# US 11,300,559 B2 · Vitamin B2 detection by mass spectrometry

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- Number: US 11,300,559 B2
- Document ID: US11300559B2
- Status: Granted
- Kind: B2
- Application number: 16/791,345
- Filed: 2020-02-14
- Published: 2020-06-11
- Granted: 2022-04-12
- Earliest claimed: 2008-12-18
- Assignees: Quest Diagnostics Investments Incorporated
- Applicants: Quest Diagnostics Investments Incorporated
- Inventors: Changming Yang; Sum Chan
- Web page: https://patentgrep.com/patent/US11300559B2

## CPC

- G01N 33/487: of liquid biological material
- G01N 30/72: Mass spectrometers {(mass spectrometers per se H01J49/00)}
- G01N 30/7233: {interfaced to liquid or supercritical fluid chromatograph (interfaces in general for introducing or extracting samples to be analysed with specially adapted mass spectrometer, see H01J49/04)}
- G01N 33/6848: {Methods of protein analysis involving mass spectrometry}
- G01N 33/82: involving vitamins {or their receptors}
- H01J 49/0027: {Methods for using particle spectrometers}
- H01J 49/0031: {Step by step routines describing the use of the apparatus (H01J49/0081 takes precedence)}
- H01J 49/0054: {by an electron beam, e.g. electron impact dissociation, electron capture dissociation}
- G01N 2030/027: {Liquid chromatography}
- Y10T 436/147777: Plural nitrogen in the same ring [e.g., barbituates, creatinine, etc.]
- 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)
- H01J: ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS (spark-gaps H01T; electric arc lamps H05B31/00; particle accelerators H05H)
- Y10T: TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION

## Abstract

Methods are described for measuring the amount of a vitamin B2 in a sample. More specifically, mass spectrometric methods are described for detecting and quantifying vitamin B2 in a sample utilizing on-line extraction methods coupled with tandem mass spectrometric techniques.

## Claims

### Claim 1

A method for determining the amount of vitamin B2 in a biological sample from a human, said method comprising:
(a) adding internal standard to the sample;
(b) purifying the sample and the internal standard by high performance liquid chromatography (HPLC);
(c) ionizing vitamin B2 and the internal standard by electrospray ionization (ESI) in positive ion mode to produce one or more ions detectable by tandem mass spectrometry;
(d) determining the amount of said one or more ions by mass spectrometry, wherein a fragment ion of the internal standard comprises a mass to charge ratio of 246.20±0.50; and
(e) determining the amount of vitamin B2 in the sample from the amount of said one or more ions of vitamin B2 in step (c).

### Claim 2 (depends on 1)

The method of claim 1, wherein said HPLC and mass spectrometry are conducted with on-line processing.

### Claim 3 (depends on 1)

The method of claim 1, wherein said biological sample comprises plasma or serum.

### Claim 4 (depends on 1)

The method of claim 1, wherein said one or more ions detectable by mass spectrometry comprise one or more ions of vitamin B2 selected from the group consisting of ions with a mass to charge ratio of 377.2±0.5 and 243.2±0.5.

### Claim 5 (depends on 1)

The method of claim 1, wherein the HPLC is a reverse-phase high performance liquid chromatography (RP-HPLC).

### Claim 6 (depends on 1)

The method of claim 1, further comprising protein precipitation.

### Claim 7 (depends on 6)

The method of claim 6, wherein an acid is used for precipitating protein.