Expired Epinephrine Maintains Chemical Concentration and Sterility
William Bradley Weir, Linda Y. Fred, Matthew Pike, Stanislav S. Rubakhin, Tyler J. Ludwig, Ashley M. Shar, Lingyang Zhu, Ann Frederick, Ike Uzoaru, Lin Wang & Jonathan V. Sweedler
To cite this article: William Bradley Weir, Linda Y. Fred, Matthew Pike, Stanislav S. Rubakhin, Tyler J. Ludwig, Ashley M. Shar, Lingyang Zhu, Ann Frederick, Ike Uzoaru, Lin Wang & Jonathan
V. Sweedler (2018): Expired Epinephrine Maintains Chemical Concentration and Sterility, Prehospital Emergency Care, DOI: 10.1080/10903127.2017.1402109
To link to this article: https://doi.org/10.1080/10903127.2017.1402109
ABSTRACT
Objectives: Epinephrine shortages affect nearly all American emergency medical services (EMS) systems. Utilization of expired epinephrine could mitigate this situa- tion in daily EMS operations. Concerns about using expired medications include sterility, potency, and potential harmful chemical decay byproducts. There are no cross-platform analyses of sterility and chemical purity of multiple sam- ples of expired parenteral epinephrine. We hypothesized that epinephrine injections will remain sterile and will retain their active ingredient’s content for more than 30 months past expiration. Methods: Six parenteral epinephrine prefilled syringes, 1 mg/10 mL, with an expiration date of January 1, 2012 had been stored in the climate controlled setting of a hospital inpatient pharmacy where they remained until they were taken for chemical or microbial analysis 30 months after expiration. An unexpired parenteral epinephrine prefilled syringe content was used as a control. Contents of three separate syringes with expired content from the same lot and one control underwent ultra-high pressure liquid chromatography-mass spectrometry (UHPLC-MS) and nuclear magnetic resonance (NMR) to determine epinephrine content and stability. In parallel, contents of another three expired epinephrine syringes were analyzed for sterility by plating on aerobic, anaerobic, and fungal media in a hospital microbiology laboratory. The aerobic plates were checked for growth in 3 days, the anaerobic in 5 days, and the fungal in 28 days. Results: UHPLC-MS and NMR showed that content of epinephrine present in the original sample remained unchanged compared to the control. There was no statistical difference in the UHPLC-MS and NMR signal amplitudes between the control and the expired samples. No chemical degradation byproducts were detected using NMR. There was no growth of any bacteria or fungus. Conclusion: Recurrent epinephrine shortages impact EMS and hospital operations in the United States. Individual administrators may be hesitant to authorize use of expired pharmaceuticals due to perceived potential compli- cations or fear of litigation. This study shows that the original parenteral epinephrine remains sterile and detectably pure more than 2.5 years after expiration. Further study of the sterility and chemical integrity of expired medications that had been subjected to the conditions of EMS vehicles may be a future research endeavor based on the aforementioned paradigm. Key words: expired epinephrine; sterility; chemical concentration.
INTRODUCTION
Medication shortages have become increasingly prob- lematic over the last decade. Following a maximum of 267 new shortages in 2011, there has been an average of 180 new drug shortages annually through 2015. Within that same time period, there have been up to 320 active drug shortages per quarter (1). While certain drug shortages have been more severe than others in extent and duration, impact on patient care is the greatest con- cern. For example, many shortages affect medications that primarily treat non-acute diseases. Other short- ages pertain to therapies utilized to manage lower com- plexity disease states or less critical symptoms. Beyond those considerations, in many shortage situations there have been pharmacotherapeutic alternatives that have allowed for patient care to proceed with minimal or no compromise.
The most concerning shortages involve medications utilized for acute, critical conditions where alternate therapies with equivalent efficacy, safety, or clinical experience are not available. An example of this is parenteral epinephrine for the treatment of anaphy- laxis or cardiac arrest (2,3). Also complicating the case of epinephrine are the various formulations available, intended for emergent administration by healthcare personnel or ease of administration by patients and family members (i.e., prefilled syringes, automatic injection devices, etc.). Situations may arise when epinephrine is available, but not in an optimal formulation for critical therapeutic contexts. We therefore decided to examine the stability of expired injectable epinephrine. If there were a future significant shortage of injectable epinephrine, the overall risk-versus-benefit ratio at that time could shift such that administration of expired product may be more prudent than denying a patient life- saving therapy. If such a shortage were to occur, knowing the extent of the physiochemical and micro- biological stability of epinephrine could be of high value.
