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US 2022/0276263 A1Published application
Mass spectrometric analysis of biomarkers
AssigneeIDEXX Laboratories, Inc.
InventorsE.A. Prabodha Ekanayaka, Jordan Haddock, Murthy V.S.N. Yerramilli
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Abstract
The present invention relates to methods for detecting and/or measuring alanine transaminase (ALT) activity, aspartate transaminase (AST) activity, alkaline phosphatase (ALP) activity, glucose levels, creatinine levels, urea levels, asymmetric dimethylarginine (ADMA) levels, and/or symmetrical dimethylarginine (SDMA) levels in a sample. The method allows all of the activities and analytes, or any combination of activities and analytes, to be measured in a single assay.
Claims20
A method for assaying for alanine transaminase activity in a sample comprising:
- (i) providing a sample suspected of containing alanine transaminase;
- (ii) contacting the sample with
- (b) α-ketoglutarate, and
- (c) pyridoxal phosphate;
- to provide an assay mixture;
- (iii) passing the assay mixture through an HPLC column to provide an eluant containing components of the assay mixture,
- (iv) subjecting at least a portion of the eluant to ionization in a mass spectrometer operated in the negative ion mode to provide a plurality of parent ions;
- (v) separating parent ions having an m/z ratio of 88 from the plurality of parent ions;
- (vi) fragmenting the parent ions having an m/z ratio of 88 to provide a plurality of daughter ions;
- (vii) separating daughter ions that have an m/z ratio of 43 from the plurality of daughter ions;
- and
- (vii) detecting the intensity daughter ions that have an m/z ratio of 43.
9 more elements
A method for assaying for aspartate transaminase activity in a sample comprising:
- (i) providing a sample suspected of containing aspartate transaminase;
- (ii) contacting the sample with
- (b) α-ketoglutarate,
- (c) pyridoxal phosphate, and
- (d) oxaloacetate decarboxylase;
- to provide an assay mixture;
- (iii) passing the assay mixture through an HPLC column to provide an eluant containing components of the assay mixture,
- (iv) subjecting at least a portion of the eluant to ionization in a mass spectrometer operated in the negative ion mode to provide a plurality of parent ions;
- (v) separating parent ions having an m/z ratio of 90 from the plurality of parent ions;
- (vi) fragmenting the parent ions having an m/z ratio of 90 to provide a plurality of daughter ions;
- (vii) separating daughter ions that have an m/z ratio of 45 from the plurality of daughter ions;
- and
- (vii) detecting the intensity daughter ions that have an m/z ratio of 45.
10 more elements
A method for assaying for alkaline phosphatase activity in a sample comprising:
- (i) providing a sample suspected of containing alkaline phosphatase;
- (ii) contacting the sample with p-nitrophenyl phosphate and, optionally, zinc sulfate, magnesium acetate, and HEDTA, to provide an assay mixture;
- (iii) passing the assay mixture through an HPLC column to provide an eluant containing components of the assay mixture;
- (iv) subjecting at least a portion of the eluant to ionization in a mass spectrometer operated in the negative ion mode to provide a plurality of parent ions;
- (v) separating parent ions having an m/z ratio of 138 from the plurality of parent ions;
- (vi) fragmenting the parent ions having an m/z ratio of 138 to provide a plurality of daughter ions;
- (vii) separating daughter ions that have an m/z ratio of 46 from the plurality of daughter ions;
- and
- (viii) detecting the intensity daughter ions that have an m/z ratio of 46 derived from the parent ion having an m/z ratio of 138.
6 more elements
A method for assaying for glucose levels in a sample comprising:
- (i) providing a sample suspected of containing glucose;
- (ii) adding
- to the sample, to provide an assay mixture;
- (iii) subjecting at least a portion of the assay mixture to ionization in a mass spectrometer operated in the negative ion mode to provide a plurality of parent ions;
- (iv) separating the parent ions having an m/z ratio of 179 and an m/z ratio of 185 from the plurality of parent ions;
- (v) fragmenting the parent ions having an m/z ratio of 179 and an m/z ratio of 185 to provide a plurality of daughter ions;
- (vi) separating daughter ions that have an m/z ratio of 119 and an m/z ratio of 123 from the plurality of daughter ions;
- and
- (vii) detecting the intensity of the daughter ions that have an m/z ratio of 119 and an m/z ratio of 123.
