Patent Publication Number: US-7718958-B2

Title: Mass spectroscopic reaction-monitoring method

Description:
CROSS-REFERENCE TO RELATED APPLICATION 
   This application is a continuation-in-part (CIP) of U.S. patent application Ser. No. 11/561,131, entitled “ELECTROSPRAY-ASSISTED LASER DESORPTION IONIZATION DEVICE, MASS SPECTROMETER, AND METHOD FOR MASS SPECTROMETRY”, filed on Nov. 17, 2006. 

   BACKGROUND OF THE INVENTION 
   1. Field of the Invention 
   The invention relates to amass spectroscopic method, more particularly to a mass spectroscopic reaction-monitoring method. 
   2. Description of the Related Art 
   For a liquid sample undergoing a chemical reaction, composition thereof varies over time. State of the chemical reaction can be monitored by monitoring the presence/absence of substances in the liquid sample and quantity changes of the substances. 
   Various methods, such as classical analysis, Ultraviolet (UV), Nuclear Magnetic Resonance (NMR), Infrared (IR), and neon spectroscopic analyses, have conventionally been used individually or in combination for monitoring chemical reactions. However, time-consuming steps such as separation and purification are required. In addition, instantaneous monitoring of the chemical reaction cannot be conducted. In other words, relative quantities of various substances in a liquid sample cannot be acquired, and the growth and decline of the quantity of each of the substances over a certain period of time cannot be determined. 
   Although Electrospray Ionization (ESI) mass spectrometry can be used to monitor chemical reactions by making the liquid sample an electrospray solution, the following shortcomings occur as the composition of the liquid sample may be very complicated:
         1. it is difficult to remove the liquid portion of the droplets formed by electrospraying (e.g., if the liquid sample is non-volatile);   2. the chemical reaction under monitor is easily affected by the addition of other solvents into the liquid sample for speeding up the removable of the liquid portion of the droplets, and by the addition of acidic substances into the liquid sample for enhancing ionizing efficiency of analytes in the liquid sample, thereby adversely affecting the credibility of the results obtained; and   3. it is prone to misinterpret the obtained mass spectrum when the liquid sample contains salt.       

   SUMMARY OF THE INVENTION 
   Therefore, the object of the present invention is to provide a mass spectroscopic method for monitoring a chemical reaction in a liquid sample that can be conducted with ease, convenience, and speed, and that is capable of revealing quantity variations of reactants, intermediate products and final products involved in the chemical reaction. 
   According to the present invention, there is provided a mass spectroscopic reaction-monitoring method that includes the steps of:
         a) forcing sequentially generated charge-laden liquid drops to move towards a receiving unit of a mass spectrometer along a traveling path;   b) exposing to a laser beam a region that is to be formed of a liquid sample surface, the laser beam being transmitted from an overhead laser beam directing member and having an irradiation energy sufficient to cause analytes present behind the liquid sample surface relative to the laser unit to be desorbed to fly along at least one flying path;   c) introducing a liquid sample to the region so as to form the liquid sample surface at successive points of time that are respectively spaced a plurality of predetermined intervals apart, the liquid sample containing at least one reactant that undergoes an ongoing chemical reaction as a first one of the analytes to form at least one product that co-exist therewith as a second one of the analytes; and   d) positioning the liquid sample surface relative to the laser beam at each of the successive points of time such that the at least one flying path intersects the traveling path to enable at least one of the coexisting first and second analytes to be occluded in at least one of the charge-laden liquid drops to thereby form at least a corresponding one of first and second ionized analytes.       

   
     BRIEF DESCRIPTION OF THE DRAWINGS 
     Other features and advantages of the present invention will be come apparent in the following detailed description of the preferred embodiment with reference to the accompanying drawings, of which: 
       FIG. 1  is a schematic view of a mass spectrometer for implementing the preferred embodiment of a mass spectroscopic reaction-monitoring method according to the present invention; 
       FIGS. 2(   a ) to  2 ( c ) are single-scan mass spectra obtained for exemplary method 1; 
       FIGS. 3(   a ) to  3 ( c ) are single-scan mass spectra obtained for exemplary method 2; 
       FIG. 4  is a chromatograph constructed for two representative m/z signals in exemplary method 2, 
       FIGS. 5(   a ) to  5 ( c ) are average mass spectra obtained for exemplary method 3; 
       FIGS. 6(   a ) to  6 ( g ) are chromatographs respectively constructed for seven representative m/z signals in exemplary method 3; and 
       FIG. 7  is an average mass spectrum obtained for comparative example 1. 
   

   DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT 
     FIG. 1  illustrates a mass spectrometer  1  for implementing the preferred embodiment of a mass spectroscopic reaction-monitoring method according to the present invention. The mass spectrometer  1  includes a sample stage  11 , a receiving unit  12 , a detector  13 , an electrospray unit  14 , a voltage supplying member  15 , and a laser unit  16 . 
   The sample stage  11  permits placement of a liquid sample thereon. 
   The receiving unit  12  is disposed to admit therein ionized analytes that are derived from the liquid sample, and includes a mass analyzer  121 . The mass analyzer  121  is formed with a conduit  122  for receiving the ionized analytes to be analyzed by the mass analyzer  121 . 
   The detector  13  is disposed to receive signals generated by the mass analyzer  121  as a result of analyzing the ionized analytes so as to generate a mass spectroscopic analysis result. In this embodiment, the mass spectroscopic analysis result includes at least one mass spectrum and/or a chromatograph. 
   The electrospray unit  14  includes a reservoir  141  for accommodating a liquid electrospray medium  142 , and a nozzle  143  disposed downstream of the reservoir  141 . The nozzle  141  is configured to sequentially form liquid drops of the electrospray medium  142  thereat, and is spaced apart from the conduit  122  of the mass analyzer  121  of the receiving unit  12  in a longitudinal direction so as to define a traveling path. 
   The voltage supplying member  15  is disposed to establish between the nozzle  143  of the electrospray unit  14  and the mass analyzer  121  of the receiving unit  12  a potential difference which is of an intensity such that the liquid drops are forced to leave the nozzle  143  as charge-laden ones for heading toward the conduit  122  of the mass analyzer  121  along the traveling path. 
   The laser unit  16  is capable of transmitting a laser beam  161 , which is directed by an overhead laser beam directing member  162  to irradiate a region that is to be formed of a liquid sample surface such that, upon irradiation, at least one analyte present behind the liquid sample surface relative to the overhead laser beam directing member  162  is desorbed to fly along at least one flying path. Preferably, the liquid sample is a liquid drop  2  (as that illustrated in  FIG. 1 ), and the liquid sample surface is the surface-tensed area of the liquid drop. Alternatively, the liquid sample is contained in an open reaction cell (not shown) that is disposed on the sample stage  11 , and the liquid sample surface is a level of the liquid sample in the open reaction cell. 
   The mass spectroscopic reaction-monitoring method will now be described with reference to the mass spectrometer  1  illustrated in  FIG. 1 . 
   First, sequentially generated charge-laden liquid drops are forced to move towards the receiving unit  12  of the mass spectrometer  1  along the traveling path. In this embodiment, the sequentially generated charge-laden liquid drops are formed by the electrospray unit  14  at the nozzle  143  thereof, and are forced to move towards the mass analyzer  121  of the receiving unit  12  by the electrospray unit  14  under the electric field generated by the voltage supplying member  5 . 
   Second, the region that is to be formed of the liquid sample surface is exposed to the laser beam  161 . In this embodiment, the laser beam  161  is emitted from the laser unit  16 , and has an irradiation energy sufficient to cause the analytes present behind the liquid sample surface relative to the overhead laser beam directing member  162  to be desorbed to fly along the at least one flying path. 
   Third, a liquid sample  2  is introduced to the region so as to form the liquid sample surface at successive points of time that are respectively spaced a plurality of predetermined intervals apart. The liquid sample  2  contains at least one reactant that undergoes an ongoing chemical reaction as a first one of the analytes to form at least one product that co-exist therewith as a second one of the analytes. 
   Fourth, the liquid sample surface is positioned relative to the laser beam  161  at each of the successive points of time to render the at least one flying path to intersect the traveling path so as to enable at least one of the coexisting first and second analytes to be occluded in at least one of the charge-laden liquid drops to thereby form at least a corresponding one of first and second ionized analytes. 
   Subsequently, a plurality of mass spectra are obtained for the plurality of successive points of time. Each of the mass spectra is obtained through analyzing the at least a corresponding one of the first and second ionized analytes which correspond to the liquid sample introduced at a corresponding one of the successive points of time. 
   Next, first and second representative mass-to-charge ratio (m/z) signals which respectively characterize the first and second analytes are preferably selected from the plurality of mass spectra. 
   Finally, a reaction rate of the chemical reaction is determined based on changes of intensities respectively for the first and second representative mass-to-charge ratio signals with reference to corresponding elapses of the predetermined time intervals. 
   Preferably, a suitable matrix is added to the liquid sample for conducting the mass spectroscopic analysis. 
