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• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/92—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving lipids, e.g. cholesterol, lipoproteins, or their receptors
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
  • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
  • G01N33/6848—Methods of protein analysis involving mass spectrometry
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
  • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
• • H—ELECTRICITY
  • H01—ELECTRIC ELEMENTS
  • H01J—ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS
  • H01J49/00—Particle spectrometers or separator tubes
  • H01J49/0027—Methods for using particle spectrometers
  • H01J49/0031—Step by step routines describing the use of the apparatus
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
  • G01N2333/90—Enzymes; Proenzymes
  • G01N2333/902—Oxidoreductases (1.)
  • G01N2333/90245—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
  • G01N2333/90248—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14) with NADH or NADPH as one of the donors, and incorporation of one atom of oxygen 1.14.13
  • G01N2333/90251—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14) with NADH or NADPH as one of the donors, and incorporation of one atom of oxygen 1.14.13 with a definite EC number (1.14.13.-)
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2400/00—Assays, e.g. immunoassays or enzyme assays, involving carbohydrates
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2800/00—Detection or diagnosis of diseases
  • G01N2800/04—Endocrine or metabolic disorders
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2800/00—Detection or diagnosis of diseases
  • G01N2800/06—Gastro-intestinal diseases
  • G01N2800/065—Bowel diseases, e.g. Crohn, ulcerative colitis, IBS
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2800/00—Detection or diagnosis of diseases
  • G01N2800/38—Pediatrics
  • G01N2800/385—Congenital anomalies

