Detection of the GH analog somatrogon in sports drug testing: Immunological approaches and LC-HRMS/MS.

Due to the presumed lipolytic and anabolic properties, the misuse of human growth hormone (hGH) and its synthetic analogs in sports is prohibited both in- and out-of-competition. Within this research project, the detectability of somatrogon, a recombinant fusion glycoprotein of 22 kDa hGH and the C-terminal peptide (CTP) of the human chorionic gonadotropin (hCG) ß-subunit, with current WADA-approved doping control assays for hGH and hCG was investigated. For that purpose, cross-reactivity tests and a somatrogon administration study were conducted, and only "Kit 2" of the GH isoform differential immunoassays proved applicable to the detection of somatrogon administration in serum. In urine, the immunoassay specific for total hCG yielded presumptively positive findings for several post-administration samples, which can probably be attributed to the presence of an immunoreactive fragment of the hCG ß-subunit. As the detectability of somatrogon with these approaches was found to be limited, a highly specific detection assay (LOD: 10 ng/mL) for the drug in serum samples was developed by using affinity purification with GH receptor (GHR)-conjugated magnetic beads, proteolytic digestion, and liquid chromatography high-resolution tandem mass spectrometry (LC-HRMS/MS). Following optimization, the approach was comprehensively characterized, and authentic post-administration serum samples were successfully analyzed as proof-of-concept, indicating a detection window of at least 96 h. Consequently, the presented method can be employed to confirm the presence of somatrogon in serum samples, where only "Kit 2" of the currently used immunoassay kits yielded an abnormally high Rec/Pit ratio.

Development of an LC-HRMS/MS Method for Quantifying Steroids and Thyroid Hormones in Capillary Blood: A Potential Tool for Assessing Relative Energy Deficiency in Sport (RED-S).

Relative energy deficiency in sport (RED-S) is a condition that arises from persistent low energy availability (LEA), which affects the hypothalamic-pituitary axis and results in alterations of several hormones in both male and female athletes. As frequent blood hormone status determinations using venipuncture are rare in sports practice, microsampling offers promising possibilities for preventing and assessing RED-S. Therefore, this study aimed at developing a liquid chromatography-high-resolution tandem mass spectrometry (LC-HRMS/MS) method for quantifying relevant steroids and thyroid hormones in 30 µL of capillary blood obtained using Mitra® devices with volumetric absorptive microsampling technology (VAMS®). The results of the study showed that all validation criteria were met, including a storage stability of more than 28 days in a frozen state (-18 °C) and 14 days at room temperature (20 °C). The validated assay provided precise (<12%) and accurate (<13%) results for all the target analytes. Furthermore, as a proof of concept, autonomously collected VAMS® samples from 50 female and male, healthy, active adults were analyzed. The sensitivity of all analytes was adequate to quantify the decreased hormone concentrations in the RED-S state, as all authentic samples could be measured accordingly. These findings suggest that self-collected VAMS® samples offer a practical opportunity for regular hormone measurements in athletes and can be used for early RED-S assessment and progress monitoring during RED-S recovery.

Presentation of microstructural diffusion components by color schemes in abdominal organs.

PURPOSE: Development of a color scheme representation to facilitate the interpretation of tri-exponential DWI data from abdominal organs, where multi-exponential behavior is more pronounced. METHODS: Multi-exponential analysis of DWI data provides information about the microstructure of the tissue under study. The tri-exponential signal analysis generates numerous parameter images that are difficult to analyze individually. Summarized color images can simplify at-a-glance analysis. A color scheme was developed in which the slow, intermediate, and fast diffusion components were each assigned to a different red, green, and blue color channel. To improve the appearance of the image, histogram equalization, gamma correction, and white balance were used, and the processing parameters were adjusted. Examples of the resulting color maps of the diffusion fractions of healthy and pathological kidney and prostate are shown. RESULTS: The color maps obtained by the presented method show the merged information of the slow, intermediate, and fast diffusion components in a single view. A differentiation of the different fractions becomes clearly visible. Fast diffusion regimes, such as in the renal hilus, can be clearly distinguished from slow fractions, such as in dense tumor tissue. CONCLUSION: Combining the diffusion information from tri-exponential DWI analysis into a single color image allows for simplified interpretation of the diffusion fractions. In the future, such color images may provide additional information about the microstructural nature of the tissue under study.

Detection of sgRNA via SHERLOCK as Potential CRISPR Related Gene Doping Control Strategy.

