Abstract:
Provided herein is a sample injection method that enables efficient injection of a trace sample solution while reducing the measurement time. A sample solution is injected into a sample loop with air layers disposed on both sides of the sample solution, and the total amount of the sample solution, including the air layers, is injected into a detector. The start and the end of data collection are determined from the detection signal intensity changes that occur upon the air layers being injected into the detector, and the velocity of the flowing liquid is increased to reduce the measurement time. A washing solution is injected after the air layer to improve the washing efficiency and reduce the washing time.

Description:
TECHNICAL FIELD 
       [0001]    The present invention relates to a sample injection device that injects a sample into a mass spectrometer, and particularly to a sample injection device for mass spectrometers that uses a flowing solvent for the injection of a sample solution by a flow injection method. 
       BACKGROUND ART 
       [0002]    A mass spectrometer (MS) is an analyzer used for high-sensitivity measurements of trace chemical species components contained in liquid or gas components. Mass spectrometers are used for the qualitative and quantitative analyses of trace chemical species contained in various types of sample solutions such as biofluids (e.g., serum, urine, and tissue extract), and environmental samples (e.g., river water, and industrial drainage water). 
         [0003]    MS measurements of a solution sample commonly use a LC-MS or a CE-MS with a sample injection device connected online to separating means such as a high performance liquid chromatograph (HPLC) and a capillary electrophoresis (CE) device. In separating means such as LC and CE, a sample solution is injected into a continuous stream of a solvent in a flow path. The target chemical species in the sample solution are separated from contaminating components at a separating unit disposed downstream of the flow path, and injected into the MS. The chemical species injected in the MS are ionized by an ionization source, and separated and detected according to the mass. The ionization source used to ionize the target chemical species in MS uses atmospheric ionization as represented by electrospray ionization (ESI). The MS connected online to the separating means involves separation of the target chemical species from contaminating components, and enables high sensitivity and high accuracy analysis. 
         [0004]    In the analysis of biofluids, the sample solution is usually available only in trace amounts. A trace sample solution can be injected into LC-MS by using a method that measures the sample solution by filling it in a small-volume sample loop. However, the method requires the sample solution in several times the volume of the sample loop. In another method, a syringe installed in a sample injection device is used to measure and inject a sample solution into a sample loop. However, the sample solution becomes diluted during the injection process as it mixes with the solvents disposed on the both sides of the sample solution, and the liquid amount with the measurement component increases. The lowered concentration of the measurement component leads to poor detection sensitivity in concentration-dependent detectors such as MS, and causes a proportional increase in measurement time. 
         [0005]    As a means to efficiently inject a trace sample solution into a HPLC or a LC-MS, a method is proposed in which a sample solution is sent to a sample loop by being sandwiched between bubbles to reduce the dilution of the sample solution by solvent. For example, PTL 1 and PTL 2 describe sandwiching a sample solution between bubbles, and sending only the sample solution to a sample loop to reduce the loss by the diffusion of the sample solution. 
         [0006]    Flow injection analysis (FIA) is a non-separatory technique that enables quick analysis. FIA is an analytical method in which a reaction reagent solution is constantly passed through a capillary of about 0.5 mm, and a solution sample is injected into the continuous stream to detect the reaction product chemical species or derivatives thereof of interest with a downstream detector (see, for example, Non PTL 1 and PTL 2). The advantages of FIA include the low cost of the analyzer, simple procedures for fast and high sensitivity measurements, and easy automation. The detection commonly uses an spectrophotometer. However, FIA-MS that uses MS is also used in applications that require high sensitivity analysis, for example, such as in environment detection, and measurements of biological components. For the injection of a trace liquid sample in FIA, for example, PTL 3 describes a method in which a sample solution and air are alternately disposed in a capillary, and these are injected into a detector flow cell to reduce the diffusion of the sample solution or the dilution by washing solution as might occur during the injection. 
       CITATION LIST 
     Patent Literature 
       [0000]    
       
         PTL 1: JP-A-62-50659 
         PTL 2: Japanese Patent No. 2573678 
         PTL 3: JP-A-7-159415 
       
     
       Non Patent Literature 
       [0000]    
       
         NPL 1: H. B. Kim et al.; Analytical Science, 16, 871-876, 2000. 
