Optimization of a robust and reliable FITC labeling process for CE-LIF analysis of pharmaceutical compounds using design of experiments strategy.

Fluorescence, especially laser induced fluorescence (LIF), is a powerful detection technique thanks to its specificity and high sensitivity. The use of fluorescence detection hyphenated to separation technique often requires the labeling of analytes with suitable fluorescent dye, such as FITC for the labeling of molecules presenting amino groups. Nevertheless, the labeling of analytes could be a tedious, time consuming and a non-robust step of the analytical workflow. In this context, the objective of the present work was to propose a robust and reliable FITC labeling process. Primary and secondary amino compounds (i.e. synthetic cathinones) were selected as model compounds because they are representative of a large proportion of pharmaceutical small molecules. Based on prior knowledge, DoE combined with multivariate statistical modeling was performed to optimize the process. Reaction time and pH of reaction buffer were highlighted as the most critical parameters to control the process. The study showed also the benefit of short reaction time to maximize the labeling efficiency. Indeed, optimal condition was defined as reaction time of 32 min with ratio between FITC and analytes of 40.4 and the buffer reaction pH of 9.7. In addition, variance component analysis was integrated to the DoE to estimate the variability of process and to evaluate its applicability for quantitative purpose. These chemometric approaches helped to develop an efficient labeling process able to reach high sensitivity for CE-LIF analysis (i.e. 10 nM) with good precision (i.e. intermediate precision values lower or close to 5 %).

Development of a sensitive MEKC-LIF method for synthetic cathinones analysis.

Synthetic cathinones are phenylalkylamine compounds related to natural cathinone from Catha edulis leaves. Due to their sympathomimetic effects comparable to common illicit drugs, these substances are mainly drugs of abuse and constitute the second most frequently seized group of new psychoactive substances. In order to ensure their regulation and to promote public health, reliable analytical tools are required to track these substances. In the present study, we developed a CE hyphenated to laser-induced fluorescence detection method to demonstrate its suitability to perform fast and cost-effective synthetic cathinones analysis. Fourteen compounds including isobaric compounds and position isomers were selected to encompass the large panel of chemical structures. To separate the FITC-labeled analytes (presenting the same negative charge and close mass to charge ratios), MEKC separation mode was selected. Method selectivity was not suitable using common surfactants. In this context, alkyl polyethylene glycol ether surfactants were successfully used as neutral surfactant to overcome this analytical challenge. The effect of surfactant nature on separation performances and migration behaviors of the analytes was also studied. Optimal BGE composition included 75 mM borate buffer at pH 9.3 and 0.4 mM of C12E10 surfactant. Final MEKC separation conditions were proposed to analyze a large panel of synthetic cathinones. This method helped to reach a sensitivity with LOD from 0.1 to 0.4 nM (pg/mL order).