Apprehensions about gene doping have grown consistently due to advancements in gene engineering techniques, particularly with the emergence of clustered regularly interspaced short palindromic repeats/CRISPR-associated (CRISPR/Cas)-based tools. These tools not only provide unprecedented possibilities for illicit performance enhancement by athletes but also offer new avenues for the detection of gene doping through biosensing of nucleic acids. Hence, pursuing on a previous study, an analytical method based on reverse transcriptase-recombinase polymerase amplification (RT-RPA) and subsequent qualitative nucleic acid detection by means of Specific High Sensitive Enzymatic Reporter UnLOCKing (SHERLOCK) was optimized for the direct detection of sgRNA associated with Streptococcus pyogenes in serum. Detection device, assay parameters, and sample handling were adjusted, to overcome previously determined assay limitations. The conducted method characterization confirmed the methods' specificity and increased detection sensitivity from 100 pM to 1 fM sgRNA in 100 µL of serum. Furthermore, reanalysis of in vivo mouse administration samples collected in a previous proof-of-concept study was conducted with successful identification of sgRNA in all anticipated postadministration samples within the 24-h collection period. Those findings support the applicability of the refined analytical procedure for the detection of illegal doping attempts via ribonucleoprotein-based CRISPR/Cas application through sgRNA identification, offering a new potential doping control strategy for CRISPR related gene doping.

Urinary metabolites indicative of the administration of hypoxen monitored by liquid chromatography-high resolution/accurate mass tandem mass spectrometry.

Hypoxen, a poly(dihydroxyphenylene) thiosulfonate-based drug, has been investigated concerning its effect on mitochondrial respiration and the utilization of lactate, especially in the context of strenuous exercise. Since 2023, patterns of use regarding hypoxen amongst the athletic population are monitored by the World Anti-Doping Agency (WADA) and its accredited anti-doping laboratories, necessitating information on suitable urinary markers indicative of the administration of hypoxen. In this exploratory study, urine samples collected post-administration of 1.5 and 2.0 g of hypoxen were analyzed by means of liquid chromatography-high resolution/high mass accuracy (tandem) mass spectrometry, which allowed for the identification of eight analytes that were plausibly attributable to metabolites of hypoxen. The identified species were assigned to the unconjugated species of S-(2,2',5,5'-tetrahydroxy-[1,1'-biphenyl]-3-yl) sulfurothioate and its glucuronide and additional tentatively identified analytes comprising a mercaptobenzene core structure. Including the identified markers into routine doping control analytical procedures enabled the detection of hypoxen use in athletes' doping control samples, thus contributing relevant information to WADA's monitoring program.

Challenging the gold standard: the limitations of molecular assays for detection of Mycobacterium tuberculosis heteroresistance.

OBJECTIVES: Heteroresistant infections are defined as infections in which a mixture of drug-resistant and drug-susceptible populations are present. In Mycobacterium tuberculosis (M. tb), heteroresistance poses a challenge in diagnosis and has been linked with poor treatment outcomes. We compared the analytical sensitivity of molecular methods, such as GeneXpert and whole genome sequencing (WGS) in detecting heteroresistance when compared with the 'gold standard' phenotypic assay: the agar proportion method (APM). METHODS: Using two rounds of proficiency surveys with defined monoresistant BCG strains and mixtures of susceptible/resistant M. tb, we determined the limit of detection (LOD) of known resistance associated mutations. RESULTS: The LOD for rifampin-R (RIF-R) detection was 1% using APM, 60% using GeneXpert MTB/RIF, 10% using GeneXpert MTB/RIF Ultra and 10% using WGS. While WGS could detect mutations beyond those associated with RIF resistance, the LOD for these other mutations was also 10%. Additionally, we observed instances where laboratories did not report resistance in the majority population, yet the mutations were present in the raw sequence data. CONCLUSION: The gold standard APM detects minority resistant populations at a lower proportion than molecular tests. Mycobacterium bovis BCG strains with defined resistance and extracted DNA from M. tb provided concordant results and can serve in quality control of laboratories offering molecular testing for resistance. Further research is required to determine whether the higher LOD of molecular tests is associated with negative treatment outcomes.

Pharmacokinetic Profile of Caffeine and Its Two Main Metabolites in Dried Blood Spots After Five Different Oral Caffeine Administration Forms-A Randomized Crossover Study.

