Use of the theoretical collision cross section ("CCS") in sample identification

Abstract

Methods for mass spectrometry, which include the following: Predictions of one or more first reaction products that may result from subjecting an analyte to one or more reactions of interest, Calculating one or more first masses or mass / charge ratios and one or more first ion mobility values, collision cross sections or interaction cross sections of at least some first reaction products that can be generated under first conditions using the one or more first reaction products, Calculating one or more second masses or mass / charge ratios and one or more second ion mobility values, collision cross sections or interaction cross sections of at least some second reaction products that can be generated under two different conditions using one or more first reaction products, Generating third ions from a sample and experimentally determining one or more third masses or mass / charge ratios and one or more third ion mobility values, collision cross sections or interaction cross sections of at least some of the third ions under the first conditions, Generating fourth ions from the sample and experimentally determining one or more fourth masses or mass / charge ratios and one or more fourth ion mobility values, collision cross sections or interaction cross sections of at least some of the fourth ions under the second conditions, and Comparing the first and second masses or mass / charge ratios with the third and fourth masses or mass / charge ratios and / or comparing the first and second ion mobility values, collision cross sections or interaction cross sections with the third and fourth ion mobility values, collision cross sections or interaction cross sections to confirm the presence and / or absence of one or more first reaction products in the sample.

Description

AREA OF INVENTION

[0001] The present invention relates generally to mass spectrometry and in particular to methods for mass spectrometry and mass spectrometers. BACKGROUND

[0002] In metabolite identification (“ID”) experiments, a drug can be administered to an organism and, at a later time, a biological sample can be taken and analyzed for the presence and quantity of metabolites of that drug.

[0003] The biotransformation of target drugs in the body is part of the natural elimination process, which generally produces inactive metabolites during detoxification. However, biotransformation can sometimes lead to metabolites that are themselves toxic (bioactivation). The pharmaceutical industry is required by regulatory authorities to identify all metabolites.

[0004] The identification of metabolites is therefore an important and often time-consuming step in the development of pharmaceutical compounds.

[0005] It is common to predict typical metabolomic products of pharmaceutical compounds in a given biological system using in silico calculations and biochemical knowledge.

[0006] For example, metabolites from the oxidation, reduction, hydrolysis, cyclization, and decyclization of a parent drug can be predicted given a drug structure and knowledge of the biological system. Conjugation, such as methylation, sulfonation, acetylation, glucuronidation, glutathione conjugation, and glycine conjugation, is also commonly expected, and potential metabolites can be hypothetically predicted.

[0007] In known mass spectrometric metabolite identification experiments, the structure, elemental composition, and therefore the exact mass of each expected or proposed metabolite are used to identify these compounds if they exist in the analyzed biological sample.

[0008] However, it may not be possible to identify the isomeric forms of a particular metabolite based solely on the mass-to-charge ratio. Due to the complexity of the biological sample matrix, mass interferences can also lead to misidentification.

[0009] WO 2011 / 128703 A1 (Micromass) discloses a method for identifying a sample compound, wherein a theoretical collision cross-sectional area value is calculated for each of at least two known compounds, a collision cross-sectional area value for the sample compound is measured, and the measured value is compared with the theoretical values ​​to identify which of the at least two known compounds the sample compound is most similar to.

[0010] US 2013 / 0218478 A1 discloses a method and apparatus for identifying and / or characterizing a sample that may contain two or more isomeric or isobaric compounds, such as hydroxylated metabolites. The method includes modeling a multitude of possible structures for each of the two or more known isomeric or isobaric compounds, calculating a theoretical collision cross-section for each modeled structure, and averaging the calculated values ​​for each known compound to obtain a theoretical collision cross-section value for each known compound. A traveling wave ion mobility cell is used to measure a collision cross-section value for the sample compound, and the measured value is then compared with the theoretical values ​​to determine which of the two or more known compounds the sample compound is most similar to.

[0011] It is desirable to provide an improved method for mass spectrometry. SUMMARY

[0012] According to one aspect, a mass spectrometry procedure is provided which includes the following: Predictions of one or more first reaction products that may result from subjecting an analyte to one or more reactions of interest, Calculating one or more first masses or mass / charge ratios and one or more first ion mobility values, collision cross sections or interaction cross sections of at least some first reaction products that can be generated under first conditions using the one or more first reaction products, Calculating one or more second masses or mass / charge ratios and one or more second ion mobility values, collision cross sections or interaction cross sections of at least some second reaction products that can be generated under two different conditions using one or more first reaction products, Generating third ions from a sample and experimentally determining one or more third masses or mass / charge ratios and one or more third ion mobility values, collision cross sections or interaction cross sections of at least some of the third ions under the first conditions, Generating fourth ions from the sample and experimentally determining one or more fourth masses or mass / charge ratios and one or more fourth ion mobility values, collision cross sections or interaction cross sections of at least some of the fourth ions under the second conditions, and Comparing the first and second masses or mass / charge ratios with the third and fourth masses or mass / charge ratios and / or comparing the first and second ion mobility values, collision cross sections or interaction cross sections with the third and fourth ion mobility values, collision cross sections or interaction cross sections to confirm the presence and / or absence of one or more first reaction products in the sample.

[0013] One embodiment relates to the use of the theoretically calculated collision cross section (“CCS”) of usual expected reaction products in a reaction of interest in the form of metabolites to confirm the presence of a particular reaction product in the form of a particular metabolite or metabolite omer.

