# US 11,442,073 B2 · Methods and systems for measuring serotonin in a sample

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- Number: US 11,442,073 B2
- Document ID: US11442073B2
- Status: Granted
- Kind: B2
- Application number: 17/088,129
- Filed: 2020-11-03
- Published: 2021-02-18
- Granted: 2022-09-13
- Earliest claimed: 2015-03-03
- Assignees: Laboratory Corporation of America Holdings
- Applicants: Laboratory Corporation of America Holdings
- Inventors: Matthew Lee Francis Crawford; Yvonne Zamorano Wright; Russell Philip Grant
- Web page: https://patentgrep.com/patent/US11442073B2

## CPC

- G01N 33/942: {Serotonin, i.e. 5-hydroxy-tryptamine}
- G01N 33/49: Blood {(chemical methods for determining blood cell populations G01N33/5094; chemical analysis of blood groups or blood types G01N33/80)}
- G01N 33/6848: {Methods of protein analysis involving mass spectrometry}
- G01N 2030/8813: {biological materials}
- G01N 2458/15: Non-radioactive isotope labels, e.g. for detection by mass spectrometry
- G01N 2800/222: Platelet 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 are methods and systems for measuring serotonin in a sample using liquid chromatography and mass spectrometry.

## Claims

### Claim 1

A method for determining amount of serotonin released by a biological sample, the method comprising:
incubating at least one sample comprising the biological sample, heparin and donor platelets for a period of time to release serotonin from the donor platelets, thereby generating at least one incubated sample;
chromatographically separating serotonin from other components in the at least one incubated sample using liquid chromatography; and
analyzing the chromatographically separated serotonin by tandem mass spectrometry to determine the amount of released serotonin in the at least one sample relative to a total amount of serotonin available in the donor platelets.

### Claim 2 (depends on 1)

The method of claim 1, wherein the analyzing includes ionizing the chromatographically separated serotonin using electrospray ionization, atmospheric pressure chemical ionization, or atmospheric pressure photoionization.

### Claim 3 (depends on 1)

The method of claim 1, wherein the tandem mass spectrometry uses a quadrupole mass spectrometer.

### Claim 4 (depends on 3)

The method of claim 3, wherein the quadrupole mass spectrometer is a triple quadrupole mass spectrometer.

### Claim 5 (depends on 4)

The method of claim 4, wherein the analyzing includes:
detecting intact serotonin ion in first quadrupole;
fragmenting the intact serotonin ion in second quadrupole to yield one or more serotonin fragment ions; and,
detecting the one or more serotonin fragment ions in third quadrupole.

### Claim 6 (depends on 1)

The method of claim 1, wherein the analyzing comprises ionizing the chromatographically separated serotonin to produce one or more serotonin ions, comprising at least one of a precursor ion of 160.1±0.5 mass-to-charge ratio, or a product ion of 115.1±0.5, 132.1±0.5, 105.1±0.5, or 89.1±0.5 mass-to-charge ratio.

### Claim 7 (depends on 1)

The method of claim 1, wherein the at least one sample is at least two samples, wherein a first one or more of the at least two samples comprises heparin at a first concentration, and a second one or more of the at least two samples comprises heparin at a second concentration different from the first concentration.

### Claim 8 (depends on 7)

The method of claim 7, wherein the first concentration of heparin is 0.001 to 1 U/mL, and the second concentration of heparin is 50 to 1000 U/mL.

### Claim 9 (depends on 1)

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

### Claim 10 (depends on 9)

The method of claim 9, wherein the serum sample or the plasma sample is obtained from a heparin-treated subject, or a subject suspected of having heparin-induced thrombocytopenia (HIT).

### Claim 11 (depends on 1)

The method of claim 1, wherein the donor platelets are washed and partially purified prior to the incubating the at least one sample.

### Claim 12 (depends on 1)

The method of claim 1, wherein the donor platelets are serotonin-incubated donor platelets.

### Claim 13 (depends on 12)

The method of claim 12, wherein the serotonin-incubated donor platelets are incubated with serotonin prior to the incubating the at least one sample.

### Claim 14 (depends on 12)

The method of claim 12, wherein the serotonin-incubated donor platelets are incubated with stable isotopically-labeled serotonin prior to the incubating the at least one sample.

### Claim 15 (depends on 1)

The method of claim 1, further comprising partially purifying the at least one incubated sample prior to the chromatographically separating serotonin from the other components in the at least one incubated sample.

### Claim 16 (depends on 1)

The method of claim 1, wherein the liquid chromatography includes reverse phase column chromatography.

### Claim 17 (depends on 1)

The method of claim 1, wherein the liquid chromatography includes using two or more liquid chromatography columns in parallel, where the two or more liquid chromatography columns are connected inline to a single mass spectrometer.

### Claim 18

A system for determining amount of serotonin released by a biological sample, the system comprising:
a station for incubating at least-one sample comprising the biological sample, heparin and donor platelets for a period of time to release serotonin from the donor platelets, thereby generating at least one incubated sample;
a station for chromatographically separating serotonin from other components in the at least one incubated sample using liquid chromatography; and
a station for analyzing the chromatographically separated serotonin by tandem mass spectrometry to determine amount of released serotonin in the at least one sample relative to a total amount of serotonin available within the donor platelets.

### Claim 19 (depends on 18)

The system of claim 18, wherein the station for analyzing the chromatographically separated serotonin by tandem mass spectrometry comprises a quadrupole mass spectrometer.

### Claim 20 (depends on 19)

The system of claim 19, wherein the quadrupole mass spectrometer is a triple quadrupole mass spectrometer.

### Claim 21 (depends on 18)

The system of claim 18, wherein the station for chromatographically separating serotonin comprises at least one reverse phase column.

### Claim 22 (depends on 18)

The system of claim 18, wherein the station for chromatographically separating serotonin comprises two or more liquid chromatography columns in parallel, wherein the two or more liquid chromatography columns are connected inline to a single mass spectrometer.

### Claim 23 (depends on 18)

The system of claim 18, further comprising a station for partially purifying the at least one incubated sample prior to chromatographically separating serotonin from other components in the at least one incubated sample.

### Claim 24 (depends on 18)

The system of claim 18, wherein the at least one sample is at least two samples, wherein a first one or more of the at least two samples comprises heparin at a first concentration, and a second one or more of the at least two samples comprises heparin at a second concentration different from the first concentration.

### Claim 25 (depends on 24)

The system of claim 24, wherein the first concentration of heparin is 0.001 to 1 U/mL, and the second concentration of heparin is 50 to 1000 U/mL.