Regella Venkata Rama Prabhakara Sastry
◽
Chidambaram Subramanian Venkatesan
◽
Bhetanabhotla Sarveswara Sastry
◽
Singaram Sathiyanarayanan
◽
Sanapati Murali
Background:
Four major degradation products (1-4) of pralatrexate injection were formed
under hydrolytic and light stress conditions. The impurities 1 and 2 were the potential photo degradation
products and the impurities 3 and 4 were the potential hydrolytic degradation products.
Objective:
To prepare and characterize the novel degradation impurities 1, 2, 3 and 4 of pralatrexate
injection using NMR, HR MS and IR techniques; and to develop and validate stability indicating analytical
reverse phase HPLC-UV method for quantitative simultaneous determination of potential
degradation impurities, related substances of pralatrexate and pralatrexate active in pralatrexate liquid
formulation.
Methods:
Gradient HPLC-UV method was developed for the quantification of degradation impurities,
related substances and pralatrexate in pralatrexate injection. The separation was achieved on C18 column
(250 mm X 4.6 mm, 5µm) using a mobile phase composed of sodium dihydrogen phosphate
monohydrate in water (pH 3.0; 0.01M) and methanol. The components were monitored by the UVvisible
detector at 242 nm with a flow rate of 1.0 mL/min.
Results:
The method validation parameters such as accuracy, selectivity, linearity, LOD, LOQ, precision,
ruggedness and robustness were demonstrated successfully for pralatrexate and its degradation
impurities. The stability-indicating capability of the developed HPLC method was demonstrated by
adequate separation of all potential pralatrexate related substances from pralatrexate stressed drug
product samples.
Conclusion:
The developed stability indicating HPLC method was found to be suitable for the simultaneous
quantitative determination of potential degradation impurities and related substances of pralatrexate
and pralatrexate active in pralatrexate liquid formulation.