Rapid screening of polysaccharide‐based plasma volume expanders dextran and hydroxyethyl starch in human urine by liquid chromatography–tandem mass spectrometry

Abstract

The increasing number of samples and target substances in doping control requires continuously improved screening methods, combining high-throughput analysis, simplified sample preparation, robustness and reliability. Hence, a rapid screening procedure based on liquid chromatography-electrospray ionization-tandem mass spectrometry with in-source collision-induced dissociation was developed. The detection of the polysaccharide-based plasma volume expanders dextran and hydroxyethyl starch (HES) in human urine was established without further sample preparation. The in-source fragmentation strategy of the approach represented a valuable tool in the analysis of the polysaccharide-based compounds, allowing the use of tandem mass spectrometry. After direct injection of urine specimens, analytes were chromatographically separated on a monolithic reverse-phase column and detected via multiple reaction monitoring of diagnostic ions at detection limits of 10 microg/mL for HES and 30 microg/mL for dextran. Validation was performed regarding the parameters specificity, linearity, precision (8-18%) and accuracy (77-105%) and the method was applied to the investigation of approximately 400 doping control samples and seven dextran and two hydroxyethyl starch post-administration samples. The approach demonstrated its capability as a rapid screening tool for the detection of dextran and hydroxyethyl starch and represents an alternative to existing screening procedures since time consuming hydrolysis or derivatization steps were omitted.

High‐throughput screening for various classes of doping agents using a new ‘dilute‐and‐shoot’ liquid chromatography‐tandem mass spectrometry multi‐target approach

“…On the basis of modern and powerful analytical instruments consisting of liquid chromatographs coupled to sensitive mass spectrometers – for example, triple‐quadrupole, time‐of‐flight (TOF) or Fourier transform (OrbiTrap) instruments – many new multi‐target assays have been developed providing reliable detection of prohibited substances at the required limits. Currently, different screening procedures are utilized for the analysis of diuretics, beta2‐agonists, stimulants, narcotics, and plasma volume expanders (PVE) . With the ongoing progress in the field of liquid chromatography and triple quadrupole mass spectrometry, new generation instruments have become accessible with the possibility of enhanced scan speed and scan‐to‐scan polarity switching.…”

“…Using a high‐sensitive next‐generation triple‐quadrupole mass spectrometer, a comprehensive multi‐target approach was developed that, for the first time, enables a combined screening of diuretics, beta2‐agonists, narcotics, stimulants, and their sulfo‐conjugates and PVEs. One main achievement was the implementation of the PVEs dextran and HES, commonly analyzed in time‐consuming stand‐alone procedures . Moreover, analyzing intact sulfo‐conjugates of stimulating agents enables screening for numerous prohibited compounds and avoids complex sample preparation and hydrolysis steps.…”

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“…To solve this problem, a threshold of 500 µ g · mL −1 has been proposed for screening purposes [9]. Indeed, several negative samples, analyzed with this method, showed an isCID signal from endogenous oligosaccharides with 10-100 fold lower signals than the suggested threshold (Figure 6b).…”

“…Among the LC-MS methods described for the detection of PVEs in urine, those from Guddat [9] and Kolmonen [13] were based on in-source fragmentation (isCID) to generate low mass fragment ions from the sugar backbone and can be analyzed with a “dilute-and-shoot” strategy.…”

The Analytical Chemistry of Drug Monitoring in Athletes

“…Typical procedures to detect the presence of β2-agonists (19,20), hormone antagonists and modulators (21)(22)(23)(24), diuretics and masking agents (25)(26)(27)(28)(29), stimulants (30)(31)(32)(33), narcotics (31,34), cannabinoids, glucocorticosteroids (35,36), and β-blockers (31) have been published by the WADA-recognized laboratories. I focus here on three prohibited substance classes: anabolic agents, protein and peptide hormones, and oxygen-transport enhancement.…”