[0014] The embodiment increases the specificity of compound identification analysis in the manner of metabolite identification analysis, in order to reduce the probability of misidentification of reaction products or metabolites.

[0015] Although it is known to use ion mobility in combination with mass spectrometry to increase the peak capacity of analytical instruments and to eliminate some interferences that would otherwise lead to misidentification of reaction products or metabolites, conventional approaches generally do not determine the collision cross section of the reaction products or metabolites and therefore do not use it as an additional identification criterion.

[0016] This embodiment enables the identification and quantification of the isomeric forms of each reaction product or metabolite, which cannot be determined solely from the mass-to-charge ratio. Although isomers can be separated by ion mobility spectrometry (IMS), the presence of a particular isomer cannot be confirmed without theoretically calculating its collision cross-section. The only alternative would be to synthesize every possible reaction product, metabolite, or isomer and generate experimental results that could be compared with those obtained from the biological sample. This is practically impossible in most laboratories.

[0017] Furthermore, according to one embodiment and in contrast, for example, to WO 2011 / 128703 A1 (Micromass), ions are generated from the analyte under first conditions and second different conditions, and / or ions generated from the sample are analyzed under first experimental conditions and second different experimental conditions. The determined values ​​of mass, mass-to-charge ratio, ion mobility, collision cross-section, and / or interaction cross-section are compared to confirm the presence and / or absence of one or more reaction products of interest in the sample.

[0018] Accordingly, this embodiment allows for more specific confirmation of the presence of reaction products of interest, reduces false positive identification, and increases the accuracy of identification.

[0019] It will therefore be understandable that an improved method for mass spectrometry will be provided.

[0020] According to one embodiment, the method further comprises the following: Determining a first mass or mass / charge ratio difference between one or more first masses or mass / charge ratios and one or more second masses or mass / charge ratios, Determining a second mass or mass / load ratio difference between one or more third masses or mass / load ratios and one or more fourth masses or mass / load ratios, Comparing the first and second mass or mass / charge ratio difference to confirm the presence and / or absence of one or more first reaction products in the sample.

[0021] According to one embodiment, the method further comprises the following: Determining a first ion mobility, collision cross-section or interaction cross-section difference between one or more first ion mobility values, collision cross-sections or interaction cross-sections and one or more second ion mobility values, collision cross-sections or interaction cross-sections, Determining a second ion mobility, collision cross-section or interaction cross-section difference between one or more third ion mobility values, collision cross-sections or interaction cross-sections and one or more fourth ion mobility values, collision cross-sections or interaction cross-sections, Comparing the first and second ion mobility, collision cross-section or interaction cross-section difference to confirm the presence and / or absence of one or more first reaction products in the sample.

[0022] According to one embodiment, the method comprises the following: Calculating one or more of the first additional physicochemical or other properties of at least some of the first ions, Calculating one or more additional physicochemical or other properties of at least some of the second ions, experimental determination of one or more third additional physicochemical or other properties of at least some of the third ions, experimental determination of one or more fourth additional physicochemical or other properties of at least some of the fourth ions and Comparing the first and second additional physicochemical or other properties with the third and fourth additional physicochemical or other properties to confirm the presence and / or absence of one or more first reaction products in the sample.

[0023] According to one embodiment, the method further comprises the following: Determining a first additional physicochemical or other property difference between one or more first additional physicochemical or other properties and one or more second additional physicochemical or other properties, Determining a second additional physicochemical or other property difference between one or more third additional physicochemical or other properties and one or more fourth additional physicochemical or other properties, Comparing the first and second additional physicochemical or other property differences to confirm the presence and / or absence of one or more first reaction products in the sample.

[0024] According to one embodiment, the one or more additional physicochemical or other properties include the isotope ratio pattern, peak shape, peak width, peak slope, number of peaks and / or peak kurtosis.

[0025] According to one embodiment, the one or more reactions comprise one or more biological and / or chemical reactions.

[0026] According to one embodiment, the one or more reactions comprise one or more biotransformation and / or metabolomic reactions.

[0027] According to one embodiment, the one or more reactions comprise one or more of the following: (i) oxidation, (ii) reduction, (iii) hydrolysis, (iv) cyclization, (v) decyclization, (vi) conjugation, (vii) methylation, (viii) sulfonation, (ix) acetylation, (x) glucuronidation, (xi) glutathione conjugation and / or (xii) glycine conjugation.

[0028] According to one embodiment, the one or more first reaction products comprise one or more metabolites of the analyte.

[0029] According to one embodiment, the analyte comprises a pharmaceutical compound.

[0030] According to one embodiment, the one or more reactions comprise one or more organic or catalytic reactions.

[0031] According to one embodiment, the sample comprises one or more secondary reaction products resulting from subjecting an analyte to one or more reactions.

[0032] One embodiment is provided, wherein: the reaction products include ions and / or precursor ions and / or isomeric ions and / or fragment, product or adduction ions and / or conformer ions, which can be generated directly or indirectly from one or more of the first reaction products, and / or The generated ions include ions and / or precursor ions and / or isomeric ions and / or fragment, product or adduction ions and / or conformer ions, which are generated directly or indirectly from the sample.