         NPL 2: K. Kameyama et al.; Biophysical Journal, 90, 2164-2169, 2006. 
       
     
       SUMMARY OF INVENTION 
     Technical Problem 
       [0012]    The separation capability of LC-MS and CE-MS suffers when bubbles enter the separation unit where column separation or electrophoresis takes place. In this case, electrophoresis can no longer be performed properly, and a problem is posed for the analysis. PTL 1 and PTL 2 address this problem by not retaining the bubbles on the both sides of the sample solution in a sample loop so as to prevent entry of the bubbles in the analysis flow path. This means that the sample solution is partially present also on the outside of the sample loop, and that this portion of the sample solution on the outside of the sample loop becomes washed and wasted without being used for analysis. PTL 3 achieves efficient displacement of a sample solution. However, the sample solution sandwiched between air layers is also not used for analysis, and wasted. 
         [0013]    It is accordingly an object of the present invention to provide a sample injection method for MS whereby a trace sample solution can be fully injected while also reducing the measurement time. 
       Solution to Problem 
       [0014]    In order to achieve the foregoing object, the present invention provides a sample injection device that is configured from sample drawing means, a sample loop, flow path switching means, and solvent delivering means, and that injects a sample into a detector in a stream of a solvent. The sample is drawn and injected into the sample loop with air layers disposed on both sides of the sample, and the total sample amount, including the air layers, is injected into the detector. 
       Advantageous Effects of Invention 
       [0015]    The present invention uses air layers that are disposed on both sides of a sample. This reduces the sample diffusion in the flow path, and increases the signal intensity of the sample at a detecting section. Signal intensity changes due to the air layer are detected to enable an easy transition to the washing step, and the measurement time is reduced. 
     
    
     
       BRIEF DESCRIPTION OF DRAWINGS 
         [0016]      FIG. 1  is a diagram representing the configuration of an automatic analyzer according to an embodiment of the present invention. 
           [0017]      FIG. 2  is a diagram representing the flow path in a sample injection section according to the embodiment of the present invention. 
           [0018]      FIG. 3  is a schematic diagram representing inside of a sample loop with the injected extracted sample solution and air layers. 
           [0019]      FIG. 4  is a diagram representing the measurement result according to the embodiment of the present invention. 
           [0020]      FIG. 5  is a diagram representing the measurement result according to a conventional method. 
           [0021]      FIG. 6  is a diagram representing the configuration of an automatic analyzer according to another embodiment of the present invention. 
           [0022]      FIG. 7  is a diagram representing the flow path in a sample injection section according to another embodiment of the present invention. 
       
    
    
     DESCRIPTION OF EMBODIMENTS 
       [0023]    Embodiments of the present invention are described in detail below. It should be noted that the present invention is in no way limited by the following embodiments. 
       First Embodiment 
       [0024]    An automatic analyzer according to an embodiment of the present invention is described below with reference to  FIG. 1 . Specifically, an automatic analyzer is described that is intended to automatically and continuously analyze trace components contained in biofluids such as serum and urine, and that includes a solid phase extracting mechanism for performing solid phase extraction as a pretreatment, a sample injection device for passing an extracted sample solution, and a MS equipped with an ESI ionization source. More specifically, the following describes an example of an analysis of the immunosuppressant tacrolimus contained in a whole blood sample. 
         [0025]    The automatic analyzer shown in  FIG. 1  is configured from a sample holder  102  on which sample containers  101  with a dispensed biofluid to be subjected to a solid phase extraction process are disposed; a processing section  104  that sequentially performs a solid phase extraction process with a solid phase extraction cartridge  103 ; a reagent installation unit  106  on which reagent containers  105  for various reagents such as a washing solution  203  and an eluent used for an extraction process are disposed; a sample dispensing mechanism  107  by which the biofluid dispensed in the sample container  101  is dispensed into the solid phase extraction cartridge  103 ; a reagent dispensing mechanism  108  by which the reagent in the reagent container  105  is dispensed into the solid phase extraction cartridge  103 ; a solid phase extraction processing section  109  that performs a solid phase extraction process; a extract container holder  111  on which extract containers  110  are disposed; a holder  112  for expendables such as the solid phase extraction cartridge  103  and the extract container  110 ; a sample injection section  113  that dispenses the extracted sample solution in the extract containers  110 , and passes the extracted sample solution to an ionization source  114 ; and a mass spectrometry section  115  in which the components ionized in the ionization source  114  are subjected to mass spectrometry. 
