Quantitative analysis of metabolites in complex biological samples using ion-pair reversed-phase liquid chromatography–isotope dilution tandem mass spectrometry

A rapid, sensitive and selective ion-pair reversed-phase liquid chromatography–electrospray ionization isotope dilution tandem mass spectrometry (IP-LC–ESI-ID-MS/MS) was developed for quantitative analysis of free intracellular metabolites in cell cultures. As an application a group of compounds involved in penicillin biosynthesis pathway of Penicillium chrysogenum cells, such as penicillin G (PenG), 6-aminopenicillanic acid (6-APA), benzylpenicilloic acid (PIO), ortho-hydroxyphenyl acetic acid (o-OH–PAA), phenylacetic acid (PAA), 6-oxopipeidine-2-carboxylic acid (OPC), 8-hydroxypenicillic acid (8-HPA), l-α-(δ-aminoadipyl)-l-α-cystenyl-d-α-valine (ACV) and isopenicillin N (IPN) were chosen. 13C-labeled analogs of the metabolites were added to the sample solutions as internal standards (I.S.). Sample mixtures were analyzed without any sample pretreatment. No extraction recovery check was needed because I.S. was added to the cell samples before extraction process. The method showed excellent precision (relative standard deviation (RSD) ≤ 11%, except for PIO and 8-HPA) in present of interferences from sample matrix. Limits of quantification (LOQs) for all metabolites were below 1 μM level.