Effects of Time and Storage Conditions on the Chemical and Microbiologic Stability of Diluted Buprenorphine for Injection

Abstract

Buprenorphine is a partial μ-opioid agonist used for analgesia. Due to the small size of laboratory rodents, buprenorphine HCl is typically diluted 10- or 20-fold with a sterile diluent, such as saline, for accurate dosing. Protocols for preparing and storing diluted buprenorphine vary by institution, and little published information is available regarding stability and beyond-use dating of specific buprenorphine preparations. The purpose of this study was to determine the chemical and microbiologic stability of diluted buprenorphine stored for a maximum of 180 d. Buprenorphine HCl was diluted 1:10 into sterile bacteriostatic saline by using aseptic technique. Diluted samples were stored in glass vials or plastic syringes, protected from light, and maintained at refrigerated or room temperature for as long as 180 d. Aerobic and anaerobic cultures on all stored samples were negative for bacterial and fungal growth. According to HPLC analysis, diluted buprenorphine stored in glass vials experienced less than 10% loss when stored for 180 d at either refrigerated or room temperature. However, the concentration of buprenorphine stored in syringes declined rapidly to more than 80% loss at room temperature and 28% loss in the refrigerator after 180 d. According to the results of this study, diluted buprenorphine stored in glass vials retains more than 90% of the initial concentration and is microbiologically stable for 180 d. However, our data suggest that, regardless of the duration, storing diluted buprenorphine in plastic syringes is inadvisable.

Buprenorphine is a synthetic partial μ opioid agonist commonly used for the prevention and treatment of pain in laboratory animals. Only one FDA-approved veterinary formulation of buprenorphine, which is labeled for subcutaneous use in cats, is available in the United States.⁶ Therefore, laboratory animal veterinarians often use the approved human injectable formulation, buprenorphine HCl (0.324 mg/mL, equivalent to 0.3 mg/mL buprenorphine), for pain management in other species. Recommended doses of buprenorphine are 0.05–0.1 mg/kg SC or IP every 4 to 12 h for mice and 0.01–0.05 mg/kg SC or IP every 4 to 12 h for rats.^2,6 These doses would require an extremely small volume of 0.3 mg/mL buprenorphine (for example, 4 to 8 μL for a 25-g mouse), so this formulation is usually diluted 10- to 20-fold into a sterile diluent to increase dosing accuracy. Dilution of a drug in this fashion is considered pharmaceutical compounding.⁹

The United States Pharmacopeia (USP) General Chapter 797 describes the conditions and practices necessary to prevent harm to patients when compounding sterile preparations. In particular, inadvertent microbial contamination of a compounded sterile preparation (CSP) poses a significant risk to patients. In addition, degradation of the active ingredients in a CSP might result in decreased efficacy and put patients at risk due to the formation of potentially toxic degradation products.⁹ These are important considerations when diluting buprenorphine, especially given that this analgesic agent is packaged as a single human dose that lacks preservative and is intended for immediate use. For these reasons, it is essential to adhere to strict aseptic technique when compounding a sterile preparation and to assign a beyond-use date (BUD) to the CSP. The BUD is calculated from the date of compounding and is defined as the date or time after which the CSP shall not be stored or transported.⁹ A BUD is not the same as an expiration date, which is determined by the manufacturer and applies only to a specific formulation in its unopened container and stored at the labeled environmental conditions.¹

Storage protocols for diluted buprenorphine vary among institutions, with storage of compounded drug in vials, blood collection tubes, or syringes at refrigerated or room temperature and with recommended maximal durations that range from 24 h to 6 mo. The variety of methods for dilution and storage of injectable buprenorphine may be due, in part, to the lack of published data regarding the stability of this preparation. In fact, few articles address the stability or beyond-use dating for any compounded preparation containing buprenorphine.^3,5 When a BUD for a specific compounded preparation has not been established, the USP states that an experimental stability study may be performed to assess the physical, chemical, microbiologic, toxicologic, and therapeutic stability of a CSP. The purpose of the current study was to determine the effects of storage conditions and time on the sterility and concentration of diluted buprenorphine. We hypothesized that samples prepared aseptically would remain sterile and that the concentration of buprenorphine would fall below 90% (a minimum potency specification listed in the USP⁹ for many drugs) over a period of 180 d under all conditions tested.

