Abstract
Metronidazole is indicated for the treatment of trichomoniasis, amebiasis, and anaerobic bacterial infections. The dosage regimen of metronidazole needs to be individualized in the treatment of trichomoniasis, in patients with hepatic impairment, and in pediatric as well as geriatric patients. A review of the therapeutic uses of metronidazole reveals the need for flexibility in dosing. This flexibility is readily achieved using an oral liquid dosage form. However, no commercial liquid dosage form of metronidazole currently exists. Metronidazole is commercially available only as 250-mg and 500-mg film-coated tablets. An extemporaneously compounded suspension from pure drug powder or commercial tablets would provide a convenient option to meet unique patient needs. The purpose of this study was to determine the physicochemical and microbiological stability of extemporaneously compounded metronidazole suspensions in PCCA SuspendIt. This base is a sugar-free, paraben-free, dye-free, and gluten-free thixotropic vehicle containing a natural sweetener obtained from the monk fruit. The study design included two metronidazole concentrations to provide stability documentation over a bracketed concentration range for eventual use by compounding pharmacists. A robust stability-indicating ultra-performance liquid chromatographic assay for the determination of the chemical stability of metronidazole in PCCA SuspendIt was developed and validated. Suspensions of metronidazole were prepared in PCCA SuspendIt at 25-mg/mL and 50-mg/mL concentrations, selected to represent a range within which the drug is commonly dosed. Samples were stored in plastic amber prescription bottles at two temperature conditions (5°C and 25°C). Samples were assayed initially and on the following time points (days): 7, 14, 28, 42, 59, 90, 122, and 180. Physical data such as pH, viscosity, and appearance were also noted. Microbiological stability was also tested. All measurements were obtained in triplicate. A stable extemporaneous product is defined as one that retains at least 90% of the initial drug concentration throughout the sampling period and is protected against microbial growth. The study showed that metronidazole concentrations did not go below 97% of the label claim (initial drug concentration) at both temperatures studied. No microbial growth was observed. Viscosity and pH values also did not change significantly. This study demonstrates that metronidazole is physically, chemically, and microbiologically stable in PCCA SuspendIt for 180 days in the refrigerator and at room temperature, thus providing a viable, compounded alternative for metronidazole in a liquid dosage form, with an extended beyond-use-date to meet patient needs.
Introduction
Oral liquid preparations are well suited for special populations that may experience difficulty in swallowing solid dosage forms, such as tablets and capsules. In addition, liquid dosage forms offer the benefit of flexibility by providing convenient and accurate dosing options that can be customized for pediatric and geriatric patients. However, palatable liquid dosage forms of various drugs are often not commercially available, or they may be in short supply. In such situations, the compounding pharmacist is uniquely qualified to meet patient needs by extemporaneously compounding oral liquids using pure drug powder or tablets/capsules. Aqueous vehicles are frequently used to prepare compounded oral liquids, raising the possibility of chemical drug degradation and microbial growth. In addition, the pure drug powder and the tablet/capsule excipients are often insoluble in water; therefore, the resulting product will be a suspension, instead of a solution. It is the responsibility of the compounding pharmacist to prepare a suspension that is physically, chemically, and microbiologically stable throughout its period of use. Physical stability is achieved by formulating a homogeneous suspension that does not cake upon standing and re-disperses easily upon shaking. The pharmacist must also obtain validated chemical and microbiological stability information in order to assign accurate beyond-use dates (BUDs) to compounded preparations.
Metronidazole is indicated for the treatment of trichomoniasis, amebiasis, and anaerobic bacterial infections.1 The dosage regimen of metronidazole needs to be individualized in the treatment of trichomoniasis, in patients with hepatic impairment, and in pediatric as well as geriatric patients. A review of the therapeutic uses of metronidazole reveals the need for flexibility in dosing. However, metronidazole is commercially available only as 250-mg and 500-mg film-coated tablets. No manufactured liquid dosage form of metronidazole currently exists. An extemporaneously compounded suspension from pure drug powder or commercial tablets would provide a flexible, customizable option to meet unique patient needs with convenient and accurate dosing options for adult and pediatric patients.
