Development of a stable isotope dilution LC–MS assay for the quantitation of multiple polyethylene glycol (PEG) homologues to be used in permeability studies

• Stable isotope dilution assays for polyethylene glycol homologues were developed.
• PEG homologues were purified and quantified by quantitative NMR.
• [13C42H4] and [13C82H8]PEG homologues were synthesized as internal standards.
• Homologues were sensitively quantified from PEG 400, 1500, 3000 and 4000 mixtures.
• PEG 400, 1500, 3000 and 4000 were quantified in human and mural urine after dosing of these mixtures.

A new quantitation method based on a multiple stable isotope dilution assay (SIDA) was developed for polyethylene glycol (PEG) homologues from PEG mixtures with average molecular weights (MW) of 400, 1500, 3000 and 4000 Da in urine. Seven [13C42H4] and two [13C82H8]PEG homologues were synthesized and served as labelled internal standards for SIDA. PEG oligomers were resolved by reversed phase high performance liquid chromatography (RP-HPLC) coupled to mass spectrometry (MS) in multiple ion (MI) scan modus. Very low limits of detection (LODs) in a range of 0.4–12 ng/mL were achieved for the single homologues. Higher PEG homologues showed increased LODs and LOQs and less effective recovery (77–87%) than PEG with lower molecular masses (95–121%). Precision (relative standard deviation) varied between 3 and 13% and showed no dependence of the chain length. The method was successfully applied to human and mice urine samples. Beside an accurate quantitation of single PEG homologues it was possible to show an alteration in the MW distribution in urine samples compared to the dosed PEG solutions. The highest MW, with which a PEG can pass the intestinal wall (so called “cut off”) for humans appeared to be higher than for mice.