Stress stability study of simeprevir, a hepatitis C virus inhibitor, using feasible TLC-spectro-densitometry: application to pharmaceutical dosage form and human plasma

Abstract

Simeprevir is one of the newest direct action anti-hepatitis C drugs. In the present work, a simple, highly selective and stability-indicating, high-performance thin-layer chromatography (HPTLC) method is proposed and validated for the assay of simeprevir both in pharmaceutical dosage form and spiked human plasma. The method used silica gel 60 F₂₅₄ coated HPTLC aluminum plates as the stationary phase. The mobile phase system was ethyl acetate-hexane-methanol (5 : 4 : 1, v/v/v). The wavelength for both detection and quantitation was set at 288 nm. This system was found to give a compact spot of simeprevir; the retardation factor (R _F) value was 0.67 ± 0.02. The guidelines of the International Conference on Harmonization were followed to validate the proposed analytical method, and the results were acceptable. The calibration curve was linear over the range of 80-1000 ng per spot. The limit of detection was 19.0 ng per spot, and the limit of quantitation was 57.0 ng per spot. The drug was subjected to various stress conditions including hydrolytic, oxidative and UV-induced resulting in varying degrees of degradation. The results showed that the proposed method could efficiently separate the degradation products from the intact drug and allow its satisfactory quantitation. The proposed method was employed successfully for the accurate and reproducible analysis of the pharmaceutical preparation and human plasma containing the drug. The proposed method's precision and accuracy were statistically similar to those of a reported method.

Fig. 1. The chemical structure of simeprevir (SMV).

Fig. 2. A 3D chromatogram for the calibration of SMV, using the optimized solvent system “ethyl acetate–hexane–methanol” in the ratios (5.0 : 4.0 : 1.0, v/v/v), and wavelength of TLC scanner was set at 288 nm.

Fig. 3. A 3D chromatogram for evaluation of the accuracy of investigated analytical procedure for determination of SMV at five concentration levels with three replicate measurements.

Fig. 4. Two-dimensional TLC-densitograms of simeprevir; (A) standard solution (500 ng per spot), (B) extract of pharmaceutical dosage form (500 ng per spot), (C) plasma sample spiked with (500 ng per spot), using solvent system containing “ethyl acetate–hexane–methanol” in the ratios (5.0 : 4.0 : 1.0, v/v/v), and wavelength of TLC scanner was set at 288 nm.

Fig. 5. A 2D chromatogram of (A) 500 ng per spot SMV, (B) 1.0 M HCl acid-induced degradation, (C) 1.0 M NaOH base-induced degradation, (D) oxidative degradation with 30% v/v H₂O₂, (E) UV light degradation products, and (F) neutral hydrolysis degradation products.

Fig. 6. Possible pathways for the hydrolytic and oxidative degradations of SMV. Compound (1) was previously suggested as the oxidative product.