Metronidazole is an odorless white to yellow or cream crystal-line powder or crystals.2 Metronidazole has a very bitter taste and is slightly soluble in water, having an aqueous solubility of about 10 mg/mL at 20°C. Its solubility in water has been reported to be 8.3 mg/mL, 8.8 mg/mL, and 11.4 mg/mL at 20°C, 26°C, and 30°C, respectively. Interestingly, solubility in a suspension formulation at 25°C was found to be only 6 mg/mL – a possible indication that the precipitate acts as nuclei to “seed” further precipitation. In ethanol, the solubility of metronidazole is about 5 mg/mL.
The pH of a saturated aqueous solution of metronidazole is 5.8. Metronidazole has a pKa of 2.6. Metronidazole is stable in air but may darken upon exposure to light. The decomposition profile of metronidazole shows a pH-independent region in the range of 3.9 to 6.6, with maximum stability reported in the range of 5.1 to 5.6. The rate of decomposition increases as the environment becomes more alkaline, particularly above pH 8.
The degradation of metronidazole tablets/pure powder has been previously investigated in traditional vehicles such as sodium carboxymethylcellulose, simple syrup, cherry syrup, chocolate syrup, veegum, Ora-Sweet and Ora-Plus.3–10 These studies generally provide documentation for short-term drug stability, ranging from 60 to 90 days. BUDs as short as 4.3 days at 25°C have also been reported. An official United States Pharmacopeia (USP) monograph for Metronidazole Benzoate Compounded Oral Suspension containing the equivalent of 50 mg/mL metronidazole in the commercial vehicle Ora-Blend also exists.11 The BUD is not to exceed 90 days after the date on which it was compounded when stored at 2°C to 8°C or controlled room temperature. No documentation regarding long-term metronidazole stability in the newer thixotropic vehicles is available. Therefore, stability studies of metronidazole in contemporary vehicles, such as PCCA SuspendIt, are required to establish accurate BUDs up to six months for use by the compounding pharmacist, and for eventual adoption as an official monograph by the United States Pharmacopeial Convention, Inc.
The purpose of the current study was to determine the physicochemical and microbiological stability of metronidazole in PCCA SuspendIt, a new suspending agent developed by the Professional Compounding Centers of America (PCCA), Houston, Texas, for use by compounding pharmacists. PCCA SuspendIt, a unique, hypoallergenic, oral suspending vehicle with special thixotropic properties, is sugar free, paraben free, dye free, and gluten free and contains a natural sweetener obtained from the monk fruit. It thickens upon standing to minimize settling of any insoluble drug particles and becomes fluid upon shaking to allow convenient pouring during administration to the patient. It utilizes a natural patented thixotropic agent which is ideal for the compounding of oral liquids. It has been found to form uniform suspensions with a wide variety of active pharmaceutical ingredients.12
The current study was conducted to develop and validate a stability-indicating ultra-performance liquid chromatographic (UPLC) assay for metronidazole in PCCA SuspendIt, and to evaluate the chemical, physical, and microbiological six-month stability of the extemporaneous preparations.
The study design included two metronidazole concentrations to provide stability documentation over a bracketed concentration range for eventual use by compounding pharmacists.
Suspensions of metronidazole were prepared in PCCA SuspendIt at 25 mg/mL and 50 mg/mL concentrations to represent a range in which the drug is commonly dosed. Samples were stored in plastic amber prescription bottles at two temperature conditions (5°C and 25°C). Samples were assayed initially and at pre-determined time intervals over a six-month period. Physical data such as pH, viscosity, and appearance were also noted. All measurements were obtained in triplicate. Samples were also tested for microbiological stability. A stable extemporaneous product is defined as one that retains at least 90% of the initial drug concentration throughout the sampling period and is protected against microbial growth. The goal was to provide a viable, compounded alternative for metronidazole in a thixotropic liquid dosage form with an extended BUD to meet patient needs.
