Reverse-phase high pressure liquid chromatography methods for measuring amino acids, ammonium, and glutathione concentrations in biological samples
AssigneeChildren's National Medical Center
InventorsGary CUNNINGHAM, Marshall SUMMAR
Life of this patent
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Abstract
A fast and accurate reverse-phase high pressure liquid chromatography (“RP-HPLC”) method for detecting amino acids in small volumes (e.g. less than 50 µL) of a biological sample, such as plasma. An assay for the simultaneous determination of ammonium and primary amino acids using RP-HPLC in samples such as plasma. A method for calculating intercellular volumes from a cell lysate to which a known volume and concentration of a non-naturally occurring amino acid is added.
Claims30
A method for detecting amino acids in a sample volume of 50 µL or less comprising:
- (a) removing proteins from a sample,
- (b) derivatizing the sample obtained from (a) by contacting it with O-Phthalaldehyde (OPA),
- (c) injecting the derivatized sample from (b) into a RP-HPLC column to produce fractions, and
- (d) detecting RP-HPLC resolved fractions or components as they elute from the RP-HPLC column by ultraviolet illumination.
A method for simultaneous determination of ammonium and primary amino acids in a same sample comprising:
- (a) removing solids from a sample,
- (b) derivatizing the sample obtained from (a) by contacting it with O-Phthalaldehyde (OPA),
- (c) injecting the derivatized sample from (b) into a RP-HPLC column to produce fractions, and
- (d) detecting RP-HPLC resolved fractions or components as they elute from the RP-HPLC column by ultraviolet illumination.
A method for calculating an intercellular volume of cells used to produce a cell lysate, comprising:
- (a) producing a cell lysate,
- (b) adding a known concentration of a non-naturally occurring amino acid to the cell lysate to produce a sample for RP-HPLC,
- (c) derivatizing the sample obtained from (b) by contacting it with O-Phthalaldehyde (OPA),
- (d) injecting the derivatized sample from (c) into a RP-HPLC column to produce fractions,
- (e) detecting RP-HPLC resolved fractions or components as they elute from the RP-HPLC column by ultraviolet illumination;
- and
- (f) calculating an average intracellular volume of the cells used to produce the lysate by comparison of the concentration of the non-naturally occurring amino acid and the detected concentrations of one or more analytes with the concentration of the non-naturally occurring amino acid, thereby calculate an average cell volume of the cells used to produce the cell lysate.
Dependencies are inferred from the claim text.