Anal. Chem. 1996, 68, 4076-4079
Mapping of Pharmaceutical Degradants of Gabitril Using Radiotracers Henrik Olsen,* Christian Foged, Liselotte Hansen, and Peter G. Nielsen
Analytical Laboratories, Health Care Discovery & Development, Novo Nordisk A/S, Novo Park Alle` , DK-2760 Ma˚løv, Denmark
Gabitril is a new antiepileptic drug. HPLC/UV analysis indicates that stress-induced degradation of Gabitril tablets is very complex, leading to many degradation products. Strategies for alleviating the mass balance of the degradation using radiolabeled Gabitril are proposed. The extraction efficiency of [14C]Gabitril and its degradation products from the tablet formulation were determined after storage under various conditions. Degradation products greater than 0.1% were located by HPLC combined with fraction collection and radioactivity counting. The UV response factors of degradants relative to the Gabitril signal could be estimated by correlating UV and radioactivity profiles. All pharmaceutical products degrade to some extent upon storage. Since the formation of degradation products can severely affect the safety and efficacy of the pharmaceutical product, monitoring and understanding the degradation processes is an area of increasing attention in the pharmaceutical industry. Consequently, adequate analytical methods must be developed and applied to assure the quality of the products.1,2 Drug products are controlled by a stability-indicating assay procedure for assessing the potency of the product. Further, chromatographic methods are used to monitor the individual degradation products. The sum of the peak areas of drug degradants should match with the observed loss of strength in the preparations; in other words, mass balance should be achieved. If mass balance cannot be achieved, then it is necessary to investigate whether all the degradants are actually being extracted from the tablets during the procedure(s), and if these degradants are quantified correctly in the chromatographic method(s).3 Radiolabeling provides a very sensitive and specific method of locating and measuring particular compounds. It has thus become a preferred method for mapping the metabolic pathways of compounds in biological systems.4 In the present study, we show how radiolabeled material can be used to highlight the degradation pathways and mass balance in nonbiological systems. To our knowledge, this represents the first example of a study of this kind. The new antiepileptic drug, Gabitril (1),5 which has been found to degrade in a very complex manner,6,7 and its (1) (2) (3) (4)
ICH Guideline: Impurities in new drug substances, Final, May 5, 1995. ICH Guideline: Impurities in new drug products, Draft #4, August 10, 1995. Notices. Fed. Regist. 1993, 58, (No. 72) 21088. Hawkins, D. R. Applications of Isotopes in Drug Metabolism. In Isotopes: Essential Chemistry & Applications; Elvidge J. A., Jones J. R., Eds.; The Chemical Society, Burlington House: London, 1979; pp 232-275. (5) Pharm. Business News 1995, 8 Sept, 19. (6) Callen G.; Chorghade M. S.; Lee E. C.; Nielsen P. G.; Pedersen H.; Rustum A. Heterocycles 1994, 39 (1), 293-303.
4076 Analytical Chemistry, Vol. 68, No. 22, November 15, 1996
radiolabeled analogue, [14C]Gabitril (2), have been used for these studies.
EXPERIMENTAL SECTION Chemicals. [14C]Gabitril (2) was obtained from Abbott Laboratories (North Chicago, IL). The tracer was purified at Novo Nordisk A/S by reverse-phase liquid chromatography to a radiochemical purity of >99%, determined by radio-HPLC. The tracer was diluted with unlabeled material to produce a bulk material with the required specific radioactivity. Reference standard materials of Gabitril (1, (R)-(-)-N-[4,4-bis(3-methylthien-2-yl)but3-enyl]nipecotic acid hydrochloride) and bisthiophene ketone (bis(3-methylthien-2-yl) ketone) were prepared by Novo Nordisk A/S, Ma˚løv, Denmark. Formulations. The formulation was a melt granulation process conducted in laboratories approved for handling of isotopes by the Pharmacy Department, Novo Nordisk A/S. The tablets were prepared using a Diaf TM-20 for compression. Gabitril in two different formulations was used for the experiments. Thus, the determination of the extraction efficiency was done with 4 mg of Gabitril in 180 mg tablets with a specific activity of about 3.5 × 104 DPM/tablet. Mass balance and response factor studies were done using 5 mg of Gabitril in 160 mg tablets with a specific activity of about 1.3 × 106 DPM/tablet. Analogue unlabeled tablets and placebo tablets were also prepared. Extraction Procedure. Bulk product and tablets were extracted with a mixture of methanol and 0.1 N sodium hydroxide (1:1 v/v) solution using magnetic stirring for 60 min. HPLC Analysis and Fractionation. A Macherey-Nagel Nucleosil 5 µm, 4.0 mm × 12.5 cm C-18 column was used for monitoring the degradation of [14C]Gabitril upon storage. The mobile phase was composed of 0.1% TFA (v/v) in Milli-Q water and acetonitrile programmed from 0 to 95%. The UV detection was set at 250 nm and flow rate at 1 mL/min. A sample volume of 20 or 50 µL was injected for UV measurements. A 95 µL volume was injected when collecting samples for radioactivity measure(7) Novo Nordisk A/S, unpublished work. S0003-2700(96)00551-3 CCC: $12.00
© 1996 American Chemical Society
Table 1. Content of Radioactivity (%) in Tablet Filtrate and Excipient Residue after 5 and 13 Months of Storage at Various Conditions and HPLC Assay after 13 Months 5 months conditionsa -22 °C 15 °C 30 °C 40 °C 40 °C/75 RH a
Figure 1. Superimposed HPLC chromatograms of Gabitril and degraded (40 °C/75% RH for 6 months) Gabitril in the tablet formulation.