[0033] One embodiment is provided, wherein: the first conditions include one or more initial pre-ionization, ionization or post-ionization conditions and The second set of different conditions includes one or more second pre-ionization, ionization, or post-ionization conditions.

[0034] According to one embodiment, the first or second conditions are selected from the group consisting of the following: (i) the composition and / or concentration of a salt, doping agent, derivatizing agent, reagent, displacement reagent, overloading reagent or charge-reducing reagent added to a liquid sample prior to ionization, (ii) the composition and / or concentration of a neutral gas, doping gas, derivatizing agent gas, reagent gas, displacement reagent gas, overload reagent gas or charge-reducing reagent gas added to a gaseous or vapor phase sample prior to ionization, (iii) the composition and / or concentration of a neutral gas, reactive gas, doping gas, derivatizing agent, reagent gas, displacement reagent gas, overload reagent gas or charge-reducing reagent gas designed to interact or react with analyte ions after ionization, and (iv) the composition and / or concentration of doping ions, derivatizing ions, reagents, overloading reagents or charge-reducing reagents designed to interact or react with analyte ions after ionization.

[0035] According to one embodiment, the first or second conditions are selected from the group consisting of: (i) a condition that affects a charge state of the analytes, (ii) a condition that affects an energy level of the analytes, (iii) a condition that affects the kinetic energy of the analytes, (iv) a condition that affects the activation energy of the analytes, and (v) a condition that affects the conformational shape or nature of the analytes.

[0036] According to one embodiment, the first or second conditions are selected from the group consisting of: (i) an ionization condition of an ion source, (ii) the type of ion source used for ionizing a sample, (iii) the voltage setting of an ion source, (iv) the ionization polarity of ions generated by an ion source, (v) the flow rate of the sample supplied to an ion source, (vi) one or more liquid chromatography conditions of a liquid chromatography system, (vii) the composition of a liquid chromatography solution or liquid chromatography solvent, and (viii) the liquid chromatography flow rate.

[0037] According to one embodiment, the first or second conditions are selected from the group consisting of: (i) subjecting ions to hydrogen-deuterium exchange, (ii) one or more hydrogen-deuterium exchange conditions, (iii) subjecting ions to activation, photoactivation, dissociation or photodissociation, (iv) one or more dissociation, photodissociation, activation and / or photoactivation conditions, (v) subjecting ions to heating or RF heating, (vi) one or more heating or RF heating conditions, (vii) exposing ions to electromagnetic radiation, microwave radiation or laser radiation, (viii) one or more conditions of electromagnetic radiation, microwave radiation or laser radiation, (ix) subjecting ions to fragmentation or reaction and (x) one or more fragmentation or reaction conditions.

[0038] According to one embodiment, in the step of experimentally determining the one or more masses or mass / charge ratios, the ions are analyzed by mass.

[0039] According to one embodiment, in the step of experimentally determining one or more ion mobility values, collision cross sections or interaction cross sections, at least some of the ions are passed through an ion mobility separation device.

[0040] According to one embodiment, the first or second experimental conditions are selected from the group consisting of: (i) a voltage applied to an ion-optical component, (ii) a path traveled by analyte ions through a section of the mass spectrometer, (iii) the transit time of analyte ions through a section of the mass spectrometer, (iv) one or more pressures inside the mass spectrometer, (v) one or more temperatures inside the mass spectrometer, (vi) the composition of a gas inside the mass spectrometer, and (vii) the strength of an electric field inside the mass spectrometer.

[0041] According to one embodiment, the first or second experimental conditions are selected from the group consisting of: (i) the composition of an ion mobility separation or buffer gas, (ii) the composition of one or more additives, dopants, and / or reagents added to an ion mobility separation or buffer gas, (iii) the flow rate and / or flow direction of an ion mobility separation or buffer gas, (iv) the pressure or number density of an ion mobility separation or buffer gas, (v) the temperature inside an ion mobility separation device, (vi) the strength of an electric field inside an ion mobility separation device, (vii) the path length traveled by ions inside an ion mobility separation device.(viii) the residence time of ions within an ion mobility separation device, (ix) the initial width of an ion pulse introduced into an ion mobility separation device, and (x) the velocity, amplitude, or repetition pattern of a traveling DC voltage wave within an ion mobility separation device.

[0042] According to one embodiment, in the step of calculating the one or more masses or mass / charge ratios of the reaction products, the elemental composition and / or the exact mass of one or more of the reaction products is calculated.

[0043] According to one embodiment, the following is performed in the step of calculating one or more ion mobility values, collision cross-sections or interaction cross-sections of the reaction products: Calculating a three-dimensional structure of one or more of the reaction products and Calculating one or more of the ion mobility values, collision cross sections or interaction cross sections using the three-dimensional structure. According to one embodiment, in the step of calculating one or more ion mobility values, collision cross sections or interaction cross sections of ions, the effects of electronic interactions of the ions with a polar or polarizable ion mobility separation or buffer gas are calculated.

