Methods for quantitation of insulin levels by mass spectrometry
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.
1. A method for determining the amount of insulin and C-peptide in a sample by mass spectrometry, the method comprising:
(a) subjecting insulin and C-peptide from a sample to an ionization source under conditions suitable to generate one or more insulin and C-peptide ions detectable by mass spectrometry, wherein said one or more ions comprise one or more C-peptide fragment ions selected from the group consisting of ions with m/z of 533.3±0.5, 646.4±0.5, and 927.5±0.5; and
(b) determining the amount of one or more insulin and C-peptide ions by mass spectrometry.
2. The method of claim 1 , wherein said biological sample comprises a plasma or serum sample.
3. The method of claim 1 , wherein said ionization source is an electrospray (ESI) ionization source.
4. The method of claim 1 , wherein said sample is subjected to acidic conditions prior to mass spectrometry.
5. The method of claim 4 , wherein subjecting said sample to acidic conditions comprises subjecting said sample to formic acid.
6. The method of claim 1 , wherein said sample is subjected to basic conditions prior to mass spectrometry.
7. The method of claim 6 , wherein subjecting said sample to basic conditions comprises subjecting said sample to tris base and/or ethanol.
8. The method of claim 1 , wherein said one or more ions comprise an insulin precursor ion has a mass to charge ratio (m/z) of 968.9±0.5.
9. The method of claim 1 , wherein said one or more ions comprise one or more insulin fragment ions selected from the group consisting of ions with m/z of 136.0±0.5, 226.1±0.5, and 345.2±0.5.
10. The method of claim 1 , wherein said one or more ions comprise a C-peptide precursor ion has a mass to charge ratio (m/z) of 1007.7±0.5.
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 in positive ion mode.
18. The method of claim 1 , wherein internal standards for insulin and C-peptide are added to the sample.
19. The method of claim 18 , wherein the internal standard for insulin is bovine insulin.
20. The method of claim 19 , wherein the bovine insulin comprises a precursor ion with a mass to charge ratio (m/z) of 956.8±0.5 and fragment ions selected from the group consisting of ions with a m/z of 136.0±0.5, 226.1±0.5, and 315.2±0.5.
21. The method of claim 18 , wherein the internal standard for C-peptide is C-peptide heavy internal standard.
22. The method of claim 21 , wherein the C-peptide heavy internal standard comprises a precursor ion with a mass to charge ratio (m/z) of 1009.5±0.5 and fragment ions selected from the group consisting of ions with m/z of 540.3±0.5, 653.4±0.5, and 934.5±0.5.
23. The method of claim 1 , wherein the amount of the one or more ions determined is used to determine the amount of insulin and C-peptide in the sample.
24. The method of claim 23 , wherein the amount of insulin and C-peptide in the sample is used to determine the ratio of insulin to C-peptide.
25. A method for determining the amount of insulin and C-peptide in a sample by mass spectrometry, the method comprising:
(a) subjecting a sample to an enrichment process to obtain a fraction enriched in insulin and C-peptide;
(b) subjecting the enriched insulin and C-peptide to an ionization source under conditions suitable to generate one or more insulin and C-peptide ions detectable by mass spectrometry wherein said one or more ions comprise one or more C-peptide fragment ions selected from the group consisting of ions with m/z of 533.3±0.5, 646.4±0.5, and 927.5±0.5; and
(c) determining the amount of one or more insulin and C-peptide ions by mass spectrometry.
26. The method of claim 25 , wherein the enrichment process comprises immunocapture of insulin and C-peptide.
27. The method of claim 26 , wherein the immunocapture comprises using anti-insulin antibodies and anti-C-peptide antibodies.
28. The method of claim 27 , wherein the antibodies are monoclonal antibodies.
29. The method of claim 28 , wherein the antibodies IgG.
30. The method of claim 27 , wherein the anti-insulin antibodies and anti-C-peptide antibodies are immobilized on magnetic beads.
31. The method of claim 30 , wherein insulin and C-peptide immunocaptured on magnetic beads are washed and eluted.
32. The method of claim 25 , wherein said biological sample comprises a plasma or serum sample.
33. The method of claim 25 , wherein said ionization source is an electrospray (ESI) ionization source.
34. The method of claim 25 , wherein said sample is subjected to acidic conditions prior to mass spectrometry.
35. The method of claim 34 , wherein subjecting said sample to acidic conditions comprises subjecting said sample to formic acid.
36. The method of claim 25 , wherein said sample is subjected to basic conditions prior to mass spectrometry.
37. The method of claim 36 , wherein subjecting said sample to basic conditions comprises subjecting said sample to trizma tris base and/or ethanol.
38. The method of claim 25 , wherein said one or more ions comprise an insulin precursor ion has a mass to charge ratio (m/z) of 968.9±0.5.
39. The method of claim 25 , wherein said one or more ions comprise one or more insulin fragment ions selected from the group consisting of ions with m/z of 136.0±0.5, 226.1±0.5, and 345.2±0.5.
40. The method of claim 25 , wherein said one or more ions comprise a C-peptide precursor ion has a mass to charge ratio (m/z) of 1007.7±0.5.
41. The method of claim 25 , wherein the sample is delipidated prior to quantitation by mass spectrometry.
42. The method of claim 25 , further comprising purifying the sample prior to mass spectrometry.
43. The method of claim 42 , wherein said purifying comprises subjecting the sample to liquid chromatography.
44. The method of claim 43 , wherein liquid chromatography comprises high performance liquid chromatography (HPLC) or high turbulence liquid chromatograph (HTLC).
45. The method of claim 42 , wherein said purifying comprises subjecting a sample to solid phase extraction (SPE).
46. The method of claim 25 , wherein the mass spectrometry is tandem mass spectrometry, high resolution mass spectrometry, or high resolution/high accuracy mass spectrometry.
47. The method of claim 25 , wherein ionization is in positive ion mode.
48. The method of claim 25 , wherein internal standards for insulin and C-peptide are added to the sample.
49. The method of claim 48 , wherein the internal standard for insulin is bovine insulin.
50. The method of claim 49 , wherein the bovine insulin comprises a precursor ion with a mass to charge ratio (m/z) of 956.8±0.5 and fragment ions selected from the group consisting of ions with a m/z of 136.0±0.5, 226.1±0.5, and 315.2±0.5.
51. The method of claim 48 , wherein the internal standard for C-peptide is C-peptide heavy internal standard.
52. The method of claim 51 , wherein the C-peptide heavy internal standard comprises a precursor ion with a mass to charge ratio (m/z) of 1009.5±0.5 and fragment ions selected from the group consisting of ions with m/z of 540.3±0.5, 653.4±0.5, and 934.5±0.5.
53. The method of claim 25 , wherein the amount of the one or more ions determined is used to determine the amount of insulin and C-peptide in the sample.
54. The method of claim 53 , wherein the amount of insulin and C-peptide in the sample is used to determine the ratio of insulin to C-peptide.
55. A method for diagnosing glycemic disorders or insulin resistant syndromes in diabetic and pre-diabetic patients comprising determining the amount of insulin and C-peptide as in claim 25 .
56. A method for diagnosing diabetes comprising determining the amount of insulin and C-peptide as in claim 25 .
57. A method for distinguishing insulin-secreting tumors from exogenous insulin administration as a cause for hypoglycemia comprising determining the amount of insulin and C-peptide as in claim 25 .
58. A method for distinguishing type 1 diabetes from type 2 diabetes comprising determining the amount of insulin and C-peptide as in claim 25 .
59. A method for assessing the risk of diabetes in pre-diabetic patients comprising determining the amount of insulin and C-peptide as in claim 25 .