Method and apparatus for isotope ratio mass spectrometry

Abstract

A method of isotope ratio mass spectrometry comprising: flowing a liquid mobile phase through a separation device; reducing the flow rate of the mobile phase through the separation device for at least a portion of time that at least one molecular species is emerging from the separation device to achieve a desired isotope ratio precision, wherein the flow rate is reduced from a first rate to a second rate corresponding to a higher theoretical plate height of the separation device; and mass analyzing the molecular species that has emerged from the separation device at least while the flow rate is reduced; and determining at least one isotope ratio from the intensities of mass peaks of at least two isotopologues, wherein the mass analysis is performed with mass resolving power high enough to resolve the two most abundant mass peaks at the nominal mass of at least one of the isotopologues.

Claims

1. A method of isotope ratio mass spectrometry comprising: flowing a liquid mobile phase that contains a sample through a separation device at a first flow rate, the sample comprising at least one molecular species having an isotope ratio to be determined, wherein the first flow rate is at least 50% of an optimum flow rate corresponding to a minimum theoretical plate height of the separation device; reducing the flow rate of the liquid mobile phase flowing through the separation device from the first flow rate to a second flow rate that is lower than the first flow rate but corresponds to a higher theoretical plate height of the separation device for at least a portion of time that the at least one molecular species is emerging from the separation device, the second flow rate is selected to produce a desired isotope ratio precision; mass analyzing the at least one molecular species that has emerged from the separation device at least while the flow rate is reduced to the second flow rate; and determining from the mass analysis at least one isotope ratio of the at least one molecular species from the intensities of mass peaks of at least two isotopologues of the at least one molecular species, wherein the mass analysis is performed with mass resolving power high enough to resolve the two most abundant mass peaks at the nominal mass of at least one of the isotopologues.

2. The method of claim 1 wherein the separation device is a liquid chromatography column, size-exclusion chromatography (SEC) column, ion chromatography (IC) column, thin-layer chromatography (TLC) plate, or capillary electrophoresis (CE) system.

3. The method of claim 1 wherein the separation device is a liquid chromatography column, wherein the column has an internal diameter and wherein reducing the flow rate comprises reducing the flow rate from the first rate to the second flow rate having a value in mL/min that is less than half the value given by 0.06(internal diameter in mm).sup.2.

4. The method of claim 1 wherein the liquid mobile phase comprises an organic solvent.

5. The method of claim 1 wherein the flow rate is reduced to the second flow rate for at least substantially the whole of the time that the at least one molecular species is eluting from the separation device.

6. The method of claim 1 wherein the isotope ratio is determined with an isotope ratio precision of <20 .

7. The method of claim 1 wherein the flow rate is increased from the second flow rate once the at least one molecular species has substantially finished eluting from the separation device.

8. The method of claim 1 wherein the second flow rate is reduced by a factor of at least 5 compared to the first flow rate.

9. The method of claim 1 wherein the first flow rate is at least 80% of the optimum flow rate and the second flow rate is less than 20% of the optimum flow rate, wherein the second flow rate is reduced relative to the first rate by a factor of at least 5.

10. The method of claim 1 wherein the flow rate is reduced from the first flow rate to the second flow rate by reducing a pump speed of a pump that is flowing the liquid mobile phase through the separation device.

11. The method of claim 1 wherein the flow rate is reduced from the first flow rate to the second flow rate by splitting the flow of mobile of phase upstream of the separation device so that a reduced flow rate of mobile phase passes through the separation device.

12. The method of claim 1 wherein the mass analysis is performed by a high resolution accurate mass (HR-AM) mass spectrometer.

13. The method of claim 1 wherein the mass analysis is performed with a mass resolving power high enough to resolve the two most abundant A+1 isotopologues and/or the two most abundant A+2 isotopologues of the at least one molecular species, where A is the monoisotopic mass peak.

14. The method of claim 1 wherein the mass analysis is performed with a resolving power of at least 50,000.

15. The method of claim 1 wherein the mass analysis is performed using a mass spectrometer comprising a mass analyzer selected from: an electrostatic orbital trap mass analyzer, an FT-ICR mass analyzer, or a time of flight TOF mass analyzer.

16. The method of claim 1 wherein the mass analysis comprises ionizing the at least one molecular species before ejecting the ions to a mass analyzer for mass analysis.

17. The method of claim 16 wherein the mass analysis comprises fragmenting the ionized at least one molecular species prior to mass analysis.

18. The method of claim 17 wherein determining at least one isotope ratio of the at least one molecular species comprises determining position specific information about the determined isotope ratio.

19. The method of claim 1 wherein determining at least one isotope ratio of the at least one molecular species comprises comparing the intensity of a monoisotopic mass peak A with an A+1 mass peak or A+2 mass peak to provide an isotope ratio of a light isotope and a heavy isotope of interest.

