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.

What is claimed is: 
     
         1 . A method for determining the amount of total thiamine in a plasma or serum sample, comprising:
 (i) removing soluble protein from a plasma or serum sample;   (ii) incubating the sample from step (i) with an acid phosphatase, for not longer than about 2 hours, 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 (iii) by liquid chromatography; and   (v) determining the amount of thiamine by mass spectrometry, wherein the amount of total thiamine in said sample is determined.   
     
     
         2 . The method of  claim 1 , wherein the incubation of step (ii) is carried out at a pH of about 4.6±0.1. 
     
     
         3 . The method of  claim 1 , wherein said acid phosphatase is carried out at a temperature of about 40° C. 
     
     
         4 . The method of  claim 1 , wherein said incubation is carried out between about 1 and about 2 hours. 
     
     
         5 . The method of  claim 1 , wherein soluble protein is removed in step (i) by treating said sample with an acid. 
     
     
         6 . The method of  claim 1 , wherein said liquid chromatography comprises high performance liquid chromatography (HPLC). 
     
     
         7 . 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. 
     
     
         8 . The method of  claim 7 , 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. 
     
     
         9 . The method of  claim 8 , 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. 
     
     
         10 . The method of  claim 8 , wherein said one or more daughter ions comprise an ion having a mass/charge ratio of 144.00±1.0. 
     
     
         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 plasma or serum sample, comprising:
 (i) removing soluble protein from a plasma or serum sample;   (ii) incubating the sample from step (i) with an acid phosphatase, for not longer than about 2 hours to convert phosphorylated thiamine to thiamine;   (iii) ionizing thiamine in the sample from step (ii) into a parent ion;   (iv) fragmenting said parent ion into one or more daughter ions; and   (v) 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 the incubation of step (ii) is carried out at a pH of about 4.6±0.1. 
     
     
         17 . The method of  claim 15 , wherein said acid phosphatase is carried out at a temperature of about 40° C. 
     
     
         18 . The method of  claim 15 , wherein said incubation is carried out between about 1 and about 2 hours. 
     
     
         19 . The method of  claim 15 , wherein said parent ion has a mass/charge ratio of 265.00±1.0. 
     
     
         20 . The method of  claim 15 , wherein said one or more daughter ions comprise an ion having a mass/charge ratio of 144.0±1.0.