         [0026]    The biofluid analysis procedures by the automatic analyzer are described below. A predetermined quantity of the biofluid dispensed in the sample container  101  on the sample holder  102  is dispensed into the solid phase extraction cartridge  103  with the sample dispensing mechanism  107 . The solid phase extraction cartridge  103  with the dispensed biofluid is moved by the rotation of the processing section  104  to the position of the solid phase extraction processing section  109 . In the solid phase extraction processing section  109 , a liquid is passed in the solid phase extraction cartridge  103  containing the dispensed sample. By this process, the dispensed sample is passed in the solid phase extraction cartridge, and the measurement target component becomes retained in the solid phase of the solid phase extraction cartridge  103 . The washing solution  203  prepared in the reagent container  105  is then dispensed into the solid phase extraction cartridge  103  with the reagent dispensing mechanism  108 , and is passed to wash the solid phase extraction cartridge  103 . After the washing, the reagent dispensing mechanism  108  dispenses the eluent into the solid phase extraction cartridge  103  containing the dispensed sample, and the eluent is passed to elute the measurement target component retained in the solid phase of the solid phase extraction cartridge  103 . The resulting liquid is then collected into the extract container  110  as an extracted sample solution. 
         [0027]    The extract container  110  with the collected extracted sample solution is moved to the position of the sample injection section  113  by the rotation of the extract container holder  111 . The extracted sample solution in the extract container  110  is injected into the ionization source  114  with the sample injection section  113 . The measurement target component is ionized in the ionization source  114 , and the component is detected in the mass spectrometry section  115 . 
         [0028]    The sample injection section  113  is described below in detail with reference to  FIG. 2 . The sample injection section  113  is configured from a passing pump  202  that delivers a flowing solvent  201 ; a syringe pump  204  that draws the extracted sample solution in the extract container  110 , and the washing solution  203 ; a needle  205  that draws and sends the extracted sample solution and the washing solution  203  to the flow path; a sample loop  206  that retains the drawn extracted sample solution; a flow path switching valve  207  that connects the passing pump  202 , the syringe pump  204 , the needle  205 , and the both ends of the sample loop  206  to the ionization source  114 , and is adapted to switch the flow path to pass the retained extracted sample solution in the sample loop  206  to the ionization source  114 ; and a waste receptacle  208  that collects the liquid ejected from the needle  205 .  FIG. 2  also shows the mass spectrometry section  115  that detects the components ionized in the ionization source  114 . 
         [0029]    The flow path switching valve  207  has six connection ports, and is adapted to switch the flow paths by connecting any two adjacent ports. The flow path switching valve  207  has two switchable flow paths, Inject and Load. Referring to  FIG. 2 , Inject is the flow path indicated by solid line. Switching the flow path to Inject creates a state in which the passing pump  202  and the sample loop  206 , the sample loop  206  and the ionization source  114 , and the needle  205  and the syringe pump  204  are connected to each other. Load is the flow path indicated by dotted line in  FIG. 2 . The needle  205  is moved to dip the needle tip portion into either the extracted sample solution collected into the extract container  110 , or the washing solution  203 , and the liquid is drawn into the needle  205  by the operation of the syringe pump  204 . The needle  205  is also moved to the position of the waste receptacle  208  to eject the liquid inside the needle  205  and the flow path. 
         [0030]    The operation of the sample injection section  113  according to the present embodiment is described below with reference to  FIGS. 2 and 3 . The sample injection section  113  is in a standby state until it receives the extract container  110  containing the extracted sample solution, and the passing pump  202  passes the flowing solvent  201  to the ionization source  114  at a predetermined flow rate. Here, the flow path switching valve  207  switches the flow path to Inject position, and the flowing solvent  201  is passed to the ionization source  114  through the sample loop  206 . The syringe pump  204  repeats the drawing and the ejection of the washing solution  203  into the waste receptacle  208  to fill the flow path between the syringe pump  204  and the needle  205  with the liquid (washing solution  203 ) and remove the air. 