20. The method of claim 19 wherein the mass analysis resolves two or more A+1 isotopologues and/or two or more A+2 isotopologues.

21. The method of claim 19 wherein the light isotope is selected from .sup.12C, .sup.14N, .sup.16O, .sup.1H, .sup.32S, .sup.35Cl, .sup.79Br, .sup.28Si and the heavy isotope is selected from .sup.13C, .sup.14C, .sup.15N, .sup.17O, .sup.18O, .sup.2H, .sup.34S, .sup.37Cl, .sup.81Br, .sup.29Si, .sup.30Si.

22. The method of claim 1 wherein determining at least one isotope ratio of the at least one molecular species comprises comparing the intensity of two peaks having the same nominal mass, being either two A+1 mass peaks or two A+2 mass peaks.

23. The method of claim 1, further comprising calibrating the at least one isotope ratio determined from the intensities of mass peaks of at least two isotopologues against one or more isotope ratios for one or more known standards.

24. A method of isotope ratio mass spectrometry comprising: flowing a liquid mobile phase that contains a sample through a separation device at a first flow rate, the sample comprising at least one molecular species having an isotope ratio to be determined, wherein the first flow rate is at least 50% of an optimum flow rate corresponding to a minimum theoretical plate height of the separation device; reducing the flow rate of the liquid mobile phase flowing through the separation device from the first flow rate to a second flow rate that is lower than the first flow rate but corresponds to a higher theoretical plate height of the separation device for at least a portion of time that the at least one molecular species is emerging from the separation device, the second flow rate is selected to produce a desired isotope ratio precision; mass analyzing the at least one molecular species that has emerged from the separation device at least while the flow rate is reduced to the second flow rate; and determining from the mass analysis at least one isotope ratio of the at least one molecular species from the intensities of mass peaks of at least two isotopologues of the at least one molecular species, wherein each isotopologue mass peak used for the isotope ratio determination is resolved from at least any other mass peaks at the same nominal mass which are more than 20% of the intensity of the isotopologue mass peak.

25. An apparatus for isotope ratio mass spectrometry comprising: a separation device for separating components of a sample in a liquid mobile phase, the components of the sample comprising at least one molecular species having an isotope ratio to be determined; a mass spectrometer coupled to the separation device downstream for mass analyzing the at least one molecular species as the at least one molecular species elutes from the separation device and determining from the mass analysis at least one isotope ratio of the at least one molecular species, wherein the mass analysis is performed with mass resolving power high enough to resolve the two most abundant mass peaks at the nominal mass of at least one of the isotopologues; and a controller configured to control the flow of the liquid mobile phase through the separation device, to flow the liquid mobile phase through the separation device for a first portion of time at a first flow rate and to reduce the flow rate of the liquid mobile phase through the separation device from the first flow rate to a second flow rate lower than the first flow rate for at least a portion of time that the at least one molecular species is eluting from the separation device, the second flow rate is selected to produce a desired isotope ratio precision.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 shows a flow diagram schematically showing an embodiment of the invention.

(2) FIG. 2 shows schematically an embodiment of an apparatus for LC/MS for use in the invention.

(3) FIG. 3 shows schematically another embodiment of an apparatus for LC/MS for use in the invention.

(4) FIG. 4 shows schematically an embodiment of a mass spectrometer instrument for use in the invention.

(5) FIG. 5 shows a representative mass spectrum obtained from a sample of caffeine in an infusion MS experiment.

(6) FIG. 6 shows a measured and simulated isotope pattern of caffeine.

(7) FIG. 7 shows a plot of measured isotope ratios (196/195) of vs. known certified .sup.13C values caffeine from an infusion MS experiment.

(8) FIG. 8 shows a solvent step gradient used in an LC/MS comparative experiment.

(9) FIG. 9 shows a chromatogram and mass spectrum obtained in an LC/MS comparative experiment.

(10) FIG. 10 shows a plot of measured isotope ratios of caffeine vs. known certified .sup.13C values from a comparative LC/MS experiment.

(11) FIG. 11 shows a solvent step gradient used in an LC/MS experiment with a reduced flow step.

(12) FIG. 12 shows a chromatogram, mass spectrum and LC pump pressure v. time profile obtained in an LC/MS experiment using reduced flow.

(13) FIG. 13 shows a plot of measured isotope ratios of caffeine vs. known certified .sup.13C values from an LC/MS experiment using reduced flow.

(14) FIG. 14 shows a schematic mass spectrum displaying an isotopic mass peak pattern of isotopologues of an eluting molecular species. The solid lines represent mass peaks of isotopologues of the same molecular species whose isotope ratio is to be determined. The dotted lines represent mass peaks of external interferences.

(15) FIG. 15 shows a schematic relationship between HETP and the flow rate for an HPLC column.

