Lab-In-Syringe for automated double-stage sample preparation by coupling salting out liquid-liquid extraction with online solid-phase extraction and liquid chromatographic separation for sulfonamide antibiotics from urine

A double-stage Lab-In-Syringe automated extraction procedure coupled online to HPLC for the determination of four sulfonamides in urine has been developed. Our method is based on homogeneous liquid-liquid extraction at pH 3 using water-miscible acetonitrile with induction of phase separation by the addition of a saturated solution of kosmotropic salts MgSO4 and NaCl. The procedure allowed extraction of the moderately polar model analytes and the use of a solvent that is compatible with the used separation technique. The automated sample preparation system based on the stirring-assisted Lab-In-Syringe approach was coupled on-line with HPLC-UV for the subsequent separation of the sulfonamide antibiotics. To improve both preconcentration factor and extract cleanup, the analytes were trapped at pH 10 in an anion-exchange resin cartridge integrated into the HPLC injection loop thus achieving a double-stage sample clean-up. Analytes were eluted using an acidic HPLC mobile phase in gradient elution mode. Running the analytes separation and the two-step preparation of the following sample in parallel reduced the total time of analysis to mere 13.5 min. Limits of detection ranged from 5.0 to 7.5 mu g/L with linear working ranges of 50-5000 mu g/L (r(2) > 0.9997) and RSD <= 5% (n = 6) at a concentration level of 50 mu g/L. Average recovery values were 102.7 +/- 7.4% after spiking of urine with sulfonamides at concentrations of 2.5 and 5 mg/L followed by 5 times dilution. To the best of our knowledge, the use of Lab-In-Syringe for the automation of coupled homogeneous liquid-liquid extraction and SPE for preparation of the complex matrices suitable for separation techniques is here presented for the first time.