Detection Method for Gene Doping in a Mouse Model Expressing Human Erythropoietin from Adeno-Associated Virus Vector-9.

With the rapid development of gene therapy technology in recent years, its abuse as a method of sports doping in athletics has become a concern. However, there is still room for improvement in gene-doping testing methods, and a robust animal model needs to be developed. Therefore, the purposes of this study were to establish a model of gene doping using recombinant adeno-associated virus vector-9, including the human erythropoietin gene (rAAV9-hEPO), and to establish a relevant testing method. First, it was attempted to establish the model using rAAV9-hEPO on mice. The results showed a significant increase in erythrocyte volume accompanied by an increase in spleen weight, confirming the validity of the model. Next, we attempted to detect proof of gene doping by targeting DNA and RNA. Direct proof of gene doping was detected using a TaqMan-qPCR assay with certain primers/probes. In addition, some indirect proof was identified in RNAs through the combination of a TB Green qPCR assay with RNA sequencing. Taken together, these results could provide the foundation for an effective test for gene doping in human athletes in the future.

Rapid investigating of phase I metabolites of SR9009 in vitro horse liver microsomes via feature-based molecular networking approach: Potential applications in doping control.

SR9009, a peroxisome proliferator-activated receptor δ (PPARδ) agonist, is known for its potential benefits in energy homeostasis. It failed to receive the United States Food and Drug Administration (USFDA) approval and its illegal distribution has raised concerns. As a result, it has been classified as a prohibited substance by the World Anti-Doping Agency and the International Federation of Horseracing Authorities (IFHA). This study emphasizes the application of the in-silico molecular networking technology to analyze phase I drug metabolites in horses, distinguishing it from conventional methodologies in forensic science. Feature-based molecular networking (FBMN) analysis identified 15 metabolites, with novel major N-dealkylated metabolite (-C8H7NO4S), indicative of diverse metabolic modifications in horse liver microsomes incubation assay. Additionally, a proposed metabolic pathway of SR9009 in the in vitro assay was outlined, including the previously known dehydroxylated metabolite. Finally, the metabolic pathways included in this study were as follows: hydroxylation, dehydrogenation, N-dealkylation dihydroxylation, and combinations. Molecular networking provided insights into MS spectra connectivity, facilitating rapid interpretation and accurate detection of previously undiscovered metabolites. In conclusion, this study contributes to the understanding of SR9009 metabolism in horses and underscores the importance of advanced analytical techniques, such as molecular networking, in enhancing the accuracy and efficiency of metabolite analysis for forensic and doping control purposes.

Probing the hair detectability of prohibited substances in sports: an in vivo-in silico-clinical approach and analytical implications compared with plasma, urine, and faeces.

Hair analysis is a crucial method in forensic toxicology with potential applications in revealing doping histories in sports. Despite its widespread use, knowledge about detectable substances in hair is limited. This study systematically assessed the detectability of prohibited substances in sports using a multifaceted approach. Initially, an animal model received a subset of 17 model drugs to compare dose dependencies and detection windows across different matrices. Subsequently, hair incorporation data from the animal experiment were extrapolated to all substances on the World Anti-Doping Agency's List through in-silico prediction. The detectability of substances in hair was further validated in a proof-of-concept human study involving the consumption of diuretics and masking agents. Semi-quantitative analysis of substances in specimens was performed using ultra-performance liquid chromatography-tandem mass spectrometry. Results showed plasma had optimal dose dependencies with limited detection windows, while urine, faeces, and hair exhibited a reasonable relationship with the administered dose. Notably, hair displayed the highest detection probability (14 out of 17) for compounds, including anabolic agents, hormones, and diuretics, with beta-2 agonists undetected. Diuretics such as furosemide, canrenone, and hydrochlorothiazide showed the highest hair incorporation. Authentic human hair confirmed diuretic detectability, and their use duration was determined via segmental analysis. Noteworthy is the first-time reporting of canrenone in human hair. Anabolic agents were expected in hair, whereas undetectable compounds, such as peptide hormones and beta-2 agonists, were likely due to large molecular mass or high polarity. This study enhances understanding of hair analysis in doping investigations, providing insights into substance detectability.

Impact of low-volume blood withdrawal on hematological biomarkers for the athlete biological passport.