Materials and Methods

Study design.

Buprenorphine HCl (1-mL vials, 0.3 mg/mL; Par Pharmaceutical Companies, Spring Valley, NY) was diluted 1:10 by using 0.9% bacteriostatic saline (Hospira, Lake Forrest, IL) at 180, 150, 120, 90, 60, 30, 21, 14, 7, 3, and 1 d before analysis. At each time point, 2 vials of buprenorphine HCl were diluted to create 20 approximately 1-mL samples that were stored under 4 different conditions (n = 5 each) until analysis: 10-mL sterile clear glass vials (Hospira) or 3-mL sterile plastic syringes (Monoject, Covidien, Mansfield, MA) stored at ambient room temperature (21.4 ± 3.2 °C) or in a refrigerator (5.5 ± 4.0 °C). In an effort to control variables, buprenorphine HCl and bacteriostatic saline were from the same lot numbers from their respective manufacturers. All samples were stored in corrugated cardboard boxes to protect from light. Sterility testing and HPLC were conducted on 2 different occasions: once for samples stored short-term (1 to 21 d) and once for samples stored for 30 to 180 d. Physical characteristics, weight, and pH were assessed at the time of analysis. Five additional samples (time 0) of diluted buprenorphine HCl were prepared and placed directly into 1-mL glass sampling vials for HPLC on each day of analysis.

Sample preparation.

All manipulations were performed by the same individual (JMD) using aseptic technique in a class II, type A2 biologic safety cabinet (Labgard NU-S629-400, NuAire, Plymouth, MN). The blower motor was allowed to run for at least 10 min before items were placed in the cabinet. The work surface was sprayed with disinfectant (Quatricide-TB, Pharmacal Research Labs, Waterbury, CT) according to label directions before and after each batch of samples was prepared. The top of each vial was wiped with an alcohol prep pad. A sterile 3-mL syringe with a 22-gauge, 1-in. hypodermic needle (Monoject, Covidien) was used to draw up 1 mL buprenorphine HCl from the vial supplied by the manufacturer and dispense it into a sterile empty clear glass vial for mixing. A 12-mL syringe with a 22-gauge, 1 in. needle was used to draw up 9 mL sterile bacteriostatic saline and add it to the mixing vial. The mixing vial was inverted 3 times to mix contents. A new 12-mL syringe with a 22-gauge, 1-in. needle was used to draw up the diluted buprenorphine from the mixing vial and dispense it into 5 sterile empty clear glass vials and 5 sterile 3-mL syringes, approximately 1 mL each. The needle was replaced between each sample container and sterile syringe caps (Monoject, Covidien, Mansfield, MA) were placed on sample syringes. All samples were weighed, placed into a corrugated cardboard box, and stored either in a cabinet at ambient room temperature or in a refrigerator (5.5 ± 4.0 °C). All samples remained undisturbed in storage until the day of analysis.

Physical stability.

The pH of buprenorphine HCl was assessed twice during sample preparation (before and after dilution) and once after the storage period by placing 1 drop of the solution on a strip of pH paper (pH 4.0 to 9.0, Micro Essential Laboratory, Brooklyn, NY). Poststorage pH was assessed for 1 sample from each of the 4 storage conditions at every time point. All samples were weighed and examined for visible changes after the storage period.

Sterility testing.

