Purity profiles of pteroylglutamate reference substances by high-performance liquid chromatography

Area of Science:

• Analytical Chemistry
• Pharmaceutical Analysis

Background:

• Pteroylglutamic acid derivatives, including folic acid, leucovorin, and methotrexate, are crucial in pharmaceuticals.
• Ensuring the purity of these reference standards is vital for accurate drug analysis and quality control.

Purpose of the Study:

• To evaluate the purity of pteroylglutamic acid-type reference substances using HPLC.
• To investigate the impact of mobile phase pH on the separation of related compounds and impurities.
• To optimize the chromatographic method for improved resolution of methotrexate and its isomers.

Main Methods:

• Reversed-phase high-performance liquid chromatography (HPLC) was employed for purity analysis.
• An artificial mixture of pteroylglutamic acid derivatives and potential impurities was analyzed.
• The influence of mobile phase pH, utilizing standard buffers versus ion-pairing agents, was systematically studied.

Main Results:

• HPLC successfully determined the purity of folic acid, leucovorin calcium, and methotrexate reference standards.
• A standard buffer mobile phase provided superior separation of methotrexate from its major impurities compared to ion-pairing mobile phases.
• The method achieved separation of methotrexate from its biologically inactive 7-isomer.

Conclusions:

• Reversed-phase HPLC is a suitable technique for the purity assessment of pteroylglutamic acid derivatives.
• Standard buffer mobile phases offer advantages for resolving methotrexate from critical impurities and isomers.
• The developed method enhances the quality control of essential pharmaceutical reference standards.