METHODS
Materials
Six epinephrine prefilled 1 mg/10 mL syringes (McKesson Packaging Services Business Unit of McKesson Corporation) from the same batch, with an expiration date of January 1, 2012, were stored at room temperature in a climate-controlled setting at the Carle Foundation Hospital inpatient pharmacy. In July 2014, which was 30 months after expira- tion and 48 months after production, three of each were submitted to the Carle Foundation Hospi- tal microbiology laboratory and the University of Illinois at Urbana-Champaign chemistry laborato- ries. Each medication package was intact and the epinephrine solution within the syringes was clear and colorless. One epinephrine prefilled 1 mg/10 mL syringe, with an expiration date of August 1, 2015, was used as a controlled comparator solution for the chemical analysis. All epinephrine samples were purchased from Sigma-Aldrich Corporation (St. Louis, Missouri).
Sample Preparation
For UHPLC-MS, a Sstandard epinephrine stock solu- tion was first prepared in dimethyl sulfoxide (DMSO) at 200 mg/mL of epinephrine. The as a stock solu- tion was and serially diluted into concentrations (0.05–1.00 µg/mL) with solvent A (95/5 Ace- tonitrile/50 mM Ammonium Acetate). Expired epinephrine samples were diluted into 1 µg/mL with solvent A. Standard addition method was used to build the calibration curve in which samples were spiked with standard solution at various concentra- tions. Each sample was mixed thoroughly on a vortex between dilutions. Finally, 4 µL of solution was loaded on the system for measurement and six duplicate measurements were carried out for each solution. For NMR experiments, the epinephrine solution samples were used without any dilution.
Chemical Analysis
UHPLC-MS
Epinephrine was measured by UHPLC-MS and NMR. The EVOQ ultra-high performance liquid chromatography mass spectrometer (UHPLC-MS) system equipped with a hydrophilic interaction liq- uid chromatography (HILIC) column (100 x 2.1 mm, 2.6 mm) on-line hyphenated to EVOQ triple quad mass spectrometer (all Bruker Daltonics) was are used for analyte separation and detection. HPLC solvent A is 95/5 Acetonitrile/50 mM Ammonium Acetate pH 4, and solvent B is 50/40/10 Acetonitrile/water/50 mM Ammonium Acetate pH 4. Multiple reaction moni- toring (MRM) in positive mode was used to analyze all solutions. Epinephrine-containing solutions were analyzed with multiple reaction monitoring (MRM) approach using heated electrospray ionization con- ducted in positive mode. Electrospray voltage was set to 4500V, cone and heated probe temperature were 150°C and 450°C, respectively. Cone gas flow and probe gas flow were 10 and 15, respectively. Nebulizer gas flow was 60. Three transitions are mon- itored – m/zs 184.2 > 79.4 as quantifier, 184.2 > 166 and 184.2 > 150.9 as qualifiers. Information on epinephrine signal area is collected and statistically processed using Microsfoft Excel with mean and stan- dard deviation for six independent measurements of each sample presented. Multiple reaction monitoring (MRM) in positive mode was used to analyze all solutions.
1H-NMR
The Agilent VNMRS 750 MHz spectrometer with a 5 mm indirect-detection probe equipped with X,Y,Z- gradient capability was used for structural and quanti- tative analysis of epinephrine solutions. A total of 128 scans were used, each with a 60 degree pulse angle and 4.7 second repetition time between scans. A con- tinuous low power (2 s) helped to saturate the water signal. The chemical shifts were referenced to water peak at 5.01 ppm. The proton spectra of three expired epinephrine solution and one control epinephrine solution samples in their native buffer condition (citric acid and sodium citrate) were collected at 4oC. The pro- ton spectrum of the epinephrine standard (0.2 mg/mL) in deuterated dimethyl sulfoxide (DMSO) was con- ducted at 25oC. Sample concentrations were calculated by comparing the integration values of epinephrine solution samples with those of the epinephrine standard.
Microbiological Analysis
Using standard clinical microbiology laboratory tech- niques, 0.5 mL of epinephrine from each of three expired prefilled syringes was plated onto standard aerobic, anaerobic, and fungal culture media. These cultures were maintained in the microbiology labora- tory under appropriate temperature and atmospheric conditions for the culture type. Culture media were examined for growth daily for 3 days for aerobic bacte- ria, daily for 5 days for anaerobic bacteria, and weekly up to 28 days for fungi.
