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A radiotracer technique is a simple, fast, and sensitive technique for analyzing the integrity of clinical supply packages to water.
As part of the drug development process, packaging for clinical supplies must protect formulations for at least the duration of clinical trials. Nonetheless, tablets stored in clinical supply packages occasionally exhibit low dissolution (both for the mean and for single tablets) during stability studies.
Patty Stelzer (Pfizer)
The inconsistent nature of dissolution failure is puzzling. Low mean dissolution could indicate a possible catastrophic package failure in some cases. But, single low outlier tablet dissolution behavior (see Figure 1) suggests discrete failures during or after packaging.
Water uptake by tablets is a suspected cause of low dissolution (1). Traditional analysis by Karl Fischer (KF) titration measures total water content, but cannot properly determine the source of the water (i.e., whether it was absorbed by the tablet during or after packaging). Researchers using the KF method can differentiate only ~0.2–0.3% changes in water content because of the method's low reproducibility and sensitivity. Moreover, changes of this magnitude can be detected only after 1–2 months on stability. Any experiments using KF titration to identify the problem require extended periods of time to confirm information about water uptake.
Figure 1: Dissolution of tablets on ICH stability in foilÃ¢ÂÂfoil blisters at 40 Â°C and 75% relative humidity. This study involved packages of immediate-release, film-coated tablets (formulated of a reverse-transcriptase inhibitor and excipients prone to moisture sorption).
Thus, a simple, fast, and sensitive method for analyzing water uptake in large numbers of tablets is needed. Such a method must determine the amount of water absorbed by individual tablets inside the package. The method also must be sensitive enough to detect moisture transmission levels (estimated to be 5 nL [5 μg] of water per tablet per day) caused by the pinholes that are inherent in aluminum foil. In addition, sample preparation must be simple because large numbers of tablets must be assayed. And, results must be available quickly so that appropriate decisions about the packaging system's integrity can be made in a timely manner.
Previous studies indicate that radiotracer techniques are sensitive enough to determine the diffusion of water in polymers (2–4) and the vapor transmission rates of packaging systems (5, 6). In this article, the radiotracer method is used to estimate the water uptake of tablets placed in a confined, water-saturated environment in which a fraction of the moisture contains tritiated water (3H2O).
Packages of tablets (specially developed with an immediate-release, film-coated tablet formulation of a reverse-transcriptase inhibitor drug and several excipients prone to moisture sorption) were placed into this environment. After a period of time, the individual tablets were monitored for radioactivity. Elevated radioactivity from the tablets indicates that water was absorbed from outside the package. The total water absorbed by each tablet can be estimated by the amount of radioactivity.
Several factors (e.g., stability, density, exchange, and vapor or liquid partition) associated with the differences between tritiated and unlabeled water were taken into account in the design of the experiments. Most of these elements were related to the physical properties of tritium and tritiated water and the potential for isotope effects.
The stability of tritiated water was the first concern because tritiated water at 100% (58 Ci/mmol) undergoes self-radiolysis (i.e., radiation-induced self-decomposition) (7). At 2–19%, some degradation occurs, whereas at ~1% (0.5 Ci/mmol), tritiated water undergoes little degradation in one year (8). This study uses tritiated water at only 7.7 × 10–7% (4.7 × 10–7 Ci/mmol) levels and hence, decomposition is not a significant factor. The difference between the densities of tritiated water (99.30 mol %, 1.21215 g/cm3 at 25 °C) (9) and of water (0.99707 g/cm3 at 25 °C) (10) do not affect its use. Any exchange of tritium between labeled and unlabeled water does not adversely affect results. Sepall measured the vapor–liquid partition of tritium in tritiated water and found that the ratio of 3H:1H in the vapor phase to that ratio in the liquid phase varied from 0.90 to 0.97 over a 14–90 °C temperature range (11). The ratio was 0.94 at 50 °C, which is near the experimental conditions of this study. This bias was considered acceptable for the present application.
Materials and methods
Instrumentation and materials. Direct or static liquid scintillation counting was performed by a liquid scintillation analyzer (Tri-Carb model 2200CA, Packard Instruments, Meriden, CT). Scintillation vials were 20-mL borosilicate glass.
Tablet lots A (50 mg active drug) and B (50 mg active drug) were packaged in polyvinyl chloride (PVC) and paper blister cards and sealed in foil–foil gusseted pouches (6 tablets/card, 1 card/pouch). Tablet lots C (100 mg active drug), D (50 mg active drug), E (50 mg active drug), and F (50 mg active drug) were sealed in foil–foil blister cards (10 tablets/card).
