Stability Testing of Tetracycline HCl Capsules – AppNote

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ARTICLES, APPNOTES, FAQ (9289)
- AppNotes Library (1888)
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- Frequently Asked Questions?? (3402)
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June 14, 2012
Bill Ciccone

AppNotes Cogent Bidentate C18 / AppNotes Library / AppNotes Pharmaceuticals / AppNotes Reversed Phase Methods / ARTICLES, APPNOTES, FAQ

Robust, high-throughput Separation of API from its Degradation Products

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. In this Method, ion- pairing 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 Robustness of the Method. Retention time %RSDs for all of the analytes were < 0.1%. In addition, the post time was minimal (3 minutes).

Peaks:
1. 4-Epitetracycline
2. Tetracycline
3. 4-Epianhydrotetracycline
4. Anhydrotetracycline

Method Conditions
Column: Cogent Bidentate C18™, 4µm, 100Å
Catalog No.: 40018-75P
Dimensions: 4.6 x 75mm
Mobile Phase:
—A: DI Water / 0.1% Formic Acid
—B: Acetonitrile/ 0.1% Formic Acid
Gradient:

Time (minutes)	%B
0	10
4	30
6	80
7	10

Post Time: 3 minutes
Flow rate: 1mL / minute
Detection: UV @ 360nm (0–5 minutes) 430nm (5–7 minutes)
Sample Preparation:
—Stock Solution: 10mg capsule contents were diluted with 10mL 1.0 HCI and sonicated for 10 minutes. 1mL aliquot was diluted to 20mL with DI Water. Solution was heated at 80°C for 30 minutes.
—Working Solution: Stock Solution was then diluted to 100mL with DI Water and filtered through a 0.45µm Nylon Syringe Filter (MicroSolv Technology). Stock solution was diluted 10x using 0.01N HCI diluent before injection.

Notes: 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.

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