Method for identification of the elemental composition of species of molecules

Abstract

Methods of identification of at least one most likely elemental composition of at least one species of molecules contained in a sample and/or originated from a sample by at least one ionisation process are provided. The method includes measuring a mass spectrum of the sample and may include reducing the measured mass spectrum to a neutral mass spectrum. The method further includes determining for a peak of interest a set of candidate species of molecules which have an expected peak with a peak position within a peak position tolerance range in the corresponding measured mass spectrum or neutral mass spectrum. An identification mass spectrum is identified for each candidate species and a range of peak positions is determined of all peaks of the identification mass spectrum. Two subscores of candidate species are determined by comparing the identification spectra with the measured or neutral mass spectrum and final scores are calculated from the subscores. An elemental composition of the candidate species is determined having calculated final scores of the highest values.

Claims

1. A method of identification of one or more most likely elemental compositions of at least one species of molecules M contained in a sample and/or originated from a sample by at least one ionization process comprising the following steps: (i) measuring a mass spectrum I.sub.meas(p) of the sample with a mass spectrometer or measuring a mass spectrum I.sub.meas(p) of the sample with a mass spectrometer and reducing the measured mass spectrum I.sub.meas(p) to a neutral mass spectrum I.sub.neut(p); (ii) determining for a peak of interest C.sub.int of the measured mass spectrum I.sub.meas(p) or neutral mass spectrum I.sub.neut(p) a set S.sub.cand of candidate species of molecules M.sub.cand that is defined according to an expectation regarding which kind of species of molecules can be present in the sample from a set S.sub.inv of species of molecules M.sub.inv which have an expected peak C.sub.ex,inv in a mass spectrum corresponding to the measured mass spectrum I.sub.meas(p) or the neutral mass spectrum I.sub.neut(p) with a peak position p.sub.ex,inv within a peak position tolerance range p.sub.tol assigned to the peak of interest C.sub.int in the corresponding measured mass spectrum I.sub.meas(p) or neutral mass spectrum I.sub.neut(p); (iii) determining for each candidate species M.sub.cand of the set S.sub.cand of candidate species of molecules M.sub.cand an identification mass spectrum I.sub.id,M_cand(p) showing at least a part of an isotope distribution of the molecule M.sub.cand corresponding to the measured mass spectrum I.sub.meas(p) or the neutral mass spectrum I.sub.neut(p) and determining a range of peak positions p comprising the peak positions P.sub.id,M_cand,i of all peaks C.sub.id,M_cand,i of the identification mass spectrum I.sub.id,M_cand(p) corresponding to the measured mass spectrum I.sub.meas(p) or the neutral mass spectrum I.sub.neut(p) of all candidate species M.sub.cand of the set S.sub.cand of candidate species of molecules M.sub.cand; (iv) comparing the measured mass spectrum I.sub.meas(p) or the neutral mass spectrum I.sub.neut(p) with each identification mass spectrum I.sub.id,M_cand(p) of the candidate species M.sub.cand of the set S.sub.cand of candidate species of molecules M.sub.cand corresponding to the measured mass spectrum I.sub.meas(p) or the neutral mass spectrum I.sub.neut(p) in the determined range of peak positions p with a first method, which is determining a first subscore s.sub.1,M_cand for each candidate species M.sub.cand, and with a second method, which is determining a second subscore s.sub.2,M_cand for each candidate species M.sub.cand, wherein the first subscore s.sub.1,M_cand is addressing all peaks C.sub.id,M_cand,i in the identification mass spectrum I.sub.id,M_cand(p) of the candidate species M.sub.cand, which are not identified in the measured mass spectrum I.sub.meas(p) or neutral mass spectrum I.sub.neut(p), and wherein the second subscore s.sub.2,M_cand is addressing all peaks C.sub.meas,i in the measured mass spectrum I.sub.meas(p) or all peaks C.sub.neut,i in the neutral mass spectrum I.sub.neut(p), which are not identified in the identification mass spectrum I.sub.id,M_cand(p) of the candidate species M.sub.cand and calculating for each candidate species M.sub.cand of the set S.sub.cand of candidate species of molecules M.sub.cand a final score fs.sub.M_cand from the subscores S.sub.i,M_cand or calculating for each candidate species M.sub.cand of the set S.sub.cand of candidate species of molecules M.sub.cand a final score fs.sub.M_cand from the subscores s.sub.i,M_cand, for which one of both of the subscores of the first method and the second method s.sub.1,M_cand and s.sub.1,M_cand are higher than an assigned threshold value s.sub.i,th,fs for calculating the final score fs.sub.M_cand; (v) determining one or more calculated final scores fs.sub.high, k having the highest values; and (vi) determining the elemental composition of the candidate species M.sub.cand,high_k of the set S.sub.cand of candidate species of molecules M.sub.cand which have the one or more calculated final scores fs.sub.high, k of the highest values by looking up the elemental composition of the candidate species M.sub.cand,high k.

2. The method of claim 1 wherein in step (iv) by the first method to compare the measured mass spectrum I.sub.meas(p) or the neutral mass spectrum I.sub.neut(p) with each identification mass spectrum I.sub.id,M_cand(p) of the candidate species M.sub.cand as subscore s.sub.1,M_cand a pattern spectral distance is calculated.

3. The method of claim 2 wherein by the second method to compare the measured mass spectrum I.sub.meas(p) or the neutral mass spectrum I.sub.neut(p) with each identification mass spectrum I.sub.id,M_cand(p) of the candidate species M.sub.cand as subscore s.sub.2,M_cand a measured mass spectrum coverage subscore is calculated.

4. The method of claim 1 wherein in step (i) a mass spectrum I.sub.meas(p) of the sample is measured with a mass spectrometer, wherein in step (ii) for a peak of interest C.sub.int of the measured mass spectrum I.sub.meas(p) a set S.sub.cand of candidate species of molecules M.sub.cand that is defined according to the expectation regarding which kind of species of molecules can be present in the sample from a set S.sub.inv of species of molecules M.sub.inv is determined which have an expected peak.sub.ex,inv in a mass spectrum corresponding to the measured mass spectrum I.sub.meas(p) with a peak position p.sub.ex,inv within a peak position tolerance range p.sub.tol of the peak of interest C.sub.int in the measured mass spectrum I.sub.meas(p); wherein in step (iii) for each candidate species M.sub.cand of the set S.sub.cand of candidate species of molecules M.sub.cand a theoretical mass spectrum I.sub.th,M_cand(p) showing at least a part of an isotope distribution of the molecule M.sub.cand corresponding to the measured mass spectrum I.sub.meas(p) is calculated and a range of peak positions p comprising the peak positions p.sub.th,i of all peaks C.sub.th,M_cand,i of the complete theoretical mass spectra I.sub.th,M_cand(p) corresponding to the measured mass spectrum I.sub.meas(p) of all candidate species M.sub.cand of the set S.sub.cand of candidate species of molecules M.sub.cand is determined, and wherein in step (iv) the measured mass spectrum I.sub.meas(p) is compared with each theoretical mass spectrum I.sub.th,M_cand(p) of the candidate species M.sub.cand of the set S.sub.cand of candidate species of molecules M.sub.cand corresponding to the measured mass spectrum I.sub.meas(p) in the determined range of peak positions p with a first method, which is determining a first subscore s.sub.1,M_cand for each candidate Species M.sub.cand, and with a second method, which is determining a second subscore s.sub.2,M_cand for each candidate Species M.sub.cand, wherein the first subscore s.sub.1 is addressing all peaks C.sub.th,M_cand,i in the theoretical mass spectrum I.sub.th,M_cand(p) of the candidate species M.sub.cand, which are not identified in the measured mass spectrum I.sub.meas(p), and wherein the second subscore s.sub.2 is addressing all peaks C.sub.meas,i in the measured mass spectrum I.sub.meas(p), which are not identified in the theoretical mass spectrum I.sub.th,M_cand(p) of the candidate species M.sub.cand.

5. The method of claim 4 wherein with the most likely elemental composition of at least one species of molecules M originated from the sample by at least one ionization process is identified and then the most likely elemental composition of a species of molecules M.sub.s contained in a sample is derived from the identified most likely elemental composition of each of the at least one species of molecules M originated from the sample by the at least one ionization process according to the at least one ionization process.

6. The method of claim 1 wherein in step (i) a mass spectrum I.sub.meas(p) of the sample is measured with a mass spectrometer and then the measured mass spectrum I.sub.meas(p) is reduced to a neutral mass spectrum I.sub.neut(p), wherein in step (ii) for a peak of interest C.sub.int of the neutral mass spectrum I.sub.neut(p) a set S.sub.cand of candidate species of molecules M.sub.cand that is defined according to the expectation regarding which kind of species of molecules can be present in the sample from a set S.sub.inv of species of molecules M.sub.inv is determined which have an expected peak.sub.ex,inv in a mass spectrum corresponding the neutral mass spectrum I.sub.neut(p), with a peak position p.sub.ex,inv within a peak position tolerance range p.sub.tol of the peak of interest C.sub.int in the neutral mass spectrum I.sub.neut(p), wherein in step (iii) for each candidate species M.sub.cand of the set S.sub.cand of candidate species of molecules M.sub.cand a complete theoretical mass spectrum I.sub.th,M_cand(p) showing at least a part of an isotope distribution of the molecule M.sub.cand corresponding to the neutral mass spectrum I.sub.neut(p) is calculated and a range of peak positions p comprising the peak positions p.sub.th,i of all peaks C.sub.th,M_cand,i of the complete theoretical mass spectra I.sub.th,M_cand(p) corresponding to the neutral mass spectrum I.sub.neut(p) of all candidate species M.sub.cand of the set S.sub.cand of candidate species of molecules M.sub.cand is determined and wherein in step (iv) the neutral mass spectrum I.sub.neut(p) is compared with each theoretical mass spectrum I.sub.th,M_cand(p) of the candidate species M.sub.cand of the set S.sub.cand of candidate species of molecules M.sub.cand corresponding to the neutral mass spectrum I.sub.neut(p) in the determined range of peak positions p with a first method, which is determining a first subscore s.sub.1,M_cand for each candidate species M.sub.cand, and with a second method, which is determining a second subscore s.sub.2,M_cand for each candidate species M.sub.cand, wherein the first subscore s.sub.1 is addressing all peaks C.sub.th,M_cand,i in the theoretical mass spectrum I.sub.th,M_cand(p) of the candidate species M.sub.cand, which are not identified in the neutral mass spectrum I.sub.neut(p), and wherein the second subscore s.sub.2 is addressing all peaks C.sub.neut,I in the neutral mass spectrum I.sub.neut(p), which are not identified in the theoretical mass spectrum I.sub.th,M_cand(p) of the candidate species M.sub.cand.

7. The method of claim 1 wherein in step (i) a mass spectrum I.sub.meas(p) of the sample is measured with a mass spectrometer, wherein in step (ii) for a peak of interest C.sub.int of the measured mass spectrum I.sub.meas(p) a set S.sub.cand of candidate species of molecules M.sub.cand from a set S.sub.inv of species of molecules M.sub.inv is determined which have an expected peak.sub.th,int in a mass spectrum corresponding to a neutral mass spectrum I.sub.neut(p) derived by reduction of the measured mass spectrum I.sub.meas(p) with a peak position p.sub.ex,inv within a peak position tolerance range p.sub.tol assigned to the peak of interest A.sub.int in the neutral mass spectrum I.sub.neut(p) derived by reduction of the measured mass spectrum I.sub.meas(p), wherein in step (iii) for each candidate species M.sub.cand of the set S.sub.cand of candidate species of molecules M.sub.cand a complete theoretical mass spectrum I.sub.th,M_cand(p) corresponding the measured mass spectrum I.sub.meas(p) is calculated and a range of peak positions p comprising the peak positions p.sub.th,i of all peaks C.sub.th,M_cand,i of the complete theoretical mass spectra I.sub.th,M_cand(p) corresponding to the measured mass spectrum I.sub.meas(p) of all candidate species M.sub.cand of the set S.sub.cand of candidate species of molecules M.sub.cand is determined and wherein in step (iv) the measured mass spectrum I.sub.meas(p) is compared with each theoretical mass spectrum I.sub.th,M_cand(p) of the candidate species M.sub.cand of the set S.sub.cand of candidate species of molecules M.sub.cand corresponding to the measured mass spectrum I.sub.meas(p) in the determined range of peak positions p with a first method, which is determining a first subscore s.sub.1,M_cand for each candidate species M.sub.cand, and with a second method, which is determining a second subscore s.sub.2,M_cand for each candidate species M.sub.cand, wherein the first subscore s.sub.1 is addressing all peaks C.sub.th,M_cand,i in the theoretical mass spectrum I.sub.th,M_cand(p) of the candidate species M.sub.cand, which are not identified in the measured mass spectrum I.sub.meas(p), and wherein the second subscore s.sub.2 is addressing all peaks C.sub.meas,i in the measured mass spectrum I.sub.meas(p), which are not identified in the theoretical mass spectrum I.sub.th,M_cand(p) of the candidate species M.sub.cand.

8. The method of claim 7 wherein in step (ii) the position P.sub.meas,int of the peak of interest C.sub.int of the measured mass spectrum is reduced to its position p.sub.neutral,int in the neutral mass spectrum I.sub.neut(p) derived by reduction of the measured mass spectrum I.sub.meas(p) and the mass spectrum of candidate species of molecules M.sub.cand has an expected peak C.sub.ex,inv with a peak position p.sub.ex,inv within a peak position tolerance range p.sub.tol of the position p.sub.neutral,int of the peak of interest C.sub.int in the neutral mass spectrum I.sub.neut(p).

9. The method of claim 8 wherein before the measurement of the mass spectrum I.sub.meas(p) in step (i) the sample is ionized by at least one ionization process and in step (iii) for each candidate species M.sub.cand is determined an assigned ion I.sub.cand which is originated by at least one ionization process of the sample before the measurement of the mass spectrum I.sub.meas(p) and based on this assigned ions I.sub.cand for each candidate species M.sub.cand the complete theoretical mass spectrum I.sub.th,M_cand(p) corresponding the measured mass spectrum I.sub.meas(p) is calculated.

10. The method of claim 1 wherein in step (iv) a dynamic recalibration is used shifting the measured mass spectrum I.sub.meas(p) or the neutral mass spectrum I.sub.neut(p) for each candidate molecule M.sub.cand by the difference p.sub.recal of the peak positions of expected peaks and the measured peaks of peaks of the neutral mass spectrum before a subscore s.sub.i calculated.

11. The method of claim 1 wherein in step (iv) a third score that is a MS.sup.2 spectrum coverage subscore s.sub.3,M_cand is determined and used to calculate the final score fs.sub.M_cand from a summation of linear functions of the subscores S.sub.i,M_cand.