6 more elements
A method for assaying for urea levels in a sample comprising:
- (i) providing a sample suspected of containing urea;
- (ii) adding
- to the sample, to provide an assay mixture;
- (iii) subjecting at least a portion of the assay mixture to ionization in a mass spectrometer in the positive ion mode to provide a plurality of parent ions;
- (iv) separating the parent ions having an m/z ratio of 61 and an m/z ratio of 64 from the plurality of parent ions;
- (v) fragmenting the parent ions having an m/z ratio of 61 and an m/z ratio of 64 to provide a plurality of daughter ions;
- (vi) separating daughter ions that have an m/z ratio of 44 and an m/z ratio of 46 from the plurality of daughter ions;
- and
- (vii) detecting the intensity of the daughter ions that have an m/z ratio of 44 and an m/z ratio of 46.
6 more elements
A method for assaying for creatinine levels in a sample comprising:
- (i) providing a sample suspected of containing creatinine;
- (ii) adding
- to the sample, to provide an assay mixture;
- (iii) subjecting at least a portion of the assay mixture to ionization operated in a mass spectrometer in the positive ion mode to provide a plurality of parent ions;
- (iv) separating the parent ions having an m/z ratio of 114 and an m/z ratio of 117 from the plurality of parent ions;
- (v) fragmenting the parent ions having an m/z ratio of 114 and an m/z ratio of 117 to provide a plurality of daughter ions;
- (vi) separating daughter ions that have an m/z ratio of 44 and an m/z ratio of 47 from the plurality of daughter ions or separating daughter ions having an m/z ratio of 86 and 89 from the plurality of daughter ions;
- and
- (vii) detecting the intensity of the daughter ions that have an m/z ratio of 44 and an m/z ratio of 47 or detecting the intensity of the daughter ions that have an m/z ratio of 86 and an m/z ratio of 89.
6 more elements
A method for assaying for ADMA levels in a sample comprising:
- (i) providing a sample suspected of containing ADMA;
- (ii) adding
- to the sample, to provide an assay mixture;
- (iii) subjecting at least a portion of the assay mixture to ionization in a mass spectrometer operated in the positive ion mode to provide a plurality of parent ions;
- (iv) separating the parent ions having an m/z ratio of 203 and an m/z ratio of 210 from the plurality of parent ions;
- (v) fragmenting the parent ions having an m/z ratio of 203 and an m/z ratio of 210 to provide a plurality of daughter ions;
- (vi) separating daughter ions that have an m/z ratio of 46 from the plurality of daughter ions;
- and
- (vii) detecting the intensity of the daughter ions that have an m/z ratio of 46.
6 more elements
A method for assaying for SDMA levels in a sample comprising:
- (i) providing a sample suspected of containing SDMA;
- (ii) adding
- to the sample, to provide an assay mixture;
- (iii) subjecting at least a portion of the assay mixture to ionization in a mass spectrometer operated in the positive ion mode to provide a plurality of parent ions;
- (iv) separating the parent ions having an m/z ratio of 203 and an m/z ratio of 209 from the plurality of parent ions;
- (v) fragmenting the ions having an m/z ratio of 203 and an m/z ratio of 209 to provide a plurality of daughter ions;
- (vi) separating daughter ions that have an m/z ratio of 172 and an m/z ratio of 175 from the plurality of daughter ions;
- and
- (vii) detecting the intensity of the daughter ions that have an m/z ratio of 172 and an m/z ratio of 175.
