madman
Super Moderator
The quantification of a large panel of endogenous steroids in serum by LC-MS/MS represents a powerful clinical tool for the screening or diagnosis of diverse endocrine disorders. This approach has also demonstrated excellent sensitivity for the detection of testosterone misuse in the anti-doping field, especially in the female athlete population. In both situations, the use of dried blood spots (DBS) could provide a viable alternative to invasive venous blood collection. Here, the evaluation of DBS sampling for the quantification of a panel of endogenous steroids using UHPLC-MS/MS is described.
The UHPLC-MS/MS method was validated for quantitative analysis of eleven free and eight conjugated steroids and was then used for the analysis of DBS samples collected in 14 healthy women during a normal menstrual cycle (control phase) followed by a 28-days testosterone gel treatment (treatment phase). Results were compared with those obtained from the serum matrix. Satisfactory performance was obtained for all compounds in terms of selectivity, linearity, accuracy, precision, combined uncertainty, stability as well as extraction recovery, and matrix effects. In the control phase, a high correlation was observed between DBS and serum concentrations for most compounds. In the treatment phase, higher testosterone concentrations were observed in capillary than in venous DBS, suggesting a possible interference resulting from testosterone contamination on the finger(s) used for gel application.
*Steroid profiling in capillary DBS represents a simple and efficient strategy for monitoring endogenous steroid concentrations and their fluctuation in the clinical context of steroid-related disorders, or for the detection of testosterone abuse in anti-doping.
1. Introduction
The diagnosis and the monitoring of various endocrine conditions frequently depend on the quantitative analysis of endogenous steroids in the blood matrix. For many years, this analysis was performed using immunoassays allowing high throughput. However, this technique is subject to cross-reactivity and is often limited to the measurement of a single or few compounds instead of a flexible panel of substances. Actually, clinical laboratories rather use liquid chromatography-tandem mass spectrometry (LC-MS/MS) as the gold standard method to measure steroid hormones, because it offers the possibility of quantifying multiple analytes at the same time with higher specificity and selectivity [1]. Along with further extensions of the panel, e.g. phase II metabolites, LC-MS/MS is indeed a valuable approach for the study of potential alterations in steroidogenesis due to hormonal imbalances, especially in women for whom the androgenic activity is not necessarily reflected by serum testosterone level [2,3].
In the anti-doping context, testosterone misuse is currently targeted using an individual and longitudinal monitoring of urinary biomarkers of testosterone in the so-called Athlete Biological Passport (ABP) [4]. In case of abnormal values for one or several of these markers, a time-consuming and expensive analysis based on gas chromatography-combustion- isotope ratio MS (GC/C/IRMS) is performed to confirm the potential exogenous origin of testosterone and its metabolites. While the implementation of this tool improved the testosterone detection capability, various confounding factors may influence the urinary steroid profile complicating its interpretation and decreasing its sensitivity [5–7]. Furthermore, athletes rather resort to low doses of topical testosterone, which significantly reduces peaks of urinary concentrations that are difficult to discriminate from natural variability [7–9].
To overcome these limitations, endogenous steroid profiling in the serum has been proposed as a potentially complementary approach to the urinary steroid profile for the detection of testosterone misuse by athletes [9,10]. Particularly, the blood matrix is more informative than urine for the correlation between hormone concentration and the physiological responses. The longitudinal monitoring in serum has been proven particularly useful for testosterone detection in female subjects in whom menstrual fluctuations may lead to a great source of variation for urinary biomarkers disrupting their sensitivity [7,9,11].
A primary drawback of the application of serum steroid profiling is that it requires invasive venous blood sampling and sample collection by a trained phlebotomist. Moreover, these biological specimens have to be transported undercooled temperature conditions within a short timeframe, which all increase the total costs of sample collection. Dried blood spots (DBS), which are based on the transfer of a limited volume of capillary blood onto a filter paper or similar matrix, could tackle these obstacles offering a convenient and more affordable alternative. This process benefits from minimal invasiveness, simplicity of sample collection, facilitated transport and storage conditions, and reduced costs that could allow for more frequent sampling for anti-doping programs. The advent of volumetric microsampling technologies has further improved the collection of DBS for quantitative purposes. While DBS has been used for neonatal screening for decades, its applicability has been recently evaluated for SARS–CoV–2 serology assays [12], therapeutic drug monitoring [13] or alcohol abstinence [14]. In the anti-doping context, DBS has been considered as a complementary matrix for many years [15–18] and has been investigated for either direct detection of prohibited substances [19–21] or indirect detection through potential biomarkers [22–24]. In particular, a method using volumetric microsampling and GC–MS/MS was recently developed for the quantification of testosterone and eight synthetic anabolic androgenic steroids (AAS) [25]. However, this method was limited to the quantification of only one endogenous AAS (EAAS) and could therefore be hardly applied in the clinical context for the monitoring of steroidogenesis disorders such as polycystic ovary syndrome or congenital adrenal hyperplasia.
