212, P212

Establishment of a GC-MS/MS methodology for comprehensive sex steroid profiling in mouse serum

Maria Nilsson1,*, Liesbeth Vandenput2, Åsa Tivesten2, Anna-Karin Norlén1, Henrik Ryberg1, Claes Ohlsson2

1Clinical chemistry, Sahlgrenska universitetssjukhuset, 2Göteborgs universitet, Gothenburg, Sweden

Introduction: There are two major differences in sex steroid metabolism between humans and mice. (i) A substantial portion of serum sex steroids are bound to SHBG in humans, while mice do not have SHBG. (ii) Humans, but not mice, secrete substantial amounts of androgen precursors (Dehydroepiandrosterone [DHEA] and Androstenedione [A]) from the adrenal gland. Therefore, the serum levels of sex steroids and sex steroid precursors are expected to be relatively low and difficult to determine in mice. Serum levels of sex steroids in mice have routinely been analyzed using immunoassays having questionable specificity in the lower range.

Objectives: The aim of the present study was to develop a sensitive and specific GC-MS/MS assay for measurement of a comprehensive sex steroid profile in mice.

Methods: A GC-MS/MS method was used to measure serum testosterone (T), dihydrotestosterone (DHT), estradiol (E2), estrone (E1), DHEA, A and progesterone (P). After addition of isotope-labeled standards, steroids were extracted to chlorobutane, purified on a silica-column and derivatized using pentafluorobenzyl hydroxylamine hydrochloride followed by pentafluorobenzoyl chloride. Steroids were analyzed in MRM-mode with ammonia as reagent gas, and using negative chemical ionization.

Results: The lower limits of quantification (LLOQ) for T, DHT, E2, E1, DHEA, A, and P were 8, 2.5, 0.6, 1, 200, 6, and 74 pg/ml, respectively. Using ≥250 µl female mouse serum, concentrations of DHEA and E1 were under LLOQ but all other sex steroids were measureable (average levels: T 14, DHT 13, E2 2.4, A 189, and P 13900 pg/ml). In male mice, E2 and E1 were undetectable and DHEA levels were under LLOQ but all other sex steroids were reliably measured using 100 µl of serum (T 8240, DHT 168, A 207 and P 680 pg/ml).

Conclusion: Our established GC-MS/MS-based assay for the measurement of a comprehensive serum sex steroid profile in mice might be useful for the characterization of sex steroid metabolism in a variety of sex-steroid related transgenic mouse models.

Keywords: Blood, Method