Caffeine is an ergogenic substance that is consumed globally in many forms. The use of buccally absorbable formulations instead of gastrointestinal uptake has become increasingly popular over the years, especially when accelerated absorption with minimal gastrointestinal stress is desired. This study investigated the impact of five different formulations and administration routes of caffeine on the whole blood concentrations of caffeine, paraxanthine, and theobromine: caffeinated capsules, tablets, shots, pouches, and chewing gums. A uniform dose of caffeine (200 mg) was administered to 16 healthy recreational athletes (26.0 ± 2.1 years) using a randomized crossover design. Samples were taken in the form of dried blood spots at 16 different time points in a 2-hr timeframe after drug administration. The samples were analyzed using a validated liquid chromatography-tandem mass spectrometry method. The results for caffeine showed no significant differences in the overall bioavailability (area under the concentration-time curve), maximal concentration, and time to maximum concentration. However, when analyzing the bioavailability of caffeine in the first 5, 10, and 15 min, the liquid caffeine formulation was superior to other administered forms (p < .05). This indicates that caffeine solubility has a major influence on its absorption rate. In sports, the rate of caffeine absorption must be considered, not only when ingesting anhydrous caffeine, but also when choosing buccal absorption. These findings imply that general guidelines for ergogenic caffeine use should consider the formulation used and, accordingly, the corresponding route of absorption.

Recent advances in mass spectrometry for the detection of doping.

INTRODUCTION: The analysis of doping control samples is preferably performed by mass spectrometry, because obtained results meet the highest analytical standards and ensure an impressive degree of reliability. The advancement in mass spectrometry and all its associated technologies thus allow for continuous improvements in doping control analysis. AREAS COVERED: Modern mass spectrometric systems have reached a status of increased sensitivity, robustness, and specificity within the last decade. The improved sensitivity in particular has, on the other hand, also led to the detection of drug residues that were attributable to scenarios where the prohibited substances were not administered consciously but rather by the unconscious ingestion of or exposure to contaminated products. These scenarios and their doubtless clarification represent a great challenge. Here, too, modern MS systems and their applications can provide good insights in the interpretation of dose-related metabolism of prohibited substances. In addition to the development of new instruments itself, software-assisted analysis of the sometimes highly complex data is playing an increasingly important role and facilitating the work of doping control laboratories. EXPERT OPINION: The sensitive analysis and evaluation of a higher number of samples in a shorter time is made possible by the ongoing developments in mass spectrometry.

Comparator Data Characteristics and Testing Procedures for the Clinical Performance Evaluation of Continuous Glucose Monitoring Systems.

Comparing the performance of different continuous glucose monitoring (CGM) systems is challenging due to the lack of comprehensive guidelines for clinical study design. In particular, the absence of concise requirements for the distribution of comparator (reference) blood glucose (BG) concentrations and their rate of change (RoC) that are used to evaluate CGM performance, impairs comparability. For this article, several experts in the field of CGM performance testing have collaborated to propose characteristics of the distribution of comparator measurements that should be collected during CGM performance testing. Specifically, it is proposed that at least 7.5% of comparator BG concentrations are <70 mg/dL (3.9 mmol/L) and >300 mg/dL (16.7 mmol/L), respectively, and that at least 7.5% of BG-RoC combinations indicate fast BG changes with impending hypo- or hyperglycemia, respectively. These proposed characteristics of the comparator data can facilitate the harmonization of testing conditions across different studies and CGM systems and ensure that the most relevant scenarios representing real-life situations are established during performance testing. In addition, a study protocol and testing procedure for the manipulation of glucose levels are suggested that enable the collection of comparator data with these characteristics. This work is an important step toward establishing a future standard for the performance evaluation of CGM systems.

Chromatographic-mass spectrometric analysis of peptidic analytes (2-10 kDa) in doping control urine samples.

