Specific analysis of analytes using reagent compounds, labeling strategies, and mass spectrometry workflow

Abstract

Labeling reagents, sets of labeling reagents, and labeling techniques are provided for the relative quantitation, absolute quantitation, or both, of ketone or aldehyde compounds including, but not limited to, analytes comprising steroids or ketosteroids. The analytes can be medical or pharmaceutical compounds in biological samples. Methods for labeling, analyzing, and quantifying ketone or aldehyde compounds are also disclosed as are methods that also use mass spectrometry.

1 . A set of mass labels comprising two or more compounds of the general formula (II): Z—R 1 , wherein one or more of the compounds in the set of labels contains one or more heavy atom isotopes and wherein, Z represents a mass reporter group comprised of substituted or unsubstituted straight, branched or cyclic alkyl; a substituted or unsubstituted aryl; a substituted or unsubstituted hetero aryl; a substituted or unsubstituted amino; or a substituted or unsubstituted thio; and R 1  represents a terminal aminoxy group. 
     
     
         2 . The set of labels of  claim 1 , wherein R 1  is selected from the group consisting of 
       
         
           
           
               
               
           
         
       
     
     
         3 . The set of labels of  claim 1 , wherein one or more of the compounds of the general formula (II) of the set of labels is isotopically enriched with two or more heavy atoms. 
     
     
         4 . The set of labels of  claim 1 , wherein one or more of the compounds of the general formula (II) of the set of labels is isotopically enriched with three or more heavy atoms. 
     
     
         5 . The set of labels of  claim 1 , wherein the heavy atom isotopes are each independently  13 C,  15 N,  18 O,  33 S, or  34 S. 
     
     
         6 . The set of labels of  claim 1 , wherein the labels are isobaric in the unsalted or unhydrated form, and each of the isobaric labels contains one or more heavy atom isotopes. 
     
     
         7 . The set of labels of  claim 1 , wherein each label in the set of labels being differentiated from the another label in the set by a mass difference greater than about 1 amu. 
     
     
         8 . The set of labels of  claim 1 , wherein at least one of the two or more compounds is a compound of the formula:
   Y—(CH 2 ) n -ONH 2  
   
       wherein n is an integer from 1 to 100 and Y is selected from the group consisting of: 
       
         
           
           
               
               
           
         
       
     
     
         9 . The set of labels of  claim 8 , wherein n is an integer from 2 to 10. 
     
     
         10 . The set of labels of  claim 1 , wherein each of the two or more compounds individually comprises a permanently charged aminoxy reagent. 
     
     
         11 . A kit comprising the set of mass labels of  claim 1 , and one or more other assay components. 
     
     
         12 . The kit of  claim 11 , wherein the one or more other assay components comprises a buffer, a reagent, a separation column, and instructions for carrying out an assay. 
     
     
         13 . A method for mass analysis of an analyte in a sample comprising:
 derivatizing an analyte comprising an aldehyde or ketone functional group, with a labeling reagent comprising a terminal aminoxy moiety and a reporter group, to form a labeled analyte;   subjecting the labeled analyte to ionization; and   detecting the analyte by mass analysis.   
     
     
         14 . The method of  claim 13 , further comprising determining a concentration of the analyte in a sample. 
     
     
         15 . The method of  claim 13 , wherein the labeling reagent comprises a permanently charged aminoxy reagent. 
     
     
         16 . The method of  claim 13 , wherein the labeling reagent comprises a compound of the formula:
   Y—(CH 2 ) n -ONH 2  
   
       wherein n is an integer from 1 to 100 and Y is selected from the group consisting of: 
       
         
           
           
               
               
           
         
       
     
     
         17 . The method of  claim 16 , wherein n is an integer from 2 to 10. 
     
     
         18 . The method of  claim 13 , wherein the labeling reagent is selected from the group consisting of 
       
         
           
           
               
               
           
         
       
       and combinations thereof. 
     
     
         19 . The method of  claim 13 , further comprising the step of subjecting the labeled analyte to ion fragmentation to yield an ionized reporter group. 
     
     
         20 . The method of  claim 13 , wherein the step of detecting the analyte is comprised of detecting ions of the reporter group in a mass analyzer. 
     
     
         21 . The method of  claim 13 , wherein the step of detecting is comprised of detecting a first transmitted parent ion and a daughter ion fragment by parent-daughter ion transition monitoring. 
     
     
         22 . The method of  claim 13 , further comprising derivatizing an aldehyde or ketone functional group of a standard compound and measuring the relative concentration of the analyte. 
     
     
         23 . The method of  claim 13 , wherein the labeling reagent is isotopically enriched with two or more heavy atoms. 
     
     
         24 . The method of  claim 13 , wherein at least two analyte compounds are derivatized with the labeling reagent and the method further comprises the step of determining a relative concentration between at least two analytes. 
     
     
         25 . The method of  claim 13 , wherein at least two analyte compounds are derivatized with the labeling reagent and the method further comprises the step of determining an absolute concentration of at least one analyte. 
     
     
         26 . The method of  claim 13 , wherein the ionization produces structurally specific fragment ions and Q3 MRM ions, the labeling reagent is wholly or partly contained in the structurally specific fragment ions, and the method provides both sensitivity and specificity for the Q3 MRM ions. 
     
     
         27 . The method of  claim 8 , wherein the labeling reagent is isotopically enriched with two or more heavy atoms. 
     
     
         28 . The method of  claim 13 , further comprising subjecting the labeled analyte to liquid chromatographic separation prior to the ionization.