12. A method of identification of one or more most likely elemental compositions of at least one species of molecules M contained in a sample and/or originated from a sample by at least one ionization process comprising the following steps: (i) measuring a mass spectrum I.sub.meas(p) of the sample with a mass spectrometer or measuring a mass spectrum I.sub.meas(p) of the sample with a mass spectrometer and reducing the measured mass spectrum I.sub.meas(p) to a neutral mass spectrum I.sub.neut(p); (ii) determining for a peak of interest C.sub.int of the measured mass spectrum I.sub.meas(p) or neutral mass spectrum I.sub.neut(p) a set S.sub.cand of candidate species of molecules M.sub.cand that is defined according to an expectation regarding which kind of species of molecules can be present in the sample from a set S.sub.inv of species of molecules M.sub.inv which have an expected peak C.sub.ex,inv in a mass spectrum corresponding to the measured mass spectrum I.sub.meas(p) or the neutral mass spectrum I.sub.neut(p) with a peak position p.sub.ex,inv within a peak position tolerance range p.sub.tol assigned to the peak of interest C.sub.int in the corresponding measured mass spectrum I.sub.meas(p) or neutral mass spectrum I.sub.neut(p); (iii) determining for each candidate species M.sub.cand of the set S.sub.cand of candidate species of molecules M.sub.cand an identification mass spectrum I.sub.id,M_cand(p) showing at least a part of an isotope distribution of the molecule M.sub.cand corresponding to the measured mass spectrum I.sub.meas(p) or the neutral mass spectrum I.sub.neut(p) and determining a range of peak positions p comprising the peak positions p.sub.id,M_cand,i of all peaks C.sub.id,M_cand,i of the identification mass spectra I.sub.id,M_cand(p) corresponding to the measured mass spectrum I.sub.meas(p) or the neutral mass spectrum I.sub.neut(p) of all candidate species M.sub.cand of the set S.sub.cand of candidate species of molecules M.sub.cand; (iv) comparing the measured mass spectrum I.sub.meas(p) or the neutral mass spectrum I.sub.neut(p) with the identification mass spectrum I.sub.id,M_cand(p) of candidate species M.sub.cand of the set S.sub.cand of candidate species of molecules M.sub.cand corresponding to the measured mass spectrum I.sub.meas(p) or the neutral mass spectrum I.sub.neut(p) in the determined range of peak positions p with a first method, which is determining a first subscore s.sub.1,M_cand for the candidate species M.sub.cand, and with a second method, which is determining a second subscore s.sub.2,M_cand for the candidate species M.sub.cand, wherein the first subscore s.sub.1,M_cand is addressing all peaks C.sub.id,M_cand,i in the identification mass spectrum I.sub.id,M_cand(p) of the candidate species M.sub.cand, which are not identified in the measured mass spectrum I.sub.meas(p) or neutral mass spectrum I.sub.neut(p), and wherein the second subscore s.sub.2,M_cand is addressing all peaks C.sub.meas,i in the measured mass spectrum I.sub.meas(p) or all peaks C.sub.neut,i in the neutral mass spectrum I.sub.neut(p), which are not identified in the identification mass spectrum I.sub.id,M_cand(p) of the candidate species M.sub.cand and wherein at first the comparison is done for each candidate species M.sub.cand only with one method of the first method and the second method and only for candidate species M.sub.cand whose subscore M.sub.i,M_cand has a subscore within a specific number of subscores having the highest values, the comparison with the other method is done and calculating for each candidate species M.sub.cand of the set S.sub.cand of candidate species of molecules M.sub.cand a final score fs.sub.M_cand from the subscores S.sub.i,M_cand, for which both subscores of the first method and the second method s.sub.1,M_cand and s.sub.1,M_cand have been calculated or for which both subscores of the first method and the second method s.sub.1,M_cand and s.sub.1,M_cand have been calculated and one of both of the subscores of the first method and the second method s.sub.1,M_cand and s.sub.1,M_cand are higher than an assigned threshold value s.sub.i,th,fs for calculating the final score fs.sub.M_cand; (v) determining one or more calculated final scores fs.sub.high, k having the highest values; and (vi) determining the elemental composition of the candidate species M.sub.cand,high_k of the set S.sub.cand of candidate species of molecules M.sub.cand which have the one or more calculated final scores fs.sub.high, k of the highest values by looking up the elemental composition of the candidate species M.sub.cand,high k.

13. A method of identification of one or more most likely elemental compositions of at least one species of molecules M contained in a sample and/or originated from a sample by at least one ionization process comprising the following steps: (i) measuring a mass spectrum I.sub.meas(p) of the sample with a mass spectrometer or measuring a mass spectrum I.sub.meas(p) of the sample with a mass spectrometer and reducing the measured mass spectrum I.sub.meas(p) to a neutral mass spectrum I.sub.neut(p); (ii) determining for a peak of interest C.sub.int of the measured mass spectrum I.sub.meas(p) or neutral mass spectrum I.sub.neut(p) a set S.sub.cand of candidate species of molecules M.sub.cand that is defined according to an expectation regarding which kind of species of molecules can be present in the sample from a set S.sub.inv of species of molecules M.sub.inv which have an expected peak C.sub.ex,inv in a mass spectrum corresponding to the measured mass spectrum I.sub.meas(p) or the neutral mass spectrum I.sub.neut(p) with a peak position p.sub.ex,inv within a peak position tolerance range p.sub.tol assigned to the peak of interest C.sub.int in the corresponding measured mass spectrum I.sub.meas(p) or neutral mass spectrum I.sub.neut(p); (iii) determining for each candidate species M.sub.cand of the set S.sub.cand of candidate species of molecules M.sub.cand an identification mass spectrum I.sub.id,M_cand(p) showing at least a part of an isotope distribution of the molecule M.sub.cand corresponding to the measured mass spectrum I.sub.meas(p) or the neutral mass spectrum I.sub.neut(p) and determining a range of peak positions p comprising the peak positions p.sub.id,M_cand,i of all peaks C.sub.id,M_cand,i of the identification mass spectra I.sub.id,M_cand(p) corresponding to the measured mass spectrum I.sub.meas(p) or the neutral mass spectrum I.sub.neut(p) of all candidate species M.sub.cand of the set S.sub.cand of candidate species of molecules M.sub.cand; (iv) comparing the measured mass spectrum I.sub.meas(p) or the neutral mass spectrum I.sub.neut(p) with the identification mass spectrum I.sub.id,M_cand(p) of candidate species M.sub.cand of the set S.sub.cand of candidate species of molecules M.sub.cand corresponding to the measured mass spectrum I.sub.meas(p) or the neutral mass spectrum I.sub.neut(p) in the determined range of peak positions p with a first method, which is determining a first subscore s.sub.1,M_cand for the candidate species M.sub.cand, and with a second method, which is determining a second subscore s.sub.2,M_cand for the candidate species M.sub.cand, wherein the first subscore s.sub.1,M_cand is addressing all peaks C.sub.id,M_cand,i in the identification mass spectrum I.sub.id,M_cand(p) of the candidate species M.sub.cand, which are not identified in the measured mass spectrum I.sub.meas(p) or neutral mass spectrum I.sub.neut(p), and wherein the second subscore s.sub.2,M_cand is addressing all peaks C.sub.meas,i in the measured mass spectrum I.sub.meas(p) or all peaks C.sub.neut,i in the neutral mass spectrum I.sub.neut(p), which are not identified in the identification mass spectrum I.sub.id,M_cand(p) of the candidate species M.sub.cand and wherein at first the comparison is done with one method for each candidate species M.sub.cand and only if the subscore S.sub.i,M_cand of this method is higher than a threshold value S.sub.th,2.cal the comparison with the other method is done and calculating for each candidate species M.sub.cand of the set S.sub.cand of candidate species of molecules M.sub.cand a final score fs.sub.M_cand from the subscores S.sub.i,M_cand, for which both subscores of the first method and the second method S.sub.1,M_cand and S.sub.1,M_cand have been calculated or for which both subscores of the first method and the second method s.sub.1,M_cand and s.sub.1,M_cand have been calculated and one of both of the subscores of the first method and the second method s.sub.1,M_cand and s.sub.1,M_cand are higher than an assigned threshold value s.sub.i,th,fs for calculating the final score fs.sub.M_cand; (v) determining one or more calculated final scores fs.sub.high,k having the highest values; (vi) determining the elemental composition of the candidate species M.sub.cand,high_k of the set S.sub.cand of candidate species of molecules M.sub.cand which have the one or more calculated final scores fs.sub.high, k of the highest values by looking up the elemental composition of the candidate species M.sub.cand,high k.

14. A mass spectrometer able to execute the method of claim 13.

15. A mass spectrometer able to execute the method of claim 1.

16. A mass spectrometer able to execute the method of claim 12.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 shows a measured mass spectrum I.sub.meas_1(m/z) and the comparison of the measured mass spectrum I.sub.meas_1(m/z) with a theoretical mass spectrum I.sub.th,M1(m/z) of a first candidate species M1 with two different methods.

(2) FIG. 2 shows the measured mass spectrum I.sub.meas_1(m/z) also shown in FIG. 1 and the comparison of the measured mass spectrum I.sub.meas_1(m/z) with a theoretical mass spectrum I.sub.th,M2(m/z) of a second candidate species M2 with two different methods.

(3) FIG. 3 shows the measured mass spectrum I.sub.meas_1(m/z) also shown in FIG. 1 and the comparison of the measured mass spectrum I.sub.meas_1(m/z) with a theoretical mass spectrum I.sub.th,M3(m/z) of a third candidate species M3 with two different methods.

(4) FIG. 4 shows a measured mass spectrum I.sub.meas_2(m/z) and the comparison of the measured mass spectrum I.sub.meas_2(m/z) with a theoretical mass spectrum I.sub.th,M4(m/z) of a fourth candidate species M4 with two different methods.

(5) FIG. 5 shows the measured mass spectrum I.sub.meas_2(m/z) also shown in FIG. 4 and the comparison of the measured mass spectrum I.sub.meas_2(m/z) with a theoretical mass spectrum I.sub.th,M5(m/z) of a fifth candidate species M5 with two different methods.

(6) FIG. 6 shows a measured mass spectrum I.sub.meas_3(m/z) of the fragments of a molecule measured with the mass spectrum I.sub.meas_2(m/z) and the comparison of the measured mass spectrum I.sub.meas_3(m/z) with an expected mass spectrum I.sub.th,M4(m/z) of the fragments of the fourth candidate species M4.

(7) FIG. 7 shows a measured mass spectrum I.sub.meas_3(m/z) of the fragments of a molecule measured with the mass spectrum I.sub.meas_2(m/z) also shown in FIG. 6 and the comparison of the measured mass spectrum I.sub.meas_3(m/z) with an expected mass spectrum I.sub.th,M5(m/z) of the fragments of the fifth candidate species M5.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

(8) The method of invention is used for identification of a most likely elemental composition of at least one species of molecules M contained in a sample and/or originated from a sample by at least an ionization process.

(9) Preferably the method is used to identify the most likely elemental composition of molecules like herbicides, insecticides, other pesticides, lipids, soluble or suspended solids in leachates, metabolites, drugs, narcotics, pharmaceuticals, toxins, molecules in extracts and in particular metabolites derived from drugs, narcotics, pharmaceuticals and toxins having typically a mass of typically between 50 u and 2,000 u, preferably between 200 u and 700 u and particularly preferably between 300 u and 500 u. If only for specific class of species of molecules like herbicides, insecticides, pesticides, lipids, metabolites, drugs, or narcotics the elemental composition of a species of molecules shall be identified with the inventive method, then the mass range, in which the identification is possible can be wider. Then the mass range is typically between 50 u and 3,000 u, preferably between 200 u and 1,000 u and particularly preferably between 300 u and 600 u.

(10) The method of the invention is used to investigate samples. These samples may contain species of molecules which can be identified by their elemental composition.

(11) The investigated sample can be also understood by ions which are generated from the sample by at least an ionization process and their elemental composition described by their molecular formula. The ions may be preferably generated by electrospray ionization (ESI), matrix-assisted laser desorption ionization (MALDI), plasma ionization, electron ionization (EI), chemical ionization (CI) and atmospheric pressure chemical ionization (APCI). A particular preferred method of ionization is electrospray ionization. The generated ions are charged particles mostly having a molecular geometry and a corresponding molecular formula. In the context of this patent application the term species of molecules originated from a sample by at least an ionisation process shall be understood is referring to the molecular formula of an ion which is originated from a sample by at least an ionisation process. So just if ions which are originated from a sample by at least an ionisation process have one common molecular formula which is describing the elemental composition, the elemental composition of their species of the molecule, they may have a different molecular geometry.

(12) The elemental composition of a species of molecules originated from a sample by at least an ionization process is correlated with the elemental composition of the species of molecule contained in the sample by reducing the charge of the ion has to zero and changing the elemental composition accordingly to the ionization process.

(13) In detail, if a species of molecule M.sub.0 is contained in the sample and during the ionization an ion I.sub.ad.sup.q having an elemental composition of the molecule I.sub.ad and a charge q is added, then the resulting adduct is the ion [M.sub.0+I.sub.ad].sup.q. So by subtracting the charge q and the elemental composition from the ion [M.sub.0+I.sub.ad].sup.q the elemental composition species of molecule contained in the sample can be deduced.

(14) When the molecule M.sub.0 which is contained in the sample has for example the formula C.sub.xH.sub.yO.sub.z and by the ionization process an electron is added, the resulting ion has the elemental formula C.sub.xH.sub.yO.sub.z.

(15) When the molecule M.sub.0 which is contained in the sample has for example the formula C.sub.xH.sub.yO.sub.z and by the ionization process an proton H.sup.+ is added, the resulting ion has the elemental formula C.sub.xH.sub.y+1O.sub.z.sup.+.

(16) When the molecule M.sub.0 which is contained in the sample has for example the formula C.sub.xH.sub.yO.sub.z and by the ionization process a potassium ion K.sup.+ is added, the resulting ion has the elemental formula C.sub.xH.sub.yO.sub.zK.sup.+.

(17) When the molecule M.sub.0, which is contained in the sample, has for example the formula C.sub.xH.sub.yO.sub.z, and by the ionization process a double charged calcium ion Ca.sup.2+ is added, the resulting ion has the elemental formula C.sub.xH.sub.yO.sub.zCa.sup.2+.

(18) In the species of molecules all molecules have the same composition of atoms according to the molecular formula. But each atom of the molecule can occur as different isotopes. So the basic element of the organic chemistry, the carbon atom occurs in two stable isotopes, the .sup.12C isotope with a natural probability of occurrence of 98.9% and the .sup.13C isotope (having one more neutron in its atomic nucleus) with a natural probability of occurrence of 1.1%. Due to this probabilities of occurrence of the isotope particularly complex molecules of higher mass consisting of a higher number of atoms have a lot of isotopes. These isotopes have different masses resulting in a mass distribution of the isotopes, named in the content of this patent application isotope distribution (short term: ID) of the species of molecules. Each species of molecules therefore can have different masses. The different masses of the isotopes of a species of molecules and the mass distribution of the isotopesthe abundance of the isotopes of different massescan be visible in the mass spectrum of a mass spectrometer. Depending on the resolution of the mass analyser which is used to measure the mass spectrum more or less peaks of different isotopes can be found in the measured mass spectrum. Preferably the resolution is the difference in the mass to charge ratio m/z of two peaks m/z for which the two peaks can be separated in the mass spectrum. Accordingly the resolving power R of the mass analyser is defined for e peak having the mass to charge ratio m/z by the ratio:

(19) $R\left(m/z\right)=\frac{m/z}{m/z}$

(20) Preferably it is assumed that two peaks should be separated at the half maximum height of a peak, so that the resolution m/z is FWHM (full width at half maximum) of the peak. Accordingly the resolving power R of the mass analyser is then:

(21) $R\left(m/z\right)=\frac{m/z}{\mathrm{FWHM}}$

(22) Mass analyzers have typically a resolving power R of 500 to 10,000. Mass analyzers of increased resolution have typically a resolving power R of 10,000 to 50,000. High resolution mass analyzers have typically a resolving power R of 50,000 to 200,000 and ultra high resolution mass analysers have a resolving power R between 200,000 and 10,000,000.

