BENCHMARK FOR LC-MS SYSTEMS

Abstract

The present invention relates to a method of monitoring performance of a liquid chromatography-mass spectrometry (LC-MS) system, comprising the steps of: (a) performing LC with a first buffer, said first buffer comprising a defined concentration of a first compound, said performing comprising a step of eluting; (b) subjecting the eluate of said LC to electro-spray ionization; and (c) determining the amount of said first compound in said eluate by means of MS, thereby monitoring said performance; wherein said first compound (i) has either no affinity or negligible affinity to the chromatographic matrix; and (ii) is detectable by MS.

Claims

1. A method of operating a liquid chromatography-mass spectrometry (LC-MS) system, comprising the steps of: (a) performing LC with a first buffer, said first buffer comprising a defined concentration of a first compound, said performing comprising a step of eluting; (b) subjecting the eluate of said LC to electro-spray ionization; and (c) determining the amount of said first compound in said eluate by means of MS; wherein said first compound (i) has either no affinity or negligible affinity to the chromatographic matrix; and (ii) is detectable by MS.

2. A method of monitoring performance of a liquid chromatography-mass spectrometry (LC-MS) system, comprising the steps of: (a) performing LC with a first buffer, said first buffer comprising a defined concentration of a first compound, said performing comprising a step of eluting; (b) subjecting the eluate of said LC to electro-spray ionization; and (c) determining the amount of said first compound in said eluate by means of MS, thereby monitoring said performance; wherein said first compound (i) has either no affinity or negligible affinity to the chromatographic matrix; and (ii) is detectable by MS.

3. The method of claim 2, wherein said performance is selected from (a) performance of the LC system comprised in said LC-MS system, preferably performance of mixing, loading, and/or pumps; (b) performance of the electro-spray ionization device comprised in said LC-MS system, preferably performance thereof in terms of spray-stability such as fluctuations of ionization efficiency, droplet formation, and background signals; and (c) performance of the MS system comprised in said LC-MS system, preferably performance of the quadrupole and mass-analyzer such as efficiency of mass selection, mass accuracy, resolution and ion-intensities measured.

4. A method of separating or fractionating using a liquid chromatography-mass spectrometry (LC-MS) system, comprising the steps of: (a) performing LC with a first buffer, said first buffer comprising a defined concentration of a first compound, said performing comprising a step of eluting with a gradient; and (b) determining the amount of said first compound in the eluate by means of MS, thereby defining a given fraction; wherein said first compound (i) has no or negligible affinity to the chromatographic matrix; and (ii) is detectable by MS.

5. The method of claim 1, wherein said LC is gradient liquid chromatography using said first buffer, furthermore a second buffer, and optionally one or more further buffers.

6. The method of claim 5, wherein said second buffer and, if present, each of said one or more further buffers comprises a defined concentration of a second compound and, if applicable, one or more further compounds, respectively, and said method comprises the further step: (c) determining the concentration of said second compound and, if applicable, said one or more further compounds, in the eluate by means of MS; wherein said second and, if applicable, said further compound(s) (iii) has/have no or negligible affinity to the chromatographic matrix; (iv) is/are detectable by MS; and (v) is/are different from said first compound and, if applicable, from each other.

7. The method of claim 1, wherein the concentration(s) of said compound(s) is/are used (iv) to normalize quantities of analytes within or across different measurements; and/or (v) to predict elution of analytes within or across different measurements and/or are used to identify analytes within or across different measurements.

8. A kit comprising or consisting of a first compound, a second compound and optionally one or more further compounds, wherein all compounds (i) have no or negligible affinity to a chosen chromatographic matrix; (ii) are detectable by MS; and (iii) are different from each other.

9. The method of claim 6, wherein said first compound, said second compound, and, if applicable, said further compounds (iv) have similar ionization properties; (v) are differently isotope labelled forms of otherwise the same compounds; and/or (vi) are isobaric and fragment differently when the mass spectrometer is operated in fragmentation mode.

10. The kit of claim 8, wherein (a) the compounds are provided in the form of tablets comprising defined amounts of said compounds, each of said tablets being confectioned to be added to a defined volume of a corresponding buffer, respectively; and/or the kit further comprises a manual containing instructions for performing the method of operating a liquid chromatography-mass spectrometry (LC-MS) system, comprising the steps of: (b) performing LC with a first buffer, said first buffer comprising a defined concentration of a first compound, said performing comprising a step of eluting; (c) subjecting the eluate of said LC to electro-spray ionization; and (d) determining the amount of said first compound in said eluate by means of MS: wherein said first compound (i) has either no affinity or negligible affinity to the chromatographic matrix; and (ii) is detectable by MS.