         [0031]    The sample injection operation into the sample injection section  113  is started upon the extract container  110  with the collected extracted sample solution being sent to the sample injection section  113  in a standby state. First, the flow path switching valve  207  switches the flow path from Inject to Load (dotted line in  FIG. 2 ). This connects the syringe pump  204  and the needle  205  with the sample loop  206  in between. The needle  205  is then moved to above the extract container  110  where there is no liquid, and the syringe pump  204  performs a certain draw operation to draw air into the needle  205  through the needle tip (first air layer). The needle  205  is lifted down to move the tip into the extracted sample solution, and the syringe pump  204  performs a certain draw operation to draw the extracted sample solution into the needle  205  through the needle tip. The needle  205  is then lifted up to move the tip out of the extracted sample solution, and the syringe pump  204  performs a certain draw operation to draw air into the needle  205  through the needle tip (second air layer). The tip of the needle  205  is then moved into the washing solution  203 , and the syringe pump  204  performs a certain draw operation. This draws the washing solution  203  into the needle  205  through the needle tip, and, at the same time, injects the extracted sample solution between the two air layers into the sample loop  206 .  FIG. 3  is a schematic diagram inside the sample loop  206  after the sample injection operation. 
         [0032]    After the sample injection operation, the flow path switching valve  207  switches the flow path from Load to Inject, and the extracted sample solution is sent to the ionization source  114  by the operation of the passing pump  202 . Each component in the extracted sample solution is ionized in the ionization source  114 , and sent to the mass spectrometry section  115 . In the mass spectrometry section  115 , the ionized components are separated for detection according to mass-to-charge (m/z). 
         [0033]      FIG. 4  represents the time dependent changes in the signal intensity detected in the mass spectrometry section  115  according to the present embodiment. In  FIG. 4 , the horizontal axis represents the time after the switching of the flow path to Inject by the flow path switching valve  207 , and the vertical axis represents the signal intensity of the ionized tacrolimus. A 70% methanol aqueous solution containing 10 mmol/L of ammonium acetate was used as the flowing solvent  201 . The passing pump  202  had a flow rate of 100 μL/min, and the sample loop  206  had a 60 μL volume. The sample injection operation injected the first air layer (5 μL), the extracted sample solution (injected in 10 μL), the second air layer (15 μL), and the washing solution  203  (2-propanol, 30 μL) into the sample loop  206 . 
         [0034]    The tacrolimus contained in the extracted sample solution was detected in the mass spectrometry section  115  after about 16 seconds from the switching of the flow path by the flow path switching valve  207 , and the signal intensity increased almost vertically. The signal intensity showed a rapid decrease after about 21 seconds from the switching of the flow path, and the count reached zero. Another signal was immediately detected, and the signal became gradually weaker over the course of about 10 seconds until it was finally undetectable after about 35 seconds from the switching of the flow path. The signal detected in the 6 second period from 16 seconds to 21 seconds after the switching of the flow path is attributed to the extracted sample solution between the two air layers. The signal detected after 22 seconds from the switching of the flow path is due to the extracted sample solution that remained in the sample loop  206 , the ionization source  114 , and the pipe, and washed by the washing solution  203 . 
         [0035]    The rapid increase or decrease of signal intensity is due to two air layers disposed on the both sides of the extracted sample solution, preventing the extracted sample solution from being mixed and diluted with the flowing solvent  201  or the washing solution  203 . While the air layer is passing the ionization source  114 , the extracted sample solution does not exist in the ionization source  114 , and accordingly the signal intensity count was zero. A quantitative analysis of components from mass spectrometry signals typically uses the integration value of the signal intensity, specifically the peak area. A further reduction of the measurement time can be achieved by using the 0 count time of signal intensity as the reference point of a peak area in the waveform of the signal intensity obtained in the present embodiment. Specifically, the time needed to wash the flow path can be reduced by increasing the flow rate of the passing pump  202  and the velocity of the flowing solvent  201  at the time when the signal intensity has decreased to the zero count. Changing the flow rate of the passing pump  202  changes the ionization efficiency in the ionization source  114 , and the signal intensity obtained in the mass spectrometry section  115  fluctuates. However, this does not affect the result of quantification because the peak area calculations only use the signal intensity from the zero count signal intensity (air layer) to the signal intensity that has decreased to 0 count. 
