Abstract
Objective—To determine the concentration of doxycycline compounded from doxycycline hyclate tablets into liquid formulations for oral administration in veterinary species and stored for 28 days.
Design—Evaluation study.
Sample—Doxycycline hyclate tablets (100 mg) crushed and mixed with a 50:50 mixture of syrup and suspension vehicles for oral administration to produce 3 batches each of 2 doxycycline formulations: 33.3 and 166.7 mg/mL.
Procedures—Formulations were stored, protected from light, at room temperature (22° to 26°C [71.6° to 78.8°F]) and at a controlled cold temperature (refrigerated 2° to 8°C [35.6° to 46.4°F]). Doxycycline was extracted from the formulations, and concentration was measured by high-pressure liquid chromatography on days 0 (date of preparation), 1, 4, 7, 14, 21, and 28. Concentrations were compared with those of a US Pharmacopeial Convention reference standard. Formulation quality at each point was also assessed through color change, formulation consistency, and suspension uniformity.
Results—On days 0, 1, 4, and 7, the concentration of each formulation was within 90% to 110% of the reference standard (range, 93% to 109%), which was deemed acceptable. However, doxycycline concentrations had decreased dramatically by day 14 and remained low for the duration of the study period. Doxycycline concentrations on days 14, 21, and 28 were all < 20% (range, 14% to 18%) of the reference standard, and the quality of the formulations decreased as well. No effect of storage temperatures on doxycycline concentration was identified.
Conclusions and Clinical Relevance—The concentration of doxycycline, compounded from commercial tablets in the vehicles evaluated to yield doses of 33.3 and 166.7 mg/mL, cannot be assured beyond 7 days.
Doxycycline is a widely used antimicrobial in veterinary medicine, but no FDA-approved formulation is available for veterinary species in the United States. As a consequence, veterinarians usually prescribe the approved human formulation for treatment of their patients, doxycycline hyclate (100-mg tablets). Pharmacokinetic studies1–3 have yielded information to guide dose selection; however, no veterinary formulations are available that are convenient to administer to dogs, cats, or horses. For this reason, tablets are typically crushed, broken, or pulverized and compressed into gelatin capsules or crushed and mixed with various vehicles to form a liquid suspension. The suspensions have been sold through compounding pharmacies for use in animals without assurance of their strength or quality.
Liquid suspensions are attractive for use in cats because solid formulations such as tablets or capsules can cause esophageal lesions when they become trapped in the esophagus.4,5 This phenomenon has been observed in experimental studies6,7 and in feline patients.8
Doxycycline has been evaluated for the treatment of infectious and inflammatory conditions in horses.1,9 In some circumstances, long-term treatment may be necessary, requiring a convenient and stable form that can be mixed with the horse's feed or administered orally by mixing with a paste or liquid vehicle.
In the United States, doxycycline formulations are available for use in human medicine as capsules and film-coated tablets of doxycycline hyclate and doxycycline monohydrate. The antimicrobial is also commercially available as a doxycycline calcium suspension in a raspberry- or apple-flavored syrup (10 mg/mL) or doxycycline monohydrate powder for suspension (5 mg/mL) in a raspberry flavor. However, these liquid formulations are not suitable for use in some species because of low drug concentrations or poor palatability. In addition, the stability and concentration of doxycycline over time of liquid formulations for veterinary use have not been reported. Veterinary-approved formulations of doxycycline hyclate and doxycycline monohydrate are available in Europe and other countries but are not available in the United States.
