Track: Bioanalytics - Chemical - Drug Quantification - Other Methods/Techniques
Category: Poster Abstract
Development and Validation of a High-Performance Liquid Chromatography Based Analytical Assay for Determination of Naloxone Hydrochloride in Skin Permeation Studies
Purpose: Addiction to opioids is a serious national crisis and the most daunting challenge for health professionals in the country. Naloxone is an opioid antagonist, prescribed to counter the effects of opioid overdose. Presently, it is administered via intravenous, intramuscular, subcutaneous injection, or as an intranasal spray. Short duration of action―half-life of 64 min―requiring frequent drug dosing via invasive parenteral routes and variable drug absorption by intranasal route are the drawbacks associated with the current treatment options. Therefore, development of more patient compliant non-injectable formulations, providing rapid onset and sustained therapeutic effect, would facilitate widespread acceptance of naloxone across the public. Transdermal drug delivery, possessing all the stated characteristics, is an attractive alternative. Our long-term goal is to develop a transdermal system for rapid and sustained delivery of naloxone. However, a pre-requisite for evaluating the performance of any transdermal system is to have a sensitive analytical method that can selectively detect and quantify the drug, without any interference from compounds that may leach from skin during permeation studies. Thus, our preliminary goal was to develop and validate such a method that can be employed for transdermal studies. Methods: Reverse phase HPLC methods, employing different columns (Kinetex® 5 µm, 100 Ao, 250 X 4.6 mm C18; Kinetex® 5 µm Biphenyl 100 Ao, 250 X 4.6 mm C18 column; and Kinetex® 2.6 µm, 100 Ao, 100 X 2.1 mm, C18 column); mobile phase solvents (acetonitrile, methanol, deionized water, buffers with pH varying from 4-6); flow rates: 0.9 -1.1 mL/min; and column temperatures: 25-45 oC were investigated. The optimized conditions were then validated for inter- and intra-day accuracy and precision after determining the linearity range, limit of detection (LOD) and quantitation (LOQ). To ensure no interference from the skin, dermatomed porcine ear skin was minced and placed in phosphate buffered saline, pH 7.4 and shaken for 24 h, to extract any possible interfering components. The extract was filtered and analyzed with the optimized HPLC conditions. Further, naloxone standards employed for validation (0.25, 2.5, and 25 µg/mL) were stored at room temperature and 4o C and re-analyzed after 7 days. Results: The optimized method involved isocratic elution on the biphenyl column, at a ﬂow rate of 1.05 mL/min and column temperature of 40°C, after injecting 45 µL of sample and using methanol with 0.1% trifluoroacetic acid (30%), DI water (60%), and 0.2 M phosphate buffer- pH 4.0 (10%) as mobile phase and UV detection at 211 nm. Linearity was observed between 0.1-50 µg/mL. LOD and LOQ were determined to be 0.04 and 0.13 µg/mL, respectively. Inter- and intra-day accuracy and precision of the method were within the acceptable limit of ±20% at the lower limit of quantitation and ±15% at other concentrations, as per International Conference on Harmonisation guidelines. No interfering peak from the skin extract at the retention time of naloxone was observed. Further, the naloxone samples were found to be stable after storage for a week at room temperature and 4 oC. Conclusion: A reverse-HPLC based sensitive analytical method for detection and quantification of naloxone was developed and validated. The method exhibited no interference due to compounds leaching from the skin. Also, seven-day stability profile of naloxone samples at room and refrigerated conditions, was also established. Future plans involve employing the validated method for assessing the transdermal permeation of naloxone. References: Opioid Overdose Crisis (2019). NIDA. Retrieved from: https://www.drugabuse.gov/drugs- abuse/opioids/opioid-overdose-crisis. Strang, J., McDonald, R., Alqurshi, A., Royall, P., Taylor, D., & Forbes, B. (2016). Naloxone without the needle − systematic review of candidate routes for non-injectable naloxone for opioid overdose reversal. Drug and Alcohol Dependence, 163, 16–23. Yamamoto, R., Takasuga, S., Yoshida, Y., Mafune, S., Kominami, K., Sutoh, C., ... Kinoshita, M. (2012). In vitro and in vivo transdermal iontophoretic delivery of naloxone, an opioid antagonist. International Journal of Pharmaceutics, 422(1–2), 132–138. Puri, A., Murnane, K. S., Blough, B. E., & Banga, A. K. (2017). Effects of chemical and physical enhancement techniques on transdermal delivery of 3-fluoroamphetamine hydrochloride. International Journal of Pharmaceutics, 528(1–2), 452–462.