         [0036]    For comparison,  FIG. 5  represents a conventional sample injection method performed under the same measurement conditions as in the present embodiment except for the absence of the two air layers. Specifically,  FIG. 5  represents the time dependent signal intensity changes when the extracted sample solution is passed to the ionization source  114  in contact with the flowing solvent  201  and the washing solution  203 . As shown by the peak waveform in  FIG. 5 , the signal intensity gradually increased after about 10 seconds from the switching of the flow path, and became the maximum after about 24 seconds before the signal became undetectable after about 45 seconds. By comparing the measurement results of  FIG. 4  and  FIG. 5 , the signal intensity obtained in the result presented in  FIG. 4  of the present embodiment was at least two times as strong as that shown in  FIG. 5 , and the signal intensity of the component became undetectable about 10 seconds earlier. 
       Second Embodiment 
       [0037]    Another embodiment of the present invention is described below with reference to  FIGS. 6 and 7 . 
         [0038]      FIG. 6  shows an automatic analyzer that is intended to automatically and continuously analyze trace components contained in biofluids such as serum and urine, and that includes a solid phase extracting mechanism for performing solid phase extraction as a pretreatment, a sample injection device for passing an extracted sample solution, and a MS equipped with an ESI ionization source. More specifically,  FIG. 6  represents an example of an analysis of the immunosuppressant tacrolimus contained in a whole blood sample. The difference from First Embodiment is the configuration of a sample injection section  301 , and the other configuration is the same as in First Embodiment. 
         [0039]      FIG. 7  is a detailed diagram of the sample injection section  301 . The sample injection section  301  is configured from a passing pump  202  that delivers a flowing solvent  201 ; a syringe pump  204  that draws the extracted sample solution in the extract container  110 , and the washing solution  203 ; a needle  205  that is placed in the extracted sample solution and the washing solution  203  when drawing these solutions; a sample loop  206  that retains the drawn extracted sample solution; a flow path switching valve  207  that connects the passing pump  202 , the syringe pump  204 , the needle  205 , and the both ends of the sample loop  206  to the ionization source  114 , and is adapted to switch the flow path to pass the retained extracted sample solution in the sample loop  206  to the ionization source  114 ; a waste receptacle  208  that collects the liquid ejected from the needle  205 ; a washing pump  303  that delivers the washing solution  302 ; and a three-way joint  304  that connects the flow path between the passing pump  202 , the washing pump  303 , and the flow path switching valve  207 .  FIG. 7  also shows the mass spectrometry section  115  that detects the components ionized in the ionization source  114 . The configuration of the flow path switching valve  207  is the same as in First Embodiment. 
         [0040]    The operation of the sample injection section  301  according to the present embodiment is described below. The sample injection section  301  is in a standby state until the sample injection section  301  receives the extract container  110  containing the extracted sample solution, and the passing pump  202  passes the flowing solvent  201  to the ionization source  114  at a predetermined flow rate. The washing pump  303  remains inactivated with the washing solution  302  filling the flow path to the three-way joint  304 . Here, the flow path switching valve  207  switches the flow path to Inject, and the flowing solvent  201  is passed to the ionization source  114  through the sample loop  206 . The syringe pump  204  repeats the drawing and the ejection of the washing solution  203  into the waste receptacle  208  to fill the flow path between the syringe pump  204  and the needle  205  with the liquid and remove the air. 
