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Analysis of different drug extraction methods in saliva samples for LC MS / MS assays

Grant number: 20/04730-7
Support type:Scholarships in Brazil - Master
Effective date (Start): May 01, 2020
Effective date (End): November 30, 2021
Field of knowledge:Biological Sciences - Pharmacology - General Pharmacology
Principal researcher:Adriana Maria Calvo
Grantee:Viviane da Silva Siqueira
Home Institution: Faculdade de Odontologia de Bauru (FOB). Universidade de São Paulo (USP). Bauru , SP, Brazil
Associated research grant:17/12725-0 - Model of pharmakinetics/pharmacodynamics (PK/PD) on the influence of P450 genetic polymorphism (CYP2C9) of non-steroidal anti-inflammatory drugs and main metabolics from saliva samples through LCMS/MS and its role on prescription personalization, AP.JP

Abstract

The use of saliva samples in pharmacokinetic and bioequivalence studies has been successfully studied since the middle of the last century and we are obtaining expressive results in this Young Researcher aid. The concentration found in saliva corresponds to the free or unbound fraction of the vast majority of drugs, this being the most important value when studying the pharmacological or toxic action of a drug. The choice of saliva versus blood or plasma must be approached seriously, given the recognized fact that saliva offers quick and non-invasive samples, which often cause bruising, discomfort or even are not performed due to fear of patients considering the multiple collections required in pharmacokinetic studies, and when we have drugs (analytes) present in a matrix as complex as saliva, sample preparation is of crucial importance for analysis. The purpose of sample preparation is to remove interfering substances and also to enrich analytes. The extraction procedure must be highly reproducible, with a high recovery of the target analytes. In addition, an ideal sample preparation method should involve a minimum number of work steps, which should preferably be automated, to increase reliability and human interference with the result. Currently, liquid-liquid methods, with the addition of ethyl acetate and hydrochloric acid are being used with great frequency in the Laboratory of Pharmacology and Genetics of the Faculty of Dentistry of Bauru / USP, requiring several steps that take a few hours to dry the sample. to be analyzed. Miniaturization is the new trend in the area of bioanalysis. Methods that consume less time, require less work and are more environmentally friendly and economical. There is a new technique on the market for the preparation of samples online with the liquid chromatography / mass spectrometry (LC MS / MS) assay that has been used with some drugs, mainly local anesthetics. Microextraction by packaged sorbent (MEPS) is the miniaturization of conventional solid phase extraction (SPE), with the packaged bed (1-2 mg) incorporated directly into an injection syringe. MEPS technology allows the removal of components from the matrix, isolation and concentration of targeted analytes and also reduces the effects of the matrix. Sample preparation takes place in the packed bed of the column. The bed can be coated to provide selective and suitable sampling conditions. The new method appears to be very promising, easy to use, fast compared to traditionally used methods, but it has not yet been tested on non-steroidal anti-inflammatory drugs (NSAIDs) in saliva samples. This project aims to analyze and compare the effectiveness of the two drug extraction methodologies to be adopted in a rational way by the Pharmacology and Genetics laboratory of the Faculty of Dentistry of Bauru / USP in the research line of this Young Researcher Aid and which are quite complex in the development and, in particular, this master student will carry out the validation of methodologies in LC MS / MS so that they are eligible for the next research in the laboratory. (AU)