Quantification of Corticosteroids and Androgens in Serum, Utilizing Waters MassTrak™ Steroid Serum Sets 2 and Sets 3

A clinical research method for the quantification of seven endocrine steroid hormones in human serum by UPLC-MS/MS.

Benefits

• Analytical selectivity of the chromatographic clinical research method provides separation of isobaric species
• LC-MS/MS enables high sample-throughput using multi-well plate automation
• Excellent agreement (within ± 6%) to the EQA Mass spectrometer mean and in-house panel mean for testosterone, androstenedione, 17-OHP, DHEAS, cortisol, 11-deoxycortisol, and 21-deoxycortisol
• Lyophilized calibrators and QCs that reduce sample preparation time, aid in clinical research method harmonization, and assist with metrological traceability in accordance with ISO 15189:2022.

Steroid hormones encompass a large class of small molecules that play a central role in metabolic processes, such as the regulation of sexual characteristics, blood pressure, and inflammation. Enzymes that form part of the steroid biosynthetic pathway are pivotal in these metabolic processes, and their dysfunction can be examined through the correct measurement of steroid hormones in a clinical research setting.

Quantification of steroids is more beneficial using LC-MS/MS, as opposed to traditional ligand binding techniques such as immunoassay, as it can provide higher analytical sensitivity and selectivity and allows for simultaneous quantification of multiple analytes. However, many LC-MS methods lack harmonization or standardization. The Waters MassTrak Steroid Serum Sets (IVD) contains a range of steroid hormones in lyophilized serum that are metrologically traceable to the highest level of metrological traceability available, aiding laboratories in their compliance to ISO 15189, and provide confidence in the accuracy of results.

Waters MassTrak Steroid Serum Cal Set 2 is intended for the quantitative determination of androgens and progestogens in human serum to aid with monitoring of physiological markers. Whilst the MassTrak Steroid Serum Cal Set 3 is intended to be used for the quantitative determination of androgens and glucocorticoids in human serum to aid with monitoring of physiological markers.

Here we describe a clinical research method utilizing Waters MassTrak Steroid Serum Sets 2 and 3, Cals and QCs, and Waters Oasis™ PRiME HLB µElution plate technology for the extraction of testosterone, androstenedione, 17-hydroxyprogesterone (17-OHP), dehydroepiandrosterone sulfate (DHEAS), Cortisol, 11-deoxycortisol, and 21-deoxycortisol from serum samples, which has been automated using the Hamilton™ Microlab STAR Liquid Handler. Chromatographic separation was performed on a ACQUITY UPLC™ I-Class Plus (FL-I) System, using a ACQUITY UPLC HSS T3 Vanguard™ Pre-Column, and ACQUITY UPLC HSS T3 Column, accompanied by a Xevo™ TQ-S micro Mass Spectrometer (Figure 1).

Sample Preparation

For this study Waters MassTrak Steroid Serum Cal Set 2 (186010563IVD), MassTrak Steroid Serum QC Set 2 (186010564IVD), MassTrak Steroid Serum Cal Set 3 (186010565IVD), and MassTrak Steroid Serum QC Set 3 (186010566IVD) Calibrators and Quality Controls, alongside MassTrak Steroid Internal Standard Mix (186010567IVD) were prepared as per instructions.

MassTrak Steroid Serum Cal Set 2 and Set 3, QC Set 2 and Set 3 contain the following analytes in a range of concentrations.

Sample Extraction

Prior to extraction it is suggested to centrifuge all samples for 5 mins at 3000 x g. Extraction was performed using the Hamilton Microlab Star Liquid Handler. To 100 µl of sample; 25 µl of MassTrak Steroid Internal Standard Mix was added to each well of sample. (2 ng/mL of testosterone ¹³C₃, 4 ng/mL of androstenedione ¹³C₃, 10 ng/mL of 17OHP ¹³C₃, 3000 ng/mL of DHEAS ²H₆, 100 ng/mL of cortisol ¹³C₃, 4 ng/mL of 11-deoxycortisol ¹³C_3, and 10 ng/mL of 21-deoxycortisol ²H₄). 200 µl of LC-MS Grade Methanol and 550 µl of LC-MS grade Water is added to each well. The samples are mixed after each reagent addition. Samples then centrifuged for five minutes at 4000 x g.