Chemicals and Reagents
Metronidazole USP (Lot C191184); PCCA SuspendIt (Lot 8337927); PCCA Flavor, Bitter Stop (Lot C193773); PCCA Flavor, Banana Crème, Artificial (Lot C193355); PCCA Acesulfame Potassium FCC (Lot C181189); and PCCA Steviol Glycosides 95% (Lot C175764) were generously provided by PCCA. The high-performance liquid chromatographic (HPLC) grade methanol (Lot 18J194005) was purchased from VWR Chemicals (Radnor, Pennsylvania). The reagent grade formic acid (Lot 70250), sodium hydroxide (Lot 935523), hydrochloric acid (Lot 119H3484), and hydrogen peroxide (Lot MKBF1744V) were purchased from Sigma Aldrich Corporation (St. Louis, Missouri). Deionized water (18.2 MΩ) was prepared in house using the ELGA Pure Lab Classic system (ELGA LLC, Woodridge, Illinois).
Metronidazole 25-mg/mL Oral Suspension (PCCA SuspendIt) – Master Formula
| Metronidazole USP | 25.0 g | |
| PCCA Steviol Glycosides 95% | 4.0 g | |
| PCCA Acesulfame Potassium FCC | 4.0 g | |
| PCCA Flavor, Bitter Stop | 4.0 mL | |
| PCCA Flavor, Banana Créme, Artificial | 30.0 mL | |
| PCCA SuspendIt | q.s. | 1000 mL |
Metronidazole 50-mg/mL Oral Suspension (PCCA SuspendIt) – Master Formula
| Metronidazole USP | 50.0 g | |
| PCCA Steviol Glycosides 95% | 4.0 g | |
| PCCA Acesulfame Potassium FCC | 4.0 g | |
| PCCA Flavor, Bitter Stop | 4.0 mL | |
| PCCA Flavor, Banana Créme, Artificial | 30.0 mL | |
| PCCA SuspendIt | q.s. | 1000 mL |
Sample Preparation
Two suspensions, one containing 25 mg/mL, and the other containing 50 mg/mL of metronidazole in PCCA SuspendIt, were prepared by first weighing out either 25.0 grams or 50.0 grams of metronidazole, respectively, and placing the powder in a mortar. Additionally, 4 grams of acesulfame potassium, and 4 grams of steviol glycosides 95% powder were added to each mortar, and the powders were triturated together to reduce the particle size. The powders were levigated to a smooth paste using a small amount of PCCA SuspendIt along with 4 mL of Flavor, PCCA Flavor, Bitter Stop and 30 mL of PCCA Flavor, Banana Crème, Artificial. Additional PCCA SuspendIt was added to the mortar, and the contents transferred into a 1000-mL volumetric flask using a rubber spatula. This process was repeated three times to facilitate complete removal and transfer of the liquid from the mortar to the flask. The volumetric flask was filled to the mark with additional PCCA SuspendIt, vortexed on a vortex mixer, and sonicated in an ultrasonic bath for 5 minutes to remove any air bubbles. The volumetric flask was again filled to the mark with PCCA SuspendIt and placed on a magnetic stirrer.
For both drug concentrations, six 4-oz. plastic amber prescription bottles were filled with 80 mL of the prepared suspension, and six bottles were filled with 60 mL of the suspension, retaining the remainder for initial (zero day) analysis. The bottles containing 80 mL of suspension were sealed, divided into two groups of three bottles each, and stored at room temperature (25°C) in a desiccator, or under refrigerated conditions (5°C). The temperature at each storage location was monitored throughout the study. Samples from each temperature and concentration were analyzed and characterized initially on day zero, and subsequently after 7 days, 14 days, 28 days, 42 days, 59 days, 90 days, 122 days, and 180 days of storage. The bottles containing 60 mL of the suspension were shipped via overnight delivery to a contract laboratory (Eagle Analytical Services Inc., Houston, Texas) for initial microbiological testing in accordance with USP Chapter <51> Antimicrobial Effectiveness (AME) Test,13 and following 90 days and 180 days of storage at room temperature (25°C), or under refrigerated conditions (5°C).
One extra sample of 50 mg/mL metronidazole in PCCA SuspendIt was prepared using standard compounding equipment (mortar and pestle), omitting the vortexing and sonication steps. This was done to confirm content uniformity regardless of the mixing procedure. The sample had an initial drug concentration of 50.2 mg/mL ± 1.5 mg/mL, which is within the acceptable range of ±10% for drug content.
