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-12352756-B2
Publication Date
2025-07-08
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 PGD2 in a biological sample by tandem mass spectrometry. A sample is obtained from a subject, and PGD2 is purified by liquid chromatography before measurement. The disclosed approach measures endogenous prostaglandin D2 using tandem mass spectrometry.
The disclosed workflow emphasizes LC-MS/MS analysis and monitoring of precursor ions and fragment ions in tandem MS/MS. Selected reaction monitoring (SRM) is described as a mode for detecting PGD2 by tandem mass spectrometry, and stable isotope labeled PGD2 internal standards are described as optional components that support quantitation.
The document further describes assay performance and validation for PGD2 in human serum, serum/6% BSA, urine, and related sample types, including sensitivity and reportable/quantification ranges. It also includes reference interval findings, sample normalization to creatinine, selectivity and specificity testing against potential interferents, and system architecture or computer-controlled automation supporting the LC-MS/MS workflow.
Claims Coverage
The independent claim coverage centers on a tandem mass spectrometry method for determining the presence or amount of PGD2 in a biological sample using liquid chromatography purification followed by tandem mass spectrometry measurement. The inventive features include purification by liquid chromatography, measurement of PGD2 by tandem mass spectrometry, and dependent refinements such as SRM detection, stable isotope-labeled internal standards, HTLC purification, monitored precursor and fragment ion m/z values, and a defined concentration range.
Tandem mass spectrometry determination of PGD2 after liquid chromatography purification
A method for determining the presence or amount of PGD2 in a biological sample by tandem mass spectrometry, comprising obtaining a sample from a subject, performing liquid chromatography to purify the sample, and measuring PGD2 by tandem mass spectrometry.
Stable isotope labeled PGD2 internal standard
The method includes adding a stable isotope-labeled PGD2-d9 to the sample as an internal standard.
Selected reaction monitoring detection
The method further includes detecting PGD2 by tandem mass spectrometry using selected reaction monitoring (SRM) mode.
High turbulence liquid chromatography purification
The liquid chromatography uses high turbulence liquid chromatography (HTLC) to purify the sample.
Precursor and fragment ion monitoring
The method uses a precursor ion having an m/z of about 360.4 and quantitation using one or more fragment ions that include a fragment ion with an m/z of about 232.9.
Defined PGD2 concentration range
The method includes detecting PGD2 across a specified concentration range from 1.0 pg/mL to 1,000 μg/mL.
Overall, the claim coverage is a liquid chromatography-purified tandem mass spectrometry method for PGD2, further defined by SRM detection, stable isotope-labeled internal standards, optional HTLC, monitored precursor and fragment ion m/z values, and a specified quantitative detection range.
Stated Advantages
Establishment of a serum reference interval, including the 97.5th percentile value range of 1.6–57 pg/mL.
Validated performance support through extraction recovery evaluation and stability testing across autosampler storage, short-term handling, and freeze/thaw conditions.
Assessment of selectivity and specificity against potential interferents using spiking and retention time ratio criteria.
Provides specificity/selectivity and validation performance for urine and serum measurements of PGD2, including sensitivity and reportable/quantification ranges.
Supports detection with SRM mode and measurement using monitored precursor and fragment ions.
Documented Applications
Determining the presence or amount of PGD2 in a biological sample from a subject using tandem mass spectrometry with liquid chromatography purification.
Serum reference interval assessment for PGD2 in human serum, including the 97.5th percentile value range of 1.6–57 pg/mL.
Evaluation of assay performance in human serum/6% BSA using extraction recovery, autosampler stability, short-term stability, and freeze/thaw stability testing.
Selectivity and specificity evaluation for PGD2 measurement in the presence of potential interferents including PGD2 isomers/metabolites and prostaglandin-related compounds.
Determining endogenous PGD2 in urine and serum biological samples, including validation and reference interval findings with urine normalization to creatinine.
Clinical diagnostic context related to mastocytosis.
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