Mass spectrometry method for measuring thiamine in body fluid

Abstract

Provided are methods for determining the amount of total thiamine in a body fluid sample using liquid chromatography and mass spectrometry. Total thiamine is converted to free thiamine by treatment with an acid phosphatase prior to thiamine separation and quantification.

1 . A method for determining the amount of total thiamine in a body fluid sample, comprising:
 (i) removing soluble protein from said sample;   (ii) treating said sample with an acid phosphatase to convert phosphorylated thiamine to thiamine;   (iii) performing an organic solvent extraction of said sample from step (i), wherein the result of said extraction is an organic solvent phase and an aqueous phase;   (iv) purifying said thiamine from said aqueous phase of step (iii) by liquid chromatography; and   (v) determining the amount of thiamine by mass spectrometry, wherein the amount of total thiamine in said body fluid sample is determined.   
   
   
       2 . The method of  claim 1 , wherein said soluble protein is removed from said body fluid sample in step (i) by treating said sample with an acid. 
   
   
       3 . The method of  claim 1 , wherein said liquid chromatography comprises high performance liquid chromatography (HPLC). 
   
   
       4 . The method of  claim 1 , wherein step (v) comprises ionizing said thiamine to a parent ion having a mass/charge ratio of 265.00±1.0. 
   
   
       5 . The method of  claim 4 , wherein the amount of said parent ion is determined. 
   
   
       6 . The method of  claim 4 , wherein said parent ion is fragmented into one or more daughter ions and wherein the amount of one or more of said daughter ions is determined. 
   
   
       7 . The method of  claim 6 , wherein said one or more daughter ions comprises an ion having a mass/charge ratio of 144.00±1.0 or 121.94±1.0. 
   
   
       8 . The method of  claim 4 , wherein said thiamine is ionized by electrospray ionization. 
   
   
       9 . The method of  claim 6 , wherein said parent ion is fragmented by collision-induced dissociation using an inert gas. 
   
   
       10 . The method of  claim 1 , wherein said body fluid sample is plasma, serum, or whole blood. 
   
   
       11 . The method of  claim 1 , wherein an internal standard is added to said body fluid sample prior to step (i). 
   
   
       12 . The method of  claim 11 , wherein said internal standard is pyrithiamine. 
   
   
       13 . The method of  claim 12 , wherein said pyrithiamine is ionized to a parent ion having a mass to charge ratio of 259.04±1.0. 
   
   
       14 . The method of  claim 13 , wherein said pyrithiamine parent ion is fragmented into a daughter ion having a mass to charge ratio of 122.00±1.0. 
   
   
       15 . A method for determining the amount of total thiamine in a body fluid sample, comprising:
 (i) treating said sample with an acid and removing the acid-insoluble protein;   (ii) treating said sample with an acid phosphatase to convert phosphorylated thiamine to thiamine;   (iii) performing an organic solvent extraction of said sample from step (ii), wherein the result of said extraction is an organic solvent phase and an aqueous phase;   (iv) purifying said thiamine from said aqueous phase of step (ii) by high performance liquid chromatography (HPLC);   (v) ionizing said purified thiamine from step (iii) into a parent ion;   (vi) isolating said parent ion by mass spectrometry;   (vii) fragmenting said parent ion into one or more daughter ions; and   (viii) quantifying said one or more daughter ions by mass spectrometry and relating the amount of said one or more daughter ions to the concentration of total thiamine present in said body fluid sample.   
   
   
       16 . The method of  claim 15 , wherein said thiamine parent ion has a mass/charge ratio of 265.00±1.0. 
   
   
       17 . The method of  claim 16 , wherein said one or more daughter ions comprises an ion having a mass/charge ratio of 144.00±1.0 or 121.94±1.0. 
   
   
       18 . The method of  claim 15 , wherein step (v) comprises electrospray ionization and step (vii) comprises collision-induced dissociation using a inert gas. 
   
   
       19 . The method of  claim 18 , wherein the inert gas is argon. 
   
   
       20 . The method of  claim 15 , wherein an internal standard is added to said body fluid prior to step (i). 
   
   
       21 . The method of  claim 20 , wherein said internal standard is pyrithiamine. 
   
   
       22 . The method of  claim 21 , wherein said pyrithiamine is ionized to a parent ion having a mass to charge ratio of 259.04±1.0. 
   
   
       23 . The method of  claim 22 , wherein said pyrithiamine parent ion is fragmented into a daughter ion having a mass to charge ratio of 122.00±1.0. 
   
   
       24 . The method of  claim 15 , wherein said acid in step (i) is 7% perchloric acid. 
   
   
       25 . The method of  claim 15 , wherein said organic solvent in step (iii) is chloroform.