According to the AMDUCA, drug compounding for use in veterinary medicine is considered legal provided certain conditions listed in the AMDUCA extralabel drug use regulations10 are met. The USP11 lists requirements for compounding nonsterile dosage formulations. Included in these requirements is an assurance that the compounded preparation contains an acceptable range (≥ 90% and ≤ 110%) of the theoretically calculated and labeled quantity of active ingredient and has been assigned a beyond-use date that is evidence based or conforms with USP standards.11
The FDA-approved label for commercial doxycycline suspensions for oral administration in humans indicates that they should be stored at < 30°C in tight, light-resistant containers.a,b The approved label also indicates that following reconstitution with water, doxycycline monohydrate oral suspensions are stable for 2 weeks at room temperature. Crushed doxycycline tablets are reportedly stable for at least 6 days when wrapped in aluminum foil and stored at room temperature (22° to 26°C [71.6° to 78.8°F]).12 Doxycycline tablets crushed and mixed with food or drinks (chocolate pudding, regular or low-fat chocolate milk, apple juice with granulated sugar, low-fat milk, yogurt, strawberry jelly, grape jelly, and water) are stable for 24 hours at room temperature or when refrigerated at 2° to 8°C (35.6° to 46.4°F); crushed doxycycline tablets mixed with low-fat chocolate milk are stable when refrigerated for 1 week.12
The USP chapter on compounding11 states that the beyond-use date for compounded aqueous formulations (prepared from ingredients in solid form) should be “not later than 14 days if stored at controlled cold temperatures.” However, the chapter also states that “these beyond-use date limits may be exceeded when there is supporting valid scientific stability information that is directly applicable to the specific preparation.” In veterinary medicine, doxycycline hyclate is the predominant form of doxycycline prescribed, and suspensions have been prepared by veterinary compounding pharmacies that use the FDA-approved tablets and capsules for administration in humans as well as the active pharmaceutical ingredient and have been assigned beyond-use dates much longer than 14 days. The compounding pharmacies typically prepare doxycycline hyclate and monohydrate suspensions for oral administration in concentrations ranging from 20 to 200 mg/mL.
The purpose of the study reported here was to evaluate doxycycline concentrations and stability over time in 2 compounded liquid suspensions of doxycycline hyclate (33.3 and 166.7 mg/mL) as representative of the range of suspensions commonly prepared for veterinary use. Although 5 types of stability tests are described in the USP,11 we chose to examine only the chemical stability and some aspects of the physical stability (appearance and uniformity).
Materials and Methods
Compounding of doxycycline formulation—Commercially available, 100-mg doxycycline hyclate tabletsc that had been approved by the US FDA for use in humans were used for the study. Ten or 50 tablets/concentration of formulation (33.3 or 166.7 mg/mL, respectively) were crushed to a fine powder and brought to a final volume of 30 mL with a 50:50 mixture of syrupd and suspensione vehicles for oral administration to achieve the desired concentration. The vehicles met the compendial requirements of the USP National Formulary (Appendices 1 and 2).
All formulations were prepared in triplicate, yielding 3 batches of each concentration for testing. After the mixing process, the formulations were subjected to 2 storage conditions: protected from light at a controlled cold (refrigerated) temperature at 2° to 8°C or protected from light at a controlled room temperature (22° to 26°C). The samples were stored in brown plastic 100-mL bottles that met USP compendial requirements. Samples were observed for changes in quality (color change, formulation consistency, and suspension uniformity) on days 0 (date of preparation), 1, 4, 7, 14, 21, and 28.
Measurement of doxycycline concentration—Analysis of doxycycline suspensions was performed in accordance with a USP monograph for doxycycline oral suspension,13 with a modification (the samples were not subjected to forced degradation). Sample containers were shaken gently to ensure that each mixture was homogenous and uniform. Afterward, 50 μL of each suspension was transferred to a volumetric flask and mixed and diluted with 0.01N hydrochloric acid (volume calculated to achieve a nominal concentration of 150 μg of doxycycline/mL). The mixture was shaken on a mechanical agitator for 15 minutes, then analyzed by HPLC.
The percentage strength of the concentration of the formulation was calculated as follows:
where RU is the response factor for the test formulation, RS is the response factor for the reference standard solution, CU is the nominal concentration of the test formulation, CS is the concentration of the reference standard solution, and P is the potency (in μg of doxycycline/mg) of the USP doxycycline hyclate reference standard. The strength of the tabletsc used was measured by crushing 20 tablets from the same lot of tablets used for compounding and applying the procedure listed in the USP13 for measuring the strength of tablets in relation to the USP reference standard.f The percentage concentration of doxycycline in the formulation was adjusted by the strength of the tablets in the final calculation.
