Mass spectrometry assay for plasma-renin
View Patent ↗Provided are methods for measuring renin activity in a plasma sample using mass spectrometry. The methods generally involve ionizing purified angiotensin 1 from the sample and detecting the amount of angiotensin 1 ions generated. The amount of detected angiotensin 1 ions are then related to the amount of angiotensin 1 generated in the sample, which in turn is related to renin activity in the sample.
1. A method for measuring the amount of angiotensin 1 in a sample, said method comprising:
(a) ionizing angiotensin 1 from said sample to produce a precursor ion detectable by mass spectrometry wherein said precursor ion has a mass to charge ratio of 433.0±0.5;
(b) fragmenting said precursor ion to produce a fragment ion with a mass/charge ratio of 619.5±0.5;
(c) detecting the amount of one or more of the precursor and fragment ions by mass spectrometry; and
(d) using the amount of ion(s) detected to measure the amount of angiotensin 1 in said sample.
2. The method of claim 1 , wherein said method has a limit of quantitation less than or equal to 0.1 ng/mL.
3. The method of claim 1 , wherein said method further comprises purifying angiotensin 1 from said sample by high performance liquid chromatography (HPLC).
4. The method of claim 1 , wherein said method further comprises purifying angiotensin 1 from said sample with a solid-phase extraction column.
5. The method of claim 1 , wherein the amount of the angiotensin 1 ion is related to the presence or amount of angiotensin 1 in the sample by comparison to an internal standard.
6. A method for determining whether a patient exhibiting low plasma renin activity levels as determined by the method of claim 1 is a member of a subpopulation with high natural degradation of angiotensin 1, said method comprising: measuring the degree of degradation of angiotensin 1 in the sample by adding a degradation standard to the sample prior to incubation and measuring the amount of degradation standard remaining after incubation.
7. A method for measuring the amount of angiotensin 1 in a sample, said method comprising:
(a) incubating said sample premixed with water stable protease inhibitor that is not effective against renin under conditions suitable for the generation of angiotensin 1 by renin in the sample;
(b) purifying angiotensin 1 in said sample by liquid chromatography;
(c) ionizing the purified angiotensin 1 from said sample to produce one or more ions detectable by mass spectrometry;
(d) detecting the amount of the angiotensin 1 ion(s) by mass spectrometry; and
(e) using the amount of ion(s) detected to measure the amount of angiotensin 1 in said sample.
8. The method of claim 7 , wherein said method has a limit of quantitation less than or equal to 0.1 ng/mL.
9. The method of claim 7 , wherein said angiotensin 1 ions comprise an ion with a mass/charge ratio of 619.5±0.5.
10. The method of claim 7 , wherein said ionizing comprises generating a precursor ion with a mass/charge ratio of 433.0±0.5, and generating a fragment ion with a mass/charge ratio of 619.5±0.5.
11. The method of claim 7 , wherein the amount of the angiotensin 1 ion is related to the presence or amount of angiotensin 1 in the sample by comparison to an internal standard.
12. The method of claim 7 , wherein said step (b) comprises purifying angiotensin 1 from said sample with a solid-phase extraction column.
13. The method of claim 7 , wherein said water stable protease inhibitor is aminoethylbenzylsulfonyl fluoride.
14. The method of claim 7 , further comprising measuring the degree of degradation of angiotensin 1 in the sample by adding a degradation standard to the sample prior to incubation and measuring the amount of degradation standard remaining after incubation.
15. The method of claim 7 , further comprising measuring the degree of degradation of angiotensin 1 in the sample by adding a degradation standard to the sample prior to incubation and measuring the amount of breakdown products from degradation of the degradation standard in the sample after incubation.
