Marketplace Analysis of Conjugated Estrogens: Determining the Consistently Present Steroidal Content with LC-MS

Abstract

Conjugated estrogens purified from pregnant mares urine has been used as estrogen hormone replacement therapy since 1942. Previously, methods were proposed to identify and quantify the components of this complex mixture but ultimately were withdrawn due to incomplete characterization of the product and difficulties in transferring the method between laboratories. The aim of the current study is to develop a LC method that can reliably detect multiple steroidal components in conjugated estrogen tablets and measure their relative amount. The method developed was optimized for UHPLC columns, and the elution profile was analyzed using high-resolution mass spectrometry. A total of 60 steroidal components were identified using their exact m/z, product ion spectra of known, and predicted conjugated estrogen structures. These components were consistently present in 23 lots of Premarin tablets spanning two production years. The ten conjugated estrogens identified in the USP monograph and other additional estrogens reported elsewhere are among the 60 steroidal components reported here. The LC-MS method was tested in different laboratories using multiple samples, and the obtained results were reproducible among laboratories.

Keywords: conjugated estrogens; mass spectrometry; steroidal content.

Fig. 1

The extracted ion chromatogram is shown of the separation of DHES and EqS peaks with a resolution of 1.8

Fig. 2

Total ion chromatogram (top) of the conjugated estrogen compounds isolated from Premarin. Extracted ion chromatograms of ions m/z 345.0802, 347.0959, and 349.1115 (second, third, or bottom panel, respectively) showing multiple species at multiple m/z

Fig. 3

Product ion spectra of Δ^8,9-dehydroestrone-3-sulfate and equilin-3-sulfate after HCD at 75% NCE

Fig. 4

The variation in peak area was plotted for each of the top 60 components using SigmaPlot 12.3. The median value is displayed as the black bar within the box with the 25th and 75th percentiles as the lower and upper boundaries of the box, respectively, and the 10th and 90th percentiles shown as the error bars. The top panel shows the data for the 15 most abundant compounds and the peak areas are tightly controlled. The bottom panel shows the data for compounds 16–60 with the relative variation being much higher for these lower abundance peaks