Combining direct urinary injection with automated filtration and nanoflow LC-MS for the confirmatory analysis of doping-relevant small peptide hormones.

Nano-liquid chromatography (nanoLC) has proven itself as a powerful tool and its scope entails various applications in (bio)analytical fields. Operation at low (nL/min) flow rates in combination with reduced inner dimensions (ID < 100 µm), leads to significantly enhanced sensitivity when coupled with electrospray ionization-mass spectrometry (ESI-MS). Challenges that remain for the routine implementation of such miniaturized setups are related to clogging of the system and robustness in general, and thus the application of tedious sample preparation steps. To improve ruggedness, a filter placed upstream in the LC prevents particles from entering and clogging the system. This so-called online automatic filtration and filter back-flush (AFFL) system was combined with nanoLC and the direct injection principle for the sensitive confirmatory analysis of fifty different doping-relevant peptides in urine. The presented assay was fully validated for routine purposes according to selectivity and matrix interference, limit of identification (LOI), carryover, matrix effect, sample extract stability, analysis of educational external quality assessment (EQAS) samples, robustness of the online AFFL-setup and retention time stability. It was also fully compliant with the most recent minimum required performance levels (MRPL) and chromatographic/mass spectrometric identification criteria (IDCR), as imposed by the World Anti-Doping Agency (WADA). In the absence of labor-intensive sample preparation, the application of AFFL allowed for the injection of diluted urine samples without any noticeable pressure buildup in the nanoLC system. Contrary to earlier observations by our group and others, the addition of dimethylsulfoxide (DMSO) to the mobile phase did not enhance sensitivity in the presented nanoflow setup, yet was beneficial to reduce carry over. Although the robustness of the presented setup was evaluated only for the analysis of diluted urine samples, it is entirely conceivable that routine applications employing other matrices and currently running on analytical scale LC instruments could be transferred to micro/nanoLC scale systems to reach lower detection limits.

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