This study investigated the impact of low-volume blood withdrawal on the hematological biomarkers currently considered for anti-doping purposes. After baseline measurement (D - 7), a 140 mL blood withdrawal was completed (D + 0) on 12 healthy volunteers, followed by weekly monitoring for 21 days (D + 7 - 21). Each visit consisted of a full blood count (Sysmex XN-1000) and duplicate blood volume measurements by CO-rebreathing. A significant decrease in total hemoglobin mass (Hbmass) (-2.3%, p = 0.007) and red blood cell volume (RBCV) (-2.8%, p = 0.028) was reported at D + 7. Despite no atypical passport finding (ATPF) when considering the athlete biological passport adaptive longitudinal model, hemoglobin concentration ([Hb]) increased significantly at D + 21 (+3.8%, p = 0.031). Besides, ferritin (FERR) was significantly downregulated at all points following blood withdrawal, with the largest decrease occurring at D + 7 (-26.6%, p < 0.001). Regardless of the presumable effect of blood reinfusion on ABP biomarkers, these results illustrate the challenge of monitoring hematological variables for the detection of low-volume blood withdrawal. Finally, this study outlines the sensitivity of FERR to altered erythropoiesis to support the implementation of iron markers as complementary variables for the longitudinal monitoring of blood doping, despite the potential influence of confounding factors (e.g., iron supplementations).

Analysis of Potential Gene Doping Preparations for Transgenic DNA in the Context of Sports Drug Testing Programs.

Gene doping has been classified as a prohibited method by the World Anti-Doping Agency (WADA) and the International Olympic Committee (IOC) for over two decades. As gene therapeutic approaches improve and, concomitantly, safety concerns regarding clinical applications decline, apprehensions about their illicit use in elite sports continue to grow. Two products available via Internet-based providers and advertised as EPO-gene- and IGF1-gene-containing materials were analyzed for the presence of potential gene doping agents using a newly developed analytical approach, allowing for the detection of transgenic DNA corresponding to seven potential targets (EPO, FST, GH1, MSTN (Propeptide), IGF1, VEGFA, and VEGFD). Panel detection was based on a 20-plex polymerase chain reaction (PCR) followed by a single base extension (SBE) reaction and subsequent SBE product analyses via matrix-assisted time-of-flight laser desorption/ionization mass spectrometry (MALDI-TOF MS). Extracts of both products were found to contain transgenic EPO-DNA, while transgenic DNA for IGF-1 was not detected. The results were confirmed using SYBR Green qPCR with primer sets directed against EPO and IGF1 cDNA, and the CMV promotor sequence. In this case study, the detection of authentic (whilst low concentrated) transgenes, potentially intended for gene doping practices in readily available products, is reported for the first time.

Detection of extracellular hemoglobin from Arenicola marina in doping control serum samples by means of liquid chromatography and high-resolution tandem mass spectrometry.

The manipulation of blood and blood components in sports is prohibited at all times, and besides blood transfusions, also hemoglobin-based oxygen carriers (HBOCs) can be employed to artificially improve the oxygen transport capacity of the blood. But while most drug candidates based on stabilized hemoglobin (Hb) were found to be characterized by serious side effects, the natural giant extracellular Hb from the marine invertebrate Arenicola marina (lugworm) could be another candidate for transfusion medicine and cheating athletes, as it was found to be well tolerated in preclinical animal studies. Within this research project, lugworm Hb was implemented into the existing doping control detection method for bovine HBOCs based on ultrafiltration, tryptic digestion, and liquid chromatography coupled with high-resolution tandem mass spectrometry (LC-HRMS/MS). For the mass spectrometric identification of lugworm Hb, two precursor-product ion pairs for a total of four tryptic peptides originating from subunits hbA2 (T6 ), hbB1 (T3 and T6 ), and the linker chain (T16 ) were employed. The modified approach was comprehensively characterized and found to allow for the specific and sensitive detection of lugworm Hb down to concentrations of 10 µg/mL from 50 µL of serum/plasma. Therefore, it can serve as confirmation procedure for lugworm Hb following visual or electrophoretic screening. Moreover, a proof-of-concept rat administration study was conducted, and the observed detection windows of at least 4 (dose: 200 mg/kg) and 8 h (dose: 600 mg/kg) suggest that the approach can be readily employed to efficiently test in-competition doping control samples for the presence of the drug candidate.

Achieving routine application of dried blood spots for erythropoietin receptor agonist analysis in doping control: low-volume single-spot detection at minimum required performance level.

