UHPLC-HRMS Method for the Simultaneous Screening of 235 Drugs in Capillary Blood for Doping Control Purpose: Comparative Evaluation of Volumetric and Non-volumetric Dried Blood Spotting Devices.

We present a quick and simple multi-targeted analytical workflow based on ultra-high-performance liquid chromatography coupled to high-resolution mass spectrometry for the screening in dried blood spots and dried plasma spots of a wide variety of drugs with different chemical properties. Seven different microsampling devices were evaluated in view of their application for the detection of the selected target analytes in the framework of doping control analysis. The extraction of the analytes was optimized by assessing the efficacy of protocols based on ultrasonication with aqueous buffers and/or organic solvents of different polarities. Optimal recoveries were obtained by using pure methanol or mixtures of methanol/acetonitrile and methanol/isopropanol, depending on both the device and the target analytes. The method was fully validated according to both ISO17025 and the requirements of the World Anti-Doping Agency: all the analytes were clearly distinguishable from the matrix, with limits of detection in the range of 0.1-3.0 ng mL-1. Stability studies simulating the storage of samples before the analysis and in view of a possible re-analysis showed that most of the analytes were stable for at least 24 h at 50 °C and for at least 3 weeks at 25 and at 4 °C. The real applicability of the method was assessed by analyzing the samples collected after the administration of two model drugs, acetazolamide and deflazacort. The performance of the method was confirmed to be fit for purpose, and data obtained in blood can also be used to complement those available in urine, allowing to refine the knowledge concerning the pharmacokinetic profiles.

Urinary excretion profile of prednisolone and prednisone after rectal administration: Significance in antidoping analysis.

The rectal administration of glucocorticoids, as well as any injectable, and oral ones, is currently prohibited by the World Anti-Doping Agency when occurs "in competition." A reporting level of 100 ng/ml for prednisolone and 300 ng/ml for prednisone was established to discriminate the allowed and the prohibited administration. Here, the urinary excretion profiles of prednisone and prednisolone were evaluated in five volunteers in therapy with glucocorticoid-based rectal formulations containing prednisone or prednisolone caproate. The urinary levels of the excreted target compounds were determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS) following the procedure validated and currently in use in our laboratory to detect and quantitate glucocorticoids in urine. Predictably, the excretion trend of the analytes of interest were generally comparable with those obtained after oral administration, even if the excretion profile showed a broad interindividual variability, with the absorption rate and the systemic bioavailability after rectal administration being strongly influenced by the type of formulations (suppository or rectal cream, in our case) as well as the physiological conditions of the absorption area. Results showed that the target compounds were detectable for at least 30 h after drug administration. After suppository administration, prednisolone levels reached the maximum after 3 h from drug administration and then dropped below the reporting level after 15-21 h; prednisone reached the maximum after 3 h from drug administration, and then dropped below the reporting level after 12-15 h. After cream administration, both prednisone and prednisolone levels remained in a concentration below the reporting level throughout the entire monitored period.

Application of liquid chromatography coupled to data-independent acquisition mass spectrometry for the metabolic profiling of N-ethyl heptedrone.

We have investigated the metabolic profile of N-ethyl heptedrone, a new designer synthetic stimulant drug, by using data independent acquisition mass spectrometry. Phase I and phase II metabolism was studied by in vitro models, followed by liquid-chromatography coupled to mass spectrometry, to characterize and pre-select the most diagnostic markers of intake. N-ethyl heptedrone was incubated in the presence of pooled human liver microsomes. The contribution of individual enzymatic isoforms in the formation of the phase I and phase II metabolites was further investigated by using human recombinant cDNA-expressed cytochrome P450 enzymesand uridine 5'-diphospho glucuronosyltransferases. The analytical workflow consisted of liquid-liquid extraction with tert-butyl-methyl-ether at alkaline pH, performed before (to investigate the phase I metabolic profile) and after (to investigate the glucuronidation profile) enzymatic hydrolysis. The separation, identification, and determination of the compounds formed in the in vitro experiments were carried out by using liquid chromatography coupled to either high- or low-resolution mass spectrometry. Data independent acquisition method, namely sequential window acquisition of all theoretical fragment-ion spectra (SWATH®) and product ion scan were selected for high-resolution mass spectrometry, whereas multiple reaction monitoring was used for low-resolution mass spectrometry. Thirteen phase-I metabolites were isolated, formed from reactions being catalyzed mainly by CYP1A2, CYP2C9, CYP2C19 and CYP2D6 and, to a lesser degree, by CYP3A4 and CYP3A5. The phase I biotransformation pathways included hydroxylation in different positions, reduction of the ketone group, carbonylation, N-dealkylation, and combinations of the above. Most of the hydroxylated metabolites underwent conjugation reactions to form the corresponding glucurono-conjugated metabolites. Based on our in vitro observation, the metabolic products resulting from reduction of the keto group, N-dealkylation and hydroxylation of the aliphatic chain appear to be the most diagnostic target analytes to be selected as markers of exposure to N-ethyl heptedrone.

