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In this study, we developed and validated a method for the extraction, identification, and quantitation of penicillin G and its metabolites (penilloic acid and penillic acid) in a variety of citrus fruits by employing sequential liquid/liquid and solid-phase extraction techniques in conjunction with UHPLC-MS/MS. Two product ion transitions per analyte were required for identification, which contributes to a high degree of selectivity. Corrected recoveries of penicillin G using an isotopically labeled internal standard were 90-100% at fortification levels of 0.1, 0.25, 1, and 10 ng/g. Absolute recoveries for penillic acid and penilloic acid were 50-75% depending on the matrix used. The limit of detection (LOD) of penicillin G and its metabolites was found to be 0.1 ng/g when 2 g of citrus was extracted. This method is useful in determining residue levels of penicillin G and its metabolites in citrus trees infected with huanglongbing bacteria after antibiotic treatment.

A molecularly imprinted polymer (MIP) was designed for benzylpenicillin via suspension polymerization. The specific absorption of benzylpenicillin to the MIP, applied in a molecularly imprinted solid-phase extraction (MISPE), was compared to the nonspecific binding using a NIP (nonimprinted polymer without a target molecule) in a non-molecularly imprinted solid-phase extraction. This validation was performed successfully in acetonitrile solutions and milk extracts spiked with benzylpenicillin. Significant differences in absorption were observed. In acetonitrile, the recoveries using MISPE (90-95%) were a fraction higher than those in milk extracts (70-80%). The validation revealed the limit of detection and the limit of quantitation for the MISPE application in milk samples to be 0.51 and 1.02 μg/kg, respectively. In addition, comparing the results of the analysis of positive milk samples using MISPE with those using a classic sample preparation step showed a Pearson correlation of 0.989. Finally, cross reactivity tests using other antibiotics showed a certain cross reactivity, but non-β-lactams were barely bound.

A molecularly imprinted polymer (MIP) prepared using penicillin G procaine salt as the template (PENGp) and a stoichiometric quantity of urea-based functional monomer to target the single oxyanionic species in the template molecule has been applied to the development of a molecularly imprinted solid-phase extraction (MISPE) procedure for the selective preconcentration of beta-lactam antibiotics (BLAs) from environmental water samples. Various parameters affecting the extraction efficiency of the polymer have been evaluated to achieve the selective preconcentration of the antibiotics from aqueous samples and to reduce nonspecific interactions. This resulted in an MISPE-HPLC method allowing the direct extraction of the analytes from the sample matrix with a selective wash using just 10% (v/v) organic solvent. On the basis of UV detection only, the method showed good recoveries and precision, ranging between 93% and 100% (RSD 3.8-8.9%, n = 3) for tap water and between 90% and 100% (RSD 4.2-9.1%, n = 3) for river water fortified with 30 or 60 microg L-1 (50 mL samples) penicillin G, penicillin V, nafcillin, oxacillin, cloxacillin, and dicloxacillin, suggesting that this MIP can be successfully applied to the direct preconcentration of BLAs in environmental water samples.