Sampling Procedure
On each of the 8 sampling days, the bottles were removed from storage and uniformly shaken. An approximate volume of 8 mL was removed from each bottle for analysis, prior to the bottles being returned to storage. For each of the six bottles of the 25-mg/mL metronidazole samples, three 5-mL volumetric flasks were tared and approximately 20 μL of the suspensions were pipetted and weighed to the nearest 0.1 mg. The volumetric flasks were brought up to volume with mobile phase consisting of 85% formic acid (0.1%) solution and 15% methanol, giving a target sample concentration of 100 μg/mL. The six 50-mg/mL metronidazole samples were treated in a similar manner, using 10-mL tared volumetric flasks and pipetting 20 μL of the suspensions, weighed to the nearest 0.1 mg, to achieve the same target concentration of 100 μg/mL. The volumetric flasks were briefly sonicated in an ultrasonic bath to disperse the PCCA SuspendIt. The samples were then placed in a heated shaker, maintained at 40°C for 20 minutes to dissolve the drug. The samples were subsequently pipetted into 2.0-mL UPLC sampling vials, being careful not to transfer any globular-gelled PCCA SuspendIt. Transfer of the gelled PCCA SuspendIt was found to clog the analytical equipment. The samples were analyzed by UPLC using a method adapted from the literature.14,15
Ultra-performance Liquid Chromatographic Analysis
All samples were analyzed on a Waters Acquity UPLC system (Waters Corporation, Milford Massachusetts) fitted with a photodiode array detector set at 318 nm for the detection of the drug. An isocratic mobile phase containing 85% formic acid (0.1%) and 15% methanol was used at a flow rate of 0.3 mL/min. The injection volume was 3.0 microliters. An Acquity UPLC BEH C18 column was used for the separation.
A series of standards ranging between 25 μg/mL and 200 μg/mL were prepared in mobile phase from a 1-mg/mL stock solution of metronidazole in mobile phase that was prepared fresh for each sampling period. The chromatograms were acquired for the standards and samples. Using the peak areas from the metronidazole peaks at 318 nm, a least squares analysis was performed on the calibration curve, and the sample concentrations were determined.
pH and Viscosity Measurements
The suspensions were also analyzed for pH, appearance, and intrinsic viscosity. The pH of each sample was measured on a VWR Scientific pH meter (VWR, Radnor, Pennsylvania) using an Ag/AgCl combination electrode that was calibrated prior to analysis. Measurements were taken in auto mode, with each sample run in triplicate. The viscosity was determined using a Brookfield DV-III Ultra programmable cone/plate rheometer (Brookfield Engineering Laboratories Inc., Middleboro, Mississippi) fitted with a cpe-40 spindle. After the instrument was leveled and aligned, approximately 0.5 mL of sample was placed in the viscometer for measurement. Using the Rheocalc program, a viscosity protocol was selected to determine the average viscosity over a 10-second measurement period at a fixed spindle speed of 0.2 rpm, with each measurement being performed 5 times with a 2-minute rest period between measurements.
Microbiological Stability Testing
Nonsterile aqueous dosage forms have preservatives to protect them from growth of microorganisms inadvertently introduced during, or subsequent to, the compounding process.
PCCA SuspendIt contains potassium sorbate and sodium benzoate as antimicrobial preservatives. According to the USP,13 antimicrobial effectiveness must be demonstrated for aqueous, multiple-dose oral dosage forms. Challenge organisms are generally based on likely contaminants to a drug product while considering its physical attributes, formulation, and intended use. The standard battery of challenge organisms described in this test include Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Candida albicans, and Aspergillus brasiliensis. The USP provides the following criteria to determine antimicrobial effectiveness:
for bacteria, there should be no less than 1.0 log reduction from the initial count at 14 days, and no increase from the 14 days’ count at 28 days;
for yeasts and molds, there should be no increase from the initial calculated count at 14 days and 28 days.