An aliquot of each of the pre-treated samples (600 µl) was loaded into individual wells of the Oasis PRiME HLB µElution Plate (p/n: 186008052) and slowly pulled through at low vacuum (100 mbar). Consecutive washes with 150 µl of 0.1% (v/v) ammonia in 35% (v/v) methanol_(aq) and 150uL 0.1% (v/v) formic acid in 35% (v/v) methanol_(aq) were preformed to reduce potential ionic interference. Analytes were eluted into a collection plate using 30 µL of 85/15 (v/v) acetonitrile/methanol, followed by addition of 70 µL water.

The chromatographic selectivity of the column is demonstrated through the baseline resolution of isobaric steroid species; 11-deoxycortisol and 21-deoxycortisol (please see Analysis of Corticosteroids and Androgens in Serum for Clinical Research 720005999 (2017), which demonstrated the separation of 17-OHP and 21-OHP and testosterone and epitestosterone). No significant interferences were observed at the retention time of testosterone, androstenedione, 17-OHP, DHEAS, cortisol, 11-deoxycortisol, and 21-deoxycortisol, when each of these compounds were individually examined. No significant system carryover was observed from high concentration samples into subsequent blank injections. Analytical sensitivity using signal:noise (S/N) of the low calibrator of each set (C1) was >10:1 at each of calibrator 1 concentrations across several analytical runs.

The clinical research method allows for precise quantification of all analytes at low concentrations. Total precision was determined by extracting and quantifying two replicates per QC level over five days. Repeatability was assessed by analysing four replicates at each QC level in a single analytical run. All results for all analytes were <10%CV, with the majority of results <5%CV.

The clinical research method was shown to be linear for all analytes over the calibration ranges specified in Table 1 when different ratio of low and high concentration pools of the analytes were combined and analysed. Furthermore, calibration lines created using Set 2 and Set 3 were analysed over a five-day period and were linear with a co-efficient of determination of (r²) >0.999 for all analytes.

Accuracy was assessed for testosterone, androstenedione, 17-OHP, DHEAS, and cortisol through the analysis of EQA material. The data obtained over a four-day period, was compared to the mass spectrometry method mean for each EQA sample, and the mean % difference from target was tabulated (Table 6). All analytes were assessed at the low, medium, and high end of the calibration line for each MassTrak Steroid Serum Set, which yielded excellent results across the range (±10%). Due to the unavailability of EQA material for 11-deoxycortisol and 21-deoxycortisol, in house panels were used to assess the accuracy of these compounds and the low and medium range. Deming regression and Linear regression was preformed (Table 7). No Statistically significant bias was observed for each compound, with a mean method bias of ±1.3% for Set 2 and ±1.0% for Set 3.

Through this proof of performance evaluation, it has been demonstrated the MassTrak Steroid Serum Calibrator and Quality Control Sets 2 and 3 (IVD) can provide precise and accurate quantification of steroid hormones in serum. An analytically sensitive and selective clinical research method has been developed for the analysis of testosterone, androstenedione, 17-OHP, DHEAS, cortisol, 11-deoxycortisol, and 21-deoxycortisol in serum using Waters Xevo TQ-S micro (FL).

The Xevo TQ-S micro Mass Spectrometer enables this clinical research method to provide sufficient analytical sensitivity to analyse low levels of testosterone, androstenedione, 17-OHP, DHEAS, cortisol, 11- deoxycortisol, and 21-deoxycortisol by using only 100 µL of sample volume.

The assay’s accuracy (±6%) and precision (±10%) have been confirmed through comparison to EQA LC-MS/MS means, In House Panels, and QC material for testosterone, androstenedione, 17-OHP, DHEAS, cortisol, 11-deoxycortisol, and 21-deoxycortisol. The addition of automation or liquid handlers, in particular this study utilized the Hamilton Microlab STAR improves laboratory workflow and helps remove operator errors.

Disclaimer

This clinical research method is an example of an application using the instrumentation, software, and consumables described in this document. This clinical research method has not been cleared by any regulatory entity for diagnostic purposes. The end user is responsible for completion of the method development and validation. MassTrak Endocrine Steroid Calibrator and Quality Control Sets are not available for sale in all countries. For information on availability, please contact your local sales representative.

Foley D, Calton L. Analysis of Corticosteroids and Androgens in Serum for Clinical Research. Waters Application Note 720005999. May 2017.