‘Wrong‐way‐round ionization’ and screening for doping substances in human urine by high‐performance liquid chromatography/orbitrap mass spectrometry

Abstract

To free analytical resources for new classes of doping substances, such as banned proteins, maximization of the number of compounds that can be determined with high sensitivity in a single run is highly urgent. This study demonstrates an application of 'wrong-way-round ionization' for the simultaneous detection of multiple classes of doping substances without the need to switch the polarity. A screening method for the detection of 137 compounds from various classes of prohibited substances (stimulants, diuretics, β(2)-agonists, β-blockers, antiestrogens, glucocorticosteroids and anabolic agents) has been developed. The method involves an enzymatic hydrolysis, liquid-liquid extraction and detection by liquid chromatography/orbitrap mass spectrometry with wrong-way-round ionization. Up to 64% of compounds had a 10-fold lower limit of detection (LOD) than the minimum required performance limit. To compare the efficiency of conventional ionization relative to wrong-way-round ionization of doping substances in + ESI, a fortified blank urine sample at the minimum required performance limit was analyzed using two ESI approaches. All compounds were detected with markedly better S/N in a high-pH mobile phase, with the exception of acetazolamide (minimal change in S/N, < 20%).The method was validated by spiking 10 different blank urine samples at five different concentrations. Validation parameters included the LOD, selectivity, ion suppression, extraction recovery and repeatability.

Non‐targeted acquisition strategy for screening doping compounds based on GC‐EI‐hybrid quadrupole‐Orbitrap mass spectrometry: A focus on exogenous anabolic steroids

“…Thus, for some cases, the required sensitivity established by WADA cannot be achieved . Moreover, electron ionization (EI) is less susceptible to unpredictable ionization mechanisms than electrospray ionization (ESI) …”

“…Most recently, liquid chromatography with atmospheric pressure ionization interfaced to hybrid high resolution mass spectrometry (HRMS) instruments like quadrupole-time of flight (Q-TOF) and quadrupole-orbitrap analyzers have been employed in drug analysis. [4][5][6] Nevertheless, some compounds, mainly androgenic anabolic steroids (AASs), have poor ionization efficiency in atmospheric pressure ionization sources. [7][8][9] Thus, for some cases, the required sensitivity established by WADA cannot be achieved.…”

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

“…Ionization capability was subject to investigation in the development of a complementary LC‐high resolution/high accuracy MS (LC‐HRMS) method in 2012 . Using the so‐called wrong‐way‐round ionization, a total of 137 analytes belonging to the prohibited substance classes S1, S3, S4‐S7, S9, and P2 were measured in a single run (17 min) with positive ESI and HRMS.…”

Supercritical fluid (CO2) chromatography for quantitative determination of selected cancer therapeutic drugs in the presence of potential impurities

“…[15][16][17][18][19][20][21][22] One validated high performance liquid chromatography method for the determination of fulvestrant in pharmaceutical dosage forms is reported by Varanasi, using a normal phase separation on a cyano column by employing a mixture of nhexane and isopropyl alcohol as an eluent. It utilizes a mobile phase consisting of solution A (water : acetonitrile : methanol 41 : 32 : 27) and solution B consisting of (acetonitrile : methanol : water 49 : 41 : 10).…”