“Dilute-and-inject” multi-target screening assay for highly polar doping agents using hydrophilic interaction liquid chromatography high resolution/high accuracy mass spectrometry for sports drug testing

Abstract

In the field of LC-MS, reversed phase liquid chromatography is the predominant method of choice for the separation of prohibited substances from various classes in sports drug testing. However, highly polar and charged compounds still represent a challenging task in liquid chromatography due to their difficult chromatographic behavior using reversed phase materials. A very promising approach for the separation of hydrophilic compounds is hydrophilic interaction liquid chromatography (HILIC). Despite its great potential and versatile advantages for the separation of highly polar compounds, HILIC is up to now not very common in doping analysis, although most manufacturers offer a variety of HILIC columns in their portfolio. In this study, a novel multi-target approach based on HILIC high resolution/high accuracy mass spectrometry is presented to screen for various polar stimulants, stimulant sulfo-conjugates, glycerol, AICAR, ethyl glucuronide, morphine-3-glucuronide, and myo-inositol trispyrophosphate after direct injection of diluted urine specimens. The usage of an effective online sample cleanup and a zwitterionic HILIC analytical column in combination with a new generation Hybrid Quadrupol-Orbitrap® mass spectrometer enabled the detection of highly polar analytes without any time-consuming hydrolysis or further purification steps, far below the required detection limits. The methodology was fully validated for qualitative and quantitative (AICAR, glycerol) purposes considering the parameters specificity; robustness (rRT < 2.0%); linearity (R > 0.99); intra- and inter-day precision at low, medium, and high concentration levels (CV < 20%); limit of detection (stimulants and stimulant sulfo-conjugates < 10 ng/mL; norfenefrine; octopamine < 30 ng/mL; AICAR < 10 ng/mL; glycerol 100 μg/mL; ETG < 100 ng/mL); accuracy (AICAR 103.8-105.5%, glycerol 85.1-98.3% at three concentration levels) and ion suppression/enhancement effects.

Applications and Challenges in Using LC–MS/MS Assays for Quantitative Doping Analysis

“…To achieve baseline chromatographic separation of pseudoephedrine and its metabolite, norpseudoephedrine, which are ephedrine and norephedrine diastereomers, respectively, only one dilution step is needed for LC-MS or LC-MS/MS [22,42,43]. For plasma and serum samples, protein precipitation requires dilution with methanol [44] or acetonitrile [45,46,49], or LLE [47,48].…”

“…Compound identification confidence is with the isotopic abundance patterns used in mass analysis of complex biological samples. Currently, two widely used HRMS technologies, TOF and orbitrap are used for doping analysis [20,37,42,53,62] and TOFMS can be used to measure long-lasting metabolites by mass spectra. These long detection windows have been used for exogenous steroid detection.…”

“…THCA and its glucuronide conjugate are major urinary cannabinoid metabolites and the WADA threshold for THCA is 150 ng/ml. Dilute-and-shoot method development and validation for LC-MS/MS to measure urinary THCA has been described in the literature [10][11][12] and this approach permitted more rapid sample preparation with fewer steps prior to LC-MS/ THCA 150 ng/ml 15 ng/ml 10 180 ng/ml [10][11][12][13][14][15][16][17][18][19][20][21] Salbutamol 1.0 μg/ml 0.1 μg/ml 10 1.2 μg/ml [22][23][24] Formoterol 40 ng/ml 6.0 ng/ml 15 50 ng/ml [10,[25][26][27] Glycerol 4.3 mg/ml 0.65 mg/ml 15 5.3 mg/ml [28][29][30] Morphine 1.0 μg/ml 0.15 μg/ml 15 1.3 μg/ml [31][32][33][34][35][36][37][38][39][40][41] Cathine 5.0 μg/ml 0.5 μg/ml 10 6.0 μg/ml [22] Ephedrine 10 μg/ml 0.5 μg/ml 5 11 μg/ml [42][43][44][45][46][47][48][49]…”

Systematic evaluation of matrix effects in hydrophilic interaction chromatography versus reversed phase liquid chromatography coupled to mass spectrometry

“…Hydrophilic interaction chromatography (HILIC) is nowadays considered as a viable alternative to reverse-phase liquid chromatography (RPLC) for the analysis of polar and/or ionizable compounds in bioanalysis [12][13][14]. HILIC involves the use of a polar stationary phase and an aqueous-polar organic solvent mobile phase containing between 60 and 95% of an aprotic organic solvent miscible with water, usually acetonitrile (ACN).…”

Analysis of Glycerol with Isolation of Endogenous Interferences using "Dilute and Shoot" Strategy and High-Resolution Mass Spectrometry in Human Urine for Antidoping Testing

“…This method can be applied successfully in polar compounds or metabolites that are difficult to be extracted or detected. 8 This strategy was successfully demonstrated for selected compounds in diuretics, stimulants, narcotics, plasma volume expander, and beta-2-agonists. [9][10][11][12] Although "dilute and shoot" method can provide various advantages, some problems persist concerning an ionization suppression 13 or a spectral congestion.…”