[0044] According to one aspect, a mass spectrometer is provided, which includes the following: a control system that is set up and designed to perform the following: (i) Predictions of one or more first reaction products that may result from subjecting an analyte to one or more reactions of interest, (ii) Calculating one or more first masses or mass / charge ratios and one or more first ion mobility values, collision cross sections or interaction cross sections of at least some first reaction productions that can be generated under first conditions using the one or more first reaction products, and (iii) Calculating one or more second masses or mass / charge ratios and one or more second ion mobility values, collision cross sections or interaction cross sections of at least some second reaction productions that can be generated under two different conditions using one or more first reaction products, and a device that is set up and designed to perform the following: (i) Generating third ions from a sample and experimentally determining one or more third masses or mass / charge ratios and one or more third ion mobility values, collision cross sections or interaction cross sections of at least some of the third ions under the first conditions, (ii) Generating fourth ions from the sample and experimentally determining one or more fourth masses or mass / charge ratios and one or more fourth ion mobility values, collision cross sections or interaction cross sections of at least some of the fourth ions under the second conditions, wherein the control system is further set up and designed to perform the following: comparing the first and second masses or mass / charge ratios with the third and fourth masses or mass / charge ratios and / or comparing the first and second ion mobility values, collision cross sections or interaction cross sections with the third and fourth ion mobility values, collision cross sections or interaction cross sections to confirm the presence and / or absence of one or more first reaction products in the sample. BRIEF DESCRIPTION OF THE DRAWING

[0045] Various embodiments are now described only as examples and with reference to the attached drawing, wherein: Fig. Figure 1 shows a flowchart of one embodiment. DETAILED DESCRIPTION

[0046] One embodiment relates to a method in which one or more first reaction products that may result from subjecting an analyte to one or more reactions of interest are predicted. Given a proposed compound identity, probable three-dimensional gas-phase structures for expected gas-phase ions can be calculated. Molecular mechanics and quantum chemistry modeling approaches can be used to achieve this. Commercially available software such as Gaussian (www.gaussian.com) can be used for this purpose.

[0047] Once structures have been proposed, the collision cross-section can be calculated, for example, using software such as MobCal from Indiana University. See AA Shvartsburg and MF Jarrold, “An Exact Hard Spheres Scattering Model for the Mobilities of Polyatomic Ions”, Chem. Phys. Lett. 1996, 261, 86–91.

[0048] The effect of long-range electronic interactions between ions and polar or polarizable molecules in an ion mobility drift medium (buffer gas) on the apparent collision cross-section can be taken into account within these calculations, for example to ultimately calculate an interaction cross-section.

[0049] According to one embodiment, one or more mass or mass / charge ratios and one or more ion mobility values, collision cross sections or interaction cross sections (i.e., drift time, ion mobility drift time, ion mobility or differential ion mobility), optionally together with additional physicochemical or other properties of the ions (such as the isotope ratio pattern, mass / charge ratio or ion mobility peak shape, mass / charge ratio or ion mobility peak width, slope of a mass / charge ratio or ion mobility peak, number of mass / charge ratio or ion mobility peaks and / or kurtosis of a mass / charge ratio or ion mobility peak) can be calculated for reaction productions taking into account two or more different analytical conditions.The analytical conditions may include various pre-ionization, ionization and / or post-ionization conditions and / or various experimental or measurement conditions.

[0050] According to one embodiment, ions are generated from a sample and analyzed using at least two selected analytical conditions, and mass / charge ratios, collision cross-sections or interaction cross-sections, as well as additional physicochemical or other properties of the ions can be determined. The experimentally determined values ​​and the calculated values ​​can be compared to confirm the presence and / or absence of one or more reaction products of interest in the sample.

[0051] Fig. Figure 1 shows a flowchart of one embodiment.

[0052] In a first step, potential metabolites are identified. Common biotransformations are well documented, and a list of proposed target metabolites is generated. This list includes, for example, isomeric forms of a given predicted metabolite and characteristic fragment ions.

[0053] To make the identification more specific, the sample is analyzed more than once under different solution phase and / or gas phase conditions (i.e., under different pre-ionization, ionization and / or post-ionization conditions) to change the nature of the metabolites, and / or under different (experimental or measurement) conditions under which ions are analyzed.

[0054] Depending, for example, on the facilities within a particular laboratory and the flexibility of the ion source design and inlet, a range of different conditions, including the following preferred conditions, can be investigated. The following list of conditions is not exhaustive. a. Various solution and gas-phase chemistries designed to change the nature of the gas-phase ion generated from the analytes

[0055] According to embodiments in which electrospray ionization is used, adding a salt solution to the analyte current prior to ionization makes it possible to control the charge carrier associated with the analyte ion or to generate known analyte ion adducts.

[0056] For example, in a positive ion electrospray, the addition of formic acid leads to the predominant formation of protonated ions [M+nH] n+ , where n is the number of charges. The addition of sodium chloride (NaCl) predominantly leads to sundated ions [M+nNa] . n+ .

[0057] In general, adducts of the form [M+nY] n+ formed by adding a suitable soluble ionic salt, where Y = Na, K, Li, H, NH3 etc.

[0058] According to embodiments that use a negative ion mode, adducts can be formed in a similar manner. For example, adducts can be formed by adding soluble salts to [M+X] - to generate, where X = F, Cl, Br, I, NO3, etc. In a negative ion mode, [M-nH] -n -ions can be generated by adding, for example, ammonium hydroxide.

[0059] Many other embodiments for different solution-phase chemistries are being considered. According to some embodiments, more complex derivatizing agents can be used based on knowledge of the chemical reactivity of the analytes.