RESULTS
UHPLC-MS detected strong signals for all measured epinephrine transitions with good linearity of the calibration curve in 0–5 ppm range. Samples were diluted to achieve the best concentration for quan- titation. Epinephrine concentrations (mean stan- dard deviation) determined in the expired samples 1- 3 were 0.09 0.02 mg/mL, 0.11 0.02 mg/mL, and 0.10 0.02 mg/mL, respectively, and concentration of unexpired epinephrine was 0.11 0.04 mg/mL (Figure 1). No significant difference between data points was observed when technical replicates were taken into account.
1H-NMR detected six signals attributed to epinephrine in analyzed samples (Figure 2). One peak was under the water signal and was detected by two-dimensional correlation spectroscopy (2D COSY). The signals are consistent with the 1H spectrum of epinephrine in the Human Metabolome Database (HMDB, http://www.hmdb.ca/spectra/nmr_one_d/ 1066). Peaks with chemical shifts of 2.71, 3.27, 4.94, 6.84, 6.93, and 6.94 ppm were observed. The two large peaks at 2.64 and 2.78 in our samples that are not present in HMDB were from the citric buffer (epinephrine in HMDB was in water). Therefore, for quantita- tive analyzes and as confirmation of our ability to detect epinephrine samples of compound’s standard, expired, and current epinephrine injections are com- pared. Epinephrine concentrations measured by 1H- NMR for the expired samples 1–3 were 0.088 mg/mL, 0.090 mg/mL, and 0.090 mg/mL, respectively, and concentration of unexpired epinephrine sample was 0.090 mg/mL. Expired and control epinephrine sam- ples have the same NMR spectral profiles. (Figure 2). Absence of new significant peaks or disappearance of observed ones in NMR spectra indicates stabil- ity of the compound and formulation. No nore- pinephrine or metanephrine was detected. How- ever, differences in data acquisition temperature and chemical matrix (DMSO vs. water-containing buffer) between the samples and the standard likely resulted in small underestimation of epinephrine concentration.
FIGURE 1. UHPLC-MS epinephrine concentration results for sam- ples 1–3 and control.
Microbiological analysis revealed no growth of any organisms. A certified microbiology technician and a Pathologist determined there was no growth of aerobic bacteria after 3 days, no growth of anaerobic bacteria after 5 days, and no fungal growth after 28 days.
DISCUSSION
The use of expired medications has understandably been avoided. However, given the current shortages of critical resuscitation medications for which no proven substitute exists, the use of expired medications may be necessary. Epinephrine is a critical medication for treat- ment of anaphylaxis and for cardiac arrest resuscita- tion. Verified extension of expiration dates for medica- tions may play a crucial role in alleviation of shortages. This study is the first to perform bioanalytical confir- mations of preservation of active ingredients with par- allel verification of sterility post expiration. Our study analyzed prefilled epinephrine that had been in the controlled environment of an inpatient pharmacy. The expiration date set by drug manufacturers is purpose- fully a conservative estimate of the product’s stability (4,5).
Other studies have evaluated the chemical concentrations of EMS medications for a variety of environ- mental conditions such as sunlight, vibration, and temperature extremes. Many EMS medications were evaluated including atropine, ondansetron, hydro- morphone, diphenhydramine, fentanyl, midazolam, morphine, naloxone, tranexamic acid, diazepam, lorazepam, and midazolam (6–9). Results from Armenian et al. demonstrated different stability and sensitivity of the drugs to environmental factors with atropine be most unstable, becoming undetectable after 4 weeks of exposure to heat (6). Epinephrine degradation was also studied demonstrating rela- tive stability of the compound at different conditions and concentrations (10–14). For example, 64% of the drug was degraded after 12 week regiment of ele- vated temperatures at lower concentration but no changes are noted in more concentrated solution (11). Not only environmental factors but composition of epinephrine formulations including pH and presence of different preservatives/stabilizers influences stability of the drug (13, 15–18). These results indicate that epinephrine can be classified as a thermo-labile drug in certain formulations (6–9).
FIGURE 2. 1H-NMR spectra of samples 1–3 and control (sample 4). The ∗ refers to the peaks from epinephrine. The peak at 4.94 ppm under water was detected by 2D COSY spectrum (homonuclear correlation spectroscopy).
