The LC–MS/MS method used for the qualitative analysis of LGD-4033 after H-SPE was comprehensively characterized and the results are listed in Table 1. The method was found to be selective and showed good inter- and intra-day precision at different concentrations. LOD and LOI were determined at 8 pg/mL and 125 pg/mL respectively. Recovery, ion suppression, stability, carryover, and robustness showed acceptable values and were deemed adequate for this application. The method is highly linear with R2-values of > 0.999.
The collected urine samples were prepared with the developed H-SPE method. The SPE showed better extraction capabilities in comparison to liquid–liquid extraction, especially for the detection of the highly polar metabolites such as M5 and M6 (data not shown). The preceding hydrolysis with β-glucuronidase was added to ensure the maximum detection time of all metabolites, as previous studies demonstrated no increased detection times of glucurono-conjugated phase II metabolites . As only phase I metabolites were included in this study, hereinafter, all mentioned metabolites are phase I metabolites.
A total of 6 metabolic pathways led to 15 metabolites in addition to the compound LGD-4033, which could be detected in excreted urine samples after the application of micro-doses of the SARM. For all described metabolites, mass spectrometric data has already been published prior to this study [15,16,17, 21]. The previously described increase of peak intensity and detection times by measuring the formate adducts could not be observed with this method . Therefore, all analytes were detected using the deprotonated molecules [M-H]− as precursor ions. A previously described methoxylated metabolite was not detected with this method .
The detected metabolites included the epimer of LGD-4033 (M1), hydroxylated and oxidized LGD-4033 (M2), mono-hydroxylated LGD-4033 (M3), LGD-4033 after hydroxylation and ring opening of the pyrrolidine ring (M4), bis-hydroxylated LGD-4033 (M5), and tri-hydroxylated LGD-4033 (M6). The chromatographic separation of the metabolites showed between two and four isomers with different retention times (see Table 2). These might be stereo- or regioisomers, of which some have been structurally elucidated in the literature [15, 16, 21]. To facilitate the estimation of concentrations of the metabolites, the synthesis of reference material will be necessary.
All detected metabolites and their diagnostic ions used for the evaluation are listed in Table 2. The most intense product ions are indicated in bold and were chosen as target ions. Depending on the analyte, either one or two additional confirming ions were chosen. Extracted ion chromatograms of the detected analytes are shown in Fig. 1. The metabolites M2-a and M2-b elute with a very similar retention time as M5-a and M5-b. It could not yet be determined if M2-a and M2-b are true metabolites as postulated by Geldof et al. or in-source fragments of M5-a and M5-b as postulated by Cox et al. [15, 21].
Even at the low administered doses, LGD-4033 and its metabolites can be detected with the developed method for multiple days or weeks depending on the analyte. The detection times of the analytes are shown in Fig. 2. The minimum and maximum detection times of the analytes varied notably between the volunteers. M2-d, M3-b, and M4-a could not be detected in any sample after 1 µg application. M1 and M2-c could only be detected in some volunteers after 1 µg application. M2-d, M3-b, and M4-a could only be detected in some volunteers after 10 µg application. All analytes could be detected in all volunteers after 50-µg application. The analyte that shows the longest detection window is M5-a, with the earliest negative sample after 11 days (50 µg). Therefore, this metabolite is the ideal target for doping analysis. The maximum concentrations of LGD-4033 that were detected after single-dose application were 2.5–4.0 ng/mL (50 µg), 0.4–1.1 ng/mL (10 µg), and 0.05–0.1 ng/mL (1 µg). These maximum values were detected between 2 and 4 h after application in all studies. It can therefore be concluded that although the detection times of LGD-4033 and its metabolites vary notably between individuals, the excretion behavior and metabolism are quite similar in the first hours after application.
In Fig. 3, the elimination profiles of LGD-4033 after the intake of 1, 10, and 50 µg LGD-4033 are shown. A pilot study with one volunteer showed that after the ingestion of 10 ng of LGD-4033, the metabolite M5-b is detected for 72 h (data not shown).
