Derivatization of estrogens enhances specificity and sensitivity of analysis of human plasma and serum by liquid chromatography tandem mass spectrometry

Abstract

Estrogens circulate at concentrations less than 20 pg/mL in men and postmenopausal women, presenting analytical challenges. Quantitation by immunoassay is unreliable at these low concentrations. Liquid chromatography tandem mass spectrometry (LC–MS/MS) offers greater specificity and sometimes greater sensitivity, but ionization of estrogens is inefficient. Introduction of charged moieties may enhance ionization, but many such derivatives of estrogens generate non-specific product ions originating from the “reagent” group. Therefore an approach generating derivatives with product ions specific to individual estrogens was sought.Estrogens were extracted from human plasma and serum using solid phase extraction and derivatized using 2-fluoro-1-methylpyridinium-p-toluenesulfonate (FMP-TS). Electrospray in positive mode with multiple reaction monitoring using a QTrap 5500 mass spectrometer was used to quantify “FMP” derivatives of estrogens, following LC separation.Transitions for the FMP derivatives of estrone (E1) and estradiol (E2) were compound specific (m/z 362→238 and m/z 364→128, respectively). The limits of detection and quantitation were 0.2 pg on-column and the method was linear from 1–400 pg/sample. Measures of intra- and inter-assay variability, precision and accuracy were acceptable (<20%). The derivatives were stable over 24 h at 10 °C (7–9% degradation). Using this approach, E1 and E2, respectively were detected in human plasma and serum: pre-menopausal female serum (0.5 mL) 135–473, 193–722 pmol/L; male plasma (1 mL) 25–111, 60–180 pmol/L and post-menopausal female plasma (2 mL), 22–78, 29–50 pmol/L.Thus FMP derivatization, in conjunction with LC–MS/MS, is suitable for quantitative analysis of estrogens in low abundance in plasma and serum, offering advantages in specificity over immunoassay and existing MS techniques.

Spatial Localization and Quantitation of Androgens in Mouse Testis by Mass Spectrometry Imaging

“…13 C labeling is preferred to deuterium due to the potential for loss of deuterium during derivatization or fragmentation. 19 …”

Ultra‐performance liquid chromatography–tandem mass spectrometry assay for determination of plasma nomegestrol acetate and estradiol in healthy postmenopausal women

“…Huang, Cao, and Gu () developed a reversed‐phase HPLC method in rat plasma, but the sensitivity was very low (100 ng/mL), which may not be useful for pharmacokinetic studies in humans. Analysis of estrogens including E2 is analytically challenging owing to their very low circulating levels in plasma (≤50 pg/mL) and interference from endogenous isomers and other steroids present in the biological fluids (Faqehi et al, ). Accurate and precise measurement of low concentration of estrogens is essential to determine menopausal status and estrogen deficiency, and also to assess risk of osteoporosis and cardiovascular diseases (Kushnir et al, ).…”

“…Accurate and precise measurement of low concentration of estrogens is essential to determine menopausal status and estrogen deficiency, and also to assess risk of osteoporosis and cardiovascular diseases (Kushnir et al, ). Although immunoassays have been extensively used to measure low concentration levels of E2 (15–1000 pg/mL; Xin, Liang, Wang, Li, & Lin, ), they suffer from several limitations including nonspecific binding, cross reactivity, long analysis time and other analytical interferences (Faqehi et al, ; Kushnir et al, ; Nelson, Grebe, O'Kane, & Singh, ). On the other hand methods based on high‐performance liquid chromatography (HPLC) with electrochemical (Yamada, Yoshizawa, & Hayase, ) and fluorescence (Yilmaz & Kadioglu, ) detection have very low sensitivity (≥2.4 ng/mL) and require very long analysis time (≥15 min).…”

“…However, owing to the lack of ionizable groups in estrogens, derivatization is required to improve the sensitivity by introducing charged groups which can promote better ionization efficiency and fragmentation in MS and also for improved retention in chromatography. Several derivatization reagents, like 2‐fluoro‐1‐methylpyridinium‐ p ‐toluene sulfonate (Faqehi et al, ), dansyl chloride (Faqehi et al, ; Nelson et al, ; Ray, Kushnir, Bunker, Rockwood, & Meikle, ; Szarka, Nguyen, Prokai, & Prokai‐Tatrai, ; Xia et al, ; Zhang et al, ), picolonic acid and 2‐methyl‐6‐nitrobenzoic anhydride (Yamashita et al, ), have been used to analyze E2 in different biological matrices like human plasma (Faqehi et al, ; Nelson et al, ), human serum (Faqehi et al, ; Kushnir et al, ; Ray et al, ; Szarka et al, ; Yamashita et al, ), ovariectomized mouse plasma and brain (Xia et al, ) and human cell line (Zhang et al, ) by LC–MS/MS. A sensitive GC–MS method has also been reported to determine E2 from human serum after derivatization with pentafluorobenzoyl chloride in a chromatographic run time of 24 min (Fedeniuk, Boison, & MacNeil, ).…”

Molecular labels for analysis of amines and diols by spray based ionization-mass spectrometry

“…cells) are examples of possible areas of application of the methodology reported on here. Ideally, the labels used in such studies will carry a permanent charge (tetra-alkyl ammonium, pyridinium or other positive ions) so as to confer high ionization efficiency in spray-based ionization [21][22][23] . In general, labeling methods should be simple, the labels themselves easy to obtain or to synthesize, and it should be possible to implement the label analysis in a convenient manner, be it by the detection of a label-molecule conjugate or by the release of labels from a particle to serve as reporters.…”