Mass spectrometry method for measuring vitamin B6 in body fluid
First Claim
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1. A method for detecting the presence or amount of pyridoxal 5′
- -phosphate in a body fluid sample by tandem mass spectrometry, comprising;
purifying said sample, wherein purifying comprises;
(a) applying said sample to an extraction column under turbulent flow conditions whereby pyridoxal 5′
-phosphate is reversibly retained by the extraction column, wherein said extraction column comprises particles with an average diameter of between about 35 μ
m and about 50 μ
m;
(b) washing the extraction column under conditions whereby non-retained materials from the sample are washed from the column;
(c) eluting retained pyridoxal 5′
-phosphate from the extraction column;
(d) applying the eluted pyridoxal 5′
-phosphate from the extraction column to an analytical column under laminar flow conditions; and
(e) eluting 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 body fluid sample.
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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.
14 Citations
26 Claims
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1. A method for detecting the presence or amount of pyridoxal 5′
- -phosphate in a body fluid sample by tandem mass spectrometry, comprising;
purifying said sample, wherein purifying comprises; (a) applying said sample to an extraction column under turbulent flow conditions whereby pyridoxal 5′
-phosphate is reversibly retained by the extraction column, wherein said extraction column comprises particles with an average diameter of between about 35 μ
m and about 50 μ
m;(b) washing the extraction column under conditions whereby non-retained materials from the sample are washed from the column; (c) eluting retained pyridoxal 5′
-phosphate from the extraction column;(d) applying the eluted pyridoxal 5′
-phosphate from the extraction column to an analytical column under laminar flow conditions; and(e) eluting 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 body fluid sample. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15)
- -phosphate in a body fluid sample by tandem mass spectrometry, comprising;
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16. A method for detecting the amount of pyridoxal 5′
- -phosphate in a body fluid sample by tandem mass spectrometry, comprising;
(i) purifying said sample, wherein purifying comprises; (a) applying said sample to a turbulent flow liquid chromatography column under conditions whereby pyridoxal 5′
-phosphate is reversibly retained;(b) washing the turbulent flow liquid chromatography column under conditions whereby non-retained materials from the sample are washed from the column; (c) eluting retained pyridoxal 5′
-phosphate from the turbulent flow liquid chromatography column;(d) applying the eluted pyridoxal 5′
-phosphate to a high performance liquid chromatography (HPLC) column; and(e) eluting pyridoxal 5′
-phosphate from the HPLC 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. - View Dependent Claims (17, 18, 19, 20, 21, 22, 23, 24, 25, 26)
- -phosphate in a body fluid sample by tandem mass spectrometry, comprising;
Specification