US-12625117-B2 - Quality control tools for LC-MS

US12625117B2US 12625117 B2US12625117 B2US 12625117B2US-12625117-B2

Abstract

A method for identifying and/or verifying at least one analyte peak in a chromatogram of a sample for said analyte from a liquid chromatography mass spectrometer device, said method comprising: a) determining a chromatogram of the sample by acquiring a plurality of data points for quantifier signal intensities and/or qualifier signal intensities, over time; and, in case the sample comprises an internal standard, optionally acquiring a plurality of data points for internal standard quantifier signal intensities and/or internal standard qualifier signal intensities, over time; b) determining for at least a fraction of the data points acquired in step a), a ratio type; c) comparing the ratios determined in step b) to a reference; and d) identifying and/or verifying at least one analyte peak in a chromatogram based on comparison step c).

Inventors

Daniel Intelmann
Julian Michely

Assignees

ROCHE DIAGNOSTICS OPERATIONS, INC.

Dates

Publication Date: 20260512
Application Date: 20220908
Priority Date: 20200316

Claims (20)

1 . A method for identifying and/or verifying at least one analyte peak in a chromatogram of a sample for said analyte from a liquid chromatography mass spectrometer device, said method comprising the following steps: a) determining or have determined a chromatogram of the sample by performing at least one chromatography run on the sample by a liquid chromatography device configured to separate the analyte of the sample from other components of the sample for detection, coupled to a mass spectrometry device through an interface that includes an ionization source configured to generate molecular ions and transfer the molecular ions to a gas phase, and by acquiring a plurality of data points for quantifier signal intensities and/or qualifier signal intensities, over time; and, in case the sample comprises an internal standard, optionally acquiring a plurality of data points for internal standard quantifier signal intensities and/or internal standard qualifier signal intensities, over time, wherein the values of the quantifier signal intensities, qualifier signal intensities, internal standard quantifier signal intensities, and/or internal standard qualifier signal intensities are single-cycle intensities or values derived therefrom; b) determining for at least a fraction of the data points acquired in step a), a ratio type selected from the list consisting of (i) analyte quantifier to analyte qualifier ratios, (ii) internal standard quantifier to internal standard qualifier ratios, (iii) analyte quantifier to internal standard quantifier ratios, and (iv) analyte qualifier to internal standard qualifier ratios; c) comparing the ratios determined in step b) to a reference; and d) identifying and/or verifying at least one analyte peak in a chromatogram based on comparison step c).
2 . The method of claim 1 , wherein said sample comprises an internal standard and wherein the method comprises the following steps: a) providing a chromatogram of a sample by acquiring a plurality of data points for quantifier signal intensities and/or qualifier signal intensities, over time; and, optionally, acquiring a plurality of data points for internal standard quantifier signal intensities and/or internal standard qualifier signal intensities, over time; b) determining for at least a fraction of the data points acquired in step a) a ratio type selected from the list consisting of (i) analyte quantifier to analyte qualifier ratios, (ii) internal standard quantifier to internal standard qualifier ratios, (iii) analyte quantifier to internal standard quantifier ratios, and (iv) analyte qualifier to internal standard qualifier ratios; c) comparing the ratios determined in step b) to a reference; and d) identifying and/or verifying at least one analyte peak in a chromatogram based on comparison step c); or wherein said sample does not comprise an internal standard and wherein the method comprises the following steps: a) providing a chromatogram of a sample by acquiring a plurality of data points for quantifier and qualifier signal intensities over time; b) determining analyte quantifier to analyte qualifier ratios for at least a fraction of the data points acquired in step a); c) comparing the ratios determined in step b) to a reference; and d) identifying and/or verifying at least one analyte peak in a chromatogram based on comparison step c).
3 . The method of claim 1 , wherein said method further comprises step a1) determining a putative lower peak boundary and/or a putative upper peak boundary corresponding to the analyte.
4 . The method of claim 3 , wherein said method further comprises optionally determining a putative lower peak boundary and/or a putative upper peak boundary corresponding to the internal standard.
5 . The method of claim 1 , wherein said reference is or is calculated from a ratio pre-determined for the analyte and/or internal marker.
6 . The method of claim 5 , wherein the analyte is a purified analyte and/or the internal marker is a purified internal marker.
7 . The method of claim 1 , wherein said identifying and/or verifying at least one analyte peak comprises identifying and/or verifying at least one putative peak boundary of an analyte peak.
8 . The method of claim 7 , wherein said fraction of the data points for which ratios are determined in step b) comprises data points downstream and/or upstream the putative peak boundary to be identified or verified, and/or wherein said putative peak boundary is identified and/or verified in case the ratio lies within a pre-defined reference range for a pre-defined fraction of data points.
9 . The method of claim 8 , wherein said pre-defined fraction of data points comprises at least 3 out of 5 consecutive data points.
10 . The method of claim 1 , wherein said identifying and/or verifying at least one analyte peak comprises identifying and/or verifying peak identity and/or peak purity.
11 . The method of claim 10 , wherein said peak identity and/or said peak purity is verified in case the ratio lies within a pre-defined reference range.
12 . The method of claim 2 , wherein said method further comprises determining a putative analyte peak area to internal standard peak area ratio (peak area ratio), wherein said method comprises determining analyte quantifier to internal standard quantifier ratios, or analyte qualifier to internal standard qualifier ratios, and wherein said identifying and/or verifying at least one analyte peak comprises identifying and/or verifying said peak area ratio.
