Abstract

Abstract

In this technical note, a solid-phase extraction (SPE) procedure and a robust, sensitive C-MS/MS method using the SCIEX QTRAP 6500+ system enabled the accurate quantitation of 9 steroid hormones in human serum. Sub-nmol/L level sensitivity was achieved with excellent precision (ranging from 1.9 to 8.6%) and accuracy (ranging from 92.0 to 115%) at the lowest calibrator, demonstrating the quantitative performance of the assay. The signal-to-noise ratio (S/N) ranged from 14:1 to 900:1 for the panel of 9 steroid hormones at the lowest matrix calibrator measured. In addition, excellent linearity was observed across clinically relevant concentrations, with r² values above 0.99 across the calibration ranges for all the analytes.

Key-features

Key benefits of steroid hormone analysis in human serum using the QTRAP 6500+ system

• Efficient sample preparation: Steroid hormones were extracted from human serum samples using an efficient solid-phase extraction (SPE) procedure, requiring 100 μL of human serum
• Low-nmol/L level sensitivity and excellent quantitative performance: Sensitive quantitation of steroid hormones was performed with excellent precision and accuracy (6.3% and 104.2% for corticosterone, 5.0% and 115% for cortisol, 2.1% and 95.1% for androstenedione, 3.4% and 94.9% for progesterone, 5.8% and 94.8% for 11-deoxycortisol, 5.2% and 93.6% for 21-deoxycortisol, 1.9% and 93.7% for 17-OH progesterone, 4.8% and 93.1% for testosterone and 8.6% and 89.3% for DHEAS)
• Excellent linearity: Calibration curves for the 9 steroid hormones showed r² values above 0.99 across the calibration ranges for all the analytes.

Figure 1. Chromatograms of the 9 steroid hormones at the lowest calibrator level extracted from serum matrix and analyzed using the QTRAP 6500+ system. Steroids analyzed in A) positive and B) negative mode using rapid (10 ms) polarity switching from a single injection, where two MRM transitions were monitored per compound with >10 points across the peaks for all compounds.

Introduction

Introduction

Steroid hormones are essential regulators of key physiological functions such as metabolism, immune activity, and muscle maintenance. Given their structural diversity and the presence of numerous synthetic analogues, accurate quantitation of steroid profiles in biological samples is crucial. Reliable measurement supports investigations into hormone balance, metabolic function, and overall human health, providing valuable insights for clinical research applications.

Methods

Methods

Sample preparation: Sample preparation was performed using Diagnotix’s steroid panel basic reagent set (https://www.diagnotix.com/nl/lcms-uhplc/Steroids) according to the manufacturer’s specifications. 100 μL calibrators in serum matrix were used to perform the SPE procedure. This reagent set is only available in certain EU countries.

Liquid chromatography conditions: Chromatographic separation was achieved using a Phenomenex Kinetex Biphenyl column (150 x 2.1 mm, 2.6 µm, 00F-4622-AN). Mobile phases A and B from the reagent set were used. The total run time was 10 minutes at a flow rate of 800 μL/min. The injection volume was 20 μL. The LC gradient program is presented in Table 1.

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Mass spectrometry conditions: Data was collected using a QTRAP 6500+ system with an IonDrive Turbo V source and operated in both positive and negative electrospray mode using rapid (10 ms) polarity switching. The scheduled MRM algorithm was used in SCIEX OS software (version 3.1.6) to collect 10-12 data points for quantifiable data. Source and gas conditions are presented in Table 2. Compound-dependent parameters were optimized by infusion.

Table 2: Source and gas parameters for the analysis of steroid hormones in human serum using the QTRAP 6500+ system.

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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, assay precision, and accuracy statistics were automatically generated.

Results

Results and discussion

Figure 1 shows the chromatographic separation of the panel of 9 steroid hormones at the lowest calibrator level. The 10 min gradient, in combination with the column selection and mobile phase composition, resulted in separation of the panel of steroid hormones. The extracted ion chromatograms showed (S/N) values of 14:1 for corticosterone at 0.2 nmol/L, 254:1 for cortisol at 8.3 nmol/L, 58:1 for androstenedione at 0.5 nmol/L, 72:1 for progesterone at 0.4 nmol/L, 43:1 for 11-deoxycortisol at 0.4 nmol/L, 17:1 for 21-deoxycortisol at 0.3 nmol/L, 30:1 for 17-OH progesterone at 0.3 nmol/L, 51:1 for testosterone at 0.9 nmol/L and 900:1 for DHEAS at 0.2 nmol/L, 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 the various concentration levels and injected in triplicate. Linearity, precision and accuracy were assessed across the calibration ranges for each of the 9 analytes. Figure 2 shows the representative extracted ion chromatograms (XICs) for A) testosterone and B) DHEAS their respective concentration ranges (0.3-60.9 nmol/L for testosterone and 0.2-12.2 nmol/L for DHEAS). Figure 3 shows the calibration curves for the 9 steroid hormones over the analytes’ respective calibration ranges. The plots show excellent linear responses across the calibration series, with r² values greater than 0.99 for all the analytes.

Figure 2. Extracted ion chromatograms (XICs) for testosterone and DHEAS. The XICs show the signal for A) testosterone and B) DHEAS across their respective concentration ranges (0.3-60.9 nmol/L for testosterone and 0.2-12.2 nmol/L for DHEAS).

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The precision and accuracy values were calculated from 3 replicates of the lowest matrix calibrators analyzed. The precision (%CV) and % accuracy were 6.3% and 104.2% for corticosterone at 0.2 nmol/L, 5.0% and 115% for cortisol at 8.3 nmol/L, 2.1% and 95.1% for androstenedione at 0.5 nmol/L, 3.4% and 94.9% for progesterone at 0.4 nmol/L, 5.8% and 94.8% for 11-deoxycortisol at 0.4 nmol/L, 5.2% and 93.6% for 21-deoxycortisol at 0.3 nmol/L, 71.9% and 93.7% for 17-OH progesterone at 0.3 nmol/L, 4.8% and 93.1% for testosterone at 0.9 nmol/L and 8.6% and 89.3% for DHEAS at 0.2 nmol/L.

Figure 3. Linear calibration curves for the 9 steroids analyzed in positive and negative modes extracted from serum matrix. The calibration curves were run in triplicate across the measured ranges. The curves were generated using a linear regression and 1/x² weighting, resulting in r² values of >0.99 for the 9 steroid hormones.

Conclusion

Conclusion

A fast and sensitive LC-MS/MS method for the detection of 9 steroid hormones extracted from human serum samples was developed using the SCIEX QTRAP 6500+ system. The method demonstrated:

• Efficient sample preparation which consisted of a solid-phase extraction, requiring 100 μL of human serum sample
• Excellent linear responses across the calibration series consisting of 6 calibrators, with r² values greater than 0.99 across the calibration ranges for all the analytes
• Good sensitivity resulting in S/N ranging from 14:1 to 900:1 at the lowest matrix calibrator measured
• High quantitation performance of the method, resulting in excellent precision from 2.1% to 8.6% and accuracy from 89.3% to 115% across the steroid hormones

References