O5 CHEM2 Amante Eleonora - Bari Analitica 2019barianalitica2019.it/doc/O5...
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XXVIII Congress of the Analytical Chemistry Division Bari 22 – 26 September 2019 O5 CHEM2 EFFECTIVE VALIDATION OF CHROMATOGRAPHIC ANALYTICAL METHODS E. Amante 1,2 , E. Alladio 1,2 , F. Seganti 2 , A. Salomone 1,2 , M. Vincenti 1,2 , B. Desharnais 3 1 Dipartimento di Chimica, Università degli Studi di Torino, Torino, Italy 2 Centro Regionale Antidoping “A. Bertinaria”, Orbassano (TO), Italy 3 Laboratoire de sciences judiciaires et de médecine légale, Montréal, Québec, Canada The validation of an analytical method is mandatory to assess its reliability. At the present, there is not an official validation procedure, despite several organizations and scientists have tried to standardize the protocol. According to the purpose of the analysis (e.g., qualitative, quantitative) and the application field, the evaluation of specific parameters is recommended [1,2]. For the validation of quantitative methods, a feature of utmost importance is represented by the calibration. Although most instrumental systems should theoretically supply a straight correlation between concentrations and analytical signals, in reality some interfering physical and chemical phenomena may result in a deviation from the expected linear trend and/or heterogeneous distribution of data-point at different concentration levels [3]. The occurrence of heteroscedasticity is responsible of the erroneous evaluation of other parameters, among which the limit of detection (LOD). In our daily work with biological matrices, LC-MS/MS and GC-MS-based methods are continuously developed and/or updated to support the ongoing evolution of clinical and toxicological requirements. This changing scenario opens further analytical inquiries not addressed in standardized validation procedures, including practical questions that frequently need careful consideration, including (i) for how long is a calibration curve valid and reliable? (ii) Should it be tested before each working section? (iii) Is heteroscedasticity distributed along the whole range of calibration, or is it peculiar only of the highest portion of the calibration range? (iii) Is it sufficient to evaluate the validation parameters only at the lower, middle and upper levels or is it safer to consider all the calibration points? (iv) Whenever deviation from linearity occurs at the high concentration range, is it preferable to adopt a quadratic calibration model or to split the curve into two linear segments? To answer these questions, we developed an efficient and rigorous validation protocol. In practice, our approach consists in the systematic measurement of three replicates of the calibration curve in three different days. It allows the robust computation of the calibration curve (taking into consideration the heteroscedasticity and the contribution of the quadratic term), limit of detection (LOD), intra- and inter-day precision and accuracy, ion abundance repeatability, selectivity, specificity and carry over (Figure 1). Few further experiments are required for the evaluation of matrix effect, extraction recovery and retention time repeatability. The computations are performed employing the R routine developed by Desharnais et al. [4,5] and in-home built Excel sheets.
Transcript of O5 CHEM2 Amante Eleonora - Bari Analitica 2019barianalitica2019.it/doc/O5...
XXVIII Congress of the Analytical Chemistry Division Bari 22 – 26 September 2019
O5 CHEM2
EFFECTIVE VALIDATION OF CHROMATOGRAPHIC ANALYTICAL METHODS
E. Amante1,2, E. Alladio1,2, F. Seganti2, A. Salomone1,2, M. Vincenti1,2, B. Desharnais3 1Dipartimento di Chimica, Università degli Studi di Torino, Torino, Italy 2Centro Regionale Antidoping “A. Bertinaria”, Orbassano (TO), Italy 3Laboratoire de sciences judiciaires et de médecine légale, Montréal, Québec, Canada The validation of an analytical method is mandatory to assess its reliability. At the present, there is not an official validation procedure, despite several organizations and scientists have tried to standardize the protocol. According to the purpose of the analysis (e.g., qualitative, quantitative) and the application field, the evaluation of specific parameters is recommended [1,2]. For the validation of quantitative methods, a feature of utmost importance is represented by the calibration. Although most instrumental systems should theoretically supply a straight correlation between concentrations and analytical signals, in reality some interfering physical and chemical phenomena may result in a deviation from the expected linear trend and/or heterogeneous distribution of data-point at different concentration levels [3]. The occurrence of heteroscedasticity is responsible of the erroneous evaluation of other parameters, among which the limit of detection (LOD). In our daily work with biological matrices, LC-MS/MS and GC-MS-based methods are continuously developed and/or updated to support the ongoing evolution of clinical and toxicological requirements. This changing scenario opens further analytical inquiries not addressed in standardized validation procedures, including practical questions that frequently need careful consideration, including (i) for how long is a calibration curve valid and reliable? (ii) Should it be tested before each working section? (iii) Is heteroscedasticity distributed along the whole range of calibration, or is it peculiar only of the highest portion of the calibration range? (iii) Is it sufficient to evaluate the validation parameters only at the lower, middle and upper levels or is it safer to consider all the calibration points? (iv) Whenever deviation from linearity occurs at the high concentration range, is it preferable to adopt a quadratic calibration model or to split the curve into two linear segments? To answer these questions, we developed an efficient and rigorous validation protocol. In practice, our approach consists in the systematic measurement of three replicates of the calibration curve in three different days. It allows the robust computation of the calibration curve (taking into consideration the heteroscedasticity and the contribution of the quadratic term), limit of detection (LOD), intra- and inter-day precision and accuracy, ion abundance repeatability, selectivity, specificity and carry over (Figure 1). Few further experiments are required for the evaluation of matrix effect, extraction recovery and retention time repeatability. The computations are performed employing the R routine developed by Desharnais et al. [4,5] and in-home built Excel sheets.
XXVIII Congress of the Analytical Chemistry Division Bari 22 – 26 September 2019
O5 CHEM2
Figure 1. Scheme representing the validation protocol
References [1] Shabir G. and J. Valid. Technol.,2004, (10) 210. [2] Taverniers I., De Loose M. and Van Bockstaele E., TrAC - Trends Anal. Chem., 2004, (23) 535. [3] Sayago A. and Asuero A.G., Crit. Rev. Anal. Chem., 2004, (34) 133. [4] Desharnais B., Camirand-Lemyre F., Mireault P. and Skinner C.D., J. Anal. Toxicol., 2017, (41) 261. [5] Desharnais B., Camirand-Lemyre F., Mireault P. and Skinner C.D., J. Anal. Toxicol., 2017, (41) 269.
