Elucidating important factors and corresponding method optimization for sensitive detection of small peptide drugs in human urine by solid-phase extraction and UPLC-HRMS: The influence of MS scan modes, protein precipitants, and ammonium formate.

Small peptide hormones are widely used in sports as performance-enhancing substances, making it crucial to develop sensitive analytical methods for their detection in doping control analysis. Various factors significantly affect analytical sensitivity, such as the selection of ultra-performance liquid chromatography (UPLC) mobile phase, high-resolution mass spectrometry (HRMS) scanning modes, and extraction solvents for pretreatment. Herein, comparative study approach was utilized to investigate the sensitivity of each peptide analyte under both full scan and parallel reaction monitoring (PRM) modes of HRMS and assess the effects of some protein precipitants as a part of extraction solvents on solid-phase extraction (SPE). The results showed that full scan should be selected as the primary scan mode of HRMS, and the combination with PRM mode could effectively compensate for the limitations of full scan, and the addition of protein precipitants would adversely affect the detection of certain small peptide analytes. Meanwhile, influences of ammonium formate in reverse UPLC mobile phase on the charge state distribution of small peptides were investigated and elucidated. Based on these findings, a sensitive and reliable UPLC-HRMS analytical method combining full scan and PRM mode was validated for screening and confirmation of 63 small peptide analytes after SPE, with limits of detection (LODs) ranging between 0.010 and 0.473 ng/ml and limits of identification (LOIs) ranging between 0.015 and 1.512 ng/ml. Additionally, suggestions were provided for the detection of [Arg8]-vasopressin, dermorphin, and its analogues.

Gas chromatography properties and mass spectrometry fragmentation of anabolic androgenic steroids in doping control.

Aim: This study aimed to investigate the gas chromatographic properties and mass spectrometric fragmentations of anabolic androgenic steroids (AASs) after trimethylsilylated derivatization. Materials & methods: A total of 113 AASs were analyzed through gas chromatography-mass spectrometry in the full-scan mode. Results: New fragmentation pathways yielding m/z 129, 143 and 169 ions were analyzed. Based on the characteristics of the A-ring, seven classes of drugs were identified and analyzed. Conclusion: The fragmentation pathway of a new classification of 4-en-3-hydroxyl was reported for the first time. The relationship between the chemical structures of AASs and their retention time, along with their molecular ion peak abundance, was also reported herein for the first time.

A comprehensive and high-throughput screening method for multiple prohibited substances by UPLC-QE Plus-HRMS and HPLC-QQQ-MS in human urine for doping control.

Since the World Anti-Doping Agency's (WADA) Prohibited List is updated on an annual basis, screening methods must be continually adapted to align with these changes. In accordance with Technical Document-MRPL 2022, a newly combined, comprehensive, rapid and high-throughput doping control screening method has been developed for the analysis of 350 substances with different polarities in human urine using ultra-high performance liquid chromatography coupled with Q Exactive Plus Hybrid Quadrupole-Orbitrap mass spectrometer (UPLC-QE Plus-HRMS) and ultra-high performance liquid chromatography coupled with triple quadrupole mass spectrometer (UPLC-QQQ-MS). The limits of detection were in the range of 0.12-50 ng mL-1 for beta-2 agonists, hormone and metabolic modulators, narcotics, cannabinoids and glucocorticoids, 0.1-14 ng mL-1 for the manipulation of blood and blood components, beta blockers, anabolic agents and hypoxia-inducible factor (HIF) activating agents, and 2.5-100 000 ng mL-1 for substances of Appendix A, diuretics & masking agents and stimulants. The sample preparation consisted of two parts: one is the dilute & shoot part analyzed in UPLC-QQQ-MS, another is a mixture of the dilute & shoot part and a liquid-liquid extraction part of hydrolyzed human urine analyzed in UPLC-QE Plus-HRMS in full scan mode with polarity switching and parallel reaction monitoring (PRM) mode. The method has been fully validated for doping control purposes. All the substances were compliant with WADA's required 1/2 minimum requirement performance level (MRPL) or minimum reporting level (MRL), and this method was successfully used in the 2022 Beijing Winter Olympic Games and Winter Paralympic Games for anti-doping purpose.

Quantification of Ephedrine Substances in Human Urine by Ultra-Performance Liquid Chromatography-Tandem Mass Spectrometry.

