Martini V, Raffagli A, Stramesi C
… +6 more, Vignali C, Ortu S, Franceschini P, Baudone I, Bucchioni P, Morini L
Drug Test Anal
· 2026 Jun · PMID 41967834
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Environmental exposure can affect hair analysis, making decontamination a critical preanalytical step. No standardized washing protocol exists, and laboratories use different solvent combinations. This study evaluated th...Environmental exposure can affect hair analysis, making decontamination a critical preanalytical step. No standardized washing protocol exists, and laboratories use different solvent combinations. This study evaluated the impact of three common hair decontamination procedures on THC and its primary metabolite, THCCOOH, in cannabinoid-positive hair samples. Hair samples were longitudinally divided into three aliquots and subjected to three different washing protocols. After alkaline hydrolysis, THC and THCCOOH were extracted by liquid-liquid extraction and analyzed by GC-MS and GC-MS/MS (NCI), respectively. In selected cases, washing solvents were also analyzed to assess analyte loss during decontamination. THC concentrations ranged from 0.03 to 3.9 ng/mg and were significantly influenced by the washing procedure. Compared with Protocol A, mean THC losses of 18.5% and 54.2% were observed after Protocols B and C, respectively, with reductions exceeding 80.0% in several samples following Protocol C. In some cases, this decrease was sufficient to shift samples from positive to negative relative to the 0.05 ng/mg cut-off. Analysis of washing solvents confirmed substantial extraction of THC during Protocol C, particularly in the dichloromethane fraction following the aqueous wash. In contrast, THCCOOH concentrations showed minimal variability across all washing procedures, with mean losses below 10.0%. The choice of washing protocol markedly affects THC quantification in hair, potentially leading to underestimation and misclassification, whereas THCCOOH remains largely unaffected. These findings highlight the need for standardized decontamination procedures to ensure reliable and comparable results in forensic and clinical hair analysis.
Blanco-Ces M, de-Castro-Ríos A, Cobo-Golpe M
… +3 more, López-Rabuñal Á, Cruz A, Lendoiro E
Drug Test Anal
· 2026 Jun · PMID 41947718
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Hair analysis is increasingly used in forensic toxicology; however, result interpretation may be influenced by cosmetic hair treatments. Although the effects of bleaching, dyeing, and thermal straightening have already b...Hair analysis is increasingly used in forensic toxicology; however, result interpretation may be influenced by cosmetic hair treatments. Although the effects of bleaching, dyeing, and thermal straightening have already been evaluated, data on permanent chemical hair straightening are scarce, particularly under in vivo conditions. This case report evaluates the impact of an alkaline-based permanent hair straightening procedure on the determination of an endogenous compound, gamma-hydroxybutyrate (GHB), and an exogenous drug, alprazolam, in hair. Hair samples were collected from a 39-year-old woman before and 1 month after undergoing chemical hair straightening based on ammonium thioglycolate. Adjacent hair strands corresponding to identical temporal windows were analyzed. GHB was quantified in 0.5-cm hair segments using a validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) method, whereas alprazolam was analyzed in 2-cm segments using a validated LC-MS/MS method for drugs of abuse and psychoactive pharmaceuticals. All analyzed segments were positive for GHB in both samples. After the straightening procedure, endogenous GHB concentrations showed a pronounced increase compared with pretreatment levels, with fold changes ranging from approximately 6 to 43 in corresponding segments. In contrast, alprazolam concentrations in segments corresponding to the reported period of drug intake decreased by approximately 1.7- to 2-fold following chemical straightening. This case report demonstrates that permanent chemical hair straightening can produce marked and analyte-dependent effects on hair drug concentrations. These findings emphasize the importance of systematic documentation of cosmetic hair treatments and careful interpretation of hair analysis results in forensic casework, particularly when analyte concentrations are low.
