The advantages of microflow LC-MS/MS compared with conventional HPLC-MS/MS for the analysis of methotrexate from...

In support of bioanalysis, there has always been a desire to improve detection limits and reduce scale. Microflow LC (MFLC) coupled with MS accomplishes both of these goals.

As such, MFLC coupled with an MS system was used to generate bioanalytical validation data that met US FDA criteria. The MFLC-MS/MS data was compared with the same method with the use of conventional HPLC-MS/MS and a more than 14× S/N improvement was found with the MFLC-MS/MS method. Methotrexate was used as a model molecule to demonstrate the validation of the method from human plasma. The MFLC-MS/MS method was demonstrated to be accurate (±7%) and precise (12.9% at the LLOQ and a maximum of 11.6% at all other concentrations) across the dynamic range of the assay (1-1000 ng/ml) and compared well with the HPLC-MS/MS method. The MFLC bioanalytical validation was performed at a flow rate of 35 µl/min on a 0.5-mm inner diameter (I.D.) column, whereas, for the same linear velocities on the 2.0-mm I.D. column, the conventional HPLC bioanalytical validation was performed at 700 µl/min. Since the flow rate of the MFLC system is 20-times less than the HPLC system, the consumable solvent and disposal cost to perform the MFLC validation was significantly less.

MFLC-MS/MS can be used to perform bioanalytical method validations with increased MS signal, reduced source contamination and reduced solvent consumption.