DETAILED DESCRIPTION OF EMBODIMENTS

(16) In order to enable a more detailed understanding of the invention, embodiments will now be described by way of examples with reference to the accompanying drawings.

(17) Referring to FIG. 1, a method is described in relation to an LC column but it should be understood that the invention is applicable to other liquid based separation devices as described above. In a first step 102, a sample containing a number of components to be separated is injected into a liquid mobile phase in a conventional manner in an LC system. Typically, a plurality of components are to be separated. In other embodiments, a single component may be present in the sample.

(18) An apparatus as shown schematically in FIG. 2 may be used. In this embodiment, a mass spectrometer 10 is interfaced to an LC system 20. The LC system includes an autosampler 24 for holding samples to be analyzed and a pump system 28 that includes at least one pump, such as a syringe pump, to drive liquid mobile phase and a sample injector to inject a sample into the mobile phase. A preferred mass spectrometer 10 is a Q Exactive mass spectrometer from Thermo Scientific, such as the Q Exactive HF mass spectrometer, which includes an ORBITRAP mass analyzer. A preferred LC system is a Vanquish UHPLC system from Thermo Scientific. The mobile phase loaded with sample is pumped through the LC column 30. A preferred LC column is an Accucore aQ HPLC column, 2.1100 mm from Thermo Scientific. The mass spectrometer 10 and LC system 20 are typically under the control of a controller 15 that includes a computer system. The controller typically handles data acquisition from the mass spectrometer and data processing of acquired data. Data processing includes the determination of isotope ratios from the mass analysis data. The controller also controls the pump system of the LC system 20, to reduce the flow rate of the mobile phase in accordance with the invention. It will appreciated that in other embodiments, the controller for the LC system or separation device could be a separate controller from a controller for the mass spectrometer.

(19) Another apparatus for use in the invention is shown schematically in FIG. 3, described in more detail below.

(20) The sample comprises at least one component that is a molecular species having an isotope ratio to be determined. The mobile phase will typically comprise an organic solvent. If a gradient solvent is used, at least a part of the gradient will typically comprise an organic solvent. However, in other embodiments, aqueous-based, even fully aqueous, mobile phase could be used.

(21) In step 104, using the pump system the mobile phase is flowed through the LC column by a means of the pump to the mass spectrometer and one or more of the components of the sample become separated by the LC column as they elute from the column with different retention times. The eluate and mobile phase leaving the column flow to the downstream mass spectrometer for detection. In another type of embodiment, components eluting from the LC column can be collected and/or stored in one or more separate compartments and subsequently analyzed offline (e.g. in a separate mass spectrometer offline). For example. LC sample collection could be made in well plates and later analyzed by an MS by infusion or electrospray or nanospray.

(22) In preferred embodiments, the mass spectrometer is able to analyze in one acquisition cycle the full mass spectrum of each of the components eluting from the column, i.e. all mass (m/z) peaks of a compound can be recorded in a single acquisition cycle. Spectrometers that comprise high resolution and accurate mass (HR-AM) analyzers are preferred. Preferred spectrometers include mass analyzers of the following types, Fourier transform (FT) mass analyzers, electrostatic orbital traps (such as an ORBITRAP mass analyzer) which are typically FT, FT-ICR mass analyzers, and single or multi-reflection TOF mass analyzers. In such analyzers, all mass (m/z) peaks of a compound are measured during one acquisition cycle (herein referred to as measurement in parallel), i.e. from a single injection of ions into the analyzer.

(23) The mass spectrometer typically analyses the eluate over time, e.g. at set sampling intervals, to provide a mass chromatogram, i.e. a series of mass spectra measured at a series of data points across retention time. In step 106, the flow rate of the liquid mobile phase flowing through the LC column is reduced for at least a portion of the time that the molecular species having an isotope ratio to be determined emerges from the column. The flow rate in the preferred embodiment is reduced just before the molecular species of interest starts emerging from the column (e.g. at a time starting from between 1 and 100 secs before, more preferably between 1 and 50 secs before and even more preferably between 1 and 10 secs before). The rate remains reduced until at least a time when most of the molecular species has emerged from the column, such as until a time when the molecular species has substantially finished emerging from the column, or preferably until the desired precision in the isotope ratio determination has been achieved. The flow rate can remain reduced until a time after all of the molecular species has emerged from the column. The flow rate is typically reduced by a factor of at least 3, or better still at least 5 or better still at least 10, or at least 100 in some instances. The reduction factor typically has a dependence on the column size (diameter) as described above. A reduced flow rate by a factor of between 5 and 1000, or between 5 and 200, is typically employed. After the flow has been reduced in this way, the flow rate can be increased, typically to the original flow rate before the flow rate was reduced. The time at which to reduce the flow rate can be determined from a previous measurement (i.e. previous LC experiment), or it may be known from a database, or may be known a priori. Alternatively, the time at which to reduce the flow rate can be triggered by the detection of one or more mass spectral peaks of the molecular species of interest in real time as the mass spectrometer analyses the eluting species.