   The matrix is made from a material that is non-transmissible by laser. More preferably, the matrix is selected from the group consisting of gold, carbon, cobalt, iron, 2,5-dihydroxybenzoic acid (2,5-DHB), 3,5-dimethoxy-4-hydroxycinnamic acid (sinapinic acid, (SA)), α-cyano-4-hydroxycinnamic acid (α-CHC), and a combination thereof. Optionally, the matrix has a particle diameter ranging from 50 nm to 50 μm. In this embodiment of the present invention, carbon powders with particle diameter of less than 50 μm are added to the liquid sample to serve as the matrix. 
   Since the mass spectroscopic reaction-monitoring method is capable of monitoring various kinds of chemical reactions, such as organic reactions, biochemical reaction (e.g., enzyme digesting protein reactions), organic metal complexation reactions, etc., no limitation is imposed on the liquid sample used. The solution portion of the liquid sample may be an aqueous solution or an organic solution. The analytes contained in the liquid sample (i.e., those related to the reaction) may be a biochemical substance, such as protein, or an organic compound. 
   The electrospray unit  14  may operate in a “positive ion mode” (i.e., voltage level at the mass analyzer  121  is higher than that at the nozzle  143 ), or in a “negative ion mode” (i.e., voltage level at the mass analyzer  121  is lower than that at the nozzle  143 ). 
   The electrospray medium  142  preferably includes water, organic solvents, or a combination thereof. Further, in order to prevent interference due to the addition of cations such as Na +  and K +  in the electrospray medium  142 , which results in a complicated mass spectrum, the electro spray medium  142  is more preferably a solution containing a volatile liquid. For example, the electrospray medium  142  may contain one of isoacetonitrile, acetone, alcohol, and a combination thereof. More preferably, the electrospray medium  142  is alcohol. Optionally, the electrospray medium  142  contains an acid to facilitate ionization of the analytes. The acid may be selected from the group consisting of formic acid, acetic acid, trifluoroacetic acid, and a combination thereof. In the embodiments of the present invention, the electrospray medium  142  is methanol. 
   Preferably, the laser unit  16  is selected from the group consisting of an infrared (IR) laser, an ultraviolet (UV) laser, a nitrogen laser, an argon ion laser, a helium-neon laser, a carbon dioxide (CO 2 ) laser, and a garnet (Nd:YAG) laser. In one embodiment of the present invention, the laser unit  16  is an ultraviolet laser for providing an ultraviolet laser beam. 
   No limitation is imposed upon the wavelength, energy, and frequency of the laser beam  161  transmitted by the laser unit  16 , as long as the laser beam  161  is capable of desorbing at least one of the analytes from behind the liquid sample surface when the latter is irradiated thereby. For the ultraviolet laser, the pulse energy is preferably higher than 20 μJ, and more preferably between 100 μJ and 150 μJ. In this embodiment, the pulse energy of the laser beam  161  is 120 μJ, and the laser beam  161  forms a spot size of 0.5 mm 2  on the liquid sample surface. 
   U.S. patent application Ser. No. 11/561,131 may be referred to for other operational parameters related to the electrospray unit  14 , the mass analyzer  121 , and the detector  13 . 
   It should be noted herein that since hydroxyl group, primary amino group, secondary amino group, etc. are highly absorbent to infrared (IR) light, when the liquid sample contains the above substances (e.g., water, amino, etc.), the substances may serve as the matrix. Therefore, it is particularly suitable to use an infrared laser as the laser unit  16  when the liquid sample contains water. 
   Moreover, the mass spectroscopic result obtained by carrying out the mass spectroscopic reaction-monitoring method of the present invention may be an average mass spectrum for a period of time, a single-scan mass spectrum for a particular point of time, or a chromatograph for a particular analyte (used to investigate the variation of the particular analyte over time). In addition, the plurality of time intervals between the successive points of time at which the liquid sample is introduced are chosen depending on the characteristic of the reaction under monitor, and may vary according to operational conditions. 
   It should be noted herein that the preferred embodiment disclosed herein is merely presented for the purpose of illustration, and should not be taken to limit the scope of the present invention. 
   Chemicals and Equipments Used 
   Exemplary methods 1˜3 and comparative example 1 were conducted using the following chemicals:
         1. laser unit: Ultraviolet (UV) Laser model no. VSL-337i, manufactured by Laser, Science Inc. of the United States. The laser beam transmitted by the ultraviolet laser has a wavelength of 337 nm, a frequency of 10 Hz, a pulse duration of 4 ns, and a pulse energy of 120 μJ.   2. Mass Analyzer (including the Detector): Quadrupole Time-of-Flight Mass Analyzer model no. BioTOF-Q, manufactured by Bruker Dalton company of Germany.   3. methanol (MeOH): a HPLC material manufactured by Merck of Germany (also known as German Merck)   4. ethanol (EtOH): model no. 459844 manufactured by Sigma-Aldrich company of the United States   5. Chalcone: molecular weight of 208, model no. 136123, manufactured by Aldrich company of the United States   6. acetic anhydride: molecular weight of 102.03, model no. 320102, manufactured by Sigma-Aldrich company of the United States   7. 4-aminophenol: molecular weight of 109.05, model no. 10968, manufactured by Fluka company   8. NaOH: model no. SK371842 manufactured by Nihon Shiyaku Industries Ltd.   9. H 2 O 2 : concentration of 30%, model no. 31692 manufactured by Riedel-de Haën company   10. Carbon powders: model no. 4206A manufactured by Merck company of Germany; particle diameter of below 50 μm.       

