Environmental temperature variations cause degradations in epinephrine concentration and biological activity

Environmental Temperature Variations Cause Degradations in Epinephrine Concentration and Biological Activity TERRY A. GRANT, MD,* ROBERT G. CARROLL, PHD,t WILLIAM H. CHURCH, PHD,§ ANTHONY HENRY,5 N. HERAMBA PRASAD, MD,’ ABDEL A. ABDEL-RAHMAN, PHD,+ E. JACKSON ALLISON, JR, MD, MPH* This study determined the biological consequence 01 temperature induced epinephrine deqradatlon. Two dlfferant epinephrine preparations (1: 1,tttlO and l:lO,OOO) were exposed to either cold (5°C) or hot (70°C) temperaturas. The exposure occurred for p-hour periods each day in 4-, tl-, and 12-week intervals. Samples and identical controls were then chemically evaluated usinq high-pressure liquid chromatography (HPLC), and bloloqical activity 01 samples showing chemical degradation was assessed in conscious rats. Epinephrine (l:lO,OOO) underwent a significant degradation and a loss 01 concentration of the parent compound alter 8 weeks of heat treatment. By 12 weeks, 64% of the epinephrine was degraded. A smaller (30%) but significant loss of cardiovascular potency was determined by blood pressure and heart rate responses in conscious rats. The degradation of epinephrine (l:l,OOO) was not statistically significant even after 12 weeks of heat exposure. No change was noted from centml In either epinephrine concentration when exposed to cold temperatures. In conclusion, epinephrine (l:lO,OOO) deteriorates in the presence of elevated temperature and should be protected from high temperatures when carried by EMS providers. The degradation products may possess biological activity. (Am .I Emerg Med lgg4;12:31g-322. Copyright 0 1994 by W.6. Saunders Company)

Advanced Life Support (ALS) prehospital providers carry with them an assortment of medications. These medicines are subjected to a wide variety of environmental stresses,

from which hospital-based medications are protected. As the practice of emergency medicine continues to extend into the prehospital environment, emergency physicians need to evaluate long-standing hospital practices to determine if these practices are appropriate in the prehospital management of patients. Little information is available on the effect of exposure to extreme temperatures on medications and its impact on patient care. The pharmacological inserts on many medications recommend that the medications be stored between 15°C and 30°C (59°F and 86°F). The prehospital practice environment,

From the ‘Departments of Emergency Medicine, tPhysiology, SPharmacology, and SChemistry, East Carolina University, Greenville, NC. Manuscript received September 27, 1993; revision accepted December 12, 1993. Address reprint requests to Dr Carroll, Department of Physiology, School of Medicine, East Carolina University, Greenville, NC 27858-4354. Key Words: Breakdown, catecholamine, emergency medical services, heat. Copyright 0 1994 by W.B. Saunders Company 0735~6757194/1203-0014$500/O

however, often exceeds these recommendations. This results in medications being exposed to wide temperature fluc-

tuations before the printed expiration date is met. The ambient air temperature variation is exacerbated when ambulances (including air ambulances) are subject to direct sunlight and sit unventilated for extended periods of time. Uninsulated prehospital medication boxes will also make the problem worse by further concentrating the elevated temperature and then maintaining this elevated temperature as ambient temperatures decrease.’ Epinephrine stability is decreased by exposure to elevated temperature. Storage at 37°C for 6 months decreased epinephrine concentration by 50%.13 The rate of epinephrine degradation increases further as the temperature increases. Storage at 50°C caused a 25% loss within 2 months.‘1*‘2 A recent study has indicated that isoproterenol underwent an 11% loss of the parent compound after exposure to 4 weeks of temperature variability.2 The same study showed that heat also caused epinephrine to undergo a change in ionized state. The biological consequences of these changes, however, were not explored. The duration of the exposure to environmental stress is a confounding variable. Urban emergency medical service (EMS) organizations have a rapid turn-over of medicines. Rural EMS organizations, however, may store drugs for longer duration before administering them to patients. The possibility of temperature enhanced breakdown of epinephtine is also a concern for individuals who carry epinephrine 1: 1,000 for self administration in the event of anaphylaxis. This study determined the effect of cold and hot temperature exposure on the biological activity of epinephrine preparations frequently used in prehospital settings.

