Automated centrifugation-less milk deproteinization and homogenous liquid-liquid extraction of sulfonamides for online liquid chromatography.

A novel approach to the determination of sulfonamides in milk based on the Lab-In-Syringe technique is presented. The method involves automated salting-out liquid-liquid extraction of the analytes, allowing simultaneous sample deproteination without requiring centrifugation or manual sample handling. The procedural parameters, including salt type, solvent-to-sample ratio, and salt solution volume, were studied. The extracts obtained were diluted in situ and transferred to online coupled liquid chromatography for analyte separation carried out in parallel to the subsequent extraction. In this way, a sample throughput of approximately 6 h-1 was achieved. The detection limits ranged from 25 to 32 µg L-1 using a 500 µL milk sample and spectrophotometric detection. The applicability of the developed method to sample analysis was proven by recovery values ranging from 73.2% to 94.1% (86.3% on average) for milk samples of different fat content spiked at 3 µg mL-1 level. Straightforward automation of one of the most laborious preparation steps in food analysis, i.e., deproteination, was demonstrated.

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 µg/L with linear working ranges of 50-5000 µg/L (r2 > 0.9997) and RSD ≤ 5% (n = 6) at a concentration level of 50 µg/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.