   In conducting the exemplary methods 1 to 3 presented hereinbelow, after choosing a particular reaction to monitor, a group of ionized analytes that correspond to the reactants, intermediate products and final products was predicted to be detected by the mass spectroscopic reaction-monitoring method of the present invention. The prediction was made in consideration of possible combinations of the reactants, intermediate products and final products to solvents, protons (H + ), Na + , and/or other substances present in the environment (e.g., air). 
   For each of the exemplary methods, the following steps were conducted for obtaining the results thereof:
         (i) It was first observed whether signals corresponding to the predicted ionized analytes were obtained to thereby verify the presence of the reactants, intermediate products and final products for the reaction in the liquid sample at the corresponding points of time. In addition, when signals corresponding to unpredicted ionized analytes were observed, the formation of these signals was to be investigated.   (ii) A chromatograph is constructed for each of particular ionized analytes in interest, i.e., particular ones of the reactants, intermediate products and final products chosen by selecting representative mass-to-charge ratio (m/z) signals to respectively characterize the particular ionized analytes, so as to facilitate the investigation of signal intensities of the corresponding ionized analyte over time.       

   If not specified otherwise, the exemplary methods were conducted under room temperature and atmospheric pressure. The electrospray unit operated under the “positive ion mode”, and methanol was used as the electrospray medium. During the course of each of experimentation for each of exemplary methods, all components of the mass spectrometer other than the laser unit were turned on the whole time. Moreover, the “points of time” were calculated with respect to the beginning of the experiment. In addition, in each of the exemplary methods, representative m/z signals were chosen from the average of the mass spectra obtained at the different points of time, and the chromatography were constructed only for the chosen representative m/z signals. 
   &lt;Exemplary Method 1&gt; Monitoring the Tryptic Digestion of Cytochrome C 
   Prediction 
   Since trypsin is capable of cleaving the bond between arginin and lysine in proteins, it was predicted that the amount of peptide from cytochrome c would increase over time. Therefore, it can be assumed that the signals corresponding to peptide in the mass spectra obtained at the successive points of time would reveal an increase in relative intensity. In other words, it can be anticipated that the intensity of the peptide signal would approach, or even surpass, that of cytochrome c over time. 
   Procedure 
   In exemplary method 1, an aqueous solution containing a cytochrome c standard (10 −4 M) was mixed with carbon powders (8 mg/ml). Subsequently, magnetic nano-particles (provided privately) coated with trypsin (2.5 μg/μL) were added into the aqueous solution to form the liquid sample. A drop of the liquid sample was withdrawn and deposited on the sample stage  11  (shown in  FIG. 1 ) every minute starting at minute 0 for analysis conducted using the mass spectroscopic reaction-monitoring method of the present invention. 
   Three representative mass spectra obtained from the droplets respectively collected at minutes 0, 15 and 30 are illustrated in  FIG. 2(   a ) to  FIG. 2(   c ), respectively. The composition of the liquid sample used and the information related to the results obtained for exemplary method 1 are tabulated in Table 1 below. 
                                       TABLE 1                          Liquid   Matrix   Carbon Powder (0.8 mg/μL)               Sample   Reactants   Cytochrome c (10 −4 M)                   Magnetic Nano-Particles                   (concentration 2.5 μg/mL)                                         Reaction Time       Minute 0   Minute 15   Minute 30           Single-scan Mass       FIG. 2(a)   FIG. 2(b)   FIG. 2(c)           Spectrum                        
Results
 
