Methods and systems for detecting prostaglandins by LC-MS/MS
Inventors
Holmquist, Brett • Kelemen, Mary Katherine Morr
Assignees
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Publication Number
US-12025619-B2
Publication Date
2024-07-02
Expiration Date
Abstract
Disclosed are methods, systems, and computer program products for using liquid chromatography/tandem mass spectrometry (LC-MS/MS) for the analysis of endogenous biomarkers, such as PGD2, in a biological sample. More specifically, the methods, systems, and computer program products are described for detecting and quantifying the amount of an PGD2 in a sample. The quantitative analysis may be helpful in making clinical diagnoses.
Core Innovation
The invention relates to determining the presence or amount of prostaglandin D2 (PGD2) in a biological sample by tandem mass spectrometry. The workflow includes obtaining a sample from a subject, adding a stable isotope labeled PGD2 as an internal standard, performing liquid chromatography to purify the sample, and measuring PGD2 by tandem mass spectrometry. A system and a computer-program product are also described for carrying out the same overall measurement concept centered on PGD2 detection and quantitation in a biological sample.
The disclosed approach centers on stable isotope labeled PGD2 as an internal standard to support accurate determination of PGD2 presence or amount, and it incorporates defined tandem mass spectrometry detection and quantitation behavior, including using precursor and fragment ions and, in refined embodiments, selected reaction monitoring. The described analytical context also includes purification and chromatographic separation to account for sample-related interferences, together with calibration and acceptance rules, matrix effects, blank matrix and internal standard interference, and carry-over assessment during tandem mass spectrometry measurement.
The work further provides analytical validation for PGD2 in human serum, including selectivity and specificity assessment against potential interferents and validation elements such as sample collection tube type effects, calibrator freeze/thaw stability, stability across relevant testing conditions, recovery versus baseline, precision, accuracy, dilution linearity, and calibration curve performance. The described validation also includes a serum reference interval established from 140 serum samples and a reported 97.5th percentile reference range of 1.6–57 pg/mL.
Claims Coverage
The independent claims collectively cover a method, a system, and a computer-program product for determining the presence or amount of PGD2 in a biological sample. Across the independent claims, the core inventive features center on stable isotope labeled PGD2 internal standard, liquid chromatography purification or separation, and tandem mass spectrometry analysis; refinements specify SRM mode, precursor and fragment ion detection, specific m/z values, urine or serum, and a PGD2 measurement range.
Method for determining presence or amount of PGD2 by tandem mass spectrometry
A method comprising obtaining a sample from a subject, adding a stable isotope labeled PGD2 to the sample as an internal standard, performing liquid chromatography to purify the sample, and measuring the PGD2 by tandem mass spectrometry.
Workflow system for partially purifying, separating, and mass spectrometry analysis of PGD2
A system comprising a station for providing a test sample suspected of containing PGD2, a station for partially purifying PGD2 from other components in the sample, a station for chromatographically separating PGD2 from other components in the sample, and a station for analyzing the chromatographically separated PGD2 by mass spectrometry to determine the presence or amount of PGD2 in the biological sample.
Computer-program product for measuring PGD2 using stable isotope internal standards and tandem mass spectrometry
A computer-program product including instructions configured to cause one or more computers to perform actions comprising obtaining a biological sample from a subject, adding a stable isotope-labeled PGD2 to the sample as an internal standard, performing liquid chromatography, and measuring PGD2 by tandem mass spectrometry.
Selected reaction monitoring mode for PGD2 measurement
Measuring PGD2 by tandem mass spectrometry in selected reaction monitoring mode (SRM).
Precursor and fragment ion detection for internal standard-based quantitation
Detecting the internal standard by generating a precursor ion and one or more fragment ions, detecting the presence or amount of the precursor ion generated and the fragment ions generated, and relating the detected ions to the presence or amount of the internal standard.
Specific precursor ion and quantitation fragment ion m/z values
Using a precursor ion with an m/z of about 360.4 and quantitation fragment ions including a fragment ion with an m/z of about 232.9.
Biological sample as urine or serum for PGD2 measurement
Defining the biological sample as urine or serum.
Defined PGD2 measurement concentration range
Measuring the PGD2 over a range of from 1.0 pg/mL to 1,000 pg/mL.
Across the independent claims, the central claimed concept is determining the presence or amount of PGD2 in a biological sample using liquid chromatography purification and tandem mass spectrometry measurement supported by a stable isotope labeled PGD2 internal standard, with system and software embodiments implementing the same overall workflow.
Stated Advantages
Provides selectivity/specificity evaluation against potential interferents, including multiple PGD2-related prostaglandin species and isomers, using retention time ratio criteria and recovery outcomes.
Supports assay qualification/validation elements including sample collection tube type effects, calibrator freeze/thaw stability, and calibration curve accuracy/precision.
Defines tandem mass spectrometry quantification performance for PGD2 using stable-isotope internal standards and selected reaction monitoring with defined measurement ranges.
Supports stability in human serum with mean % recovery compared to baseline using an 85–115% recovery criterion across storage conditions and time points.
Provides assay stability and mean % recovery data for PGD2 in human serum across storage and temperature conditions, including discussion of samples outside an 85–115% criterion.
Provides a reference interval for normalized prostaglandin D2 concentration in serum derived from 140 serum samples (1.6–57 pg/mL, 97.5th percentile basis).
Autosampler stability is reported as meeting acceptance criteria over an approximately 3 day 22 hour 16 minute interval.
Freeze storage conditions are reported as meeting acceptance criteria within specified freezing temperature limits and a defined maximum freeze/thaw cycle.
Documented Applications
PGD2 serum assay qualification/validation, including selectivity/specificity assessment and additional validation elements such as sample collection tube type effects, calibrator freeze/thaw stability, and calibration curve accuracy/precision.
Determining the presence or amount of PGD2 in a biological sample (urine or serum) suspected of containing PGD2.
PGD2 measurement workflow in human serum, evaluated for stability/recovery under multiple storage conditions and time points.
Determining the presence or amount of PGD2 in biological samples.
Measuring PGD2 in urine and serum.
Analytical validation of PGD2 assay performance in human serum, including autosampler stability, recovery versus baseline, and freeze/thaw stability across multiple donor samples.
Assessment of stability handling conditions for PGD2 measurement, including autosampler stability and short-term plus freeze/thaw stability using acceptance criteria.
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