ments. Fractions of 30 or 60 s were collected with a LKB 2211 Superrac collector. The tablets were also analyzed for content of Gabitril using the HPLC assay. Radioactivity Measurements. The collected samples from HPLC fractionation were added to Pico-Aqua scintillation reagent and counted for 30 min on a Packard Tri-carb Model 2000CA with quench correction. The extracts were counted for 60 min. The solid residue was filtered through a 5 µm Millipore filter (Type LS), washed with 10 mL of extraction solvent, and dried. The dried filter paper was combusted using a Packard Tri-carb 306 automatic sample oxidizer. The resulting [14C]CO2 was collected by adsorption in Carbo-Sorb sorbing solution, and Permafluor E scintillation fluid was added. Stress Conditions. In the extraction efficiency studies, the tablets were stored at -20, 15, 30, and 40 °C, and 40 °C/75% relative humidity for 13 months in 15 mL open DUMA containers. In the mass balance and response factor studies, the tablets were stored at 5 and 40 °C for 6 months and at 40 °C/75% relative humidity for 9 months in 15 mL open DUMA containers. Safety Considerations. It is important that studies using radiolabeled tracers are handled in dedicated laboratories and appropriate safety actions are taken to protect persons involved with such studies. It should be noted that, in contrast to drug metabolism work, formulation experiments involving handling of the tracer material in dry form require special precautions. According to national guidelines, there will be an upper limit for the amount of the radioactivity that can be used in these studies, depending on the anticipated formulation procedure. RESULTS AND DISCUSSION A critical step in an analytical procedure involving extraction of a pharmaceutical solid dosage form is the determination of the efficiency of extraction. A standard procedure for evaluating recoveries is to add standard materials to placebo tablets. This procedure does not, however, take the effect of the manufacturing process into account. When mass balance has not been achieved, scepticism always exists as to whether the components related to the active ingredient(s) have been extracted from the insoluble tablet excipient. The new antiepileptic drug Gabitril degrades under enhanced conditions. In Figure 1, superimposed HPLC chromatograms of the drug substance in a tablet formulation before and after degradation have been depicted. It is seen from
13 months
radioactivity filtrate residue 99.7 99.7 99.2 99.5 97.9
0.3 0.3 0.8 0.5 2.1
HPLC assay (%) 100.8 93.8 91.8 88.0 63.5
radioactivity filtrate residue 99.6 99.5 99.2 98.9 96.3
0.4 0.5 0.8 1.1 3.7
RH, relative humidity.
Table 2. Percentage Recovery of Radioactivity after 5 and 13 Months of Storage at Various Conditions in DPM and as Percentage of the Total Recovery of the Reference Station (at -22 °C) 5 months
13 months
condition
total DPM
in %
total DPM
in %
-22 °C 15 °C 30 °C 40 °C 40 °C/75% RH
31 294 ( 2254 32 433 ( 2095 32 566 ( 1970 32 431 ( 2838 30 526 ( 4950
100 104 104 104 98
30 905 ( 2548 31 303 ( 1246 31 202 ( 3607 31 896 ( 1829 27 628 ( 952
100 101 101 103 89
a
RH, relative humidity.
the chromatograms that, besides the drug component, many other peaks are detected in the degraded tablet. The degradation products could only account for one-third of the loss in potency using a gradient HPLC purity method. The content of degradants was calculated using the response factor of the drug substance. Since mass balance apparently was not fulfilled, we have used radiotracer incorporated in the solid dosage form in order to determine extraction efficiencies and response factors and to examine whether any major degradation products escaped detection. [14C]Gabitril (2) tablets were prepared and stressed under various conditions for 5 and 13 months, respectively. The stressed tablets were subsequently analyzed using an assay method for the active ingredient. The analytical procedure involves an extraction step, followed by a reverse-phase HPLC analysis with UV detection at a wavelength of 250 nm. The radioactivity was determined in the filtrate after extraction and in the remaining solid excipient residues. The results have been summarized in Table 1. From Table 1, it can be concluded that a small but significant amount of the radioactivity is left in the excipient residue after the extraction step. The radioactivity in the solid residue is fairly proportional to the loss of potency determined by HPLC analysis. In the tablets stored at 40 °C/75% relative humidity for 13 months, ∼4% escapes analysis, most likely because of binding to the excipient. For lesser degraded samples, e.g., samples with a potency greater or equal to 90%,