METHODS Simulated temperature environments were developed using a standard 3 cu refrigerator and an uninsulated laboratory oven. Both units were equipped with temperature probes. An electric timer was placed on both units to allow for standard cycling of the two environments. The freezer was allowed to run for a 2-hour period, resulting in cooling to 2.X. Return to room temperature (23°C) required approximately 6 hours for a total treatment time of 8 hours. The oven was heated for 6 hours, resulting in a temperature of 70°C. Cooling to room temperature required approximately 2 hours for a total treatment time of 8 hours. These times were chosen to simulate diurnal temperature changes. The cycle was repeated once every 24 hours. Fresh medication samples were obtained from a single lot number 319

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of epinephrine 1:1,000 ampules (1 mg/mL) and epinephrine 1: 10.000 in prefilled syringes (0.1 mg/mL). Treatment groups consisted of either low- or high-temperature cycling for intervals of 4, 8, and 12 weeks. Five samples were assigned to each treatment group. Samples were added to the thermal environments at 4-week intervals, so that all treatment groups were withdrawn from the thermal environment at the same time. A control group of identical medications was maintained at room temperature. All samples were left in their original clear glass containers and protected from exposure to light. The control group and medications awaiting placement into the test situation were stored in a cabinet protected from the light. On completion of the test period, all samples were returned to the control environment until analysis was performed (approximately 2 weeks). The sealed ampules and syringes were opened. and the samples were analyzed in duplicate using high pressure liquid chromatography (HPLC) and electrochemical detection. The analysis included measurement of retention times and peak heights, evaluation for the presence of degradation products, and calculation of medication concentration in units of milligrams per milliliters. Standardized solutions of each medication were initially studied to establish normal readings. Samples were identified by a random numerical code to insure a double-blinded analysis. After the chemical analysis, the biological activity of heat-treated epinephrine 1: 10,000 was determined using instrumented conscious rats. Sprague-Dawley rats (300 to 350 g) were anesthetized with methohexital and fitted with femoral artery and femoral vein catheters. A thermistor was advanced through the left carotid artery into the thoracic aorta, and a PE50 catheter was advanced through the right jugular vein into the right atrium. Animals were allowed to recover for 48 hours before the study. The exposed catheters were connected to an infusion pump, a Cardiomax II cardiac output computer (Columbus Instruments, Columbus, OH), and a pressure transducer. Arterial pressure and heart rate were recorded on a Grass polygraph, and cardiac output was calculated from the thermodilution curves constructed by rapidly injecting 0.1 mL room temperature saline into the jugular catheter. Pilot experiments indicated that an epinephrine dose of 8 pgikg provided a peak blood pressure response. The median sample (of the five replicates in each treatment group) based on the HPLC analysis was selected for assay of biological activity. Serial 1: 1 dilutions were made to provide a dose range of 0.5 kg/kg to 8 kg/kg. Peak blood pressure and bradycardic responses were recorded after bolus injection, with 15-minute interval allowed for recovery between doses. The samples were coded, and the sequence was varied to diminish investigator bias. Complete data sets were obtained for six rats in the control, 4 week, and 8 week heat-treated samples, and for four rats in the 12-week heat-treated samples. The short duration of the pressor response to the low doses of epinephrine precluded cardiac output measurement during bolus injection. As an alternative, rats were infused continuously at the end of the dose-response curve, and cardiac output was determined during the steady-state response. Data were subjected to a one-way analysis of variances, and individual means were compared using Tukey’s test. Comparisons with a P < .05 were considered significant. All data are expressed as mean 2 standard error.

RESULTS Significant changes were found in epinephrine l:lO,OOO (0.1 mg/mL) when subjected to environmental temperature variations. Both the g-week and 1Zweek heat-exposed samples showed a significant decrease in epinephrine concentration when compared with control samples (Figure 1). At the end of 12 weeks, a 64% loss of the parent compound was noted. The sample subjected to the cold environment did not

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Time (weeks) FIGURE 1. Exposure to heat causes a time-related decrease on epinephrine 1: 10,000. Values shown are mean * SEM. *Values significantly different from 0 weeks (control). P < .05.