Kim H, Seo S, Kim A
… +3 more, Kim HS, Kim HK, Yoo GJ
Drug Test Anal
· 2026 Jun · PMID 41937464
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The manufacture and distribution of illicit drugs in the Republic of Korea are an increasing problem. We aimed to develop a rapid and reliable simultaneous analytical method for monitoring the illegal presence of anaboli...The manufacture and distribution of illicit drugs in the Republic of Korea are an increasing problem. We aimed to develop a rapid and reliable simultaneous analytical method for monitoring the illegal presence of anabolic androgenic steroids (AAS) in foods and drugs. Forty-five AAS compounds were analyzed using a validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) method in accordance with international standards. Liquid chromatography-quadrupole time-of-flight mass spectrometry (LC-QTOF/MS) has enabled the determination of representative fragment ion patterns. The validated method was applied to 95 real samples, including 11 foods, 40 dietary supplements, and 44 counterfeit drugs. The overall detection rate was 38.9%, with the highest rate observed in counterfeit drugs (81.8%), followed by dietary supplements (2.5%), whereas no AAS were detected in foods. When classified by formulation, injectables showed the highest detection rate (100.0%), followed by liquids (78.6%), tablets (58.3%), powders (20.0%), and capsules (3.3%). Frequently detected compounds included testosterone-17-propionate (0.001-119, 850.0 μg/g), testosterone-17-isocaproate (0.002-1442.7 μg/g), and nandrolone decanoate (0.01-120,023.0 μg/g). Anastrozole and exemestane, which are not currently included as LC-MS/MS target compounds, were also identified through LC-QTOF/MS. Furthermore, LC-QTOF/MS results revealed the presence of nonsteroidal compounds, including phosphodiesterase-5 inhibitors and weight-loss compounds, indicating potential health risks associated with the combined pharmacological effects of these substances. The analytical method developed in this study can be effectively used for both screening and quantification and offers practical applicability to regulatory monitoring and enhancement of public health protection.
Feng J, Wang X, Zhang M
… +2 more, Xiang P, Zhao J
Drug Test Anal
· 2026 Jun · PMID 41936393
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The nonmedical use of etomidate and its structural analogs as additives in e-cigarettes has shown a significant growth trend, and the public health and safety risks arising from the abuse of such substances have drawn wi...The nonmedical use of etomidate and its structural analogs as additives in e-cigarettes has shown a significant growth trend, and the public health and safety risks arising from the abuse of such substances have drawn widespread attention. This study comprehensively employed gas chromatography-mass spectrometry, liquid chromatography-high-resolution mass spectrometry, and nuclear magnetic resonance spectroscopy (NMR) techniques to identify a novel metomidate analog, 4-fluorometomidate, in seized cartridges for the first time. Mass spectrometry analysis revealed that under electron ionization conditions, the compound produced fragment ions at m/z 77.1, 95.1, 103.1, 123.1, and 248.1. The molecular ion (m/z 248.1) and base peak (m/z 123.1) were both 18 Da higher than the corresponding peaks (m/z 230.1 and 105.1) characteristic for metomidate. In electrospray ionization mode, its protonated molecule (m/z 249.1032) also exhibited an increase of 17.9908 Da compared to metomidate (m/z 231.1124), and the characteristic fragment at m/z 123.0607 showed an increase of 17.9909 Da relative to the fragment of metomidate (m/z 105.0698), which was overall consistent with the substitution of one hydrogen atom by a fluorine atom (△m/z 17.9905). NMR analysis confirmed that the fluorine atom is located at the 4'-position of the benzene ring.
Casati S, Ravelli A, Bergamaschi RF
… +6 more, Magliocco A, Vanerio S, Roda G, Rota P, Orioli M, Battistini A
Drug Test Anal
· 2026 Jun · PMID 41916898
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Alteration of the hair matrix by cosmetic products presents a challenge for forensic hair analysis. Oxidative treatments lead to analyte depletion and false-negative results. Currently, the degradation product of eumelan...Alteration of the hair matrix by cosmetic products presents a challenge for forensic hair analysis. Oxidative treatments lead to analyte depletion and false-negative results. Currently, the degradation product of eumelanin, 1H-pyrrole-2,3,5-tricarboxylic acid (PTCA) is being investigated as a marker for oxidative hair treatment; however, it requires the definition of the cut-off value. Recently, it has been shown that 1H-pyrrole-2,3,4,5-tetracarboxylic acid (PTeCA) also increased significantly after in vitro oxidative hair treatments. Here, our previously published LC-MS/MS method for hair PTCA has been fully validated for the simultaneous quantification of PTeCA (range 0.01-2.5 ng/mg; lower limit of quantification [LLOQ] 0.003 ng/mg). The method was applied to 3378 self-reported treated (T) and untreated (UT) hair samples (3-6 cm proximal). In addition, the in vitro formation of PTeCA was assessed in 225 UT hair samples by different professional cosmetic treatments with and without oxidative agents. In the UT group (N = 1144), PTCA was determined in about 40% of the samples with a median PTCA of 0.04 ng/mg (range 0.01-14.9 ng/mg) and PTeCA was >LLOQ in < 2% of the samples (N = 53). In the T group (N = 425), PTCA was determined in 84% of the samples with a median of 0.75 ng/mg (range 0.01-58.1 ng/mg); while the median PTeCA was 0.40 ng/mg (N = 243; range 0.02-31.2 ng/mg). Moreover, the in vitro cosmetic treatment confirmed the PTeCA formation only in oxidative conditions. Finally, a PTCA cut-off value was proposed using PTeCA as the gold standard. Our data suggest that PTeCA could be a reliable marker for detecting oxidative cosmetic treatments in the hair matrix.