[0060] For example, according to embodiments employing gas chromatography-mass spectrometry (“GC”), acylation, silylation, alkylation, and esterification can be used, wherein these are common derivatization procedures employing commercially available derivatization reagents. According to embodiments employing liquid chromatography-mass spectrometry (“LC”), Schiff base forming reagents, primary and secondary amines, and chromopores for fluorometric detection can be used, wherein these are commonly available reagents. According to these embodiments, derivatization can be performed offline prior to separation.

[0061] According to some embodiments, the derivatization of the ionized analyte can be carried out either prior to ion mobility separation or within an ion mobility separation device in the gas phase. This can be achieved, for example, by adding reactive neutral or charged species to an RF-constrained reaction cell that can be maintained at a pressure below one atmosphere.

[0062] According to one embodiment, an overcharging and / or a charge reduction can be used additionally or alternatively to predictably change the nature of the ions formed from the analyte.

[0063] According to one embodiment, the charge state of an ion can be manipulated either in the solution phase prior to ionization and / or in the gas phase, for example by overcharging and / or charge reduction techniques. For example, in embodiments using electrospray ionization (“ESI”), the addition of mNBA (“m-nitrobenzyl alcohol”), tetramethylenesulfone (“sulfolane”), or dimethyl sulfoxide (“DMSO”) can lead to an increase in the intensity of higher charged ions.

[0064] According to one embodiment, a reduction in the charge of analytes ions can be achieved, for example, by using an atmospheric pressure or sub-atmospheric pressure neutralization chamber, wherein a corona discharge or ultraviolet (UV) radiation or another energy source is used to generate reagents that can be caused to interact with the analytes ions, resulting in a reduction of the analytes' charge state. According to other embodiments, base compounds, such as triethylammonium bicarbonate or imidazole, can be added to solution prior to ionization, and / or base molecules can be introduced into the gas phase, for example, at atmospheric pressure or at a pressure below atmosphere, to interact with the analytes ions.

[0065] According to embodiments in which different ionization techniques, such as atmospheric pressure chemical ionization (“APCl”), sub-atmospheric pressure chemical ionization, matrix-assisted laser desorption ionization (“MALDI”), etc., are used, other reagents or methods for manipulating the chemical nature of the analyte ions may be used.

[0066] In all these embodiments, the nature of the generated metabolites is predictable. According to one embodiment, a single analyte can be manipulated to generate several different types of gas-phase ions, depending on the available solution-phase or gas-phase chemistry.

[0067] According to one embodiment, a solution- or gas-phase hydrogen-deuterium exchange (“HDX”) can be used. After hydrogen-deuterium exchange, the mass-to-charge ratio of a metabolite and its isotopic ratios reflect the number of exchangeable hydrogen atoms on the molecule. This can provide additional information and confirm or refute the identification of possible metabolites in the sample. According to another embodiment, other reactions can be considered and used.

[0068] According to one embodiment, a change in the charge carrier or charge state of ions can lead to significant changes in the gas-phase structure and thus in the collision cross-section, thereby causing compound-specific shifts in the ion mobility drift time and in the measured cross-section. According to another embodiment, the electronic structure of a gas-phase ion can be changed by altering the nature of the long-range electronic interactions between the ion and the polarizable or polar drift medium.

[0069] According to the preferred embodiment, a number of different alternative experiments can be considered, leading to a number of different forms of the same metabolite. The expected or predicted metabolite species can be theoretically modeled, and the theoretical ion mobility behavior can be investigated to confirm the presence or identity of this metabolite. b. Different IMS drift gas compositions

[0070] The choice of ion mobility separation drift medium can dramatically influence the separation by ion mobility separation. According to one embodiment, a polarizable drift gas, buffer gas, or drift medium containing neutral gas-phase elements, for example, with a permanent dipole moment, can be used to selectively modify the separation of different analyte ions or isomer ions of an analyte. In another embodiment, a selection of different drift gas compositions is made, and the predicted effect on analyte ion mobility can be investigated by theoretical collision cross-section calculation. Depending on the electronic structure of the analyte ions, ion mobility separation may be better in one drift gas than in another. c. Different pre-IMS or intra-IMS activation energies

[0071] According to one embodiment, the activation of an ion can, for example, be used to increase the internal temperature by an arbitrary or known amount in order to cause unfolding or a transition between conformational states. According to various embodiments, laser or other energy sources can be used to excite ions before or during ion mobility separation.

[0072] To Fig. Returning to point 1, it should be noted that in a second step 2, the theoretical collision cross-sectional values ​​for each of the predicted metabolites can be calculated under each chosen pre-ionization, ionization, post-ionization and / or experimental condition considered in the first step 1.

[0073] In a third step, the sample, which is expected to contain at least some of the predicted metabolites, can be analyzed under one or more of the pre-ionization, ionization, post-ionization, and / or experimental or measurement (e.g., mass spectrometry ("MS") and / or ion mobility separation) conditions that have been selected. In this step, mass spectrometry data, tandem mass spectrometry ("MS-MS") data, or data involving multiple fragmentation or reaction stages ("MS-MS") can be analyzed. n “), are used, are recorded.

[0074] In a fourth step 4, the presence of the metabolites proposed in the first step 1 can be confirmed using a combination of both theoretical or predicted mass spectrometry (“MS”) data (e.g., mass or mass / charge ratio, isotopic pattern, isotopic ratio(s), peak shape, peak width, peak slope, number of peaks and / or peak kurtosis) and theoretical ion mobility data (e.g., ion mobility value, collision cross section and / or interaction cross section, peak shape, peak width, peak slope, number of peaks and / or peak kurtosis).