Effects of the administration of miconazole by different routes on the biomarkers of the "steroidal module" of the Athlete Biological Passport.

This article reports the results obtained from the investigation of the influence of miconazole administration on the physiological fluctuation of the markers of the steroid profile included in the "steroidal module" of the Athlete Biological Passport. Urines collected from male Caucasian subjects before, during, and after either systemic (i.e., oral and buccal) or topical (i.e., dermal) treatment with miconazole were analyzed according to validated procedures based on gas chromatography coupled to tandem mass spectrometry (GC-MS/MS) (to determine the markers of the steroid profile) or liquid chromatography coupled to MS/MS (LC-MS/MS) (to determine miconazole urinary levels). The results indicate that only after systemic administration, the markers of the steroid profile were significantly altered. After oral and buccal administration, we have registered (i) a significant increase of the 5α-androstane-3α,17ß-diol/5ß-androstane-3α,17ß-diol ratio and (ii) a significant decrease of the concentration of androsterone, etiocholanolone, 5ß-androstane-3α,17ß-diol, and 5α-androstane-3α,17ß-diol and of the androsterone/etiocholanolone, androsterone/testosterone, and 5α-androstane-3α,17ß-diol/epitestosterone ratios. Limited effects were instead measured after dermal intake. Indeed, the levels of miconazole after systemic administration were in the range of 0.1-12.5 µg/ml, whereas after dermal administration were below the limit of quantification (50 ng/ml). Significant alteration started to be registered at concentrations of miconazole higher than 0.5 µg/ml. These findings were primarily explained by the ability of miconazole in altering the kinetic/efficacy of deglucuronidation of the endogenous steroids by the enzyme ß-glucuronidase during the sample preparation process. The increase of both incubation time and amount of ß-glucuronidase was demonstrated to be effective countermeasures in the presence of miconazole to reduce the risk of uncorrected interpretation of the results.

Simultaneous detection of different chemical classes of selective androgen receptor modulators in urine by liquid chromatography-mass spectrometry-based techniques.

Analytical procedures to detect the misuse of selective androgen receptor modulators in human urine, targeting either the parent drugs and/or their main metabolites, were developed and validated. In detail, 19 target compounds belonging to 9 different chemical classes were considered: arylpropionamide (i.e., andarine (S4), ostarine (S22), S1, S6, S9 and S23), diarylhydantoin (i.e., GLPG0492), indole (i.e., LY2452473, GSK2881078), isoquinoline-carbonyle (i.e., PF-02620414), phenyl-oxadiazole (i.e., RAD140), pyrrolidinyl-benzonitrile (i.e., LGD4033), quinolinone (i.e., LGD2226, LGD3303), steroidal (i.e., Cl-4AS-1, MK0773 and TFM-4AS-1), and tropanol (i.e., AC-262536 and ACP105) derivatives. The metabolites of the target compounds considered were enzymatically synthesized by using human liver microsomes. Sample pre-treatment included enzymatic hydrolysis followed by liquid-liquid extraction at neutral pH. The instrumental analysis was performed by ultra-high-performance liquid chromatography coupled to either high- or low-resolution mass spectrometry. Validation was performed according to the ISO 17025 and the World Anti-Doping Agency guidelines. The analyses carried out on negative samples confirmed the method's selectivity, not showing any significant interferences at the retention times of the analytes of interest. Detection capability was determined in the range of 0.1-1.0 ng/mL for the screening procedure and 0.2-1.0 ng/mL for the confirmation procedure (except for GLPG0492 and GSK2881078). The recovery was greater than 80 % for all analytes, and the matrix effect was smaller than 35 %. The method also matched the criteria of the World Anti-Doping Agency in terms of repeatability of the relative retention times (CV% < 1.0) and of the relative abundances of the selected ion transitions (performed only in the case of triple quadrupole, CV% < 15), ensuring the correct identification of all the analytes considered. Urine samples containing andarine, ostarine, or LGD4033 were used to confirm the actual applicability of the selected analytical strategies. All target compounds (parent drugs and their main metabolites) were detected and correctly identified.

UPLC-MS-Based Procedures to Detect Prolyl-Hydroxylase Inhibitors of HIF in Urine.