Peptides with a molecular mass between 2 and 10 kDa that are prohibited in elite sports usually require dedicated sample preparation and mass spectrometric detection that commonly cannot be combined with other (lower molecular mass) substances. In most instances, the physicochemical differences are too significant to allow for a generic analytical procedure. A simplification of established and comparably complex analytical approaches is therefore desirable and has been accomplished in the context of this study. With urine samples representing still the most frequently collected doping control specimens, efficient extraction of peptidic analytes from this matrix was a major goal of this method, as demonstrated for the included compounds such as insulins (human, lispro, aspart, glulisine, tresiba, glargine metabolite, bovine insulin, porcine insulin), growth hormone-releasing hormones (sermorelin, CJC-1295, tesamorelin) incl. their respective metabolites, insulin-like-growth factors (long-R3 -IGF-I, R3 -IGF-I, des1-3 -IGF-I), synacthen, gonadorelin and mechano growth factors (human MGF, MGF-Goldspink). Sample preparation and detection are controlled by five internal standards, covering all five included peptide drug categories. Nearly all requirements of the recent technical documents from the World Anti-Doping Agency (WADA) considering their minimum required performance levels (MRPL) are fulfilled, and the method was validated for its utilisation as initial testing procedure in doping controls. Finally, the approach was applied to authentic post-administration study urine samples (for insulins and gonadorelin) in order to provide proof of principle.

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 doping control sample substitutions via single nucleotide polymorphism-based ID typing.

The authenticity of a doping control sample is a key element of sports drug testing programmes. Doping control sample manipulation by providing another individual's urine or blood (instead of the tested athlete's sample) has been observed in the past and is an unequivocal violation of the World Anti-Doping Agency anti-doping rules. To determine attempts of manipulations by sample swapping, the utility of a single nucleotide polymorphism (SNP)-based sample authentication with a multi-target SNP panel was assessed. The panel comprises detection assays for 44 different SNPs, 3 gender markers and 5 quality control markers for DNA-profile determination. Sample analysis is based on a multiplex polymerase chain reaction step followed by a multiplex single base extension (SBE) reaction and subsequent SBE-product detection by MALDI-TOF MS. Panel performance was evaluated for urine and dried blood spot (DBS) samples. Urine (8 ml) and DBS (20 µl) test samples were reliably typed and matched to whole blood reference samples, while efficient typing of urine samples correlated with sample quality and input amounts. Robust profiling of urine doping control specimens was confirmed with an assay input of 12 ml. Samples can be processed in a high-throughput format with an overall assay turnaround time of approximately 11 h. SNP-based DNA typing via MALDI-TOF MS thus represents a high throughput-capable possibility for doping control sample authentication. SNP profiling of samples could offer the opportunity to complement existing steroid profile analytics to substantiate sample manipulations and to support quality control processes in high throughput routine settings.

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.

A combined top-down and bottom-up LC-HRMS/MS method for the quantification of human growth hormone in plasma and serum.

OBJECTIVE: The precise and accurate quantification of human growth hormone (GH) in plasma/ serum is crucial for the diagnosis and treatment of diseases like GH deficiency or acromegaly. However, the ligand-binding assays (LBAs) currently used for routine testing show considerable methodological variability. Here, we present a complementary, combined top-down and bottom-up LC-MS-based method to quantify (intact) GH in plasma and serum, which concurrently provides a basis for a MS-based analysis of GH in doping controls. DESIGN: Extraction of GH from plasma/ serum was accomplished by protein precipitation, followed by an immunocapture step using protein A-coupled magnetic beads and a polyclonal anti-GH antibody. The intact protein was subsequently analyzed top-down on a 2D-LC-HRMS/MS system. In addition, sample extracts were digested with trypsin and analyzed for signal peptides corresponding to 'total', 22 kDa and 20 kDa GH (bottom-up). Both assays were validated according to current guidelines and compared to the GH isoform differential immunoassay used in routine doping control analysis. GH concentrations in serum samples of healthy adults, patients with acromegaly, and in samples obtained after administration of recombinant GH were analyzed as proof-of-principle. RESULTS: The intact monomeric 22 kDa isoform of GH was selectively quantified in a representative working range of 0.5 to 10 ng/ml by top-down LC-HRMS/MS. Subsequent bottom-up analysis provided additional data on 'total' and 20 kDa GH. Top-down and bottom-up assay results for the 22 kDa isoform correlated well with the corresponding immunoassay results (R2 > 0.95). For a possible application of the method in an anti-doping context, the ratio between 22 kDa and 'total' GH was evaluated, indicating differences between the various donor groups, but only with limited significance. CONCLUSION: The top-down and bottom-up LC-HRMS/MS method developed here presents a valuable tool for the quantification of GH in plasma/ serum complementary to established LBAs used at present in clinical measurements. Albeit the examination of the GH isoform proportions by the LC-MS method does not yet allow for the assessment of GH abuse, the obtained findings provide an important basis to enable LC-MS-based GH analysis of doping control samples in the future.

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.

Clinical Performance Evaluation of Continuous Glucose Monitoring Systems: A Scoping Review and Recommendations for Reporting.