(23) In a first step of the inventive method a mass spectrum of the sample has to be measured by a mass spectrometer. In general every kind of mass spectrometer can be used known to a person skilled in the art to measure a mass spectrum of a sample. In particular it is preferred to use a mass spectrometer of high resolution like a mass spectrometer having an Orbitrap mass analyser, a FT-mass spectrometer, an ICR mass spectrometer or an MR-TOF mass spectrometer. Other mass spectrometers for which the inventive method can be applied are particularly TOF mass spectrometer, mass spectrometer with a HR quadrupole mass analyser, and mass spectrometer with an ion trap mass analyzer.

(24) Mass spectrometer with a HR quadrupole mass analyser may have a resolving power R between up to 10.000. TOF mass spectrometer typically have a resolving power R between 2,000 and 20,000. Mass spectrometer with an Orbitrap mass analyser have typically a resolving power R between 5,000 and 1,000,000. FT-mass spectrometer have typically a resolving power R between 100,000 and 5,000,000. MR-TOF mass spectrometer have typically a resolving power R between 20,000 and 100,000. ICR mass spectrometer have typically a resolving power R between 1,000,000 and 5,000,000.

(25) Typically a mass to charge ratio tolerance ratio R.sub.m/z_tol or a mass tolerance ratio R.sub.m_tol to determine the candidate species of molecules M.sub.cand in step (ii) is used between 3 ppm and 30 ppm, preferably between 5 ppm and 20 ppm and particularly preferably between 8 ppm and 15 ppm for mass spectrometers without high resolution.

(26) Mass spectrometer of high resolution with a resolving power R of 50,000 and higher have typically a mass to charge ratio tolerance ratio R.sub.m/z_tol or a mass tolerance ratio R.sub.m_tol to determine the candidate species of molecules M.sub.cand in step (ii) is used between 1 ppm and 15 ppm, preferably between 2 ppm and 10 ppm and particularly preferably between 3 ppm and 7 ppm.

(27) Mass spectrometer of ultra high resolution with a resolving power R of 200,000 and higher have typically a mass to charge ratio tolerance ratio R.sub.m/z_tol or a mass tolerance ratio R.sub.m_tol to determine the candidate species of molecules M.sub.cand in step (ii) is used between 0.5 ppm and 10 ppm, preferably between 1.5 ppm and 7 ppm and particularly preferably between 2.5 ppm and 5 ppm.

(28) The definition of the mass tolerance ratio R.sub.m_tol is the same as the mass to charge ratio tolerance ratio R.sub.m/z_tol, when the charge z is set to 1.

(29) In the following examples of the inventive method are described in detail:

(30) With the first example of the inventive method one or more most likely elemental composition of at least one species of molecules M are identified, which are originated from an investigated sample by an ionization process.

(31) In step (i) of the method of the first example a mass spectrum I.sub.meas(p) of a sample is measured with a mass spectrometer. The species of molecules M, for which its elemental composition shall be identified, is originated from the sample by the ionization of the mass spectrometer and is therefore an ion.

(32) In step (ii) of the inventive method at first a peak of interest C.sub.int is identified in the measured mass spectrum I.sub.meas(p). It is the task of the inventive method to identify the elemental composition of that species of molecules M, which has generated the peak of interest C.sub.int in the measured mass spectrum I.sub.meas(p).This is done manually by an user who wants to know from which species of molecules M the peak of interest C.sub.int is originated. The inventive methods can identify the most likely elemental composition of the species of molecules M using not only the information of the peak of interest C.sub.int but also the information of other peaks originated from the species of molecules M.

(33) Further on a set S.sub.inv of species of molecules M.sub.inv has to be defined, for which molecules M.sub.inv it has to be investigated if their isotope distribution occurs in the measured mass spectrum I.sub.meas(p) or neutral mass spectrum I.sub.neut(p).This set S.sub.inv of species of molecules M.sub.inv can be defined by a lot of criteria.

(34) Typical criteria for the set S.sub.inv of investigated species of molecules M.sub.inv being applicable to all methods encompassed by this invention are: The type of the elements X contained in the species of molecules M.sub.inv. The minimum number Min.sub.x of atoms of each element X contained in the species of molecules M.sub.inv. The maximum number Max.sub.x of atoms of each element X contained in the species of molecules M.sub.inv. A minimum value for the ratio between the number of atoms of two elements contained in the species of molecule M, e.g. the ratio H/C between the number hydrogen atoms (H) and the number of carbon atoms (C) contained in the species of molecule M.sub.inv. A maximum value for the ratio between the number of atoms of two elements contained in the species of molecule M, e.g. the ratio H/C between the number hydrogen atoms (H) and the number of carbon atoms (C) contained in the species of molecule M.sub.inv. A minimum value for a degree of unsaturation of the molecule M.sub.inv, in particular a minimum value of double-bond equivalents of the molecule M.sub.inv and/or a minimum value of rings-plus-double-bond equivalents (RDBE) of the molecule M.sub.inv. A maximum value for a degree of unsaturation of the molecule M.sub.inv, in particular a maximum value of double-bond equivalents of the molecule M.sub.inv and/or a maximum value of rings-plus-double-bond equivalents (RDBE) of the molecule M.sub.inv.

(35) Values for these criteria applicable to all methods encompassed by this invention are:

(36) The type of the elements X which may be contained in the species of molecules M.sub.inv, have been already described before. Also their minimum number Min.sub.x of atoms and their maximum number Max.sub.x of atoms.

(37) Typically the minimum value for the ratio H/C between the number hydrogen atoms (H) and the number of carbon atoms (C) contained in the species of molecule M.sub.inv is between 0.02 and 1.0, preferably between 0.05 and 0.5 and particularly preferably between 0.05 and 0.2.

(38) Typically the maximum value for the ratio H/C between the number hydrogen atoms (H) and the number of carbon atoms (C) contained in the species of molecule M.sub.inv is between 2.0 and 10.0, preferably between 3.0 and 7.5 and particularly preferably between 3.5 and 5.0.

(39) When the rings-plus-double-bond equivalents (RDBE) may be calculated according to Watson, Sparkman Introduction of Mass Spectrometry, Fourth Edition, Chapter 5, IV.F. then typically the minimum value of rings-plus-double-bond equivalents (RDBE) of the molecule M.sub.inv is between 0 and 6, preferably between 0 and 4 and particularly preferably between 0 and 2 and typically the maximum value of rings-plus-double-bond equivalents (RDBE) of the molecule M.sub.inv is between 20 and 80, preferably between 28 and 60 and particularly preferably between 34 and 50.

(40) In step (ii) of the inventive method it is determined a set S.sub.cand of candidate species of molecules M.sub.cand from the defined set S.sub.inv of species of molecules M.sub.inv which have an expected peak C.sub.ex,inv in a mass spectrum corresponding to the measured mass spectrum I.sub.meas(p) with a peak position p.sub.ex,inv within the tolerance range p.sub.tol assigned to the peak of interest C.sub.int in the corresponding measured mass spectrum I.sub.meas(p).

(41) The peak position p.sub.ex,inv of the expected peak C.sub.ex,inv has to be given by a mass spectrum which corresponds to the mass spectrum in which the peak of interest C.sub.int is identified. So if the peak of interest C.sub.int is identified in the measured mass spectrum I.sub.meas(p) the peak position p.sub.ex,inv of the expected peak C.sub.ex,inv has to be defined for a mass spectrum corresponding to the measured mass spectrum I.sub.meas(p).

(42) Preferably the peak position p in the measured mass spectrum is given by the mass to charge ratio m/z, then the peak position p.sub.ex,inv of the expected peak C.sub.ex,inv has a mass to charge value m/z.sub.ex,inv to be defined for a mass spectrum corresponding to the measured mass spectrum I.sub.meas(m/z). In particular in this case only the mass to charge value m/z.sub.ex,inv of the expected peak C.sub.ex,inv has to be known. If the mass to charge value m/z.sub.ex,inv of an expected peak C.sub.ex,inv of a species of molecules M.sub.inv is within the mass to charge tolerance range m/z.sub.tol of the peak of interest C.sub.int then the species of molecules M.sub.inv is a candidate species of molecules M.sub.cand which will be investigated further.

(43) The tolerance range p.sub.tol assigned to the peak of interest C.sub.int is defined in that mass spectrum which is corresponding to the mass spectrum in which the expected peaks C.sub.ex,inv of the species of molecules M.sub.inv used in step (ii) are known.

(44) In the inventive method of this first example is the tolerance range p.sub.tol assigned to the peak of interest C.sub.int defined in the measured mass spectrum I.sub.meas(p).

(45) The peak of interest C.sub.int has a peak position p.sub.int,meas in the measured mass spectrum I.sub.meas(p) and the tolerance range p.sub.tol is assigned to the peak of interest C.sub.int by a range around the peak position p.sub.int,meas of the peak of interest C.sub.int. Preferably the tolerance range p.sub.tol is symmetrically to the peak position p.sub.int,meas of the peak of interest C.sub.int, so that the distance between the lower endpoint of the tolerance range p.sub.tol and the peak position p.sub.int,meas of the peak of interest C.sub.int is equal to the distance between higher endpoint of the tolerance range p.sub.tol and the peak position p.sub.int,meas of the peak of interest C.sub.int.

(46) In step (iii) of the inventive method for each candidate species M.sub.cand of the set S.sub.cand of candidate species of molecules M.sub.cand determined before in step (ii) an identification mass spectrum I.sub.id,M_cand(p) is determined, which is a theoretical mass spectrum I.sub.th,M_cand(p), which is for each candidate species M.sub.cand corresponding to the measured mass spectrum I.sub.meas(p).

(47) The theoretical mass spectra I.sub.th,M_cand(p) are calculated during the execution of the method.

(48) It is possible that in both steps (ii) and (iii) for the candidate species M.sub.cand of the set S.sub.cand of candidate species of molecules M.sub.cand theoretical mass spectra I.sub.th,M_cand(p) are calculated which correspond to the measured mass spectrum I.sub.meas(p) and the theoretical mass spectra I.sub.th,M_cand(p) are used in step (iii) as identification mass spectrum I.sub.id,M_cand(p). Particularly the same theoretical mass spectra I.sub.th,M_cand(p) may be used in both steps.

(49) The theoretical mass spectra I.sub.th,M_cand(p) comprise the complete mass spectra of the candidate species of molecules M.sub._cand showing the whole isotope distribution of the molecule M.sub.cand only limited by the resolving power and signal-to-noise ratio S/N under which the identification mass spectra I.sub.id,M_cand(p) are calculated. The resolving power and signal-to-noise ratio S/N used for the calculating have values equal or very similar to the values of the mass spectrometer used to measure the measured mass spectrum I.sub.meas(p) in step (i).

(50) In step (iii) of the inventive method of this first example a range of peak positions p is determined in which the determined theoretical mass spectra I.sub.th,M_cand(p) of all candidate species M.sub.cand of the set S.sub.cand of candidate species of molecules M.sub.cand are arranged. The range of peak positions p is determined by identifying a range of peak positions which is comprising the peak positions p.sub.th,i of all peaks C.sub.th,M_cand,i of the complete theoretical mass spectra I.sub.th,M_cand(p) corresponding to the measured mass spectrum I.sub.meas(p) of all candidate species M.sub.cand of the set S.sub.cand of candidate species of molecules M.sub.cand. So in the range of peak positions p all peaks C.sub.th,M_cand,i of the theoretical mass spectra I.sub.th,M_cand(p) of all candidate species M.sub.cand of the set S.sub.cand of candidate species of molecules M.sub.cand are positioned in the range of peak positions p. The lower endpoint of the range of peak positions p is similar or below the lowest value of a peak position p.sub.th,i of any peak C.sub.th,M_cand,I of all candidate species M.sub.cand of the set S.sub.cand of candidate species of molecules M.sub.cand and the highest endpoint of the range of peak positions p is similar or above the highest value of a peak position p.sub.th,i of any peak C.sub.id,M_cand,I of all candidate species M.sub.cand of the set S.sub.cand of candidate species of molecules M.sub.cand. So it is guaranteed that in the range of peak positions p all isotope distributions of all candidate species M.sub.cand of the set S.sub.cand of candidate species of molecules M.sub.cand are completely encompassed. In the range of peak positions p each theoretical mass spectra I.sub.th,M_cand(p) of a candidate species of molecules M.sub.cand can be compared with the corresponding measured mass spectrum I.sub.meas(p) without missing any peak of the candidate species of molecules M.sub.cand existing in its theoretical mass spectra I.sub.th,M_cand(p).

(51) In step (iv) of the inventive method of the first example this comparison of the measured mass spectrum I.sub.meas(p) with each theoretical mass spectra I.sub.th,M_cand(p) of the candidate species M.sub.cand of the set S.sub.cand of candidate species of molecules M.sub.cand corresponding to the measured mass spectrum I.sub.meas(p) is executed in the determined range of peak positions p.

(52) For all candidate species M.sub.cand this comparison is done with two different methods, a first method and a second method, having a different focus on the features of measured mass spectrum I.sub.meas(p) and the theoretical mass spectra I.sub.th,M_cand(p) of the candidate species of molecules M.sub.cand.

(53) It is also possible to use more than this two methods of comparison in step (iv) of the inventive method.

(54) By the first method to compare the theoretical mass spectra I.sub.th,M_cand(p) of the candidate species of molecules M.sub.cand with the measured mass spectrum I.sub.meas(p) a first subscore s.sub.1,M_cand is determined for each candidate species M.sub.cand. This first subscore s.sub.1,M_cand of the first method is addressing all peaks C.sub.id,M_cand,i in the theoretical mass spectra I.sub.th,M_cand(p) of the candidate species M.sub.cand, which are not identified in the measured mass spectrum I.sub.meas(p). So the first method is in particular sensitive with its subscore s.sub.1,M_cand for peaks C.sub.id,M_cand,i of a theoretical mass spectra I.sub.th,M_cand(p) of a candidate species M.sub.cand, which cannot be identified in the measured mass spectrum I.sub.meas(p).

(55) When the first method recognizes that is a peak C.sub.id,M_cand,i of a candidate species M.sub.cand cannot be identified in the measured mass spectrum I.sub.meas(p), the subscore s.sub.1,M_cand of the method is reduced. In particular for each peak C.sub.id,M_cand,i of a theoretical mass spectrum I.sub.th,M_cand(p) of a candidate species M.sub.cand which cannot be identified in the measured mass spectrum I.sub.meas(p) the subscore s.sub.1,M_cand of the first method is reduced. The reduction is the same for every identified peak C.sub.id,M_cand,I. There is no reduction if the intensity of the not identified peak C.sub.id,M_cand,i is below a threshold value. In general, the first method is taking care if expected peaks of the theoretical mass spectra I.sub.th,M_cand(p) are found in the measured mass spectrum. If expected peaks are missing and in particular many expected peaks are missing this is an indicator that the candidate species M.sub.cand is not abundant which is resulting in a lower score s.sub.1,M_cand.

(56) In a preferred embodiment of the first method to compare the measured mass spectrum I.sub.meas(p) with each theoretical mass spectra I.sub.th,M_cand(p) of the candidate species M.sub.cand of the set S.sub.cand of candidate species of molecules M.sub.cand corresponding to the measured mass spectrum I.sub.meas(p) the method described in the U.S. Pat. No. 8,831,888 B2 can be used. Then the first subscore S.sub.1,M_cand is the pattern spectral distance (PSD) calculated for the elemental composition of the candidate species of molecules M.sub.cand. A preferred use of this method will be described below for the third example, which can be applied in the same way in all inventive methods, in particular in the inventive method of this first example.

