In this research study, a novel deep eutectic solvent was synthetized and used for rapid and simple ultrasound-assisted dispersive liquid–liquid microextraction based on solidifcation of foating organic drop (USA-DLLME-SFO) method. The
proposed method was applied for simultaneous separation and preconcentration of penicillin G, ampicillin, and amoxicillin
prior to their analysis by high-performance liquid chromatography with photodiode array detection (HPLC–PDA). Benzyl triethylammonium chloride (as hydrogen bond acceptor (HBA)) and decanoic acid (as hydrogen bond donor (HBD))
(1:3 mol ratio)) were synthetized and used as hydrophobic deep eutectic solvent (HP-DES) in USA-DLLME-SFO process.
Important parameters including pH, DES volume, dispersive solvent type and volume, sonication time, sample volume,
and ionic strength were investigated. Under the optimum conditions, the LODs of 0.18–0.35 µg kg−1 for aqueous solutions,
2.8–3.5 µg kg−1 for chicken meat, 1.16–2.35 µg kg−1 for honey, and 2.73–5.08 µg kg−1 for egg were acquired for penicillin G,
ampicillin, and amoxicillin. The suitable linearity of 3.0–500 µg kg−1 for penicillin G and amoxicillin and 7.5–750 µg kg−1
for ampicillin was attained for all real samples. The inter-day precision (the relative standard deviations (RSDs %)) below
4.2% was also acquired for three concentrations of 50, 100, and 200 µg kg−1 of analytes within seven replications. In order
to consider the reproducibility, the analytical process was carried out at optimum conditions within 4 days and each day for
three times. The reproducibility values of 3.1, 2.6, and 3.5% were achieved for penicillin G (Pen. G), ampicillin (Amp.), and
amoxicillin (Amox.) (100 µg kg−1), respectively. The proposed method was successfully applied for determination of the
target antibiotics in chicken meat, egg, and honey with the relative recoveries over 97.0% which confrms high capability of
the method for application in complex matrices.