Verhoeven MCM, Huls RC, van der Stelt NW
… +5 more, de Koning JC, van Grol M, Kranenburg RF, van Asten AC, Hulshof JW
Drug Test Anal
· 2026 Jun · PMID 41906427
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Illicit amphetamine synthesis is frequently conducted using the well-known Leuckart method, generating complex impurity profiles that provide forensic insights. This study reports the effect of reaction temperature and t...Illicit amphetamine synthesis is frequently conducted using the well-known Leuckart method, generating complex impurity profiles that provide forensic insights. This study reports the effect of reaction temperature and time during the first step of the Leuckart synthesis on subsequent conversion efficiency and impurity profile. Controlled syntheses were conducted at 120°C, 140°C, and 160°C, with samples collected at defined intervals. Reaction progress was monitored by gas chromatography-flame ionization detection (GC-FID), and impurity profiling followed the European harmonized method for profiling amphetamine (EHMPA) using GC-mass spectrometry (GC-MS). In addition, preparative liquid chromatography (prep-LC) followed by LC-high-resolution MS (LC-HRMS) was employed to isolate and characterize previously unidentified impurities. Results indicate that higher temperatures and longer reaction times accelerate benzyl methyl ketone (BMK) conversion, increasing the formation of N-formylamphetamine (after step 1) and amphetamine (after step 2). Lower temperatures and shorter reaction times result in incomplete conversion and the persistent presence of BMK, along with several impurities, including unsaturated ketones (3,5-diphenyl-4-methyl-3-penten-2-one and 1,5-diphenyl-4-methyl-3-penten-2-one), 1,3-dimethyl-2-phenylnaphthalene (Naphthalene 1), and 1-benzyl-3-methylnaphthalene (Naphthalene 2). Elevated temperatures favor the formation of 4-methyl-5-phenylpyrimidine (4M5PP), 4-benzylpyrimidine (4BP), N,N-bis-(‒1-phenylpropan-2-yl)formamide (DPIF), and Unknowns A2, B1, and B2, which were tentatively identified as N-[3,5-diphenyl-4-methyl-3-penten-2-yl]amine, and the cis/trans isomers of N-[1,5-diphenyl-4-methyl-3-penten-2-yl]amine, respectively. These amines form via hydrolysis of N-formylamino derivatives generated from unsaturated ketone intermediates. These findings demonstrate that reaction temperature and time strongly govern both amphetamine formation and impurity composition. The identified impurities serve as distinct chemical indicators, enabling reconstruction of illicit synthesis conditions and supporting intelligence on amphetamine production.
Gjelstad A, Holden G, Gundersen POM
… +5 more, Dehnes Y, Tvedte M, Levernæs M, Eriksson ÅK, Lauritzen F
Drug Test Anal
· 2026 Jun · PMID 41882508
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1,3-DMBA was detected in urine samples of eight athletes following a doping control, resulting in one AAF and levels below WADA's MRL in seven teammates. Investigation revealed contamination from rubber infill used on th...1,3-DMBA was detected in urine samples of eight athletes following a doping control, resulting in one AAF and levels below WADA's MRL in seven teammates. Investigation revealed contamination from rubber infill used on the indoor artificial turf, with analysis confirming 1,3-DMBA in the turf's crumb rubber originating from recycled tires.
