title: Lights and shadows of the actual European guidelines on bioanalytical method validation: the case of raltegravir.
abstract: BACKGROUND
  Recently, the European Medicines Agency (EMA) has released new guidelines on the validation of bioanalytical methods. In this work, we compared the analytical performance of 2 high-performance liquid chromatography with tandem mass spectrometry methods designed for the quantification of the antiretroviral drug raltegravir (RAL) that fully accomplish the criteria according to the new EMA guidelines.
  
  
  METHODS
  The first method was developed with the goal of separating RAL from its main metabolite, whereas in the second method, we deliberately did not discriminate the parent drug from its metabolite. After validation, both methods were used for the quantification of plasma samples from HIV-infected patients on RAL-based maintenance antiretroviral therapy.
  
  
  RESULTS
  Incurred reanalysis of samples obtained from patients receiving RAL as therapy evidenced optimal performance for both methods. Similarly, the comparison of both methods performed by the Deming test showed that they correlate significantly with each other (Pearson coefficient of regression 0.97, P < 0.0001) with no significant deviation from linearity according to the Cusum test. The Bland-Altman test, however, showed a mean difference between the 2 methods of 54.1% (limits of agreements of ±1.96 SD ranged from -163.3% to +271.5%). Such differences were significantly affected by interindividual variations in the production of RAL metabolites.
  
  
  CONCLUSIONS
  We conclude that the recently released EMA guidelines on bioanalytical method validation present some limitations related to the process of method development. To have confidence in the drug-concentration measurements, laboratories must demonstrate their performance through the participation in international proficiency testing schemes that must include patient samples.
authors.affiliation: *Unit of Clinical Pharmacology, Institute of Neuroscience, Consiglio Nazionale delle Ricerche; †Department of Biomedical and Clinical Sciences, L. Sacco University Hospital, Università degli Studi di Milano, Milan; and ‡Scientific Institute IRCCS Eugenio Medea, Bosisio Parini, Italy.
authors.forename: Sara
authors.initials: S
authors.lastname: Baldelli
authors.forename: Dario
authors.initials: D
authors.lastname: Cattaneo
authors.forename: Serena
authors.initials: S
authors.lastname: Fucile
authors.forename: Emilio
authors.initials: E
authors.lastname: Clementi
mesh_terms: D002851:Chromatography, High Pressure Liquid / Q000592:standards; D005060:Europe; D017408:Guidelines as Topic / Q000592:standards*; D006801:Humans; D011760:Pyrrolidinones / Q000032:analysis* / Q000097:blood; D000068898:Raltegravir Potassium; D053719:Tandem Mass Spectrometry / Q000592:standards*
chemical_list: D011760:Pyrrolidinones; D000068898:Raltegravir Potassium