The demand for epinephrine is increasing as it appears that the incidence of anaphylaxis is increas- ing (19). States are expanding scope of practice to allow EMT-Basics to inject epinephrine for ana- phylaxis (19). Some states require school staff and law enforcement officers to administer epinephrine. Other states, like Illinois, are mandating training of police to recognize anaphylaxis with the option to administer epinephrine. Price increases follow the increasing demand for epinephrine as supply may be limited. When faced with a shortage, temporary authorization of expired medication may not be unsafe and may be preferable to death or disability from a critical illness such as anaphylaxis or cardiac arrest. Furthermore, the increased prevalence of epinephrine in schools and among law enforcement officers may increase the likelihood that an emergency responder or a lay person is faced with using an expired medication or not treating a potentially deadly illness. We believe this research shows there is some reasonable expectation that expired parenteral epinephrine maintains chemical stability and sterility many months beyond its labeled expiration date.
Our results are consistent with the majority of previously published data on epinephrine dosages stability (10) where epinephrine content demon- strated preservation for more than 8,500 hours at 40ºC at laboratory settings (20). As our results show, stability of injectable epinephrine for 2.5 years beyond expiration date provides compelling evi- dence that the safe shelf life for prefilled syringes may extend well beyond the date listed by the manufacturer.
Our data have additional support from results of the Shelf Life Extension Program (SLEP), which has also shown that many medications are viable past their labeled expiration dates (5). The SLEP is a federal pro- gram to extend the expiration date of medications for use by the Department of Defense and the Strategic National Stockpile by routinely testing samples from the appropriate medication lots. Over 50% of the med- ications tested by the SLEP have their expiration date extended by at least a year but the program has found a significant amount of lot-to-lot variability for any spe- cific medication (5).
Private entities are currently not allowed to participate in the SLEP and the data collected by the program is not readily available for public use, which justifies studies such as ours to further evaluate the potential role of expired medications. Strengths of our study include the use of multiple samples and testing in independent certified laboratories. A limitation of our study is that the medications were stored under ideal conditions within the hospital. Others have reported that conditions in EMS vehicles frequently deviate from recommended storage conditions, which com- plicates direct application of these results to expired medications that were stored within EMS vehicles (21–23). This presents an opportunity for future research using expired medications that were sub- jected to the real-world environment of prehospital medicine.
CONCLUSION
This study shows expired epinephrine maintains chemical purity and sterility more than 30 months after the manufacturer’s listed expiration date. Therefore, if there were a future significant shortage of injectable epinephrine, the overall risk-versus-benefit ratio at that time could shift such that administration of expired product may be more prudent than denying a patient life-saving therapy.
References
1. Fox ER. Drug shortages statistics [PowerPoint slides]. 2016. [Cited 2017 Oct 24]. Available from: https://www.ashp.org/ drug-shortages/current-shortages/drug-shortages-statistics
2. Campbell RL, Li JT, Nicklas RA, Sadosty AT; Members of the Joint Task Force; Practice Parameter Workgroup. Emergency department diagnosis and treatment of anaphylaxis: a practice parameter. Ann Allergy Asthma Immunol. 2014;113:599–608. doi:10.1016/j.anai.2014.10.007. PMID: 25466802
3. Link MS, Berkow LC, Kudenchuk PJ, Halperin HR, Hess EP, Moitra VK, et al. Part 7: adult advanced cardiovascu- lar life support: 2015 American Heart Association guide- lines update for cardiopulmonary resuscitation and emergency cardiovascular care. Circulation. 2015;132(18 Suppl 2):444–64. doi:10.1161/CIR.0000000000000261.
4. American Medical Association. “Pharmaceutical Expira- tion Dates”. Report 1 of the Council on Scientific Affairs (A-01). July 25, 2001. Available from: http://www.ama- assn.org/meetings/public/annual01/csa_reports.pdf#search ’Pharmaceutical%20Expiration%20Dates.
