Measuring levels of a metabolite

1. A method for determining an amount of an analyte selected from a secosteroid metabolite and a steroid 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 analyte 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 analyte; and

(g) using the calibration curve and the determined signal intensities corresponding to the second isotopically labeled standard and the analyte to determine the amount of the analyte in the test sample.

2. The method of claim 1 , wherein the secosteroid 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 analyte 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 2 H, 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-d6).

7. The method of claim 4 , wherein the first isotopically-labeled standard is 25-hydroxyvitamin-D2 (26,26,26,27,27,27-d6).

8. The method of claim 1 , wherein the analyte 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-d3).

12. The method of claim 1 , wherein the analyte is a vitamin D2 metabolite.

13. The method of claim 12 , wherein the analyte 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 analyte 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-d3).

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 17 , 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 analyte in a test sample, comprising:

an isotopically labeled analyte derivative calibrator standard, and

an isotopically labeled analyte derivative internal standard,

wherein the calibrator and internal standards have distinct isotope content,

wherein the analyte is selected from a steroid, a secosteroid metabolite and a steroid metabolite.

22. A method for determining an amount of a steroid analyte 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 analyte 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 steroid analyte; and

(g) using the calibration curve and the determined signal intensities corresponding to the second isotopically labeled standard and the steroid analyte to determine the amount of the steroid analyte in the test sample.

23. The method of claim 22 , wherein the first isotopically-labeled standard is labeled with 2H, 13C, 15N, 18O, or a combination thereof.

24. The method of claim 22 , wherein the steroid analyte is selected from the group consisting of: 11-deoxycortisol, 21-deoxycortisol, 17-α-hydroxyprogesterone, aldosterone, 4-androstene-3,17-dione, corticosterone, deoxycorticosterone, cortisol, dehydroepiandrosterone, dehydroepiandrosterone sulfate, estradiol, estriol, estrone, progesterone, 5-α-dihydrotestosterone, testosterone, pregnenolone, 17-α-hydroxypregnenolone, 5-α-androstane-3b,17b-diol, 2-hydroxyestradiol, 4-hydroxyestradiol, high-density lipoprotein (HDL) cholesterol, low-density lipoprotein (LDL) cholesterol, and very low-density lipoprotein (VLDL) cholesterol, and the first and second isotopically labeled standards are isotopically-labeled analogs of the selected analyte.

25. The method of claim 22 , wherein the steroid analyte is testosterone and an isotopically-labeled standard is testosterone (3,4- 13 C; 2,2,4,6,6-d5).

26. The method of claim 22 , wherein the steroid analyte is 4-androstene-3,17-dione and an isotopically-labeled standard is 4-androstene-3,17-dione (3,4- 13 C; 2,2,4,6,6-d5).

27. The method of claim 22 , wherein the steroid analyte is dehydroepiandrosterone and an isotopically-labeled standard is dehydroepiandrosterone (3,4- 13 C; 2,2,3,4,4,6-d5).

28. The method of claim 22 , wherein the steroid analyte is progesterone and the isotopically-labeled standard is progesterone (3,4- 13 C; 2,2,5,6,6,17a,21,21,21-d9).