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2.5. Solid Phase Extraction (SPE) Sample solutions were applied to OASIS MCX cartridges which had previously been conditioned twice with 1?mL methanol and equilibrated with 1?mL purified water, successively. The cartridge was washed with 1?mL 2% formic acid in water (v/v). Elution was carried out by 1?mL elution mixture: acetonitrile/methanol/ammonium hydroxide (57?:?38?:?5), and the eluate was collected and evaporated to dryness using gentle stream of nitrogen. The residue was reconstituted with 100?��L methanol. For the validation procedure, Oasis MCX ��Elution Plates (30?��M, Waters) were used with an extraction plate manifold (Waters, Milford, MA, USA). Each well of the plate was conditioned two times with 200?��L of methanol and equilibrated with 100?��L of ultrapure water. The samples (prepared as explained in Section 2.4) were loaded in each well. Formic acid (2%, v/v in water) washes were then performed. [http://www.selleckchem.com/screening/kinase-inhibitor-library.html Selleckchem Kinase Inhibitor Library] Elution was done in collection plates [http://en.wikipedia.org/wiki/Isotretinoin Isotretinoin] with 50?��L of elution mixture, acetonitrile/methanol/ammonium hydroxide (57?:?38?:?5), and quickly diluted with 50?��L of formic acid 4% (v/v) in water. During the validation step, recoveries at all the levels of concentration were calculated on three consecutive days. Areas of the peaks after MCX extraction were compared to those of standard solutions. 2.6. Validation Methodology The developed UPLC method was validated using accuracy profiles concept. The Soci��t�� Fran?aise des Sciences et Techniques Pharmaceutiques (SFSTP) commissions elaborated validation guidelines to help scientists to apply harmonized regulatory recommendations [http://www.selleckchem.com/products/BI-2536.html BI 2536 manufacturer] and to validate their analytical and biopharmaceutical procedures [31�C33]. Their novel validation strategy was based on the total error (bias + standard deviation) and the accuracy profiles decision tool. Currently, this new protocol of validation becomes more attractive and knows a wider spreading among the scientific community [34�C40]. Briefly, a procedure can be qualified as acceptable if the difference between every measurement (x) of a sample and its ��true value�� (��T) is inside the acceptance limits �� (predefined by the analyst depending to the objective of the method). The probability that the results will be in these acceptance limits should be superior or equal to a probability ��. It can be translated into the following [31�C33]: Px?��T