madman
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Determination of anabolic steroids in dried blood using microsampling and gas chromatography-tandem mass spectrometry: Application to a testosterone gel administration study
Nine anabolic steroids are separated within 6.4 min and are detectable at 50 fg
VAMS dried blood exhibits good stability and better recovery over spotting card
Quantification of testosterone between serum and VAMS dried blood is in agreement
Doping with micro-dose testosterone can be caught by using 20 μL of dried blood
Abstract
Anabolic-androgenic steroids (AAS) have been the most commonly abused substances taken by not only professional sportsmen but also recreational bodybuilders. The detection of micro-dose testosterone (T) misuse is particularly challenging as it possesses pseudo-endogenous origin and is sometimes impossible to be identified in urine samples. Dried blood (DB) obtained by finger pricking has been proven to be an alternative matrix for better correlating to physiological responses. Moreover, the introduction of the volumetric absorptive microsampling (VAMS) technology allows overcoming some major limitations of spotting blood onto a filter paper card. In this work, a fast and sensitive GCMS/MS method was developed and validated for the quantification of AAS in DB collected by means of VAMS. T and the eight top abused synthetic AAS, namely nandrolone, boldenone, mesterolone, drostanolone, metenolone, metandienone, oxandrolone, and dehydrochloromethyl T were selected as the target analytes. The method based on VAMS exhibited good precision, accuracy as well as stability, and superior extraction recoveries over the punched DB spots reported in the literature. The chromatographic separation was achieved within 6.4 min and the detection limit is as little as 50 fg (i.e. able to detect 0.10 ng mL−1 in 20 μL of DB). Confirmed by forty real blood samples, the Deming regression and Bland-Altman analysis revealed that the VAMS DB could be employed for quantifying blood T level in agreement with using the serum specimen. The feasibility of the method was then successfully proven by the analysis of samples collected from a three-arm T administration trial. Our results highlighted that DB total T was a sensitive indicator for identifying transdermal micro-dosing of T. In the groups of receiving T gel administration, T concentrations could rise up to ten times higher than the baseline at 9 h after the application. As a future step, this approach is being expanded to a large cohort screening of bodybuilders at gym and ultimately may allow universal applications on monitoring sports drug misuse.
.4 Conclusions
In this work, we developed a promising and reliable approach for the determination of 9 AAS in 20 μL of DB using VAMS and GC-MS/MS. The GC-MS/MS method was able to detect 50 fg of AAS (i.e. 0.10 ng mL−1 in 20 μL of DB) within a separation time of 6.4 min. The extraction recoveries of the VAMS DB were found to be superior to that of the punched DB spots. Furthermore, good agreement between serum and VAMS DB was established for the AAS quantification via analyzing forty real samples. A proof-of-concept study was carried out with a three-arm T administration trial. We demonstrated that the DB total T could be a sensitive indicator for the identification of micro-dosing pseudo-endogenous T. In the forthcoming work, this approach is being expanded to a large cohort screening of bodybuilders at the gym and ultimately may allow universal applications on monitoring sports drug misuse.
Nine anabolic steroids are separated within 6.4 min and are detectable at 50 fg
VAMS dried blood exhibits good stability and better recovery over spotting card
Quantification of testosterone between serum and VAMS dried blood is in agreement
Doping with micro-dose testosterone can be caught by using 20 μL of dried blood
Abstract
Anabolic-androgenic steroids (AAS) have been the most commonly abused substances taken by not only professional sportsmen but also recreational bodybuilders. The detection of micro-dose testosterone (T) misuse is particularly challenging as it possesses pseudo-endogenous origin and is sometimes impossible to be identified in urine samples. Dried blood (DB) obtained by finger pricking has been proven to be an alternative matrix for better correlating to physiological responses. Moreover, the introduction of the volumetric absorptive microsampling (VAMS) technology allows overcoming some major limitations of spotting blood onto a filter paper card. In this work, a fast and sensitive GCMS/MS method was developed and validated for the quantification of AAS in DB collected by means of VAMS. T and the eight top abused synthetic AAS, namely nandrolone, boldenone, mesterolone, drostanolone, metenolone, metandienone, oxandrolone, and dehydrochloromethyl T were selected as the target analytes. The method based on VAMS exhibited good precision, accuracy as well as stability, and superior extraction recoveries over the punched DB spots reported in the literature. The chromatographic separation was achieved within 6.4 min and the detection limit is as little as 50 fg (i.e. able to detect 0.10 ng mL−1 in 20 μL of DB). Confirmed by forty real blood samples, the Deming regression and Bland-Altman analysis revealed that the VAMS DB could be employed for quantifying blood T level in agreement with using the serum specimen. The feasibility of the method was then successfully proven by the analysis of samples collected from a three-arm T administration trial. Our results highlighted that DB total T was a sensitive indicator for identifying transdermal micro-dosing of T. In the groups of receiving T gel administration, T concentrations could rise up to ten times higher than the baseline at 9 h after the application. As a future step, this approach is being expanded to a large cohort screening of bodybuilders at gym and ultimately may allow universal applications on monitoring sports drug misuse.
.4 Conclusions
In this work, we developed a promising and reliable approach for the determination of 9 AAS in 20 μL of DB using VAMS and GC-MS/MS. The GC-MS/MS method was able to detect 50 fg of AAS (i.e. 0.10 ng mL−1 in 20 μL of DB) within a separation time of 6.4 min. The extraction recoveries of the VAMS DB were found to be superior to that of the punched DB spots. Furthermore, good agreement between serum and VAMS DB was established for the AAS quantification via analyzing forty real samples. A proof-of-concept study was carried out with a three-arm T administration trial. We demonstrated that the DB total T could be a sensitive indicator for the identification of micro-dosing pseudo-endogenous T. In the forthcoming work, this approach is being expanded to a large cohort screening of bodybuilders at the gym and ultimately may allow universal applications on monitoring sports drug misuse.
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