(24) The reduction of the flow rate can be achieved in different ways. In one preferred embodiment, the speed of the pump (i.e. the pressure of the pumping) that drives the flow of mobile phase through the column is reduced. In another preferred embodiment, the flow can be split upstream of the column by activating a valve so that a portion of the mobile phase flow is diverted into a branch flow path upstream of the separation device and only a portion is passed through the column, thus providing a reduced flow rate through the column. Such a system is shown schematically in FIG. 3. In FIG. 3, the system components are generally similar to FIG. 2 so that like reference numerals denote like components. However, in the FIG. 3 set-up, a tee junction 40 has been inserted into the flow path upstream of the LC column and connected to a HPLC switching valve 50. The valve can be switched between two positions: i) a blocked port, and ii) a port connected to a flow restriction capillary leading to a waste receiver 60. If the valve is switched to position i), all of the flow goes through the column. However, when the valve is switched to position ii), only a fraction of the flow goes through the column (e.g. 10%), thereby extending the time it takes to elute a peak from the column and leading to significant peak broadening. The switching valve 50 is under the control of the controller 15.

(25) In step 108, at a point during the sample elution, the mass spectrometer records the mass spectrum of the peak of interest from the LC column corresponding to at least the molecular species having an isotope ratio to be determined. As mentioned, the mass spectrometer records the mass spectrum of the eluate as it emerges from the LC column in a sequence of data acquisition cycles taken over time. The mass spectrum is typically recorded across the range of retention times from 0 minutes (or sample injection) until at least the mass spectrum of the molecular species having an isotope ratio to be determined has been recorded across its elution peak. As described, the flow rate is reduced during the period of the elution peak of interest, which increases the peak width (or elution time, i.e. the time it spends in the eluate) for the molecular species having an isotope ratio to be determined. Thus, the total mass analysis time for the molecular species having an isotope ratio to be determined is extended by the reduction in flow rate, i.e. by increasing the number of measurement points by a factor of the ratio of the peak widths (peak width with flow reduction/peak width without flow reduction). For many of the preferred ionization methods employed in the mass spectrometer when coupling to a liquid separation like LC, such as an electrospray ionization method, the signal intensity is not significantly affected by the reduction in flow rate. Thus, the overall result is a gain in precision of the mass spectrum. Preferably, the isotope ratio precision is <20 , preferably (in order of increasing preference) <15 , or <10 , or <7 , or <5 , or <3 , or <1 , or <0.5 , or <0.1.

(26) The mass spectrum recorded includes an isotopic pattern from which it is possible to resolve a plurality of isotopic peaks, i.e. peaks due to different isotopologues of the molecular species having an isotope ratio to be determined. From analysis of the isotopically resolved mass spectrum, an isotope ratio can be determined (step 110). For example, from intensities of two or more resolved isotopic peaks in the mass spectrum, often where one is the monoisotopic peak, it is possible to determine an isotope ratio (optionally expressed as a delta () value) for an element. Preferred elements of interest to determine an isotopic ratio for are C, N, O, H, S, P, Cl, Br and Si. Some common isotope ratios to be determined include .sup.13C/.sup.12C, .sup.14C/.sup.12C, .sup.15N/.sup.14N, .sup.2H/.sup.1H, .sup.18O/.sup.16O, .sup.17O/.sup.16O or .sup.34S/.sup.32S, .sup.37Cl/.sup.35Cl, .sup.81Br/.sup.79Br, .sup.29Si/.sup.28Si, .sup.30Si, .sup.28Si, etc.