At the end of the storage period, 0.1 to 0.2 mL of each stored sample was aseptically collected by using 1-mL tuberculin syringes (BD, Franklin Lakes, NJ) with 22-gauge, 1-in. needles. These samples were pooled by time point and storage condition in 2-mL blood collection tubes (Monoject, Covidien, Mansfield, MA) and submitted to the Oregon Veterinary Diagnostic Laboratory for sterility testing. Each pooled sample was used to inoculate a whole-blood agar plate for aerobic growth, an anaerobic Brucella agar plate for anaerobic growth, a tube of brain–heart infusion broth, and a tryptic soy agar plate at room temperature for fungal growth. Cultures were held for 7 d before a final report was generated.

HPLC analysis.

All chromatographic measurements were performed using a Delta 600 solvent delivery system and model 600 controller equipped with a model 717 Plus autosampler and a model 2996 photodiode array detector (Waters, Milford, MA). The window of the autosampler door was covered with aluminum foil to minimize light exposure. The HPLC method was modeled after a previously described procedure⁵ and modified to optimize resolution of the buprenorphine peak by using available instruments. Samples were separated by using a reverse-phase Cogent 4-µm bidendate C8 column (150 × 2.1 mm, Microsolv, Leland, NC). The mobile phase consisted of 2 solvents: 10 mM ammonium acetate prepared with HPLC-grade water (solvent A) and HPLC-grade acetonitrile (solvent B). The solvent gradient was 70% solvent B in solvent A to 100% solvent B over 10 min at a rate of 0.5 mL/min. After holding for 2 min, the solvents were returned to starting conditions in 0.5 min, and the column was allowed to equilibrate for 7 min before the next injection. The injection volume was 30 µL for all samples. UV spectra in the range of 210 to 500 nm were recorded, and the 288-nm trace was used to calculate the peak area for buprenorphine. A 6-point standard concentration curve was constructed on each day of analysis by using buprenorphine HCl solid (Sigma-Aldrich, St Louis, MO) diluted in sterile bacteriostatic saline at 20%, 40%, 60%, 80%, 100%, and 120% of the expected concentration (0.03 mg/mL). The line equation from the standard concentration curve was used to calculate the concentration of buprenorphine in each sample.

Statistical analysis.

HPLC data were calculated as percentage of baseline concentration. All statistical analyses were performed by using SAS version 12.1 (SAS Institute, Cary, IL). Data were tested for normality and variance homogeneity and were shown to differ among container and temperature combinations. Therefore, time-dependent changes for each container and temperature combination were analyzed separately by using one-way ANOVA in the procedure general linear model. The Tukey test was used to adjust the P value for multiple comparisons across time within each container and temperature combination. All tests were 2-sided, and results were considered significantly different when the P value was less than 0.05.

Results

Physical stability.

All samples remained transparent, with no visible particulates or apparent change in color or viscosity throughout the entire storage period. No trends were seen in the differences between sample weights at the beginning compared with the end of the storage period. A pH of 6 was obtained for all tested samples.

Microbiologic stability.

None of the pooled samples from the short-term time points (1 to 21 d in storage) yielded any bacterial or fungal growth. Five aerobic cultures and 1 anaerobic culture from 5 of the pooled samples from the long-term time points (30 to 180 d in storage) each had a single colony of growth. These colonies were located at the far edge of the plates or outside the primary swab area. Repeat cultures of these 5 pooled samples were negative for growth. In addition, these 5 pooled samples showed no growth in primary or subcultures of brain-heart infusion broth.

Chemical stability.

Short-term storage.

Over the 21-d storage period, the buprenorphine concentration was significantly decreased by the 3-d time point for samples in syringes stored refrigerated (P = 0.01) or at room temperature (P = 0.03; Figure 1). Syringes stored at room temperature showed the greatest loss of buprenorphine, where 76.7% ± 2.5% (mean ± SEM) of the starting amount remained after 21 d. Samples stored in glass vials did not show a significant loss of buprenorphine.

Figure 1.

Buprenorphine concentration (% of initial concentration, mean ± SEM [n = 5 per group]) over time for samples stored as long as 21 d in glass vials and plastic syringes at room temperature (RT) and in the refrigerator (5 °C). Some SEM are too small to be visible.

