Methods and Systems for Measuring Serotonin in a Sample

Patent metadata

Field	Value
Publication number	US-2016258969-A1
Application number	US-201615059957-A
Country	US
Kind code	A1
Filing date	Mar 3, 2016
Priority date	Mar 3, 2015
Publication date	Sep 8, 2016
Grant date	—

Abstract

Disclosed are methods and systems for measuring serotonin in a sample using liquid chromatography and mass spectrometry.

First claim

1 . A method for determining the presence or amount of released serotonin in a sample, the method comprising:
providing a sample comprising a biological sample, donor platelets, and heparin; incubating the sample for a period of time to release serotonin from the donor platelets; chromatographically separating serotonin from other components in the incubated sample using liquid chromatography; and analyzing the chromatographically separated serotonin by mass spectrometry to determine the presence or amount of released serotonin in the sample.

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

3 . The method of claim 1 , wherein the biological sample is obtained from a heparin-treated subject.

4 . The method of claim 1 , wherein the biological sample is obtained from a subject suspected of having heparin-induced thrombocytopenia.

5 . The method of claim 1 , wherein the donor platelets are obtained from at least one presumed healthy subject.

6 . The method of claim 1 , wherein the donor platelets in the sample are washed and partially purified.

7 . The method of claim 1 , wherein the incubating step is performed at room temperature.

8 . The method of claim 1 , wherein the incubating step is performed for at least 30 minutes.

9 . The method of claim 1 , wherein the heparin in the providing step is present in an amount of from 0.001 to 1 U/mL.

10 . The method of claim 1 , wherein the heparin in the providing step is present in an amount of from 50 to 1000 U/mL.

11 . The method of claim 1 , further comprising contacting the incubated sample with an internal standard prior to the chromatographically separating step.

12 . The method of claim 11 , wherein the internal standard is a stable isotopically-labeled form of serotonin.

13 . The method of claim 12 , wherein the stable isotopically-labeled form of serotonin comprises deuterium labeled serotonin, carbon-13 labeled serotonin, nitrogen-15 labeled serotonin, oxygen-18 labeled serotonin, or combinations thereof.

14 . The method of claim 11 , wherein the internal standard is serotonin-d4.

15 . The method of claim 1 , wherein the donor platelets in the providing step were incubated with serotonin prior to the providing step.

16 . The method of claim 1 , wherein the donor platelets in the providing step were incubated with deuterium labeled serotonin, carbon-13 labeled serotonin, nitrogen-15 labeled serotonin, oxygen-18 labeled serotonin, or combinations thereof prior to the providing step.

17 . The method of claim 1 , further comprising partially purifying the incubated sample prior to the chromatographically separating step.

18 . The method of claim 17 , wherein the partially purifying step comprises centrifuging the incubated sample.

19 . The method of claim 1 , wherein using liquid chromatography includes using analytical liquid chromatography.

20 . The method of claim 19 , wherein using analytical liquid chromatography includes using a reverse phase column.

21 . The method of claim 1 , wherein using liquid chromatography includes using at least one column.

22 . The method of claim 1 , wherein using 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.

23 . The method of claim 22 , wherein using two or more liquid chromatography columns in parallel includes introducing the incubated sample to the two or more liquid chromatography columns at staggered times.

24 . The method of claim 1 , wherein the analyzing step includes ionizing serotonin using an ionization technique selected from the group consisting of: electrospray ionization, atmospheric pressure chemical ionization, and atmospheric pressure photoionization.

25 . The method of claim 1 , wherein the analyzing step includes detecting serotonin using a quadrupole mass spectrometer.

26 . The method of claim 25 , wherein the quadrupole mass spectrometer is a triple quadrupole mass spectrometer.

27 . The method of claim 26 , wherein the analyzing step includes: detecting intact serotonin ion in the first quadrupole; fragmenting intact serotonin ion in the second quadrupole to yield one or more serotonin fragment ions; and detecting the one or more serotonin fragment ions in the third quadrupole.

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

29 . A method for determining the presence or amount of released serotonin in a sample, the method comprising:
providing a sample comprising a biological sample, heparin, and serotonin-incubated platelets; incubating the sample for a period of time to release serotonin from the platelets; chromatographically separating serotonin from other components in the incubated sample using liquid chromatography; and analyzing the chromatographically separated serotonin by mass spectrometry to determine the presence or amount of released serotonin in the sample relative to the total amount of serotonin available within the donor platelets.

30 . A method for determining the presence or amount of released stable isotopically labeled serotonin in a sample, the method comprising:
providing a sample comprising a biological sample, heparin, and stable isotopically labeled serotonin-incubated platelets; incubating the sample for a period of time to release stable isotopically labeled serotonin from the platelets; chromatographically separating stable isotopically labeled serotonin from other components in the incubated sample using liquid chromatography; and analyzing the chromatographically separated stable isotopically labeled serotonin by mass spectrometry to determine the presence or amount of released stable isotopically labeled serotonin in the sample relative to the total amount of isotopically labeled serotonin available within the donor platelets.

31 . The method of claim 30 , wherein the stable isotopically labeled serotonin is deuterium labeled serotonin, carbon-13 labeled serotonin, nitrogen-15 labeled serotonin, oxygen-18 labeled serotonin, or combinations thereof prior to the providing step.

32 . A method of generating a report useful for diagnosing a disease or condition associated with abnormal platelet activation, the method comprising:
(a) providing a sample comprising a biological sample, donor platelets, and heparin; (b) incubating the sample for a period of time to release serotonin from the donor platelets; (c) chromatographically separating serotonin from other components in the incubated sample using liquid chromatography; (d) analyzing the chromatographically separated serotonin by mass spectrometry to determine the amount of released serotonin in the sample relative to the total amount of serotonin available within the donor platelets; and (e) generating a report that recites the percentage release of serotonin in the sample.

33 . The method of claim 32 , wherein the disease or condition is heparin-induced thrombo

Assignees

• Laboratory Corp America Holdings

Inventors

• Crawford Matthew Lee Francis
• Wright Yvonne Zamorano
• Grant Russell Philip

Classifications

• G01N2800/222

  Platelet disorders · CPC title

• G01N2458/15

  Non-radioactive isotope labels, e.g. for detection by mass spectrometry · CPC title

• G01N33/942 Primary

  Serotonin, i.e. 5-hydroxy-tryptamine · CPC title

• G01N33/6848

  Methods of protein analysis involving mass spectrometry · CPC title

• G01N2030/8813

  biological materials · CPC title