(27) The isotope ratio determination in the present invention can be based on the intensities, for example on comparing (i.e. finding the ratio of) the intensities, of mass peaks of isotopologues of the molecular species, in which the isotopologues have different nominal mass (typically differing by one or two nominal mass units). Typically, the monoisotopic peak (for the monoisotopic isotopologue) in the mass spectrum of the at least one molecular species having an isotope ratio to be determined, e.g. the [M+H].sup.+ peak in a mass spectrum obtained from an electrospray source, can provide an isotope abundance for the particular light isotope of interest (e.g. .sup.12C, .sup.14N, .sup.16O, .sup.1H, .sup.32S) and an A+1 peak or A+2 peak (where A is the monoisotopic mass peak) for an A+1 isotopologue or A+2 isotopologue can provide an isotope abundance for the particular heavy isotope of interest (e.g. .sup.13C, .sup.15N, .sup.18O, .sup.2H, .sup.34S). The isotope ratio determination may thus comprise comparing the intensity of a monoisotopic mass peak A with an A+1 mass peak or A+2 mass peak to provide an isotope ratio of a light isotope and a heavy isotope of interest. The high resolving power of the mass spectrometer, especially a spectrometer comprising an ORBITRAP mass analyzer, has been found able to resolve a particular A+1 peak (or A+2 peak) of an isotopologue having the particular heavy isotope of interest (.sup.13C, or .sup.15N, or .sup.18O, or .sup.2H, or .sup.34S etc.), i.e. resolve that particular isotopologue peak from other A+1 nominal mass peaks (or A+2 nominal mass peaks) of isotopologues having another heavy isotope. Each isotopologue mass peak used for the isotope ratio determination should preferably be resolved from at least any other mass peaks at the same nominal mass which are more than 20%, or more preferably more than 10%, or most preferably more than 5%, of the intensity of the isotopologue mass peak. For example, in an embodiment of the invention the resolved isotope peak pattern of A+1 peaks shows that a .sup.13C isotopologue peak can be sufficiently resolved from a .sup.15N isotopologue peak or .sup.2H peak. Further, the resolved isotope peak pattern of A+2 peaks shows that an .sup.18O isotopologue peak can be sufficiently resolved from a .sup.13C.sub.2 isotopologue peak or a .sup.13C.sup.15N isotopologue peak. Thus, the invention enables analysis of multiply substituted isotopologues as well as singly substituted isotopologues. The mass analysis therefore preferably resolves the monoisotopic peak from A+1 and A+2 peaks. The mass analysis further preferably resolves two or more A+1 isotopologues and/or two or more A+2 isotopologues from each other.

(28) In some embodiments, the isotope ratio determination in the present invention can be based on the intensities, for example on comparing (i.e. finding the ratio of) the intensities, of mass peaks of isotopologues of the molecular species, in which the isotopologues have the same nominal mass. For example, the isotope ratio determination can be based on a ratio of the .sup.15N isotopologue peak to the .sup.13C isotopologue peak.

(29) The mass analysis is preferably performed with a sufficient resolving power (R) to resolve mass peaks of isotopologues of the at least one molecular species, for example to resolve the two most abundant mass peaks at the nominal mass of at least one of the isotopologues used to determine the isotope ratio (preferably at the nominal masses of each of the isotopologues used to determine the isotope ratio). The two most abundant mass peaks that are resolved at the same nominal mass are typically two isotopologues (since the liquid separation step will typically mean that other molecular species are not present having mass peaks within 2 m/z units of the isotopologues used to determine the isotope ratio). The two most abundant mass peaks at the nominal mass are preferably peaks that if not resolved would each contribute significantly to the isotope ratio to be determined (e.g. by contributing >20%, or >10%, or >5% of the peak intensity of the isotopologue at the nominal mass). The mass analysis is preferably performed with a mass resolving power high enough to resolve the two most abundant A+1 isotopologues and/or the two most abundant A+2 isotopologues (where A is the monoisotopic mass peak).

(30) Referring to FIG. 14, there is shown a schematic mass spectrum displaying an isotopic mass peak pattern of isotopologues of an eluting molecular species. The solid lines represent mass peaks of isotopologues of the same molecular species whose isotope ratio is to be determined. The dotted lines represent mass peaks of external interferences from other molecular species. A monoisotopic peak P1 of nominal mass A is shown, which is resolved from a small external interference mass peak i1 at the same nominal mass A. An isotopologue peak P2 is also indicated at nominal mass A+1, which is to be used together with the monoisotopic peak P1 to find an isotope ratio of the molecular species, i.e. the ratio of the intensity of the P2 (A+1) peak to the P1 (A) peak, which can be expressed as a delta value. The P2 A+1 isotopologue peak is resolved from two other A+1 isotopologue peaks P3 and P4 as well as an external interference peak i2. It can be seen that at least the two most abundant A+1 mass peaks, P2 and P4, are mass resolved from each other. It is particularly important that P2 is resolved from P4 since P4 is more than 20% of the intensity of the P2 peak and so would significantly affect the isotope ratio determination. It is also preferable that P2 is resolved from P3 since P3 is more than 10% of the intensity of the P2 peak. As an example, the A+1 peak, P2, could be the .sup.13C isotopologue and together with the monoisotopic peak P1 could be used to determine the .sup.13C/.sup.12C ratio. In addition, the A+1 peak, P3, could be the .sup.15N isotopologue and together with the monoisotopic peak P1 could be used to determine the .sup.15N/.sup.14N ratio. Similarly, with the A+2 nominal mass peaks, there are shown isotopologue peaks P5, P6, P7 and P8 and an external interference peak i3, which are all mass resolved from each other and may be used with the monoisotopic peak P1 to determine isotope ratios, e.g. of isotopes differing by two nominal mass units such as .sup.34S/.sup.32S, or .sup.18O/.sup.16O, or of multiply substituted isotopologues such as .sup.13C.sub.2 or .sup.13C.sup.15N. Importantly, at least the two most abundant A+2 mass peaks, P6 and i3, are mass resolved from each other.

