Measuring estrogens at very low concentrations has long been a challenge in clinical and research laboratories. Traditional immunoassays often lack the necessary specificity, whereas conventional liquid chromatography-tandem mass spectrometry (LC–MS/MS) methods have historically required tedious derivatization to achieve the desired sensitivity. Now, an optimized workflow, developed by Marin and her team, demonstrates that picogram-level quantitation of estrone (E1) and estradiol (E2) can be achieved without derivatization, providing a more streamlined and consistent analytical approach.
“A single LC–MS/MS method that can quantitate estrogens for the majority of samples can allow labs to use a single platform for estrogen quantitation,” explains Marin, who specializes in analytical method development. She adds that “improved selectivity, sensitivity, and precision afforded by LC–MS/MS, without derivatization, provides a more consistent, cohesive workflow for clinical research samples.”
Optimizing the MS: Mobile Phase Chemistry and High-Sensitivity Hardware
Historically, achieving the necessary low limits of quantitation (LOQs) for E1 and E2 required derivatization to enhance ionization efficiency. Marin and her team optimized multiple aspects of the LC–MS/MS workflow to enable detection down to approximately 5 pg/mL for E1 and 10 pg/mL for E2.
Working in analytical science?
Register for a FREE Separation Science account to subscribe to the Separation Science Newsletter.
Marin notes that achieving this level of detection required a concerted effort: “Multiple components of this workflow are required together to yield the pg/mL detection of E1 and E2.” Specifically, she highlights that a more sensitive triple quadrupole mass spectrometer, the ammonium fluoride mobile phase modifier, and clean extracts using a specialized sample preparation solution were all necessary to achieve the LOQs achieved in this method.
Ammonium fluoride, Marin explains, plays a crucial role in boosting the MS signal: “Ammonium fluoride facilitates the deprotonation of molecules like estrogens and promotes the formation of
[M−H]− ions, resulting in a stronger MS signal.” The additive also helps “suppress matrix effects and improve selectivity, reducing interferences from other structurally similar compounds.”
Selective Separation: Biphenyl Chemistry Meets Streamlined SLE
The chromatographic separation was achieved using a biphenyl-type stationary phase, which provided superior resolution of E1 and E2 compared with conventional C18 or pentafluorophenyl (F5) phases.
“Estrogens are some of the only hormones with an aromatic ring,” Marin notes. She explains that this is key to the improved separation: “The π−π interactions between E1, E2, and the biphenyl stationary phase provide the best retention and resolution between the structurally similar E1 and E2.”
Sample cleanliness also proved critical. The team selected supported liquid extraction (SLE) as the sample preparation technique. Marin points out that “SLE provides cleaner samples and removes endogenous matrix interferences like phospholipids,” and compared to simpler methods, “the cleaner extracted samples provide increased sensitivity for steroid analysis when compared to protein precipitation.” Furthermore, she notes that SLE offers “a protocol that has fewer steps and requires less method development than solid phase extraction (SPE).”
Ensuring Analytical Rigor: LOQs and Selectivity Guardrails
“We determined our LOQs by looking at the signal-to-noise ratio and observing linearity and accuracy at the lower concentrations”, states Marin. These detection limits provide adequate quantitation for most population ranges reported in the clinical literature, which she cites as “40−200 pg/mL for E1 and 40−350 pg/mL for E2,” concluding, “this method provides adequate quantitation for the majority of the population from underivatized samples.”
To maintain selectivity and guard against potential interferences, the team monitored ion ratios. “Ion ratios >20% would be a possible indication of a near-eluting interfering species,” explains Marin. They also included isotopically labeled internal standards, which “should compensate for inaccuracies in quantitation caused by endogenous interfering compounds.”
Bench-to-Bench: Key Steps for Successful Method Adoption
For laboratories considering adoption, Marin emphasizes that “the most important aspect is performing the SLE extraction using the sample preparation solution correctly.” She highlights a specific, critical step: “The five-minute wait time after the sample has been applied to the sorbent is crucial for proper extraction.”
“In addition, using two aliquots of elution solvent, as opposed to a larger, single fraction, generally provides better recovery and improved precision of extracted samples,” Marin advises.
Throughput will depend on factors such as automation, instrument multiplexing, and evaporation efficiency. Marin clarifies that she “can't give a specific throughput ” but notes that “bottlenecks usually occur during sample evaporation, actual LC-MS/MS analysis, and data review.” To reduce carryover, she recommends several steps:
- Prevent pipette tips from touching the sides of plates or vials.
- Ensure no sample spattering occurs during loading, elution, or evaporation.
- Utilize a strong autosampler wash solvent.
Implementing these practices can significantly improve the accuracy and reliability of your results.
Expanding Applicability
While this method provides excellent coverage of typical adult hormone ranges, Marin notes that derivatization may still be necessary for extremely low concentrations, such as those encountered in pediatric or postmenopausal studies. For such cases, she suggests the path to pivot is straightforward: “Most methods use post-extraction, pre-LC-MS/MS derivatization with something like dansyl chloride or 2-dimethylimidazole-5-sulfonyl chloride to increase detector response for E1 and E2 for quantitation below 10 pg/mL.”
Ultimately, this work highlights the ongoing development of LC–MS/MS as a quantitative tool for hormone analysis. By refining mobile-phase chemistry, chromatographic selectivity, and sample cleanup, laboratories can achieve robust, high-sensitivity estrogen quantification without the added complexity of derivatization—a practical advance that simplifies workflows while maintaining analytical rigor.