An accurate quantitative method for four prohibited ephedrine substances in human urine has been established, based on ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS). The quantitative bias caused by pretreatment operations and matrix effects was reduced by the dilute and shoot pretreatment method. The good separation of isomers was achieved with the advantages of the UPLC instrument and Agilent Poroshell 120 EC-C8 UPLC column. Stable quantitative ions were selected during the analysis with MS/MS. The result of the method validation experiment showed an excellent linearity between 50% and 200% threshold concentration with a correlation coefficient (r2) greater than 0.999. The coefficient of variation at the limit of quantification and threshold was <20% and 10%, respectively. The uncertainty was below the maximum uncertainty specified in the technical document of the World Anti-Doping Agency (WADA). The analytical result using this method has passed the WADA-external quality assessment scheme. The anti-doping laboratory has applied the method in routine tests and reported adverse analytical finding.

Metabolic Profiles of 5F-MDMB-PICA in Human Urine, Serum and Hair Samples Using LC-Q Exactive HF-MS.

In 2020, 5F-MDMB-PICA (5F-MDMB-2201) was one of the most common synthetic cannabinoids (SCs) identified in drugs seized by the Beijing Drug Control Agency, and it was categorized as Schedule II by the United Nations Office on Drugs and Crime in March 2020. It is difficult to detect 5F-MDMB-PICA in biological matrices due to its fast metabolic rate in vivo. In this work, 5F-MDMB-PICA metabolic profiles were investigated by liquid chromatography--quadrupole exactive high field orbitrap mass spectrometer (LC-Q Exactive HF MS), with accurate mass measurements in human urine, serum and pubic hair. To obtain intact metabolites, solid-phase extraction for urine and serum and direct ultrasonic extraction for pubic hair were applied to clean the samples without enzymatic hydrolysis. The differences in 5F-MDMB-PICA metabolism in the three different matrices were compared for the first time to determine the best detection biomarkers for monitoring 5F-MDMB-PICA misuse. Urine samples were determined to be the preferred biological material for identifying 5F-MDMB-PICA abuse. Forty-seven intact metabolites were detected in human urine, the ester hydrolyzed with glucuronidated metabolite in urine samples can be used as the primary biomarker to identify drug misuse. Fifteen metabolites were found in serum samples. Ester hydrolysis was considered to be the major metabolic pathway, and a large number of metabolites were involved with it. Zero metabolites apart from the parent drug were detected in pubic hair samples. Twenty-eight new metabolites and their metabolic pathways were characterized and tentatively identified by LC-QE-HF-MS, and a new potential biomarker (M5 ester hydrolysis + propionic acid) was also identified.

Doping control analysis of small peptides in human urine using LC-HRMS with parallel reaction monitoring mode: screening and confirmation.

This study described a reliable analytical method, which combines solid-phase extraction (SPE) with liquid chromatography-high resolution mass spectrometry (LC-HRMS) employing the parallel reaction monitoring (PRM) mode, for screening 41 small peptides and 3 non-peptide growth hormone secretagogues in human urine. Additionally 36 small peptides and 3 non-peptide growth hormone secretagogues were also confirmed in the same way. For the whole screening procedure, the PRM mode was applied to the HRMS detection of small peptides, which reduces the background noise from matrix compounds to a large extent and thus improves the selectivity and reliability of the peptide analytes. Meanwhile, competent chromatographic separation was achieved within a total runtime of 14 minutes, indicating an improvement in the detection efficiency. Moreover, the PRM mode could also be applied to the confirmation procedure due to its strong identification power with a low risk of generating false positives or negatives and good selectivity. Validation was performed according to the relevant World Anti-Doping Agency (WADA) criteria, including selectivity and reliability, limit of detection (LOD), limit of identification (LOI), recovery, extraction stability and carryover. The LODs of the peptide analytes ranged between 0.20 ng mL-1 and 0.92 ng mL-1 in urine, while their LOIs ranged between 0.20 ng mL-1 and 2.00 ng mL-1, which met the corresponding Minimum Required Performance Levels (MRPLs) as defined by WADA. The developed method furnished the rapid and sensitive detection of small peptides in urine for more than 5000 samples with no false-positive or false-negative, indicating that it is an eligible method for doping control analysis.

Enantiomeric analysis of clenbuterol in Chinese people by LC-MS/MS to distinguish doping abuse from meat contamination.