The 25-mg/mL and 50-mg/mL samples of metronidazole suspensions were sent to a contract laboratory (Eagle Analytical Services Inc.) for initial microbiological testing in accordance with the USP Chapter <51> AME Test, and following 90 days and 180 days of storage at room temperature (25°C), and under refrigerated conditions (5°C).
Forced-Degradation Study
The analytical method was demonstrated to be stability indicating by subjecting metronidazole samples to accelerated degradation and determining if any degradants interfered with the analytical peak of metronidazole. These forced degradations included caustic, acidic, peroxide, and ultraviolet (UV) light degradation. For the caustic degradation, 0.5 mL of 5N NaOH was added to a 10-mL volumetric flask containing either 200 μL of a 5-mg/mL stock solution of metronidazole in mobile phase, or approximately 20 mg of a test formulation containing 50 mg/mL metronidazole in PCCA SuspendIt. The sample was heated to 60°C for 1 hour, followed by neutralization of both the standard and sample with 2.5 mL of a 1N HCl solution. The flasks were then brought up to volume with mobile phase. For the acidic degradation, the stock solution or formulation was mixed with 0.5 mL of a 1N HCl solution and stored at room temperature for 1 hour. The acidic solution was then neutralized with 0.1 mL of the 5N NaOH solution and filled to volume with mobile phase. The peroxide degradation was accomplished in an analogous manner by mixing the sample or stock solution with 483.3 μL of deionized water and 16.7 μL of a 30% hydrogen peroxide solution, resulting in a 1% peroxide solution. Forced degradation by UV light was achieved by placing either a 5-mg/mL stock solution, or a small amount of the 50-mg/mL formulation in 10-mL volumetric flasks in a Millipore UV sterilizer (Catalog No. XX6370000, Billerica; Massachusetts) for 1 hour. The samples were placed on the blue sample plate of the sterilizer, exposing them to the upper two UV tubes only. This provided UV light exposure of approximately 0.63 mW/cm2.16 In all cases, the volumetric flasks were filled to the mark with mobile phase prior to analysis.
Results and Discussion
Metronidazole formed a bright yellow, translucent suspension in PCCA SuspendIt at the 25-mg/mL and 50-mg/mL drug concentrations, with the 50-mg/mL suspension having a slightly more opaque appearance. The bright yellow color of the suspensions was due to the artificial banana crème flavor. No observable change in appearance was noted throughout the duration of the study. The pH of the samples displayed no significant changes over the test period (TABLE 1). The pH measurements were consistent (5.02 to 5.17), averaging 5.13 for all the samples.
TABLE 1.
MEASUREMENTS OF pH OF METRONIDAZOLE IN PCCA SUSPENDIT.
| 25 MG/ML | 50 MG/ML | |||
|---|---|---|---|---|
| TIME | 5°C | 25°C | 5°C | 25°C |
| Day 0 | 5.16 ± 0.01 | 5.16 ± 0.01 | 5.16 ± 0.01 | 5.16 ± 0.01 |
| Day 7 | 5.15 ± 0.03 | 5.16 ± 0.01 | 5.17 ± 0.01 | 5.17 ± 0.01 |
| Day 14 | 5.15 ±0.01 | 5.14 ± 0.02 | 5.15 ± 0.01 | 5.14 ± 0.01 |
| Day 28 | 5.13 ± 0.01 | 5.15 ± 0.01 | 5.14 ± 0.01 | 5.11 ± 0.01 |
| Day 42 | 5.14 ± 0.01 | 5.11 ± 0.01 | 5.15 ± 0.01 | 5.11 ± 0.01 |
| Day 59 | 5.13 ± 0.01 | 5.07 ± 0.01 | 5.14 ± 0.01 | 5.08 ± 0.01 |
| Day 90 | 5.13 ± 0.02 | 5.07 ± 0.02 | 5.14 ± 0.01 | 5.09 ± 0.01 |
| Day 122 | 5.15 ± 0.02 | 5.02 ± 0.01 | 5.15 ± 0.02 | 5.07 ± 0.02 |
| Day 180 | 5.11 ± 0.01 | 5.13 ± 0.01 | 5.13 ± 0.01 | 5.16 ± 0.01 |
The initial suspension viscosity of both concentrations was found to be quite high, ranging from 3834 to 12752 centipoises (cP) for the 25-mg/mL and 50-mg/mL batches, respectively (TABLE 2). This high viscosity required a modification of the viscosity program. The shear rate was reduced from 16 rpm to 0.2 rpm. Following the initial (day zero) measurement, the suspensions appeared to exhibit significant dilatancy or shear thickening, making repeat measurements difficult. Therefore, only the initial viscosity at each concentration and temperature condition was measured and averaged across the triplicate samples. The rational was that the initial measurement represented the actual steady-state viscosity of the sample in the bottle at any given sample period and was representative of the intended use by patients. The viscosity levels for all samples were observed to be adequate for maintaining a uniformly dispersible suspension. Based on the USP Chapter <51> AME Test, the results confirmed that the preservative system in PCCA SuspendIt successfully prevented growth of the challenge organisms (TABLE 3), thus conferring the desired microbiological stability.
