A sampling procedure for quantitative metabolomics in Penicillium chrysogenum based on cold aqueous methanol quenching was re-evaluated and optimized to reduce metabolite leakage during sample treatment. The optimization study included amino acids and intermediates of the glycolysis and the TCA-cycle. Metabolite leakage was found to be minimal for a methanol content of the quenching solution (QS) of 40% (v/v) while keeping the temperature of the quenched sample near -20°C. The average metabolite recovery under these conditions was 95.7% (±1.1%). Several observations support the hypothesis that metabolite leakage from quenched mycelia of P. chrysogenum occurs by diffusion over the cell membrane. First, a prolonged contact time between mycelia and the QS lead to a somewhat higher extent of leakage. Second, when suboptimal quenching liquids were used, increased metabolite leakage was found to be correlated with lower molecular weight and with lower absolute net charge. The finding that lowering the methanol content of the quenching liquid reduces metabolite leakage in P. chrysogenum contrasts with recently published quenching studies for two other eukaryotic micro-organisms. This demonstrates that it is necessary to validate and, if needed, optimize the quenching conditions for each particular micro-organism. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s11306-011-0367-3) contains supplementary material, which is available to authorized users.