   As shown in  FIG. 2(   a ), all three of the apparent signals observed originate from cytochrome c, and no signal corresponding to peptide is observed. As shown in  FIG. 2(   b ) and  FIG. 2(   c ), signals corresponding to peptide are observed, and are denoted by “●”. In addition, the relative intensities of the peptide signals in  FIG. 2(   c ) are higher than those in  FIG. 2(   b ), and are closer to the relative intensities of the signals corresponding to cytochrome c. 
   It is verified by the results that the mass spectroscopic reaction-monitoring method of the present invention is capable of monitoring the progress of a biochemical reaction. 
   &lt;Exemplary Method 2&gt; Monitoring Epoxidation Reaction of Chalcone 
   Prediction 
   The chemical reaction in interest is illustrated in the figure below: 
   
     
       
       
           
           
       
     
   
   where the molecular weight of chalcone, serving as the reactant, is 208, and the molecular weight of the product is 224. In addition, since the liquid sample contains Na +  ions, it was predicted that the signals (represented by corresponding m/z values) corresponding to the ionized analytes tabulated in Table 2 would be detected. 
                       TABLE 2               m/z value   Ionized Analyte                  209   Chalcone + H +         231   Chalcone + Na +         247   Product + Na +                      
Procedure
 
   In exemplary method 2, a liquid sample containing 3 ml of EtOH, 24 mg of carbon powders and 75 mg of chalcone (i.e., the reactant in exemplary method 2) was disposed in an open reaction cell. Starting from minute 0.4, the liquid sample surface (i.e., level of the liquid sample in the open reaction cell) was irradiated by a laser beam. At minute 1.8, 0.5 ml of H 2 O 2  aqueous solution was added into the liquid sample. At minute 2.26, 0.5 ml of 5% NaOH aqueous solution was added into the liquid sample, and a large amount of bubbles were observed. The mass spectroscopic analysis was conducted for a total of 5.5 minutes. 
   Results 
   Three single-scan mass spectra were chosen for illustration purposes and are shown in  FIGS. 3(   a )˜ 3 ( c ), and a chromatograph illustrated in  FIG. 4  was constructed for two representative m/z signals selected. Information related to the results of exemplary method 2 is tabulated in Table 3 below. 
   
