Sensitive quantitation of estrogens in human serum
Abstract
In this technical note, the combination of a rapid sample preparation procedure consisting of a liquid-liquid extraction with a robust and sensitive LC-MS/MS method using the SCIEX QTRAP 6500+ system enabled sensitive quantitation of estrogens extracted from human serum. Excellent linearity was observed across the calibration series (1-1000 pg/mL), with r2 values greater than 0.99 for all three estrogens. Low-level sensitivity was achieved at the lowest calibrator (1 pg/mL) with signal-to-noise ratios (S/N) of 55:1 for estrone [E1}, 4:1 for estradiol [E2], and 10:1 for estriol [E3]. In addition, the method showed excellent precision (%CV) and % accuracy (4.6% and 98.5% for estrone [E1], 9.2% and 102.9% for estradiol [E2], 18.4% and 104.0% for estriol [E3]] at the lowest calibrator (1 pg/mL), demonstrating the quantitative performance of the assay.
Key benefits of estrogens analysis from human serum using the QTRAP 6500+ system
- Rapid sample preparation: Estrogens were extracted from human serum samples using a liquid-liquid extraction (LLE), evaporation, and reconstitution
- Chromatographic separation: Optimized LC conditions enabled baseline separation of the estrogens
- Low pg/mL level sensitivity and excellent quantitative performance: Sensitive quantitation of estrogens was performed with excellent precision (4.6%, 9.2%, and 18.4% for Estrone [E1], Estradiol [E2], and Estriol [E3]) at the lowest calibrator (1 pg/mL)
- Excellent linearity: Calibration curves for the estrogens showed r2 values above 0.99 for all three estrogens across the calibration series (1-1000 pg/mL)
Introduction
Estrogens, a group of steroid hormones including estradiol, estrone and estriol, are central to the regulation of reproductive function, secondary sexual characteristics and bone health. These hormones are synthesized primarily in the ovaries and circulate throughout the body, exerting effects on multiple tissues. Due to their potent biological activity and structural similarity to other endogenous and synthetic compounds, accurate quantification of estrogens in biological matrices is essential in research settings.
Methods
Sample preparation: Estrogens were extracted from charcoal-stripped serum using a liquid-liquid extraction (LLE), evaporation, and reconstitution.
Liquid chromatography: Chromatographic separation was achieved using a Phenomenex Kinetex C8 column (50 x 2.1 mm, 2.6 µm, 00B-4497-AN). Mobile phase A was ammonium fluoride in water and mobile phase B was ammonium fluoride in methanol. The total run time was 12 minutes.
Mass spectrometry: Data was collected using a QTRAP 6500+ system with an IonDrive Turbo V ion source and operated in electrospray ionization (ESI) negative mode. The Scheduled MRM algorithm was used in SCIEX OS software (version 3.1.6) to collect 10-12 data points for quantifiable data. Compound-dependent parameters were optimized by infusion.
Data processing: Data processing was performed using SCIEX OS software (version 3.1.6). Peak integration was achieved using the MQ4 algorithm. Quantitative analysis was conducted in the Analytics module of SCIEX OS, where calibration curves, concentration calculations, and assay precision statistics were automatically generated.
Results and discussion
Figure 1 shows the chromatographic separation of estrogens into a control, charcoal-stripped serum sample at final concentrations of 1 pg/mL. The 12 min gradient, in combination with the column selection and mobile phase composition, resulted in baseline separation of the estrogens. The extracted ion chromatograms showed S/N of 55:1, 4:1, and 10:1 for Estrone [E1], Estradiol [E2], and Estriol [E3], respectively, at the lowest matrix calibrator measured, calculated using the peak-to-peak algorithm in SCIEX OS.
The quantitative performance of the method was investigated by injecting a series of calibrator samples spiked at concentrations from 1-1000 pg/mL for Estrone [E1}, Estradiol [E2], and Estriol [E3], respectively. Linearity, precision and accuracy were assessed across the calibration range (1-1000 pg/mL). Figure 2 shows the calibration curves for estrogens over the calibration range. The plots show excellent linear responses across the calibration series, with r2 values greater than 0.99 for all the analytes.
The precision and accuracy values were calculated from 3 replicate injections of the lowest matrix calibrators analyzed (1 pg/mL). The precision (%CV) and % accuracy were 4.6% and 98.5% for estrone [E1}, 9.2% and 102.9% for estradiol [E2], 18.4% and 104.0% for estriol [E3] at the lowest calibrator (1 pg/mL).
Conclusion
A fast and sensitive LC-MS/MS method for the detection of estrogens extracted from human serum samples was
developed. The method demonstrated:
- Fast sample preparation which consisted of a liquid-liquid extraction
- Excellent linear responses across the calibration series, with r2 values greater than 0.99 for all analytes
- Good sensitivity resulting in S/N of 55:1 for estrone [E1}, 4:1 for estradiol [E2], and 10:1 for estriol [E3] at the lowest
calibrator (1 pg/mL) - High quantitative performance of the method, resulting in excellent precision and accuracy at the lowest calibrator (1 pg/mL)