The tritiated water (Amersham, Buckinghamshire, UK) had a labeled concentration of 5 mCi/mL. At the time of use, the activity was 4.3 mCi/mL (9.5×109 disintegrations per minute [dpm]). The water was diluted with deionized unlabeled water to an activity of 21 μCi/mL (4.7 × 107 dpm/mL) and placed into a pan at the base of a 30 × 30 × 27-cm, airtight cabinet (stainless steel and glass, VWR International, West Chester, PA). The cabinet was placed into a convection oven (Blue M, Williamsport, PA) set at 40 °C.
A liquid scintillation cocktail (LSC) (Ready Safe, Beckman Instrument, Inc., Fullerton, CA) was used in the static liquid scintillation counter. All other reagents and chemicals were from standard sources and used as received.
Methods. The packages containing tablets were placed on plastic shelves in the cabinet and were undisturbed until they were removed for analysis. The oven was turned off approximately one hour before the samples were removed to allow them to cool. External and internal oven surfaces were monitored for radioactivity throughout the experiment to ensure no leakage from the inner cabinet occurred. The outside of the pouch was swabbed (GF/B 37-mm diameter filter, Whatman plc, Middlesex, UK) and checked for activity before opening. This procedure ensured the tablets were not exposed to tritiated water upon removal for analysis. Tablets were transferred to individual scintillation vials and dispersed in unlabeled water (1.5 mL for 50 mg tablets, 2.0 mL for 100 mg tablets) after which scintillation fluid (20 mL) was added. The blister cards were always oriented in the same manner for each analysis to correlate tablet location and water uptake. Tablet samples were mixed by inversion and vortex, and then centrifuged for 5 min at 2000 rpm to settle the solids. The vials were read in the scintillation counter.
Quenching studies to determine interferences caused by the tablet matrix were performed by preparing samples containing various concentrations of 3H2O (activity) with and without tablets present. Quenching (i.e., an interference by a substance in the sample that causes a reduction in the efficiency of the scintillation process) was determined as the difference in activity between the two sample sets.
This technique's detection limit primarily depends on the water radioactivity (activity) used to generate moisture in the test environment. Figure 2 shows the relationship between the activity of the water and the quantitation limit. As the activity of the water increases, the quantitation limit decreases. The activity needed to achieve a 5-μg water level per tablet is approximately 4.7 × 107 dpm/mL or 21 μCi/mL. It also is important to note that this figure is the amount of water absorbed, not the total water present. Because of the excipients' water content, one tablet typically contains ~10 mg of water, as measured by KF titration.
Figure 2: This plot shows that as the radioactivity of the water increases, the quantitation limit decreases.
To some extent, quenching is likely to occur in any scintillation-counting experiment. As Figure 3 shows, the presence of the tablet matrix reduced the efficiency of the process by 28%.
Figure 3: This plot shows the quenching effect of the tablet matrix. Sample preparations containing a tablet matrix had an average relative response of 0.72 Â±0.025 compared with samples without a matrix.
Foil–foil gusseted pouch. The first package evaluated was the PVC and paper blister card inside a foil–foil gusseted pouch. Packages from lots A and B were placed into the titrated water environment and tested during one week (see Figure 4). After one day, tritiated water could be determined in each tablet tested. The offset in the measured absorption occurred because the samples were placed in the oven at the same time as the tritiated water (i.e., before equilibrium was reached between the 3H2O and the internal environment). After the offset, absorption was linear. The rate of water uptake was 0.13 mg/day/tablet (r2 = 0.991) for lot A and 0.12 mg/day/tablet (r2 = 0.996) for lot B. The tablets within a given pouch showed good consistency in water uptake. The relative standard deviations at each timepoint were less than 3% for lot B, whereas the relative standard deviations at each timepoint were less than 9% for lot A.
Figure 4: Foil over wrapped PVC blister packages of immediate-release, film-coated tablets (formulated of a reverse-transcriptase inhibitor and excipients prone to moisture sorption) were placed into a confined, water-saturated environment containing a fraction of tritiated water and were monitored for radioactivity. This plot shows the amount of water absorbed versus time, as determined by the radiotracer technique. Each point is the mean of three individual tablets. Linear regression lines and error bars representing Â± 1 standard deviation are plotted for each lot. The relative standard deviations at each timepoint were less than 3% for lot B, whereas the relative standard deviations at each timepoint were less than 9% for lot A.
Foil–foil blister cards with no secondary overwrap. The second investigation evaluated foil–foil blister cards with no secondary overwrap. For these experiments, relative amounts of water absorbed by individual tablets were of interest rather than the overall rate of water transmission of the package. More than 300 tablets were tested, but because the method was simple, analysis time required only one day. Figure 5 illustrates the amount of water absorbed by individual tablets after 16 days in the humidity chamber. In several instances, one tablet did not disperse well when the water was added as part of the sample preparation procedure. One would expect that the dissolution characteristics of such tablets would be poor. For example, tablet 10 in lot C on the blister card E showed poor dispersion. Radiotracer analysis showed that the tablet absorbed ~90 mg of water. In other cases, tablets absorbed so much water that they broke apart upon removal from the blister card.