(31) Different work flows can be used for determining the isotope ratio from the mass spectrum. An example of one work flow for determining the isotope ratio from the mass spectrum is given by the following steps. 1. For each spectrum acquired (this includes those spectra at retention times where no peak is eluting from the column) determine the peak intensities of the two isotope peaks of interest. This is typically done by taking the highest intensity point within a narrow mass window around the accurate mass of the peak of interest. For mass analyzers other than FT based, peak integration may the method of choice instead of height of the peak. 2. Optional step: Determine the average background intensity of the two mass peaks in an area of the chromatogram where no peak of interest and no peaks interfering with the masses of interest elute. This may be an average, geometrical average, or any other similar method of determining such a value. This step may include outlier analysis and removal. This step may include other steps (like drift testing) to make sure selected part of the chromatogram is suitable for background determination. 3. Optional step: For each spectrum across the eluted peak, subtract the average background intensities from the peaks of interest. 4. For each spectrum, determine the isotope ratio by dividing the intensity of the first (optionally background subtracted) isotope peak of interest by the intensity of the other peak of interest. 5. Optional step: Use statistical tools to validate and improve the determined ratios. For example: i) analyze and remove outliers, ii) determine whether there is any time trend (instrumental drift) in the data, iii) plot the ratios vs. peak intensities to eliminate any dependency on peak intensity, etc. 6. Determine the average isotope ratio and the standard error associated with the data set based on the data set obtained from step 4 (or optionally from step 5).

(32) In an alternative mode of data evaluation, the spectra could be averaged first, then background subtracted and final isotope ratio be determined as the ratio of the two peaks of interest in the averaged spectrum.

EXPERIMENTAL EXAMPLES

(33) A number of experimental examples, non-limiting on the scope of the invention, will now be described to aid understanding of the invention.

(34) Sample and Reference Materials

(35) Caffeine was used as a model compound to demonstrate the effectiveness of the invention. Three certified isotopic reference materials of caffeine were obtained from USGS: USGS61, USGS62, USGS63 (Schimmelmann, A., Qi, H., Coplen, T. B., Brand, W. A., Fong, J., Meier-Augenstein, W., Kemp, H. F., Toman, B., Ackermann, A., Assonov, S. and Aerts-Bijma, A. T., 2016. Organic Reference Materials for Hydrogen, Carbon, and Nitrogen Stable Isotope-Ratio Measurements: Caffeines, n-Alkanes, Fatty Acid Methyl Esters, Glycines, I-Valines, Polyethylenes, and Oils. Analytical chemistry, 88(8), pp. 4294-4302). An additional caffeine sample was obtained from Sigma Aldrich (Sigma Aldrich C-8960, Lot #30K0169) and characterized using a Delta V mass spectrometer couples to an EA Isolink elemental analyzer. The certified .sup.13C reference values (for USGS61-63) and measured value (for the sample from Sigma Aldrich, herein denoted BRE001) are listed in Table 1.

(36) TABLE-US-00001 TABLE 1 Certified reference values and measured values for .sup.13C Sample Name .sup.13C [] USGS61 35.05 USGS62 14.97 USGS63 1.17 BRE001 39.21

Example 1Infusion Measurements (Comparative Example)

(37) Sample solutions of approx. 10 g/ml of the samples listed in table 1 in methanol/water 50:50 (v:v) were made and directly infused into a standard Thermo Scientific Q Exactive HF ORBITRAP mass spectrometer from Thermo Fisher Scientific. A schematic of the instrument, which is a preferred mass spectrometer for use in the invention, is shown in FIG. 4. The mass spectrometer 200, comprises an electrospray ion source 202 which generates ions that enter an RF lens 204 before being guided by the active beam guide ion optics 206 to a quadrupole mass filter 208. A mass isolation window can be set by the mass filter to transmit ions of the desired mass to a downstream ion trap (C-Trap) 210, where ions can be accumulated before ejection of the ions to the ORBITRAP mass analyzer 212 for mass analysis. If required, the ions can be transmitted through the C-trap 210 to a downstream higher energy collision dissociation (HCD) cell 214 where ions can be fragmented before being returned to the C-trap 210 and subsequently mass analyzed in the ORBITRAP mass analyzer 212.

(38) The spectrometer was operated using the following parameters: Infusion using a syringe pump Mass isolation range of the spectrometer 190-200 m/z Resolving power=240 k @ m/z 200 10 microscans were utilised, i.e. 10 scans in the ORBITRAP mass analyzer were performed for each transient subjected to Fourier transformation Target AGC value 1E6 15 min total data acquisition, which results in approx. 160 scans per data file (with 10 microscans utilized per scan).