TABLE 2.
VISCOSITY (cP) MEASUREMENTS OF METRONIDAZOLE IN PCCA SUSPENDIT.
| 25 MG/ML | 50 MG/ML | |||
|---|---|---|---|---|
| TIME | 5°C | 25°C | 5°C | 25°C |
| Day 0 | 3834 ± 192 | 3834 ± 192 | 12752 ± 733 | 12752 ± 733 |
| Day 7 | 2861 ± 578 | 3766 ± 1682 | 3363 ± 661 | 2447 ± 165 |
| Day 14 | 3030 ± 127 | 3308 ± 227 | 2709 ± 204 | 3679 ± 255 |
| Day 28 | 2818 ± 442 | 2725 ± 335 | 3619 ± 430 | 3368 ± 91 |
| Day 42 | 3123 ± 708 | 3286 ± 631 | 3085 ± 490 | 3886 ± 170 |
| Day 59 | 2970 ± 318 | 3504 ± 286 | 3210 ± 366 | 3161 ± 955 |
| Day 90 | 3123 ± 680 | 3608 ± 573 | 3395 ± 378 | 2943 ± 231 |
| Day 122 | 3156 ± 578 | 3025 ± 536 | 3243 ± 9 | 2987 ± 745 |
| Day 180 | 3417 ± 408 | 3334 ± 511 | 3134 ± 218 | 3706 ± 509 |
TABLE 3.
UNITED STATES PHARMACOPEIA CHAPTER <51> ANTIMICROBIAL EFFECTIVENESS TEST OF METRONIDAZOLE IN PCCA SUSPENDIT AFTER 180 DAYS.
| MICROBE | 25MG/ML | 50 MG/ML | ||
|---|---|---|---|---|
| 5°C | 25°C | 5°C | 25°C | |
| Aspergillus brasiliensis | Pass | Pass | Pass | Pass |
| Candida albicans | Pass | Pass | Pass | Pass |
| Pseudomonas aeruginosa | Pass | Pass | Pass | Pass |
| Staphylococcus aureus | Pass | Pass | Pass | Pass |
| Escherichia Coli | Pass | Pass | Pass | Pass |
The UPLC method utilized in the study clearly separated any peak associated with the PCCA SuspendIt from the analytical peak of the metronidazole (FIGURE 1A). The analytical wave-length of 318 nm was chosen to eliminate any potential interference from other formulation components (FIGURE 1B). A C18 column was chosen for optimal drug retention and good peak separation. The column efficiency for the metronidazole separation was 16253, and the tailing factor was 1.23. The calibration curves displayed excellent linearity, with a correlation coefficient greater than 0.999 over the observed concentration range for all the calibration curves (FIGURES 2 AND 3). Forced-degradation studies revealed that peaks associated with the degradants had much shorter retention times than the metronidazole peak and showed no interference with its analytical peak (FIGURES 4 AND 5). There was no overlap of any forced-degradation peaks with the peak for the metronidazole. In addition, there was no interference from any degradation peaks associated with PCCA SuspendIt.
FIGURE 1A.