Background: Erythropoietin receptor agonists (ERAs) are substances prohibited in sports and currently monitored in urine and blood. There is a great interest in new matrices like dried blood spots (DBSs). Method: A direct method for the detection of ERAs in DBSs using one single spot of 25 µl has been optimized and validated. Results: Limits of detection close or equal to those required by the World Anti-Doping Agency for serum/plasma samples were achieved, using a volume 20-times lower. All analytes were stable for at least 90 days at room temperature. Conclusion: Method performance was comparable to the requirements established for blood samples and, thus, monitoring of ERAs is reliable in DBSs in the context of doping control.

Insulin-mimetic peptides in sports drug testing.

Because of its influence on carbohydrate metabolism and, at the same time, anti-catabolic effects, the misuse of the peptide hormone insulin and its synthetic analogs is prohibited in sports at all times according to the regulations of the World Anti-Doping Agency (WADA). The biological effects of insulin and its analogs are mediated through binding to the insulin receptor, which was also found to be activated by different peptides structurally largely unrelated to insulin. Such insulin-mimetic peptides or selective-insulin receptor modulators (SIRMs) represent a novel class of potential performance-enhancing agents, which is currently not explicitly mentioned on the WADA Prohibited List. Within this research project, advanced solid-phase extraction (SPE) and liquid chromatography coupled to high-resolution tandem mass spectrometry (LC-HRMS/MS) were employed to develop a fast, reliable, and specific assay for the detection of the insulin-mimetic peptides S597 and S519 from plasma. Method validation demonstrated a detection limit of 0.5 ng/mL and successfully illustrated the applicability of the approach to routine sports drug testing programs. Moreover, sophisticated and comprehensive in vitro metabolism experiments were conducted, and several metabolic degradation products were identified, which will enhance the information generated from future analyses of doping control samples.

Using Proteins As Markers for Anabolic Steroid Abuse: A New Perspective in Doping Control?

Drug toxicity is a major concern and has motivated numerous studies to elucidate specific adverse mechanisms, with acetaminophen being the favorite candidate in toxicology studies. Conversely, androgenic anabolic steroids (AASs) also represent a severe public health issue in sports for elite and non-elite athletes. Supraphysiological dosages of AASs are associated with various adverse effects, from cardiovascular to neurological repercussions including liver dysfunction. Yet, few studies have addressed the toxicity of anabolic steroids, and a significant amount of work will be needed to elucidate and understand steroid toxicity properly. This Perspective suggests ideas on how proteomics and liquid chromatography coupled with high-resolution tandem mass spectrometry (LC-HRMS/MS) can contribute to (1) pinpoint serum proteins affected by substantial doses of anabolic steroids that would represent interesting novel candidates for routine testing and (2) provide additional knowledge on androgenic anabolic steroid toxicity to help raise awareness on the harmful effects.

Detection of synthetic analogues of insulin-like growth factor 1 in different biological fluids by liquid chromatography quadrupole time-of-flight mass spectrometry: comparison of different immunoaffinity protocols.

Insulin-like growth factor 1 analogues are prohibited in sport for their ability to enhance athletic performance in several sport disciplines. Their detection presents several analytical challenges, mainly due to the minimum required performance limits fixed by the World Anti-Doping Agency. Here, we are presenting analytical workflows to detect IGF-1 and its analogues in different biological matrices. Several off-line immunocapture techniques and protocols were comparatively evaluated. Separation and detection were performed by using standard flow reverse-phase liquid chromatography coupled to a time-of-flight mass spectrometer. The best recoveries were obtained using magnetic beads or pipette tips functionalized with protein A. The analytical workflows were fully validated for qualitative determinations: all the target analytes were clearly distinguishable from the interference of the matrices, with limits of detection and identification in the range of 0.05-0.30 ng/mL in urine and 0.5-2.0 ng/mL in serum/plasma. The extraction efficiency proved to be repeatable (CV% < 10) with recoveries higher than 50%. Intra- and inter-day precision were found to be smaller than 10 and 15%, respectively. The method was successfully applied to the analysis of authentic matrix samples containing the target peptides at the minimum required performance limits, proving that the method developed can be successfully applied to detect and identify IGF-1 analogues for doping control purposes in all the matrices selected. The analytical workflow developed here to detect the target peptides in different matrices can be readily implemented in anti-doping laboratories and has the potential to be adapted for the simultaneous analysis of different similarly sized peptide hormones of doping relevance.

Administration and detection of gene therapy in horses: A systematic review.

