Procedure for the Quantitative Determination of Mixtures of Nucleic Acid Components Based on Multivariate Spectrophotometric Acid−Base Titrations

Abstract

A new procedure for the quantitative determination of mixtures of nucleic acid components, based on continuous spectrophotometric acid-base titrations and multivariate curve resolution, is proposed. The procedure simultaneously takes into account the spectroscopic and acid-base properties of the compounds, which leads to a higher selectivity. Furthermore, quantitative determination of an analyte in a complex mixture is performed using a synthetic solution as standard containing only the analyte of interest. An intrinsic difficulty in the analysis of spectrometric titration data is the presence of rank deficiency due to closure for the mixtures of two or more compounds. An additional problem can be encountered in some mixtures if species spectra or species concentration profiles are practically identical (rank overlap). However, even in the presence of these rank difficulties, accurate quantitation with prediction errors lower than 5% was obtained. The presence of unknown and uncalibrated interferences in the samples does not affect the quantitative determination of the analyte of interest. The proposed procedure was successfully applied to the analysis of real samples (pharmaceuticals) using synthetic external standards.

Resolution of overlapped peaks of amino acid derivatives in capillary electrophoresis using multivariate curve resolution based on alternating least squares

“…However, the resolution of such a system was not satisfactory. This is a common feature in other examples using MCR-ALS [34,41]. The unsuccessful resolution of that augmented matrix was attributed to the strong overlap of data.…”

“…This procedure has been successfully applied to other cases with strongly overlapped data [41]. The idea behind rank augmentation by this matrix augmentation was that the CE-DAD data for the standard was mathematically detected to be different to the CE-DAD data of the mixture (in a certain way, differences for the components of the mixture were enhanced by the addition of the standard).…”

“…Even in these cases, the augmented matrices obtained were still rankdeficient. Although rank deficiency hinders the complete resolution of all chemical components present, it does not affect the resolution of the analyte of interest [41].…”

A second-order standard addition method based on the data treatment by calculation of variation matrix of kinetic systems analyzed by MCR-ALS

“…Moreover, the second-order algorithms avoid the need of using a large number of calibration sample since a single calibration sample, that contains the analyte of interest, is sufficient. 8 Suitable algorithms for analyzing second-order data are parallel factor analysis (PARAFAC), 9 the generalized rank annihilation method (GRAM), 10 rank annihilation factor analysis (RAFA), 11,12 direct trilinear decomposition (DTLD), 13 bilinear least squares (BLLS), 14 alternating trilinear decomposition (ATLD) 15 and its variants (self-weighted alternating trilinear decomposition (SWATLD), 16 alternating penalty trilinear decomposition (APTLD) 17 and multivariate curve resolution-alternating least squares (MCR-ALS). 18 It was shown that MCR-ALS is a powerful tool for the species resolution and the quantitative determination of many types of unresolved chemical mixtures especially in kinetic systems.…”

“…21 Such situation occurs quite frequently when, for instance, at the beginning of a chemical reaction or process, more than one component already exists or parallel reactions take place. 21,8 This problem will also occur when the reaction of one of the component immediately takes place and produces a constant signal during the time range of the experiments. 21 Variation matrix is one of the concepts proposed for handling the rank deficiency problem in parallel reaction systems.…”