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Abstract Introduction:
Whether ingested, inhaled, injected or otherwise applied, drugs with systemic mechanism of action are at some point present in blood and then in urine, with which they are excreted. And however popular, so called conventional matrices are also troublesome. Collection of blood is invasive, while obtaining urine is either intrusive when performed under supervision or else burdened with high risk of adulteration. Such inconveniences shift interests of doping control and law enforcement towards alternative matrices. Among them, oral fluid (OF) stands out as a direct filtrate of blood, well resembling its composition. Easy to collect in transparent manner, OF offers very promising and reliable opportunity for determination of recent substance use. Solid phase microextraction (SPME) provides sample preparation for optimal testing performance with its economically friendly device reusability, environmentally friendly low organic solvent consumption and user friendly simple protocol. High performance liquid chromatography (HPLC) is already well-established separation method for complex biological samples, while tandem mass spectrometry (MS) grants excellent selectivity and sensitivity as a detection method.
Objectives:
The main objective was to confirm that OF could be introduced as a matrix for routine drug testing with analytical approach based on combining the benefits of SPME and LC-MS methods.
Methods:
Presented method utilized SPME in fiber format (C18 sorbent), reversed phase HPLC (PFP column) and triple quadrupole MS (Shimadzu LCMS-8060). All experiments were performed with OF samples spiked with 46 various drugs from the World Anti-Doping Agency’s (WADA) Prohibited List covering 7 different classes and broad range of proprieties. SPME fibers were evaluated in terms of extraction efficiency and prevalence of carry-over effect. In addition series of comparisons helped establish optimal solvent for sorbent preconditioning, essential parameters of extraction process (agitation rate, pH value) and most fitting desorption solvent. Permission to conduct experiments was given by the local Bioethics Committee.
Results:
Optimized protocol was validated and fully complies with requirements of Food and Drug Administration and WADA. Best results were obtained with SPME preconditioning in acetonitrile/water (50/50, v/v) for 44 of 46 drugs, extraction pH value of 7 for 26 of 46 and desorption to acetonitrile/water/formic acid (80/19,9/0,1) for 25 of 46 in terms of efficiency and 40 of 46 substances for lowest carry-over effect.
Conclusion:
Oral fluid was confirmed as a potential matrix for drug testing and presented SPME-LC-MS method was proved to be suitable for antidoping or forensic applications. In addition, use of only the commercially available materials enables its introduction to everyday laboratory practice.
Acknowledgement:
The authors would like to acknowledge NCRD Poland (grant LIDER/44/0164/L-9/NCBR/2018), Shimpol and Supelco for their support. |
= Discovery stage.
= Translation stage.
= Clinically available.