Definitions

• isotopically enriched quaternary aminoxy reagents disclosed herein can be used to make internal standards for each analyte of interest by a simple reaction with a clean standard.
• internal standards for a panel of biomarkers can be created by labeling known concentrations of a plurality of biomarkers analytes with isotopically variants of a quaternary aminoxy reagent disclosed herein (i.e., labeling each biomarker with a different isotopically variant of the QAO reagent).
• Such internal standards can be added to test samples to allow absolute quantifications of biomarkers of interest.
• the methods in accordance with the present teachings can be employed for highly sensitive and specific analysis of ketosterol biomarkers that allow for detection of the ketosteroid bile acid precursors from a variety of samples, including samples having a low volume and complex biological matrices.
• ketosterol bile acid precursors are analyzed, such as one or more of 7a-cholesten-3-one (7aC4), 5a-cholestan-3-one, 4-cholesten-3-one, 7a,12a-dihydroxy-4-cholesten-3-one, 7a-hydroxy-5p-cholestan-3-one, and 7cc,12a-dihydroxy-5p- cholestan-3-one (7a,12a-C4).
• FIG. 8 is a concentration curve using d7 7aC4 as IS (squares) and deuterated reagent as IS (diamonds).
• the QAO reagent as described herein substantially improves the ionization efficiency of these ketosterol bile acid precursors and provides significantly lower LLOQ than without the QAO reagent. This method provides for analysis using sample volume as low as 3 - 5 pL.
• n 2, 3, 4, 5, or 6 and Y has the structure:
• X is an anion
• n is 2 - 4 and in other embodiments, n is 3.
• Y is -N(CH 3 ) 3 ⁇ .
• m is an integer between 1 and 12 or an integer between 1 and 5.
• each R4 is independently H or a Ci - Ci 2 alkyl which is branched or straight chain, or each R4 is independently H or a Ci - C 6 alkyl which is branched or straight chain. In some embodiments, each R4 is the same.
• the signature ion fragment can comprise, in some embodiments, a fragment ion having a mass of 152.0.
• the signature ion fragment can comprise, in some embodiments, a fragment ion having a mass of 443.0.
• the signature ion fragment can comprise, in some embodiments, a fragment ion having a mass of 161.2.
• isobaric tags can be used.
• sets of isobaric labels may comprise one, or more heavy atom isotopes.
• a set of isobaric labels can have an identical or specifically defined range of aggregate masses but can have a primary reporter ion or charged analyte of a different measurable mass.
• a set of isobaric reagents enables both qualitative and quantitative analysis of ketosterol biomarker analyte compounds using mass spectroscopy.
• Quantitation can be enabled by relative or absolute measurement of the signal derived from one or more analytes and standards.
• the positive charge can be transferred to the analyte which functions as the fragment ion to be detected by mass spectrometry.
• isobaric ketosteroid in the biological sample may have a similar Q1/Q3 MRM transition
• the isobaric ketosteroids can share the same fragmentation pattern with the analyte in order to appear as interference.
• the isobaric ketosteroid is preferably chromatographically separated from the analyte.
• the concentration of multiple ketosterol bile acid precursor compounds in one or more samples can be determined in a multiplex fashion, for example, by combining two or more labeled analyte compounds to produce a combined sample and subjecting the combined sample to PDITM, and monitoring the analyte or reporter ions of two or more of labeled precursor compounds.
• PDITM can be performed on any suitable mass analyzer known in the art, including a mass analyzer system comprising a first mass separator, and ion fragmentor and a second mass separator.
• the transmitted parent ion m z range of a PDITM scan (selected by the first mass separator) is selected to comprise a m/z value of one or more of the labeled analyte compounds and the transmitted daughter ion m/z range of a PDITM scan (selected by the second mass separator) is selected to comprise a m/z value one or more of the reporter ions corresponding to the tag of the transmitted labeled analyte compound.
• the one or more ketosterol bile acid precursor samples are labeled with one or more of tags selected from a set of mass differential tags so that within the same experimental measurement: (i) multiple ketosterol bile acid precursor-containing compounds from different samples (e.g., a control, treated) can be compared and/or quantified; (ii) multiple concentration measurements can be determined on the same ketosterol bile acid precursor compound from the same sample; and (iii) different isolates of a clinical sample can be evaluated against a baseline sample.
• the measured ion signals of one or more of the reporter ions corresponding to one or more of the one or more labeled standard compounds in the combined sample determines the concentration of one or more of the labeled analyte compounds. Determining the concentration of a labeled analyte compound is based at least on a comparison of the measured ion signal of the corresponding fragment ion to the measured ion signal of one or more fragment ions corresponding to one or more of the one or more labeled standard compounds in the combined sample.
• a sample comprising a plurality of different ketosterol bile acid precursors can be treated with different QAO reagents to label each of two or more of the ketosterol bile acid precursors with a different one of said reagents.
• the labeled sample can then be analyzed using mass spectrometry, e.g., LC/MSMS, to quantitate the labeled ketosterol bile acid precursors.
• mass spectrometry e.g., LC/MSMS
• such analysis of the sample can be performed in a single run of the spectrometer.
• the ketosterol bile acid precursors can be steroid or steroid-like analytes.
• the analysis of ketosterol bile acid precursors can comprise generating reporter ions, e.g., via a high-energy collision in a mass spectrometer, and utilizing the intensity or the peak area of the reporter ions for quantitation.
• the QAO reagents shown above can undergo neutral loss during high energy collisions (MSMS) leaving a charged analyte species as the reporter ion, and the reporter ion can then be subjected to MS 3 analysis.
• the QAO reagents can generate a tag fragment upon a high energy collision, and the tag fragment can then be subjected to MS 3 analysis.
• a plurality of mass spectrometry (MS) tagging agents can be used for labeling one or more ketosterol bile acid precursors and/or adducts thereof formed in accordance with the present teachings.
• the aminoxy tagging agents can fragment well to provide intense reporter ions.
• the aminoxy tagging agents can comprise tagging agents that are specifically designed for mass spectrometry and according to some embodiments, the aminoxy tagging agent is specifically designed for a Multiple Reaction Monitoring (MRM) assay.
• MRM Multiple Reaction Monitoring
• an aminoxy tagging agent can be used to tag an adduct generated by labeling a ketosterol bile acid precursor.
• the fragment ion that is the Q3 signature ion is selected to comprise structural fragments with an attached derivatization reagent, or a part of the reagent, both the sensitivity and selectivity can be enhanced. The chances that a compound with exactly the same Q1/Q3 transition would be detected and create background noise interference are very low. The only possibility for a similar Q1/Q3 MRM transition would be the existence of an isobaric ketosteroid in the biological sample. The isobaric ketosteroid would have to share the same fragmentation pattern with the analyte in order to appear as interference. In such a rare scenario, the isobaric ketosteroid can be chromatographically separated from the analyte.
• One or more of the quadrupoles in a triple quadrupole mass spectrometer can be configurable as a linear ion trap (e.g., by the addition of end electrodes to provide a substantially elongate cylindrical trapping volume within the quadrupole).
• the first quadrupole selects the parent ion.
• the second quadrupole is maintained at a sufficiently high collision gas pressure and voltage so that multiple low energy collisions occur causing some of the parent ions to fragment.
• the third quadrupole is selected to trap fragment ions and, after a fill time, transmit the selected daughter ion to a detector by pulsing an end electrode to permit the selected daughter ion to exit the ion trap.
• Desired fill times can be determined, e.g., based on the number of fragment ions, charge density within the ion trap, the time between elution of different signature peptides, duty cycle, decay rates of excited state species or multiply charged ions, or combinations thereof.
• the supports can be reacted with different samples thereby labeling the analytes of a sample with the isobaric tag associated with the respective support.
• Analysis of ketosterol bile acid precursors from different samples can be contacted with different supports and thus labeled with different reporter/linker combinations.
• a set of isobaric tags can comprise compounds of formula (I) or (II), or a salt or a hydrate form thereof.
• a daughter ion of an isobaric tag that can be used to distinguish between members of the set can be a reporter ion of the isobaric tag or charged analyte.
• a set of isobaric tags can be used to label ketosterol bile acid precursors and produced labeled compounds that are substantially chromatographically indistinguishable, but which produce signature ions following CID.
• the masses of the individual members of a set of mass labels can be identical or different. Where the individual isotopic substitutions are the same, the masses can be identical. Differences in selecting individual atoms for the heavy or light element incorporated into a specific label of the set can also yield mass differences based on the specific atomic weights of the isotopically enriched substituents.
• Salts of compounds having a carboxylic acid, or other acidic functional group can be prepared by reacting the compound with a suitable base, for example, a hydroxide base. Accordingly, salts of acidic functional groups may have a countercation, such as sodium, potassium, magnesium, calcium, etc.
• a plasma sample (5 ⁇ ,) was combined with 80 iL (210 ⁇ g) QAO reagent in MeOH

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• 2013
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