(W1030-10-56) Validation and Application of an LC-MS/MS Method to Evaluate In Vitro Permeation of Naloxone Nasal Spray Using Artificial Substrate

Purpose: Naloxone nasal spray (NNS) is used as an effective emergency treatment for opioid overdose to save lives. Naloxone is an opioid antagonist that reverses respiratory depression due to opioid overdose. The objective of this study was to validate a developed LC-MS/MS analytical method for quantification of naloxone along with its impurities: naloxone n-oxide and noroxymorphone in the permeation samples based on the solvent used in the in vitro permeation study, e.g., Dulbecco's Phosphate buffered saline (DPBS).

Methods: An Agilent ULTIVO QQQ LC/TQ coupled with an Infinity II 1260 autosampler was used for quantification of naloxone and its impurities (naloxone n-oxide and noroxymorphone) in the permeation samples. Naloxone-d5 was used as internal standard to compensate variability in analytes response. Agilent InfinityLab Poroshell analytical column equipped with an InfinityLab Poroshell guard column was used for sample analysis. Samples were ionized using positive electrospray ionization (ESI). The LC analytical column temperature was maintained at 30°C, and initial flow rate was 0.5 mL/min. Autosampler temperature was maintained at 4°C and sample acquisition time was ~5.0 minutes. Vitrocell cloud alpha 12 system was used for aerosolization and dosing of NNS onto the artificial substrate, e.g., Nuclepore track-etched membrane. To deposit dose of naloxone on the membrane, NNS was nebulized through a 4 to 6 micron vibrating mesh nebulizer which was connected to the cloud system. The in vitro permeation pattern of deposited naloxone on the artificial membrane was investigated using a flow through cells assembly. DPBS fortified with Calcium and Magnesium was used as the permeation medium in flow through cells and was pumped at a flow rate of 50 uL/min. Permeation samples across the membranes were collected at predetermined time points over a period of 2 hours. Concentration of naloxone and its impurities in the collected permeation samples were quantified using the in-house developed and validated LC-MS/MS analytical method.

Results: The analytical method featured a linearity range of 0.25 ng/mL - 20 ng/mL in DPBS and documented a sensitivity of 0.25 ng/mL (lower limit of quantitation). All compounds demonstrated linearity correlation coefficients r² > 0.99 (Figure 1). Figure 2 showing specificity for all the compounds including naloxone, naloxone-d5, naloxone-n-oxide, and noroxymorphone. The accuracy for naloxone, naloxone n-oxide, and noroxymorphone were 95.7% - 116.5 %, 96.2% – 113.6 %, and 96.5% – 118.5 %, respectively. All compounds displayed acceptable precision with %RSD < 15%. In vitro permeation test (IVPT) samples were stable for 7 days when stored at 4°C, if not processed freshly. The permeation results in Figure 3 showing that amount of naloxone permeated across the membranes plateaued (0.237 mg/cm²) after 45-60 minutes due to drug depletion from the membrane surface over time. Maximum flux of naloxone was attained within 5 minutes of permeation.

Conclusion: A sensitive, selective, and high throughput analytical method has been developed and validated for quantitation of naloxone and its impurities, naloxone n-oxide and noroxymorphone in DPBS using LC-MS/MS. The analytical method was designed according to the FDA bioanalytical guidance for specificity, linearity, range (0.25 ng/mL - 20 ng/mL), accuracy, precision, limit of detection, limit of quantitation (0.25 ng/mL), and stability. This method was successfully applied to determine the amount of naloxone and its impurities permeated in IVPT samples in DPBS.

Acknowledgements: Disclaimer: This abstract reflects the views of the authors and should not be construed to represent FDA’s views or policies.

Figure 1: Calibration curve featuring linearity and range for A) Naloxone, B) Naloxone-N-Oxide, and C) Noroxymorphone in DPBS.

Figure 2: Specificity for A) Naloxone, B) Naloxone-D5, C) Naloxone-N-Oxide, and D) Noroxymorphone in DPBS.

Figure 3: Cumulative amount of Naloxone permeated in DPBS across the Nuclepore track-etched membrane (n=4).