Simple Isocratic Method for API and Main Impurity

Formulations of Doxycycline may contain a Methacycline Impurity.  As such, it is crucial for analytical methods to be able to separate these two peaks. Using this simple isocratic Method, the baseline resolution is obtained for this pair.

1. Methacycline
2. Doxycycline
* Impurities in doxycycline standard

Method Conditions
Column: Cogent UDC Cholesterol™, 4μm, 100Å
Catalog No.: 69069-15P
Dimensions: 4.6 x 150mm
Mobile Phase:
A: 73% DI Water / 0.1% TFA
B: 27% Acetonitrile / 0.1% TFA
Injection vol.: 10 µL
Flow rate: 1.0mL / minute
Detection: UV @ 350nm
Temperature: 25˚C
Samples: 1.0mg / mL stock Solutions of Doxycycline HCL and Methacycline HCL reference standards were prepared in a diluent of 50/50 Solvent A / Solvent B. 100μL of each solution was transferred to a new vial and diluted with 800μL diluent. Individual standard dilutions were also prepared for peak identity confirmation.
t0: 1.8 minutes

Note: Doxycycline is an antibiotic used for treatment of various bacterial infections such as Lyme disease, Rocky Mountain Spotted Fever, and others. Methacycline is a synthetic precursor to Doxycycline and therefore is a known impurity in Doxycycline formulations.


No 311 Doxycycline and Methacycline Analysis with HPLC pdf   0.3 Mb