Gene therapy uses genetic modification of cells to produce a therapeutic effect. Defective or missing genes can be repaired or replaced, or gene expression can be modified using a variety of technologies. Repair of defective genes can be achieved using specialized gene editing tools. Gene addition promotes gene expression by introducing synthetic copies of genes of interest (transgenes) into cells where they are transcribed and translated into therapeutic proteins. Protein production can also be modified using therapies that regulate gene expression. Gene therapy is currently prohibited in both human and equine athletes because of the potential to induce production of performance-enhancing proteins in the athlete's body, also referred to as "gene doping." Detection of gene doping is challenging and necessitates development of creative, novel analytical methods for doping control. Methods for detection of gene doping must be specific to and will vary depending on the type of gene therapy. The purpose of this paper is to present the results of a systematic review of gene editing, gene therapy, and detection of gene doping in horses. Based on the published literature, gene therapy has been administered to horses in a large number of experimental studies and a smaller number of clinical cases. Detection of gene therapy is possible using a combination of PCR and sequencing technologies. This summary can provide a basis for discussion of appropriate and inappropriate uses for gene therapy in horses by the veterinary community and guide expansion of methods to detect inappropriate uses by the regulatory community.

The effects of iron injection on blood doping biomarkers in dried blood spots.

Iron supplementation is not considered as a doping method; however, it can affect the levels of several biomarkers of the hematologic module of the athlete biological passport (ABP), such as the reticulocyte percentage (%RET) and hemoglobin (HGB) level. Thus, iron injection could be a confounding factor in antidoping analyses. Previous studies have suggested that the HGB level and the expression levels of reticulocyte-related-mRNAs, such as 5'-aminolevulinate synthase 2 (ALAS2) and carbonic anhydrase 1 (CA1), could be promising biomarkers for the ABP and detectable in dried blood spots (DBSs). Therefore, in this study, we examined the impact of iron injection on the levels of these potential biomarkers in DBSs. Reticulocyte-related-mRNAs analyses were performed by RT-qPCR. Ferritin level in DBS was measured with enzyme-linked immunosorbent assay (ELISA) method. Notably, there were no significant effects of iron supplementation on the levels of ALAS2 and CA1 mRNAs but by contrast, the %RET and immature reticulocyte fraction (IRF) measured in whole blood increased significantly following iron injection. As expected, iron supplementation increased the ferritin level significantly in both serum and DBS samples. In conclusion, these findings reinforce the specificity of reticulocyte-related mRNAs in DBSs as biomarkers of blood doping to target in antidoping analyses.

EPO and the athlete biological passport: Hematological results from a placebo-controlled, boosting and microdose EPO administration in male recreational athletes.

Hematological results in the context of the Athlete Biological Passport (ABP) from a placebo-controlled EPO administration study are provided here. Twelve participants administered eight subcutaneous boosting doses of epoetin alfa (at 40 IU/kg) over the course of 20 days. After a 10-day washout period, the same volunteers administered six microdoses (900 IU), intravenously, over 13 days. A blinded placebo cohort followed the same dosing pattern, administering saline instead of EPO. All participants supplemented with oral iron, daily, throughout the entirety of the study. In the EPO cohort, as expected, significant changes from baseline were identified in IRF, RET#, RET%, RDW, HCT, HGB, and RBC. No meaningful changes were identified in the placebo cohort population. From the ABP perspective, atypical passport findings (ATPF) were identified in 49% of the samples collected during the boosting and initial washout phases, and 24% of the samples during the microdosing and final washout phases. ATPFs from this cohort were flagged as late as Day 70, the final day of the study. Only a single ATPF was identified from all samples collected from the placebo cohort. ABPs from all volunteers in the study are provided as an avenue to visually convey differences in magnitude and timing of the hematological changes caused by EPO on the individual level. These data are expected to provide important content for Athlete Passport Management Units and ABP expert panels alike.

Stereoisomers in sports drug testing: Analytical strategies and applications.