Doxycycline assay—Concentrations of doxycycline in the processed samples were quantitatively determined by HPLC through the use of a modified method developed in our laboratory.1,2 The analytic reference standard of doxycycline hyclate was obtained from the USP.f The doxycycline reference standard was dissolved in distilled water to make up a stock solution with a doxycycline concentration of 1 mg/mL. This solution was prepared fresh for each assay and stored in the refrigerator and protected from light. For each assay, the reference standard solution was further diluted in 0.01N hydrochloric acid to a doxycycline concentration of 150 μg/mL for HPLC injection.
The mobile phase for HPLC analysis consisted of 25% acetonitrile, 65% 0.1M oxalic acid, and 10% methanol. All solvents were HPLC grade, and the water used for buffer was distilled. All solvents were filtered through a 0.22-μm filter and degassed prior to use. The HPLC system consisted of a quaternary solvent delivery systemg set at a flow rate of 1 mL/min, an autosampler,h and a UV detectori set at a wavelength of 350 nm. The resulting chromatograms were integrated with a computer program.j A C8 reverse-phase columnk was used for separation and kept at a constant temperature of 40°C (104.0°F).
All experimental and reference standard samples were prepared identically. Twenty microliters of each prepared sample was injected into the HPLC system. Retention time for peak of interest was 4.19 to 4.21 minutes. Guidelines published by the USP14 were used for method validation.
Statistical analysis—Mean values and coefficients of variation were calculated for the 3 batches of each concentration of compounded formulation. A concentration was deemed acceptable when the compounded preparations contained ≥ 90% but ≤ 110% of the theoretically calculated and labeled quantity of active ingredient per unit weight or volume.
Results
The mean strength of the doxycycline tablets was 98% of the amount of doxycycline suggested on the product label. The percentage concentration of doxycycline in the compounded preparations was adjusted by the strength of the tablets in the final calculations and was summarized (Table 1). From days 0 through 7, the doxycycline concentration in the compounded formulations was within the predefined acceptable range of 90% to 110% of the reference standard concentration (range, 93% to 109%; Figure 1). During that period, the appearance of the formulation was uniform in color and texture. However, after 7 days of storage, the quality of the compounded formulations decreased, as determined by the lack of uniformity in consistency, visible separation of the phases in the bottle, and a dark brown-colored band that appeared near the bottom of the container. These changes coincided with the decrease in doxycycline concentration of the formulation. After day 7, the doxycycline concentration in all samples was far below 90% (range, 14% to 18%) of the reference standard concentration.
Percentage strength of doxycycline in suspensions prepared in vehicles for oral administration at concentrations of 33.3 mg/mL (A) and 166.7 mg/mL (B) on the date of preparation (day 0) and at various points afterward. Suspensions were prepared from crushed tablets and stored, protected from light, at room temperature (22° to 26°C; black bars) or in a refrigerator (2° to 8°C; gray bars) for 28 days.
Citation: Journal of the American Veterinary Medical Association 242, 12; 10.2460/javma.242.12.1674
Mean (coefficient of variation) percentage strength of 2 doxycycline suspensions stored at room temperature (22° to 26°C) or in a refrigerator (2° to 8°C) on the date of preparation (day 0) and at various points afterward.
| Suspension | Storage | Day 0 | Day 1 | Day 4 | Day 7 | Day 14 | Day 21 | Day 28 |
|---|---|---|---|---|---|---|---|---|
| 33.3 mg/mL | Room temperature | 109.1 (10.3%) | 97.7 (1.2%) | 94.8 (5.8%) | 93.4 (0.5%) | 15.8 (1.6%) | 14.7 (3.8%) | 14.1 (4.2%) |
| Refrigeration | 99.6 (1.6%) | 101.2 (1.7%) | 96.5 (0.7%) | 99.9 (1.0%) | 17.1 (1.5%) | 15.9 (2.1%) | 16.9 (2.0%) | |
| 166.7 mg/mL | Room temperature | 102.9 (2.9%) | 104.3 (1.8%) | 101.8 (1.4%) | 102.0 (0.7%) | 18.7 (1.2%) | 16.8 (2.0%) | 16.9 (0.8%) |
| Refrigeration | 104.0 (4.4%) | 100.2 (2.9%) | 95.3 (1.8%) | 100.7 (2.2%) | 17.9 (1.8%) | 17.2 (1.8%) | 17.2 (3.7%) |
Each value represents the mean of 3 replicates.