seem to be affected compared with the control group (data not shown). Twelve weeks of heat cycling significantly reduced the mean arterial pressure response (when compared with untreated epinephrine samples) in two of the five doses tested (Figure 2). The baroreceptor mediated drop in heart rate after epinephrine infusion was similarly attenuated (Figure 3). The data for the 4- and 8-week heat cycling treatments decreased between these extremes, but were not significantly different from control (data not shown). The slope at the individual animal dose-response curves was used to calculate the dose required to obtain a 45 mm Hg pressor response (ED-50 in control epinephrine samples). The 8- and 12-week heat-treated samples required a signifi-

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Epinephrine (ug/kg) FIGURE 2. Heat treatment of epinephrine decreased the peak blood pressure response observed in conscious rats. N = 6 for control; N = 4 for the heat-treated group. Values shown are mean 2 SEM. *Values significantly different from corresponding control dose, P < .05.

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Epinephrine (ug/kg) FIGURE 3. Heat treatment attenuated the bradycardic response to bolus epinephrine injection. N = 6 for control; N = 4 for the heat-treated group. Values shown are mean k SEM. *Values significantly different from corresponding control dose, P < .05.

cantly higher dose to achieve the 45 mm Hg response (Figure 4). The changes in cardiac output after epinephrine infusion were not significant for any group as a result of large interanimal variation (data not shown). Epinephrine 1: 1,000 (1 mg/mL) did not show a significant degradation during a 12-week period while subjected to heat. Although a change was noted from the control sample in the concentration of degradation product, there was not a significant change in the concentration of the parent product. An effect of cold temperature was not apparent compared with the control group (data not shown). DISCUSSION The most important finding of this study is that thermal stress causes epinephrine to degrade, with a consequent loss of biological potency. Heat exposure caused a time-

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Duration of heat exposure FIGURE 4. Heat exposure increased the dose needed to achieve a 45 mm Hg pressure response in conscious rats. N = 6 for control; N = 4 for the heat-treated group. Values shown are mean 2 SEM. *Values significantly different from control, P < .05.

dependent breakdown of epinephrine l:lO,OOO, with more than 50% of the parent compound being lost after a 1Zweek period. With the advent of EMS more than 2 decades ago, administration of medications outside of the controlled hospital environment has become commonplace. As paramedic systems grow nationwide, more medications are being stored in medication boxes in EMS vehicles. In 1985, Palmer et al showed that the temperature inside a paramedic medication box ranged from 26.6”C to 4OS”C, whereas outside ambient air temperature ranged only from 23.9”C to 34&C.’ These medications are being subjected to extremes of temperature in closed vehicles. Often, these medications are kept almost until their expiration dates in closed containers in ambulances. In some settings, these medications may be exchanged at the hospital pharmacy before their expiration dates. The heat-exposed medications may then be placed in hospital supplies for use before the listed expiration date. This practice is perhaps more prevalent in rural EMS systems. The stability of these medications and the physiological consequences of heat-induced degradation then become hospital, as well as prehospital, concerns. In 1989, Valenzuela et al studied the thermal stability of prehospital medications.’ Four identical sets of 23 medications were kept in a simulated environment for 4 weeks. Inside the medication box, the temperatures ranged from 26°C to 38°C. At the end of each week, one set was removed and analyzed by gas chromatography-mass spectrometry for evidence of degradation products. At week 3 of exposure, changes were noted in the buffer compound in which epinephrine was dissolved. The biological consequences of these changes were not determined. In practice, these medications may be kept in EMS vehicles for much longer than 4 weeks, especially in rural areas. The effects of long-term exposure to temperature variations need to be addressed. Our study represents an extreme temperature stress, chosen to represent a worse case scenario. Ambient temperatures in the South and Southwest can exceed 38°C for days at a time. Temperatures in closed vehicles exposed to the sun, however, will significantly exceed ambient temperatures. Additionally, storage of drugs in a closed, secure box may further increase the thermal stress. Eventhough our choice of temperature is an extreme case, it is still appropriate for evaluation of the safety of medications stored outside of the hospital. Much has been written and discussed regarding the improper storage of sublingual nitroglycerin tablets and problems related to the loss of medication potency.3-5 In 1987, Sullivan et al noted that ionizing radiation had no detrimental effect on cardiac resuscitation medications (atropine, dopamine, epinephrine, and isoproterenol) and concluded that these medications may be safely stored in diagnostic radiology rooms without loss of potency.6 This type of scrutiny needs to extend to all of the ALS prehospital medications. Epinephrine is an endogenous catecholamine produced primarily in the adrenal medulla, with a wide variety of clinical uses. Epinephrine is poorly soluble in water, but readily forms water soluble salts in the presence of acids. Injectable solutions are buffered to maintain a pH of 2.5 to 5.0. Epinephrine solutions are colorless; however, oxidation of the catechol nucleus will impart a pink color that progresses to a

