Purpose: Epinephrine autoinjectors (EAI) are recommended for the treatment of anaphylaxis. Epinephrine is a chiral molecule where only the L(-) configuration demonstrates biological activity, and the D(+) configuration is not considered biologically active. Inactive epinephrine (D-form) is present as an impurity or formed as a byproduct of active epinephrine degradation. Our purpose is to develop a chiral HPLC method to evaluate epinephrine content in the EAIs and the degradation products including the D/L enantiomer ratio. Methods: Separating chiral compounds is important as each enantiomer poses different pharmacokinetic and pharmacological activities, as active epinephrine is ten times more potent than the inactive. Chiral separation of epinephrine enantiomers was performed on an ORpak CDBS-453 4.6 x 150 mm column (Shodex) using an Agilent HPLC. The mobile phase was composed of a 95%:5% (v/v) aqueous/acetonitrile mixture. The aqueous phase is a 200 mM sodium chloride solution containing prepared 0.05% glacial acetic acid. A flow rate of 0.5 mL/min was used in isocratic mode and the column was cooled to 10°C. The injection volume was set to 10 µL, and UV detection was monitored at 260 nm. Results: The chiral separation technique offers good selectivity and low limits of quantification. Each sample was examined over a 20-minute run and the peaks for L(-) and D(+) epinephrine appear around 7.75 minutes and 8.23 minutes, respectively. The chiral HPLC chromatogram peak areas for each enantiomer were used to calculate the corresponding concentrations. Pure standards of L(-) epinephrine (99%, Thermo Scientific) and D(+) epinephrine (Santa Cruz Biotechnology, Inc.) were used to generate calibration curves that correlate the peak areas to concentrations. Conclusion: The method was used for the identification and quantification of the D(+) and L(-) racemic ratio of epinephrine in expired epinephrine autoinjectors (EAIs). References: Kassel L, Jones C, Mengesha A. Epinephrine drug degradation in autoinjector products. Journal of Allergy and Clinical Immunology: In Practice 2019; 7:2491-3. Fukushima T, Murayama K, Santa T, Homma H, Imai K. Enantiomeric separation of d-/l-norepinephrine and -epinephrine by high-performance liquid chromatography with a β-cyclodextrin type chiral stationary phase. BIOMED CHROMATOGR 1998; 12:1-3.
