On-line column coupled isotachophoresis-capillary zone electrophoresis hyphenated with tandem mass spectrometry in drug analysis: Varenicline and its metabolite in human urine

•New hyphenation was based on two-dimensional CE (ITP-CZE) and tandem MS (QqQ).•2D-CE mode provided on-line sample preparation (preconcentration, sample clean-up).•ITP-CZE–ESI-QqQ combination was approved by excellent performance parameters.•Application area involves trace (pg mL−1) drugs in unpretreated complex matrices.•We proposed routine clinical method for varenicline and its metabolite in urine.

A new highly advanced analytical approach, based on two-dimensional column coupled CE (ITP-CZE) hyphenated with tandem mass spectrometry (MS/MS, here triple quadrupole, QqQ) was developed, evaluated and applied in biomedical field in the present work. Capillary isotachophoresis (ITP) coupled on-line with capillary zone electrophoresis (CZE) used in hydrodynamically closed separation system was favorable for increasing the sample load capacity, increasing the analyte concentration, and removing the deteriorative highly conductive major matrix constituents. These factors considerably reduced the concentration limits of detection (cLOD) and external sample preparation (comparing to single column CZE), and, by that, provided favorable conditions for the mass spectrometry (enhanced signal to noise ratio, reproducibility of measurements, working life of MS). Here, the CZE–ESI combination provided more effective interfacing than ITP–ESI resulting in both a higher obtainable intensity of MS detection signal of the analyte as well as reproducibility of measurements of the analyte’s peak area. The optimized ITP-CZE–ESI-QqQ method was successfully evaluated as for its performance parameters (LOD, LOQ, linearity, precision, recovery/accuracy) and applied for the direct identification and ultratrace (pg mL−1) determination of varenicline and, in addition, identification of its targeted metabolite, 2-hydroxy-varenicline, in unpretreated/diluted human urine. This application example demonstrated the real analytical potential of this new analytical approach and, at the same time, served as currently the most effective routine clinical method for varenicline.

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