Measuring levels of a metabolite
View Patent ↗Described herein are methods for determining an amount of an analyte in a test sample. The methods involve preparing a calibration curve using standard samples containing an isotopically-labeled standard in a biological matrix.
1. A method for determining an amount of a vitamin D metabolite in a test sample, the method comprising:
(a) providing a set of calibrator samples, each calibrator sample comprising a different amount of a first isotopically-labeled standard in a biological matrix,
(b) providing to each calibrator sample a selected constant amount of a second isotopically labeled standard, the first and second isotopically labeled standards having distinct isotope content, and wherein the first and second isotopically labeled standards are isotopically-labeled vitamin D metabolite derivatives;
(c) subjecting the calibrator samples comprising the first and second isotopically-labeled standards to mass spectrometry to determine signal intensities corresponding to the first and second isotopically-labeled standards;
(d) generating a calibration curve based on ratios of the determined signal intensities of the first and second isotopically-labeled standards as a function of the amount of the first isotopically-labeled standard in the calibrator samples;
(e) contacting a test sample with the selected constant amount of the second isotopically-labeled standard;
(f) subjecting the test sample comprising the second isotopically-labeled standard to mass spectrometry to determine signal intensities corresponding to the second isotopically labeled standard and the vitamin D metabolite, and
(g) using the calibration curve and the determined signal intensities corresponding to the second isotopically labeled standard and the vitamin D metabolite to determine the amount of the vitamin D metabolite in the test sample.
2. The method of claim 1 , wherein the vitamin D metabolite is selected from 25-hydroxyvitamin D3, 1-hydroxyvitamin D3, 1, 25-dihydroxyvitamin D3, 25-hydroxyvitamin D2, 1α-hydroxyvitamin D2, and 24-hydroxyvitamin D2.
3. The method of claim 1 , wherein the vitamin D metabolite is a vitamin D3 metabolite and the first isotopically-labeled standard is selected from isotopically labeled 1, 25-dihydroxyvitamin D3 and isotopically-labeled 25-hydroxyvitamin-D3.
4. The method of claim 1 , wherein the first isotopically-labeled standard is labeled with 2H, 13 C, 15 N, 18 O, or a combination thereof.
5. The method of claim 4 , wherein the first isotopically-labeled standard is selected from labeled 1α,25-dihydroxyvitamin D3, d 6 -labeled 25-hydroxyvitamin-D3, d 3 -labeled 1, 25-dihydroxyvitamin D3 and d 3 -labeled 25-hydroxyvitamin-D3.
6. The method of claim 5 , wherein the first isotopically-labeled standard is 25-hydroxyvitamin-D3 (26,26,26,27,27,27-d 6 ).
7. The method of claim 4 , wherein the first isotopically-labeled standard is 25-hydroxyvitamin-D2 (26,26,26,27,27,27-d 6 ).
8. The method of claim 1 , wherein the vitamin D metabolite is a vitamin D3 metabolite and the second isotopically-labeled standard is selected from isotopically labeled 1, 25-dihydroxyvitamin D3 and isotopically-labeled 25-hydroxyvitamin-D3.
9. The method of claim 1 , wherein the second isotopically-labeled standard is labeled with deuterium.
10. The method of claim 9 , wherein the second isotopically-labeled standard is selected from d 6 -labeled 1, 25-dihydroxyvitamin D3, d 6 -labeled 25-hydroxyvitamin-D3, d 3 -labeled 1, 25-dihydroxyvitamin D3 and d 3 -labeled 25-hydroxyvitamin-D3.
11. The method of claim 10 , wherein the second isotopically-labeled standard is 25-hydroxyvitamin-D3 (6,19,19-d 3 ).
12. The method of claim 1 , wherein the vitamin D metabolite is a vitamin D2 metabolite.
13. The method of claim 12 , wherein the vitamin D metabolite is a vitamin D2 metabolite and the first isotopically-labeled standard is selected from 1,25-dihydroxyvitamin D2 and 25-hydroxyvitamin-D2.
14. The method of claim 12 , wherein the vitamin D metabolite is a vitamin D2 metabolite and the second isotopically-labeled standard is selected from 1,25-dihydroxyvitamin D2 and 25-hydroxyvitamin-D2.
15. The method of claim 14 , wherein the second isotopically-labeled standard is 25-hydroxyvitamin-D2 (6,19,19-d 3 ).
16. The method of claim 1 , wherein the mass spectrometry comprises a technique selected from LC-MS and LC-MS/MS.
17. The method of claim 1 , wherein the test sample is a biological sample.
18. The method of claim 14 , wherein the biological sample is selected from the group consisting of blood, serum, plasma, saliva, and urine.
19. The method of claim 18 , wherein the blood is provided dried on a paper matrix.
20. The method of claim 1 , wherein first and second isotopically-labeled standards each comprise a different number of isotopic labels comprising the same isotope.
21. A kit for determining an amount of an vitamin D metabolite in a test sample, comprising:
an isotopically labeled calibrator standard, and
an isotopically labeled internal standard,
wherein the first and second isotopically labeled standards are isotopically-labeled vitamin D metabolite derivatives; and
wherein the calibrator and internal standards have distinct isotope content.