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Liquid chromatography-mass spectrometry applications for quantification of endogenous sex hormones
Amy Gravitte, BS; Timothy Archibald, PharmD; Benjamin Kennard, BS; Allison Cobble; Stacy Brown, PhD
ABSTRACT
Liquid chromatography, coupled with tandem mass spectrometry, presents a powerful tool for the quantification of the sex steroid hormones 17-β estradiol, progesterone, and testosterone from biological matrices. The importance of accurate quantification with these hormones, even at endogenous levels, has evolved with our understanding of the role these regulators play in human development, fertility, and disease risk and manifestation. Routine monitoring of these analytes can be accomplished by immunoassay techniques, which face limitations on specificity and sensitivity or using gas chromatography-mass spectrometry. LC-MS/MS is growing in capability and acceptance for clinically relevant quantification of sex steroid hormones in biological matrices and is able to overcome many of the limitations of immunoassays. Analyte specificity has improved through the use of novel derivatizing agents, and sensitivity has been refined through the use of high-resolution chromatography and mass spectrometric technology. This review highlights these innovations, among others, in LC-MS/MS steroid hormone analysis captured in the literature over the last decade.
1. Introduction to sex steroid hormone LC-MS
Endogenous sex hormones, such as testosterone and estrogen, play vital roles in the coordination and normal development of both male and female sex characteristics. These hormones are important chemical messengers derived from cholesterol and undergo synthesis following complex metabolic pathways, as shown in Figure 1 (Galligan, 2018). The hormonal milieu of endogenous sex hormones has been thought to play a role in multiple disease processes, such as infertility, osteoporosis, breast cancer, prostate cancer, and endometrial cancer (Hankinson and Tworoger, 2011). Recently, testosterone levels have been linked to increased SARS-CoV-2 transmission in males; therefore, examining these complex hormones has become important in epidemiologic and metabolomic investigations (Montopoli, 2020). Their correct identification is paramount to clinical practice and patient care.
Prior to advancements in high-performance liquid chromatography-mass spectrometry (HPLC-MS), the predominant methods to measure endogenous sex hormones were conventional and/or direct radioimmunoassay (RIAs). However, these aforementioned methods have come with many limitations. For example, conventional RIAs are costly, time-consuming, require large sample volumes, and are subject to antibody cross-reactivity (Stanczyk, 2010). Conversely, direct RIAs often overestimate measurements due to lack of specificity of antibodies, lack reliability/sensitivity to detect low-level steroid hormones such as 17-β estradiol, and direct RIAs can only measure one analyte at a time (Tate and Ward, 2004). The previously described limitations of RIAs have rendered their utility largely to analytes of single steroids or steroid metabolites present in high volumes, rather than steroid profiles or samples of closely related steroids (Abraham, 1975).
2. Testosterone bioanalysis
3. Estradiol bioanalysis
4. Progesterone bioanalysis
5. Matrices and sample preparation
5.1 Testosterone analysis from serum and plasma
5.2 Other matrices used in testosterone quantification
5.3 Highly sensitive assays for determining 17-β estradiol in serum and plasma
5.4 Alternative matrices for 17-β estradiol quantification
5.5 Saliva and other matrices for progesterone quantification
6. The role of derivatization in sex steroid LC-MS
6.1 Estradiol
6.2 Testosterone and progesterone
7. Chromatography considerations for quantification of steroid sex hormones by LCMS
7.1 Column choice and instrumental configuration
7.2 Mobile phase and separation conditions
8. Mass spectrometric considerations
8.1 Testosterone
8.2 Estradiol
8.3 Progesterone
Discussion
Testosterone, estradiol, and progesterone are endogenous sex hormones that are critical for the development and maintenance of male and female sex characteristics. Measuring these hormones accurately and with high sensitivity is necessary to monitor the health of children, pre-and post-menopausal females, males, and animals in veterinary care. Immunoassays have been used to measure endogenous hormones for decades; however, immunoassays can only measure one analyte at a time, and cannot compete with the accuracy and sensitivity of newer methods, most notably, HPLC coupled with tandem mass spectrometry (LC-MS/MS). Mass spectrometry is increasingly being recognized as a “gold standard” tool for the measurement of testosterone, estradiol, and progesterone in biological matrices in the endocrinology community (Conklin, 2020; Rosner, 2007). This technique allows for simultaneous quantification of testosterone, estradiol, and progesterone, as well as other steroid hormones and metabolites. Human serum is the most widely used matrix for the LC-MS/MS quantification of endogenous sex hormones, followed by plasma. In fact, NIST methods for all three hormones utilize serum as the matrix (Tai, 2005; Tai, 2006; Tai, 2007). The most variable aspect of LC-MS/MS among testosterone, estradiol, and progesterone is sample preparation. While testosterone and progesterone run well in positive ion mode following extraction (LLE, SPE), derivatization is often a key step for estradiol analysis. While estradiol can run in negative ion mode underivatized, higher sensitivity achieved in positive mode is predicated upon derivatization for this analyte (Denver, 2019). Furthermore, the need for high sensitivity is most relevant to estradiol, which can be found at clinically relevant low pg/mL levels in some patients. The lipophilicity of these hormones leads them to separation in reversed-phase mode, most often with C18 columns. Other phases, like biphenyl, are growing in popularity.
