Ultra high performance supercritical fluid chromatography coupled with tandem mass spectrometry for screening of doping agents. I: Investigation of mobile phase and MS conditions.

The conditions for the analysis of selected doping substances by UHPSFC-MS/MS were optimized to ensure suitable peak shapes and maximized MS responses. A representative mixture of 31 acidic and basic doping agents was analyzed, in both ESI+ and ESI- modes. The best compromise for all compounds in terms of MS sensitivity and chromatographic performance was obtained when adding 2% water and 10mM ammonium formate in the CO2/MeOH mobile phase. Beside mobile phase, the nature of the make-up solvent added for interfacing UHPSFC with MS was also evaluated. Ethanol was found to be the best candidate as it was able to compensate for the negative effect of 2% water addition in ESI- mode and provided a suitable MS response for all doping agents. Sensitivity of the optimized UHPSFC-MS/MS method was finally assessed and compared to the results obtained in conventional UHPLC-MS/MS. Sensitivity was improved by 5-100-fold in UHPSFC-MS/MS vs. UHPLC-MS/MS for 56% of compounds, while only one compound (bumetanide) offered a significantly higher MS response (4-fold) under UHPLC-MS/MS conditions. In the second paper of this series, the optimal conditions for UHPSFC-MS/MS analysis will be employed to screen >100 doping agents in urine matrix and results will be compared to those obtained by conventional UHPLC-MS/MS

Ultra high performance supercritical fluid chromatography coupled with tandem mass spectrometry for screening of doping agents. II: Analysis of biological samples.

The potential and applicability of UHPSFC-MS/MS for anti-doping screening in urine samples were tested for the first time. For this purpose, a group of 110 doping agents with diverse physicochemical properties was analyzed using two separation techniques, namely UHPLC-MS/MS and UHPSFC-MS/MS in both ESI+ and ESI- modes. The two approaches were compared in terms of selectivity, sensitivity, linearity and matrix effects. As expected, very diverse retentions and selectivities were obtained in UHPLC and UHPSFC, proving a good complementarity of these analytical strategies. In both conditions, acceptable peak shapes and MS detection capabilities were obtained within 7min analysis time, enabling the application of these two methods for screening purposes. Method sensitivity was found comparable for 46% of tested compounds, while higher sensitivity was observed for 21% of tested compounds in UHPLC-MS/MS and for 32% in UHPSFC-MS/MS. The latter demonstrated a lower susceptibility to matrix effects, which were mostly observed as signal suppression. In the case of UHPLC-MS/MS, more serious matrix effects were observed, leading typically to signal enhancement and the matrix effect was also concentration dependent, i.e., more significant matrix effects occurred at the lowest concentrations

Toward a Scalable Synthesis and Process for EMA401 Part III: Using an Engineered Phenylalanine Ammonia Lyase Enzyme to Synthesize a Non-natural Phenylalanine Derivative

A process using engineered phenylalanine ammonia lyase (PAL) enzymes was developed as part of an alternative route to a key intermediate of olodanrigan (EMA401). In the first part of the manuscript, the detailed results from a screening for the optimal reaction conditions are presented, followed by the discussion of several work-up strategies investigated. In the PAL catalyzed reaction, 70–80% conversion of a cinnamic acid derivative to the corresponding phenylalanine derivative could be achieved. The phenylalanine derivative was subsequently telescoped to a Pictet-Spengler reaction with formaldehyde and the corresponding tetrahydroisoquinoline derivative was isolated in 60–70% yield with >99.9:0.1 er. Based on our screenings, carbonate/carbamate buffered ammonia at 9–10 M NH3 concentration and pH 9.5–10.5 were found as the optimal conditions. Enzyme loadings down to 2.5wt% (E:S 1:40 w/w) could be achieved and substrate concentrations between 3–9 v/w (1.17–0.39 M) were found to be compatible with the reaction conditions. A temperature gradient was applied in the final process: a pre-equilibrium was established at 45 °C, before making use of the temperature-dependence of the entropy term with subsequent cooling to 20 °C and achieving maximum conversion. This temperature gradient also allowed balancing enzyme stability (low at 45 °C, high at 20 °C) with activity (high at 45 °C, low at 20 °C) in order to achieve optimal conversion (low at 45 °C, high at 20 °C). From the various work-up operations investigated, a sequence consisting of denaturation of the enzyme, followed by NH3/CO2 removal by distillation, acidification and telescoping to the subsequent Pictet-Spengler cyclization was our preferred approach. The process presented in this study is a more sustainable, shorter and more cost effective alternative to the previous process