In addition to the single-dose studies, multi-dose studies were performed to simulate the daily intake of LGD-4033 and investigate its potential influence on metabolite ratios and accumulation behavior. In Fig. 4, the minimum and maximum detection times of LGD-4033 and its metabolites are shown. M3-b and M4-a could not be detected in any sample after multiple 1 µg applications. M1, M2-c, M2-d, and M6-a could only be detected in some volunteers after multiple 1 µg applications. All analytes included in this study were detected in all volunteers’ urine samples after multiple 10 µg and 50 µg applications. For M5-b, no maximum detection time for the multiple 50 µg applications can be determined because M5-b was detected in all samples until the end of the study 1004 h and 1128 h after the last ingestion of LGD-4033.
In Fig. 5, the elimination profiles of LGD-4033 after the intake of five doses of 1, 10, and 50 µg LGD-4033 are shown. Five distinct maxima could be detected after each ingestion with tmax of 2–5 h (50 µg), 2–8 h (10 µg), and 2–13 h (1 µg). No considerable accumulation of LGD-4033 after multiple ingestions of micro-doses can be observed. The metabolites M1, M2a, M2d, M3b, M4c, M4d, and M5a show some accumulation after multiple doses (data not shown). The maximum concentrations of LGD-4033 that were detected after multi-dose application were 4.3–6.4 ng/mL (50 µg), 0.7–1.6 ng/mL (10 µg), and 0.05–0.2 ng/mL (1 µg). As with the single-dose application, the inter-personal variability between volunteers is high.
A particularity of the metabolism of LGD-4033 is the relative metabolite ratio of isomers that changes over time. As most metabolites are excreted in more than one isomeric form (see Table 2), the ratio between the different isomers can be used to estimate a possible time span since ingestion of LGD-4033. The main goal of this study is to contribute analytical data for best-possible result management in support of differentiating a recent intake of (or exposure to) low amounts of LGD-4033 as opposed to a tail end excretion of a pharmacologically relevant amount of the drug candidate. As the mere concentration of LGD-4033 in a sample does not provide sufficient information, the combined metabolite ratio and LGD-4033 concentration determined in doping control urine samples can be compared to data shown in this study to probe for scenarios compatible with micro-doses. The analyte pair that shows the most promising results is LGD-4033 and its epimer M1. Although M5-a and M5-b also show a ratio shift over time and offer the longest detection times, the ratio of M5-a and M5-b could not be employed for this approach as its increase is followed by a decline at a later stage of drug elimination, which prevents a reliable interpretation of the time line (data not shown).
M1 was synthesized by oxidation of the alcohol moiety of LGD-4033 utilizing a Dess-Martin reaction and subsequently reducing the resulting carbonyl moiety. This yielded a mixture of LGD-4033 and M1, which was separated on a preparative LC (see Methods—“Synthesis of M1”). The structure of M1 was confirmed as identical to LGD-4033 by 1H-NMR spectroscopy and the shift in retention time of 0.2 min in the LC–MS/MS method supports the identity of M1 as the epimer of LGD-4033.
The ratio M1/LGD-4033 is shown in Fig. 6a for all excretion study samples in which LGD-4033 could be positively identified using the mass spectrometric identification criteria (see Methods—“Assay characterization”). This approach reduces outliers, as the ratio tends to be unstable at low concentrations. To better simulate the likely contamination scenario of the daily intake of small amounts of LGD-4033, the ratio values of the multi-dose study were also included in the underlying data. With each new ingestion, the time was set back to 0 h. As some volunteers showed slightly increased ratios after multiple applications, the values between 0 and 4 h after a repeated ingestion were not included in the data to minimize this effect. The resulting data was tested for linear correlation with a resulting Pearson’s correlation coefficient of 0.87, a coefficient of determination of 0.76, and a p-value of < 0.001. The slope was determined at 0.0091 and the y-intercept at 0.018.