[0075] Metabolites can be identified by comparing measured mass-to-charge ratio values ​​with theoretical mass-to-charge ratio values ​​based on the predicted elemental composition. Additionally, theoretical and experimental isotope ratios and / or theoretical and experimental production mass-to-charge ratios and intensities can be compared.

[0076] According to the preferred embodiment, in addition to this mass-to-charge ratio information, theoretical collision cross-sectional data can be compared with experimental data to increase the degree of specificity and confidence in the identification. In this step, the isomeric form of the metabolite can also be identified. This information is not available using the mass-to-charge ratio alone.

[0077] As described above, to increase the specificity of the identification, the sample can be analyzed more than once under different conditions, such as solution phase or gas phase conditions, which can change the nature of the analyte ions formed, and / or under different experimental conditions, which can change the nature of the IMS separation.

[0078] According to one embodiment, the sample can be analyzed using at least two drift gas compositions, and the experimental and theoretical shifts of the collision cross-section can be compared. According to another embodiment, the use of a volatile polar dopant can yield drift time shifts that are highly specific for a given ionic structure. According to yet another embodiment, theoretical calculations can yield the magnitude of the expected shift in the apparent collision cross-section and be used to increase the specificity of metabolite identification.

[0079] Additionally or alternatively, the metabolite can be modified by altering its solution or gas-phase chemistry. For example, according to some embodiments, the structure or collision cross-section of a protonated ion can be very different from the structure and collision cross-section of a sodiated ion, or an ion with a different charge carrier, adduct, or derivatization modification. This modification can relate to the stereochemistry or electronic structure of the ion, and the experimentally observed shift can then be compared with the theoretically calculated shift to increase the specificity of metabolite identification.

[0080] According to one embodiment, a mass or mass / charge ratio difference and / or an ion mobility, collision cross-section, or interaction cross-section difference between ions generated or measured under different analytical conditions can be used to confirm the presence and / or absence of one or more reaction products of interest in the sample. In particular, according to one embodiment, the drift time difference of analyte ions caused to separate over time in the presence of buffer gases having different compositions is measured. This approach is particularly advantageous in that it is considerably more robust to changes in the conditions of the ion mobility separator than using an absolute drift time measurement. As a result, the approach according to this embodiment leads to a significant improvement in accuracy.

[0081] According to one embodiment, one or more additional physicochemical or other properties of the ions, such as the isotopic pattern, isotopic ratio or isotopic ratio pattern, peak shape, peak width, peak slope, number of peaks and / or peak kurtosis, for example a mass / charge ratio or ion mobility peak, can be calculated and experimentally determined and used to confirm the presence and / or absence of one or more reaction products of interest in the sample, for example by comparing the calculated and experimentally determined values.

[0082] For example, peak shape, width, and optionally the mass-to-charge ratio can be used to further increase the specificity of the identification. It may be the case, for instance, that a particular ion peak is fragmented into two or more ion peaks under different conditions, or that a shoulder or asymmetry, etc., might occur. This could be due, for example, to the existence of a protomer, etc., and / or the existence of a different structure (e.g., collision cross-section), and / or different electronic interactions with a (polar or polarizable) buffer gas.

[0083] According to one embodiment, properties of one or more experimentally determined ion peaks, such as width, slope, or kurtosis, can be determined, for example, using peak shape fitting. The corresponding theoretical values ​​can be calculated, for example, by factoring in the device parameters and conditions (resolution, etc.) to determine one or more expected peak shapes.

[0084] Although the foregoing embodiment has been described in relation to the identification of metabolites, the same procedure can be applied to different types of analysis, where the starting material is known and productions can be proposed.

[0085] For example, in organic synthesis or resynthesis, the starting materials and the target structure are known, and the structures of possible impurities formed in the reaction processes can be hypothetically assumed. According to one embodiment, the theoretical cross-sections of these possible products are calculated and used to confirm the presence of these compounds in the manner of the embodiments discussed above.

[0086] Another embodiment consists of catalytic processes that include known starting materials and proposed products, which can be confirmed by theoretical collision cross-sectional calculations in the manner of the embodiments discussed above. Although the present invention has been described with reference to preferred embodiments, those skilled in the art will understand that various modifications to the form and details can be made without deviating from the scope of protection of the invention as set out in the attached claims.

