madman
Super Moderator
Abstract
Stable isotope dilution (SID) methodology coupled with liquid chromatography-tandem mass spectrometry (LC-MS) is rapidly becoming the gold standard for measuring estrogens in serum and plasma due to improved specificity, high accuracy, and the ability to conduct a more comprehensive analysis. A general consideration of the problems associated with measuring estrogens and two detailed derivatization methods are described in this chapter. These methods quantify estrogens and their metabolites in serum and plasma samples using this state-of-the-art technology, which is applicable to the routine clinical laboratory.
1. Introduction
Estrogen measurement plays an important role in the clinical evaluation of many endocrine disorders as well as in research on the role of hormones in human biology and disease. The predominant methodologies used to quantify circulating levels of estrogens were conventional radioimmunoassays (RIAs) or direct enzyme immunoassays (EIAs) (Blair, 2010; Stanczyk, Jurow, & Hsing, 2010). Mainly these techniques were chosen due to their low cost and routine nature. These assays afford good sensitivity but often lack specificity due to the cross-reaction of antibodies used in the assays with other steroids or unknown matrix components (Denver, Khan, Homer, MacLean, & Andrew, 2019; Ketha, Girtman, & Singh,2015). This problem is particularly marked when measuring lower levels. This led to the Endocrine Society issuing a consensus statement recommending avoidance of immunoassays for steroid hormone assays (Handelsman & Wartofsky, 2013). Furthermore, the inter-laboratory variability of immunoassays has caused significant problems when interpreting epidemiologic studies (Shackleton, 2010).
Over the decade, significant advances in liquid chromatography coupled with multiple reaction monitoring-mass spectrometry (LC–MRM/MS) have provided a solution to these problems. LC–MS can overcome potential problems of cross-reactivity that usually occur in immunoassay-based methodology (Keevil, 2016; Stanczyk & Clarke, 2010). Outstandingly, LC–MS methods make it possible to quantify multiple estrogens in a single analytical run, which allows for more comprehensive analyses to be conducted (Fuhrman, Xu, Falk, Dallal, & Veenstra, 2014; Wang, Rangiah, Mesaros, Snyder, & Vachani, 2015). When studying a family of related estrogens they may fragment to give the same ion, thus, spiking in isotope-labeled internal standards (INSTDs) at the beginning of sample preparation as well as improved efficient chromatographic separation of the isomers is important to avoid isobaric interference and ensure accurate quantification(Blair, 2010; Wang, Mesaros, & Blair, 2016). However, it is still challenging to quantify circulating estrogens and their metabolites where ultra-high sensitivity is required, such as serum from older men, children, postmenopausal women, and women receiving aromatase inhibitors for breast cancer treatment. Many approaches to enhance the sensitivity of estrogen quantification through derivatization have been reported (Mesaros, Wang, &Blair, 2014). The first approach, which we have pioneered (Singh, Gutierrez, Xu, & Blair, 2000), involved the preparation of an electron-capturing pentafluorobenzyl (PFB) derivative of the estrogen-3-hydroxyl groups coupled with the use of electron-capture atmospheric pressure chemical ionization (ECAPCI)-MS. This derivatization approach makes the quantification of estrogens in the low to pg/mL range in plasma samples possible. The second approach involved the use of estrogen derivatives that enhance the electrospray ionization (ESI) signal and therefore improve overall sensitivity during LC-ESI/MS analysis. This approach was exemplified by the derivatization of estrogen phenolic moiety to a dansyl ester (Xu et al., 2007). The third LC–MS approach involved the preparation of pre-ionized (quaternized) derivatives so that protonation of the estrogen derivative is not required (Wang et al., 2016). Therefore, the suppression of ionization in the ESI source of the mass spectrometer is minimized. In Section 4, we elaborate on this approach using Girard P (GP) reagent for the analysis of estrone (E1) and its metabolites in serum samples from postmenopausal women by an LC–MRM/MS method (Rangiah, Shah, Vachani, Ciccimaro, & Blair, 2011). Furthermore, a novel pre-ionized derivatization procedure to detect estradiol (E2) and its metabolites as preionized N-methyl pyridinium-3-sulfonyl (NMPS) derivatives is described as well (Wang et al., 2015). The LLOQ of 1.0 fg on column with 1 μL injection volume makes it very powerful to absolutely quantify estrogens and their metabolites in serum samples from postmenopausal women and older men. We anticipate that the use of pre-ionized estrogen derivatives will help conserve important plasma and serum samples, as it will be possible to conduct high-sensitivity analysis using sample volumes.