In this study, we developed a UHPLC-MS/MS method for the simultaneous determination of eleven free (testosterone, epitestosterone, androstenedione, dehydroepiandrosterone (DHEA), 5-dihydrotestosterone (DHT), progesterone, 17-hydroxyprogesterone, cortisol, corticosterone, deoxycorticosterone, and 11-deoxycortisol) and eight conjugated (glucuro-conjugated androsterone and etiocholanolone, sulfoconjugated testosterone, DHEA, androsterone, etiocholanolone, epiandrosterone, and dehydroandrosterone) steroids in DBS matrix. Following validation according to World-Anti Doping Agency (WADA) requirements, the method was applied to the analysis of DBS samples collected from healthy eumenorrheic women during a normal menstrual cycle followed by a 28-days T gel treatment and results were compared with those of serum.
5. Conclusion
In summary, a fit-for-purpose UHPLC-MS/MS method was developed and validated for the quantification of a panel of steroids in DBS. This method could be applied to anti-doping as a complementary approach for the longitudinal monitoring of steroid profile and detection of testosterone administration in the ABP allowing for more frequent sampling and for targeting blood (serum) and urine sample collection that would be used for a full steroid profile and for confirmatory GC/C/IRMS analysis. The increased sampling frequency would provide a better estimation of natural baseline variability of a given athlete and would provide a better resolution of a possible doping picture [34]. This approach could also be employed for the monitoring of steroid-related pathologies in the clinical context. Indeed, for patients requiring regular medical visits or for whom venipuncture is complicated (neonates, elderly patients), DBS collected with Tasso-M20 or finger prick with HemaXis DB10 at home or on-site could be a valuable alternative to classical serum collection.
The UHPLC-MS/MS method was validated for quantitative analysis of eleven free and eight conjugated steroids and was then used for the analysis of DBS samples collected in 14 healthy women during a normal menstrual cycle (control phase) followed by a 28-days testosterone gel treatment (treatment phase). Results were compared with those obtained from the serum matrix. Satisfactory performance was obtained for all compounds in terms of selectivity, linearity, accuracy, precision, combined uncertainty, stability as well as extraction recovery, and matrix effects. In the control phase, a high correlation was observed between DBS and serum concentrations for most compounds. In the treatment phase, higher testosterone concentrations were observed in capillary than in venous DBS, suggesting a possible interference resulting from testosterone contamination on the finger(s) used for gel application.
*Steroid profiling in capillary DBS represents a simple and efficient strategy for monitoring endogenous steroid concentrations and their fluctuation in the clinical context of steroid-related disorders, or for the detection of testosterone abuse in anti-doping.
1. Introduction
The diagnosis and the monitoring of various endocrine conditions frequently depend on the quantitative analysis of endogenous steroids in the blood matrix. For many years, this analysis was performed using immunoassays allowing high throughput. However, this technique is subject to cross-reactivity and is often limited to the measurement of a single or few compounds instead of a flexible panel of substances. Actually, clinical laboratories rather use liquid chromatography-tandem mass spectrometry (LC-MS/MS) as the gold standard method to measure steroid hormones, because it offers the possibility of quantifying multiple analytes at the same time with higher specificity and selectivity [1]. Along with further extensions of the panel, e.g. phase II metabolites, LC-MS/MS is indeed a valuable approach for the study of potential alterations in steroidogenesis due to hormonal imbalances, especially in women for whom the androgenic activity is not necessarily reflected by serum testosterone level [2,3].
