Utilizing the New Xevo™ TQ Absolute XR Mass Spectrometer for the LC-MS/MS Analysis of Serum Estrogens for Clinical Research

Significance of the topic

Accurate measurement of circulating estrogens 17β-estradiol (E2) and estrone (E1) at low concentrations is critical for clinical research in endocrinology, oncology, reproductive medicine and population studies. Conventional immunoassays often lack the required sensitivity and specificity at low pg/mL levels and published LC-MS/MS protocols frequently rely on large sample volumes, extensive extraction or chemical derivatization. Demonstrating a robust, high-sensitivity LC-MS/MS workflow that uses small sample volumes and minimal sample handling addresses a persistent analytical bottleneck and supports broader adoption of mass-spectrometry-based steroid testing in research and specialized clinical settings.

Objectives and study overview

• Evaluate the analytical sensitivity, precision, accuracy and robustness of the Xevo TQ Absolute XR triple quadrupole mass spectrometer for quantifying serum E2 and E1 in a clinical research context.
• Demonstrate a simplified liquid–liquid extraction (LLE) sample preparation compatible with UHPLC separation that avoids derivatization and uses only 250 µL of sample.
• Compare method performance against an external quality assessment (UK NEQAS) and report limits of quantification, linearity and matrix impact.

Methodology

Calibrators and QC: Certified reference standards for E2 and E1 and corresponding stable isotope-labeled internal standards were used. Calibrators were prepared in stripped human serum (surrogate matrix). Calibration ranges were 2–1000 pg/mL for E2 and 1–1000 pg/mL for E1. QC levels were prepared at 3, 10 and 700 pg/mL.

Sample preparation: To 250 µL of serum, 20 µL of internal standard solution was added. LLE was performed using 1 mL of hexane:ethyl acetate (85:15, v:v) with 10 minutes mixing, centrifugation and transfer of 700 µL of the organic layer. Extracts were evaporated to dryness, reconstituted in 20 µL methanol + 30 µL water and mixed prior to injection. The approach intentionally avoids derivatization.

Chromatography and MS settings: UHPLC separation was achieved on a phenyl stationary phase (Waters CORTECS Phenyl column) maintained at 50 °C using a water/methanol gradient containing ammonium fluoride as additive. Total chromatographic run time was ~4.75 minutes with an injection-to-injection cycle ≈5.75 minutes. Detection used multiple reaction monitoring (MRM) in electrospray negative ionization with a capillary voltage of 2.2 kV and unit-resolution settings (0.7 FWHM) for both MS1 and MS2 transitions.

Used instrumentation

• Xevo TQ Absolute XR triple quadrupole mass spectrometer (Waters) with StepWave ion guide and eXtended Dynamic Range (XDR) capability.
• ACQUITY UPLC I-Class FL System (Waters) for UHPLC separations.
• CORTECS Phenyl LC column (Waters) operated at 50 °C.
• Reagents and consumables included certified reference standards, deuterated/stable-isotope internal standards, hexane, ethyl acetate, methanol, ammonium fluoride and stripped human serum (surrogate matrix).

Main results and discussion

• Linearity and calibration: Calibration curves were linear across the stated ranges with correlation coefficients r2 > 0.999 for both E2 and E1 across multiple runs.
• Precision: Within-run and total precision for low, mid and high QC extracts were ≤3.2% CV across five occasions and five replicates per level, demonstrating excellent repeatability and reproducibility.
• Analytical sensitivity and LLOQ: For E1, acceptable performance (%CV ≤20, bias ≤15%, S/N >10:1) was achieved at 1.0 pg/mL (3.7 pmol/L). For E2, although %CV and bias criteria were met at 1.0 pg/mL, the peak-to-peak S/N ≥10:1 criterion was not consistently reached, so the practical LLOQ was set to 3.0 pg/mL (11.1 pmol/L). Despite that, blank stripped serum was distinguishable from 1 pg/mL E2 spikes when using the 250 µL sample volume.
• Matrix effects: Evaluation using sera from six individual donors showed some ion suppression/enhancement when comparing peak areas, but isotope-labeled internal standards effectively compensated for these matrix effects, producing reliable calculated concentrations.
• Accuracy vs EQA: Comparison of 39 UK NEQAS E2 samples showed close agreement with the external scheme. Deming regression yielded y = 1.17 + 1.012x and Bland–Altman analysis produced a mean bias of −2.50%, consistent with accurate quantification suitable for research use.
• Throughput and robustness: The short UHPLC cycle (~5.75 min) combined with the StepWave ion guide and XDR functionality supports robust quantitation over a broad dynamic range and improved sample throughput without complex sample chemistry.

Benefits and practical applications

• Low sample volume requirement (250 µL) enables testing in studies with limited sample availability (pediatric, longitudinal cohorts).
• Simplified sample preparation (single-step LLE, no derivatization) reduces hands-on time, lowers reagent use and minimizes potential sources of variability.
• High sensitivity permits quantification of E1 and E2 in low-concentration clinical research settings (postmenopausal, endocrine disruption studies) with good precision and accuracy.
• Good agreement with an external quality assessment program supports method credibility for research laboratories and can facilitate adoption in specialized clinical testing environments.

Future trends and potential applications

• Extension to expanded steroid panels: The demonstrated sensitivity and robustness make the platform suitable for multiplexed steroid profiling (androgens, corticosteroids) from the same small-volume extracts.
• Automation and high-throughput workflows: Integrating automated LLE or online extraction could further increase throughput and consistency for large cohort studies.
• Standardization and clinical translation: Continued participation in EQA schemes and inter-laboratory comparisons can help drive method harmonization and potential uptake into regulated clinical assays where appropriate.
• Ultra-trace and special-population studies: Enhanced sensitivity supports research in pediatric endocrinology, environmental exposure assessment and studies requiring detection of very low hormone concentrations.
• Isotope-dilution and reference-material based approaches combined with robust triple-quadrupole platforms will underpin future efforts toward traceable, quantitative steroidomics.

Conclusion

The Xevo TQ Absolute XR mass spectrometer combined with a short UHPLC separation and a straightforward LLE protocol provides a sensitive, precise and accurate method for quantifying serum E2 and E1 for clinical research. Key advantages are low sample volume, avoidance of derivatization, strong precision (≤3.2% CV), linearity across wide concentration ranges (r2 > 0.999) and practical LLOQs of 3.0 pg/mL for E2 and 1.0 pg/mL for E1 under the stated acceptance criteria. Matrix effects are manageable with isotope-labeled internal standards and method results compare well with an independent EQA scheme.

References

• Farrell D., Hammond G., Wardle R. Utilizing the New Xevo TQ Absolute XR Mass Spectrometer for the LC-MS/MS Analysis of Serum Estrogens for Clinical Research. Waters Corporation. Application Brief. Published May 20, 2026.