Selective separation, detection of zotepine and mass spectral characterization of degradants by LC-MS/MS/QTOF

Abstract

A simple, precise, accurate stability-indicating gradient reversed-phase high-performance liquid chromatographic (RP-HPLC) method was developed for the quantitative determination of zotepine (ZTP) in bulk and pharmaceutical dosage forms in the presence of its degradation products (DPs). The method was developed using Phenomenex C₁₈ column (250 mm×4.6 mm i.d., 5 µm) with a mobile phase containing a gradient mixture of solvents, A (0.05% trifluoroacetic acid (TFA), pH=3.0) and B (acetonitrile). The eluted compounds were monitored at 254 nm; the run time was within 20.0 min, in which ZTP and its DPs were well separated, with a resolution of >1.5. The stress testing of ZTP was carried out under acidic, alkaline, neutral hydrolysis, oxidative, photolytic and thermal stress conditions. ZTP was found to degrade significantly in acidic, photolytic, thermal and oxidative stress conditions and remain stable in basic and neutral conditions. The developed method was validated with respect to specificity, linearity, limit of detection, limit of quantification, accuracy, precision and robustness as per ICH guidelines. This method was also suitable for the assay determination of ZTP in pharmaceutical dosage forms. The DPs were characterized by LC-MS/MS and their fragmentation pathways were proposed.

Keywords: Bulk drugs and formulations; Characterization; ESI-Q-TOF-MS; Stability-indicating RP–HPLC method; Zotepine.

Fig. 1

Chemical structure of zotepine.

Fig. 2

Typical HPLC chromatograms of (A) ZTP std, (B) acid hydrolysis, (C) base hydrolysis, (D) neutral hydrolysis, (E) oxidation, (F) photolytic and (G) thermal conditions.

Fig. 3

LC–ESI–MS/MS spectra of (A) [M+H]⁺ ions (m/z 332) of ZTP at 15 eV, (B) [M+H]⁺ ions (m/z 348) of Z1 at 15 eV, (C) [M+H]⁺ ions (m/z 318) of Z2 at 15 eV and (D) [M+H]⁺ ions (m/z 261) of Z3 at 15 eV.

Scheme 1

Proposed fragmentation mechanism for protonated ZTP (m/z 332).

Scheme 2

Proposed structures of DPs (Z1–Z3) formed under various stress conditions.

Scheme 3

Proposed fragmentation mechanism for degradation product Z1(m/z 348).

Scheme 4

Proposed fragmentation mechanism for degradation product Z2 (m/z 318).

Scheme 5

Proposed fragmentation mechanism for degradation product Z3 (m/z 261).