(57) By the second method to compare the theoretical mass spectra I.sub.th,M_cand(p) of the candidate species of molecules M.sub.cand with measured mass spectrum I.sub.meas(p) a second subscore s.sub.2,M_cand is determined for each candidate species M.sub.cand, wherein the second subscore s.sub.2,M_cand is addressing all peaks C.sub.meas,i in the measured mass spectrum I.sub.meas(p), which are not identified in the theoretical mass spectrum I.sub.th,M_cand(P of the candidate species M.sub.cand. So the second method is in particular sensitive in its subscore s.sub.2,M_cand for peaks C.sub.meas,i in the measured mass spectrum I.sub.meas(p), which cannot be identified in the theoretical mass spectra I.sub.th,M_cand(p) of the candidate species of molecules M.sub.cand.

(58) When the second method recognizes that a peak C.sub.meas,i in the measured mass spectrum I.sub.meas(p) cannot be identified in the theoretical mass spectra I.sub.th,M_cand(p) of the candidate species of molecules M.sub.cand, the subscore s.sub.2,M_cand of the method is reduced. In particular for each peak C.sub.meas,i in the measured mass spectrum I.sub.meas(p) which cannot been identified in the theoretical mass spectra I.sub.th,M_cand(p) of the candidate species of molecules M.sub.cand the subscore s.sub.2,M_cand of the second method is reduced. The reduction is in the same way for every not identified peak C.sub.meas,i or C.sub.neut,i. The reduction depends on the intensity of the not identified peak C.sub.meas,i found in the measured mass spectrum I.sub.meas(p). There is no reduction if the intensity of the not identified peak C.sub.meas,i is below a threshold value. In general, the second method is taking care if measured peaks in the measured mass spectrum I.sub.meas(p) are explained by in the theoretical mass spectrum I.sub.th,M_cand(p) of a candidate species M.sub.cand. If measured peaks are missing and in particular measured peaks of high intensity are missing this is an indicator that the candidate species M.sub.cand is not abundant which is resulting in a lower score s.sub.2,M_cand.

(59) For all candidate species M.sub.cand of the set S.sub.cand of candidate species of molecules M.sub.cand, preferably all candidate species M.sub.cand of the set S.sub.cand of candidate species of molecules M.sub.cand, further in step (iv) of the inventive method a final score fs.sub.M_cand is calculated from the subscores s.sub.i,M_cand.

(60) In inventive method of this first example in step (iv) of the inventive method a final score fs.sub.M_cand is calculated from the subscores s.sub.i,M_cand only for all candidate species M.sub.cand, for which one of both of the subscores of the first method and the second method s.sub.1,M_cand and s.sub.1,M_cand are higher than an assigned threshold value s.sub.i,th,fs for calculating the final score fs.sub.M_cand. By this criteria the calculation of final scores fs.sub.M_cand is avoided, which due to the value of one subscore have no chance to belong to the final scores fs.sub.high,k having the highest values. The threshold values s.sub.i,th,fs can have fixed values or can be derived from a ranking of the candidate species M.sub.cand according to a subscore s.sub.i,M_cand. Then the value of the subscore s.sub.i,M_cand of the candidate species M.sub.cand on a specific rank is defining the threshold values s.sub.i,th,fs.

(61) The final score fs.sub.M_cand is calculated in the first example from a summation of functions which are only depending on one subscore s.sub.i,M_cand.

(62) Because only the subscores s.sub.1,M_cand and s.sub.2,M_cand are used then the final score fs.sub.M_cand of the first example is given by:
fs.sub.M_cand=f(s.sub.1,M.sub.cand)+g(s.sub.2,M.sub.cand)

(63) In a particular preferred embodiment of the inventive method of the first example the final score fs.sub.M_cand is calculated by a summation of linear functions of the subscores s.sub.i,M_cand. Each function is defined by a weighting factor fi assigned to each subscore s.sub.i,M_cand. Then the final score fs.sub.M_cand is given by:
fs.sub.M_cand=f.sub.1*s.sub.1,M.sub.cand+f.sub.2*s.sub.2,M.sub.cand

(64) When the final score fs.sub.M_cand has been calculated for all candidate species M.sub.cand of the set S.sub.cand of candidate species of molecules M.sub.cand, in step (v) of the inventive method the final scores fs.sub.high,k from all final score fs.sub.M_cand of all candidate species M.sub.cand of the set S.sub.cand of candidate species of molecules M.sub.cand are determined, which have the highest values. The number N of final scores fs.sub.high,k having the highest values is defined as default value before the inventive method is used. So at the end N final scores fs.sub.high,k having the highest values are identified.

(65) Then in the nest step (vi) of the inventive method the elemental composition of the candidate species M.sub.cand,high_k of the set S.sub.cand of candidate species of molecules M.sub.cand is determined which have the N calculated final scores fs.sub.high,k of the highest values. This done by looking up the elemental composition of the candidate species M.sub.cand, if it is realised that the final scores fs.sub.M_cand belongs to the final scores fs.sub.high,k of the highest values. The elemental compositions of these N candidate species M.sub.cand are then listed with their final score fs.sub.M_cand in a table and shown on a display.

(66) With the second example of the inventive method the most likely elemental composition of at least one species of molecules M is identified, which are contained in an investigated sample.

(67) In step (i) of the method of the second example a mass spectrum I.sub.meas(p) of a sample is measured with a mass spectrometer. Because during this measurement only ions are detected and shown in the mass spectrum I.sub.meas(p) which have been originated by ab ionization process, the measured mass spectrum I.sub.meas(p) has to be reduced to a neutral mass spectrum I.sub.neut(p) according to the ionization process. In this neutral mass spectrum I.sub.neut(p) peaks C.sub.meas,M,i according the isotope distribution of the species of molecules M contained in the investigated sample can are identified.

(68) In step (ii) of the inventive method at first a peak of interest C.sub.int is identified in the neutral mass spectrum I.sub.neut(p). It is the task of the inventive method to identify the elemental composition of that species of molecules M, which has generated the peak of interest C.sub.int in the neutral mass spectrum I.sub.neut(p).This is done manually by an user who wants to know from which species of molecules M the peak of interest C.sub.int is originated. The inventive methods can identify the most likely elemental composition of the species of molecules M using not only the information of the peak of interest C.sub.int but also the information of other peaks originated from the species of molecules M.

(69) Further on a set S.sub.inv of species of molecules M.sub.inv has to be defined, for which has to be investigated if their isotope distribution occurs in the measured mass spectrum I.sub.meas(p) or neutral mass spectrum I.sub.neut(p). This set S.sub.inv of species of molecules M.sub.inv can be defined by a lot of criteria already explained above.

(70) In step (ii) of the inventive method a set S.sub.cand of candidate species of molecules M.sub.cand is determined from the defined set S.sub.inv of species of molecules M.sub.inv which have an expected peak C.sub.ex,inv in a mass spectrum corresponding to the neutral mass spectrum I.sub.neut(p) with a peak position p.sub.ex,inv within the tolerance range p.sub.tol assigned to the peak of interest C.sub.int in the corresponding neutral mass spectrum I.sub.neut(p).

(71) The peak position p.sub.ex,inv of the expected peak C.sub.ex,inv has to be given by a mass spectrum which corresponds the mass spectrum in which the peak of interest C.sub.int is identified. So if the peak of interest C.sub.int is identified in the neutral mass spectrum I.sub.neut(p) the peak position p.sub.ex,inv of the expected peak C.sub.ex,inv has to be defined for a mass spectrum corresponding to the neutral mass spectrum I.sub.neut(p).

(72) Preferably the peak position p in the neutral mass spectrum I.sub.neut(p) is given by the mass to charge ratio m/z, then the peak position p.sub.ex,inv of the expected peak C.sub.ex,inv has a mass to charge value m/z.sub.ex,inv to be defined for a mass spectrum corresponding to the neutral mass spectrum I.sub.neut(m/z). In particular in this case only the mass to charge value m/z.sub.ex,inv of the expected peak C.sub.ex,inv has to be known. If the mass to charge value m/z.sub.ex,inv of an expected peak C.sub.ex,inv of a species of molecules M.sub.inv is within the mass to charge tolerance range m/z.sub.tol of the peak of interest C.sub.int then the species of molecules M.sub.inv is a candidate species of molecules M.sub.cand which will be investigated further.

(73) The tolerance range p.sub.tol assigned to the peak of interest C.sub.int is defined in the mass spectrum which is corresponding to the mass spectrum in which the expected peaks C.sub.ex,inv of the species of molecules M.sub.inv used in step (ii) are known.

(74) In the inventive method of this second example is the tolerance range p.sub.tol assigned to the peak of interest C.sub.int is defined in the neutral mass spectrum I.sub.neut(p).

(75) The peak of interest C.sub.int has a peak position p.sub.int,neut in the neutral mass spectrum I.sub.neut(p) and the tolerance range p.sub.tol is assigned to the peak of interest C.sub.int by a range around the peak position p.sub.int,neut of the peak of interest C.sub.int. Preferably the tolerance range p.sub.tol is symmetrically to the peak position p.sub.int,neut of the peak of interest C.sub.int, so that the distance between the lower endpoint of the tolerance range p.sub.tol and the peak position p.sub.int,neut of the peak of interest C.sub.int is equal to the distance between higher endpoint of the tolerance range p.sub.tol and the peak position p.sub.int,neut of the peak of interest C.sub.int.

(76) In step (iii) of the inventive method for each candidate species M.sub.cand of the set S.sub.cand of candidate species of molecules M.sub.cand determined before in step (ii) an identification mass spectrum I.sub.id,M_cand(p) is determined, which is a theoretical mass spectrum I.sub.th,M_cand(p), which is for each candidate species M.sub.cand corresponding to the neutral mass spectrum I.sub.neut(p).

(77) The theoretical mass spectra I.sub.th,M_cand(p) is calculated during the execution of the method.

(78) It is possible that in both steps (ii) and (iii) for the candidate species M.sub.cand of the set S.sub.cand of candidate species of molecules M.sub.cand theoretical mass spectra I.sub.th,M_cand(p) are calculated which correspond to the neutral mass spectrum I.sub.neut(p) and the theoretical mass spectra I.sub.th,M_cand(p) are used in step (iii) as identification mass spectrum I.sub.id,M_cand(p). Particularly the same theoretical mass spectra I.sub.th,M_cand(p) may be used in both steps.

(79) The theoretical mass spectra I.sub.th,M_cand(p) comprise the complete mass spectra of the candidate species of molecules M.sub._cand showing the whole isotope distribution of the molecule M.sub.cand only limited by the resolving power and signal-to-noise ratio S/N under which the identification mass spectra I.sub.id,M_cand(p) are calculated. The resolving power and signal-to-noise ratio S/N used for the calculating have values equal or very similar to the values of the mass spectrometer used to measure the measured mass spectrum I.sub.meas(p) in step (i).

(80) In step (iii) of the inventive method of this second example a range of peak positions p is determined in which the determined theoretical mass spectra I.sub.th,M_cand(p) of all candidate species M.sub.cand of the set S.sub.cand of candidate species of molecules M.sub.cand are arranged. The range of peak positions p is determined by identifying a range of peak positions which is comprising the peak positions p.sub.th,i of all peaks C.sub.th,M_cand,i of the complete theoretical mass spectra I.sub.th,M_cand(p) corresponding to the neutral mass spectrum I.sub.neut(p) of all candidate species M.sub.cand of the set S.sub.cand of candidate species of molecules M.sub.cand. So in the range of peak positions p all peaks C.sub.th,M_cand,i of the theoretical mass spectra I.sub.th,M_cand(p) of all candidate species M.sub.cand of the set S.sub.cand of candidate species of molecules M.sub.cand are positioned in the range of peak positions p. The lower endpoint of the range of peak positions p is similar or below the lowest value of a peak position p.sub.th,i of any peak C.sub.th,M_cand,I of all candidate species M.sub.cand of the set S.sub.cand of candidate species of molecules M.sub.cand and the highest endpoint of the range of peak positions p is similar or above the highest value of a peak position p.sub.th,i of any peak C.sub.id,M_cand,I of all candidate species M.sub.cand of the set S.sub.cand of candidate species of molecules M.sub.cand. So it is guaranteed that in the range of peak positions p all isotope distributions of all candidate species M.sub.cand of the set S.sub.cand of candidate species of molecules M.sub.cand are completely encompassed. In the range of peak positions p each theoretical mass spectra I.sub.th,M_cand(p) of a candidate species of molecules M.sub.cand can be compared with the corresponding neutral mass spectrum I.sub.neut(p) without missing any peak of the candidate species of molecules M.sub.cand existing in its theoretical mass spectra I.sub.th,M_cand(p).

(81) In step (iv) of the inventive method of the second example this comparison of the neutral mass spectrum I.sub.neut(p) with each theoretical mass spectra I.sub.th,M_cand(p) of the candidate species M.sub.cand of the set S.sub.cand of candidate species of molecules M.sub.cand corresponding to the neutral mass spectrum I.sub.neut(p) is executed in the determined range of peak positions p.

(82) For all candidate species M.sub.cand this comparison is done with two different methods, a first method and a second method, having a different focus on the features of the neutral mass spectrum I.sub.neut(p) and the theoretical mass spectra I.sub.th,M_cand(p) of the candidate species of molecules M.sub.cand.

(83) It is also possible to use more than these two methods of comparison in step (iv) of the inventive method.

(84) By the first method to compare the theoretical mass spectra I.sub.th,M_cand(p) of the candidate species of molecules M.sub.cand with neutral mass spectrum I.sub.neut(p) a first subscore s.sub.1,M_cand is determined for each candidate species M.sub.cand. This first subscore S.sub.1,M_cand of the first method is addressing all peaks C.sub.id,M_cand,i in the theoretical mass spectra I.sub.th,M_cand(p) of the candidate species M.sub.cand, which are not identified in the neutral mass spectrum I.sub.neut(p). So the first method is in particular sensitive in its subscore s.sub.1,M_cand for peaks C.sub.id,M_cand,i of a theoretical mass spectra I.sub.th,M_cand(p) of a candidate species M.sub.cand, which cannot be identified in the neutral mass spectrum I.sub.neut(p).

(85) When the first method recognizes that a peak C.sub.id,M_cand,i of a candidate species M.sub.cand cannot be identified in the neutral mass spectrum I.sub.neut(p), the subscore s.sub.1,M_cand of the method is reduced. In particular for each peak C.sub.id,M_cand,i of a theoretical mass spectra I.sub.th,M_cand(p) of a candidate species M.sub.cand which cannot been identified in the neutral mass spectrum I.sub.neut(p) the subscore s.sub.1,M_cand of the first method is reduced. The reduction is for every identified peak C.sub.id,M_cand,I the same. There is no reduction if the intensity of the not identified peak C.sub.id,M_cand,i is below a threshold value. In general, the first method is taking care if expected peaks the theoretical mass spectra I.sub.th,M_cand(p) are found in the measured mass spectrum. If expected peaks are missing and in particular a lot of expected peaks are missing this is an indicator that the candidate species M.sub.cand is not abundant which is resulting in a lower score s.sub.1,M_cand.

