Comparison of chemometric methods for the screening of comprehensive two-dimensional liquid chromatographic analysis of wine.

Two chemometric methods are compared for the rapid screening of comprehensive two-dimensional liquid chromatographic (LC×LC) analysis of wine. The similarity index and Fisher ratio methods were both found to be able to distinguish geographical variability and to determine potentially significant peaks for further quantitative and qualitative study. An experimental data set consisting of five different wine samples and multiple simulated data sets were analyzed in the investigation of the screening methods. Several statistical analyses are employed in the understanding and verification of the results from the similarity index and Fisher ratio methods. The sum rank difference (SRD) method was used to compare the rankings of the two different methods as applied to the different data sets and to determine the amount of variability associated with the ranking of the peak differences. The major advantage the similarity index method offers is that it is an unsupervised method; no a priori knowledge of the samples (i.e., class identification) is required, while the Fisher ratio method is supervised. Both methods are rapid and require little user intervention other than the determination of a threshold for inclusion/exclusion of compounds from further analysis.

Chemometric analysis of targeted 3DLC-DAD data for accurate and precise quantification of phenytoin in wastewater samples.

A variety of pharmaceuticals have been found in various water systems, including wastewater treatment effluent. Due to the possible environmental and human health implications, it is important to be able to quickly and reliably quantify the amount of pharmaceuticals and personal care products that may be present in such samples. To this end, a new chromatographic analysis technique involving three dimensions of liquid chromatography, including selective comprehensive separations in the second and third dimensions, was applied to the analysis of a wastewater treatment plant effluent (WWTPE) sample using both standard addition and external calibrations. Iterative key set factor analysis alternating least squares with the application of both sample and spectral selectivity constraints was used to resolve the phenytoin peak at a concentration corresponding to about 40 parts-per-trillion using UV absorbance detection. Both the precision and accuracy of the method are investigated. We determined that the concentration of phenytoin in WWTPE using selective three dimensional liquid chromatography with diode array detection was 42 ± 1 ng/L, after resolution from an unknown interferent. The estimated concentration was not significantly different from that obtained by the reference 2DLC/MS/MS method, but was four and a half times more precise.

Factors that affect quantification of diode array data in comprehensive two-dimensional liquid chromatography using chemometric data analysis.

There has been a tremendous increase in research on comprehensive two dimensional LC (LC×LC); however, to date, the central analytical issue, quantification, has received only minimal attention. It is vital to the further development of LC×LC that a greater understanding of the specific factors affecting peak quantification in LC×LC be attained. This work focuses on the following factors: data complexity, retention time shifting, dynamic range issues, chromatographic and spectral peak overlap and difficulties related to background signal removal. The above mentioned factors that affect peak quantification are investigated using fourteen replicate analyses of a urine sample, representing the effects of such factors when analyzing samples in complex matrices. We demonstrate that quantification of LC×LC data is improved following implementation of chemometric techniques that minimized the deleterious effects on quantification due to chromatographically overlapped peaks, retention time shifting and background signal interference. The chemometrically resolved data shows a 2.5-fold increase in precision of quantification over the quantification of the raw data. It is also demonstrated that the method quantifies sixteen peaks that were not visually evident prior to chemometric analysis. The purpose of this paper is to determine the impact of these issues on the effectiveness of LC×LC as a technique for the quantitative analysis of complex samples.

Chemometric Resolution and Quantification of Four-Way Data Arising from Comprehensive 2D-LC-DAD Analysis of Human Urine.

Two-dimensional liquid chromatography (LC×LC) is quickly becoming an important technique for the analysis of complex samples, owing largely to the relatively high peak capacities attainable by this analytical technique. With the increase in the complexity of the sample comes a corresponding increase in the complexity of the collected data. Thus the need for chemometric methods capable of resolving and quantifying such data is ever more urgent in order to obtain the maximum information available from the data. To this end, we have developed a chemometric method that combines iterative key set factor analysis and multivariate curve resolution-alternating least squares analysis with a spectral selectivity constraint that is shown to be capable of resolving chromatographically rank deficient, non-multilinear data. (Spectrally rank deficient compounds can only be quantified if the peaks having the same spectra are chromatographically resolved.) Over 50 chromatographic peaks were found in a relatively small section of a LC×LC-diode array data set of replicate urine samples (a four-way data set) using the developed method. The relative concentrations for 34 of the 50 peaks were determined with % RSD values ranging from 0.09 % to 16 %.