SAMPLE CHROMATOGRAPHIC RUN OF METRONIDAZOLE STANDARD (BLUE) AND METRONIDAZOLE (ORANGE) IN PCCA SUSPENDIT USING AN ANALYSIS WAVELENGTH OF 318 NM.
FIGURE 1B.
SAMPLE ABSORPTION SPECTRUM OF METRONIDAZOLE SHOWING THE ABSORPTION MAX AT 318 NM.
FIGURE 2.
OVERLAY OF CHROMATOGRAPHIC RUNS OF METRONIDAZOLE STANDARD SOLUTIONS.
FIGURE 3.
CALIBRATION CURVE FOR ULTRA-PERFORMANCE LIQUID CHROMATOGRAPHIC ANALYSIS OF METRONIDAZOLE (RANGE: 25 μG/ML TO 200 μG/ML).
FIGURE 4.
CHROMATOGRAPHIC RUNS OF METRONIDAZOLE STANDARD (BLUE), WITH CAUSTIC DEGRADATION (BLACK), ACIDIC DEGRADATION (ORANGE), OXIDATIVE DEGRADATION (GREY), AND ULTRAVIOLET LIGHT DEGRADATION (VIOLET).
FIGURE 5.
CHROMATOGRAPHIC RUNS OF METRONIDAZOLE IN PCCA SUSPENDIT; STANDARD (BLUE), WITH CAUSTIC DEGRADATION (BLACK), ACIDIC DEGRADATION (ORANGE), OXIDATIVE DEGRADATION (GREY), AND ULTRAVIOLET LIGHT DEGRADATION (VIOLET).
Using a ±10% criterion as a means of determining drug degradation, no significant degradation of the metronidazole was found over the 180-day test period (TABLES 4 AND 5; FIGURES 6 AND 7). Drug concentrations were above 97% of initial values, and no degradation was observed for both concentrations and at both temperature conditions studied. No color change of the samples was observed throughout the test period (FIGURE 8).
TABLE 4.
METRONIDAZOLE CONCENTRATION (MG/ML) IN PCCA SUSPENDIT.
| 25 MG/ML | 50 MG/ML | |||
|---|---|---|---|---|
| TIME | 5°C | 25°C | 5°C | 25°C |
| Day 0 | 25.47 ± 0.39 | 25.47 ± 0.39 | 50.72 ± 0.40 | 50.72 ± 0.40 |
| Day 7 | 25.05 ± 0.30 | 25.22 ± 0.24 | 50.41 ± 0.98 | 50.90 ± 0.86 |
| Day 14 | 25.36 ± 0.45 | 25.65 ± 1.04 | 50.96 ± 0.29 | 51.11 ± 0.43 |
| Day 28 | 25.16 ± 0.94 | 25.67 ± 0.66 | 50.75 ± 0.46 | 51.00 ± 0.33 |
| Day 42 | 25.44 ± 0.85 | 25.41 ± 0.86 | 50.33 ± 0.78 | 50.35 ± 0.82 |
| Day 59 | 25.58 ± 0.73 | 24.92 ± 1.06 | 51.00 ± 0.70 | 51.19 ± 1.07 |
| Day 90 | 26.03 ± 0.83 | 25.23 ± 0.58 | 51.65 ± 1.52 | 51.03 ± 0.82 |
| Day 122 | 25.24 ± 0.37 | 24.76 ± 0.17 | 51.14 ± 0.50 | 50.83 ± 1.35 |
| Day 180 | 25.10 ± 0.48 | 25.88 ± 0.80 | 51.60 ± 0.35 | 50.94 ± 0.87 |
TABLE 5.
