Quantitative Determination of Estrone Sulfate at Low pg/mL in Human Plasma by LC-MS-MS
Authors
Christopher J.L. Buggé, Anders Ljungqvist, Michael P. Sullivan
Introduction
Estrone Sulfate is a metabolite of Estrone/Estradiol which are used in hormone replacement therapy. A sensitive assay was required to determine estrone sulfate in human plasma at low pg/mL concentrations to support clinical trials.
Experimental
Chemicals
Reference estrone sulfate and its tetradeuterated internal standard were obtained commercially. All other chemicals were AR grade and all solvents were HPLC grade.
Standards/Solutions
Stock solutions of estrone sulfate were prepared in acetonitrile/water 1:1, as were intermediate, calibration spiking and internal standard solutions. Calibration spiking solutions were prepared at 75, 150, 300, 600, 1200, 2400, 3380, and 3750 pg/mL. Quality control (QC) were prepared from different weighings of drug than the calibration spiking standards, at 225, 1000 and 3000 pg/mL.
LC-MS-MS
The extract was injected directly onto a Sciex API 4000 LC-MS-MS in negative ion TurboIonspray mode using a C-18 column (4.0 x 23 mm) and a mobile phase of ammonium acetate methanol. Estrone sulfate eluted at 1.0 min at a flow rate of 1.2 mL/min, and the 349.1>269.1 transition was monitored in MRM mode with its tetradeuterated internal standard. Total time run was 2 min.
Results
The linear quantitative range was established and validated from 75 to 3750 pg/ml for estrone sulfate over 3 days. Calibration curves and quality control samples were prepared from human plasma that had been cleansed of endogenous estrone sulfate. In this way, a matrix most similar to the authentic study plasma was used, and more accurate quantitative data can be obtained (compared to subtraction of endogenous levels of analyte from calibration and QC samples). Precision and accuracy were determined to be acceptable after evaluating LLOQ samples and QC samples over three days of validation (see tables).
Extraction recovery was found to be 90%.
Specificity of the assay was determined by injection of six lots of blank plasma, and injection of a solution of over-the-counter drugs at 100 ug/mL. In all cases, no interferences were seen other than peaks from endogenous estrone sulfate.
Stability of estrone sulfate was established in plasma after 5 freeze/thaw cycles, 24h room temperature exposure and 1 month at –70°C.
Matrix effect was evaluated by analyzing 6 lots of plasma, and measuring the endogenous concentrations; the same 6 lots of plasma were also fortified with estrone sulfate at 1200 pg/mL and analyzed; analytical recovery was calculated by subtracting the individual endogenous concentrations from the spiked samples and expressing the amount remaining as a percentage of 1200 pg/mL. In this way the absolute accuracy of the method could be evaluated with authentic plasma. The results were within +/- 15% of the theoretical amount added for all 6 lots, indicating the rugged nature of the assay.
Conclusions
This method was successfully used to determine concentrations of estrone sulfate in human plasma samples generated from clinical trials. The method proved to be sufficiently rugged even at the low concentrations (75 pg/ml) required. The simple extraction and short run time provided an assay with a fast sample throughput.