Semi-preparative high-resolution recycling liquid chromatography

A semi-preparative high-resolution system based on twin column recycling liquid chromatography was built. The integrated system includes a binary pump mixer, a sample manager, a two-column oven compartment, two low-dispersion detection cells, and a fraction manager (analytical). It addresses challenges in drug/impurity purification, which involve several constraints simultaneously: (1) small selectivity factors (α < 1.2, poor resolution), (2) mismatch of elution strength between the sample diluent and the eluent causing severe band fronting or tailing, (3) diluent-to-eluent mismatch of viscosity causing viscous fingering and unpredictable band deformation, (4) low abundance of the impurity relative to the active pharmaceutical ingredient (API) (<1/100), and (5) yield and purity levels to be larger than 99% and 90%, respectively. The prototype system was tested for the preparation of a trace impurity present in a concentrated solution of an API, estradiol. The ultimate goal was to collect ∼1 mg of impurity (>90% purity) for unambiguous structure elucidation by liquid state nuclear magnetic resonance (NMR 600 MHz and above). First, the particle size (3.5 μm) used to pack the 4.6 mm × 150 mm long twin columns is selected so that the speed-resolution of the recycling process is maximized at 4000 psi pressure drop. Next, the production rate of the process is also maximized by determining the optimum number (7) of cycles and the corresponding largest sample volume (160 μL) to be injected. Finally, the process is fully automated by programming the time events related to (1) sample cleaning, (2) transfer of the targeted impurity from one to the second twin column, and (3) impurity collection. The process was tested without interruption during one week for the collection of a trace impurity (α = 1.166, strong acetonitrile-methanol sample diluent, concentration ∼2 mg/L) from a concentrated (10 g/L) stock solution (60 mL total) of estradiol. The process enriches the impurity content relative to the API by about a factor ∼5000. For the lack of a sufficient collected amount (∼120 μg only) of the pure impurity (purity 50% only), NMR experiments could not provide reliable results. Instead, the combination of LC-MS (single ion monitoring) and UV absorption spectra (λ_max shift) revealed that the targeted impurity was likely the low-abundant enol tautomeric form of the ketone estrone, a possible intermediate or by-product of the synthesis reaction of estradiol.