Method for the direct detection and/or quantification of at least one compound with a molecular weight of at least 200
Inventors
Perello Bestard, Joan • Maraschiello de Zuani, Ciriaco • Lentheric, Irene • Mendoza de las Heras, Paula • Tur Espinosa, Fernando • Tur Tur, Eva • Encabo Alarcon, Maximo • Martin Becerra, Eva • Benito Amengual, Maria de Mar • Isern Amengual, Bernat
Assignees
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Publication Number
US-12130296-B2
Publication Date
2024-10-29
Expiration Date
Abstract
The present invention relates to method for the direct detection and/or quantification of at least one compound with a molecular weight of at least 200, wherein the compound to be detected and/or quantified is a chemically complex molecule, wherein said chemically complex molecule is substituted with at least two groups R, wherein each R group means independently —OH, —OP(O)(OH)2 or —P(O)(OH)2, with the proviso that at least two R are independently selected from —P(O)(OH)2 and —OP(O)(OH)2, wherein the compound or compounds to be detected and/or quantified are within a biological matrix, wherein said biological matrix is a biological fluid, a biological tissue, stomach contents, intestine contents, stool sample or a culture cells, wherein the method comprises performing a chromatography and identifying the retention time and/or the intensity of the signal by means of a mass or radioactivity detector.
Core Innovation
A method is provided for analyzing a composition comprising a phosphorus containing compound having a molecular weight of at least 200 daltons, where the composition comprises the phosphorus containing compound together with its impurities. The method introduces at least one standard sample together or sequentially with a test sample into a stream of a polar solvent system comprising potassium hydroxide (KOH) or a solvent mixture comprising at least one polar solvent comprising KOH, and passes the standard sample and test sample through a single anion-exchange chromatography column.
The single anion-exchange chromatography column contains particles of a cross-linked polystyrene resin. The test sample comprises a solution or a slurry containing the composition, and the amount of phosphorus containing compound in the composition is above 60% by weight. The phosphorus containing compound is selected from bisphosphonate or polyphosphonate, a hexametaphosphate, a C3–C7 cycloalkyl substituted compound with at least two —R groups where each —R group is —OH, —OP(O)(OH)2 or —P(O)(OH)2, ions or salts thereof, or a combination of these.
The described approach supports direct detection and quantification in chemically complex phosphorus-containing molecules in biological matrices, using retention time and signal intensity measured with a mass detector or a radioactivity detector after chromatography. The document links the analytical readout to the separating function of the anion-exchange chromatography and includes analytical options such as mass spectrometry modes and post-column derivatization with UV detection, enabling assay and impurities/metabolite profiling for phosphorus-containing species such as inositol polyphosphates and related species.
Claims Coverage
The document provides one independent claim. Across that independent claim, the core coverage is anchored by three inventive feature groupings: the KOH-based polar solvent stream, the single anion-exchange chromatography column containing a cross-linked polystyrene resin, and the constrained phosphorus-containing compound selection.
KOH-based polar solvent stream with standard and test samples
A method comprising introducing at least one standard sample together or sequentially with a test sample into a stream of a polar solvent system comprising potassium hydroxide (KOH) or a solvent mixture comprising at least one polar solvent comprising KOH, wherein the method analyzes a composition comprising a phosphorus containing compound having a molecular weight of at least 200 daltons and comprising the phosphorus containing compound together with its impurities.
Single anion-exchange chromatography column with cross-linked polystyrene resin
Passing the standard sample and test sample through a single anion-exchange chromatography column containing particles of a cross-linked polystyrene resin, wherein the test sample comprises a solution or a slurry containing the composition, and wherein the amount of phosphorus containing compound in the composition is above 60% by weight.
Constrained phosphorus-containing compound selection
Wherein the phosphorus containing compound is selected from bisphosphonate or polyphosphonate; a hexametaphosphate; a C3–C7 cycloalkyl substituted compound with at least two —R groups where each —R group is —OH, —OP(O)(OH)2 or —P(O)(OH)2, with at least two —R groups independently selected from —P(O)(OH)2 and —OP(O)(OH)2; ions or salts thereof; or a combination thereof.
Overall, the independent claim covers analysis of phosphorus-containing compounds present above 60% by weight by running a standard and a test sample through a single anion-exchange chromatography column packed with a cross-linked polystyrene resin, using a polar KOH-containing solvent system, and restricting the analyte class to the enumerated phosphorus-containing compounds.
Stated Advantages
Enables direct detection and quantification in chemically complex phosphorus-containing molecules in biological matrices based on retention time and signal intensity measured with a mass detector or a radioactivity detector after chromatography.
Enables assay and impurity/metabolite profiling of phosphorus-containing species such as inositol polyphosphates and related phosphorus-containing compounds.
Supports analysis using multiple analytical readout options, including mass spectrometry detection modes and post-column derivatization with UV detection.
Documented Applications
Analyzing phosphorus-containing compounds in biological matrices, including biological fluids, biological tissue, stomach/intestine contents, stool samples, and culture cells.
Analysis in pharmaceutical/API or related formulations for quantification and impurity profiling.
Impurity and metabolite profiling, including metabolite profiling in hepatocytes and profiling of phosphorus-containing species such as inositol polyphosphates and related species.
Use in assay contexts that include protein precipitation and solid-liquid separation/centrifugation within the described workflow for sample handling.
Direct quantification of inositol polyphosphate species and ATP in rat, dog, and human plasma, urine, and culture contexts, including bioequivalence study measurements for IP6.
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