Research Article Evaluation of matrix effects in analysis of estrogen using liquid chromatography– tandem mass spectrometry Matrix effects of different biological samples, including phosphate-buffered saline–bovine serum albumin (PBS-BSA), gelded horse serum, mouse serum, and mouse brain, were investigated for the determination of 17a- and b-estradiol using derivatization with dansyl chloride prior to LC-MS/MS. Matrix effects were evaluated based on the slopes of regression lines plotted from results obtained in biological matrices versus pure standard solutions. Such plots indicate the enhancement or suppression of signal based on the presence of a particular biological fluid for a particular method. The matrix effects from PBS-BSA were similar to those of mouse serum. In contrast, analyses performed from horse serum and mouse brain yielded significant ion suppression, especially for 17b- estradiol. Precipitation during derivatization was observed when pre-concentrated samples were processed with ethyl acetate as an extraction solvent. This was overcome with the use of methyl tert-butyl ether; however, matrix effects from this preparation were still present, evidenced by signal suppression and poor linearity in the standard curve. This work affirms that caution should be taken in the transfer of methods for use with different biological matrices, especially in the case where surrogate matrices are necessary for calibration purposes. Keywords: Biological samples / Estradiol / Matrix effects / Electrospray / LC-MS/MS DOI 10.1002/jssc.201100219 1 Introduction Estrogens play an important role in regulating secondary sexual characteristics as well as reproductive functions in females [1]. They have been widely used as hormone therapy in the treatment of hormone balance (www.webmd.com/ menopause/guide/menopause-hormone-therapy (accessed October 12, 2010)) and recently recognized for their neuroprotective effects [2, 3]. The beneficial effects of estrogens on the brain have been demonstrated in many studies [4–7]. The most active estrogen, 17b-estradiol (17b-E2), has been reported to reduce ischemic damage in focal cerebral ischemia when administered before or even after the injury, within a narrow therapeutic window of o3h [7]. Although 17a-E2, an epimer of 17b-E2, is considered less active since it binds less vigorously to estrogen receptors [8], it has recently received renewed attention. 17a-E2 is a less feminizing estrogen but still provides positive neuroprotection [8, 9]. The use of a less or non-feminizing estrogen as neuroprotective therapy can be advantageous in reducing the complications of feminizing side effects. For this reason, 17a-E2 can be a good candidate in the treatment of neurodegenerative diseases, along with 17b-E2. The analysis of these two estradiols is becoming a key component in any pre-clinical or clinical research program on neuroprotective activities of estrogens. Analysis of estrogens, in general, is of course important given their wide range of biological activities. High-performance liquid chromatography (LC) coupled to electrospray ionization–mass spectrometry (ESI-MS) has become a common chemical analysis technique for use in estrogen bioanalysis [10–15]. However, trace-level analysis of physiological samples by ESI-MS is a challenge due to ion suppression or enhancement caused by the co-elution of interfering species from complex biological matrices [16–19]. There are two main issues associated with matrix effects. The first is the variation of matrix composition and Hien P. Nguyen 1à Li Li 1 Imam S. Nethrapalli 2 Ningning Guo 2 C. Dominique Toran-Allerand 2 David E. Harrison 3 C. Michael Astle 3 Kevin A. Schug 1 1 Department of Chemistry and Biochemistry, University of Texas at Arlington, Arlington, TX, USA 2 Department of Pathology and Cell Biology, Columbia University College of Physicians and Surgeons, New York, NY, USA 3 The Jackson Laboratory, Bar Harbor, ME, USA Received March 28, 2011 Revised May 9, 2011 Accepted May 9, 2011 Abbreviations: PE, process efficiency; RE, recovery; ME, matrix effect; 17b-E2, 17b-estradiol; MTBE, methyl tert-butyl ether; LLE, liquid–liquid extraction à Additional correspondence: Dr. Hien P. Nguyen E-mail: hien.nguyen@mavs.uta.edu Correspondence: Dr. Kevin A. Schug, Department of Chemistry and Biochemistry, University of Texas at Arlington, 700 Plane- tarium Pl., Box 19065, Arlington, TX 76019-0065, USA E-mail: kschug@uta.edu Fax: 11-87-272-3808 & 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jss-journal.com J. Sep. Sci. 2011, 34, 1781–1787 1781