Methods for quantitation of insulin levels by mass spectrometry

Abstract

Methods are described for determining the amount of insulin in a sample. Provided herein are mass spectrometric methods for detecting and quantifying insulin and C-peptide in a biological sample utilizing enrichment and/or purification methods coupled with tandem mass spectrometric or high resolution/high accuracy mass spectrometric techniques. Also provided herein are mass spectrometric methods for detecting and quantifying insulin and b-chain in a biological sample utilizing enrichment and/or purification methods coupled with tandem mass spectrometric or high resolution/high accuracy mass spectrometric techniques.

That which is claimed is: 
     
         1 . A method for determining the amount of an insulin analog in a sample by mass spectrometry, the method comprising:
 (a) immunocapturing an insulin analog;   (b) subjecting the immunocaptured insulin analog to an ionization source under conditions suitable to generate one or more insulin analog ions detectable by mass spectrometry; and   (c) determining the amount of one or more insulin analog ions by mass spectrometry.   
     
     
         2 . The method of  claim 1 , wherein the insulin analog is selected from aspart (NOVOLOG®), lispro (HUMALOG®), glulisine (APIDRA®), detemir (LEVEMIR®), degludec (TRESIBA®), glargine (LANTUS®), and NPH (HUMULIN R®/NOVOLIN N®). 
     
     
         3 . The method of  claim 1 , wherein the insulin analog comprises a precurson ion with a mass to charge ratio (m/z) selected from 1162.4±0.5, 1011.2±0.5, 987.2±0.5, 971.5±0.5, 968.7±0.5, and 956.8±0.5. 
     
     
         4 . The method of  claim 1 , wherein the insulin analog comprises one or more fragment ions with a mass to charge ratio (m/z) selected from 136.0±0.5, 1179.0±0.5, 175.0±0.5, 454.4±0.5, 357.2±0.5, 219.0±0.5, 226.0±0.5, 660.8±0.5, 199.0±0.5, 346.2±0.5, 328.2±0.5, 217.3±0.5, 226.1±0.5, and 315.2±0.5. 
     
     
         5 . The method of  claim 1 , wherein said sample comprises a plasma or serum sample. 
     
     
         6 . The method of  claim 1 , wherein said ionization source is an electrospray (ESI) ionization source. 
     
     
         7 . The method of  claim 1 , wherein said sample is subjected to acidic conditions prior to mass spectrometry. 
     
     
         8 . The method of  claim 7 , wherein subjecting said sample to acidic conditions comprises subjecting said sample to formic acid. 
     
     
         9 . The method of  claim 1 , wherein said sample is subjected to basic conditions prior to mass spectrometry. 
     
     
         10 . The method of  claim 9 , wherein subjecting said sample to basic conditions comprises subjecting said sample to trizma and/or ethanol. 
     
     
         11 . The method of  claim 1 , wherein the sample is delipidated prior to quantitation by mass spectrometry. 
     
     
         12 . The method of  claim 1 , further comprising purifying the sample prior to mass spectrometry. 
     
     
         13 . The method of  claim 12 , wherein said purifying comprises subjecting the sample to liquid chromatography. 
     
     
         14 . The method of  claim 13 , wherein liquid chromatography comprises high performance liquid chromatography (HPLC) or high turbulence liquid chromatograph (HTLC). 
     
     
         15 . The method of  claim 12 , wherein said purifying comprises subjecting a sample to solid phase extraction (SPE). 
     
     
         16 . The method of  claim 1 , wherein the mass spectrometry is tandem mass spectrometry, high resolution mass spectrometry, or high resolution/high accuracy mass spectrometry. 
     
     
         17 . The method of  claim 1 , wherein ionization is electrospray ionization (ESI). 
     
     
         18 . The method of  claim 1 , wherein ionization is in positive ion mode. 
     
     
         19 . The method of  claim 1 , wherein the immunocapture comprises using anti-insulin antibodies or anti-insulin analog antibodies. 
     
     
         20 . The method of  claim 19 , wherein the antibodies are monoclonal antibodies. 
     
     
         21 . The method of  claim 20 , wherein the antibodies IgG. 
     
     
         22 . The method of  claim 19 , wherein the antibodies are immobilized on magnetic beads. 
     
     
         23 . The method of  claim 22 , wherein insulin analogs immunocaptured on magnetic beads are washed and eluted.