PERCENT OF METRONIDAZOLE IN PCCA SUSPENDIT RELATIVE TO DAY ZERO SAMPLE.
| 25 MG/ML | 50 MG/ML | |||
|---|---|---|---|---|
| TIME | 5°C | 25°C | 5°C | 25°C |
| Day 0 | 100.0 ± 2.2 | 100.0 ± 2.2 | 100.0 ± 1.1 | 100.0 ± 1.1 |
| Day 7 | 98.4 ± 1.9 | 99.0 ± 1.8 | 99.4 ± 2.1 | 100.4 ± 1.9 |
| Day 14 | 99.6 ± 2.3 | 100.7 ± 4.3 | 100.5 ± 1.0 | 100.8 ± 1.2 |
| Day 28 | 98.8 ± 4.0 | 100.8 ± 3.0 | 100.1 ± 1.2 | 100.6 ± 1.0 |
| Day 42 | 99.9 ± 3.7 | 99.8 ± 3.7 | 99.2 ± 1.7 | 99.3 ± 1.8 |
| Day 59 | 100.4 ± 3.3 | 97.8 ± 4.4 | 100.6 ± 1.6 | 100.9 ± 2.3 |
| Day 90 | 102.2 ± 3.6 | 99.1 ± 2.7 | 101.8 ± 3.1 | 100.6 ± 1.8 |
| Day 122 | 99.1 ± 2.1 | 97.2 ± 1.6 | 100.8 ± 1.3 | 100.2 ± 2.8 |
| Day 180 | 98.5 ± 2.4 | 101.6 ± 3.5 | 101.7 ± 1.1 | 100.4 ± 1.9 |
FIGURE 6.
CHANGE IN DRUG CONCENTRATION OVER 180 DAYS ON LEFT AXIS FOR THE 25-MG/ML SAMPLES OF METRONIDAZOLE IN PCCA SUSPENDIT STORED AT 5°C (BLUE) AND 25°C (ORANGE); AND RELATIVE CHANGE IN PERCENT ON RIGHT AXIS AS COMPARED TO INITIAL CONCENTRATION [A/AO = DRUG CONTENT AT TIME T (A) OVER INITIAL DRUG CONTENT (AO) X100].
FIGURE 7.
CHANGE IN DRUG CONCENTRATION OVER 180 DAYS ON LEFT AXIS FOR THE 50-MG/ML SAMPLES OF METRONIDAZOLE IN PCCA SUSPENDIT STORED AT 5°C (BLUE) AND 25°C (ORANGE); AND RELATIVE CHANGE IN PERCENT ON RIGHT AXIS AS COMPARED TO INITIAL CONCENTRATION [A/AO = DRUG CONTENT AT TIME T (A) OVER INITIAL DRUG CONTENT (AO) X100].
FIGURE 8. PHYSICAL APPEARANCE OF METRONIDAZOLE FORMULATIONS IN PCCA SUSPENDIT AT DAY 0 (PICTURE I), DAY 14 (PICTURE II), DAY 90 (PICTURE III), DAY 180 (PICTURE IV).
Picture I: Left to right are 25-mg/mL and 50-mg/mL samples, respectively. Pictures II through IV: Left to right are 25 mg/mL refrigerated, 25 mg/mL room temperature, 50 mg/mL refrigerated, and 50 mg/mL room temperature, respectively.
Conclusion
A robust stability-indicating UPLC assay method for the determination of metronidazole in PCCA SuspendIt was developed and validated. This assay was used to determine the chemical stability of the 25-mg/mL and 50-mg/mL concentrations of metronidazole in PCCA SuspendIt at 5°C and 25°C. Drug concentration did not go below 97% of the label claim (initial drug concentration) at both concentrations and both temperature conditions studied. The pH values did not change significantly. The viscosity of the suspensions was sufficient to allow easy re-dispersal of the drug particles upon shaking. Content uniformity was maintained due to the thixotropic nature of the vehicle. The preservative system in PCCA SuspendIt successfully protected the suspensions from growth of challenge microorganisms per the USP Chapter <51> AME Test. This study demonstrates that metronidazole is physically, chemically, and microbiologically stable in PCCA SuspendIt for 180 days in the refrigerator and at room temperature, thus providing a viable, compounded alternative for metronidazole in a liquid dosage form, with an extended BUD to meet patient needs. The study further provides stability documentation over a bracketed metronidazole concentration range between 25 mg/mL and 50 mg/mL, allowing compounding pharmacists more flexibility in customizing their formulations.
Acknowledgment
This study was sponsored by the Professional Compounding Centers of America, and supported in part by the NIH Grant 2U54MD007595-lI, DHHS Grant #D34HP00006 and the Louisiana Cancer Research Consortium.
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