Validation of bioanalytical procedures is performed pursuant to current regulatory requirements provided by the Guideline for Evaluation of Medicinal Products (Volume 1. — Federal State Budgetary Institution Scientific Center for Expert Evaluation of Medicinal Products, 2013), Guideline on bioanalytical Method Validation EMEA/CHMP/EWP/192217/2009, FDA Guidance on Bioanalytical Method Validation (2013) and considering the Rules for Conducting Bioequivalence Studies of Drug Products on the Territory of EAEU (according to the draft decision of the Council of the Eurasian Economic Commission "Concerning Approval of the Rules for Conducting Bioequivalence Studies of Drug Products on the Territory of Eurasian Economic Union").

Validation of analytical procedure is performed regarding the following main parameters:

• selectivity;
• evaluation of lower limit of quantitation, LLOQ;
• linearity;
• accuracy;
• precision;
• carry-over effect;
• matrix effect;
• recovery;
• dilution integrity;
• evaluation of minimal values of analyte and internal standard (IS) response;
• stability;
• back-conversion of metabolites, if applicable.

Development and validation of analytical procedure is performed by the experts of Quinta-Analytica Yaroslavl LLC individually for each study.

Particular attention is paid to the following parameters of analytical procedure:

• appropriate method of sample preparation: protein precipitation, solid phase extraction, liquid-liquid extraction, depending on target concentration range and physical and chemical characteristics of analytes;
• selection of chromatographic columns and components of mobile phase providing best analytical characteristics (sensitivity, selectivity, linearity etc.), including robustness of procedure under its long-term use for analysis of large number of samples; two-dimensional chromatographic methods are preferred;
• selection of conditions for detection: type of ionization (electrospray ionization (ESI) with thermal focusing, atmospheric pressure chemical ionization (APCI), dual ion source (DUIS)); selection of MRM-transitions providing best sensitivity and selectivity of procedure;
• evaluation of effect of biological matrix components on efficacy of analyte ionization, possibility to minimize matrix effect;
• selection of internal standard that provides maximum accuracy of quantitative determination, and that is identical to analyte in physical and chemical characteristics and differs from the analyte by mass spectrum only;
• if in the process of the analyte’s biotransformation produce and can convert back into parent compound during storage of samples or during analytical procedures (glucuronides, N-oxides, lactones etc.), then special precautions are taken to minimize back-conversion and eliminate the influence of this process on the results obtained;
• evaluation of stability of analyte in biological matrix and in solutions; stability testing of analyte in biological matrix (short-term stability, long-term stability in two temperature settings: up to –20°С and up to –70°С, stability upon freezing/thawing) is compulsory before the clinical part of the study; if instability of the analyte is detected, stabilization procedure is developed and special conditions for analytical procedures and sample storage are selected;
• in case if combination pharmaceutical forms are tested, independent validation tests are performed to evaluate the effect of combined presence of several substances in analyte samples on the results obtained.