Vigato C, Zancanaro F, Ghezzo N
… +1 more, Frison G
Drug Test Anal
· 2026 May · PMID 41875468
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A subject, admitted to an addiction treatment unit, was occupationally exposed to solvent mixtures and paint thinners containing aliphatic alcohols, while being a coachbuilder. He repeatedly tested positive for ethyl glu...A subject, admitted to an addiction treatment unit, was occupationally exposed to solvent mixtures and paint thinners containing aliphatic alcohols, while being a coachbuilder. He repeatedly tested positive for ethyl glucuronide (EtG) in weekly urine immunoassays performed in a hospital laboratory. However, he denied any alcohol consumption. Therefore, presumptive positive urine samples, as well as chest and pubic hair samples, were submitted to targeted liquid chromatography-high-resolution accurate mass-Orbitrap-mass spectrometry (LC-HRAM-Orbitrap-MS) analysis, after derivatization of analytes with N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide (EDC). MS acquisition was carried out in full scan positive-ion mode, followed by data-dependent MS and parallel reaction monitoring confirmation modes, according to an inclusion list of accurate mass values calculated from the elemental composition of MH ion species of several derivatized EtG homologs. All urine samples turned out to be EtG negative. Instead, methyl, propyl, butyl, and hexyl glucuronides were detected at varying levels depending on the exposure and/or inhalation conditions (product types, amounts, etc.), and urine sampling times. As expected, chest hair and pubic hair were EtG negative too, whereas the same other alcohol glucuronides were detected in both matrices, with higher abundances in pubic hair. Cross-reaction of EtG homologs, in this case due to exposure to chemicals containing several aliphatic alcohols, explains the false-positive urine immunoassay results. Furthermore, LC-HRAM-Orbitrap-MS analyses show that EtG homologs, like EtG, can be incorporated and detected in a variety of hair samples.
Drug Test Anal
· 2026 May · PMID 41850764
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As of 2025, the distribution of products claiming to contain so-called semisynthetic cannabinoids has continued. In most cases, the names of tetrahydrocannabinol (THC) analogs contained in these products are labeled; how...As of 2025, the distribution of products claiming to contain so-called semisynthetic cannabinoids has continued. In most cases, the names of tetrahydrocannabinol (THC) analogs contained in these products are labeled; however, the actual components are often unknown. In this study, we identified the compounds in products that claim to contain THC analogs. Gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-photodiode array-mass spectrometry (LC-PDA-MS) were used to analyze six products. Seven compounds were isolated from products via silica gel column chromatography, and their structures were determined by H, C NMR, and various two-dimensional NMR techniques, including H-H correlation spectroscopy, heteronuclear multiple quantum coherence, heteronuclear multiple-bond correlation, and nuclear Overhauser effect spectroscopy. The compounds detected in products A and B were identified as Δ-THCM and Δ-THCM. From product C, 9(R)-hexahydrocannabiphorol methyl ether (9(R)-HHCPM) was identified. The compounds isolated from product D were identified as 2-allyl-Δ-THC. Δ-THC methyl carbonate and Δ-iso-THC methyl carbonate were isolated and identified from product E. The compounds isolated from product F were identified as 10(S)-Hydroxy-9(R)-HHC. This study is the first report on THC analogs having methylcarbonated hydroxyl groups at the C1 position of THC in commercial products. The newly detected THC analogs are potential health hazards if used in general; there are no toxicity data for any of these compounds. In addition, with their unregulated synthesis and the by-product/residues that might have concerns exist. Thus, there are concerns regarding the distribution of products containing new THC analogs.
Angelis YS, Goula O, Kiousi P
… +1 more, Sakellariou P
Drug Test Anal
· 2026 May · PMID 41834284
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Tetrahydro sulfate metabolites are well-established long-term biomarkers of methyltestosterone use that can be detected intact by LC-MS/MS. However, their identification using product ion spectra under collision-induced...Tetrahydro sulfate metabolites are well-established long-term biomarkers of methyltestosterone use that can be detected intact by LC-MS/MS. However, their identification using product ion spectra under collision-induced dissociation conditions in negative ion mode is an analytical challenge under the provisions of the WADA TD2023IDCR. In this study, six out of eight potential tetrahydro-methyltestosterone sulfate metabolites were microscale-synthesized to facilitate both the structural elucidation of the detected metabolites and the development of direct identification methods. Their identification was based on their GC-MS/(MS) analysis after TMS derivatization or LC-HRMS/(MS) analysis following derivatization of the free 17β-hydroxy group with carbonyldiimidazole (CDI), producing 17β-OH-imidazole carbamate derivatives. The resulting derivatives were detectable in both negative and positive ion modes, enabling their identification through characteristic product ion spectra. Urine samples from two 17α-methyltestosterone excretion studies were analyzed using these methods, and detection/identification time windows of intact sulfate metabolites were estimated under TD2023IDCR and compared with those obtained from GC-MS/(MS) analysis of the glucuronide fraction after hydrolysis. Overall, the inclusion of the tetrahydro-methyltestosterone sulfate metabolites significantly extends the detection time window for methyltestosterone abuse. Still, the established identification time window was similar to, or shorter than, that derived from the glucuronide fraction analysis.
