This is an Application Brief and does not contain a detailed Experimental section.
For research use only. Not for use in diagnostic procedures.
Two of the key biologically active estrogens in non-pregnant humans are 17β-estradiol (E2) and estrone (E1). Most E1 is derived from androstenedione, while E2 is primarily produced in the ovaries and testes through the aromatization of testosterone.
It is best to measure both compounds, because E2 can be metabolized to E1 and E1 can be converted to E2.
The need to measure down to low concentrations in certain clinical research applications is the most significant challenge in analyzing E2 and E1. Some current immunoassay techniques lack analytical sensitivity and, more often, selectivity. Published LC-MS/MS methods employ large sample volumes and require complex sample extraction, typically including derivatization.
Figure 1. ACQUITY™ UPLC™ I-Class FL IVD and Xevo TQ Absolute IVD Mass Spectrometer. Image Credit: Waters Advanced Diagnostics
The Xevo TQ Absolute IVD Mass Spectrometer features StepWave™ XS ion transfer optics and an Xtended Dynamic Range (XDR), offering improved robustness, analytical sensitivity, and reliability over six orders of linear dynamic range.
Using this powerful instrument in conjunction with a simple sample preparation method and UPLC™ estrogen separation yields a highly selective, analytically sensitive method suitable for the analysis of E2 and E1 in clinical research environments.
Experimental
Sample preparation and UPLC-MS/MS analysis
E2 and E1 certified reference solutions were purchased from Merck (Gillingham, UK), along with their stable-labeled internal standards (13C3).
Calibrators were then prepared in a surrogate matrix of MSG4000 stripped serum purchased from Golden West Biologicals (Temecula, CA).
The calibration ranges for E2 and E1 were 2-1000 pg/mL and 1-1000 pg/mL, respectively.
QC samples were also prepared in a surrogate matrix of MSG4000 stripped human serum (purchased from Golden West Biologicals) at a rate of 3.2 pg/mL, 10.5 pg/mL, and 700 pg/mL.
Methanol, water, and ethyl acetate were purchased from Fisher Scientific (Loughborough, UK), while hexane and ammonium fluoride were also purchased from Merck.
Conventional mass units (pg/mL) can be converted to SI units (pmol/L) by multiplying by 3.671 for E2, and by 3.699 for E1.
A total of 20 μL of internal standard was added to 250 μL of sample, and this was mixed. Liquid/liquid extraction was performed by adding one mL of 85:15 (v:v) hexane: ethyl acetate and mixing this thoroughly for a 10-minute period.
Samples were centrifuged at 4000 g for a total of five minutes before transferring 700 μL of the top organic layer into a 96-well plate containing 1 mL glass inserts (Waters p/n: 186000855).
The samples were evaporated to dryness, then reconstituted in 20 μL of methanol and 30 μL of distilled water.
Following this, the samples were injected onto an ACQUITY UPLC I-Class FL System and Xevo TQ Absolute IVD Mass Spectrometer, with a water/ methanol/ ammonium fluoride gradient and a Waters™ CORTECS™ Phenyl Column (Waters p/n: 186008319) (Table 1).
Table 1. Gradient Table for the separation of E2 and E1, having a total run time of 4.75 minutes. Source: Waters Advanced Diagnostics
Table 2 features Multiple Reaction Monitoring (MRM) transitions, quantifier (quan), and qualifier (qual), for the detection of all analytes. These results were achieved using a capillary voltage of 2.2 kV in electrospray negative-ion mode and resolution settings of 0.7 FWHM for MS1 and MS2.
Table 2. MRM parameters of E2 and E1 quantifier ions, qualifier ions, and their internal standards. Source: Waters Advanced Diagnostics
Results and discussion
Chromatographic separation of E2 and E1 was achieved with an injection-to-injection time of around 5.5 minutes. Figure 2 shows a typical chromatogram.
Figure 2. Typical Chromatograms of E2 and E1 extracted from a UK NEQAS sample at 21.0 pg/mL and 34.0 pg/mL, respectively. Image Credit: Waters Advanced Diagnostics
Calibration curves were linear over the described calibration ranges, with correlation coefficients >0.99 and %bias within ±15 % (±20 % for Calibrator 1) for both E2 and E1 across eight occasions.
Precision performance was evaluated by extracting and analyzing five replicates of the low, mid, and high QC samples on five occasions. Total and within-run precisions were determined to be ≤5.9 % CV for all steroid hormones for all concentration levels (Table 3).
Table 3. Precision Performance Summary of E2 and E1 Extracted and Analyzed on the ACQUITY UPLC I-Class FL and Xevo TQ Absolute IVD. Source: Waters Advanced Diagnostics
Analytical sensitivity was evaluated by spiking MSG4000 stripped serum from Golden West Biologicals at low concentration levels. Ten replicates of each sample were extracted before being analyzed on each of four occasions.
It was possible to achieve %CVs of ≤20 % and biases of ≤15 % at 1.0 pg/mL for E2 and E1, but it was not possible to achieve a signal-to-noise ratio (peak to peak) of ≥10:1 at this level for E2. An LLoQ of 3 pg/mL was therefore selected for this analyte.
Figure 3 features typical chromatograms of E2 and E1 at the defined concentrations. An LLoQ of 1 pg/mL was not achieved, but it was possible to differentiate a blank-stripped serum sample from the same serum spiked with 1 pg/mL of E2.
Figure 3. Example Chromatograms of a blank and low-level samples in stripped serum for E2. Image Credit: Waters Advanced Diagnostics
Matrix effect investigations were completed using serum from six different individuals. The endogenous peak areas were quantified separately, with post-spiked samples at low- and high-concentration levels adjusted using the mean peak areas to allow comparison with solvent-spiked samples.
Some enhancement and suppression in the matrix factor results were noted when observing the peak areas, but the internal standard successfully compensated for these (Table 4).
Table 4. Matrix Factor Summary for E2 and E1. Source: Waters Advanced Diagnostics
Accuracy was evaluated by analyzing 40 UK NEQAS E2 samples. Calculated concentrations were compared with mass spectrometry laboratory-trimmed means. Table 5 and Figure 4 show the correlation for E2, highlighting excellent agreement with the EQA scheme.
Table 5. Accuracy Summary for E2. Source: Waters Advanced Diagnostics
Figure 4. Deming Fit and Bland Altman Plots for Comparison with the UK NEQAS E2 Scheme. Image Credit: Waters Advanced Diagnostics
Conclusion
The Xevo TQ Absolute IVD showed excellent quantitative performance and analytical sensitivity for the analysis of 17 β-estradiol and estrone in a clinical research setting. A number of performance characteristics were achieved for the method, including:
- Calibration curves with correlation coefficients (r2) of >0.99 for E2 and E1 for all runs.
- Total and within-run precision results of ≤5.9 % CV.
- Analytical sensitivity concentrations of 3.0 pg/mL (11.1 pmol/L) for E2 and 1.0 pg/mL (3.7 pmol/L) for E1.
- A %CV of ≤20 %, bias of ≤15 %, and S: N (ptp) of >10:1.
It was also possible to distinguish a blank stripped serum sample from the same matrix sample spiked with one pg/mL of E2 from just 250 µL of the sample. This was achieved without the need for derivatization.
It was also noted that little to no ion suppression was seen from six individuals when comparing control samples to calculated concentrations.
The method was also shown to be accurate when compared against the UK NEQAS E2 scheme, with a Deming Fit of y = 0.971x + 4.874 and a Bland-Altman Mean Bias of -4.15 %.
About Waters Advanced Diagnostics
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