Chromatography

Liquid chromatography tandem mass spectrometry (LC-MS) was carried out with a Shimadzu Prominence HPLC system and SCIEX 6500 mass spectrometer. An aliquot of 10 µl of each sample was injected onto an Kinetix 2.6 µm F5 100 x 2.1 mm column at 40^oC for gradient separation. Mobile phases were: A, ddH₂O with 0.1% Formic Acid; and B, Acetonitrile with 0.1% Formic Acid. Gradient schedule starts at 40% B and is linearly increased to 97% B at 6 minutes, then increased to 100% B at 6.20 mins, then held at 100% B until 8 minutes, and then returned to 40% B at 8.1 at minutes and re-equilibration until 9 minutes. Time per sample injection was 9 minutes with a flow rate is 350µL/min.

Extraction Protocols:

Extraction protocols for sample processing are dependent on the matrices involved. Please see extraction methods section for more specific instructions.

Derivatization Protocol:

Samples were loaded on to individual 30 mg/1 ml Polymeric Strata-X Solid Phase Extraction cartridge columns (Phenomenex, Torrance, CA). Samples were further washed with 1 ml of MilliQ H₂O and 1 ml of 40% methanol followed by elution with 1 ml of methanol. Sample eluents were dried under a gentle stream of N₂. 50 µl of 100mM sodium bicarbonate (pH 10.5) and 50 µl of 1 mg/ml dansyl-chloride in acetone were added to samples. Samples were incubated at 60^oC for 10 minutes to facilitate dansyl-chloride reaction with estradiol. Samples were dried once more under a gentle stream of N₂.

References

Resources

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