Long-term storage.

Over the 180-d storage period, the buprenorphine concentration decreased significantly by the 30-d time point for samples in syringes stored refrigerated and at room temperature (89.3% ± 1.7% and 60.5% ± 2.9% remaining, respectively; P < 0.0001; Figure 2). The concentration of buprenorphine stored in syringes continued to decrease with age throughout the 180-d evaluation period, with 72.0% ± 1.7% remaining in samples stored in the refrigerator and just 17.3% ± 2.9% remaining when stored at room temperature. The chromatograms for several samples that were stored in syringes at room temperature for 120 to 180 d contained additional peaks that increased in area with sample age (Figure 3). For samples stored in glass vials, buprenorphine concentration was significantly decreased by the 30-d time point when refrigerated (95.7% ± 0.5% remaining, P < 0.0001) and by the 60-d time point at room temperature (94.2% ± 0.3% remaining, P < 0.0001). Overall, storage in vials for as long as 180 d led to less than 10% loss in buprenorphine content regardless of storage temperature.

Figure 2.

Buprenorphine concentration (% of initial concentration, mean ± SEM [n = 5 per group]) over time for samples stored from 30 to 180 d in glass vials and plastic syringes at room temperature (RT) and in the refrigerator (5 °C). Some SEM are too small to be visible.

Figure 3.

Chromatograms of buprenorphine (0.3 mg/mL) diluted 1:10 with 0.9% bacteriostatic saline on (A) the day of dilution and (B) after storage for 180 d in a syringe at room temperature. Absorbance wavelength, 288 nm.

Discussion

This study was designed to replicate, to the extent possible, the methods of diluting and storing buprenorphine that are commonly practiced at many laboratory animal facilities and to determine the effects of storage conditions and time on the sterility and concentration of stored samples. The precautions that we used to prevent microbial contamination, such as aseptic handling and working in a biologic safety cabinet, were selected because of their potential to be applied at most research institutions. Sterile filtration was not performed, because it is not currently standard practice in veterinary clinical medicine when using pharmaceutical-grade products. We believe this experimental design resulted in a ‘best-case’ scenario for each of the storage conditions tested.

A single bacterial colony grew on each of 6 plates (5 aerobic, 1 anaerobic) from 5 pooled samples of the long-term storage group. Given the location of these colonies at the far edge of the plates and outside the primary swab area, as well as the lack of growth on confirmatory testing, we concluded that these colonies were likely due to environmental contaminants from the bacteriology lab. The absence of growth on bacterial and fungal cultures suggests that the diluted buprenorphine remained sterile for 180 d under the tested storage conditions.

Two key aspects of the study design resulted in minimal risk for microbial contamination. First, sample preparation was performed by using strict aseptic technique in a biologic safety cabinet. Whereas this scenario does not conform to the air-quality standards for compounding sterile preparations set forth by the USP,⁹ it was the best achievable air quality given the standard equipment available in a working vivarium. Second, by using several dilution events to create samples of different ages (rather than mixing several stock solutions and drawing samples from them for repeated analyses as they aged), the number of times the samples were manipulated was minimized, thus decreasing the number of opportunities for contamination. In clinical practice, the use of a stored drug would likely result in repeated excursions from its storage environment and increased risk for microbial contamination.

