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.

Graphical abstract

Summary points

• 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.

• In vitro 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.

Keywords: :

• bioanalytical method validation
• co-estimation
• LC–MS/MS
• levormeloxifene and raloxifene
• pharmacokinetics
• postmenopausal osteoporosis

Author contributions

D Chauhan: writing – original draft, methodology, investigation, formal analysis, data curation. D Maity: investigation. P Yadav: data curation, formal analysis, writing – review and editing. S Vishwakarma and A Agarwal: visualization. M Chourasia: formal analysis. J Gayen: conceptualization, supervision, writing – review and editing.

Financial disclosure

The authors are thankful to the Department of Science and Technology (DST), New Delhi, India, for financial support in the form of the DST-Inspire fellowship as well as the Director, CSIR-CDRI, Lucknow, for their assistance in carrying out this work. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

Competing interests disclosure

The authors have no competing interests or relevant affiliations with any organization or entity with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

Writing disclosure

No writing assistance was utilized in the production of this manuscript.

Ethical conduct of research

The Institutional Animal Ethics Committee approved the protocols (no: LAF/2023/60 and IAEC/2021/58) ensuring that all animal investigations were conducted in compliance with ethical standards.

Acknowledgments

The authors would like to thank A Vishwakarma of the SAIF division for their valuable support in flow cytometry analysis, and the Laboratory Animal Facility of CSIR-CDRI; the CSIR-CDRI communication number is 10756.