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brown color.’ Medication inserts caution that discolored solutions or those that contain a precipitate must not be used. None of the solutions in this study underwent an apparent discoloration or precipitation, in spite of a marked degradation of epinephrine. HPLC has been shown to be an accurate and reproducible analytical too18*9 and can differentiate epinephrine in the presence of degradation products.” The dramatic reduction of epinephrine 0.1 mg/mL during the 12-week study period is most disconcerning and may carry with it significant patient care concerns. The two preparations of epinephrine showed a marked difference in susceptibility to thermal breakdown. Epinephtine I:1000 did not show a significant loss of parent compound, but a time-dependent increase in a potential breakdown products was noted. It is possible that a difference in preservatives may account for this observation. The l:lO,OOO epinephrine preparation has 1 mg/mL sodium metabisulfite as an antioxidant and a citric acid/sodium citrate buffer to maintain an acidic pH. The 1: 1000 epinephrine has a higher concentration of bisulfite (2.5 mg/mL) and is not pH buffered. Church et al have identified the breakdown product as epinephrine sulfonic acid, which is formed in the presence of the metabisulfide antioxidant. I4

CONCLUSION Epinephrine 0.1 (1: 10,000) intravenous solution is adversely affected when exposed to periods of elevated temperatures. A degradation of the medication is noted by 8 weeks of temperature exposure consistent with storage in a closed ambulance sitting in a hot environment. Care must be taken to prevent this environmental exposure. Frequent rotation of epinephrine to ensure protection of the medication may also be useful. The authors studies.

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REFERENCES 1. Palmer RG, Zimmerman J, Brown PC, et al: Altered states: The influence of temperature on prehospital drugs. J Emerg Med Serv 1985;10(12):29-31 2. Valenzuela TD, Crisis EA, Hammargren WM, et al: Thermal stability of prehospital medications. Ann Emerg Med 1989;18: 173-176 3. Cummings AJ. Martin BK: Deterioration of nitroglycerin tablets. J Pharm Sci 1968;57:893 4. Fusari SA: Nitroglycerin sublingual tablets: Stability of conventional tablets. J Pharm Sci 1973;62:122-129 5. Rottman SJ, Larmon B, Mannix T: Chemical stability of sublingual nitroglycerin tablets carried on paramedic vehicles. Am J Emerg Med 1988;6(6):681-683 6. Sullivan DJ, Hubbard LB, Broadbent MV, et al: The effect of ionizing radiation on advanced life support medications. Ann Emerg Med 1987;16:662-665 7. American Society of Hospital Pharmacists: American Hospital Formulary Service Drug Information. Bethesda, MD, 1990 8. Williams DH: Ion-pair high performance liquid chromatography of terbutaline and catecholamine with aminophylline in intravenous solutions. J Pharm Sci 1982;71(8):956-958 9. Waraszkiewicz SM, Milan0 EA, DiRubio R: Stabilityindicating higher performance liquid chromatographic analysis of lidocaine hydrochloride and lidocaine hydrochloride with epinephrine injectable solutions. J Pharm Sci 1981;70(11):12151218 10. Fu CC, Sibley MJ: Quantitative high-pressure liquid chromatographic determination of epinephrine in pharmaceutical formulations. J Pharm Sci 1977;66(3):425-426 11. Kelly JR, Dalm GW: Stability of epinephrine in dental anesthetic solutions: Implications for autoclave sterilization and elevated temperature storage. Military Med 1985;150:112-114 12. Fry BW, Ciarlone AE: Concentrations of vasoconstrictors in local anesthetics change during storage in cartridge heaters. J Dent Res 1980;59:1163 13. Fry BW, Ciarlone AE: Storage at body temperature alters concentration of vasoconstrictors in local anesthetics, J Dent Res 1980;59:1069 14. Church WH, Hu SS, Henry AJ: Effect of heating conditions on epinephrine concentration in 1 :lO,OOO injectable cartridges. Am J Emerg Med (in press)