Aim: Follow-up investigations are often required for clenbuterol-positive cases. A method to distinguish doping abuse from meat contamination was developed. Materials & methods: A total of 26 volunteers were recruited to ingest clenbuterol contaminated-pork and clenbuterol tablets. Results: For 20 volunteers, after ingestion of contaminated-pork, R-(-)/S-(+)-clenbuterol ratio was <1.0, while the value was >1.0 after taking clenbuterol tablets. However, after taking clenbuterol tablets, some ratio points of the other six volunteers were between 0.9 and 1.0. A case of an abnormal cold and fever, which returned to normal after recovery, was also reported firstly. Conclusion: A change in R-(-)/S-(+)-clenbuterol was reported in the Chinese population initially. A ratio of 0.9 was recommended in doping related cases for the Chinese population.

Doping control analysis of 13 steroids and structural-like analytes in human urine using Quadrupole-Orbitrap LC-MS/MS with parallel reaction monitoring (PRM) mode.

Anabolic androgenic steroids (AASs) and structural-like substances are commonly prohibited substances found in doping control studies that can be difficult to accurately detect. In the present study, 11 AASs and 2 structural-like substances that are commonly detected were examined. Currently, such analytes are detected using low resolution GC-MS/MS or LC-MS/MS, with detection not always possible. Herein, the high resolution Quadrupole-Orbitrap liquid chromatography-tandem mass spectrometry LC-MS/MS system Q Exactive was utilized to increase the specificity. This approach was then combined for the first time with parallel reaction monitoring (PRM) during the screening procedure. The results confirmed high specificity, with the LODs of all 13 analytes being at least 25-fold lower than corresponding MRPLs as defined by WADA. Furthermore, the extraction recoveries were above 70% and the intra- and inter-day precisions were lower than 15%. This approach was successfully applied to analyze over 10,000 samples with no false-positive or false-negative results, thus suggesting that Quadrupole-Orbitrap LC-MS/MS when combined with PRM is an effective method for doping control analysis.

New long term metabolite in human urine for metenolone misuse by liquid chromatography quadrupole time-of-flight mass spectrometry.

In this study, metenolone metabolic profiles were investigated. Metenolone was administered to one healthy male volunteer. Liquid-liquid extraction and direct-injection were applied to processing urine samples. Urinary extracts were analyzed by liquid chromatography quadrupole time-of-flight mass spectrometry (LC-QTOFMS) using full scan and product ion scan with accurate mass measurement for the first time. Due to the lack of useful fragment ion for structural elucidation, GC-MS instrumentation was employed to obtain structural details of the trimethylsilylated phase I metabolite released after hydrolysis, and the EI mass spectrum was always informative in steroidal structure studies owing to more useful fragment ions than the ESI mass spectrum. 16 metabolites including 6 glucuronide and 9 unreported sulfate conjugates were characterized and tentatively identified. All the metabolites were evaluated in terms of how long they could be detected. The sulfate conjugate S6 (1-methylen-5α-androst-3,17-dione-2ξ-sulfate) was considered to be a new long term metabolite for metenolone misuse that could be detected 40 days by liquid-liquid extraction and up to 30 days by direct-injection analysis after oral administration.

New potential biomarkers for mesterolone misuse in human urine by liquid chromatography quadrupole time-of-flight mass spectrometry.

In this paper, mesterolone metabolic profiles were investigated carefully. Mesterolone was administered to one healthy male volunteer. Urinary extracts were analyzed by liquid chromatography quadruple time-of-flight mass spectrometry (LC-QTOFMS) for the first time. Liquid-liquid extraction was applied to processing urine samples, and dilute-shoot analyses of intact metabolites were also presented. In LC-QTOFMS analysis, chromatographic peaks for potential metabolites were hunt down by using the theoretical [M-H](-) as target ions in full scan experiment, and their actual deprotonated ions were analyzed in targeted MS/MS mode. Ten metabolites including seven new sulfate and three glucuronide conjugates were found for mesterolone. Because of no useful fragment ion for structural elucidation, gas chromatography-mass spectrometry instrumentation was employed to obtain structural details of the trimethylsilylated phase I metabolite released after solvolysis. Thus, their potential structures were proposed particularly by a combined MS approach. All the metabolites were also evaluated in terms of how long they could be detected, and S1 (1α-methyl-5α-androst-3-one-17ß-sulfate) together with S2 (1α-methyl-5α-androst-17-one-3ß-sulfate) was detected up to 9 days after oral administration, which could be the new potential biomarkers for mesterolone misuse.

New clostebol metabolites in human urine by liquid chromatography time-of-flight tandem mass spectrometry and their application for doping control.

