Enhanced oral bioavailability of levormeloxifene and raloxifene by nanoemulsion: simultaneous bioanalysis using liquid chromatography-tandem mass spectrometry

Abstract

Aim & objective: Levormeloxifene (L-ORM) and raloxifene (RAL) are selective estrogen receptor modulators used in the treatment of postmenopausal osteoporosis and breast cancer. Here, we developed and validated a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the simultaneous estimation of both drugs. Materials & methods: A quality-by-design (QbD) approach was used for the optimization of the nanoemulsion, and US FDA guidelines were followed for method validation. Results: Multiple reaction monitoring transitions were used for L-ORM (459.05→98.50), RAL (475.00→112.02) and internal standard (180.10→110.2). Analytes were resolved in a C18 column with 80:20 v/v% acetonitrile (ACN), 0.1% formic acid in triple-distilled water as a mobile phase. The developed method was linear over a concentration range of 1-600 ng/ml. Pharmacokinetic results of free L-ORM-RAL and the L-ORM-RAL nanoemulsion showed C_max of free L-ORM - 70.65 ± 16.64, free RAL 13.53 ± 2.72, L-ORM nanoemulsion 65.07 ± 14.0 and RAL-nanoemulsion 59.27 ± 17.44 ng/ml. Conclusion: Future findings will contribute to the treatment of postmenopausal osteoporosis and breast cancer using L-ORM and RAL.

Keywords: LC–MS/MS; bioanalytical method validation; co-estimation; levormeloxifene and raloxifene; pharmacokinetics; postmenopausal osteoporosis.

Figure 1.

Typical multiple reaction monitoring chromatograms. (A) Blank plasma samples. (B) Blank plasma spiked with IS (zero sample). (C) L-ORM and RAL spiked plasma with IS at a lower limit of quantification. (D) L-ORM and RAL spiked plasma with IS at a high-concentration quality control level. (E) Oral pharmacokinetic sample of L-ORM–RAL, spiked with IS. IS: Internal standard; L-ORM: Levormeloxifene; RAL: Raloxifene.

Figure 2.

Incorporating Box–Behnken design software for developing contour plots and response surface plots. (A) Plotting contour size. (B) 3D response surface graphic showing how size and its interactions affect Y1. (C) Contour plot of ζ-potential. (D) 3D response surface plot showing how interactions and ζ-potential affect Y2. (E) PDI contour plot. (F) 3D response surface map that illustrates how PDI affects Y3. (G & H) Desirability function of Box–Behnken design at independent variables A, B and C with optimized contour plot and 3D surface response at Y1, Y2 and Y3.PDI: Polydispersity index.

Figure 3.

Applicability of the developed liquid chromatography-tandem mass spectrometry method. (A) Implementing the proposed methodology to assess the formulation loaded with L-ORM and RAL. (a) Procedure for preparing nanoemulsions. (b & c) Characterization of the nanoformulation. (d & e) Liquid chromatography-tandem mass spectrometry (LC–MS/MS) was used to examine the loading efficiency and entrapment efficiency of the produced formulation. (B) In vitro quantitative and qualitative assessment of FITC-free and FITC-loaded nanoemulsion cellular consumption. (a) The FACS histogram shows cells that were treated with free FITC solution, control cells and nanoemulsions loaded with FITC. (b) A bar graph that compares the treatment with free FITC solution versus the FITC nanoemulsion. (c) Study of cellular consumption following an 8-h incubation. FITC: Fluorescein isothiocyanate; L-ORM: Levormeloxifene; NE: Nanoemulsion; RAL: Raloxifene.