Discussion
The present study showed that doxycycline hyclate (100-mg film-coated tablets) suspended in a vehicle for oral administration at concentrations of 33.3 and 166.7 mg/mL retained its quality and strength of ≥ 90% of the original tablets for 7 days at controlled room temperature and controlled cold (refrigerated) temperature. After 7 days of storage, however, the color, consistency, and uniformity of the formulation deteriorated, coinciding with a dramatic decrease in doxycycline concentration. This decrease in quality was evident as a dark band near the bottom of the container, which likely reflected a settling of doxycycline.
The USP chapter15 on stability considerations in dispensing practice lists factors that may affect compounded product stability. These include hydrolysis in the presence of water, epimerization (occurs with tetracyclines at pH > 3), decarboxylation, dehydration, oxidation, and photochemical decomposition as well as effects on product temperature and pH. The chapter also lists color change as a sign that a formulation might be unstable. We did not explore which of these factors might have caused degradation of doxycycline in our formulations > 7 days after preparation. The vehicles used in the formulations contained many excipients, and it was not possible to test each one individually to determine whether any were responsible for a reaction that would cause a color change or loss in concentration. The compounded formulations degraded similarly whether stored at room temperature or refrigerated. Therefore, temperature did not play a role in the decrease in doxycycline concentration or quality. All samples were protected from light, thereby minimizing the possibility of photodegradation. Because the opening in the top of the bottles was small and the bottles were uncapped only for a few seconds, any influence that opening the bottles momentarily to obtain samples might have had on the study results was deemed negligible.
The only published report12 on stability of compounded doxycycline formulations was by investigators at the FDA, who determined that crushed doxycycline tablets were stable when mixed with various foods and drinks for 24 hours or with low-fat chocolate milk for 1 week while refrigerated. The concentrations tested in that study were much lower (5 mg/mL) than the concentrations used in the present study. No other published data are available to guide veterinary practitioners and compounding pharmacists on the beyond-use date for compounded doxycycline formulations. On the basis of our findings, we recommend that clinicians who intend to use compounded formulations with assigned beyond-use dates exceeding 7 days after preparation require evidence of the products’ stability as obtained through valid scientific methods.
All veterinarians are advised to review the rules for legal use of compounded medications prior to using, prescribing, or dispensing such medications for patients. Compounding is considered legal only when federal and state rules are followed.16 Compounding from FDA-approved drugs is permitted when a veterinarian believes the format of an approved drug needs to be altered to adequately medicate a nonfood animal with a diagnosed medical condition. In other words, a valid veterinarian-client-patient relationship must exist, the patient must have a medical condition for which a prescribed medication is needed, and the veterinarian must conclude that a compounded drug and not a preexisting one is needed for the animal.
ABBREVIATIONS
| HPLC | High-pressure liquid chromatography |
| USP | US Pharmacopeial Convention |
Vibramycin calcium (doxycycline calcium oral suspension, USP) oral suspension syrup, Pfizer, New York, NY.
Vibramycin monohydrate (doxycycline monohydrate, USP; 5 mg/mL) for oral suspension, Pfizer, New York, NY.
Doxycycline hyclate tablets, 100 mg (generic), TEVA Pharmaceuticals, Sellersville, Pa.
Vehicle for oral solution, USP-NF (Ora-Sweet), OraMedix Inc, Lancaster, Calif.
Vehicle for oral suspension, USP-NF (Ora-Plus), OraMedix Inc, Lancaster, Calif.
Doxycycline for oral suspension, US Pharmacopeial Convention, Rockville, Md.
Agilent 1100 Series solvent delivery system, Agilent Technologies, Wilmington, Del.
Agilent 1100 Series autosampler, Agilent Technologies, Wilmington, Del.
Agilent 1100 Series variable wavelength detector, Agilent Technologies, Wilmington, Del.
Agilent 1100 Series Chemstation 2D software, Agilent Technologies, Wilmington, Del.
C8 column, Zorbax SB-C8, 4.6 mm × 15 cm, Agilent Technologies, Wilmington, Del.
References
1. Davis JL, Salmon JH, Papich MG. Pharmacokinetics and tissue distribution of doxycycline after oral administration of single and multiple doses in horses. Am J Vet Res 2006; 67: 310–316.