Stable isotope dilution (SID) methodology coupled with liquid chromatography-tandem mass spectrometry (LC-MS) is rapidly becoming the gold standard for measuring estrogens in serum and plasma due to improved specificity, high accuracy, and the ability to conduct a more comprehensive analysis. A general consideration of the problems associated with measuring estrogens and two detailed derivatization methods are described in this chapter. These methods quantify estrogens and their metabolites in serum and plasma samples using this state-of-the-art technology, which is applicable to the routine clinical laboratory.
1. Introduction
Estrogen measurement plays an important role in the clinical evaluation of many endocrine disorders as well as in research on the role of hormones in human biology and disease. The predominant methodologies used to quantify circulating levels of estrogens were conventional radioimmunoassays (RIAs) or direct enzyme immunoassays (EIAs) (Blair, 2010; Stanczyk, Jurow, & Hsing, 2010). Mainly these techniques were chosen due to their low cost and routine nature. These assays afford good sensitivity but often lack specificity due to the cross-reaction of antibodies used in the assays with other steroids or unknown matrix components (Denver, Khan, Homer, MacLean, & Andrew, 2019; Ketha, Girtman, & Singh,2015). This problem is particularly marked when measuring lower levels. This led to the Endocrine Society issuing a consensus statement recommending avoidance of immunoassays for steroid hormone assays (Handelsman & Wartofsky, 2013). Furthermore, the inter-laboratory variability of immunoassays has caused significant problems when interpreting epidemiologic studies (Shackleton, 2010).
Over the decade, significant advances in liquid chromatography coupled with multiple reaction monitoring-mass spectrometry (LC–MRM/MS) have provided a solution to these problems. LC–MS can overcome potential problems of cross-reactivity that usually occur in immunoassay-based methodology (Keevil, 2016; Stanczyk & Clarke, 2010). Outstandingly, LC–MS methods make it possible to quantify multiple estrogens in a single analytical run, which allows for more comprehensive analyses to be conducted (Fuhrman, Xu, Falk, Dallal, & Veenstra, 2014; Wang, Rangiah, Mesaros, Snyder, & Vachani, 2015). When studying a family of related estrogens they may fragment to give the same ion, thus, spiking in isotope-labeled internal standards (INSTDs) at the beginning of sample preparation as well as improved efficient chromatographic separation of the isomers is important to avoid isobaric interference and ensure accurate quantification(Blair, 2010; Wang, Mesaros, & Blair, 2016). However, it is still challenging to quantify circulating estrogens and their metabolites where ultra-high sensitivity is required, such as serum from older men, children, postmenopausal women, and women receiving aromatase inhibitors for breast cancer treatment. Many approaches to enhance the sensitivity of estrogen quantification through derivatization have been reported (Mesaros, Wang, &Blair, 2014). The first approach, which we have pioneered (Singh, Gutierrez, Xu, & Blair, 2000), involved the preparation of an electron-capturing pentafluorobenzyl (PFB) derivative of the estrogen-3-hydroxyl groups coupled with the use of electron-capture atmospheric pressure chemical ionization (ECAPCI)-MS. This derivatization approach makes the quantification of estrogens in the low to pg/mL range in plasma samples possible. The second approach involved the use of estrogen derivatives that enhance the electrospray ionization (ESI) signal and therefore improve overall sensitivity during LC-ESI/MS analysis. This approach was exemplified by the derivatization of estrogen phenolic moiety to a dansyl ester (Xu et al., 2007). The third LC–MS approach involved the preparation of pre-ionized (quaternized) derivatives so that protonation of the estrogen derivative is not required (Wang et al., 2016). Therefore, the suppression of ionization in the ESI source of the mass spectrometer is minimized. In Section 4, we elaborate on this approach using Girard P (GP) reagent for the analysis of estrone (E1) and its metabolites in serum samples from postmenopausal women by an LC–MRM/MS method (Rangiah, Shah, Vachani, Ciccimaro, & Blair, 2011). Furthermore, a novel pre-ionized derivatization procedure to detect estradiol (E2) and its metabolites as preionized N-methyl pyridinium-3-sulfonyl (NMPS) derivatives is described as well (Wang et al., 2015). The LLOQ of 1.0 fg on column with 1 μL injection volume makes it very powerful to absolutely quantify estrogens and their metabolites in serum samples from postmenopausal women and older men. We anticipate that the use of pre-ionized estrogen derivatives will help conserve important plasma and serum samples, as it will be possible to conduct high-sensitivity analysis using sample volumes.
