RESUMEN
This paper presents the recommendations of the Global Bioanalytical Consortium Harmonization Team on method transfer, partial validation, and cross validation. These aspects of bioanalytical method validation, while important, have received little detailed attention in recent years. The team has attempted to define, separate, and describe these related activities, and present practical guidance in how to apply these techniques.
Asunto(s)
Bioensayo/métodos , Cromatografía Liquida/métodos , Espectrometría de Masas/métodos , Guías de Práctica Clínica como Asunto , Estudios de Validación como Asunto , Bioensayo/normas , Cromatografía Liquida/normas , Espectrometría de Masas/normasRESUMEN
Fludarabine and cyclophosphamide are anticancer agents mainly used in the treatment of hematologic malignancies. We have developed and validated an assay using high-performance liquid chromatography (HPLC) coupled with electrospray ionization tandem mass spectrometry for the quantification of fludarabine in combination with cyclophosphamide in human heparin and human EDTA plasma. Sample pre-treatment consisted of a protein precipitation with cold acetonitrile (-20 degrees C) using 250 microL of plasma. Separation was performed on an Extend C18 column (150 x 2.1 mm i.d.; 5 microm) with a stepwise gradient using 1 mM ammonia solution and acetonitrile at a flow rate of 400 microL/min. The analytical run time was 12 min. The triple quadrupole mass spectrometer was operated in the positive ion mode and multiple reaction monitoring was used for drug quantification. The method was validated over a concentration range of 1 to 100 ng/mL for fludarabine and cyclophosphamide in human heparin and human EDTA plasma. The coefficients of variation were <13.9% for inter- and intra-day precisions. Mean accuracies were also within the designated limits (+/-15%). The analytes were stable in plasma, processed extracts and in stock solution under all relevant conditions.