Supplementary Text

For on-column experiments, the following chromatography conditions were used. Two Shimadzu UFLCXR 50326 LC-20AD pumps (Kyoto, Japan) and a Leap Technologies PAL HTC-xt Sample Handler (Carrboro, North Carolina) were coupled to an AB Sciex Triple Quad QTRAP 5500 LC-MS/MS mass spectrometer. The mobile phase consists of (A) water and (B) methanol at a flow rate of 200 μl/min; 10% ammonium hydroxide in acetonitrile was added post-column at a flow-rate of 6 μl/min. LC Parameters are as follows: 0-1 min 60% B, 1.10-5 min 70-75% B, 5.10-8 min 85% B, 8.10-12 min 60% B. The stationary phase consists of a C18 column (Phenomenex Kinetex 2.6 μm C18 100 Å, 100 x 3.0 mm) protected by a C18 guard cartridge (Phenomenex SecurityGuard cartridge for C18 HPLC).

Supplementary Figure Legends

Supplementary Figure 1 Negative ESI MS/MS Q-TOF of [D1]-E2. The hydroxyl group on C17 can readily exchange with the deuterium in MeOD, D2O and NaOD to form a labeled [D1]-E2 compound. Inset MS of the labeling experiment demonstrated that a deuterium exchanged with a proton on [M-H]- m/z 271 which resulted in [M+D1-H]- m/z 272. Approximately half of the E2 is unlabeled and serves as a control for comparison with [D1]-E2 (spectra identical to Figure 1A). CID of [M+D1-H]- m/z 272 at CE 55. Note that both m/z 183-m/z 184 and m/z 169-m/z 170 are present in approximately equal abundance indicating an alternative fragmentation pathway involving the deuterium from the D-ring hydroxyl group and suggesting hydrogen rearrangement.

Supplementary Figure 2 MSn Ion Trap experiments of infused 100 ng/μl E2. A MS2 of m/z 271 results in a similar spectra to those acquired from MS/MS Q-TOF and LC-MS/MS. The difference in product ion abundance is likely attributed to the different collision gases utilized in each instrument (helium in the ion trap and nitrogen in the QTRAP). B MS3 (271à269), C MS3 (271à253), D MS3 (271à183), E MS3 (271à225), F MS3 (271à213), G MS3 (271à225) spectra show no significant formation of m/z 183. Additional MS3 experiments (271à237, 271à209, 271à223, 271à197) performed in the QTRAP (data not shown) resulted in no significant m/z 183 or m/z 169. H MS3 (271à183).

Supplementary Figure 3 Negative ESI MS/MS of estrone (E1) and estriol (E3) on column. CID spectra of E1 A and [D4]-E1 B from 100 pg of E1 on column, fragmented at CE 55-the same energy used to fragment E1 and E3 in our quantitative assay. Note the shift of m/z 183 (E1) to m/z 187 ([D4]-E1) indicating all 4 deuteriums are in the final product ion structure. CID Spectra of E3 C and [D4]-E3 D generated from 500 pg of E3 on column, fragmented at CE 55. Note the shift of m/z 183 (E3) to m/z 187 ([D4]-E3) indicating all 4 deuteriums are in the final m/z 187 product ion structure.

Supplementary Figure 4 QTRAP MS3 of 271/269/287à183à and 271/269/287à169àon column. MS3 spectra of A E2 m/z 271à183à, B E1 m/z 269à183à, C E3 m/z 287à183à from 500 pg on column from CE 55 to fragment m/z 271 and an excitation energy (AF2 of 0.1) to further fragment the product ions m/z 169 or m/z 183. Additional experiments with different AF2 (0.01, 0.05, 0.1, 0.3 and the maximum 0.5) yielded similar results (Data not shown). The data indicate m/z 183 and m/z 169 are stable product ions due to the lack of ions fragmented from them.

Supplementary Table 1. High Resolution Measurements

Isotope / Elemental
Composition / Monoisotopic Mass
(Calculated) / Measured
Mass / PPM
Difference
E2 / C18H23O2 / 271.1698 / 271.1700 / 0.7
[D1]-E2 / C18H22D1O2 / 272.1761 / 272.1762 / 0.4
[D4]-E2 / C18H19D4O2 / 275.1949 / 275.1947 / 0.7
[13C6]-E2 / C1213C6H23O2 / 277.1899 / 277.1898 / 0.4
E2 / C13H11O / 183.0810 / 183.0813 / 1.6
[D1]-E2 / C13H10D1O / 184.0873 / 184.0865 / 4.3
[D4]-E2 / C13H7D4O / 187.1061 / 187.1062 / 0.5
[13C6]-E2 / C1013C3H11O / 186.0911 / 186.0910 / 0.5
E2 / C12H9O / 169.0653 / 169.0654 / 0.6
[D1]-E2 / C12H8D1O / 170.0716 / 170.0709 / 4.1
[D4]-E2 / C12H7D2O / 171.0779 / 171.0779 / 0.0
[13C6]-E2 / C1013C2H9O / 171.0720 / 171.0724 / 2.3