# US 11,906,510 B2 · Metabolic profiling by reverse-phase/ion-exchange mass spectrometry

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- Number: US 11,906,510 B2
- Document ID: US11906510B2
- Status: Granted
- Kind: B2
- Application number: 17/207,295
- Filed: 2021-03-19
- Published: 2021-11-25
- Granted: 2024-02-20
- Earliest claimed: 2020-03-20
- Assignees: Stanford Junior University
- Applicants: The Board of Trustees of the Leland Stanford Junior University
- Inventors: Anthony T. Le
- Web page: https://patentgrep.com/patent/US11906510B2

## CPC

- G01N 33/5038: {involving detection of metabolites per se}
- G01N 33/92: involving lipids, e.g. cholesterol {, lipoproteins, or their receptors (steroid hormones G01N33/743)}
- G01N 30/06: Preparation
- G01N 30/461: {with serial coupling of separation columns}
- G01N 30/72: Mass spectrometers {(mass spectrometers per se H01J49/00)}
- G01N 33/6806: {Determination of free amino acids}
- G01N 2030/027: {Liquid chromatography}
- G01N 2560/00: Chemical aspects of mass spectrometric analysis of biological material
- G01N 2800/04: Endocrine or metabolic disorders
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)

## Abstract

Disclosed herein are methods of analyzing a biological sample comprising: separating components of the biological sample via reversed-phase (RP) chromatography to obtain an elute; subjecting the elute to separation via ion-exchange (IEX) chromatography or mixed-mode IEX chromatography; and detecting the separated compounds to determine the components of the biological sample. Also disclosed are devices comprising a reversed-phase (RP) chromatography column in communication with an ion-exchange (IEX) chromatography column or mixed-mode IEX chromatography column, wherein there is no switching valve between the columns.

## Claims

### Claim 1

A method of analyzing a biological sample comprising:
separating components of the biological sample via reversed-phase (RP) chromatography to obtain an elute;
subsequently, subjecting the elute to separation via ion-exchange (IEX) chromatography or mixed-mode IEX chromatography; and
detecting separated compounds to determine the components of the biological sample;
wherein:
the separating and the subjecting are conducted with one aqueous to organic solvent gradient; and
there is no switching valve between the RP chromatography and IEX chromatography.

### Claim 2 (depends on 1)

The method of claim 1, wherein the biological sample is from a subject having or suspected of having a metabolic disruption.

### Claim 3 (depends on 1)

The method of claim 1, wherein the biological sample is a plasma sample or a urine sample.

### Claim 4 (depends on 1)

The method of claim 1, wherein the biological sample comprises lipids, carbohydrates, and metabolic intermediates.

### Claim 5 (depends on 1)

The method of claim 1, wherein the biological sample comprises polar and non-polar metabolites.

### Claim 6 (depends on 1)

The method of claim 1, wherein the detecting step is performed using mass spectrometry.

### Claim 7 (depends on 1)

The method of claim 1, wherein the detecting step includes qualitative analysis.

### Claim 8 (depends on 1)

The method of claim 1, wherein isomers of metabolites in the biological sample are separated.

### Claim 9 (depends on 1)

The method of claim 1, wherein the biological sample comprises methylmalonic acid, glutamine, glycine, citrulline, methionine, alloisoleucine, arginine, creatine, carnitine, propionylcarnitine, stearoylcarnitine, 2-methylcitric acid, propionylglycine, 2-methylbutyrylglycine, 3-methylcrotonylglycine, adipic acid, suberic acid, and glutaric acid and wherein the detecting separated compounds comprises detecting the methylmalonic acid, glutamine, glycine, citrulline, methionine, alloisoleucine, arginine, creatine, carnitine, propionylcarnitine, stearoylcarnitine, 2-methylcitric acid, propionylglycine, 2-methylbutyrylglycine, 3-methylcrotonylglycine, adipic acid, suberic acid, and glutaric acid.

### Claim 10 (depends on 1)

The method of claim 1, wherein the biological sample comprises plasma; and wherein the step of detecting comprises detecting 88 metabolites; and wherein the method is conducted in 20 minutes or less.

### Claim 11 (depends on 1)

The method of claim 1, wherein the biological sample comprises urine, and wherein the step of detecting comprises detecting 82 metabolites; and wherein the method is conducted in 20 minutes or less.