Automatic analyses of quantitative dried material spots by on-line coupling of sequential injection to high-performance liquid chromatography: A proof-of-concept.

BACKGROUND: The development and integration of platforms for reliable handling of dried material spots (DMSs) as a front-end to advanced analytical instrumentation continues to pose methodological challenges. Although robotic platforms have been developed for autonomous manipulation of dried blood spot (DBS) cards, these systems exhibit limited flexibility regarding ancillary unit operations and remain incompatible with quantitative DMS (qDMS) devices.

RESULTS: Leveraging off-the-shelf fluidic components using advanced liquid handling, this proof-of-concept work exploits the versatility of sequential injection-high-performance liquid chromatography (SI-HPLC) coupling for automatic processing and analysis of DMSs. The flow setup features a multi-position rotary valve as the core of the SI manifold connected to two 3D-printed 60-position racks. This hyphenation enables flexible in-vial processing of various DBS, qDBS, qDMS, and volumetric absorptive microsampling (VAMS) samples by controlled dispensing of microliter volumes of methanol for the spot rehydration, protein precipitation, and analyte elution, followed by mixing and homogenization of the eluate with water for the HPLC compatibility. Demonstrated on model non-steroidal anti-inflammatory drugs, the fully synchronized SI-HPLC setup enabled entirely unsupervised analysis of up to 120 DMS samples with triplicate HPLC injections per eluate (8.2 min each). Using a matrix-matched calibration based on spiking 10 μL of analyte-free blood, multiple qDBSs were eluted with 135 μL of methanol, followed by the eluate composition adjustment with 135 μL of water for HPLC compatibility prior to reversed-phase HPLC analysis. Under these conditions, calibrations were linear over 15 - 60, 0.8 - 12, 0.8 - 6, and 6.2 - 100 μg mL-1 ranges, and LODs were 5.7, 0.3, 0.3, and 1.4 μg mL-1 for ibuprofen, ketoprofen, diclofenac, and naproxen, respectively. The method precision was 2.3 - 12.7% RSD for intra-day and 9.3 - 13.2% RSD for inter-day experiments. The reliability of the proposed automatic SI-HPLC setup was demonstrated for qDBSs with therapeutic naproxen levels, with no statistically significant concentration differences at the 0.05 level when compared to manual qDBS processing with off-line analysis by capillary electrophoresis.

SIGNIFICANCE: For the first time, the proposed analytical concept enables automatic handling and analysis of various DMS and qDMS samples, while using standard off-the-shelf SI and HPLC components and freely available control software.