Measuring levels of a metabolite

1. A method for determining an amount of 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,

(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, wherein first and second isotopically-labeled standards are labeled steroid 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 metabolite, and

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

2. The method of claim 1 , wherein the first isotopically-labeled standard is labeled with 2H, 13 C, 15 N, 18 O, V or a combination thereof.

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

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

5. The method of claim 2 , wherein the isotopically-labeled steroid derivative standard is dehydroepiandrosterone (3,4- 13 C; 2,2,3,4,4,6-d5).

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

7. The method of claim 1 , wherein the second isotopically-labeled standard is labeled with deuterium.

8. The method of claim 1 , wherein the steroid 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 derivatives of the selected steroid.

9. The method of claim 1 , wherein the mass spectrometry comprises a technique selected from LC-MS and LC-MS/MS.

10. The method of claim 1 , wherein the test sample is a biological sample.

11. The method of claim 10 , wherein the biological sample is selected from the group consisting of blood, serum, plasma, saliva, and urine.

12. The method of claim 11 , wherein the blood is provided dried on a paper matrix.

13. The method of claim 1 , wherein first and second isotopically-labeled standards each comprise a different number of isotopic labels comprising the same isotope.

14. A kit for determining an amount of a steroid metabolite in a test sample, comprising:

an isotopically labeled steroid derivative calibrator standard, and

an isotopically labeled steroid derivative internal standard,

wherein the calibrator and internal standards have distinct isotope content.