Drug Test Anal
· 2026 May · PMID 41833302
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3,4-Methylenedioxymethamphetamine (MDMA) remains unapproved for therapeutic use despite the promising results of MDMA-assisted psychotherapy. There is a need to better understand the safety, pharmacokinetics, and toxicol...3,4-Methylenedioxymethamphetamine (MDMA) remains unapproved for therapeutic use despite the promising results of MDMA-assisted psychotherapy. There is a need to better understand the safety, pharmacokinetics, and toxicology of possible MDMA-based prodrugs. Like lisdexamfetamine, amino acid prodrugs of MDMA may enable more controlled systemic exposure, but their metabolic activation pathways and metabolites are not known yet. This study investigated the bioactivation and metabolism of the MDMA prodrugs, MDMA-tryptophan (MDMA-Trp), MDMA-lysine (MDMA-Lys), and MDMA-glycine (MDMA-Gly), in zebrafish embryos (ZE), pooled human liver S9 fraction (pHLS9), pooled fresh human whole blood (pFHWB), and human urine after microdosing (HMD). It elucidated mechanistic activation routes and identified screening targets relevant for drug testing and safety assessment. In ZE, MDMA-Trp underwent hydroxylation and N-dealkylation prior to amide cleavage, indicating a stepwise bioactivation pathway that differs from direct conversion observed for the other prodrugs. All three prodrugs were cleaved to MDMA in ZE, pHLS9, and HMD, with known MDMA metabolites additionally formed in ZE and pHLS9, whereas no metabolites were detected in pFHWB, suggesting that amide cleavage is not mediated in blood under the tested conditions. Unique urine screening targets were identified only for MDMA-Trp, while biomarkers for MDMA-Lys and MDMA-Gly consisted of MDMA and known MDMA metabolites. This study demonstrated conversion of amino acid prodrugs to MDMA in pHLS9- and ZE-based systems and in humans after microdosing, but not in blood. There is a need for further studies such as their pharmacokinetic profiles in humans.
So YM, Kwok WH, Yuen SMS
… +2 more, Wong COL, Ho ENM
Drug Test Anal
· 2026 May · PMID 41814125
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Despite the recent success in introducing supramolecular solvents (SUPRAS)-based extraction to drug analysis, its application and robustness in day-to-day regular urine testing have yet to be demonstrated. Moreover, the...Despite the recent success in introducing supramolecular solvents (SUPRAS)-based extraction to drug analysis, its application and robustness in day-to-day regular urine testing have yet to be demonstrated. Moreover, the applicability of SUPRAS in equine doping control testing remains unexplored. In this work, we have successfully developed for the first time a simple, rapid, inexpensive, and environmentally friendly SUPRAS extraction method for analyzing 76 prohibited substances of different classes (selective androgen receptor modulators, hypoxia-inducible factor prolyl hydroxylase inhibitors, angiotensin II receptor antagonists, benzodiazepines, etc.) in hydrolyzed horse urine with liquid chromatography-mass spectrometry (LC/MS) for detection. The developed 1,2-hexanediol-based SUPRAS-LC/MS method has been fully validated, and its applicability and robustness in day-to-day testing of horse urine have also been demonstrated. This work marks a significant milestone in the advancement of green and sustainable drug testing methodology in equine sports, offering a novel approach to address one of the complexities inherent in equine doping control.
Drug Test Anal
· 2026 May · PMID 41812645
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Prohibited gene editing in horses (either in embryos or via cell culture and cloning) can result in both desired and undesired outcomes. If left undetected, changes can proliferate within the population in subsequent gen...Prohibited gene editing in horses (either in embryos or via cell culture and cloning) can result in both desired and undesired outcomes. If left undetected, changes can proliferate within the population in subsequent generations, posing a major threat to welfare and breed integrity.