11. The method of claim 1, wherein said first compound, said second compound and, if applicable, said one or more further compounds furthermore (iii) do not directly interact with the analytes to be analyzed while preferably improve ionization of said analytes; (iv) are fluorescent; and/or (v) can be used as a mass standard or calibrant for mass calibration in MS.

12. The method of claim 1, wherein said first compound, said second compound and, if applicable, said one or more further compounds are independently selected from hydrophilic amino acids such as glutamine, asparagine, glutamic acid and aspartic acid, chemicals with a hydrophilic properties or isotope labelled forms of these compounds.

13. The method of claim 1, wherein the chromatographic matrix is selected from reversed phase materials such as C4, CB, C18 and styrene divinyl benzene (SOB), or wherein the chromatographic matrix is selected from hydrophilic interaction (HILIC) materials.

14. The method of claim 1, wherein the analytes are or include proteins, polypeptides, peptides, nucleotides, metabolites, and/or small molecules.

15. The method of claim 1, wherein said first buffer is an aqueous buffer and said second buffer is an organic buffer, preferably comprising acetonitrile, methanol and/or DMSO.

16. The kit of claim 8, wherein said first compound, said second compound, and, if applicable, said further compounds (iv) have similar ionization properties; (v) are differently isotope labelled forms of otherwise the same compounds; and/or (vi) are isobaric and fragment differently when the mass spectrometer is operated in fragmentation mode.

17. The kit of claim 8, wherein said first compound, said second compound and, if applicable, said one or more further compounds furthermore (iv) do not directly interact with the analytes to be analyzed while preferably improve ionization of said analytes; (v) are fluorescent; and/or (vi) can be used as a mass standard or calibrant for mass calibration in MS.

18. The kit of claim 8, wherein said first compound, said second compound and, if applicable, said one or more further compounds are independently selected from hydrophilic amino acids such as glutamine, asparagine, glutamic acid and aspartic acid, chemicals with a hydrophilic properties or isotope labelled forms of these compounds.

19. The kit of claim 8, wherein the chromatographic matrix is selected from reversed phase materials such as C4, CB, C18 and styrene divinyl benzene (SOB), or wherein the chromatographic matrix is selected from hydrophilic interaction (HILIC) materials.

20. The kit of claim 8, wherein the analytes are or include proteins, polypeptides, peptides, nucleotides, metabolites, and/or small molecules.

21. The kit of claim 8, wherein said first buffer is an aqueous buffer and said second buffer is an organic buffer, preferably comprising acetonitrile, methanol and/or DMSO.

Description

[0061] The Figures show:

[0062] FIG. 1: Linear Gradient (Proxeon EASY-nLC II HPLC System). Unstable electrospray was observed by single scan changes in percentage B.

[0063] FIG. 2: Malfunctioning UHPLC (Proxeon EASY-nLC 1000 UHPLC System) with a leak at rotor valve B.

[0064] FIG. 3: Effect of pre-column dead volumes on the HPLC gradient. Backmixing of dead-volumes before the packed bed leads to imprecise buffer mixing (lower dotted line). Reduction of the dead volume can improve precision and accuracy of the desired gradient (continuous line).

[0065] The Examples illustrate the invention.

EXAMPLE 1

Methods

[0066] Spike-in stock solutions (1.000) were prepared by desolving 146.2 mg of non-labelled L-(+)-Glutamine and 147.2 mg of heavy isotope labelled L-Glutamine (5-13C, 99%) in 50 ml HPLC grade water each. Heavy-labelled ions were added to the aqueous HPLC buffer A (0.1% formic acid) and non-labelled ions were mixed with the organic buffer B (80% acetonitrile, 0.1% formic acid). The ions were then observed in mass spectrometric scans on Orbitrap mass spectrometers at the m/z of 147.07669 (buffer B) and 148.08044 (buffer A). Evaluation of HPLC performance was evaluated using linear 120 min gradients of 5% buffer B to 95% buffer B at 250 nl/min flow (FIGS. 1 and 2). Dead-volume backmixing effects were observed applying a stepped gradient increasing by 1% percent buffer B within 1 min and maintaining the percentage B for 4 min (FIG. 3). The percentage of buffer B was calculated by dividing the intensity of the B ion (m/z 147.07669) by the combined intensities A and B.

EXAMPLE 2

Results

[0067] The observed ions resulted in accurate percentage B read-outs (FIG. 1-3). Unstable spray and droplet formations were observed as brief jumps in the percentage read-out (FIG. 1, unstable spray at retention times 14, 16, 23, 24, 29, 34, 38 min; droplet formation at retention time 60 min). A malfunctioning in the HPLC system could be visualized because desired concentrations of percentage B were not achieved in the first minutes of the gradient (FIG. 2), aiding in trouble-shooting and fixing of the LC system. Additionally backmixing in pre-column dead-volumes could be observed using a stepped gradient (FIG. 3). The reduction of the dead volume improves accuracy of percentage B in an observable range.