The concentration of buprenorphine stored in clear glass vials for as long as 180 d at both refrigerated and room temperatures remained above 90% of the calculated concentration at the time of dilution. In contrast, HPLC data for diluted buprenorphine stored in syringes showed a rapid decline in concentration, falling to nearly 90% in just 1 d at room temperature and in 3 d in the refrigerator. The concentration of buprenorphine stored in plastic syringes continued to decline with age until the end of the 180-d observation period. On 2 occasions, the concentration of buprenorphine stored long-term in syringes was increased compared with the preceding time point (Figure 2). The apparent increase in concentration is likely due to variation in the starting concentration of samples from separate dilution events. Interestingly, additional peaks in the chromatograms of long-term syringe-stored samples increased in area with the age of the samples (Figure 3). This increase in peak area of the early-eluting peaks also occurred in saline-only controls stored in plastic syringes, indicating that additional molecules leached into the solution from some component of the syringe. Potential explanations for the loss of buprenorphine stored in plastic syringes over time include degradation and adsorption. Degradation of the buprenorphine molecule could result from a chemical reaction, such as oxidation,¹⁰ or an interaction with leached substances from syringe components.⁴ Another possibility is that the buprenorphine molecule adsorbed to one or more of the syringe components, decreasing its concentration in solution. Adsorption to surfaces usually occurs when functional groups within a molecule interact with binding sites on the surface of the container. In fact, adsorption is the most frequently reported drug interaction with plastic materials.⁷ Determining the specific cause(s) of the rapid decline in buprenorphine concentration when stored in syringes requires further investigation with advanced methods, such as high-resolution mass spectroscopy. Regardless of the mechanism, our data support that plastic syringes are inappropriate storage containers for diluted buprenorphine, even for a short period of time.

The USP provides guidance on determining BUD in General Chapters 795 and 797 for nonsterile and sterile preparations, respectively. The compounder needs to consider the physical and chemical stability of the compounded preparation and, for sterile preparations, the risk for microbiologic contamination. If stability information is unavailable, a water-containing preparation, such as the diluted buprenorphine in this study, may be assigned a BUD of not more than 14 d when stored at refrigerated temperature.⁹ However, a shorter BUD may be indicated for a CSP depending on its Microbial Contamination Risk Category. Buprenorphine HCl for injection is packaged in a single-dose container with no preservative. It is intended for immediate-use, and the USP states that immediate-use CSP must be used within 1 h from the start of the compounding procedure and that any unused portion must be discarded appropriately.⁹ Our study's focus is not to challenge or disprove USP guidelines, given the expert opinion that formulated these founding concepts. However, our study does show that using careful handling, sterile supplies, and aseptic technique in a biologic safety cabinet resulted in the absence of detectable microbiologic contamination of diluted buprenorphine stored for as long as 180 d. It is important to note that the risk for contamination increases every time the container is punctured with a needle or opened. Although the compounded buprenorphine in our current study remained uncontaminated and above 90% of the initial concentration for 180 d when stored in glass vials, multiple entrances into such vials for clinical use should be a consideration and warrant a decrease in the assigned BUD. A good thing to remember when compounding any medication and deciding how long to maintain it for use is the ‘loved-one test,’⁸ that is, would one be comfortable administering the compounded preparation to one's own parent, child, or spouse? If the answer is no, then the preparation, handling, and storage conditions and duration of the compounded product should be improved.

In summary, the dilution of a manufactured drug is considered pharmaceutical compounding. As such, a BUD should be assigned according to available information regarding the stability of the specific preparation, general recommendations listed in the USP, and the risk of microbiologic contamination for sterile preparations. Buprenorphine diluted 1:10 with 0.9% bacteriostatic saline and stored continuously in glass vials at refrigerated and room temperatures in the dark remained above 90% of initial concentration and microbiologically stable for 180 d. The concentration of diluted buprenorphine stored in plastic syringes decreased rapidly at both refrigerated and room temperatures. Therefore, we consider it inadvisable to use plastic syringes as storage containers for this compounded preparation. Further investigation is needed to determine the toxicologic and therapeutic stability (efficacy) of diluted buprenorphine. Sterile, single-dose injectable formulations of buprenorphine are commercially available in countries outside the United States. We believe the results from our study may apply to those formulations as well, given the similarity in composition. However, because multidose formulations often contain preservatives, our findings should not be generalized to include dilutions of such formulations. In addition, preparations of buprenorphine diluted into sterile water or 5% dextrose in water require separate inquiries into their stability characteristics to support longer BUD than those recommended by the USP.