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

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 4000Da 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 0.4-12ng/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.