In this study, clostebol metabolic profiles were investigated carefully. Clostebol was administered to one healthy male volunteer. Urinary extracts were analyzed by liquid chromatography quadrupole time-of-flight mass spectrometry (MS) using full scan and targeted MS/MS techniques with accurate mass measurement for the first time. Liquid-liquid extraction and direct injection were applied to processing urine samples. Chromatographic peaks for potential metabolites were found by using the theoretical [M-H](-) as target ion in full scan experiment, and their actual deprotonated ions were analyzed in targeted MS/MS mode. Fourteen metabolites were found for clostebol, and nine unreported metabolites (two free ones and seven sulfate conjugates) were identified by MS, and their potential structures were proposed based on fragmentation and metabolism pathways. Four glucuronide conjugates were also first reported. All the metabolites were evaluated in terms of how long they could be detected and S1 (4ξ-chloro-5ξ-androst-3ξ-ol-17-one-3ξ-sulfate) was considered to be the long-term metabolite for clostebol misuse detected up to 25 days by liquid-liquid extraction and 14 days by direct injection analysis after oral administration. Five conjugated metabolites (M2, M5, S2, S6 and S7) could also be the alternative biomarkers for clostebol misuse.

A Sensitive Dilute-and-Shoot Approach for the Simultaneous Screening of 71 Stimulants and 7 Metabolites in Human Urine by LC-MS-MS with Dynamic MRM.

A novel, reliable and sensitive liquid chromatography-tandem mass spectrometry (LC-MS-MS) method was developed with dynamic multiple reaction monitoring (dMRM) mode for the simultaneous screening of 71 stimulants and 7 metabolites in human urine using unsophisticated MS instruments (Agilent triple-quadruple 6410 B mass spectrometer). With a known retention time of an analyte, dMRM algorithm monitors each MRM transition only around its expected retention time. Therefore, dMRM enables the maximization of dwell times and provides much higher sensitivity and reproducibility than the conventional multiple reaction monitoring mode (cMRM). After precipitation of protein, the urine sample was injected into LC-MS-MS system directly without sample pre-concentration. For comparison, cMRM and dMRM acquisitions were performed under the same chromatographic conditions. The result showed that the signal response and quality of the chromatograms for each stimulant improved significantly with dMRM over cMRM. The method has been fully validated giving limits of detection (0.1-25 ng/mL) satisfactory for its application to anti-doping analysis. The repeatability of the concentrations and the retention times are good both for intra- and for inter-day experiments (%CV of concentrations always <20 and %CV of retention times <0.5). The method also afforded satisfactory results in terms of accuracy, matrix effect and specificity.

Structural elucidation of new urinary tamoxifen metabolites by liquid chromatography quadrupole time-of-flight mass spectrometry.

In this study, tamoxifen metabolic profiles were investigated carefully. Tamoxifen was administered to two healthy male volunteers and one female patient suffering from breast cancer. Urinary extracts were analyzed by liquid chromatography quadruple time-of-flight mass spectrometry using full scan and targeted MS/MS techniques with accurate mass measurement. Chromatographic peaks for potential metabolites were selected by using the theoretical [M + H](+) as precursor ion in full-scan experiment and m/z 72, 58 or 44 as characteristic product ions for N,N-dimethyl, N-desmethyl and N,N-didesmethyl metabolites in targeted MS/MS experiment, respectively. Tamoxifen and 37 metabolites were detected in extraction study samples. Chemical structures of seven unreported metabolites were elucidated particularly on the basis of fragmentation patterns observed for these metabolites. Several metabolic pathways containing mono- and di-hydroxylation, methoxylation, N-desmethylation, N,N-didesmethylation, oxidation and combinations were suggested. All the metabolites were detected in the urine samples up to 1 week.

An improved liquid chromatography-tandem mass spectrometric method to quantify formoterol in human urine.

Formoterol is a new threshold substance in the prohibited list 2012 according to World Anti-Doping Agency. Extracted by ethyl acetate using formoterol-D6 as internal standard, formoterol underwent a constant flow rate gradient elution separation in reversed-phase liquid chromatography. Subsequently, mass spectrometry analysis was conducted by tandem mass spectrometry in the multiple reaction monitoring mode. According to the proposed method, a calibration curve was constructed in the range of 0.2-500 ng/mL (r(2) = 1) with a limit of quantification 0.2 ng/mL. The mean extracted recovery assessed at three different concentrations (1, 30 and 100 ng/mL) was more than 80%. The method was validated by the analysis of three quality control samples from World Association of Anti-Doping Scientists. In conclusion, the developed and validated method was sensitive, accurate and precise for the quantification of formoterol in human urine for doping control purposes.