(39) FIG. 5 shows a representative mass spectrum obtained from the BRE001 sample of caffeine infused to the Q Exactive HF mass spectrometer. The monoisotopic caffeine peak corresponding to the [M+H].sup.+ ion is labelled at 195.09 and an A+1 peak at 196.09. Due to the high resolution of the instrument, a closer look at the mass spectrum of the isotope peaks shows that the .sup.13C isotope peak at 196.09 is well resolved from the .sup.15N isotope peak at 196.08, but not from the .sup.17O isotope peak as shown in FIG. 6. However, the natural abundance of .sup.17O is negligible compared to the variations in .sup.13C abundance, such that this is quite acceptable for the study.

(40) The top row of spectra in FIG. 6 show the measured isotope pattern of caffeine and demonstrate the resolving power. The lower row of spectra represent a simulation of the spectra of caffeine to show the good correspondence with the measured data. The peak annotations in FIG. 6 indicate the type of caffeine isotopologue (e.g. 15N, 13C, 13C2, etc.).

(41) All of the samples were measured sequentially using a standard/sample bracketing sequence with USGS61 as the bracketing standard. Table 2 shows the results of the measurements with the standard denoted as Type STD and each of the samples denoted as Type UNK. FIG. 7 shows the plot of the measured isotope ratios (196/195) vs. the known certified .sup.13C values and also shows the linear calibration curve. The standard error of each single measurement in the infusion experiment was approximately 0.5-0.6% and the accuracy of the method was approx. 2-2.5.

(42) TABLE-US-00002 TABLE 2 .sup.13C () Ratio Certified .sup.13C () Sequence Sample Type (measured) Value (calculated) 1 BRE001 UNK 0.0853857 39.21 36.47 2 USGS61 STD 0.0855069 35.05 3 BRE001 UNK 0.0855094 39.21 35.34 4 USGS61 STD 0.0855632 35.05 5 USGS62 UNK 0.0874976 14.97 12.08 6 USGS61 STD 0.0855020 35.05 7 USGS63 UNK 0.0885502 1.17 0.29 8 USGS61 STD 0.0856490 35.05 9 BRE001 UNK 00855340 39.21 36.39

Example 2LC/MS Measurements without Reduced Flow (Comparative Example)

(43) Sample solutions of 10 g/ml (+/0.02 g/ml) of the samples listed in table 1 in methanol/water 20:80 were used in the experiment. An LC/MS system as shown schematically in FIG. 2 was used with the following LC setup: Thermo Fisher Scientific Vanquish LC system with autosampler and quaternary pump 10 l injection (corresponds to 100 ng injected) Thermo Fisher Scientific Accucore aQ column; 2.1100 mm, 2.6 mm particle size Solvent A: water, 0.1% formic acid (FA), 2 mM ammonium acetate Solvent B: methanol, 0.1% FA, 2 mM ammonium acetate Solvent Gradient 100% A (0-0.5 min); 50% A/50% B (0.5-3.0 min); 20% A/80% B (3.0-3.3 min); 100% A (3.3-5 min). FIG. 8 shows the solvent step gradient used in the experiment.

(44) The mass spectrometer was operated using the following parameters: Mass scan/isolation range 190-200 m/z Resolving power=240 k @ m/z 200 1 microscan per transient processed Target AGC value 1E6 4 min total data acquisition

(45) The acquisition and data evaluation strategy employed the following: Sample/standard bracketing USGS1 as the reference/standard 8 repetitions of the bracketing set (49 injections in total) Data evaluation used USGS1 measurements to correct drift Direct calculation of values with no additional corrections

(46) FIG. 9 shows a typical chromatogram and mass spectrum. With the LC peak width of caffeine of approx. 0.5 min, this resulted in approx. 40 scans across the LC peak. Table 3 shows the results of the measurements and FIG. 10 shows the plot of the measured isotope ratios vs. the known certified .sup.13C values together with the linear calibration curve. The standard error of each single measurement was approximately 4-6. Accuracy of the method was approx. 5.