(86) In a preferred embodiment as the first method to compare the neutral mass spectrum I.sub.neut(p) with each theoretical mass spectra I.sub.th,M_cand(p) of the candidate species M.sub.cand of the set S.sub.cand of candidate species of molecules M.sub.cand corresponding to the neutral mass spectrum I.sub.neut(p) the method described in the U.S. Pat. No. 8,831,888 B2 can be used. Then the first subscore S.sub.1,M_cand is the pattern spectral distance (PSD) calculated for the elemental composition of the candidate species of molecules M.sub.cand. A preferred use of this method will be described below for the third example, which can be applied in the same way in all inventive methods, in particularly in the inventive method of this second example.

(87) By the second method to compare the theoretical mass spectra I.sub.th,M_cand(p) of the candidate species of molecules M.sub.cand with neutral mass spectrum I.sub.neut(p) a second subscore s.sub.2,M_cand is determined for each candidate species M.sub.cand, wherein the second subscore s.sub.2,M_cand is addressing all peaks C.sub.meas,i in the neutral mass spectrum I.sub.neut(p), which are not identified in the theoretical mass spectrum I.sub.th,M_cand(p) of the candidate species M.sub.cand. So the second method is in particular sensitive in its subscore s.sub.2,M_cand for peaks C.sub.meas,i in the neutral mass spectrum I.sub.neut(p), which cannot be identified in the theoretical mass spectra I.sub.th,M_cand(p) of the candidate species of molecules M.sub.cand.

(88) When the second method recognizes that a peak C.sub.meas,i in the neutral mass spectrum I.sub.neut(p) cannot be identified in the theoretical mass spectra I.sub.th,M_cand(p) of the candidate species of molecules M.sub.cand, the subscore s.sub.2,M_cand of the method is reduced. In particular for each peak C.sub.meas,i in the neutral mass spectrum I.sub.neut(p) which cannot be identified in the theoretical mass spectra I.sub.th,M_cand(p) of the candidate species of molecules M.sub.cand the subscore s.sub.2,M_cand of the second method is reduced. The reduction is done for every not identified peak C.sub.neut,i in the same way. The reduction depends on the intensity of the not identified peak C.sub.neut,i found in the neutral mass spectrum I.sub.neut(p). There is no reduction if the intensity of the not identified peak C.sub.neut,i is below a threshold value. In general, the second method is taking care if measured peaks in the neutral mass spectrum I.sub.neut(p) are explained by in the theoretical mass spectrum I.sub.th,M_cand(p) of a candidate species M.sub.cand. If peaks are missing and in particular peaks of high intensity are missing this is an indicator that the candidate species M.sub.cand is not abundant which is resulting in a lower score s.sub.2,M_cand.

(89) For all candidate species M.sub.cand of the set S.sub.cand of candidate species of molecules M.sub.cand further in step (iv) of the inventive method a final score fs.sub.M_cand is calculated from the subscores s.sub.i,M_cand.

(90) In inventive method of this second example in step (iv) of the inventive method a final score fs.sub.M_cand is calculated from the subscores s.sub.i,M_cand only for all candidate species M.sub.cand, for which one of both of the subscores of the first method and the second method s.sub.1,M_cand and s.sub.1,M_cand are higher than an assigned threshold value s.sub.i,th,fs for calculating the final score fs.sub.M_cand. By this criteria the calculation of final scores fs.sub.M_cand is avoided, which due to the value of one subscore have no chance to belong to the final scores fs.sub.high,k having the highest values. The threshold values s.sub.i,th,fs are derived from a ranking of the candidate species M.sub.cand according to a subscore s.sub.i,M_cand. Then the value of the subscore s.sub.i,M_cand of the candidate species M.sub.cand on a specific rank is defining the threshold value s.sub.i,th,fs.

(91) The final score fs.sub.M_cand is calculated in the first example from a summation of functions only depending on one subscore s.sub.i,M_cand.

(92) Because only the subscores s.sub.1,M_cand and s.sub.2,M_cand are used then the final score fs.sub.M_cand of the first example is given by:
fs.sub.M_cand=f(s.sub.1,M.sub.cand)+g(s.sub.2,M.sub.cand)

(93) In a particular preferred embodiment of the inventive method of the first example the final score fs.sub.M_cand is be calculated from a summation of linear functions of the subscores s.sub.i,M_cand. Each function is defined by a weighting factor fi assigned to each subscore s.sub.i,M_cand. Then the final score fs.sub.M_cand is given by:
fs.sub.M_cand=f.sub.1*s.sub.1,M.sub.cand+f.sub.2*s.sub.2,M.sub.cand

(94) When the final score fs.sub.M_cand has been calculated for all candidate species M.sub.cand of the set S.sub.cand of candidate species of molecules M.sub.cand, in step (v) of the inventive method the final scores fs.sub.high from all final score fs.sub.M_cand of all candidate species M.sub.cand of the set S.sub.cand of candidate species of molecules M.sub.cand is determined, which has the highest value.

(95) Then in the next step (vi) of the inventive method the elemental composition of the candidate species M.sub.cand,high of the set S.sub.cand of candidate species of molecules M.sub.cand is determined which has the final scores fs.sub.high of the highest value. This is done by looking up the elemental composition of the candidate species M.sub.cand, if it is realised that the final scores fs.sub.M_cand is the final scores fs.sub.high of the highest value. The elemental composition of this candidate species M.sub.cand is then listed in a table of all molecules M contained in the investigated sample, for which the elemental composition has been identified with the inventive method of the second example, and shown on a display.

(96) A third example of the inventive method is identifying the most likely elemental compositions of at least one species of molecules M contained in an investigated sample.

(97) In step (i) of the third example of the inventive method mass spectrum I.sub.meas(m/z) of the investigated sample is measured with a mass spectrometer. In this mass spectrum the peak position p is directly given by a mass to charge ratio of ions detected in a mass spectrometer.

(98) If a mass spectrum of a sample shall be measured with a mass spectrometer, it is necessary to ionize to material of the sample, in particular the molecules M contained in the sample with an ionization process.

(99) In step (ii) of the inventive method at first a peak of interest C.sub.int in the measured mass spectrum I.sub.meas(m/z) is identified. This can be done manually by a user or according to some defined criteria like the intensity of the peak and the mass to charge ratio of the peak.

(100) Then the mass to charge ratio m/z.sub.meas,int of the peak of interest C.sub.int of the measured mass spectrum is reduced to its mass to charge ratio m/z.sub.neutral,int in the neutral mass spectrum I.sub.neut(m/z) derived by reduction of the measured mass spectrum I.sub.meas(m/z) according to the ionization process. To get the mass to charge ratio m/z.sub.neutral,int of the peak of interest C.sub.int in the neutral mass spectrum I.sub.neut(m/z) the mass to charge ratio m/z in the measured mass spectrum I.sub.meas(m/z) of ionized species of molecules M, the ions I, has to be shifted to its mass to charge ratio m/z.sub.neutral,int in the neutral mass spectrum I.sub.neut(m/z) of the neutral species of molecules M according to the mass shift, which has happened to the molecules M contained in the sample due to the ionization process.

(101) Further on a set S.sub.inv of species of molecules M.sub.inv has to be defined, for which molecules M.sub.inv it has to be investigated if the isotope distribution of their ions I.sub.in, occurs in the measured mass spectrum I.sub.meas(p).This set S.sub.inv of species of molecules M.sub.inv can be defined by a lot of criteria scribed above and according to the expectation which kind of species of molecules can be present in the investigated sample.

(102) In step (ii) of the inventive method it is determined a set S.sub.cand of candidate species of molecules M.sub.cand from the defined set S.sub.inv of species of molecules M.sub.inv which have an expected peak C.sub.ex,inv in a mass spectrum corresponding to the neutral mass spectrum I.sub.neut(m/z) with a mass to charge ratio m/z.sub.ex,inv within a mass to charge tolerance range m/z.sub.tol assigned to the peak of interest C.sub.int in the corresponding neutral mass spectrum I.sub.neut(m/z).

(103) The peak position p.sub.ex,inv of the expected peak C.sub.ex,inv has to be given by a mass spectrum which corresponds to the mass spectrum in which the peak of interest C.sub.int is identified.

(104) If the peak position p is the neutral mass spectrum I.sub.neut(m/z) is given by the mass to charge ratio m/z, then the peak position p.sub.ex,inv of the expected peak C.sub.ex,inv has a mass to charge value m/z.sub.ex,inv to be defined for a mass spectrum corresponding to the neutral mass spectrum I.sub.neut(m/z). In particular in this case only the mass to charge value m/z.sub.ex,inv of the expected peak C.sub.ex,inv has to be known. If the mass to charge value m/z.sub.ex,inv of a species of molecules M.sub.inv is within the mass to charge tolerance range m/z.sub.tol of the peak of interest C.sub.int then the species of molecules M.sub.inv is a candidate species of molecules M.sub.cand which will be investigated further.

(105) The expected peaks C.sub.ex,inv,I of species of molecules M.sub.inv are deduced from theory and it is possible to calculate a theoretical mass spectrum I.sub.th,M_inv. Methods to do these calculations are well known.

(106) The theoretical mass spectrum I.sub.th,M_inv(p) is a mass spectrum corresponding to a neutral mass spectrum I.sub.neut(m/z). It can be a calculated mass spectrum according to the expected isotope distribution of the molecule M.sub.inv. During the calculation the resolving power of the mass spectrometer measuring the measured mass spectrum I.sub.meas(m/z) can be taken into account. But also the expected centroid of an isotope of the candidate species M.sub.inv can be taken into account, which is a peak pattern. The theoretical mass spectrum I.sub.th,M_inv(m/z) can be also be a mass spectrum of the species of molecules M.sub.inv, which is stored in a database after it has been calculated. Because the elemental composition of species of molecules M contained in the sample shall be identified, which is a uncharged and therefore neutral molecule, a theoretical mass spectrum I.sub.th,M_inv(m/z) of the species of molecules M.sub.inv is used, which corresponds a the neutral mass spectrum I.sub.neut(m/z), which is achieved by the reduction of the measured mass spectrum I.sub.meas(m/z).

(107) In the step (ii) of the inventive method it is not necessary to know the mass spectrum I.sub.,M_inv(p) of the species of molecules M.sub.inv of the set S.sub.inv. The candidate species of molecules M.sub.cand must only have a peak position p.sub.ex,inv within the tolerance range p.sub.tol assigned to the peak of interest C.sub.int. Therefore it is only necessary to know the mass spectrum I.sub.M_inv(p) of the species of molecules M.sub.inv of the set S.sub.inv in the tolerance range p.sub.tol assigned to the peak of interest C.sub.int.

(108) It is also possible that for the species of molecules M.sub.inv the isotope distribution is deduced from a theoretical mass spectrum. By the peak pattern of the isotope distribution for each isotope n is identified in the mass spectrum a peak C.sub.ID,inv,n is given having a mass to charge ratio m/z.sub.ID,inv,n correlated to the abundance of the isotope n. Then the peaks C.sub.ID,inv,n of the isotope distribution of the species of molecules M.sub.inv can be used as expected peaks C.sub.ex,inv of the species of molecules M.sub.inv in step (ii) of the inventive method.

(109) A theoretical mass spectrum I.sub.th,M_inv(p) can be a calculated mass spectrum according to the expected isotope distribution of the molecule M.sub.inv. This calculation can be done for a complete expected isotope distribution or only for a part if the isotope distribution. The calculation can be limited to isotopes having an abundance higher than a threshold value. The calculation can be limited to a specific number of isotopes having the highest abundance and/or having the lowest mass in the mass spectrum.

(110) The tolerance range m/z.sub.tol assigned to the peak of interest C.sub.int is defined in the mass spectrum which is corresponding to the mass spectrum in which the expected peaks C.sub.ex,inv of the species of molecules M.sub.inv used in step (ii) are known. Because the elemental composition of species of molecules M.sub.cand contained in the sample shall be identified with the inventive method of the third example, the tolerance range m/z.sub.tol assigned to the peak of interest C.sub.int is defined in the neutral mass spectrum I.sub.neut(m/z).

(111) If the peak of interest C.sub.int is identified in the neutral mass spectrum I.sub.neut(m/z), the peak of interest C.sub.int has a mass to charge ratio m/z.sub.int,neut in the neutral mass spectrum I.sub.neut(m/z) and the tolerance range m/z.sub.tol is assigned to the peak of interest C.sub.int by a range around the peak position m/z.sub.int,neut of the peak of interest C.sub.int. Preferably the tolerance range m/z.sub.to, is symmetrically to the peak position m/z.sub.int,neut of the peak of interest C.sub.int, so that the distance between lower endpoint of the tolerance range m/z.sub.tol and the mass to charge ratio m/z.sub.int,neut of the peak of interest C.sub.int is equal to the distance between higher endpoint of the tolerance range m/z.sub.tol and the mass to charge ratio m/z.sub.int,neut of the peak of interest C.sub.int.

(112) Because the peak of interest C.sub.int is identified in the measured mass spectrum I.sub.meas(p), at first the mass to charge to charge ratio m/z.sub.int,meas of the peak of interest C.sub.int in the measured mass spectrum I.sub.meas(m/z) is identified and then reduced to the mass to charge ratio m/z.sub.int,neut of the peak of interest C.sub.int in the neutral mass spectrum I.sub.neut(m/z) due to the knowledge of the at least one ionization process applied to the investigated sample in the mass spectrometer before the measured mass spectrum I.sub.meas(m/z) has been measured. Then the tolerance range m/z.sub.tol is assigned to the peak of interest C.sub.int by a range around the peak position m/z.sub.int,neut of the peak of interest C.sub.int as described before.

(113) The identification mass spectra I.sub.id,M_cand(m/z) can be theoretical mass spectra I.sub.th,M_cand(m/z) of the candidate species of molecules. The theoretical mass spectra I.sub.th,M_cand(m/z) can be calculated during the execution of the method or can be calculated before and stored in a database. This database may be available in a storage of the used mass spectrometer or available via an internet connection in an external storage or a cloud system.

(114) In the inventive method of the third example in step (iii) for the candidate species M.sub.cand of the set S.sub.cand of candidate species of molecules M.sub.candtheoretical mass spectra I.sub.th,M_cand(m/z) are calculated which correspond to the measured mass spectrum I.sub.meas(m/z) and in step (ii) before for the candidate species M.sub.cand of the set S.sub.cand of candidate species of molecules M.sub.cand theoretical mass spectra I.sub.th,M_cand(m/z) may be calculated which correspond to the neutral mass spectrum I.sub.neut(m/z) wherein the theoretical mass spectra I.sub.th,M_cand(m/z) correspond to the measured mass spectrum I.sub.meas(m/z) are used in step (iii) as identification mass spectrum I.sub.id,M_cand(m/z).

(115) So the determination of identification mass spectra I.sub.id,M_cand(m/z) in step (iii) is done by the calculation theoretical mass spectra I.sub.th,M_cand(m/z) or the identification of the identification mass spectra I.sub.id,M_cand(m/z) in databases.

(116) in step (iii) for each candidate species M.sub.cand is determined an assigned ion I.sub.cand which is originated by at least one ionization process of the sample before the measurement of the mass spectrum I.sub.meas(m/z) and based on this assigned ions I.sub.cand for each candidate species M.sub.cand the complete theoretical mass spectrum I.sub.th,M_cand(m/zp) corresponding the measured mass spectrum I.sub.meas(m/z) is calculated or identified in databases.