16. A method for measuring renin activity in a sample, said method comprising,
(a) incubating the sample premixed with a water stable protease inhibitor that is not effective against renin under conditions suitable for the generation of angiotensin 1 by renin in the sample;
(b) purifying angiotensin 1 by liquid chromatography;
(c) ionizing the purified angiotensin 1 from said sample to produce one or more ions detectable by mass spectrometry;
(d) detecting the amount of the angiotensin 1 ion(s) by mass spectrometry;
(e) using the amount of ion(s) detected to measure the amount of angiotensin 1 in said sample; and
(f) using the amount of angiotensin 1 measured in said sample to calculate renin activity in the sample.
17. The method of claim 16 , wherein said liquid chromatography is high performance liquid chromatography (HPLC).
18. The method of claim 16 , wherein said method has a limit of quantitation less than or equal to 0.1 ng/mL.
19. The method of claim 16 , wherein said angiotensin 1 ions comprise an ion with a mass/charge ratio of 619.5±0.5.
20. The method of claim 16 , wherein said ionizing comprises generating a precursor ion with a mass/charge ratio of 433.0±0.5, and generating a fragment ion with a mass/charge ratio of 619.5±0.5.
21. The method of claim 16 , wherein said purifying angiotensin 1 in step (b) comprises purifying angiotensin 1 with a solid-phase extraction column.
22. The method of claim 16 , wherein said water stable protease inhibitor is aminoethylbenzylsulfonyl fluoride.
23. The method of claim 16 , wherein the presence or amount of the angiotensin 1 ion is related to the presence or amount of angiotensin 1 in the sample by comparison to an internal standard.
24. The method of claim 16 , further comprising measuring the degree of degradation of angiotensin 1 in the sample by adding a degradation standard to the sample prior to incubation and measuring the amount of degradation standard remaining after incubation.
25. The method of claim 16 , further comprising measuring the degree of degradation of angiotensin 1 in the sample by adding a degradation standard to the sample prior to incubation and measuring the amount of breakdown products from degradation of the degradation standard in the sample after incubation.
26. A method for measuring renin activity in a sample, said method comprising,
(a) incubating the sample under conditions suitable for the generation of angiotensin 1 by renin in the sample;
(b) purifying angiotensin 1 in said sample by liquid chromatography;
(c) ionizing the purified angiotensin 1 from said sample to produce one or more ions detectable by mass spectrometry;
(d) detecting the amount of the angiotensin 1 ion(s) by mass spectrometry, said ions comprising an ion with a mass/charge ratio of 619.5±0.5; and
(e) using the amount of ion(s) detected to measure the amount of angiotensin 1 in said sample; and
(f) using the amount of angiotensin 1 measured in said sample to calculate renin activity in the sample.
27. The method of claim 26 , wherein said ionizing comprises generating a precursor ion with a mass/charge ratio of 433.0±0.5, and generating a fragment ion with a mass/charge ratio of 619.5±0.5.
28. The method of claim 26 , wherein said method has a limit of quantitation less than or equal to 0.1 ng/mL.
29. The method of claim 26 , wherein incubating in step (a) comprises incubating in the presence of a water stable protease inhibitor that is not effective against renin.
30. The method of claim 29 , wherein said water stable protease inhibitor is aminoethylbenzylsulfonyl fluoride.
31. The method of claim 26 , wherein said purified angiotensin 1 is purified by high performance liquid chromatography (HPLC).
32. The method of claim 26 , wherein said method further comprises purifying angiotensin 1 from said sample with a solid-phase extraction column.
33. The method of claim 26 , wherein the presence or amount of the angiotensin 1 ion is related to the presence or amount of angiotensin 1 in the sample by comparison to an internal standard.
34. The method of claim 26 , further comprising measuring the degree of degradation of angiotensin 1 in the sample by adding a degradation standard to the sample prior to incubation and measuring the amount of degradation standard remaining after incubation.
35. The method of claim 26 , further comprising measuring the degree of degradation of angiotensin 1 in the sample by adding a degradation standard to the sample prior to incubation and measuring the amount of breakdown products from degradation of the degradation standard in the sample after incubation.