This article presents newly developed screening and confirmation analytical procedures to detect the misuse of nine prolyl-hydroxylase inhibitors of the hypoxia-inducible factor: daprodustat, desidustat, FG2216, IOX2, IOX4, JNJ-42041935, molidustat, roxadustat and vadadustat, targeting either the parent drugs and/or their main metabolite(s). For the sample pretreatment, different extraction protocols and technologies were evaluated. The instrumental analysis was performed by ultra-high-performance liquid chromatography coupled to either high- or low-resolution mass spectrometry. The chromatographic separation was performed on a C18 column, employing water and acetonitrile, both containing 0.1% formic acid, as mobile phase. Detection was achieved using as analyzer either a triple quadrupole or an Orbitrap, with positive and negative electrospray ionization and different acquisition modes. Validation of the procedures was performed according to the ISO 17025 and World Anti-Doping Agency guidelines. The methods do not show any significant interference at the retention times of the analytes of interest. The extraction efficiency was estimated to be greater than 75% for all analytes and the matrix effect smaller than 35%. Detection capability was determined in the range of 0.25-2.0 for the screening procedure and in the range of 0.5-2.0 ng/mL for the confirmation procedure, that is, in a range of concentration small enough to reveal the abuse of the compounds considered, in case they are used as performance-enhancing agents. The repeatability of the relative retention times (CV% < 0.5) and of the relative abundances of the selected ion transitions, considered only in the case of triple quadrupole (CV% < 15), was confirmed to be fit for purpose to ensure the unambiguous identification of all the target analytes in human urine. The applicability of the newly developed methods was verified by the analysis of urine samples containing molidustat, roxadustat or daprodustat. The developed procedures enabled to detect the compounds under investigation and their main metabolites.

Urinary excretion profile of prednisone and prednisolone after different administration routes.

The urinary excretion profile of prednisolone and prednisone after both systemic (i.e., oral) and topical (i.e., ocular and intranasal) administration was studied by liquid chromatography coupled to mass spectrometry, also to select the most appropriate marker(s) of intake for doping control purposes. Urines were collected from ten subjects every 3 h before and after the administration of therapeutic doses of pharmaceutical formulations containing either prednisone or prednisolone. Samples were subjected to enzymatic hydrolysis (performed for the investigation on the glucuronide profile) followed by liquid/liquid extraction with tert-butylmethylether in alkaline conditions. The chromatographic separation was carried out on C18 column, employing as mobile phases ultrapurified water and acetonitrile, both containing 0.1% of formic acid. Detection was achieved using as mass spectrometric analyzer a triple quadrupole, with positive ion electrospray ionization and multiple reaction monitoring as acquisition mode. After both systemic and topical use, the compounds excreted in urine in higher concentration were prednisone, prednisolone and 20ß-dihydro-prednisolone followed by 20α-dihydro-prednisolone and 20α/ß-dihydro-prednisone. All were excreted mainly as unconjugated compounds, with a maximum of excretion in the first 3-9 h after the administration. After systemic use, prednisone and prednisolone were both detectable for at least 24 h in concentrations ranging from 5 to 500 ng/mL and from 5 to 900 ng/mL respectively. Whereas, after topical administration, prednisone and prednisolone were detectable for at least 18 h in concentrations ranging from 5 to 140 ng/mL and from 5 to 50 ng/mL respectively.

Detection of 5α-reductase inhibitors by UPLC-MS/MS: Application to the definition of the excretion profile of dutasteride in urine.

An analytical procedure based on ultra-performance liquid chromatography-mass spectrometry was developed to screen and to confirm dutasteride and its metabolites in human urine. Sample preparation included an enzymatic hydrolysis followed by solid-phase extraction using the strong cation exchange cartridges OASIS® MCX. The chromatographic separation was carried out on C18 column, employing as mobile phases ultra purified water and acetonitrile, both containing 0.1% formic acid. Detection was achieved using a triple quadrupole as a mass spectrometric analyzer, with positive ion electrospray ionization and multiple reaction monitoring as acquisition mode. The analytical procedure developed was validated according to ISO 17025 and World Anti-Doping Agency guidelines. The extraction efficiency was estimated to be greater than 75% for both dutasteride and its hydroxylated metabolites. Detection capability was determined in the range of 0.1-0.4 ng/mL. Specificity and repeatability of the relative retention times (CV% < 0.5) and of the relative abundances of the characteristic ion transitions selected (CV% < 10) were confirmed to be fit for purpose to ensure the unambiguous identification of dutasteride and its metabolites in human urine. The developed method was used to characterize the urinary excretion profile of dutasteride after both chronic and acute administration of therapeutic doses. After chronic administration, dutasteride and its hydroxylated metabolites were easily detected and confirmed. After acute administration, instead, only the two hydroxylated metabolites were detected for 3-4 days.