Analytics employed in modern doping controls are designed to cover an extensive range of rather diverse classes of substances, all of which are banned in sport according to the list of prohibited substances and methods of doping, resulting from their potential to be performance-enhancing and/or harmful to health. Many of these bioactive substances or their metabolites are chiral, which are comprehensively characterized and, if appropriate analytical approaches are applied, can be clearly identified. In sports drug testing, the enantiomeric composition of relevant compounds is not considered in all instances, although differences of isomers concerning their biological activity have been established. To date, the separation of stereoisomers in doping controls is only applied for selected target compounds, but with the development of efficient chiral chromatographic stationary phases, the added value of information on e.g. racemic shifts during the metabolic biotransformation reactions of drugs has been recognized. The immense variability of the substance classes represents however a major challenge, especially because both 'classic' doping agents belonging to the category of lower molecular mass molecules (e.g. stimulants, ß2-agonists, betablockers, corticoids, etc.) as well as larger molecules from the category of peptides and proteins necessitate consideration. In the present (mini)review, the current status of analytical techniques in the field of doping control analysis of stereoisomers is highlighted and critically reviewed.

Simultaneous detection of 93 anabolic androgenic steroids in dietary supplements using gas chromatography tandem mass spectrometry.

In recent years, anabolic androgenic steroids (AASs) have been frequently detected as undeclared ingredients in dietary supplements, where the adverse analytical findings (AAFs) were obtained from analysis of athletes' urine samples after ingestion. In our present study, a GC-MS/MS method for simultaneous detection of 93 anabolic steroids was developed. The chromatographic and mass spectrometric conditions were optimized, and selective reaction monitoring (SRM) mode was adopted to obtain the necessary sensitivity. The whole sample analysis process was completed within 23 min, and the limit of detection (LOD) was 0.5-4 ng.g-1 for solid samples and 0.1-0.8 ng.mL-1 for liquid samples. This method was verified according to World Anti-Doping Agency (WADA) regulations. In addition, the method was found to be specific, accurate. The developed method was then applied to a routine analysis of more than 300 liquid and solid dietary supplements, and one testosterone-positive sample was found. Three suspected drugs, (4-hydroxyandrostenedione, DHEA, and 6-Br androstenedione) were found in three dietary supplements obtained from the Internet through the pretreatment method of this study. This study provides a high-throughput method for screening and monitoring the ingredients of supplements and their subsequent harm to public health.

Comprehensive metabolic study of nicotine in equine plasma and urine using liquid chromatography/high-resolution mass spectrometry for the identification of unique biomarkers for doping control.

Nicotine is classified as a stimulant, and its use is banned in horse racing and equestrian sports by the International Federation of Horseracing Authorities and the Fédération Équestre Internationale, respectively. Because nicotine is a major alkaloid of tobacco leaves, there is a potential risk that doping control samples may be contaminated by tobacco cigarettes or smoke during sample collection. In order to differentiate the genuine doping and sample contamination with tobacco leaves, it is necessary to monitor unique metabolites as biomarkers for nicotine administration and intake. However, little is known about the metabolic fate of nicotine in horses. This is the first report of comprehensive metabolism study of nicotine in horses. Using liquid chromatography/electrospray ionization high-resolution mass spectrometry, we identified a total of 17 metabolites, including one novel horse-specific metabolite (i.e., 4-hydroxy-4-(3-pyridyl)-N-methylbutanamide), in post-administration urine samples after nasoesophageal administration of nicotine to three thoroughbred mares; eight of these compounds were confirmed based on reference standards. Among these metabolites, N-hydroxymethylnorcotinine was the major urinary metabolite in equine, but it could only be tentatively identified by mass spectral interpretation due to the lack of reference material. In addition, we developed simultaneous quantification methods for the eight target analytes in plasma and urine, and applied them to post-administration samples to establish elimination profiles of nicotine and its metabolites. The quantification results revealed that trans-3'-hydroxycotinine could be quantified for the longest period in both plasma (72 h post-administration) and urine (96 h post-administration). Therefore, this metabolite is the most appropriate monitoring target for nicotine exposure for the purpose of doping control due to its long detection times and the availability of its reference material. Further, we identified trans-3'-hydroxycotinine as a unique biomarker allowing differentiation between nicotine administration and sample contamination with tobacco leaves.

Qualitative and quantitative analysis of ephedrine stimulants in urine by ultra-performance liquid chromatography-tandem mass spectrometry.

RATIONALE: Ephedrine analogues are stimulants that are explicitly required to be quantified and characterized in the Anti-Doping Prohibited List of the World Anti-Doping Agency. Given the difficulty of distinguishing diastereoisomers, the qualitative and quantitative analyses of ephedrine diastereoisomers are difficult. METHODS: An ultra-performance liquid chromatography-tandem mass spectrometry (UHPLC/MS/MS) method was developed to detect five ephedrine analogues, and two pairs of diastereoisomers were identified using this method. The samples were analyzed qualitatively and quantitatively using a tandem mass spectrometer with an electrospray ionization source in multiple reaction detection mode after one-step dilution. RESULTS: The effective detection limits of this method were below 0.5 ng/mL. A matrix effect (range: 83.4% to 102%) was observed in quality control samples. The intra- and inter-day precision was lower than 9.16% and 8.60%, respectively, and the accuracy was within ±8.0%. CONCLUSIONS: The method is efficient, accurate, stable and sensitive, and fully meets the requirements for the detection of ephedrine substances in stimulants.