Figure 5: Foilâfoil blister card packages of immediate-release, film-coated tablets (formulated of a reverse-transcriptase inhibitor and excipients prone to moisture sorption) were placed into a confined, water-saturated environment containing a fraction of tritiated water and were monitored for radioactivity. This illustration shows individual tablet water absorption data for the foilâfoil blister cards after 16 days in the humidity chamber.
Leakage mechanisms. Although one might expect the water vapor transmission rate (WVTR) through aluminum foil to be zero, all packages leak (12). Several possible leakage mechanisms in foil could cause a nonzero WVTR. Some moisture can transfer through pinholes in the foil. The term pinhole is very misleading because the average diameter of such holes is very small (less than 25 μm) (13). The frequency of pinholes is highly dependent on the thickness of the aluminum foil. Foil thicknesses of 6, 9, and 25 μm have ~4000 pinholes/m2, 200 pinholes/m2, and essentially no pinholes, respectively. The foil used for this packaging application had a thickness of 9 μm, so for an entire 10 × 20-cm blister card, one would expect ~7–8 pinholes. Even if one assumes the collective maximum diameter of all pinholes is 25 μm, the total area still represents an extremely small region of the foil. In addition, the aluminum foil is coated with a polymer layer that had a thickness of ~1 μm, so any water that transfers through the pinhole regions must also permeate through the polymeric coating (14).
A second possible mechanism for water transfer is permeation through the polymeric sealing area used to join the two foil surfaces, but the WVTR through adequately sealed regions was expected to be extremely low. Although moisture can permeate throughout the perimeter of the package, the cross-sectional width of the polymeric coating exposed to the environment is very small (~2 μm), and any water must permeate across the width of the package edge (~1 cm) before it reaches the inside of the foil package.
On the basis of previous discussion, one would expect that if the foil was adequately sealed and had no gaps in the sealing area, a very small WVTR would be observed from moisture transfer through pinhole regions and through the polymeric coating in the sealing area. Because of these two sources, the WVTR is not easily measured with conventional methods. It can, however, be measured with the sensitive moisture technique presented in this article and is consistent with the observation that low levels of titrated water were found in all packages examined in these studies. In any case, the WVTR represents an acceptable leakage rate because it is insignificant with respect to the packaging system's moisture requirements for this product.
As a final mechanism, water transfer can occur through gaps at the foil's edges if the package is inadequately sealed. Careful visual inspection of the blister cards showed very small channels present at the outer edges, although the presence of these channels was not necessarily a predictor of leakage because many channels did not appear to cross through to tablet cavities. The foil surfaces are coated with a polymer that adheres the two sides together during the packaging operation and it is believed that the channels were caused by insufficient pressure or heat, or a combination of both, when the two surfaces were joined. This mechanism is consistent with the observations made in this article. For paper-backed PVC blisters inside the foil overwrap, the transmission of water through the foil is rate limiting. But once inside the overwrap, moisture equilibrates evenly among the tablets through the PVC blisters. This finding is evidenced by the low relative standard deviation of moisture values (typically 5%) among individual tablets in a given package and low mean dissolution at some stability time points.
For foil–foil blisters, each tablet was isolated in its own blister. Rapid water uptake was only possible when a channel penetrated the entire width of the package edge. Many tablets, even entire cards, had very low moisture uptake (<0.1 mg/tablet). Other cards showed only a few tablets with high water content (>50 mg/tablet) whereas all others had low values (0.1–1 mg/tablet). This finding helps explain Figure 1, in which some dissolution time points exhibited one or two low values and other values were acceptable.
The radiotracer technique is a fast, sensitive method for determining the integrity of packages to moisture. In this study, the method provided a quantitation limit of 5 nL (5 μg) (see Figure 2) of water. In addition, moisture uptake was quantified in previously packaged individual dosage units. Although none of the packages tested were impervious to moisture, some allowed significantly high amounts (>50 mg) of water to enter. This effect was traced to inadequately sealed edges in the foil–foil packaging. The wide variation in moisture sorption among individual tablets suggested a cause for the inconsistent behavior of the dissolution results. Radiotracer methods should be a valuable tool for investigating other moisture-mediated phenomena for which high variability is observed.
Don L. Theis* is a senior principal scientist and Brian R. Rohrs, John D. Stodola, and Steven J. Borchert are associate research fellows, all at the Pfizer Global Research and Development Unit of Pfizer, Inc., 7000 Portage Road, Kalamazoo, MI 49009, tel. 269.833.6982, firstname.lastname@example.org@aol.com
*To whom all correspondence should be addressed.
Submitted: May 11, 2005. Accepted: May 24, 2005.
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