(117) The identification mass spectra I.sub.id,M_cand(m/z) are complete mass spectra of the candidate species of molecules M.sub._cand showing the whole isotope distribution of the molecule only limited by the resolution and signal-to-noise ratio S/N under which the identification mass spectra I.sub.id,M_cand(m/z) are calculated.

(118) The identification mass spectra I.sub.id,M_cand(p) may be comprise only peaks of isotopes having an abundance higher than a threshold value.

(119) In step (iii) of the inventive method further a range of mass to charge ratios p is determined in which the determined identification mass spectra I.sub.id,M_cand(m/z) of all candidate species M.sub.cand of the set S.sub.cand of candidate species of molecules M.sub.cand are arranged. The range of mass to charge ratios p is determined by identifying a range of mass to charge ratios which is comprising the mass to charge ratios m/z.sub.th,M_cand,i of all peaks C.sub.th,M_cand,i of the complete identification mass spectra I.sub.id,M_cand(m/z) corresponding to the measured mass spectrum I.sub.meas(m/z) of all candidate species M.sub.cand of the set S.sub.cand of candidate species of molecules M.sub.cand. So in the range of mass to charge ratios p all peaks C.sub.th,M_cand,i of the identification mass spectra I.sub.id,M_cand(m/z) of all candidate species M.sub.cand of the set S.sub.cand of candidate species of molecules M.sub.cand are positioned in the range of mass to charge ratios p. The lower endpoint of the range of mass to charge ratios p is similar or below the lowest value of a mass to charge ratio P.sub.th,M_cand,i of any peak C.sub.th,M_cand,i of all candidate species M.sub.cand of the set S.sub.cand of candidate species of molecules M.sub.cand and the highest endpoint of the range of mass to charge ratios p is similar or above the highest value of a mass to charge ratio p.sub.th,M_cand,i of any peak C.sub.id,M_cand,I of all candidate species M.sub.cand of the set S.sub.cand of candidate species of molecules M.sub.cand. Because the identification mass spectra I.sub.id,M_cand(m/z) are a complete mass spectra of the candidate species of molecules M.sub.cand it is guaranteed that in the range of mass to charge ratios p all isotope distributions of all candidate species M.sub.cand of the set S.sub.cand of candidate species of molecules M.sub.cand are completely encompassed. In the range of mass to charge ratios p each identification mass spectrum I.sub.id,M_cand(m/z) of a candidate species of molecules M.sub.cand can be compared with the corresponding measured mass spectrum I.sub.meas(m/zp) without missing any peak of the candidate species of molecules M.sub.cand existing in its identification mass spectrum I.sub.id,M_cand(m/z).

(120) In step (iv) of the inventive method this comparison of the measured mass spectrum I.sub.meas(m/z) with each identification mass spectrum I.sub.id,M_cand(m/z) of the candidate species M.sub.cand of the set S.sub.cand of candidate species of molecules M.sub.cand corresponding to the measured mass spectrum I.sub.meas(m/z) is executed in the determined range of mass to charge ratios p.

(121) In the third example of the inventive method for each candidate species M.sub.cand of the set S.sub.cand of candidate species of molecules M.sub.cand determined before its identification mass spectrum I.sub.id,M_cand(m/z) corresponding to the measured mass spectrum I.sub.meas(m/z) is compared with the measured mass spectrum I.sub.meas(m/z).

(122) For all candidate species M.sub.cand this comparison is done with two different methods, a first method and a second method, having a different focus on the features of measured mass spectrum I.sub.meas(m/z) and the identification mass spectra I.sub.id,M_cand(m/z) of the candidate species of molecules M.sub.cand.

(123) It is also possible to use more than this two methods of comparison in step (iv) of the inventive method.

(124) By the first method to compare the identification mass spectra I.sub.id,M_cand(m/z) of the candidate species of molecules M.sub.cand with measured mass spectrum I.sub.meas(m/z) it is determined a first subscore s.sub.1,M_cand for each candidate species M.sub.cand. This first subscore s.sub.1,M_cand of the first method is addressing all peaks C.sub.id,M_cand,i in the identification mass spectrum I.sub.id,M_cand(m/z) of the candidate species M.sub.cand, which are not identified in the measured mass spectrum I.sub.meas(m/z). So the first method is in particular sensitive in its subscore s.sub.1,M_cand for peaks C.sub.id,M_cand,i of an identification mass spectrum I.sub.id,M_cand(m/z) of a candidate species M.sub.cand, which cannot be identified in the measured mass spectrum I.sub.meas(m/z).

(125) When the first method recognizes that is a peak C.sub.id,M_cand,i of an identification mass spectrum I.sub.id,M_cand(m/z) of a candidate species M.sub.cand cannot been identified in the measured mass spectrum I.sub.meas(m/z), the subscore s.sub.1,M_cand of the method is reduced. In particular for each peak C.sub.id,M_cand,i of an identification mass spectrum I.sub.id,M_cand(m/z) of a candidate species M.sub.cand which cannot been identified in the measured mass spectrum I.sub.meas(m/z) the subscore s.sub.1,M_cand of the first method is reduced. The reduction can be is for every not identified peak C.sub.id,M_cand,I the same. There is no reduction if the intensity of the not identified peak C.sub.id,M_cand,i is below a threshold value. In general, the first method is taking care if expected peaks the identification mass spectrum I.sub.id,M_cand(m/z) are found in the measured mass spectrum. If expected peaks are missing and in particular a lot of expected peaks are missing this is an indicator that the candidate species M.sub.cand is not abundant which is resulting in a lower score s.sub.1,M_cand.

(126) In a preferred embodiment as the first method to compare the measured mass spectrum I.sub.meas(m/z) with each identification mass spectrum I.sub.id,M_cand(m/z) of the candidate species M.sub.cand of the set S.sub.cand of candidate species of molecules M.sub.cand corresponding to the measured mass spectrum I.sub.meas(m/z) the method described in the U.S. Pat. No. 8,831,888 B2 can be used. Then the first subscore S.sub.1,M_cand is the pattern spectral distance (PSD) calculated for the elemental composition of the candidate species of molecules M.sub.cand. More details how to use this method in a favourable manner to improve further the inventive manner are described below showing measurement examples and the use of the method of the third example.

(127) By the second method to compare the identification mass spectra I.sub.id,M_cand(m/z) of the candidate species of molecules M.sub.cand with measured mass spectrum I.sub.meas(m/z) it is determined a second subscore s.sub.2,M_cand for each candidate species M.sub.cand, wherein the second subscore s.sub.2,M_cand is addressing all peaks C.sub.meas,i in the measured mass spectrum I.sub.meas(m/zp), which are not identified in the identification mass spectrum I.sub.id,M_cand(m/z) of the candidate species M.sub.cand. So the second method is in particular sensitive in its subscore s.sub.2,M_cand for all peaks C.sub.meas,i in the measured mass spectrum I.sub.meas(p), which cannot be identified in the identification mass spectra I.sub.id,M_cand(m/z) of the candidate species of molecules M.sub.cand.

(128) When the second method recognizes that is a peak C.sub.meas,i in the measured mass spectrum I.sub.meas(m/z) cannot been identified in the identification mass spectra I.sub.id,M_cand(m/z) of the candidate species of molecules M.sub.cand, the subscore s.sub.2,M_cand of the method is reduced. For each peak C.sub.meas,i in the measured mass spectrum I.sub.meas(m/z) which cannot been identified in the identification mass spectra I.sub.id,M_cand(m/z) of the candidate species of molecules M.sub.cand the subscore s.sub.2,M_cand of the second method is reduced. The reduction is for every not identified peak C.sub.meas,i in the same way. The reduction depends on the intensity of the not identified peak C.sub.meas,i found in the measured mass spectrum I.sub.meas(m/z). There is no reduction if the intensity of the not identified peak C.sub.meas,i is below a threshold value. In general, the second method is taking care if measured peaks in the measured mass spectrum I.sub.meas(m/z) are explained by in the identification mass spectrum I.sub.id,M_cand(m/z) of a candidate species M.sub.cand. If measured peaks are missing and in particular measured peaks of high intensity are missing or a lot of measured peaks are missing this is an indicator that the candidate species M.sub.cand is not abundant which is resulting in a lower score s.sub.2,M_cand.

(129) For all candidate species M.sub.cand of the set S.sub.cand of candidate species of molecules M.sub.cand further in step (iv) of the inventive method a final score fs.sub.M_cand is calculated from the subscores s.sub.i,M_cand.

(130) In the inventive method of the third example the final score fs.sub.M_cand can be calculated from a summation of only functions (e.g. f and g), which depend only on one subscore s.sub.i,M_cand. If only the subscores s.sub.1,M_cand and s.sub.2,M_cand are used then the final score fs.sub.M_cand is given by:
fs.sub.M_cand=f(s.sub.1,M.sub.cand)+g(s.sub.2,M.sub.cand)

(131) In an inventive method of the third example the final score fs.sub.M_cand can be calculated from a summation of linear functions of the subscores s.sub.i,M_cand. Each function is defined by a weighting factor fi assigned to each subscore s.sub.i,M_cand. If only the subscores s.sub.1,M_cand and s.sub.2,M_cand are used then the final score fs.sub.M_cand is given by:
fs.sub.M_cand=f.sub.1*s.sub.1,M.sub.cand+f.sub.2*S.sub.2,M.sub.cand

(132) When the final score fs.sub.M_cand has been calculated for the candidate species M.sub.cand of the set S.sub.cand of candidate species of molecules M.sub.cand, in step (v) of the inventive method the final score fs.sub.high from the final score fs.sub.M_cand of all candidate species M.sub.cand of the set S.sub.cand of candidate species of molecules M.sub.cand is determined, which has the highest value. So at the end the final score fs.sub.high having the highest values is identified.

(133) Then in the nest step (vi) of the inventive method the elemental composition of the candidate species M.sub.cand,high of the set S.sub.cand of candidate species of molecules M.sub.cand is determined which has the final scores fs.sub.high of the highest value. This is done by looking up the elemental composition of the candidate species M.sub.cand, if it is realised that the final scores fs.sub.M_cand is the final scores fs.sub.high of the highest value. The elemental composition of this candidate species M.sub.cand can then be listed with or without their final score fs.sub.M_cand in a table together with other candidate species M.sub.cand identified to be contained in the sample and shown on a display.

(134) It will now be shown on the basis of two examples of the measured mass spectra, how the inventive method and in particular the inventive method of the third example works and identifies the most likely composition of species of molecules M contained in an investigated samples in a much better way. Further on by the examples more details of the inventive method are explained, which can be used in every inventive method encompassed by the described invention:

(135) In the first measurement example a sample is investigated which is comprising the pesticide Imidachloprid as one of various pesticides. The species of molecules Imidachloprid has the molecular formula C.sub.9H.sub.10ClN.sub.5O.sub.2. The sample is introduced into a Orbitrap Elite mass spectrometer and ionized by electrospray ionisation before measuring a mass spectrum.

(136) A portion of the measured mass spectrum I.sub.meas_1(m/z) is shown in the FIGS. 1 to 3. In a range of mass to charge ratio range p defined below 7 peaks are identified, the peaks C.sub.m1,1, C.sub.m1,2 . . . C.sub.m1,7.

(137) If now the inventive method of the third example is applied to identify species of molecules M, at first a peak of interest C.sub.int has to be identified for which the elemental composition of the corresponding molecule M.sub.int has to be identified. In this example the peak of interest C.sub.int is the highest peak of an expected isotope distribution, the measured peak C.sub.m1,1. The set S.sub.inv of investigated species M.sub.inv is to limited by same criteria as described above when applying the inventive method of the third example: the used criteria are:

(138) The molecule may contain only the elements: H, C, N, O, S, P, CI, Br, F, Si, I

(139) The minimum number of atoms contained in the molecule for all of these elements is 0.

(140) Maximum number Max.sub.H of H atoms: 180

(141) Maximum number Max.sub.C of C atoms: 80

(142) Maximum number Max.sub.N of N atoms: 30

(143) Maximum number Max.sub.O of 0 atoms: 30

(144) Maximum number Max.sub.S of S atoms: 5

(145) Maximum number Max.sub.p of P atoms: 2

(146) Maximum number Max.sub.Cl of Cl atoms: 4

(147) Maximum number Max.sub.Br of Br atoms: 3

(148) Maximum number Max.sub.I of I atoms: 1

(149) Maximum number Max.sub.F of F atoms: 10

(150) Maximum number Max.sub.Si of Si atoms: 1

(151) Minimum value of the R/C ratio: 0.1

(152) Maximum value of the R/C ratio: 4.0

(153) Minimum value of RDBE: 0

(154) Maximum value of RDBE: 40

(155) To determine the set S.sub.cand of candidate species of molecules M.sub.cand form the set S.sub.inv in step (ii) to the peak of interest C.sub.m1,1 is assigned a mass to charge ratio range m/z.sub.tol which is correlated with a mass to charge tolerance of 5 ppm That means that every candidate species of molecules M.sub.cand has to have an expected peak C.sub.ex,inv within a mass to charge range tolerance of 5 ppm related to the mass to charge ratio value m/z.sub.m1,1 of the peak of interest C.sub.m1,1. Because the mass to charge ratio value m/z.sub.m1,1 is roughly 256.1 u, the mass to charge ratio range m/z.sub.to has a value of 0.00256 u and because this range is symmetric every candidate species of molecules has to have a peak whose mass to charge ratio should not differ from that of the peak of interest C.sub.m1,1 more than 0.00128 u.

(156) For the identified candidate species of molecules M.sub.cand a theoretical mass spectrum is calculated as identification mass spectrum I.sub.id,M_cand(m/z) taking into account the resolving power and peak shape of the used mass analyser and from the identification mass spectrum I.sub.id,M_cand of all candidate species M.sub.cand of mass to charge ratio range m/z is determined comprising all peaks C.sub.id,M_cand,i of the identification mass spectrum I.sub.id,M_cand of all candidate species M.sub.cand.

(157) Then in step (iv) these identification mass spectra I.sub.id,M_cand are compared with the measured mass spectrum I.sub.meas_1(m/z) in this mass to charge ratio range m/z. The first method to compare both spectra of the method to calculate a pattern spectral distance described in the U.S. Pat. No. 8,831,888 B2 as subscore S1, M_cand. The method is applied by allowing an mass to charge ratio error of 5 ppm of the expected peaks of the identification mass spectra I.sub.id,M_cand and an intensity error of 30% of the expected peak of the identification mass spectra I.sub.id,M_cand. The pattern spectral distance (PSD) is based on the assumption to identify the expected peaks of identification mass spectra I.sub.id,M_cand. Therefore a penalty value for the spectral distance (SD) of a non-identified expected peak of the identification mass spectra I.sub.id,M_cand(m/z) is given, which is defined to be 1. Due to this penalty for expected peaks of the identification mass spectra I.sub.id,M_cand(m/z) the method of the pattern spectral distance here is addressing to missing peaks in the identification mass spectra I.sub.id,M_cand not identified in the measured spectrum mass spectrum I.sub.meas_1(m/z)

(158) The results of the comparison with the method of the pattern spectral distance are shown in the FIGS. 1 to 3 for the candidate molecules M.sub.cand:

(159) FIG. 1 M1: C.sub.9H.sub.9F.sub.4NO.sub.3

(160) FIG. 2 M2: C.sub.9H.sub.10ClN.sub.5O.sub.2

(161) FIG. 3 M3: CsH.sub.10N.sub.5O.sub.3P

(162) For each expected peak of the identification mass spectra I.sub.id,M_cand of these molecules there is given a rectangle identification box by the allowed mass to charge ratio error of 5 ppm of the expected peaks (x direction, no appropriate dimension used for clarity) and the intensity error of 30% of the expected peaks (y direction). In FIG. 2 for the expected peaks C.sub.M2,3 and C.sub.M2,4 of the molecule M2 the rectangle identification boxes are labelled. If no measured peak is lying in a rectangle identification box of an expected peak, the expected peak is missing and penalized in calculating the score s.sub.1,M2. Only expected peaks which have an intensity of below a threshold of 3 times the S/N ratio of the measured mass spectrum I meas 1(m/z) are not penalized if they are missing.

