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Stability Testing of Tetracycline HCl Capsules
Robust, high-throughput separation of API from its degradation products
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printable Application Sheet
Peaks:
- 4-epitetracycline
- tetracycline
- 4-epianhydrotetracycline
- anhydrotetracycline
Note: Tetracycline is a broad-spectrum antibiotic widely used in both human and veterinary medicine. It is known to degrade primarily by two
pathways: Dehydration and epimerization. Epimerization at the carbon-4 position leads to an inactive and non-toxic degradation product [1].
However, the anhydro form and its epimer are reported to be toxic in vivo and have been implicated in the development of Fanconi Syndrome
[2, 3]. Therefore, it is crucial to have a reliable analytical method for discriminating between tetracycline and its major degradation
products. The degradation procedure used here was adapted from Pena et al. [4].
- B.-A. Hoener, T.D. Sokoloski, L.A. Mitscher, L. Malspeis, J. Pharm. Sci. 63 (1974) 1901–1904.
- M. Kühne, G. Hamscher, U. Körner, D. Schedl, S.Wenzel, Food Chem. 75 (2001) 423–429.
- J. Montoliu, M.T Carrera, A. Darnell, L. Revert, Brit. Med. J. 283 (1981) 1576–1577.
- A. Pena, L.P. Palilis, C.M. Lino, M.I. Silveira, A.C. Calokerinos, Anal. Chim. Acta. 405 (2000) 51–56.
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Method Conditions
| Column |
Cogent Bidentate C18™, 4µm, 100A |
| Catalog No. |
40018-75P |
| Dimensions |
4.6 x 75 mm |
| Solvents |
A: DI water + 0.1% formic acid
B: acetonitrile + 0.1% formic acid |
| Gradient |
| time (min.) | %B | time (min.) | %B |
|
| 0 | 10 | 6 | 80 |
| 4 | 30 | 7 | 10 |
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| Post time |
3 min. |
| Flow rate |
1.0 mL/min. |
| Sample |
USP grade tetracycline HCl capsule extract (degraded).
Stock Solution: 10.0 mg capsule contents were diluted with 10 mL 1.0 N HCl and sonicated for 10 min. 1 mL aliquot was
diluted to 20 mL with DI water. Solution was heated at 80°C for 30 min. Solution was then diluted to 100 mL with DI water and
filtered through a 0.45 µm nylon membrane HPLC filter (MicroSolv Technology Corp., Eatontown, NJ, USA).
Working Solution: Stock solution was diluted 10x using 0.01 N HCl diluent. |
| Detection |
UV 360 nm (0–5 min) 430 nm (5–7 min) |
Discussion
Figure A shows the chromatogram obtained from a single injection of the degraded tetracycline capsule extract.Tetracycline is well-resolved from its
three main degradation products, the identities of which were confirmed by individual standards under non-degrading conditions. Amine-containing
analytes such as these often require the use of an ion-pairing agent in the mobile phase in order to reduce peak tailing from silanolic interactions.
However, ion-pairing agents often lead to poor reproducibility and long equilibration times due to slow uptake and release of these agents from the
column. TYPE-C Silica™ based columns have most of the surface silanols replaced, and therefore ionpairing agents are not necessary to obtain good
peak shapes. Figure B shows an overlay of five sequential injections of the degraded tetracycline solution, illustrating the good repeatability of the
method. Retention time %RSDs for all of the analytes were < 0.1%. In addition, the post time was minimal (3 min).
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