     
       
         
             
             
             
             
             
           
             
                 
               TABLE 3 
             
             
                 
                 
             
             
                 
                 
               Figure 
                 
                 
             
             
                 
               Result Type 
               No. 
               Time 
               m/z signal 
             
             
                 
                 
             
           
          
             
                 
               Mass Spectra 
               FIG. 3(a) 
               Minute 2 
               209, 231 
             
             
                 
                 
               FIG. 3(b) 
               Minute 
               209, 231, 247 
             
             
                 
                 
                 
               2.75 
             
             
                 
                 
               FIG. 3(c) 
               Minute 
               209, 231, 247 
             
             
                 
                 
                 
               4.0 
             
             
                 
               Chromatograph 
               FIG. 4 
               Minutes 
               209 
             
             
                 
                 
                 
               0~5.5 
               (reactant-related), 
             
             
                 
                 
                 
                 
               247 
             
             
                 
                 
                 
                 
               (product-related) 
             
             
                 
                 
             
          
         
       
     
   
   As shown in  FIGS. 3(   a )˜ 3 ( c ), the signal with m/z=209 that corresponds to “chalcone+H + ” comes out as the strongest in intensity, and as the reaction progresses over time, the intensity of the signal with m/z=247 that corresponds to “product+Na + ” surpasses that of “chalcone+H + ”. This verifies the decline of the reactant, i.e., chalcone, and the growth of the product as the reaction progresses over time. 
   As shown in  FIG. 4 , it is obvious that at minute 1.8, the reactant signal with m/z=209 abruptly increases in intensity. It is assumed that this is due to a higher level of the liquid sample surface relative to the electrospray unit  14  (i.e., closer to the charge-laden liquid drops) attained by the addition of the H 2 O 2  aqueous solution thereto, resulting in the increased ionizing efficiency of the analytes desorbed from behind the liquid sample surface. 
   &lt;Exemplary Method 3&gt; Monitoring Reaction between 4-aminophenol and acetic anhydride 
   Prediction 
   The mechanisms of the reaction between 4-aminophenol and acetic anhydride are illustrated in the following figures: 
   
     
       
       
           
           
       
     
   
   where the molecular weight of 4-aminophenol (hereinbelow referred to as reactant 1) is 109.05, the molecular weight of acetic anhydride (hereinbelow referred to as reactant 2) is 102.03, the molecular weight of the intermediate product is 211.08, and the molecular weight of the final product is 151.06. In addition, it was predicted that the signals (represented by corresponding m/z values) corresponding to the ionized analytes tabulated in Table 4 would be detected, where the predicted m/z values are in whole numbers. 
                               TABLE 4               m/z       m/z           value   Ionized Analyte   value   Ionized Analyte                  110   reactant 1 + H +     125   reactant 2 + Na +         120   reactant 2 + H 2 O   152   final product + H +         222   dimer of reactant 2 +   212   intermediate           H 2 O       product + H +         227   dimer of reactant 2 +           Na +                      
Procedure
 
   In exemplary method 3, 30 μl of ethanol solution containing 4-aminophenol with 1.0*10 −2 M concentration and carbon powders with 8 mg/ml concentration was used as the liquid sample, and was disposed in an open reaction cell. Starting from minute 0.2, the liquid sample surface (i.e., level of the liquid sample in the open reaction cell) was irradiated by a laser beam. At minute 0.5, 30 μl of acetic anhydride was added into the liquid sample. The mass spectroscopic analysis was conducted for a total of 2.3 minutes. 
   Results 
   Three average mass spectra were chosen for illustration purposes and are illustrated in  FIGS. 5(   a )˜ 5 ( c ), and seven chromatograph shown in  FIGS. 6(   a )˜ 6 ( g ) were constructed for seven representative m/z signals selected. Information related to the results of exemplary method 3 is tabulated in Table 5 below. 
   
     
       
         
             
             
             
             
             
           
             
                 
               TABLE 5 
             
             
                 
                 
             
             
                 
                 
               Figure 
                 
                 
             
             
                 
               Result Type 
               No. 
               Time 
               m/z signal 
             
             
                 
                 
             
           
          
             
                 
               Mass Spectra 
               FIG. 5(a) 
               Minute 
               110.1, 142.1 
             
             
                 
                 
                 
               0.2~0.5 
               (reactant 
             
             
                 
                 
                 
                 
               1-related) 
             
             
                 
                 
               FIG. 5(b) 
               Minute 
               110.1 (reactant 
             
             
                 
                 
                 
               0.5~1.4 
               1-related), 120.1, 
             
             
                 
                 
                 