Quantitative analysis of glycerol levels in human urine by liquid chromatography-tandem mass spectrometry.

Glycerol has the latent capacity to act as a plasma volume expander and disguise blood doping practices. Therefore, it has been prohibited in sports as a masking agent by the World Anti-Doping Agency (WADA) since January 2010 and a urinary threshold (1mg/mL) was recommended recently [1]. The purpose of this study was to establish and validate a novel quantitative method for the determination of urinary glycerol concentrations using a liquid chromatography-tandem mass spectrometry approach. This simple yet highly specific method made use of the derivatization of glycerol by benzoyl chloride in aqueous solution at 40°C followed by analysis via LC-ESI-MS/MS without sample pre-concentration or cleanup. The assay was linear over the concentration range of 1.0-1000µg/mL for glycerol in human urine. The lower limit of detection (LLOD) and lower limit of quantitation (LLOQ) were 0.3µg/mL and 1.0µg/mL, respectively. The intra- and inter-day precision of the method at three concentration levels (3, 500 and 900µg/mL) was less than 12.2%. The method also afforded satisfactory results in terms of accuracy, derivatization yield, extraction recovery, matrix effect and specificity. The method has been successfully applied to the detection of glycerol in "Quality Assurance Program" samples provided by the World Association of Anti-Doping Scientists (WAADS) and routine doping-control samples in our laboratory.

Mass spectrometric identification and characterization of new clomiphene metabolites in human urine by liquid chromatography-quadrupole time-of-flight tandem mass spectrometry.

Clomiphene, a selective estrogen receptor modulator, is prohibited by World Anti Doping Agency (WADA) out-of-competition and in-competition. As it is extensively metabolized, further investigation of clomiphene metabolic profile will be essential to routine anti-doping analysis. The metabolic pathway and the different metabolites of clomiphene in human urine collected from three healthy volunteers during 1 week were studied by liquid chromatography-quadrupole time-of-flight mass spectrometry (LC-QTOFMS) based on accurate mass measurement. Seven unreported metabolites were identified and characterized, and all of the newly found urinary metabolites belonged to a new metabolic pathway (hydrogenation). An approach for the metabolism study of clomiphene and its analogs by LC-QTOFMS was presented. Two metabolites, 3,4-dihydroxy-dihydro-clomiphene (m/z 440.1991) and 3,4-dihydroxy-dihydro-deethyl-clomiphne (m/z 412.1674), are the potential biomarkers for monitoring oral administration of clomiphene in doping control.

Mass spectrometric identification and characterization of new fluoxymesterone metabolites in human urine by liquid chromatography time-of-flight tandem mass spectrometry.

In this study fluoxymesterone urinary profiles were investigated by liquid chromatography quadrupole time-of-flight tandem mass spectrometry (LC-QTOFMS) with accurate mass measurement. Twelve metabolites including the parent drug were detected in two fluoxymesterone positive control urine samples. Three parameters were employed for evaluation of the accuracy of the chemical formulae in positive full scan experiment, which contained error between actual and calculated mass weights of prontonated and isotopic molecules together with abundance match between prontonated and isotopic molecules. The 13 analytes were determined with mass accuracy less than 1.1 ppm and isotopic abundance match more than 94 marks. Based on the ionization, CID fragmentation, the accurate mass of the product ion and comparison of the accurate mass weight and retention time with reference standard, fluoxymesterone and its 12 metabolites containing three unreported ones were detected. The chemical structures of three unreported metabolites were identified as: 9-fluro-17ß-ol-17-methyl-11-en-5α-androstan-3-one (F13), 9-fluro-17ß-ol-17-methyl-11-en-5ß-androstan-3-one (F8) and 9-fluro-17ß-ol-17-methyl-5-androstan-3,6,11-trione, and meanwhile a dihydroxylated metabolite (F12), 6,16-dihydroxylated fluoxymesterone, was also detected in human urine, which was previously reported to be available only in equine urine.

Efficient and green synthesis of bis(indolyl)methanes catalyzed by ABS in aqueous media under ultrasound irradiation.

Ultrasound-promoted synthesis of bis(indolyl)methanes catalyzed by ABS via the reaction of indole or N-methylindole with aromatic aldehyde was carried out in excellent yields in aqueous media at 23-25°C, providing a simple and efficient synthesis of these compounds.