In addition to the micro-dose data, one set of previously collected excretion study urine samples collected after the ingestion of 10 mg of LGD-4033 was re-analyzed with the developed method . The M1/LGD-4033 ratios of these samples are depicted in red in Fig. 6a. These data points lie within the limits estimated from the micro-dose data, suggesting that the M1/LGD-4033 is dose-independent, but more controlled excretion studies would be necessary to corroborate that claim.
The data points were combined into ratio ranges and visualized using boxplots (Fig. 6b). The highest and lowest values in each range were set as limits for the estimation of the time passed since the ingestion of LGD-4033. Limitation of this approach is the inter-individual variation of the ratio/time relationship, which appears to increase with the time elapsed since ingestion. The limits were devised with the data of 30 excretion studies conducted with 13 different volunteers. Due to the low number of data points for M1/LGD-4033 ratios > 0.5, the informative value of the approach is reduced.
In addition to the time passed since the ingestion, the dose that was ingested can be roughly estimated by comparing the determined urinary LGD-4033 concentration to the range of concentrations measured in the micro-dose studies using the time range estimated from the M1/LGD-4033 abundance ratio. As the time ranges are large and the urinary concentrations of the target analytes vary between individuals, a substantial error of this approach cannot be excluded. Limitations of this approach include the small sample size of 30 studies conducted with 13 male volunteers. Age, weight, and athletic ability were not considered in the evaluation of the data. Nonetheless, this data is an important first step in estimating time and dose of LGD-4033 intake, contributing to decision-making processes in cases of AAFs.
Example of time and dose estimation
An exemplary scenario of the application of the method is presented here. A urinary concentration of 0.2 ng/mL of LGD-4033 with a M1/LGD-4033 ratio of 0.12 is detected in a routine doping control sample of an athlete. The question whether the athlete unknowingly ingested LGD-4033 through a daily consumed DS is raised, and analyses of the product reveal a contamination that results in a daily intake of approximately 10 µg of LGD-4033. Using the measured M1/LGD-4033 ratio and the correlation shown in Fig. 6, the intake of LGD-4033 is estimated to have occurred between 4 and 30.5 h before urine sample collection. Further, to estimate the dose of LGD-4033 that was ingested, the concentration of the sample is compared to the concentration range detected during the excretion studies. The concentrations of LGD-4033 in post-administration urine samples (10 µg of LGD-4033) collected between 4 and 30.5 h are found between 0.006 and 0.8 ng/mL, hence supporting the scenario of a DS being the source of the SARM ingested by the athlete.
As the analytical capabilities in doping control laboratories have improved in the past years, the retrospectivity and thus the necessity to differentiate between deliberate doping and unintentional exposure to prohibited substances have substantially increased. Here, human micro-dose elimination studies can provide critical data for decision-making processes in anti-doping. In this study, the elimination behavior of LGD-4033 after oral application of microgram amounts was examined by performing controlled elimination studies and analyzing collected urine samples with a developed LC–MS/MS method, which was validated for the qualitative detection of LGD-4033 in accordance with the WADA criteria. In addition to LGD-4033 itself, 15 metabolites were detected in human urine. Information on the elimination behavior, as well as the detection times of the different metabolites, was generated. The abundance ratio of the isomeric analytes LGD-4033 and M1 was shown to exhibit a linear relationship with the time passed since the ingestion. Therefore, using the analyte ratio in an unknown sample, an estimation about the time point of drug exposure/administration is possible. By comparing the urinary LGD-4033 concentration of the unknown sample to the concentration range of the micro-dose data, a rough estimation of the administered dose can also be made. The limitation of this approach is the low number of samples providing the data that compose the interpretation scheme. For more robust information, additional elimination studies with a larger number of volunteers and further dosing (and alternative routes of administration) appear warranted.