Method for the Direct Detection and/or Quantification of at Least One Compound with a Molecular Weight of at Least 200

摘要

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.

主权项

1. 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, including ions or salts thereof, 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 for the direct detection and/or quantification comprises at least the following steps:
i) preparing at least one standard sample of the chemically complex molecule to be detected and/or quantified; ii) introducing the standard sample into the stream of a solvent system; wherein the solvent system is a polar solvent or a solvent mixture comprising at least one polar solvent. iii) passing the sample and the solvent system through at least one column wherein the column is essentially filled of smalls particles of a stationary phase while maintaining the pressure of the system between 5 and 1500 atm; iv) indentifying the retention time and/or quantifying the intensity of the signal of when the chemically complex molecule is eluated, by means of a mass or radioactivity detector; v) preparing a sample of the chemically complex molecule from a biological matrix comprising the chemically complex molecule to be detected and/or quantified, wherein the process to prepare the sample comprises at least dissolving totally or partially the biological matrix to form a solution or a slurry, and, if necessary treating the solution or the slurry to remove particles in suspension; vi) repeating the steps (ii) and (iii) with the sample prepared in the step (v); together or sequentially with the standard; and vii) detecting and/or quantifying the presence of the chemically complex molecule by comparison of retention time and/or intensity of signal of the standard sample or the standard samples, or by comparison of retention time and/or intensity of signal obtained from previous studies or from literature, wherein the detector capable to detect the eluation of the chemically complex molecule is a mass spectrometer: a tandem mass spectrometer (or triple quadrupole); a single quadrupole; working under any of the following operating modes: selected ion monitoring (SIM); multiple reaction monitoring (MRM); selected reaction monitoring (SRM) and SCAN (negative/positive); or a combination of them.