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Abstract Introduction:
Corticosteroids includes 6 steroid hormones of Cortisol, Corticosterone, 11-Deoxycorticosterone (11-DOC), 11-Deoxycortisol(11-DOCL), Testosterone and Aldosterone, which are involved in a wide range of physiological processes.
Due to low concentration and strong interference of Aldosterone, the reported sample-preparation method required a large volume of plasma (500uL) enrichment to achieve the LLOQ 10pg/mL. However, it’s difficult to achieve enough volume for every plasma sample. To obtain the required sensitivity needed for this assay with the preparation of only 50uL plasma, microflow LC/MS was utilized. Using this technique, the sensitivity of 6 compounds were enabled to improve 5-15X than normal UPLC flow rate. This enhanced level of sensitivity allows for the accurate determination of 6 steroid hormones from a starting volume of only 50uL plasma.
Methods:
Standards, QC’s and blanks were prepared by spiking in a known concentration of 6 steroid hormones and Internal standard (Corticosterone-d8) into 50 µL of human plasma. After samples were extracted by liquid/liquid extraction (LLE) method, the supernatant was evaporated to dryness and the residue was reconstituted in 100 µL of 20% methanol.
Chromatographic separation was performed on a Eksigent HaloTM C18 (0.5×50mm, 2.7um, 90A). Mobile phase A consisted of 2mM ammonium acetate and Mobile phase B consisted of 100% methanol. Mass spectrometric detection was conducted on a QTRAP®5500 mass spectrometer, operating with the polarity switch of positive and negative ion mode.
Preliminary results:
A method was developed for the quantification of 6 steroid hormones in human plasma using liquid/liquid extraction with only 50µL plasma, followed by reversed phase microflow chromatography and tandem quadrupole mass spectrometry. The separation was performed by applying a gradient from 20% to 90% B over 11 minutes at a flow rate of 5 μL/min. During method development, it was determined that 5 μL/min of micro flow rate gave 5-15X greater sensitivity over the typical standard 400 μL/min flow rate.
Using this approach, a calibration curve was prepared by spiking known concentrations of 6 steroid hormones into human plasma. The calibration range was linear from 0.01 to 10 ng/mL for Aldosterone in negative mode with an r value of 0.9980. At the same time, the calibration curve of other 5 steroid hormones in positive mode are linear from 0.1-100ng/mL with r value from 0.9912 to 0.9988. In order to determine the precision of this method, 3 replicate QC samples were prepared at 3 concentration levels and extracted. The precision of 6 steroid hormones in QC samples for each concentration was determined to be <15%.
Novel Aspect: Utilization of microflow LC/MS/MS to achieve Corticosteroid quantitation at low pg/mL levels in significantly reduced volume of plasma.
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= Discovery stage.
= Translation stage.
= Clinically available.
