Determination of rhodamine and tetramethylrhodamine-5-maleimide by ultra high performance liquid chromatography

Nanoscale materials present unquestionable advantages, however there is a need to understand how the nanoscale influences the toxicological properties of materials, as well as the possibility of exposure. Nowadays, we are still facing the lack of tools and methodologies available to easily track the movement and position of engineered nanomaterials (ENMs) from nanocomposites. Elemental analysis via atomic absorption spectrometry or inductively coupled plasma mass spectroscopy are the most commonly used techniques for tracking and detecting nanoclays. However, these methods lack the ability to track single or clustered nanoclay particles and their positions, which is an important feature. Recently fluorescent labeling has been proposed as a simple method to track particle at nanoscale. In this method fluorescent compound (tag) bonds covalently to the nanoclay allowing to track them. The main objective of this study was to develop an analytical method by Ultra High Performance Liquid Chromatographic (UHPLC) to determine simultaneously rhodamine and tetramethylrhodamine-5-maleimide, two potential labels of MMT. Due to the lack of information regarding the analytical behaviour of the selected fluorescent tags, there was the need of carrying out numerous tests to develop an UHPLC method that allowed determining the selected fluorescent tags. Different conditions of the methods were tested including chromatographic column, column temperature, mobile phase composition, mobile phase gradient and detectors conditions were tested. UHPLC was used because it allows reducing the time of analysis and to obtain well defined peaks and low quantification limits. Fluorescence detector and diode array detector (DAD) were compared as well as different solvents. The results of the calibration curves of rhodamine and tetramethylrhodamine-5-maleimide, prepared in ethanol and methanol, presented excellent determination coefficients in all cases, indicating suitability for quantification. Better response was obtained with fluorescence detector, allowing working at lower concentrations.This work was supported by the research project “Labelling and tracking of nanoclay from food packaging nanocomposites: a food safety issue – NanoPack4Food” (2014DAN1019) under the Cooperative Programme of the Agreement on Scientific Cooperation between National Research Council of Italy (CNR) and Foundation for Science and Technology of Portugal (FCT)N/