5. Lyon RC, Taylor JS, Porter DA, Prasanna HR, Hussain AS. Stability profiles of drug products extended beyond labeled expiration dates. J Pharm Sci. 2006;95(7):1549–60. doi:10.1002/jps.20636. PMID: 16721796
6. Armenian P, Campagne D, Stroh G, Tallman CI, Zeng WZD, Lin T, Gerona RR. Hot and cold drugs: National Park Service med- ication stability at the extremes of temperature. Prehosp Emerg Care. 2017;21:378–85. doi:10.1080/10903127.2016.1258098. PMID: 28059581
7. de Guzman R, Polykratis IA, Sondeen JL, Darlington DN, Cap AP, Dubick MA. Stability of tranexamic acid after 12-week storage at temperatures from 20C to 50C. Prehosp Emerg Care. 2013;17:394–400. doi:10.3109/10903127.2013.792891. PMID: 23734991
8. McMullan JT, Jones E, Barnhart B, Denninghoff K, Spaite DW, Zaleski E, Silbergleit R, Treatment NE. Degradation of benzo- diazepines after 120 days of EMS deployment. Prehosp Emerg Care. 2014;18:368–74. doi:10.3109/10903127.2013.869642. PMID: 24548058
9. McMullan JT, Pinnawin A, Jones E, Denninghoff K, Siewart N, Spaite DW, Zaleski E, Silbergleit R, Treatment NE. The 60-day temperature-dependent degradation of midazolam and lorazepam in the prehospital environment. Prehosp Emerg Care. 2013;17:1–7. doi:10.3109/10903127.2012.722177. PMID: 23148574
10. Parish HG, Bowser CS, Morton JR, Brown JC. A sys- tematic review of epinephrine degradation with expo- sure to excessive heat or cold. Ann Allergy Asthma Immunol. 2016;117:79–87. doi:10.1016/j.anai.2016.04.006. PMID: 27221065
11. Grant TA, Carroll RG, Church WH, Henry A, Prasad NH, Abdel-Rahman AA, Allison, EJ, Jr. Environmental tempera- ture variations cause degradations in epinephrine concentra- tion and biological activity. Am J Emerg Med. 1994;12:19–22. doi:10.1016/0735-6757(94)90148-1.
12. Church WH, Hu SS, Henry AJ. Thermal degradation of injectable epinephrine. Am J Emerg Med. 1994;12:306–9. doi:10.1016/0735-6757(94)90145-7. PMID: 8179736
13. Hoellein L, Holzgrabe U. Ficts and facts of epinephrine and norepinephrine stability in injectable solutions. Int J Pharm. 2012;434:468–80. doi:10.1016/j.ijpharm.2012.05.017. PMID: 22613065
14. Rachid O, Simons FE, Wein MB, Rawas-Qalaji M, Simons KJ. Epinephrine doses contained in outdated epinephrine auto-injectors collected in a Florida allergy practice. Ann Allergy Asthma Immunol. 2015;114:354–6 e1. doi:10.1016/j.anai.2015.01.015. PMID: 25707326
15. Britz-McKibbin P, Wong J, Chen DDY. Analysis of epinephrine from fifteen different dental anesthetic formulations by capillary electrophoresis. Journal of Chromatography A. 1999;853:535–40. doi:10.1016/S0021-9673(99)00689-5. PMID: 10486763
16. Lee IP, Burton, H. Thermal-stability of various epinephrine formulations. Drug Dev Ind Pharm. 1981;7:397–403. doi:10.3109/03639048109057719.
17. Kannan V, Irish P, Bergren M. Epinephrine formulations. US 09119876, Sep 1 2015.
18. Kannan V, Irish P, Bergren M. Epinephrine formulations. US 09295657, Mar 29 2016.
19. Brasted ID, Dsailey MW. Basic life support access to injectable epinephrine across the United States. Prehosp Emerg Care. 2017;21(4):442–7. doi:10.1080/10903127.2017.1294224. PMID: 28339320
20. Zeisel U, Bauer M, Tas P, Apfel C, Sefrin P. Temperature sta- bility of epinephrine under various storage conditions. Anasth Intensivmed. 1998;39:139–45.
21. Brown LH, Krumperman K, Fullagar CJ. Out-of-hospital med- ication storage temperatures: a review of the literature and directions for the future. Prehosp Emerg Care. 2004;8(2):200–6. PMID: 15060857
22. Helm M, Castner T, Lampl L. Environmental temperature stress on drugs in prehospital emergency medical service. Acta Anaesthesiol Scand. 2003;47(4):425–9. doi:10.1034/j.1399- 6576.2003.00062.x. PMID: 12694141
23. Johansen RB, Schafer NC, Brown PI. Effect of extreme temperatures Epinephrine bitartrate on drugs for prehospital ACLS. Am J Emerg Med. 1993;11(5):450–2. doi:10.1016/0735-6757(93)90080-U. PMID: 8363680.