Purification of biological samples and validation of bioanalytical HPLC methods for analysis of drugs and their metabolites

Abstract

Purification of biological matrix prior to HPLC analysis has been complex procedure and source of great variability of analytical results. The most used biological matrixes used for analysis are plasma, serum, urine and saliva and it has been advisable to use the simplest procedure for purification of these samples. Biological matrixes are complex and variability of its content is the main problem in development of bioanalytical methods. Namely, plasma and urine samples contain large number of endogenous compounds in concentrations much larger than concentration of investigated analyte. The concentrations of investigated analytes are often in very low concentrations and its structure can be very similar to structure of some endogenous compounds. Due to this problem, purification and concentration of biomatrix is one of the most important steps in development of bioanalytical methods. For bioanalytical methods the most important parameters are reliability and repeatability of the analytical results. Validation of bioanalytical chromatographic methods can be conducted according to The International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH), Food and Drug Administration (FDA) and European Medicines Agency (EMA). During the validation process selectivity, limit of detection (LOD), lower limits of quantification (LLOQ), range, linearity, precision, accuracy, stability and efficacy of biological sample purification have to be investigated.

Priprema biološkog materijala pre HPLC analize predstavlja kompleksnu proceduru koja je obično izvor velike varijabilnosti dobijenih analitičkih rezultata. Najčešće analizirane telesne tečnosti jesu plazma, serum, urin i saliva, i poželjno je primeniti što jednostavniju proceduru pri pripremi navedenih uzoraka. Kompleksnost i varijabilnost sastava biološkog materijala predstavlja jedan od glavnih problema u razvoju bioanalitičkih metoda. Plazma i urin sadrže veliki broj endogenih komponenata prisutnih u koncentracijama koje su obično veće od koncentacije samog leka i/ili njegovih metabolita. Osim što se lekovi i/ili njihovi metaboliti često nalaze u malim koncentracijama, njihova struktura može biti slična strukturi nekih endogenih komponenata. Imajući ovo u vidu, prečišćavanje i koncentrisanje biološkog materijala je jedan od najvažnijih koraka u razvoju bioanalitičke metode. Bez obzira sa kojim se uzorcima biološkog materijala radi, važno je voditi računa da se tokom analize dobiju pouzdani i ponovljivi rezultati. Validacija bioanalitičkih hromatografskih metoda izvodi se prema preporukama The International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) regulative, Food and Drug Administration (FDA) i European Medicines Agency (EMA). U okviru postupka validacije ispituje se selektivnost, donja granica detekcije (LD), donja granica određivanja (eng. lower limits of quantification, LLOQ), opseg, linearnost, preciznost, tačnost, stabilnost i efikasnost postupka prečišćavanja uzoraka biološkog materijala.