Mass spectrometry method for measuring vitamin B6 in body fluids

• US 9,234,901 B2
• Filed: 08/11/2011
• Issued: 01/12/2016
• Est. Priority Date: 06/14/2007
• Status: Active Grant

• Current Assignee: Quest Diagnostics Investments Incorporated (Quest Diagnostics Incorporated)
• Original Assignee: Quest Diagnostics Investments Incorporated (Quest Diagnostics Incorporated)
• Inventors: Jiang, Qibo, Chan, Sum, Reitz, Richard
• Primary Examiner(s): Fritchman, Rebecca M
• Application Number:
  US13/208,295
• Publication Number:
  US 20110297825A1
• Time in Patent Office: 1,615 Days
• Field of Search: 436/96, 436/518, 436/536, 250/282, 250/288
• US Class Current: 1/1
• CPC Class Codes: G01N 33/49 Blood chemical methods for determining blood cell populations G01N33/5094; chemical analysis of blood groups or blood types G01N33/80
• CPC Class Codes: G01N 33/82 involving vitamins or their receptors
• CPC Class Codes: H01J 49/0031 Step by step routines describing the use of the apparatus H01J49/0081 takes precedence
• CPC Class Codes: H01J 49/004 Combinations of spectrometers, tandem spectrometers, e.g. MS/MS, MSn
• CPC Class Codes: Y10T 436/145555 Hetero-N

Abstract

Provided are methods of detecting the presence or amount of the active form of vitamin B6, pyridoxal 5′-phosphate, in a body fluid sample using tandem mass spectrometry coupled with liquid chromatography.

Claims

1. A method for detecting the presence or amount of pyridoxal 5′

-phosphate in a body fluid sample by tandem mass spectrometry, comprising;

(i) purifying said sample, wherein purifying comprises;

(a) applying a body fluid sample and a first solvent to an extraction column under conditions suitable to reversibly retain pyridoxal 5′

-phosphate on said extraction column;

(b) applying a second solvent to said extraction column under conditions suitable to elute retained pyridoxal 5′

-phosphate from the extraction column, wherein said first and second solvents are different;

(c) applying eluted pyridoxal 5′

-phosphate from the extraction column to an analytical column for chromatographic separation; and

(d) applying a third solvent to said analytical column to elute pyridoxal 5′

-phosphate from the analytical column;

(ii) generating a parent ion of said pyridoxal 5′

-phosphate from said purified sample;

(iii) generating one or more daughter ions of said parent ion; and

(iv) detecting the presence or amount of one or more said ions generated in step (ii) or step (iii) or both, and relating the detected ions to the presence or amount of said pyridoxal 5′

-phosphate in said sample.

2. The method of claim 1, wherein the extraction column is a mixed mode anion exchange polymer column, and the analytical column is a C-8 analytical column.

3. The method of claim 2, wherein the extraction column comprises particles of about 50 μ

m, and the analytical column comprises particles of about 3.5 μ

m.

4. The method of claim 1, wherein steps (a)-(e) are performed in an online automated fashion.

5. The method of claim 4, wherein steps (ii)-(iv) are performed in an online automated fashion with steps (a)-(e).

6. The method of claim 1, wherein said parent ion has a mass/charge ratio of 248.03+/−

1.

7. The method of claim 6, wherein said one or more daughter ions comprise a daughter ion with a mass/charge ratio of 150.00+/−

1.

8. The method of claim 1, wherein said sample comprises plasma.

9. The method of claim 1, wherein step (ii) is performed by electrospray ionization.

10. The method of claim 1, wherein step (iii) is performed by Collision-Induced Dissociation using a neutral gas.

11. The method of claim 1, wherein an internal standard is added to the body fluid sample prior to step (i).

12. The method of claim 11, wherein the internal standard comprises 2-hydroxy-6-methylpyridine-3-carboxylic acid.

13. The method of claim 12, wherein the parent ion of the internal standard generated in step (ii) has a mass to charge ratio of 154.04+/−

1.

14. The method of claim 12, wherein the daughter ion of the internal standard generated in step (iii) has a mass to charge ratio of 136.00+/−

1.

15. The method of claim 1, wherein said extraction column is a turbulent flow liquid chromatography column.

16. The method of claim 15, wherein said turbulent flow liquid chromatography column is a high turbulent flow liquid chromatography column.

17. The method of claim 1, wherein said analytical column is an high performance liquid chromatography column.

18. The method of claim 17, wherein steps (ii)-(iv) are performed in an online automated fashion with steps (a)-(e).