(163) Because the measured peaks C.sub.m1,2 and C.sub.m1,3 have nearly the same mass to charge ratio, the area around the mass to charge ratio 257 u is enlarged in all three figures to show in detail the rectangle identification boxes of the expected peaks of the identification mass spectra I.sub.id,M_cand(m/z) at this mass to charge ratio.

(164) In table 1 is shown the calculated results for the candidate species M.sub.cand having the highest PSD values as score s.sub.1,M_cand.

(165) TABLE-US-00001 TABLE 1 Matched Missed Final PSD = expected expected score Ssubcore Rank Molecular formula isotopes isotopes s.sub.1 s.sub.1 1 C9 H9 F4 N O3 2 0 70.06 70.06 2 C7 H6 F N7 O3 3 0 67.62 67.62 3 C8 H10 N5 O3 P 3 0 59.90 59.90 4 C9 H18 Cl N O S2 4 1 45.10 45.10 .fwdarw. 5 C9 H10 Cl N5 O2 6 0 42.73 42.73 6 C8 H18 N O2 P S2 2 1 40.95 40.95 7 C7 H11 F6 N S 2 1 40.67 40.67 8 C3 H11 Cl N9 O P 4 1 38.56 38.56 9 C6 H12 F2 N5 P S 3 1 37.22 37.22 10 C6 H12 Cl F2 N5 Si 4 2 34.07 34.07

(166) Molecule M1 has rank 1, molecule M2 has rank 5 and molecule M3 has rank 3.

(167) In the FIGS. 1 to 3 is shown at first the master isotope, which has to fit the peak of interest C.sub.m1,1. The expected intensity of master isotope is set to be the same as the intensity of the peak of interest C.sub.m1,1 to normalize the intensity of the measured mass spectrum I.sub.meas_1(m/z) and the intensity of identification mass spectra I.sub.id,M_cand(m/z). Further it is shown which measured peaks C.sub.m1,i match with an expected peak C.sub.MX,j of the identification mass spectra I.sub.id,MX of the molecule MX (X=1,2,3) and its rectangle identification box. When a measured peaks C.sub.m1,i match with an expected peak C.sub.MX,j then the rectangle identification box is shown with a continuous line showing that an theoretical expected isotope is matched by the measured mass spectrum. When the expected intensity of an expected peak C.sub.MX,j is below the threshold of 3 times of the S/N ratio the minimum allowed expected intensity of an expected peak C.sub.MX,j is extended to 0. If no measured peaks C.sub.m1,i matches with such an expected peak C.sub.MX,j by lying in the rectangle identification box, then the rectangle identification box is shown with a dotted line showing that there is an optional expected peak C.sub.MX,j which is not matched but also not penalized due to the small value of the expected intensity of an expected peak C.sub.MX,j.

(168) It can be seen in the FIGS. 1 to 3, that for all three molecules M1, M2 and M3 the measured peaks C.sub.m1,i match with all expected peak C.sub.MX,j of the identification mass spectra I.sub.id,MX of the molecule MX (X=1,2,3) and its rectangle identification box, which otherwise would be penalized. There are only optional expected peak C.sub.MX,j which are not matched. But the number of matched measured peaks C.sub.m1,1 is different. 6 peaks C.sub.m,1, C.sub.m1,2, C.sub.m1,3, C.sub.m1,4, C.sub.m1,5 and C.sub.m1,6 of the measured mass spectrum I.sub.meas_1(m/z) match the expected peak C.sub.M2,j of the identification mass spectra I.sub.id,M2(m/z) of the molecule M2. 3 peaks C.sub.m1,1, C.sub.m1,2 and C.sub.m1,3 of the measured mass spectrum I.sub.meas_1(m/z) match the expected peak C.sub.M3,j of the identification mass spectra I(m/z).sub.id,M3 of the molecule M3. Only the two peaks C.sub.m1,1 and C.sub.m1,3 of the measured mass spectrum I.sub.meas_1(m/z) match the expected peak C.sub.M1,j of the identification mass spectra I(m/z).sub.id,M1 of the molecule M3. Particular the peak C.sub.m1,2 of the measured mass spectrum I.sub.meas_1(m/z) is not matching rectangle identification box of the expected peak C.sub.M1,2 of the identification mass spectra I.sub.id,M1 because the intensity of the measured peak C.sub.m1,2 is too high, so that this measured peak cannot be identified by the identification mass spectra I.sub.id,M1 of the molecule M1. Also the measured peaks C.sub.m1,4, C.sub.m1,5 and C.sub.m1,6 are not identified by the identification mass spectra I.sub.id,M1(m/z) of the molecule M1, which was possible by the identification mass spectra I.sub.id,M1(m/z) of the molecule M2. This shows that despite the highest PSD value of the molecule M1 this molecule may not the species of molecules contained in the sample. To improve the assignment of the measured peaks to the expected peaks further in the example of the measured mass spectrum I.sub.meas_1(m/z) an optional process of the inventive method is applied, a dynamic recalibration.

(169) In this process the mean value of the difference m/z.sub.recal of the mass to charge value of the expected peaks C.sub.M_cand,i and measured peaks c.sub.m1,j, which are assigned to each other is determined for each candidate molecule. The difference m/z.sub.recal is then added to each mass to charge value of the whole measured mass spectrum I.sub.meas_1(m/z) and then for the so shifted measured mass spectrum I.sub.shift_1(m/z)=I.sub.meas_1(m/z+m/z.sub.recal) the pattern spectral distance is calculated again resulting in the score s.sub.1,shift,M_cand.

(170) In table 2 is shown the calculated results for the candidate species M.sub.cand having the highest PSD values as score s.sub.1,shift,M_cand after the dynamic recalibration.

(171) TABLE-US-00002 TABLE 2 Matched Missed Final PSD = expected expected score Subcore Rank Molecular formula isotopes isotopes s.sub.1, shift s.sub.1, shift 1 C8 H10 N5 O3 P 3 0 84.85 84.85 2 C9 H9 F4 N O3 2 0 81.17 81.17 .fwdarw. 3 C9 H10 Cl N5 O2 6 0 77.28 77.28 4 C7 H6 F N7 O3 3 0 68.63 68.63 5 C9 H18 Cl N O S2 4 1 48.33 48.33 6 C7 H16 F N3 P2 S 3 1 45.62 45.62 7 C6 H12 F2 N5 P S 3 1 42.55 42.55 8 C8 H18 N O2 P S2 2 1 41.70 41.70 9 C7 H11 F6 N S 2 1 41.26 41.26 10 C7 H16 Cl F N3 P Si 4 2 40.00 40.00

(172) Now molecule M1 has rank 2, molecule M2 has rank 3 and molecule M3 has rank 1.

(173) But for molecule M3 the measured peaks C.sub.m1,4, C.sub.m1,5 and C.sub.m1,6 are not identified by the identification mass spectra I.sub.id,M3(m/z).

(174) Therefore in step (iii) of the third example of the inventive method the identification mass spectra I.sub.id,M_cand(m/z) are compared with the measured mass spectrum I.sub.meas_1(m/z) with a second method, which is addressing all those peaks C.sub.m1,i, which are not identified in the identification mass spectra I.sub.id,M_cand.

(175) The method is using an measured mass spectrum coverage score s.sub.2,M_cand. The score is given by the ratio, how much of the intensity of the measured peaks C.sub.m1,i in the mass to charge ratio range m/z is identified by an identification mass spectrum I.sub.id,M_cand(m/z). This is done by assigning to each measured peak C.sub.m1,i its centroid intensity I.sub.m1,i.

(176) For the measured mass spectrum I.sub.meas_1(m/z) shown in the FIGS. 1 to 3. In the mass to charge ratio range m/z 7 peaks are identified, the peaks C.sub.m1,1, C.sub.m1,2 . . . C.sub.m1,7 having the centroid intensities I.sub.m1,1, I.sub.m1,2 . . . I.sub.m1,7. If now only a subset C.sub.m1,a of these peaks C.sub.m1,a is identified by an identification mass spectra I.sub.id,M_cand of a candidate species M.sub.cand, at first the centroid intensities of these subset is summed up and then divided by the summed up centroid intensities I.sub.m1,1, I.sub.m1,2 . . . I.sub.m1,7 of all peaks C.sub.m1,1, C.sub.m1,2 . . . C.sub.m1,7 identified in the measured mass spectrum I.sub.meas_1(m/z).

(177) In this third example of the inventive method it is already defined by the first method to compare the identification mass spectra I.sub.id,M_cand(m/z) with the measured mass spectrum I.sub.meas_1(m/z), which measured peaks C.sub.m1,i in the mass to charge ratio range m/z are identified by an identification mass spectra I.sub.id,M_cand(m/z). If for example for a species of molecules M.sub.ex only the measured peaks C.sub.m1,1, C.sub.m1,3, C.sub.m1,4 and C.sub.m1,6 are identified by an identification mass spectra I.sub.id,M_ex(m/z), then the measured mass spectrum coverage score s.sub.2,M_ex is calculated by the formula:

(178) $s_{2,\mathrm{M\_ex}}=\frac{I_{m1,1}+I_{m1,3}+I_{m1,4}+I_{m1,6}}{\underset{k=1}{\overset{7}{.Math.}}{I}_{m1,k}}$

(179) In table 3 is shown the calculated results for the candidate species M.sub.cand having the highest final score fs.sub.M_cand calculated from PSD values as score s.sub.1,shift,M_cand after the dynamic recalibration and measured mass spectrum coverage (MS coverage) score s.sub.2,M_cand.

(180) TABLE-US-00003 TABLE 3 MS Matched Missed Final PSD = coverage expected expected score Subscore score Rank Molecular formula isotopes isotopes fs s.sub.1, shift S.sub.2 .fwdarw.1 C9 H10 Cl N5 O2 6 0 91.47 77.28 92.26 2 C9 H18 Cl N O S2 4 1 88.79 48.33 91.04 3 C7 H16 Cl F N3 P Si 4 2 88.35 40.00 91.04 4 C6 H12 Cl F2 N5 Si 4 2 88.21 37.28 91.04 5 C8 H18 Cl N O2 S Si 4 3 87.93 31.94 91.04 6 C3 H11 Cl N9 O P 4 1 82.52 38.40 84.97 7 C8 H10 N5 O3 P 3 0 71.63 84.85 70.90 8 C7 H6 F N7 O3 3 0 70.78 68.63 70.90 9 C9 H9 F4 N O3 2 0 70.54 81.17 69.95 10 C7 H16 F N3 P2 S 3 1 69.57 45.62 70.90

(181) The final score fs.sub.M_cand of the candidate species of molecules M.sub.cand is calculated by the formula:

(182) $f_{S_{\mathrm{M\_cand}}}=\frac{0.05*s_{1,\mathrm{shift},M_{\mathrm{cand}}}+0.9*S_{2,\mathrm{M\_cand}}}{0.05+0.9}$

(183) In table 3 it is shown, the species of molecules M2 has the highest final score and that the species of molecules M1 and M3 have only the ranks 9 and 7. By using the second method of comparison which is taking into account all peaks of the measured mass spectrum I.sub.meas_1(m/z) which are not identified in the identification mass spectra I.sub.id,M_cand(m/z) of a candidate species of molecules M.sub.cand now the species of molecules M2 has been identified to have the most likely elemental composition from which the isotope distribution comprising the peak of interest C.sub.int is originated. Only by the use of the second method of comparison it is possible to realize that a lot of measured peaks in the measured mass spectrum I.sub.meas_1(m/z) have not been identified by the identification mass spectra I.sub.id,M_cand(m/z) of species of molecules M2 and M3. Only the appropriate combination of different scores and an appropriate formula of to calculate the final score improves the identification of the most likely formula of a species of molecules from its isotope distribution measured by a mass spectrometer.

(184) This identified elemental composition as the most likely elemental composition of a molecule contained in the sample can be stored and displayed for further use. In this example it is shown that the inventive method alone is able to identify the correct elemental composition of the species of molecules Imidachloprid having the molecular formula C.sub.9H.sub.10ClN.sub.5O.sub.2 contained in the investigated sample.

(185) In the second measurement example a Sulfentrazone sample is investigated. The species of molecules Sulfentrazone has the molecular formula C.sub.11H.sub.10Cl.sub.2F.sub.2N.sub.4O.sub.3S. The sample is introduced after infusion into a Q Exactive Orbitrap mass spectrometer and ionized by electrospray ionisation before measuring a mass spectrum.

(186) A portion of the measured mass spectrum I.sub.meas_2(m/z) is shown in the FIGS. 4 and 5. In a range of mass to charge ratio range p defined below 10 peaks are identified, the peaks C.sub.m2,1, C.sub.m2,2 . . . C.sub.m2,10.

(187) If now the inventive method of the third example is applied to identify species of molecules M, at first a peak of interest C.sub.int has to be identified for which the elemental composition of the corresponding molecule M.sub.int has to be identified. In this example the peak of interest C.sub.int is the like mostly the highest peak of an expected isotope distribution, the measured peak C.sub.m2,1. The set S.sub.inv of investigated species M.sub.inv is to limited by same criteria as described above when applying the inventive method of the third example: the used criteria are:

(188) The molecule may contain only the elements: H, C, N, O, S, P, Cl, Br, F, Si, I

(189) The minimum number of atoms contained in the molecule for all of these elements is 0.

(190) Maximum number Max.sub.H of H atoms: 180

(191) Maximum number Max.sub.C of C atoms: 80

(192) Maximum number Max.sub.N of N atoms: 30

(193) Maximum number Max.sub.O of 0 atoms: 30

(194) Maximum number Max.sub.S of S atoms: 5

(195) Maximum number Max.sub.P of P atoms: 2

(196) Maximum number Max.sub.Cl of Cl atoms: 4

(197) Maximum number Max.sub.Br of Br atoms: 3

(198) Maximum number Max.sub.I of I atoms: 1

(199) Maximum number Max.sub.F of F atoms: 10

(200) Maximum number Max.sub.Si of Si atoms: 1

(201) Minimum value of the R/C ratio: 0.1

(202) Maximum value of the R/C ratio: 4.0

(203) Minimum value of RDBE: 0

(204) Maximum value of RDBE: 40

(205) To determine the set S.sub.cand of candidate species of molecules M.sub.cand form the set S.sub.inv in step (ii) to the peak of interest C.sub.m2,1 is assigned a mass to charge ratio range m/z.sub.tol which is correlated with a mass to charge tolerance of 5 ppm That means that every candidate species of molecules M.sub.cand has to have an expected peak C.sub.ex,inv within a mass to charge range tolerance of 5 ppm related to the mass to charge ratio value m/z.sub.m2,1 of the peak of interest C.sub.m2,1. Because the mass to charge ratio value m/z.sub.m1,1 is roughly 387 u, the mass to charge ratio range m/z.sub.tol has a value of 0.00387 u and because this range is symmetric every candidate species of molecules has to have a peak whose mass to charge ratio should not differ from that of the peak of interest C.sub.m2,1 more than 0.001935 u.

