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

<b>Aim &amp; objective:</b> 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. <b>Materials &amp; methods:</b> A quality-by-design (QbD) approach was used for the optimization of the nanoemulsion, and US FDA guidelines were followed for method validation. <b>Results:</b> 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. <b>Conclusion:</b> Future findings will contribute to the treatment of postmenopausal osteoporosis and breast cancer using L-ORM and RAL. To coestimate levormeloxifene and raloxifene, an LC–MS/MS method has been developed and validated as per the US FDA industry guidance and bioanalytical method validation standards. A C18 Phenomenex Luna column (7.5 × 4.6 mm, 3 μm) was used to resolve both moieties. The mobile phase consisted of 80 v/v% acetonitrile and 20% v/v 0.1% formic acid in triple-distilled water at a flow rate of 0.5 ml/min. According to the FDA guidelines, the developed method was validated for a variety of characteristics, including linearity, precision and accuracy, lower limit of quantification and stability tests. The final nanoformulation was optimized using the Box–Behnken design technique. The final optimized formulation had a particle size of 94.01 ± 1.79 nm, a ζ-potential of -22.20 ± 0.10 mV and a polydispersity index of 0.24 ± 0.002 mV. The pharmacokinetic profile of free drug was compared with that of the developed nanoformulation, suggesting improved bioavailability. <i>In vitro</i> cellular uptake studies revealed a higher uptake of the developed nanoformulation as compared with free fluorescein isothiocyanate solution. The applicability of the developed LC–MS/MS method was presented by performing oral pharmacokinetic profiling of the levormeloxifene and raloxifene coloaded nanoformulation in female Sprague Dawley rats.