Similarities in methodological parameters allow for the simultaneous quantification of many steroid hormones beyond the three discussed in this review. The most comprehensive method of steroid hormone quantification by LC-MS/MS included in this review achieved accurate measurement of more than 100 steroids simultaneously, including testosterone, estradiol, and progesterone (Gaikwad, 2013). Several investigators quantified estradiol, testosterone, and progesterone in serum, in addition to up to 17 other analytes (Desai, 2014; Genangeli, 2017; Lee, 2016; Qin, 2020). Similarly, Häkkinen et al quantified over 20 analytes simultaneously from endometrial tissue (Häkkinen, 2018). Urine was also a good matrix to capture simultaneous quantification of estradiol, testosterone, and progesterone, plus many additional analytes (Kaabia, 2018; Son, 2018; Zhou, 2019). The examples of simultaneous steroid hormone measurement mentioned above, as well as the examples of ultra-sensitive single steroid hormone quantification discussed in the main text, contribute to the field’s understanding of best practices for the quantification of testosterone, estradiol, and progesterone. LC-MS/MS will likely be the standard for hormone quantification for the foreseeable future, though researchers will continue to push the limits of accuracy and sensitivity to maximize clinical relevance.
Conclusions
The importance of sensitivity and selectivity for hormone bioanalysis cannot be overstated, especially as our understanding of the roles estradiol, testosterone, and progesterone play in normal development and disease manifestation evolves. One aspect of future development in this field will likely be the growth of multi-component assays that include not only the hormones that are the focus of this review but also their metabolites and synthetic precursors. Such assays produce highly complex data sets, suited to the fields of metabolomics and steroidomics, which are developing at a rapid pace. Moving forward, the field of steroid bioanalysis is also likely to see increased respect for standardization for routine clinical analysis. The issue of clinical analysis of hormones was significant enough to attract the attention of the CDC, who since 2007 has been working toward more standardization in hormone quantification through the HoSt program. Many of the assays cited in this review springboard from the CDC work in this area, utilizing reference materials and methodology developed and validated by CDC scientists. For both future directions, continued improvements in sensitivity needed to envelop all patient groups will hinge on advancements in and optimum utilization of LC-MS sample preparation, chromatography, and instrumentation.
Amy Gravitte, BS; Timothy Archibald, PharmD; Benjamin Kennard, BS; Allison Cobble; Stacy Brown, PhD
ABSTRACT
Liquid chromatography, coupled with tandem mass spectrometry, presents a powerful tool for the quantification of the sex steroid hormones 17-β estradiol, progesterone, and testosterone from biological matrices. The importance of accurate quantification with these hormones, even at endogenous levels, has evolved with our understanding of the role these regulators play in human development, fertility, and disease risk and manifestation. Routine monitoring of these analytes can be accomplished by immunoassay techniques, which face limitations on specificity and sensitivity or using gas chromatography-mass spectrometry. LC-MS/MS is growing in capability and acceptance for clinically relevant quantification of sex steroid hormones in biological matrices and is able to overcome many of the limitations of immunoassays. Analyte specificity has improved through the use of novel derivatizing agents, and sensitivity has been refined through the use of high-resolution chromatography and mass spectrometric technology. This review highlights these innovations, among others, in LC-MS/MS steroid hormone analysis captured in the literature over the last decade.
1. Introduction to sex steroid hormone LC-MS
Endogenous sex hormones, such as testosterone and estrogen, play vital roles in the coordination and normal development of both male and female sex characteristics. These hormones are important chemical messengers derived from cholesterol and undergo synthesis following complex metabolic pathways, as shown in Figure 1 (Galligan, 2018). The hormonal milieu of endogenous sex hormones has been thought to play a role in multiple disease processes, such as infertility, osteoporosis, breast cancer, prostate cancer, and endometrial cancer (Hankinson and Tworoger, 2011). Recently, testosterone levels have been linked to increased SARS-CoV-2 transmission in males; therefore, examining these complex hormones has become important in epidemiologic and metabolomic investigations (Montopoli, 2020). Their correct identification is paramount to clinical practice and patient care.
Prior to advancements in high-performance liquid chromatography-mass spectrometry (HPLC-MS), the predominant methods to measure endogenous sex hormones were conventional and/or direct radioimmunoassay (RIAs). However, these aforementioned methods have come with many limitations. For example, conventional RIAs are costly, time-consuming, require large sample volumes, and are subject to antibody cross-reactivity (Stanczyk, 2010). Conversely, direct RIAs often overestimate measurements due to lack of specificity of antibodies, lack reliability/sensitivity to detect low-level steroid hormones such as 17-β estradiol, and direct RIAs can only measure one analyte at a time (Tate and Ward, 2004). The previously described limitations of RIAs have rendered their utility largely to analytes of single steroids or steroid metabolites present in high volumes, rather than steroid profiles or samples of closely related steroids (Abraham, 1975).