In the anti-doping context, testosterone misuse is currently targeted using an individual and longitudinal monitoring of urinary biomarkers of testosterone in the so-called Athlete Biological Passport (ABP) [4]. In case of abnormal values for one or several of these markers, a time-consuming and expensive analysis based on gas chromatography-combustion- isotope ratio MS (GC/C/IRMS) is performed to confirm the potential exogenous origin of testosterone and its metabolites. While the implementation of this tool improved the testosterone detection capability, various confounding factors may influence the urinary steroid profile complicating its interpretation and decreasing its sensitivity [5–7]. Furthermore, athletes rather resort to low doses of topical testosterone, which significantly reduces peaks of urinary concentrations that are difficult to discriminate from natural variability [7–9].
To overcome these limitations, endogenous steroid profiling in the serum has been proposed as a potentially complementary approach to the urinary steroid profile for the detection of testosterone misuse by athletes [9,10]. Particularly, the blood matrix is more informative than urine for the correlation between hormone concentration and the physiological responses. The longitudinal monitoring in serum has been proven particularly useful for testosterone detection in female subjects in whom menstrual fluctuations may lead to a great source of variation for urinary biomarkers disrupting their sensitivity [7,9,11].
A primary drawback of the application of serum steroid profiling is that it requires invasive venous blood sampling and sample collection by a trained phlebotomist. Moreover, these biological specimens have to be transported undercooled temperature conditions within a short timeframe, which all increase the total costs of sample collection. Dried blood spots (DBS), which are based on the transfer of a limited volume of capillary blood onto a filter paper or similar matrix, could tackle these obstacles offering a convenient and more affordable alternative. This process benefits from minimal invasiveness, simplicity of sample collection, facilitated transport and storage conditions, and reduced costs that could allow for more frequent sampling for anti-doping programs. The advent of volumetric microsampling technologies has further improved the collection of DBS for quantitative purposes. While DBS has been used for neonatal screening for decades, its applicability has been recently evaluated for SARS–CoV–2 serology assays [12], therapeutic drug monitoring [13] or alcohol abstinence [14]. In the anti-doping context, DBS has been considered as a complementary matrix for many years [15–18] and has been investigated for either direct detection of prohibited substances [19–21] or indirect detection through potential biomarkers [22–24]. In particular, a method using volumetric microsampling and GC–MS/MS was recently developed for the quantification of testosterone and eight synthetic anabolic androgenic steroids (AAS) [25]. However, this method was limited to the quantification of only one endogenous AAS (EAAS) and could therefore be hardly applied in the clinical context for the monitoring of steroidogenesis disorders such as polycystic ovary syndrome or congenital adrenal hyperplasia.
In this study, we developed a UHPLC-MS/MS method for the simultaneous determination of eleven free (testosterone, epitestosterone, androstenedione, dehydroepiandrosterone (DHEA), 5-dihydrotestosterone (DHT), progesterone, 17-hydroxyprogesterone, cortisol, corticosterone, deoxycorticosterone, and 11-deoxycortisol) and eight conjugated (glucuro-conjugated androsterone and etiocholanolone, sulfoconjugated testosterone, DHEA, androsterone, etiocholanolone, epiandrosterone, and dehydroandrosterone) steroids in DBS matrix. Following validation according to World-Anti Doping Agency (WADA) requirements, the method was applied to the analysis of DBS samples collected from healthy eumenorrheic women during a normal menstrual cycle followed by a 28-days T gel treatment and results were compared with those of serum.
5. Conclusion
In summary, a fit-for-purpose UHPLC-MS/MS method was developed and validated for the quantification of a panel of steroids in DBS. This method could be applied to anti-doping as a complementary approach for the longitudinal monitoring of steroid profile and detection of testosterone administration in the ABP allowing for more frequent sampling and for targeting blood (serum) and urine sample collection that would be used for a full steroid profile and for confirmatory GC/C/IRMS analysis. The increased sampling frequency would provide a better estimation of natural baseline variability of a given athlete and would provide a better resolution of a possible doping picture [34]. This approach could also be employed for the monitoring of steroid-related pathologies in the clinical context. Indeed, for patients requiring regular medical visits or for whom venipuncture is complicated (neonates, elderly patients), DBS collected with Tasso-M20 or finger prick with HemaXis DB10 at home or on-site could be a valuable alternative to classical serum collection.
