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Abstract INTRODUCTION
High-throughput analysis of serum concentrations of testosterone, androstenedione (A4), and dehydroepiandrosterone (DHEA) plays a crucial role in the diagnosis and management of various endocrine disorders. Although testosterone is the primary androgen, other androgenic hormones are important when evaluating conditions such as polycystic ovarian syndrome (PCOS), adrenal insufficiency, and disorders involving hyperandrogenism or hypoandrogenism1,2. These hormones, mainly produced by the gonads and adrenal glands, are essential for the regulation of sexual development, reproductive functions and metabolic processes1,2. Accurate and efficient quantification of these steroids in serum is essential for detecting hormonal imbalances. High-throughput techniques, particularly steroid immunoassays, have become the standard for measuring these hormones due to their sensitivity and ability to process large sample volumes. However, these methods are often limited by the specificity of the antibodies for epitopes, which can lead to cross-reactions with structurally similar steroids, thereby affecting the accuracy of the measurements. Overcoming these limitations through advanced analytical techniques, such as laser diode thermal desorption coupled to mass spectrometry (LDTD-MS/MS), is essential to ensure accurate diagnostics and effective treatment strategies.
OBJECTIVES
To facilitate screening and diagnosis of endocrine disorders, a rapid analytical method to separate testosterone and DHEA, isobaric compound, a specific APCI ionisation, extraction method and a LDTD-MS/MS analytical method was developed. This approach aims to improve clinical decision-making by providing timely results while maintaining high sensitivity and precision across a broad range of samples.
METHODS
A calibration curve and quality controls are spiked into an artificial matrix consisting of 20 mg/mL BSA in PBS. To prepare the samples, 12.5 µL of internal standards and 250 µL of samples are mixed at 1000 rpm for 5 seconds. Then, 1000 µL of a Hexane/MTBE (90:10) mixture is added, followed by mixing at 1000 rpm for 30 seconds and centrifugation at 14,000 rpm for 4 minutes. The upper layer (500 µL) is transferred to a borosilicate tube (10x75mm) and evaporated to dryness. The samples are reconstituted with 100 µL of an Acetonitrile:Water (1:1) mixture, and 4 µL are spotted onto a LazWell96 plate for LDTD-MS/MS analysis.
Testosterone and DHEA are isobaric compounds, and to enhance selectivity between them, an ammonia solution is infused into the carrier gas to form an adduct. The infusion of ammonia into the LDTD carrier gas is optimized to maximize the signal of both adduct and protonated molecules. Ammonium hydroxide (1% in water) is introduced into the Luxon at a rate of 2 µL/min, with a carrier gas flow of 4.5 L/min. This process selectively generates ammonium adducts on DHEA. Under these ionization conditions, specific primary masses are targeted for all analytes, and the mass spectrometer operates in positive ionization mode, using a designated MRM transition for detection.
RESULTS
Specificity is confirmed by running separate calibration curves for each analyte, monitoring all MS/MS transitions. Data indicates no detectable interferences between the analytes. The solvents used in the liquid-liquid extraction (LLE) process are optimized to minimize unwanted noise in MS/MS transitions. Calibration curves range from 100 to 10,000 pg/mL for testosterone, and from 200 to 10,000 pg/mL for DHEA and androstenedione. A set of fortified quality controls (QCs) is prepared in serum matrix with low levels of testosterone, androstenedione, and DHEA. The peak area to internal standard (IS) ratio is used to normalize the signal. For linearity evaluation, the coefficient of correlation must exceed 0.995, with obtained values ranging from 0.99390 to 0.99876 for testosterone, androstenedione, and DHEA across six runs. Precision is evaluated by ensuring that the %CV for quality control samples (QCL, QCM, QCH) remains below 15%. For inter-assay precision, the %CV was below 11.4% for all QCs. Accuracy for testosterone ranged from 101.5% to 105.6%, while A4 ranged from xx% to yy%, and DHEA ranged from 99.7% to 104.9%. Using LDTD-MS/MS technology, sample stability, both in the extracted (wet stability) and dry state on the Lazwell plate (dry stability), is evaluated rather than injector stability (as in LC-MS/MS). Precision values for these stability assessments ranged from 0.9%CV to 10.9%, and accuracy ranged from 98.2% to 106% for both dry and wet stability. Finally, cross-validation with LC-MS/MS was performed, and the percentage differences between the two methods were all below 20% for the evaluated matrices.
CONCLUSION
The selective high-throughput shotgun analysis of steroids, utilizing specific APCI adduct formation and optimized liquid-liquid extraction (LLE) with LDTD-MS/MS, provides excellent specificity, precision, and accuracy for the quantification of testosterone, androstenedione, and DHEA in serum. Cross-validation with LC-MS/MS further confirms the robustness and comparability of the two techniques, ensuring consistent and reliable hormone analysis across different platforms.
REFERENCES
1. Frank Z. Stanczyk, Diagnosis of hyperandrogenism: Biochemical criteria, Best Practice & Research Clinical Endocrinology & Metabolism, Volume 20, Issue 2, 2006, Pages 177-191, ISSN 1521-690X, https://doi.org/10.1016/j.beem.2006.03.007.
2. Nassar GN, Leslie SW. Physiology, Testosterone. In: StatPearls. StatPearls Publishing, Treasure Island (FL); 2023. PMID: 30252384.
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= Discovery stage. (53.14%, 2025)
= Translation stage. (22.33%, 2025)
= Clinically available. (24.53%, 2025)