6 more elements
A method for assaying for alanine transaminase activity, aspartate transaminase activity, alkaline phosphatase activity, glucose levels, urea levels, creatinine levels, ADMA levels, and SDMA levels in a sample comprising:
- (i) providing a sample suspected of containing one or more of:
- alanine transaminase, aspartate transaminase, alkaline phosphatase, glucose, urea, creatinine, ADMA, and SDMA;
- (ii) dividing the sample into a first portion and a second portion;
- (iii) contacting the first portion with (a) a substrate for alanine transaminase, wherein the substrate for alanine transaminase comprises a first isotopic label and (b) a substrate for aspartate transaminase, wherein the substrate for aspartate transaminase comprises a second isotopic label;
- (iv) contacting the second portion with a substrate for alkaline phosphatase;
- (v) adding isotopically labelled glucose comprising a third isotopic label;
- isotopically labelled urea comprising a fourth isotopic label, isotopically labelled creatinine comprising a fifth isotopic label, isotopically labelled ADMA comprising a sixth isotopic label, and isotopically labelled SDMA comprising a seventh isotopic label to at least one of the first portion and the second portion;
- (vi) combining the first portion and the second portion to provide an assay mixture;
- (vii) passing a first quantity of the assay mixture through an HPLC column to provide a first eluant containing components of the assay mixture;
- (viii) analyzing a portion of the first eluant with a mass spectrometer, wherein the mass spectrometer is operated in the negative ion mode, to:
- (a) generate and detect an ion formed from a metabolite resulting from the action of the alanine transaminase on the substrate for alanine transaminase, wherein the metabolite resulting from the action of the alanine transaminase on the substrate for alanine transaminase comprises the first isotopic label,
- (b) generate and detect an ion formed from a metabolite resulting from the action of the aspartate transaminase on the substrate for aspartate transaminase, wherein the metabolite resulting from the action of the aspartate transaminase on the substrate for aspartate transaminase comprises the second isotopic label,
- (c) generate and detect an ion formed from a metabolite resulting from the action of the alkaline phosphatase on the substrate for alkaline phosphatase,
- (d) generate and detect an ion formed from the glucose,
- (e) generate and detect an ion formed from the isotopically labelled glucose, wherein the ion formed from the isotopically labelled glucose comprises the third isotopic label;
- (ix) passing a second quantity of the assay mixture through an HPLC column to provide a second eluant containing the components of the assay mixture;
- (x) analyzing a portion of the second eluant with a mass spectrometer, wherein the mass spectrometer is operated in the positive ion mode, to:
- (a) generate and detect an ion formed from the urea,
- (b) generate and detect an ion formed from the isotopically labelled urea, wherein the ion formed from the isotopically labelled urea comprises the fourth isotopic label,
- (c) generate and detect an ion formed from the creatinine,
- (d) generate and detect an ion formed from the isotopically labelled creatinine, wherein the ion formed from the isotopically labelled creatinine comprises the fifth isotopic label,
- (e) generate and detect an ion formed from the ADMA,
- (f) generate and detect an ion formed from the isotopically labelled ADMA, wherein the ion formed from the isotopically labelled ADMA comprises the sixth isotopic label,
- (g) generate and detect an ion formed from the SDMA, and
- (h) generate and detect an ion formed from the isotopically labelled SDMA, wherein the ion formed from the isotopically labelled SDMA comprises the seventh isotopic label.
22 more elements
A method for assaying for alanine transaminase activity, aspartate transaminase activity, alkaline phosphatase activity, glucose levels, urea levels, creatinine levels, ADMA levels, and SDMA levels in a sample comprising:
- (i) providing a sample suspected of containing one or more of:
- alanine transaminase, aspartate transaminase, alkaline phosphatase, glucose, urea, creatinine, ADMA, and SDMA;
- (ii) dividing the sample into a first and a second portion;
- (iii) contacting the first portion with:
- (a) α-ketoglutarate,
- (b) pyridoxal phosphate,
- (e) oxaloacetate decarboxylase, and
- to provide a first reaction mixture;
- (iv) contacting the second portion with:
- and
- (v) optionally zinc sulfate, magnesium acetate, and HEDTA, to provide a second reaction mixture;
- (vi) adding:
- to at least one of the first reaction mixture and the second reaction mixture;
- (vii) combining the first reaction mixture and the second reaction mixture to provide an assay mixture;
- (viii) passing a first quantity of the assay mixture through an HPLC column to provide a first eluant containing components of the assay mixture;
- (ix) analyzing at least a portion of the first eluant with a mass spectrometer, wherein the mass spectrometer is operated in the negative ion mode, to provide a first plurality of parent ions;
- (x) separating parent ions having an m/z ration of 88, 90, 138, 179, and 185 from the first plurality of parent ions;
- (xi) fragmenting the parent ions having an m/z ratio of 88, 90, 138, 179, and 185 to provide a first plurality of daughter ions;
- (xii) separating daughter ions having an m/z ratio of 43, 45, 46, 119, and 123 from the first plurality of daughter ions;
- (xiii) detecting the intensity of the daughter ions that have an m/z ratio of 43, 45, 46, 119, and 123;
- (xiv) passing a second quantity of the assay mixture through an HPLC column to provide a second eluant containing components of the assay mixture;
- (xv) analyzing at least a portion of the second eluant with a mass spectrometer, wherein the mass spectrometer is operated in the positive ion mode, to provide a second plurality of parent ions;
- (xvi) separating parent ions having an m/z ration of 61, 64, 114, 117, 203, 209, and 210 from the second plurality of parent ions;
- (xvii) fragmenting the parent ions having an m/z ratio of 61, 64, 114, 117, 203, 209, and 210 to provide a second plurality of daughter ions;
- (xviii) separating daughter ions having an m/z ratio of 44, 46, 47, 86, 89, 172, and 175 from the second plurality of daughter ions;
- and
- (xix) detecting the intensity of the daughter ions that have an m/z ratio of 44, 46, 47, 86, 89, 172, and 175.