Claims

[1] Methods of mass spectrometry which include: Predictions of one or more first reaction products that may result from subjecting an analyte to one or more reactions of interest, Calculating one or more first masses or mass / charge ratios and one or more first ion mobility values, collision cross sections or interaction cross sections of at least some first reaction products that can be generated under first conditions using the one or more first reaction products, Calculating one or more second masses or mass / charge ratios and one or more second ion mobility values, collision cross sections or interaction cross sections of at least some second reaction products that can be generated under two different conditions using one or more first reaction products, Generating third ions from a sample and experimentally determining one or more third masses or mass / charge ratios and one or more third ion mobility values, collision cross sections or interaction cross sections of at least some of the third ions under the first conditions, Generating fourth ions from the sample and experimentally determining one or more fourth masses or mass / charge ratios and one or more fourth ion mobility values, collision cross sections or interaction cross sections of at least some of the fourth ions under the second conditions, and Comparing the first and second masses or mass / charge ratios with the third and fourth masses or mass / charge ratios and / or comparing the first and second ion mobility values, collision cross sections or interaction cross sections with the third and fourth ion mobility values, collision cross sections or interaction cross sections to confirm the presence and / or absence of one or more first reaction products in the sample. [2] The method of claim 1, which further comprises: Determining a first mass or mass / charge ratio difference between one or more first masses or mass / charge ratios and one or more second masses or mass / charge ratios, Determining a second mass or mass / load ratio difference between one or more third masses or mass / load ratios and one or more fourth masses or mass / load ratios, Comparing the first and second mass or mass / charge ratio difference to confirm the presence and / or absence of one or more first reaction products in the sample. [3] The method of claim 1 or 2, further comprising: Determining a first ion mobility, collision cross-section or interaction cross-section difference between one or more first ion mobility values, collision cross-sections or interaction cross-sections and one or more second ion mobility values, collision cross-sections or interaction cross-sections, Determining a second ion mobility, collision cross-section or interaction cross-section difference between one or more third ion mobility values, collision cross-sections or interaction cross-sections and one or more fourth ion mobility values, collision cross-sections or interaction cross-sections, Comparing the first and second ion mobility, collision cross-section or interaction cross-section difference to confirm the presence and / or absence of one or more first reaction products in the sample. [4] A method according to any of the preceding claims, wherein the method comprises: Calculating one or more of the first additional physicochemical or other properties of at least some of the first ions, Calculating one or more additional physicochemical or other properties of at least some of the second ions, experimental determination of one or more third additional physicochemical or other properties of at least some of the third ions, experimental determination of one or more fourth additional physicochemical or other properties of at least some of the fourth ions and Comparing the first and second additional physicochemical or other properties with the third and fourth additional physicochemical or other properties to confirm the presence and / or absence of one or more first reaction products in the sample. [5] The method of claim 4, which further comprises: Determining a first additional physicochemical or other property difference between one or more first additional physicochemical or other properties and one or more second additional physicochemical or other properties, Determining a second additional physicochemical or other property difference between one or more third additional physicochemical or other properties and one or more fourth additional physicochemical or other properties, Comparing the first and second additional physicochemical or other property differences to confirm the presence and / or absence of one or more first reaction products in the sample. [6] Method according to claim 4 or 5, wherein one or more additional physicochemical or other properties include the isotopic pattern, isotopic ratio, peak shape, peak width, peak slope, number of peaks and / or peak kurtosis. [7] Method according to any of the preceding claims, wherein the one or more reactions comprise one or more biological and / or chemical reactions. [8] Method according to any of the preceding claims, wherein the one or more reactions comprise one or more biotransformation and / or metabolomic reactions. [9] A method according to any one of the preceding claims, wherein the one or more reactions comprise one or more of the following: (i) oxidation, (ii) reduction, (iii) hydrolysis, (iv) cyclization, (v) decyclization, (vi) conjugation, (vii) methylation, (viii) sulfonation, (ix) acetylation, (x) glucuronidation, (xi) glutathione conjugation and / or (xii) glycine conjugation. [10] Method according to any of the preceding claims, wherein the one or more first reaction products comprise one or more metabolites of the analyte. [11] Method according to any of the preceding claims, wherein the analyte comprises a pharmaceutical compound. [12] Method according to any one of claims 1 to 7, wherein the one or more reactions comprise one or more organic or catalytic reactions. [13] Method according to any of the preceding claims, wherein the sample comprises one or more second reaction products resulting from subjecting an analyte to one or more reactions. [14] Method according to any one of the preceding claims, wherein: the reaction products include ions and / or precursor ions and / or isomeric ions and / or fragment, product or adduction ions and / or conformer ions, which can be generated directly or indirectly from one or more of the first reaction products, and / or The generated ions include ions and / or precursor ions and / or isomeric ions and / or fragment, product or adduction ions and / or conformer ions, which are generated directly or indirectly from the sample. [15] Method according to any one of the preceding claims, wherein: the first conditions include one or more initial pre-ionization, ionization or post-ionization conditions and The second set of different conditions includes one or more second pre-ionization, ionization, or post-ionization conditions. [16] A method according to any of the preceding claims, wherein the first or the second conditions are selected from the group consisting of: (i) the composition and / or concentration of a salt, doping agent, derivatizing agent, reagent, displacement reagent, overloading reagent or charge-reducing reagent added to a liquid sample prior to ionization, (ii) the composition and / or concentration of a neutral gas, doping gas, derivatizing agent gas, reagent gas, displacement reagent gas, overload reagent gas or charge-reducing reagent gas added to a gaseous or vapor phase sample prior to ionization, (iii) the composition