Low-copy transgene detection using nested digital polymerase chain reaction for gene-doping control.

Gene doping is prohibited for fair competition in human and horse sports. One style of gene doping is the administration of an exogeneous gene, called a transgene, to postnatal humans and horses. Although many transgene detection methods based on quantitative polymerase chain reaction (PCR), including real-time PCR and digital PCR, have been recently developed, it remains difficult to reliably detect low-copy transgenes. In this study, we developed and validated a nested digital PCR method to specifically detect low-copy transgenes. The nested digital PCR consists of (1) preamplification using conventional PCR and (2) droplet digital PCR detection using a hydrolysis probe. Using 5, 10, 20, 60 and 120 transgene copies as template, 496.0, 1089.7, 1820.7, 4313.3 and 7840.0 copies per microlitre, respectively, were detected using our nested digital PCR. Although high concentrations of phenol, proteinase K, ethanol, EDTA, heparin and genomic DNA all inhibited preamplification, their effects on the digital PCR detection were limited. Once preamplification was successful, even substitution of bases within the primers and probes had minimal effects on transgene detection. The nested digital PCR developed in this study successfully detected low-copy transgenes and can be used to perform a qualitative test, indicating its usefulness in the prevention of false positives and false negatives in gene-doping detection.

Procollagen type III amino-terminal propeptide and insulin-like growth factor I as biomarkers of growth hormone administration.

The acceptance in 2012 by the World Anti-Doping Agency (WADA) of the biomarker test for human growth hormone (hGH) based on procollagen type III amino-terminal propeptide (P-III-NP) and insulin-like growth factor I (IGF-I) was perhaps the first time that such a method has been used for forensic purposes. Developing a biomarker test to anti-doping standards, where the strict liability principle applies, is discussed. An alternative WADA-accepted approach is based on the measurement of different hGH isoforms, a method that suffers from the very short half-life of hGH limiting the detection period. Modification or withdrawal of the immunoassays, on which the biomarker measurements largely depend, has necessitated revalidation of the assays, remeasurement of samples and adjustment of the decision limits above which an athlete will be assumed to have administered hGH. When a liquid chromatography coupled mass spectrometry (LC-MS) method became a reality for the measurement of IGF-I, more consistency of results was assured. Measurement of P-III-NP is still dependent on immunoassays although work is underway to develop an LC-MS method. The promised long-term detection time for the biomarker assay does not appear to have been realised in practice, and this is perhaps partly the result of decision limits being set too high. Nevertheless, more robust assays are needed before a further adjustment of the decision limit is warranted. In the meantime, WADA is considering using P-III-NP and IGF-I as components of a biomarker passport system recording data from an individual athlete, rather than the population. Using this approach, smaller perturbations in the growth hormone (GH) score would mandate an investigation and possible action for hGH administration.

Screening for gene doping transgenes in horses via the use of massively parallel sequencing.

Throughout the history of horse racing, doping techniques to suppress or enhance performance have expanded to match the technology available. The next frontier in doping, both in the equine and human sports areas, is predicted to be genetic manipulation; either by prohibited use of genome editing, or gene therapy via transgenes. By using massively-parallel sequencing via a two-step PCR method we can screen for multiple doping targets at once in pooled primer sets. This method has the advantages of high scalability through combinational indexing, and the use of reference standards with altered sequences as controls. Custom software produces transgene-specific amplicons from any Ensembl-annotated genome to facilitate rapid assay design. Additional scripts batch-process FASTQ data from experiments, automatically quality-filtering sequences and assigning hits based on discriminatory motifs. We report here our experiences in establishing the workflow with an initial 31 transgene and vector feature targets. To evaluate the sensitivity of parallel sequencing in a real-world setting, we performed an intramuscular (IM) administration of a control rAAV vector into two horses and compared the detection sensitivity between parallel sequencing and real-time qPCR. Vector was detected by all assays on both methods up to 79 h post-administration, becoming sporadic after 96 h.