Enhanced high-performance liquid chromatography method for the determination of retinoic acid in plasma. Development, optimization and validation.
When determining endogenous compounds in biological samples, the lack of blank or analyte-free matrix samples involves the use of alternative strategies for calibration and quantitation. This article deals with the development, optimization and validation of a high performance liquid chromatography method for the determination of retinoic acid in plasma, obtaining at the same time information about its isomers, taking into account the basal concentration of these endobiotica. An experimental design was used for the optimization of three variables: mobile phase composition, flow rate and column temperature through a central composite design. Four responses were selected for optimization purposes (area under the peaks, quantity of peaks, analysis time and resolution between the first principal peak and the following one). The optimum conditions resulted in a mobile phase consisting of methanol 83.4% (v/v), acetonitrile 0.6% (v/v) and acid aqueous solution 16.0% (v/v); flow rate of 0.68 mL min(-1) and an column temperature of 37.10 °C. Detection was performed at 350 nm by a diode array detector. The method was validated following a holistic approach that included not only the classical parameters related to method performance but also the robustness and the expected proportion of acceptable results lying inside predefined acceptability intervals, i.e., the uncertainty of measurements. The method validation results indicated a high selectivity and good precision characteristics that were studied at four concentration levels, with RSD less than 5.0% for retinoic acid (less than 7.5% for the LOQ concentration level), in intra and inter-assay precision studies. Linearity was proved for a range from 0.00489 to 15.109 ng mL(-1) of retinoic acid and the recovery, which was studied at four different fortification levels in phuman plasma samples, varied from 99.5% to 106.5% for retinoic acid. The applicability of the method was demonstrated by determining retinoic acid and obtaining information about its isomers in human and frog plasma samples from different origins.
Laboratorio de Desarrollo Analítico y Quimiometría (LADAQ), Cátedra de Química Analítica I, Facultad de Bioquímica y Ciencias Biológicas, Universidad Nacional del Litoral-CONICET, C.C. 242, S3000ZAA Santa Fe, Argentina.
Carla M
CM
Teglia
Departamento de Química y Física, Área de Química Analítica, Campus de Excelencia Internacional Agroalimentario CeiA3, Universidad de Almería, 04120 La Cañada de San Urbano, Almería, Spain.
María D
MD
Gil García
Departamento de Química y Física, Área de Química Analítica, Campus de Excelencia Internacional Agroalimentario CeiA3, Universidad de Almería, 04120 La Cañada de San Urbano, Almería, Spain.
María Martínez
MM
Galera
Laboratorio de Desarrollo Analítico y Quimiometría (LADAQ), Cátedra de Química Analítica I, Facultad de Bioquímica y Ciencias Biológicas, Universidad Nacional del Litoral-CONICET, C.C. 242, S3000ZAA Santa Fe, Argentina. Electronic address: hgoico@fbcb.unl.edu.ar.
Héctor C
HC
Goicoechea
D000818:Animals; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006801:Humans; D007536:Isomerism; D057230:Limit of Detection; D015203:Reproducibility of Results; D014212:Tretinoin / Q000097:blood* / Q000737:chemistry
D014212:Tretinoin
Endogenous compounds; Isomers; Plasma; Retinoic acid; Validation