               (reactant 
               125.1, 222.2, 
             
             
                 
                 
                 
               2 added) 
               227.1 (all reactant 
             
             
                 
                 
               FIG. 5(c) 
               Minute 
               2-related), 212.2 
             
             
                 
                 
                 
               1.4~2.0 
               (intermediate 
             
             
                 
                 
                 
                 
               product-related), 
             
             
                 
                 
                 
                 
               152.1 (final 
             
             
                 
                 
                 
                 
               product-related) 
             
             
                 
               Chromatograph 
               FIG. 6(a) 
               Minute 
               110.1 (reactant 
             
             
                 
                 
                 
                 0~2.2 
               1-related) 
             
             
                 
                 
               FIG. 6(b) 
                 
               120.1 (reactant 
             
             
                 
                 
                 
                 
               2-related) 
             
             
                 
                 
               FIG. 6(c) 
                 
               125.1 (reactant 
             
             
                 
                 
                 
                 
               2-related) 
             
             
                 
                 
               FIG. 6(d) 
                 
               222.2 (reactant 
             
             
                 
                 
                 
                 
               2-related) 
             
             
                 
                 
               FIG. 6(e) 
                 
               227.1 (reactant 
             
             
                 
                 
                 
                 
               2-related) 
             
             
                 
                 
               FIG. 6(f) 
                 
               212.2 (intermediate 
             
             
                 
                 
                 
                 
               product-related) 
             
             
                 
                 
               FIG. 6(g) 
                 
               152.1 (final 
             
             
                 
                 
                 
                 
               product-related) 
             
             
                 
                 
             
          
         
       
     
   
   As shown in  FIGS. 5(   a )˜ 5 ( c ), the signal with m/z=110.1 that corresponds to “reactant 1+H + ” comes out as the strongest in intensity, and as the reaction progresses over time, the intensities of the signals originated from reactant 2 or various products surpass that of “reactant 1+H + ”. This verifies the decline of reactant 1 and the growth of the products. In  FIG. 5(   c ), i.e., during minute 1.4˜2.0, the intensities of the reactant 2-related signals with m/z values of 120.1, 125.1, 222.2, 227.1 are obviously higher than those of the reactant 1-related signals and the product-related signals. It is assumed that this is due to the acidic nature of reactant 2 and the fact that the exemplary method 3 was conducted under the “positive ion mode”, resulting in a higher ionization rate of reactant 2. As shown in  FIG. 6(   f ) and  FIG. 6(   g ), the intermediate product and the final product were produced starting at minute 0.5 upon the addition of reactant 2. It is apparent from  FIG. 6(   f ) that the intensity of the intermediate product-related signal abruptly increases at minute 0.5, and gradually decreases over time, thereby verifying the growth and decline of an intermediate product in a chemical reaction. 
   &lt;Comparative Case 1&gt; Conducting Mass Spectroscopic Analysis on acetic anhydride Using Electrospray Ionization (ESI) Methods 
   ESI Analysis was conducted on acetic anhydride (i.e., reactant 2 in exemplary method 3), and the obtained mass spectrum is illustrated in  FIG. 7 . The main ion peaks observed in  FIG. 7  respectively have m/z values of 120.1, 125.0, 222.1 and 227.1, which are also observed in  FIGS. 5(   b )˜ 5 ( c ). This verifies the credibility of the reactant 2-related signals detected in exemplary method 3. 
   With reference to the results described hereinabove with respect to the exemplary methods, it is evident that the mass spectroscopic reaction-monitoring method according to the present invention has the ability to conduct instantaneous analysis on a liquid sample, and to monitor an ongoing reaction in the liquid sample by observing the differences among the results obtained at successive points of time. In addition, the present invention is applicable to various kinds of liquid samples, including aqueous, organic, biochemical solutions, etc. Moreover, the mass spectroscopic reaction-monitoring method is capable of eliminating the shortcomings presented in the prior art by using EST mass spectrometry. 
   While the present invention has been described in connection with what is considered the most practical and preferred embodiment, it is understood that this invention is not limited to the disclosed embodiment but is intended to cover various arrangements included within the spirit and scope of the broadest interpretation so as to encompass all such modifications and equivalent arrangements.