13 . The method of claim 1 , wherein said fraction of the data points for which ratios are determined in step b) comprises data points between the putative lower peak boundary and the putative upper peak boundary.
14 . The method of claim 1 , wherein said method comprises identifying and/or verifying at least one putative peak boundary of an analyte peak, a peak purity, and/or a putative peak area ratio, comprises determining analyte quantifier to internal standard quantifier ratios or analyte qualifier to internal standard qualifier ratios, and further comprises determining the distribution of said ratios.
15 . The method of claim 14 , wherein at least one putative peak boundary of an analyte peak, a peak purity, and/or a putative peak area ratio is identified and/or verified in case the distribution of said ratios over a multitude of n data points meets a pre-defined acceptance criterion.
16 . The method of claim 15 , wherein n is at least two data points.
17 . The method of claim 15 , wherein n is at least three data points.
18 . The method of claim 15 , wherein n is at least four data points.
19 . The method of claim 15 , wherein n is at least five data points.
20 . The method of claim 15 , wherein n is at least seven data points.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS This application is a continuation of International Patent Application No. PCT/EP2021/056470, filed 15 Mar. 2021, which claims priority to European Patent Application No. 20163293.2, filed 16 Mar. 2020, the disclosures of which are hereby incorporated by reference in their entirety. TECHNICAL FIELD The disclosure relates to a method for identifying and/or verifying at least one analyte peak in a chromatogram of a sample from a liquid chromatography mass spectrometer device, to a method of quality control applying the aforesaid method, and to systems and computer program products related thereto. BACKGROUND For LC-MS assays, ratios of peak areas are the common way for obtaining calculations or verifications. They are part of several international guidelines for validation of mass spectrometric assays, such as those by the CLSI (Clinical and Laboratory Standards Institute), the EMA (European Medicines Agency), or the GTFCh (German society for toxicological and forensic chemistry). For the quality assurance of an assay, non-extracted system suitability tests with spiked compounds, measured before the analytical run, have to fulfill acceptance requirements, such as minimal absolute peak areas or maximal retention time deviation from a target value. Within the analytical run, quality control (QC) samples are then tested with a certain frequency and the calculated result checked versus an acceptance range. Moreover, retention time, peak width (given by the retention time difference between the peak boundaries), absolute peak area of the internal standard (ISTD), and the quantifier/qualifier peak area ratio of the analyte are usually monitored in each sample and should fulfill acceptance requirements of maximal deviations or certain cut-offs values. However, most of these parameters are based on peak integration. Peak boundaries, detection of interferences, and calculation of the result are all directly dependent on the peak integration. However, noisy background (e.g., due to aged MS source) or tailing peaks (e.g., due to aged LC column) can lead to improper peak integration. Determination of peak boundaries is usually defined as the outer points of retention times of the integrated peak and thus consequently affected by improper peak integration. The peak area ratio of two different transitions of the same analyte (e.g., quantifier/qualifier ratio) is used for verifying the peak identity and to exclude interferences. This principle is due to the fact that the peak area ratio of different analyte's transitions deviates around a fixed value which is independent from the analyte concentration. However, this value cannot only be altered by detection artefacts of individual mass transitions, i.e., interferences, but also by improper peak integration. A clear differentiation of both issues is not possible by monitoring these peak area ratios alone and thus frequently requires manual peak review by an expert. In case of the peak areas of the analyte and the internal standard (ISTD), the ratio thereof (analyte/ISTD ratio) leads to the calculation of the result, i.e., the desired concentration. As the result calculation is directly dependent on peak integration, failures thereof will affect the result immediately. Furthermore, failures in the peak integration are quite common due to immature integration procedures and these failures cannot automatically be distinguished from the respective system failures. Therefore, manual peak review by an expert is often needed. This peak review on the other hand strongly depends on the experience of the operator and increases human workload. Alternative procedures with parameters independent from the peak integration are therefore desirable for verification. Nonetheless, there is still a need in the art for means and methods assisting in ensuring correctness of peak identification and result calculation in LC-MS measurements. SUMMARY In accordance with one embodiment of the present disclosure, a method for identifying and/or verifying at least one analyte peak in a chromatogram of a sample for said analyte from a liquid chromatography mass spectrometer device is provided, said method comprising the following steps: a) determining a chromatogram of the sample by acquiring a plurality of data points for quantifier signal intensities and/or qualifier signal intensities, over time; and, in case the sample comprises an internal standard, optionally acquiring a plurality of data points for internal standard quantifier signal intensities and/or internal standard qualifier signal intensities, over time; b) determining for at least a fraction of the data points acquired in step a), a ratio type selected from the list consisting of (i) analyte quantifier to analyte qualifier ratios, (ii) internal standard quantifier to internal standard qualifier ratios, (iii) analyte quantifier to internal standard quantifier ratios, and (iv) analyte qualifier to intern