces. The engine  14  is further configured to determine a retention time corresponding to the analyte parameter set. 
         [0043]      FIG. 5  sets out the steps of the method, referred to generally as  500 , carried out by the spectrometer system  10  during an analysis period. Typically, before the analysis period is commenced, the analytes of interest are determined (for which the sample  21  is being analyzed) (Block  502 ). Typically, for each analyte of interest, a designated precursor ion  204  and a plurality of corresponding designated ion fragments  206  are stored in analyte parameter sets  202  in the analyte parameter data  200 . The corresponding retention time window  208  for each parameter set  202 , is also determined and stored (Block  504 ). 
         [0044]    As will be understood, the analyte parameter sets  202  (designated precursor ion  204  and designated ion fragments  206 , together with the corresponding retention time window  208 ) for numerous analytes of interest may be previously calculated and stored as a library of data in the data storage  17 , and simply indexed and retrieved by the user and the CPU  12  utilizing the I/O device  16 . 
         [0045]    The user will then typically input a command to commence an analysis period (typically via the I/O device  16 ), upon receipt of which the analysis engine  14  is programmed to initiate the analysis period (Block  506 ). 
         [0046]    When the analysis period is commenced, the ion source  20  is activated to commence the emitting of ions from the sample  21  (which may be the commencement of the LC phase as outlined above) (Block  508 ). As will be understood, the sample may, for example, include bodily fluid taken from a test subject, which fluid often includes both drug metabolites of interest, as well as irrelevant endogenous ions from the test subject. 
         [0047]    The system  10  is then configured to selectively filter the emitted ions for the designated precursor ions  204  during the corresponding retention time windows  208  (Block  510 ). As will be understood, the CPU  12 /analysis engine  14  is programmed to cause the rod set Q 1  to selectively filter the ions received from the ion source  20  for the designated precursor ions  204 . 
         [0048]    The filtered ions  204  are then received by the fragmentation module/rod set Q 2  and fragmented (Block  512 ). The fragments are then received by the Q 3  rod set, which is controlled by the analysis engine  14  to scan or filter for the corresponding designated ion fragments  206  (Block  514 ). Such designated ion fragments  206  (if any) are permitted to impact the detector  22 . If the detector  22  detects a designated ion fragment  206  (Block  516 ), the analysis engine  14  is programmed to store corresponding data in the data storage  17 . As will be understood, the filtering, fragmenting, filtering and detecting steps of Blocks  510 - 516  are typically performed substantially simultaneously for multiple analyte parameter sets  202  which happen to share overlapping retention time windows  208 . 
         [0049]    The process  500  cycles through the various steps  510 - 516  until the analysis period is complete and ion emission is terminated. 
         [0050]    The analysis engine  14  determines a designated fragment chromatographic trace (eg.  300 A,  300 B,  300 C) for a plurality of and typically each designated ion fragment  206  (Block  518 ). Such traces will be effected subsequent to the expiry of the retention time window  208  for a particular parameter set  202 ,  202 ′ (which may be during or following the analysis period). As noted, the traces (eg.  300 A,  300 B,  300 C) may simply comprise the collection of data points represented by the chromatographic charts illustrated in  FIGS. 3A ,  3 B and  3 C, and may not be required to be a separate step. 
         [0051]    For each analyte parameter set  202 ,  202 ′, the analysis engine  14  determines a combined chromatographic trace  400  corresponding to a non-linear combination of a plurality of the designated fragment chromatographic traces (eg.  300 A,  300 B,  300 C) (Block  520 ). The analysis engine  14  may be configured to determine the combined chromatographic trace  400  for an analyte parameter set by, for each point in time during the corresponding retention time window, multiplying the values of each corresponding data point in each such designated fragment chromatographic trace (eg.  300 A,  300 B,  300 C). Thus, for example, the value of point  450  at the time 23.17 minutes in the combined chromatographic trace  400  is determined by multiplying together the corresponding values  350 A,  350 B,  350 C (all at the time of 23.17 minutes) in the designated fragment chromatographic traces  300 A,  300 B,  300 C. 
         [0052]    The analysis engine  14  may then determine a retention time for the analyte parameter set  202 ′, and correspondingly for the designated precursor ion  204 ′ (Block  522 ). Typically, the engine  14  determines the retention time by detecting a dominant peak  410  in the combined chromatographic trace  400 . Since all designated fragments (eg.  206 A,  206 B,  206 C) in an analyte parameter set (eg.  202 ′) should share the same retention time (and should hence have a non-zero intensity value in each designated fragment chromatographic trace  300 A,  300 B,  300 C), by multiplying the data point values in the traces (eg.  300 A,  300 B,  300 C) together, it is expected that the largest value/dominant peak corresponds to the retention time. 
         [0053]    Thus for example, as can be seen by referring to the peak  310 A (at 23.03 minutes) in  FIG. 3A  and the peak  410  in  FIG. 4  (at 21.80 minutes), the peak  310 A is misleading and reflects interfering matrix components. The corresponding value at 23.03 minutes in  FIG. 4  (close to point  450  at 23.17 minutes) is close to zero. 
         [0054]    The analysis engine  14  may then quantify the designated precursor ion  204 ′ (Block  524 ). Typically, the engine  14  determines the quantity by integrating a dominant peak (eg.  310 B,  310 C) in a designated fragment chromatographic trace (eg.  300 A,  300 B,  300 C), which corresponds to the determined retention time (determined in Block  522 ). Alternatively, quantitiation may be determined by integrating the dominant peak  410  in the combined chromatographic trace  400 , as will be understood. 
         [0055]    As will be understood, the controller  12  may generate a report identifying the determined retention time, one or more of the chromatographic traces  300 A,  300 B,  300 C,  400 , quantities of the various designated ions  204  and hence the presence or absence of the corresponding analytes of interest (Block  526 ). 
         [0056]    Thus, while what is shown and described herein constitute preferred embodiments of the subject invention, it should be understood that various changes can be made without departing from the subject invention, the scope of which is defined in the appended claims.