UHPLC-MS/MS method for the quantitation of penicillin G and metabolites in citrus fruit using internal standards

•Development and validation of LC-MS/MS method to identify and quantitate penicillin G and two of its metabolites, penillic acid and penilloic acid, in citrus fruit.•Improvement of a previous method by incorporating isotopically labeled internal standards, namely, penillic acid-D5, and penilloic acid-D5.•Method enhancement and precision of measurements.

Penicillin G has been applied to citrus trees as a potential treatment in the fight against Huanglongbing (HLB). Here, we have developed and validated a method to identify and quantitate penicillin G and two of its metabolites, penillic acid and penilloic acid, in citrus fruit using ultra high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). This method improves upon a previous method by incorporating isotopically labeled internal standards, namely, penillic acid-D5, and penilloic acid-D5. These standards greatly enhanced the accuracy and precision of our measurements by compensating for recovery losses, degradation, and matrix effects. When 2 g of citrus fruit sample is extracted, the limits of detection (LOD) were determined to be 0.1 ng/g for penicillin G and penilloic acid, and 0.25 ng/g for penillic acid. At fortification levels of 0.1, 0.25, 1, and 10 ng/g, absolute recoveries for penillic and penilloic acids were generally between 50-70%. Recoveries corrected with the isotopically labeled standards were approximately 90-110%. This method will be useful for the identification and quantitation of drug residues and their degradation products using isotopically labeled standards and UHPLC-MS/MS.