Jai J, Gozdzialski L, Wallace B
… +2 more, Gill CG, Hore D
Drug Test Anal
· 2026 May · PMID 41785878
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Community-based drug checking services are challenged in their ability to reliably detect low concentration adulterants that are increasingly present in the illicit drug supply. Spectral signatures from commonly used fie...Community-based drug checking services are challenged in their ability to reliably detect low concentration adulterants that are increasingly present in the illicit drug supply. Spectral signatures from commonly used field instruments such as infrared spectrometers require careful analysis to identify characteristic features in a complex mixture. In this study, we train neural network models for the detection of bromazolam and para-fluorofentanyl, using infrared absorption data collected at a point-of-care drug checking service. The neural network models classified the two components with an F1-score of 0.88 for bromazolam and 0.89 for para-fluorofentanyl. In comparison, a random forest model optimized using the same data set had an F1-score of 0.66 for bromazolam and 0.76 for para-fluorofentanyl. This demonstrates that neutral networks are excellent candidates for such complex drug detection applications and outperform other machine learning-based approaches.
Greif M, Frömel T, Wagner S
… +2 more, Huhn C, Pütz M
Drug Test Anal
· 2026 May · PMID 41785505
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In clandestine laboratories, amphetamine is predominantly synthesized via the Leuckart route. In recent years, a trend is observed: Capacities of illicit production facilities for synthetic drugs in Europe increased and...In clandestine laboratories, amphetamine is predominantly synthesized via the Leuckart route. In recent years, a trend is observed: Capacities of illicit production facilities for synthetic drugs in Europe increased and fewer small-scale laboratories are encountered by police and customs authorities. One of the designer pre-precursors currently applied is methyl α-phenylacetoacetate (MAPA), which can be converted into the key synthesis educt benzyl methyl ketone by acidic hydrolysis. Besides replacements of former designer pre-precursors due to scheduling (e.g., α-phenylacetoacetonitrile [APAAN]), another trend for synthesis is observed since 2019, namely, the alkaline hydrolysis of the reaction intermediate N-formylamphetamine into the free amphetamine base during the second step of the Leuckart route instead of the previously predominantly applied acidic hydrolysis using concentrated hydrochloric acid. In this study, 28 samples of products and production waste seized from a dismantled large-scale amphetamine laboratory in Germany that used MAPA as designer pre-precursor and the modified alkaline Leuckart step two were analyzed by a nontargeted liquid chromatography-high-resolution mass spectrometry approach for tentative identification of specific markers for the use of MAPA and the alkaline hydrolysis of N-formylamphetamine. After peak picking, 23 features were identified as suspects and further seven new features were tentatively identified. These seven potential marker compounds appeared to be indicative for the pre-precursor conversion step and were found in production waste and in amphetamine base product samples. Additionally, there were hints for the formation of high molecular weight compounds during the modified Leuckart step two.
Obratov D, Sutehall S, Liu L
… +2 more, Zhongying Z, Pitsiladis Y
Drug Test Anal
· 2026 Apr · PMID 41785503
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Recombinant human erythropoietin (rHuEPO) is often misused in endurance sports due to its potent erythropoietic effects. While transcriptomic biomarkers hold promise for detecting rHuEPO use beyond conventional testing w...Recombinant human erythropoietin (rHuEPO) is often misused in endurance sports due to its potent erythropoietic effects. While transcriptomic biomarkers hold promise for detecting rHuEPO use beyond conventional testing windows, many proposed gene markers may also respond to physiological stimuli such as exercise or altitude. This study compared 153 previously reported rHuEPO-responsive genes in whole blood with transcripts identified during exercise (GEPREP database) and high-altitude exposure (four independent studies). For the exercise dataset, gene-level statistical outputs were obtained directly from the GEPREP database, while biological relevance was calculated using Cohen's d. Analyses of altitude and rHuEPO datasets followed the original statistical procedures described in each study. Among the 153 rHuEPO-responsive genes, 94 overlapped with altitude and 34 with exercise. However, 50 genes remained unaffected by either exercise or altitude stimuli. Enriched in post-translational regulation and intracellular transport pathways, these genes represent promising candidate transcriptomic markers of rHuEPO administration. This work provides a refined gene panel that reduces the likelihood of false positives and requires further experimental validation before integration into RNA-based detection tests.