The use of different approaches for design and results presentation of studies for the clinical performance evaluation of continuous glucose monitoring (CGM) systems has long been recognized as a major challenge in comparing their results. However, a comprehensive characterization of the variability in study designs is currently unavailable. This article presents a scoping review of clinical CGM performance evaluations published between 2002 and 2022. Specifically, this review quantifies the prevalence of numerous options associated with various aspects of study design, including subject population, comparator (reference) method selection, testing procedures, and statistical accuracy evaluation. We found that there is a large variability in nearly all of those aspects and, in particular, in the characteristics of the comparator measurements. Furthermore, these characteristics as well as other crucial aspects of study design are often not reported in sufficient detail to allow an informed interpretation of study results. We therefore provide recommendations for reporting the general study design, CGM system use, comparator measurement approach, testing procedures, and data analysis/statistical performance evaluation. Additionally, this review aims to serve as a foundation for the development of a standardized CGM performance evaluation procedure, thereby supporting the goals and objectives of the Working Group on CGM established by the Scientific Division of the International Federation of Clinical Chemistry and Laboratory Medicine.

Identification and synthesis of (Z)-3'-hydroxy clomiphene as a new potential doping-relevant metabolite of clomiphene.

A recent study addressed the possibility of unintentional ingestion of clomiphene through residues in chicken eggs. The method developed here helped distinguish between microdose intake of (E/Z)-clomiphene citrate and consumption of clomiphene-containing eggs by the urinary pattern of four mono-hydroxylated clomiphene metabolites. However, reanalyses of doping-control samples, which showed an adverse analytical finding for clomiphene, revealed a hydroxy clomiphene (HC) isomer that was not found after microdose intake or after consumption of clomiphene-containing eggs and could not be assigned to any of the available reference compounds. The aim of the present follow-up study was to identify this HC isomer and to characterize this metabolite with respect to its potential properties as long-term metabolite in doping controls. METHODS: (E/Z)-3'-HC and (E/Z)-4'-HC were synthesized involving the McMurry reaction. An ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed and optimized after a derivatization step with dansyl chloride to separate eight HC isomers. Using this method, urine samples from a controlled clomiphene administration study were analyzed, in which male study participants received therapeutic doses of clomiphene for 30 days and collected urine samples for up to 8 months. Thus, isomer-specific HC elimination profiles could be monitored. RESULTS: The metabolite previously found in doping-control samples was identified as (Z)-3'-HC. The elimination profiles of the different HCs confirmed previous results, with (Z)-3-HC being the most abundant urinary hydroxy metabolite shortly after administration. A new finding was that the data suggest that (Z)-3'-HC is excreted at higher relative concentrations only several weeks after drug intake. CONCLUSION: These findings might be of particular importance in sport drug testing as they can assist in the decision-making process to distinguish between intentional doping and inadvertent exposure to clomiphene via food contamination.

Investigations into Annona fruit consumption as a potential source of dietary higenamine intake in the context of sports drug testing.

Higenamine is prohibited in sports as a ß2 -agonist by the World Anti-Doping Agency. As a key component of a great variety of plants, including the Annonaceae family, one aim of this research project was to evaluate whether the ingestion of Annona fruit could lead to higenamine adverse analytical findings. Single-dose administration studies including three Annona species (i.e., Annona muricata, Annona cherimola, and Annona squamosa) were conducted, leading to higenamine findings below the established minimum reporting level (MRL) of 10 ng/mL in urine. In consideration of cmax values (7.8 ng/mL) observed for higenamine up to 24 h, a multidose administration study was also conducted, indicating cumulative effects, which can increase the risk of exceeding the applicable MRL doping after Annona fruit ingestion. In this study, however, the MRL was not exceeded at any time point. Further, the major urinary excretion of higenamine in its sulfo-conjugated form was corroborated, its stability in urine was assessed, and in the absence of reference material, higenamine sulfo-conjugates were synthesized and comprehensively characterized, suggesting the predominant presence of higenamine 7-sulfate. In addition, the option to include complementary biomarkers of diet-related higenamine intake into routine doping controls was investigated. A characteristic urinary pattern attributed to isococlaurine, reticuline, and a yet not fully characterized bismethylated higenamine glucuronide was observed after Annona ingestion but not after supplement use, providing a promising dataset of urinary biomarkers, which supports the discrimination between different sources of urinary higenamine detected in sports drug testing programs.