Tandem mass spectrometry method for the genetic screening of inborn errors of metabolism in newborns

Abstract

A method for genetic screening of inborn errors of metabolism in a newborn by tandem mass spectrometry is provided. The method comprises the steps of: (1) placing a blood spot from said newborn into a microtiter plate well; (2) adding a solution of polar solvent to said well so as to reconstitute enzymes in said blood spot and so as to extract polar analytes, thereby forming a sample; (3) incubating said sample for a predetermined period of time, so as to extract metabolites and allow enzyme activity to take place; (4) adding a second solution to said sample so as to stop enzymatic reactions and extract remaining analytes; (5) mixing said sample for a predetermined period of time; (6) measuring said sample by tandem mass spectrometry so as to identify analyte concentration and enzyme activity; and (7) using said analyte concentration and said enzyme activity to determine whether said newborn is affected by any inborn errors of metabolism. The solution of polar solvent may contain at least one of the following: an internal standard for a polar analyte, an internal standard for a non-polar analyte, an internal standard for an enzymatic product, a substrate for at least one enzyme, and a buffer. The second solution may contain at least one of the following: an internal standard for a polar analyte, an internal standard for a non-polar analyte, an internal standard for an enzymatic product, an internal standard for at least one substrate, and a buffer.

1 . A method for genetic screening of inborn errors of metabolism in a newborn by tandem mass spectrometry, said method comprising the steps of: 
 (a) obtaining a dried blood sample containing at least one enzyme, at least one relatively polar metabolic analyte, and at least one relatively non-polar metabolic analyte;    (b) adding a solution of a relatively polar solvent to said dried blood sample so as to reconstitute said at least one enzyme in said dried blood sample and so as to extract said at least one relatively polar analyte therefrom, thereby forming a first solution;    (c) incubating said first solution with a substrate for said at least one enzyme so as to create an enzymatic product;    (d) adding a solution of a relatively non-polar solvent to said first solution so as to stop the reaction of said at least one enzyme and so as to extract said at least one relatively non-polar analyte, so as to form a second solution; and    (e) measuring said second solution by tandem mass spectrometry so as to assay the presence or concentration of said at least one relatively polar analyte, said enzymatic product, and said at least one relatively non-polar analyte.    (f)) using said analyte concentration and enzyme activity to determine whether said newborn is affected by any inborn errors of metabolism    
     
     
         2 . The method according to  claim 1  wherein said solution of a relatively polar solvent additionally comprises at least one component selected from the group consisting of an internal standard for said at least one relatively polar analyte, an internal standard for said enzymatic product to be measured, a substrate for said at least one enzyme, an internal standard for said at least one relatively non polar analyte, and a buffer; or the method according to  claim 1  wherein said solution of a relatively polar solvent additionally comprises at least one component selected from the group consisting of a substrate for said at least one enzyme and a buffer.  
     
     
         3 . The method according to  claim 2  wherein at least one of said internal standard for said at least one relatively non-polar analyte, at least one of said internal standard for said at least one relatively polar analyte, at least one of said internal standard for said at least one enzymatic product to be measured, or a substrate for said at least one enzyme in said relatively polar solvent is labeled.  
     
     
         4 . The method according to  claim 1  wherein said solution of a relatively non-polar solvent additionally comprises at least one component selected from the group consisting of: an internal standard for said at least one relatively non-polar analyte, an internal standard for said at least one substrate, and a buffer; or the method according to  claim 1  wherein said solution of a relatively non-polar solvent additionally comprises at least one component selected from the group consisting of an internal standard for said at least one relatively polar analyte, an internal standard for said enzymatic product to be measured, an internal standard for said at least one relatively non polar analyte, an internal standard for said at least one substrate, and a buffer.  
     
     
         5 . The method according to  claim 4  wherein at least one of said internal standard for said at least one relatively non-polar analyte, at least one of said internal standard for said at least one relatively polar analyte, at least one of said internal standard for said at least one enzymatic product to be measured, at least one of said internal standard for said at least one substrate in said relatively non-polar solvent is labeled.  
     
     
         6 . The method according to  claim 1  additionally comprising the step of adding at least one detergent to said sample prior to mixing said relatively polar and relatively non-polar solutions.  
     
     
         7 . A method for genetic screening of inborn errors of metabolism in a newborn by tandem mass spectrometry, said method comprising the steps of: 
 (a) placing a blood spot sample from said newborn into a microtiter plate well so as to form a sample, said microtiter plate well treated with at least one pretreater;    (b) incubating said sample for a predetermined period of time;    (c) measuring said sample by tandem mass spectrometry so as to determine analyte concentration and enzyme activity; and    (d) using said analyte concentration and enzyme activity to determine whether said newborn is affected by any inborn errors of metabolism.    
     
     
         8 . The method according to  claim 7  additionally comprising the step of: adding a solution of a relatively polar solvent to said well so as to reconstitute said at least one enzyme and so as to extract said at least one relatively polar analyte therefrom, thereby forming a first solution.  
     
     
         9 . The method according to  claim 7  additionally comprising the step of: incubating said first solution with a substrate for said at least one enzyme so as to create an enzymatic product.  
     
     
         10 . The method according to  claim 7  additionally comprising the step of: adding a solution of a relatively non-polar solvent to said first solution so as to stop the reaction of said at least one enzyme and so as to extract said at least one relatively non-polar analyte, so as to form a second solution  
     
     
         11 . The method according to  claim 7  additionally comprising the step of: mixing said second solution for a predetermined period of time.  
     
     
         12 . The method according to  claim 7  wherein said at least one pretreater is selected from the group consisting of: an internal standard for a polar analyte, an internal standard for a non-polar analyte, an internal standard for an enzymatic product to be measured, a substrate, a detergent, and a buffer.  
     
     
         13 . The method according to  claim 7  wherein at least one of said at least one pretreater is labeled.