         [0041]    The sample injection operation is started upon the extract container  110  being sent to the sample injection section  301  in a standby state. First, the flow path switching valve  207  switches the flow path from Inject (solid line in  FIG. 7 ) to Load (dotted line in  FIG. 7 ). This connects the syringe pump  204  and the needle  205  with the sample loop  206  in between. The needle  205  is then moved to above the extract container where there is no liquid, and the syringe pump  204  performs a certain draw operation to draw air into the needle  205  through the needle tip (first air layer). The needle  205  is lifted down to move the tip into the extracted sample solution, and the syringe pump  204  performs a certain draw operation to draw the extracted sample solution into the needle  205  through the needle tip. The needle  205  is then lifted up to move the tip out of the extracted sample solution, and the syringe pump  204  performs a certain draw operation to draw air into the needle  205  through the needle tip (second air layer). The tip of the needle  205  is then moved into the washing solution  203 , and the syringe pump  204  performs a certain draw operation. This draws the washing solution  203  into the needle  205  through the needle tip, and, at the same time, injects the extracted sample solution between the two air layers into the sample loop  206 . 
         [0042]    At the completion of the sample injection operation, the flow path switching valve  207  switches the flow path from Load to Inject, and the extracted sample solution is sent to the ionization source  114  by the operation of the passing pump  202 . Each component in the extracted sample solution is ionized in the ionization source  114 , and sent to the mass spectrometry section  115 . In the mass spectrometry section  115 , the ionized components are separated for detection according to mass-to-charge (m/z). 
         [0043]    The extracted sample solution is sent to the ionization source  114  by being sandwiched between the two air layers. Accordingly, as shown in  FIG. 4 , the signal intensity rapidly increases upon the transition from the air layer to the extracted sample solution in the ionization source, and rapidly decreases upon the transition from the extracted sample solution to the air layer after a certain time period. The rapid signal intensity decrease is determined by signal processing, and washing of the flow path is started. Specifically, the washing pump  303  is operated to pass the washing solution  302 . The washing solution  302  reaches the ionization source  114  through the flow path switching valve  207  and the sample loop  206 . Preferably, the washing solution  302  uses a solvent with a strong dissolving power for the contaminating components and the drugs contained in the whole blood in the extracted sample solution. In the present embodiment, the primary contaminating component of the whole blood is the lipid. Because the tacrolimus is a hydrophobic agent, an organic solvent such as 2-propanol and acetone may be used for the washing solution  302 . 
         [0044]    Because the washing solution  302  is injected with the washing pump  303  and the three-way joint  304  in the middle of the flow path, the flow rate can be increased in the flow path from the three-way joint  304 . This increases the velocity of the washing solution  302 , and the washing time can be reduced as in First Embodiment in which the flow rate of the passing pump  202  is increased to increase the velocity of the washing solution. Injecting a mixture of the washing solution  302  and the flowing solvent  201  into the ionization source  114  changes the ionization efficiency in the ionization source  114 , and the signal intensity obtained in the mass spectrometry section  115  fluctuates. However, this does not affect the result of quantification because the peak area calculations only use the signal intensity from the zero count signal intensity (air layer) to the signal intensity that has decreased to 0 count. 
         [0045]    In the present embodiment, the three-way joint  304  is installed between the passing pump  202  and the flow path switching valve  207 . However, the three-way joint  304  may be installed between the flow path switching valve  207  and the ionization source  114  to further reduce the washing time, provided that it is certain that the residual contaminating components or drugs occur in the ionization source  114 . 
       REFERENCE SIGNS LIST 
       [0000]    
       
           101 : Sample container 
           102 : Sample holder 
           103 : Solid phase extraction cartridge 
           104 : Processing section 
           105 : Reagent container 
           106 : Reagent installation unit 
           107 : Sample dispensing mechanism 
           108 : Reagent dispensing mechanism 
           109 : Solid phase extraction processing section 
           110 : Extract container 
           111 : Extract container holder 
           112 : Holder 
           113 : Sample injection section 
           114 : Ionization source 
           115 : Mass spectrometry section 
           201 : Flowing solvent 
           202 : Passing pump 
           203 : Washing solution 
           204 : Syringe pump 
           205 : Needle 
           206 : Sample loop 
           207 : Flow path switching valve 
           208 : Waste receptacle 
           301 : Sample injection section 
           302 : Washing solution 
           303 : Washing pump 
           304 : Three-way joint