Comprehensive analysis by liquid chromatography Q‐Orbitrap mass spectrometry: Fast screening of peptides and organic molecules

Abstract

The number of substances nominally listed in the prohibited list of the World Anti-Doping Agency increases each year. Moreover, many of these substances do not have a single analytical target and must be monitored through different metabolites, artifacts, degradation products, or biomarkers. A new analytical method was developed and validated for the simultaneous analysis of peptides and organic molecules using a single sample preparation and LC-Q-HRMS detection. The simultaneous analysis of 450 target molecules was performed after cleanup on a mixed-mode solid-phase extraction cartridge, combined with untreated urine. The cleanup solvent and reconstitution solvent were the most important parameters for achieving a comprehensive sample preparation approach. A fast chromatographic run based on a multistep gradient was optimized under different flows; the detection of all substances without isomeric coelution was achieved in 11 minutes, and the chromatographic resolution was considered a critical parameter, even in high-resolution mass spectrometry detection. The mass spectrometer was set to operate by switching between positive and negative ionization mode for FULL-MS, all-ion fragmentation, and FULL-MS/MS . The suitable parameters for the curved linear trap (c-trap) conditions were determined and found to be the most important factors for the development of the method. Only FULL-MS/MS enables the detection of steroids and peptides at concentrations lower than the minimum required performance levels set by World Anti-Doping Agency (1 ng mL ). The combination of the maximum injection time of the ions into the c-trap, multiplexing experiments, and loop count under optimized conditions enabled the method to be applied to over 10 000 samples in only 2 months during the 2016 Rio Summer Olympic and Paralympic Games. The procedure details all aspects, from sample preparation to mass spectrometry detection. FULL-MS data acquisition is performed in positive and negative ion mode simultaneously and can be applied to untargeted approaches.

Annual banned‐substance review: Analytical approaches in human sports drug testing

“…The use of liquid chromatography (LC) combined with HRMS and tandem mass spectrometry (MS/MS) as ITP has been established and utilized since years, and the extent of compounds covered by this methodology has been constantly growing. Recently, Sardela et al presented an assay allowing the screening of a total of 450 analytes in one analytical run, including 33 anabolic agents (or respective metabolites) . Here, urine was subjected to enzymatic hydrolysis followed by weak cation exchange/mixed mode solid‐phase extraction (SPE).…”

“…Recently, Sardela et al presented an assay allowing the screening of a total of 450 analytes in one analytical run, including 33 anabolic agents (or respective metabolites). 36 Here, urine was subjected to enzymatic hydrolysis followed by weak cation exchange/mixed mode solid-phase extraction (SPE). The extract was finally topped-up with diluted but otherwise untreated urine prior to injection into the LC-MS(/MS) system to include also conjugated metabolites and ionic compounds (such as meldonium) that are not easily extracted in urine via general approaches.…”

Determination of adulterants in whey protein food supplements by liquid chromatography coupled to Orbitrap high resolution mass spectrometry

“…A comparison between the theoretical and experimental precursor molecular mass values was evaluated in the identification of molecule structures. The instrumental conditions used were established according to anti-doping control (Sardela et al, 2018).…”

Zebrafish (Danio rerio) water tank model for the investigation of drug metabolism: Progress, outlook, and challenges

“…For the analysis of JWH-073 and selegiline by LC-HRMS, the method used was the same described by Sardela et al 18 The LC system was a …”

“…The extraction procedure for the LC-HRMS analysis of ZWT aliquots for the study of SIB, selegiline, and JWH-073 was adapted from Sardela et al 18 Briefly, 3 mL aliquots from all ZWTs were added to buspirone solution (internal standard, 50 ng/mL) and 7propylteophiline, 750 μL of pH 7.0 phosphate buffer and 50 μL of βglucuronidase solution from E. coli. The tubes were shaken and placed in a water bath at 50°C for 1 hour.…”