Robust and Fast UV–HPLC Method for Biotransformation Analysis of Azecines

Abstract

Dibenzoazecines are a new class of drug candidates for the treatment of schizophrenia. Compared to the drugs currently used in therapy, the azecines have a novel mechanism of action. Thus, they have the potential to cause fewer side effects compared to the standard therapy with a constant high neuroleptic potency. This theory was substantiated by comparative in vivo tests with haloperidol and risperidone. Seventeen new azecine derivatives have already been tested furthermore of stability, physicochemical parameters, pharmacokinetics including esterase cleavage, stability in simulated gastrointestinal fluid, stability at different pH values and determination of octanol/water-partition coefficients. For these substances, class is still a lack of information concerning the metabolism. Therefore, the present study investigated and developed a reliable and reproducible gradient reversed-phase HPLC–UV method to determinate the lead structure LE404 alongside emerging metabolites in compliance with international requirements like ICH guidelines and the European Pharmacopoeia. Up to now, there is no innovative method suitable for such a determination. Chromatographic separations were achieved with a phenomenex™ Gemini column (5 µm C18 110 Å, 250 × 4.60 mm) using a mixture of acetonitrile/potassium dihydrogen phosphate buffer (4 mmol L−1, pH 2.5) as mobile phase. The gradient method flow rate was 1.0 mL min−1, and UV detection was made at 220 nm. The optimized HPLC method was found to be specific, accurate, reproducible and robust for determination of LE404.

HPLC–UV Monitored Photostability-Test of LE404 and Identification of the Degradation Products via NMR and LC–HRMS

“…In addition, the mobile phase was degassed by soni cation. The gradient elution was as described as in [15]. Separations were achieved using a C18 reversed-phase column (phenomenex™ Gemini 5 µm C18 110 Å, 250 × 4.60 mm, Phenomenex Inc., Germany) and a precolumn (phenomenex™ Security Guard Cartridge Gemini C18, 4 × 3 mm, Phenomenex Inc., Germany).…”

“…This indicates the degradation of the drug. The development of HPLC-method to monitor degradation was already validated according to ICH standards [15]. LE404 is to be considered as photosensitive, because its content decreases by 6.55% during the required illumination.…”

“…The solution of DP1 remained colorless over the entire duration of the experiment. The decomposition was investigated using the previously published HPLC method [15], which was also used in the ICH photostability test. Figure 3 shows the HPLC-chromatograms of the forced degradation of LE404 and DP1.…”

“…The reaction conditions and the work-up are described in supplementary data le section S2.1. A semi-preparative isolation, as described in [15] would have been an option, but would have required a larger quantity chemicals and solvents compared to the direct reaction with H 2 O 2 . So far, DP2 could only be obtain by irradiation with daylight.…”

“…Initial tests already indicated that LE404 decomposes under the in uence of natural sunlight [15]. So far, the decomposition behavior has only been observed for the solid in daylight up to a period of 21 d [15].…”