24 more elements
A method for assaying for one or more selected from the group consisting of alanine transaminase activity, aspartate transaminase activity, alkaline phosphatase activity, glucose levels, urea levels, creatinine levels, ADMA levels, and SDMA levels in a sample comprising:
- (A) providing a sample suspected of containing one or more of:
- alanine transaminase, aspartate transaminase, alkaline phosphatase, glucose, urea, creatinine, ADMA, and SDMA;
- (B) dividing the sample into at least a first portion and a second portion;
- (C) contacting at least one of:
- (i) the first portion with (a) a substrate for alanine transaminase, wherein the substrate for alanine transaminase comprises a first isotopic label and (b) a substrate for aspartate transaminase, wherein the substrate for aspartate transaminase comprises a second isotopic label;
- and
- (ii) the second portion with a substrate for alkaline phosphatase;
- (D) adding at least one of isotopically labelled glucose comprising a third isotopic label;
- isotopically labelled urea comprising a fourth isotopic label, isotopically labelled creatinine comprising a fifth isotopic label, isotopically labelled ADMA comprising a sixth isotopic label, isotopically labelled SDMA comprising a seventh isotopic label to the first portion or the second portion;
- (E) if there is a first portion and a second portion, combining the first portion and the second portion to provide an assay mixture;
- (F) passing at least one of
- (i) a first quantity of the assay mixture through an HPLC column to provide a first eluant containing components of the assay mixture, and
- (a) analyzing a portion of the first eluant with a mass spectrometer, wherein the mass spectrometer is operated in the negative ion mode, to generate and detect at least one of:
- an ion formed from a metabolite resulting from the action of the alanine transaminase on the substrate for alanine transaminase, wherein the metabolite resulting from the action of the alanine transaminase on the substrate for alanine transaminase comprises the first isotopic label,
- an ion formed from a metabolite resulting from the action of the aspartate transaminase on the substrate for aspartate transaminase, wherein the metabolite resulting from the action of the aspartate transaminase on the substrate for aspartate transaminase comprises the second isotopic label,
- an ion formed from a metabolite resulting from the action of the alkaline phosphatase on the substrate for alkaline phosphatase,
- an ion formed from the glucose, and
- an ion formed from the isotopically labelled glucose, wherein the ion formed from the isotopically labelled glucose comprises the third isotopic label;
- and
- (ii) a second quantity of the assay mixture through an HPLC column to provide a second eluant containing the components of the assay mixture, and
- (a) analyzing at least a portion of the second eluant with a mass spectrometer, wherein the mass spectrometer is operated in the positive ion mode, to generate and detect at least one of:
- an ion formed from the urea,
- an ion formed from the isotopically labelled urea, wherein the ion formed from the isotopically labelled urea comprises the fourth isotopic label,
- an ion formed from the creatinine,
- an ion formed from the isotopically labelled creatinine, wherein the ion formed from the isotopically labelled creatinine comprises the fifth isotopic label,
- an ion formed from the ADMA,
- an ion formed from the isotopically labelled ADMA, wherein the ion formed from the isotopically labelled ADMA comprises the sixth isotopic label,
- an ion formed from the SDMA, and
- generate and detect an ion formed from the isotopically labelled SDMA, wherein the ion formed from the isotopically labelled SDMA comprises the seventh isotopic label.
26 more elements
Dependencies are inferred from the claim text.