and / or concentration of a neutral gas, reactive gas, doping gas, derivatizing agent, reagent gas, displacement reagent gas, overload reagent gas or charge-reducing reagent gas designed to interact or react with analyte ions after ionization, and (iv) the composition and / or concentration of doping ions, derivatizing ions, reagents, overloading reagents or charge-reducing reagents designed to interact or react with analyte ions after ionization. [17] Method according to any of the preceding claims, wherein the first or the second conditions are selected from the group consisting of: (i) a condition which affects a charge state of the analytes, (ii) a condition which affects an energy level of the analytes, (iii) a condition which affects the kinetic energy of the analytes, (iv) a condition which affects the activation energy of the analytes, and (v) a condition which affects the conformational shape or nature of the analytes. [18] Method according to any one of the preceding claims, wherein the first or the second conditions are selected from the group consisting of: (i) an ionization condition of an ion source, (ii) the type of ion source used for ionizing a sample, (iii) the voltage setting of an ion source, (iv) the ionization polarity of ions generated by an ion source, (v) the flow rate of the sample supplied to an ion source, (vi) one or more liquid chromatography conditions of a liquid chromatography system, (vii) the composition of a liquid chromatography solution or liquid chromatography solvent, and (viii) the liquid chromatography flow rate. [19] A method according to any one of the preceding claims, wherein the first or the second conditions are selected from the group consisting of: (i) subjecting ions to hydrogen-deuterium exchange, (ii) one or more hydrogen-deuterium exchange conditions, (iii) subjecting ions to activation, photoactivation, dissociation, or photodissociation, (iv) one or more dissociation, photodissociation, activation, and / or photoactivation conditions, (v) subjecting ions to heating or RF heating, (vi) one or more heating or RF heating conditions, (vii) exposing ions to electromagnetic radiation, microwave radiation, or laser radiation, (viii) one or more conditions of electromagnetic radiation, microwave radiation, or laser radiation, (ix) subjecting ions to fragmentation or reaction, and (x) one or more fragmentation or reaction conditions. [20] Method according to one of the preceding claims, wherein in the step of experimentally determining the one or more masses or mass / charge ratios the ions are mass-analyzed. [21] Method according to one of the preceding claims, wherein in the step of experimentally determining one or more ion mobility values, collision cross sections or interaction cross sections at least some of the ions are passed through an ion mobility separation device. [22] Method according to any of the preceding claims, wherein the first or the second experimental conditions are selected from the group consisting of: (i) a voltage applied to an ion-optical component, (ii) a path traveled by analyte ions through a section of the mass spectrometer, (iii) the transit time of analyte ions through a section of the mass spectrometer, (iv) one or more pressures inside the mass spectrometer, (v) one or more temperatures inside the mass spectrometer, (vi) the composition of a gas inside the mass spectrometer and (vii) the strength of an electric field inside the mass spectrometer. [23] Method according to any one of the preceding claims, wherein the first or the second experimental conditions are selected from the group consisting of: (i) the composition of an ion mobility separation or buffer gas, (ii) the composition of one or more additives, one or more dopants and / or one or more reagents added to an ion mobility separation or buffer gas, (iii) the flow rate and / or the flow direction of an ion mobility separation or buffer gas, (iv) the pressure or number density of an ion mobility separation or buffer gas, (v) the temperature inside an ion mobility separation device, (vi) the strength of an electric field inside an ion mobility separation device, (vii) the path length traveled by ions inside an ion mobility separation device, (viii) the residence time of ions inside an ion mobility separation device, (ix) the initial width of an ion pulse introduced into an ion mobility separation device, and (x) the velocity, amplitude, or repetition pattern of a traveling DC voltage wave inside an ion mobility separation device. [24] Method according to any of the preceding claims, wherein in the step of calculating the one or more masses or mass / charge ratios of the reaction products the elemental composition and / or the exact mass of one or more of the reaction products is calculated. [25] Method according to one of the preceding claims, wherein in the step of calculating one or more ion mobility values, collision cross sections or interaction cross sections of the reaction productions the following is carried out: Calculating a three-dimensional structure of one or more of the reaction products and Calculating one or more of the ion mobility values, collision cross sections or interaction cross sections using the three-dimensional structure. [26] Method according to one of the preceding claims, wherein in the step of calculating one or more ion mobility values, collision cross sections or interaction cross sections of ions the effects of electronic interactions of the ions with a polar or polarizable ion mobility separation or buffer gas are calculated. [27] Mass spectrometer, which includes the following: a control system that is set up and designed to perform the following: (i) Predictions of one or more first reaction products that may result from subjecting an analyte to one or more reactions of interest, (ii) Calculating one or more first masses or mass / charge ratios and one or more first ion mobility values, collision cross sections or interaction cross sections of at least some first reaction productions that can be generated under first conditions using the one or more first reaction products, and (iii) Calculating one or more second masses or mass / charge ratios and one or more second ion mobility values, collision cross sections or interaction cross sections of at least some second reaction productions that can be generated under two different conditions using one or more first reaction products, and a device that is set up and designed to perform the following: (i) Generating third ions from a sample and experimentally determining one or more third masses or mass / charge ratios and one or more third ion mobility values, collision cross sections or interaction cross sections of at least some of the third ions under the first conditions, (ii) Generating fourth ions from the sample and experimentally determining one or more fourth masses or mass / charge ratios and one or more fourth ion mobility values, collision cross sections or interaction cross sections of at least some of the fourth ions under the second conditions, wherein the control system is further set up and designed to perform the following: comparing the first and second masses or mass / charge ratios with the third and fourth masses or mass / charge ratios and / or comparing the first and second ion mobility values, collision cross sections or interaction cross sections with the third and fourth ion mobility values, collision cross sections or interaction cross sections to confirm the presence and / or absence of one or more first reaction products in the sample.

Images