Ultra-high sensitivity analysis of estrogens for special populations in serum and plasma by liquid chromatography–mass spectrometry: Assay considerations and suggested practices

Abstract

Estrogen measurements play 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. It remains an analytical challenge to quantify estrogens and their metabolites in specimens from special populations including older men, children, postmenopausal women and women receiving aromatase inhibitors. Historically, immunoassays have been used for measuring estrogens and their metabolites in biological samples for risk assessment. However, the lack of specificity and accuracy of immunoassay-based methods has caused significant problems when interpreting data generated from epidemiological studies and across different laboratories. Stable isotope dilution (SID) methodology coupled with liquid chromatography-selected reaction monitoring-mass spectrometry (LC-SRM/MS) is now accepted as the ‘gold-standard’ to quantify estrogens and their metabolites in serum and plasma due to improved specificity, high accuracy, and the ability to monitor multiple estrogens when compared with immunoassays. Ultra-high sensitivity can be obtained with pre-ionized derivatives when using triple quadruple mass spectrometers in the selected reaction monitoring (SRM) mode coupled with nanoflow LC. In this review, we have examined the special issues related to utilizing ultra-high sensitivity SID LC-SRM/MS-based methodology to accurately quantify estrogens and their metabolites in the serum and plasma from populations with low estrogen levels. The major issues that are discussed include: sample preparation for both unconjugated and conjugated estrogens, derivatization, chromatographic separation, matrix effects, and assay validation.

Vaginal microbiota and genitourinary menopausal symptoms: a cross-sectional analysis

“…An ultrasensitive stable isotope dilution liquid chromatography/selected reaction monitoring/mass spectrometry (LC/SRM/MS) assay was used to measure total and unconjugated estradiol and estrone. 21 The limit of detection for each estrogen using 0.5 mL of serum was 0.156 pg/mL and linear standard curves were obtained up to 20 pg/mL.…”

“…21 We measured both estradiol and estrone, in both conjugated and unconjugated forms, giving a more comprehensive picture of estrogen levels when compared with previous studies. We found that unconjugated serum estrone was associated with a Lactobacillus- dominant microbial community, though not with any particular Lactobacillus species or with quantity of L. crispatus or L. iners.…”

Validation of highly sensitive simultaneous targeted and untargeted analysis of keto-steroids by Girard P derivatization and stable isotope dilution-liquid chromatography-high resolution mass spectrometry

“…The main challenge of steroid analysis in special populations remains sensitivity, to allow quantitation of low abundance hormones within the constraint of feasible biospecimen consumption [22]. Limits of detection at the sub-pg on column range with a sample sparing method using only 100 μL of serum should provide adequate sensitivity for analysis of unconjugated steroids in even special populations with low levels of androgens.…”

Occurrence and reproductive roles of hormones in seminal plasma

“…Eventual binding to transport proteins can be completely eliminated by extraction to an organic solvent. The most common solvents used in LLE are methyl tert -butyl ether, diethyl ether, ethyl acetate, dichloromethane or mixtures of organic solvents [ 88 ]. These might be useful in reducing matrix effects, since ionized compounds, such as salts or phospholipids, do not partition into the organic layer [ 89 ].…”

“…Matrix effects have been considered as the Achilles heel in LC-MS analysis [ 118 ]. To assess the matrix effects, three different strategies currently exist: (1) post-column infusion, (2) post-extraction addition, and (3) a comparison of the slopes of calibration curves [ 88 ]. Validation of analytical methods and evaluations of matrix effects in seminal plasma are more complicated because there is not as much seminal fluid material in comparison with e.g.…”