2. Testosterone bioanalysis
3. Estradiol bioanalysis
4. Progesterone bioanalysis
5. Matrices and sample preparation
5.1 Testosterone analysis from serum and plasma
5.2 Other matrices used in testosterone quantification
5.3 Highly sensitive assays for determining 17-β estradiol in serum and plasma
5.4 Alternative matrices for 17-β estradiol quantification
5.5 Saliva and other matrices for progesterone quantification
6. The role of derivatization in sex steroid LC-MS
6.1 Estradiol
6.2 Testosterone and progesterone
7. Chromatography considerations for quantification of steroid sex hormones by LCMS
7.1 Column choice and instrumental configuration
7.2 Mobile phase and separation conditions
8. Mass spectrometric considerations
8.1 Testosterone
8.2 Estradiol
8.3 Progesterone
Discussion
Testosterone, estradiol, and progesterone are endogenous sex hormones that are critical for the development and maintenance of male and female sex characteristics. Measuring these hormones accurately and with high sensitivity is necessary to monitor the health of children, pre-and post-menopausal females, males, and animals in veterinary care. Immunoassays have been used to measure endogenous hormones for decades; however, immunoassays can only measure one analyte at a time, and cannot compete with the accuracy and sensitivity of newer methods, most notably, HPLC coupled with tandem mass spectrometry (LC-MS/MS). Mass spectrometry is increasingly being recognized as a “gold standard” tool for the measurement of testosterone, estradiol, and progesterone in biological matrices in the endocrinology community (Conklin, 2020; Rosner, 2007). This technique allows for simultaneous quantification of testosterone, estradiol, and progesterone, as well as other steroid hormones and metabolites. Human serum is the most widely used matrix for the LC-MS/MS quantification of endogenous sex hormones, followed by plasma. In fact, NIST methods for all three hormones utilize serum as the matrix (Tai, 2005; Tai, 2006; Tai, 2007). The most variable aspect of LC-MS/MS among testosterone, estradiol, and progesterone is sample preparation. While testosterone and progesterone run well in positive ion mode following extraction (LLE, SPE), derivatization is often a key step for estradiol analysis. While estradiol can run in negative ion mode underivatized, higher sensitivity achieved in positive mode is predicated upon derivatization for this analyte (Denver, 2019). Furthermore, the need for high sensitivity is most relevant to estradiol, which can be found at clinically relevant low pg/mL levels in some patients. The lipophilicity of these hormones leads them to separation in reversed-phase mode, most often with C18 columns. Other phases, like biphenyl, are growing in popularity.
Similarities in methodological parameters allow for the simultaneous quantification of many steroid hormones beyond the three discussed in this review. The most comprehensive method of steroid hormone quantification by LC-MS/MS included in this review achieved accurate measurement of more than 100 steroids simultaneously, including testosterone, estradiol, and progesterone (Gaikwad, 2013). Several investigators quantified estradiol, testosterone, and progesterone in serum, in addition to up to 17 other analytes (Desai, 2014; Genangeli, 2017; Lee, 2016; Qin, 2020). Similarly, Häkkinen et al quantified over 20 analytes simultaneously from endometrial tissue (Häkkinen, 2018). Urine was also a good matrix to capture simultaneous quantification of estradiol, testosterone, and progesterone, plus many additional analytes (Kaabia, 2018; Son, 2018; Zhou, 2019). The examples of simultaneous steroid hormone measurement mentioned above, as well as the examples of ultra-sensitive single steroid hormone quantification discussed in the main text, contribute to the field’s understanding of best practices for the quantification of testosterone, estradiol, and progesterone. LC-MS/MS will likely be the standard for hormone quantification for the foreseeable future, though researchers will continue to push the limits of accuracy and sensitivity to maximize clinical relevance.
Conclusions
The importance of sensitivity and selectivity for hormone bioanalysis cannot be overstated, especially as our understanding of the roles estradiol, testosterone, and progesterone play in normal development and disease manifestation evolves. One aspect of future development in this field will likely be the growth of multi-component assays that include not only the hormones that are the focus of this review but also their metabolites and synthetic precursors. Such assays produce highly complex data sets, suited to the fields of metabolomics and steroidomics, which are developing at a rapid pace. Moving forward, the field of steroid bioanalysis is also likely to see increased respect for standardization for routine clinical analysis. The issue of clinical analysis of hormones was significant enough to attract the attention of the CDC, who since 2007 has been working toward more standardization in hormone quantification through the HoSt program. Many of the assays cited in this review springboard from the CDC work in this area, utilizing reference materials and methodology developed and validated by CDC scientists. For both future directions, continued improvements in sensitivity needed to envelop all patient groups will hinge on advancements in and optimum utilization of LC-MS sample preparation, chromatography, and instrumentation.