(206) For the identified candidate species of molecules M.sub.cand a theoretical mass spectrum is calculated as identification mass spectrum I.sub.id,M_cand(m/z) and from the identification mass spectrum I.sub.id,M_cand of all candidate species M.sub.cand of mass to charge ratio range m/z is determined comprising all peaks C.sub.id,M_cand,i of the identification mass spectrum I.sub.id,M_cand of all candidate species M.sub.cand.

(207) Then in step (iv) these identification mass spectra I.sub.id,M_cand are compared with the measured mass spectrum I.sub.meas_2(m/z) in this mass to charge ratio range m/z. The first method to compare both spectra of the method to calculate a pattern spectral distance described in the U.S. Pat. No. 8,831,888 B2 as subscore S.sub.1,M_cand. The method is applied by allowing an mass to charge ratio error of 5 ppm of the expected peaks of the identification mass spectra I.sub.id,M_cand and an intensity error of 30% of the expected peak of the identification mass spectra I.sub.id,M_cand. The pattern spectral distance (PSD) is based on the assumption to identify the expected peaks of identification mass spectra I.sub.id,M_cand. Therefore a penalty value for the spectral distance (SD) of a non-identified expected peak of the identification mass spectra I.sub.id,M_cand(m/z) is given, which is defined to by 1. Due to this penalty for expected peaks of the identification mass spectra I.sub.id,M_cand(m/z) the method of the pattern spectral distance here is addressing to missing peaks in the identification mass spectra I.sub.id,M_cand not identified in the measured spectrum mass spectrum I.sub.meas_2(m/z)

(208) The results of the comparison with the method of the pattern spectral distance are shown in the FIGS. 4 to 5 for the candidate molecules M.sub.cand:

(209) FIG. 4 M4: C.sub.9H.sub.12Cl.sub.2F.sub.3N.sub.2O.sub.5P

(210) FIG. 5 M2: C.sub.11H.sub.10Cl.sub.2F.sub.2N.sub.4O.sub.3S

(211) For each expected peak of the identification mass spectra I.sub.id,M_cand of these molecules there is given a rectangle identification box by the allowed mass to charge ratio error of 5 ppm of the expected peaks (x direction, no appropriate dimension used for clarity) and the intensity error of 30% of the expected peaks (y direction). In FIG. 4 for the expected peaks C.sub.M4,4 and C.sub.M4,5 of the molecule M4 the rectangle identification boxes are labelled. If no measured peak is lying in an rectangle identification box of expected peak, the expected peak is missing and penalized in calculating the score s.sub.1,M4. Only expected peaks which have an intensity of below a threshold of 3 times of the S/N ratio of the measured mass spectrum I.sub.meas_2(m/z) are not penalized if they are missing.

(212) In table 4 is shown the calculated results for the candidate species M.sub.cand having the highest PSD values as subscore s.sub.1,M_cand.

(213) TABLE-US-00004 TABLE 4 Matched Missed Final PSD = expected expected score subscore Rank Molecular formula isotopes isotopes s.sub.1 s.sub.1 1 C9 H12 Cl2 F3 N2 O5 P 8 0 82.99 82.99 2 C9 H7 F7 N2 O3 P2 3 0 79.00 79.00 3 C12 H17 Cl2 I N2 8 0 78.78 78.78 4 C8 H4 F6 N2 O9 3 0 77.88 77.88 5 C8 H3 F8 N4 O3 P 3 0 75.84 75.84 6 C7 H18 F I N2 O3 P2 3 0 74.81 74.81 7 C10 H16 Cl2 F2 O5 P2 7 0 73.72 73.72 8 C10 H17 I N2 O2 P2 3 0 73.21 73.21 9 C14 H6 Cl2 F6 N2 8 0 71.12 71.12 .fwdarw. 10 C11 H10 Cl2 F2 N4 O3 S 8 0 68.95 68.95

(214) Molecule M4 has rank 1 and molecule M5 has rank 10.

(215) In the FIGS. 4 and 5 is shown at first the master isotope, which has to fit the peak of interest C.sub.m2,1. The expected intensity of master isotope is set to be the same as the intensity of the peak of interest C.sub.m2,1 to normalize the intensity of the measured mass spectrum I.sub.meas_2(m/z) and the intensity of identification mass spectra I.sub.id,M_cand(m/z). Further it is shown which measured peaks C.sub.m2,i match with an expected peak C.sub.MX,j of the identification mass spectra I.sub.id,MX of the molecule MX (X=4,5) and its rectangle identification box. When a measured peaks C.sub.m2,i match with an expected peak C.sub.MX,j then the rectangle identification box is shown with a continuous line showing that an theoretical expected isotope is matched by the measured mass spectrum. When the expected intensity of an expected peak C.sub.MX,j is below the threshold of 3 times of the S/N ratio the minimum allowed expected intensity of an expected peak C.sub.MX,j is extended to 0. If no measured peaks C.sub.m2,i matches with such an expected peak C.sub.MX,j by lying in the rectangle identification box, then the rectangle identification box is shown with a dotted line showing that there is an optional expected peak C.sub.MX,j which is not matched but also not penalized due to the small value of the expected intensity of an expected peak C.sub.MX,j.

(216) It can be seen in the FIGS. 4 and 5, that for both molecules M4 and M5 the measured peaks C.sub.m2,i match with all expected peak C.sub.MX,j of the identification mass spectra I.sub.id,MX of the molecule MX (X=1,2,3) and its rectangle identification box, which otherwise would be penalized. There are only optional expected peak C.sub.MX,j which are not matched. The number of matched measured peaks C.sub.m2,I is for both species of molecules M4 and M5 the same. 8 peaks C.sub.m2,1, C.sub.m2,2, C.sub.m2,3, C.sub.m2,4, C.sub.m,5, C.sub.m2,6, C.sub.m2,7 and C.sub.m2,9 of the measured mass spectrum I.sub.meas_2(m/z) match the expected peak C.sub.M4,j of the identification mass spectra I.sub.id,M4(m/z) of the molecule M4. 8 peaks C.sub.m2,1, C.sub.m2,2, C.sub.m2,3, C.sub.m2,4, C.sub.m,5, C.sub.m2,6, C.sub.m2,7 and C.sub.m2,9 of the measured mass spectrum I.sub.meas_2(m/z) match the expected peak C.sub.M5,j of the identification mass spectra I(m/z).sub.id,M5 of the molecule M5.

(217) Then in step (iii) of the third example of the inventive method the identification mass spectra I.sub.id,M_cand(m/z) are compared with the measured mass spectrum I.sub.meas_2(m/z) with a second method, which is addressing all those measured peaks C.sub.m2,i, which are not identified in the identification mass spectra I.sub.id,M_cand.

(218) The method is using an measured mass spectrum coverage score s.sub.2,M_cand as already described in the first measurement example before. There score is given by the ratio, how much of the intensity of the measured peaks C.sub.m2,i in the mass to charge ratio range m/z is identified by an identification mass spectra I.sub.id,M_cand(m/z). This is done by assigning to each measured peak C.sub.m2,i its centroid intensity I.sub.m2,i.

(219) In this third example of the inventive method it is already defined by the first method to compare the identification mass spectra I.sub.id,M_cand(m/z) with the measured mass spectrum I.sub.meas_2(m/z), which measured peaks C.sub.m2,i in the mass to charge ratio range m/z are identified by an identification mass spectra I.sub.id,M_cand(m/z).

(220) In table 5 is shown the calculated results for the candidate species M.sub.cand having the highest final score fs.sub.M_cand calculated from PSD values as subscore S.sub.1,M_cand and the measured mass spectrum coverage (MS coverage) score s.sub.2,M_cand.

(221) TABLE-US-00005 TABLE 5 MS Matched Missed Final PSD = coverage expected expected score Subscore score Rank Molecular formula isotopes isotopes fs s.sub.1 S.sub.2 1 C9 H12 Cl2 F3 N2 O5 P 8 0 97.74 82.99 98.56 2 C12 H17 Cl2 I N2 8 0 97.54 78.78 98.58 3 C14 H6 Cl2 F6 N2 8 0 97.13 71.12 98.58 .fwdarw.4 C11 H10 Cl2 F2 N4 O3 S 8 0 97.02 68.95 98.58 5 C8 H8 Cl2 F4 N4 O5 8 0 96.96 68.20 98.56 6 C13 H7 Cl2 F2 N6 P 8 0 96.90 66.57 98.58 7 C10 H16 Cl2 F2 O5 P2 7 0 96.89 73.72 98.18 8 C13 H8 Cl2 N4 O6 8 0 96.62 61.41 98.58 9 C9 H12 Cl2 F4 N4 S2 8 0 96.58 60.61 98.58 10 C14 H11 Cl2 F N4 P2 8 0 96.55 59.92 98.58

(222) The final score fs.sub.M_cand of the candidate species of molecules M.sub.cand is calculated by the formula:

(223) $f_{S_{\mathrm{M\_cand}}}=\frac{0.05*s_{1,\mathrm{shift},M_{\mathrm{cand}}}+0.9*S_{2,\mathrm{M\_cand}}}{0.05+0.9}$

(224) In table 5 it is shown, the species of molecules M4 has the highest final score and that the species of molecules M5 has only the rank 4. The final score values fs.sub.M4 and fs.sub.M5 have only a small difference.

(225) Therefore an option of the inventive method is used to distinguish which of both molecules M4 and M5 has the more likely elemental composition. A third subscore s.sub.3,M_cand is determined for each candidate species of molecules M.sub.cand by a further fragmentation experiment. The ions having the mass to charge ratio of the peak of interest C.sub.int are isolated and fragmented by a known fragmentation process in the fragmentation experiment of then the mass spectrum (MS.sup.2 spectrum) of the fragments is detected.

(226) In the FIGS. 6 and 7 the measured MS.sup.2 spectrum of the ions I.sub.meas_3(m/z) having the mass to charge ratio of the peak of interest C.sub.int of the mass spectrum shown in the FIGS. 4 and 5 is shown.

(227) Then the fragments shown in this measured MS.sup.2 spectrum are compared with an MS.sup.2 identification spectrum of each candidate species of molecules M.sub.cand resulting in the subscore s.sub.3. This MS.sup.2 identification spectrum of each candidate species of molecules M.sub.cand is given due to the theoretical knowledge about the fragmentation during the used fragmentation process.

(228) The method is using an measured MS.sup.2 spectrum coverage score S.sub.3,M_cand which is the same as the measured mass spectrum coverage score, but now applied to the MS.sup.2 spectrum. There score is given by the ratio, how much of the intensity of the measured peaks C.sub.m3,i in the mass to charge ratio range m/z is identified by an identification mass spectra I.sub.id,M_cand(m/z). This is done by assigning to each measured peak C.sub.m3,i its centroid intensity I.sub.m3,i.

(229) For the MS.sup.2 mass spectrum I.sub.meas_3(m/z) shown in the FIGS. 4 and 5 a lot of the peaks of fragments are shown. If now only a subset C.sub.m3,a of these peaks C.sub.m3,i is identified by an MS.sup.2 identification mass spectrum I.sub.id_MS2,M_cand of an candidate species M.sub.cand, at first the centroid intensities of these subset is summed up and then divided by the summed up centroid intensities I.sub.m3,i of all peaks C.sub.m3,i identified in the measured MS.sup.2 mass spectrum I.sub.meas_3(m/z).

(230) In table 6 is shown the calculated results for the candidate species M.sub.cand having the highest final score fs.sub.M_cand calculated from PSD values as subscore S.sub.1,M_cand, the measured mass spectrum coverage (MS coverage) score s.sub.2,M_cand and the measured MS.sup.2 spectrum coverage score s.sub.3,M_cand.

(231) TABLE-US-00006 TABLE 6 MS MS.sup.2 Matched Missed Final PSD = coverage coverage expected expected score Subscore score score Rank Molecular formula isotopes Isotopes fs s.sub.1 S.sub.2 S.sub.3 .fwdarw.1 C11 H10 Cl2 F2 N4 O3 S 8 0 97.93 68.95 98.58 99.66 2 C12 H11 Cl2 F2 N2 O4 P 8 0 97.19 52.32 98.56 99.21 3 C11 H14 Cl2 F N2 O4 P Si 7 0 96.99 61.99 98.16 98.39 4 C10 H10 Cl2 F2 N4 O4 Si 7 2 96.83 48.30 98.16 99.27 5 C9 H17 Cl F2 N2 O2 S4 7 0 96.63 39.26 98.18 99.59 6 C14 H9 Cl2 F N4 O2 S 8 0 96.46 55.91 98.58 96.70 7 C9 H16 Cl2 F3 N2 O P S Si 7 2 96.44 46.90 98.16 98.31 8 C10 H13 Cl2 F4 N2 O P S 7 0 96.40 44.83 98.16 98.39 9 C13 H9 Cl2 F N4 O3 Si 7 0 96.03 54.17 98.16 96.39 10 C12 H14 Cl2 F N2 O3 P S 7 0 95.96 57.83 98.18 95.77

(232) The final score fs.sub.M_cand of the candidate species of molecules M.sub.cand is calculated by the formula:
fs.sub.M_cand=0.05*s.sub.1,M_cand+0.9*s.sub.2,M_cand+0.05*s.sub.3,M_cand

(233) In table 6 it is shown, the species of molecules M4 is not ranked in the highest final scores and that the species of molecules M5 has now the rank 1. Due to the MS.sup.2 spectrum coverage score s.sub.3,M_cand there is now a big different in the final scores of both molecules. The explanation for this is, that the MS.sup.2 identification mass spectrum I.sub.id_MS2,M4 of the species of molecules M4 does not fit with the measured MS.sup.2 mass spectrum I.sub.meas_3(m/z). In the FIGS. 4 and 5 it is shown which fragments of the measured MS.sup.2 mass spectrum I.sub.meas_3(m/z) can be explained by the MS.sup.2 identification mass spectra I.sub.id_MS2,M5 and I.sub.id_MS2,M4. For the species of molecules M4 the important measured peaks C.sub.m3,A and C.sub.m3,B of high intensity are not explained, which is resulting in a lower measured MS.sup.2 spectrum coverage score s.sub.3,M4. For the species of molecules M4 the important measured peaks C.sub.m3,A and C.sub.m3,B of high intensity are explained resulting in a high measured MS.sup.2 spectrum coverage score s.sub.3,M5 and accordingly the highest final score fs.sub.M5.

(234) This identified elemental composition as the most likely elemental composition of a molecule contained in the sample can be stored and displayed for further use. In this example it is shown that the inventive method alone using the option to take into account a further score from a measured MS.sup.2 mass spectrum is able to identify the correct elemental composition of the species of molecules Sulfentrazone having molecular formula C.sub.11H.sub.10Cl.sub.2F.sub.2N.sub.4O.sub.3S.

(235) By repeating the procedure of the steps of the inventive method one or more most likely elemental composition of several species of molecules M can be identified when the identification is done in every repetition for another peak of interest C.sub.int.

(236) The inventive method is preferably executed by at least one processor of a controlling system, preferentially of the mass spectrometer used to measure the mass spectrum I.sub.meas(p), a local computer or a computer in a computer network or processors in a cloud system.

(237) To the content of this description of the invention belong also all embodiments which are combinations of the before mentioned embodiments of the invention. So all embodiments are encompassed which comprise a combinations of features described just for single embodiments before.