2. Bidgood TL, Papich MG. Comparison of plasma and interstitial fluid concentrations of doxycycline and meropenem following constant rate intravenous infusion in dogs. Am J Vet Res 2003; 64: 1040–1046.
3. Papich MG, Riviere JE. Tetracycline antibiotics. In: Riviere JE, Papich MG, eds. Veterinary pharmacology and therapeutics. 9th ed. Ames, Iowa: Wiley-Blackwell Publishing, 2009;1524.
4. Graham JP, Lipman AH, Newell SM, et al. Esophageal transit of capsules in clinically normal cats. Am J Vet Res 2000; 61: 655–657.
5. Westfall DS, Twedt DC, Steyn PF, et al. Evaluation of esophageal transit of tablets and capsules in 30 cats. J Vet Intern Med 2001; 15: 467–470.
6. Carlborg B, Densert O, Lindqvist C. Tetracycline induced esophageal ulcers. A clinical and experimental study. Laryngoscope 1983; 93:184–187.
7. Carlborg B, Densert O. Esophageal lesions caused by orally administered drugs: an experimental study in the cat. Eur Surg Res 1980; 12: 270–282.
8. Frowde PE, Battersby IA, Whitley NT, et al. Oesophageal disease in 33 cats. J Feline Med Surg 2011; 13: 564–569.
9. Schnabel LV, Papich MG, Watts AE, et al. Orally administered doxycycline accumulates in synovial fluid compared to plasma. Equine Vet J 2010; 42: 208–212.
10. US FDA Center for Veterinary Medicine. Chapter 6, Subchapter 600, Sec. 608.400—compounding of drugs for use in animals, July 2003. In: Compliance policy guide: compliance policy guidance for FDA staff and industry. Rockville, Md: FDA, 2003.
11. USP. Chapter 795: pharmaceutical compounding—nonsterile preparations. In: United States Pharmacopeia. 34th revision. Rockville, Md: US Pharmacopeial Convention, 2010.
12. Sadrieh N, Brower J, Yu L, et al. Stability, dose uniformity, and palatability of three counterterrorism drugs—human subject and electronic tongue studies. Pharm Res 2005; 22: 1747–1756.
13. USP. Doxycycline for oral suspension. In: United States Pharmacopeia. 34th revision. Rockville, Md: US Pharmacopeial Convention, 2010.
14. USP. Chapter 1225: validation of compendial procedures. In: United States Pharmacopeia. 34th revision. Rockville, Md: US Pharmacopeial Convention, 2010.
15. USP. Chapter 1191: stability considerations in dispensing practice. In: United States Pharmacopeia. 34th revision. Rockville, Md: US Pharmacopeial Convention, 2010.
16. AVMA. Compounding FAQs for veterinarians. Available at: www.avma.org/KB/Resources/FAQs/Documents/Compounding%20FAQs%20for%20Veterinarians.pdf. Accessed Dec 25, 2012.
Appendix 1
Composition of a syrup vehicled (ph, 4.0 to 5.0) for oral drug administration.
| Ingredient | Amount |
|---|---|
| Sucrose | 80 mg |
| Glycerin | 5 g |
| Sorbitol | 5 g |
| Sodium phosphate, dibasic | 120 mg |
| Citric acid | 200 mg |
| Potassium sorbate | 100 mg |
| Methylparaben | 100 mg |
| Purified water | Quantity sufficient to make 100 mL |
Beyond-use date, ≤ 6 months after preparation.
Appendix 2
Composition of a suspension vehiclee (ph, 4.0 to 5.0) for oral drug administration.
| Ingredient | Amount |
|---|---|
| Microcrystalline cellulose | 800 mg |
| Xanthan gum | 200 mg |
| Carrageenan | 150 mg |
| Carboxymethylcellulose sodium | 25 mg |
| Citric acid | 250 mg |
| Dibasic sodium phosphate | 120 mg |
| Simethicone | 0.1 mL |
| Potassium sorbate | 100 mg |
| Methylparaben | 100 mg |
| Purified water | Quantity sufficient to make 100 mL |
Beyond-use date, ≤ 6 months after preparation.