(47) TABLE-US-00003 TABLE 3 .sup.13C () StdErr .sup.13C () Sample (measured) () Stdev () Certified Value USGS61 35.05 N/A N/A 35.05 USGS62 17.44 1.72 4.85 14.97 USGS63 1.56 1.59 4.51 1.17 BRE001 37.14 0.58 1.65 39.21

Example 3LC/MS Measurements with Reduced Flow

(48) Sample solutions of 10 g/ml (+/0.02 g/ml) of samples listed in table 1 in methanol/water 20:80 were used in the experiment. An LC/MS system as shown schematically in FIG. 3 was used with the following LC setup: Thermo Fisher Scientific Vanquish LC with autosampler and quaternary pump 10 l injection (corresponds to 100 ng injected) Thermo Fisher Scientific Accucore aQ column; 2.1100 mm, 2.6 mm particle size Solvent A: water, 0.1% FA, 2 mM ammonium acetate Solvent B: methanol, 0.1% FA, 2 mM ammonium acetate Gradient 100% A (0-0.5 min); 50% A/50% B (0.5-8.5 min); 20% A/80% B (8.5-9.0 min); 100% A (9.0-11.0 min) (step gradient) Divert valve switching at t=2.75 min and t=8.00 min

(49) FIG. 11 shows the solvent step gradient used in the experiment. In this experiment a reduced flow setup was used. As shown in FIG. 3, a tee was inserted into the flow path before the LC column and connected to a HPLC switching valve. The valve could be switched to either of two positions: i) a blocked port, or ii) a port connected to a flow restriction capillary going to waste. If the valve was switched to position i), all flow went through the column. When switched to position ii), only a fraction of the flow went through the column, extending the time taken to elute a peak from the column. This lead to significant peak broadening. In this case, peak width was extended from 0.5 min to approx. 6 minutes. Due to the characteristics of the electrospray ionization method, the signal intensity was not significantly affected by the drop in the flow rate, effectively extending the analysis time and multiplying the number of measurement points by the ratio of the peak widths. It will be appreciated that the reduced flow rate could also be implemented using a set-up as shown in FIG. 2 by reducing the pump speed (pressure) for the required duration. The reduced flow rate approach gave a much better precision of the isotope ratio measurement.

(50) The mass spectrometer was operated using the following parameters: Mass scan/isolation range 190-200 m/z Resolving power=240 k @ m/z 200 1 microscan per transient processed Target AGC value 1E6 4 min total data acquisition

(51) The acquisition and data evaluation strategy employed the following: Sample/standard bracketing USGS1 as reference/standard 8 repetitions of bracketing set (49 injections total) Data evaluation using USGS1 measurements to correct drift Direct calculation of values; no additional corrections

(52) FIG. 12 shows a typical chromatogram, mass spectrum and LC pump pressure v. time profile. With the LC peak width of caffeine increased to approx. 6 min, this resulted in approx. 400 scans across the LC peak. Table 4 shows the results of the measurements and FIG. 13 shows the plot of the measured isotope ratios vs. the known certified .sup.13C values together with the linear calibration curve. The standard error of each single measurement was approximately 0.7-0.9 and the accuracy of the method was approx. 2. It can be seen that the reduced flow approach of the invention enables reliable determination of precise and accurate isotope ratios of molecular species using high resolution mass spectrometry (e.g. ORBITRAP mass spectrometry) coupled to liquid chromatography or another liquid separation.

(53) TABLE-US-00004 TABLE 4 .sup.13C () StdErr .sup.13C () Sample (measured) () Stdev () Certified Value USGS61 35.05 N/A N/A 35.05 USGS62 14.45 0.79 2.25 14.97 USGS63 1.36 0.77 2.18 1.17 BRE001 37.86 0.64 1.81 39.21

(54) Herein the term mass to charge ratio (m/z) in relation to the mass analysis means any quantity of the mass analysis related to m/z, for example mass, time (e.g. flight time in a TOF mass analysis), frequency (e.g. ion oscillation frequency in a Fourier transform mass analysis) etc.

(55) The use of any and all examples, or exemplary language (for instance, such as, for example and like language) provided herein, is intended merely to better illustrate the invention and does not indicate a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

(56) As used herein, including in the claims, unless the context indicates otherwise, singular forms of the terms herein are to be construed as including the plural form and vice versa. For instance, unless the context indicates otherwise, a singular reference herein including in the claims, such as a or an means one or more.

(57) Throughout the description and claims of this specification, the words comprise, including, having and contain and variations of the words, for example comprising and comprises etc., mean including but not limited to, and are not intended to (and do not) exclude other components.

(58) The present invention also covers the exact terms, features, values and ranges etc. in case these terms, features, values and ranges etc. are used in conjunction with terms such as about, around, generally, substantially, essentially, at least etc. (i.e., about 3 shall also cover exactly 3 or substantially constant shall also cover exactly constant).

(59) The term at least one should be understood as meaning one or more, and therefore includes both embodiments that include one or multiple components. Furthermore, dependent claims that refer to independent claims that describe features with at least one have the same meaning, both when the feature is referred to as the and the at least one.

(60) Any steps described in this specification may be performed in any order or simultaneously unless stated or the context requires otherwise.

(61) All of the features disclosed in this specification may be combined in any combination, except combinations where at least some of such features and/or steps are mutually exclusive. In particular, the preferred features of the invention are applicable to all aspects of the invention and may be used in any combination. Likewise, features described in non-essential combinations may be used separately (not in combination).