43. LC-MSMS Method Development for Steroid Analysis at Low Detection Limits
Poster: Mon 2:00-3:00PM
Hua-fen Liu
Hua-fen Liu, AB SCIEX
Beth Fernandez, AB SCIEX
Renee Huang, AB SCIEX
Tania Sasaki, AB SCIEX
Dave Lavarato,AB SCIEX
Lisa Sapp, AB SCIEX
Elliott Jones, AB SCIEX
Steroid analysis is critical for research into a number of common endocrine disorders and also applied as biomarker for numbers of diseases. Major challenges of steroids analysis include sensitivity, selectivity, and large sample volume. Using liquid chromatography mass spectrometer to analyze steroids at low level has been becoming a new trend to overcome these challenges.

Three major steroid methods will be presented including: testosterone for low level detection; estrogens for pg/mL detection; steroids panel including 12 major steroids for general screening.

Testosterone, the major androgenic hormone in humans, is commonly measured for its excess or deficiency. The concentration of testosterone in women, children and men undergoing anti-androgen therapy are typically less than 50ng/dL (500pg/mL). A simple one step liquid-liquid extraction method was developed using 200uL serum with 10 pg/mL detection limit and less than 10% CV. The method has been verified for robustness in different laboratories. Good correlation was observed between the results obtained from this method and other reference results.

Estrogens are a group of steroid compounds, named for their importance in the estrous cycle, and functioning as the primary female sex hormone. Estrogens detection has been a challenging task due to accurate low pg/mL level detection is needed. Multiple complex sample preparation including derivatization is often used to achieve the detection limits desired. We developed a two steps liquid-liquid extraction without derivatization method coupling with mobile phase modifier to improve the ionization to achieve less than 5pg/mL quantification limit with less than 10%CV.

Steroidogenesis is the process wherein desired forms of steroids are generated by transformation of other steroids. The LC-MSMS method of analysis 12 major steroids products in one method was desired. One major challenge of the assay is that the 12 steroids have a wide concentration of interest range from ug/mL to pg/mL. The cross interference from high concentration steroid analytes to low concentration analytes and isomer separation are evaluated and considered in the method. Simple protein precipitation was used for sample preparation. The method of 12 steroids at corresponding concentration of interested range was developed and verified.

For research use only. Not intended for any animal or human therapeutic or diagnostic use. With this addition Regulatory Affairs approves.
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