Instability of the DNPH-formaldehyde derivative in the certified reference material BCR-551 stored at −70 °C but not at −20 °C

Abstract

During the post-certification stability monitoring of the certified reference material (CRM) BCR-551 (DNPH derivatives dissolved in acetonitrile), a decreased concentration of one of the analytes of this CRM, the DNPH-formaldehyde derivative, was detected in reference samples (stored at −70 °C), while the concentration of normal “on-sale” samples (stored at −20 °C) remained stable. This behaviour is contrary to the expectation of better stability at lower temperatures. Apparently, the DNPH-formaldehyde derivative reacts with dinitrophenylhydrazine (DNPH) remaining from the synthesis phase to produce two new substances. These substances have been identified as C₁₃H₁₂N₈O₈ (substance 1) and C₂₀H₁₆N₁₂O₁₂ (substance 2) which, based on their structure, are suggested to be produced consecutively: DNPH + DNPH-formaldehyde derivative → substance 1 and substance 1 + DNPH-formaldehyde derivative → substance 2. Since acetonitrile freezes at −45 °C, reference samples are frozen at −70 °C, while normal samples are still liquid at −20 °C. We believe that this leads to a cryo-concentration of the solutes above the eutectic point and thus to an increased reaction rate in the reference samples. This case demonstrates that care should be taken when extrapolating stability results towards conditions that never have been tested, especially if phase transitions are involved, even at temperature as low as −70 °C. Furthermore, a slower degradation rate at lower temperatures can be overcompensated by a higher concentration due to cryo-concentration above the eutectic temperature.