Chromatogram simulation by area reproduction

A modified Poisson function has been developed for the simulation of chromatographic peaks. The proposed model is shown to have the property of exactly recreating the experimentally determined peak area. Model parameters are obtained directly from the experimental peak, and overlapping peaks are deconvoluted such that the area sum of overlapping peaks is kept unchanged. The method was applied to real, complex chromatograms.     

Combined Kalman filtering and steepest descent minimization for quantitative analysis of unresolved misaligned chromatographic peaks. The resolution of alternariol and altenuisol mycotoxins in mixtures by HPLC

Summary The potential of a computational approach for the quantitative resolution of seriously overlapping chromatographic peaks when there is loss of collinearity between the pure component peaks and the mixture peak has been explored. The program makes iterative use of the Kalman filter algorithm for resolving the mixture peak with the component peaks aligned according to some values of the position parameters, and of a steepest descent minimization procedure to find the optimal alignment. This combined procedure has been applied to the quantitive resolution of the HPLC chromatograms of alternariol and altenuisol mycotoxins in synthetic mixtures and in real samples.     

Single- and multi-channel detection for generalized quantitative analysis in cases of unresolved chromatographic peaks

Computerized quantification of components under overlapping chromatographic peaks is done by calibration of chromatograms against component mixtures. For conventional (single-channel) detectors, the limitations of earlier methods based on ordinary multiple regression, can be circumvented by data reduction with the aid of principal component analysis with the partial least-squares approach. Simulation studies show that the method can be applied even when there is severe peak overlap, unstable baseline, noisy chromatograms or non-linear detector response. Advantages in the quantification of fused peaks by means of multichannel detectors are outlined. Present limitations on the quantitative evaluation of several overlapping component peaks from a single spectro-chromatogram by means of the partial least-squares method combined with multiple regression on the pure component spectra, are discussed with respect to practical high-performance liquid chromatography.     

Software for Microprocessor Controlled HPLC: Peak Area Integration

Abstract

Programs are described for determination of peak areas and peak retention times from the chromatographic data obtained by a dual-microprocessor data handling microcomputer (DHC). The programs provide the necessary equipment testing and calibration routines for an accurate reproduction of a recorded chromatogram, and they are written to be merged with the data acquisition programs to provide a true “real-time” integrator. The integration is performed with baseline stabilization and automatic peak splitting. These features make the integrator applicable to gradient elution chromatography, as well as for the integration of complex chromatograms with overlapping peaks. The integrated chromatogram can be displayed with the limits of integration for each peak. Results of peak area integration of simple and complex chromatograms demonstrate satisfactorily accurate and consistant results that are independent of chromatographic conditions and shape of the peaks.     

Colachrom,a command language for chromatographic data processing

A command language for the interactive computer processing of chromatograms is described. Report data files, resulting from a peak evaluation program, are processed with single-line commands. Reference peaks for quantitative evaluation or for retention index calculations can be specified, compound names or response factors be assigned, etc. Customer-specific reports can be generated. Several commands operate on the unreduced digitized chromatograms, e.g., repeat peak evaluation with modified peak search parameters or with a user-corrected baseline. Commands can be combined into procedures for semi-automatic processing of series of chromatograms. Various forms of interactive graphical presentations supplement the command language.     

Quantitative Auswertung von Gas-Chromatogrammen partiell getrennter Stoffgemisch

A simple method is described for the qualitative and quantitative analysis of gas chromatograms with considerably overlapping peaks, based on the assumption of known retention indices and peak shapes. Its application to mixtures of deuteriated hydrocarbons is indicated. The extent to which the results are reproducible is shown to be about the same as that of high-resolution gas chromatography or mass spectrometry.     

Introducing HYPERMET-PC for automatic analysis of complex gamma-ray spectra

In short time activation analysis prompt gamma-activation analysis and in high rate -ray spectroscopy in general, the shape parameters for peaks and back ground usually vary, rendering spectrum evaluation codes based on a fixed shape calibration unsuitable. An interactive version of the well-known, fully automatic -ray spectrum analysis code HYPERMET has been developed in C ++ for the IBM-PC. It runs under MS-DOS, in conventional memory, and can handle up to 16k-channel spectra, recorded with CANBERRA's System 100 and AccuSpec and with ORTEC's ACE plug-in MCA cards. A Windows-like graphics environment is provided with mouse controlled pull-down menus, pop-up windows and rubber band expansion. All basic features of HYPERMET such as fully automatic peak search, nonlinear fitting of multiplets with automatically adjusted Gaussian peak widths exponential tails and a complex background function have been retained. All details of the fitting procedure are recorded in a data base, hence any fitted region can be retrieved and modified interactively, even after a fully automatic spectrum evaluation. The program also provides an output peak list in SAMPO90 format for further processing. The latter format is widely used in a number of sample analysis programs such as KAYZERO a software package fork ₀ standardization in neutron activation analysis.     

Accurate mass filtering of ion chromatograms for metabolite identification using a unit mass resolution liquid chromatography/mass spectrometry system

Acceleration of liquid chromatography/mass spectrometric (LC/MS) analysis for metabolite identification critically relies on effective data processing since the rate of data acquisition is much faster than the rate of data mining. The rapid and accurate identification of metabolite peaks from complex LC/MS data is a key component to speeding up the process. Current approaches routinely use selected ion chromatograms that can suffer severely from matrix effects. This paper describes a new method to automatically extract and filter metabolite-related information from LC/MS data obtained at unit mass resolution in the presence of complex biological matrices. This approach is illustrated by LC/MS analysis of the metabolites of verapamil from a rat microsome incubation spiked with biological matrix (bile). MS data were acquired in profile mode on a unit mass resolution triple-quadrupole instrument, externally calibrated using a unique procedure that corrects for both mass axis and mass spectral peak shape to facilitate metabolite identification with high mass accuracy. Through the double-filtering effects of accurate mass and isotope profile, conventional extracted ion chromatograms corresponding to the parent drug (verapamil at m/z 455), demethylated verapamil (m/z 441), and dealkylated verapamil (m/z 291), that contained substantial false-positive peaks, were simplified into chromatograms that are substantially free from matrix interferences. These filtered chromatograms approach what would have been obtained by using a radioactivity detector to detect radio-labeled metabolites of interest.     

Parametric studies of matched filters to enhance the signal-to-noise ratios of LC-MS-MS peaks

Chromatographic parameters of reference signals employed in matched filter methods have been studied using numerical experiments to improve the signal-to-noise (S/N) ratios of small liquid chromatography (LC) peaks obtained with electrospray tandem mass spectrometers (MS-MS). These parameters include the width, shape, and S/N ratios of chromatographic peaks used as the reference signal profiles. Our results show the effect of reference peak widths on improving the S/N ratio of chromatographic peaks; the influence of reference peak shapes is negligible. To verify simulation results, various reference signals, including analyte peaks of high concentration standards, internal standard peaks, and artificial Gaussian peaks of different widths, have been employed to enhance signal peaks on real liquid chromatography-tandem mass spectrometry (LC-MS-MS) chromatograms via matched filter methods. Our experimental results demonstrate that the S/N ratio enhancement of chromatographic peaks agree with the simulation predictions. These findings, therefore, suggest that regardless of peak shape, a well-smooth peak with a width close to that of the analyte peak is an adequate reference signal, when matched filter methods are used to improve LC-MS-MS chromatograms. Nevertheless, all methods processed LC-MS-MS peaks in this study do not achieve the ideal improvement ratios estimated with simulation results. We attribute this deficiency to spike-like noise, which have considerable low frequency components riding on LC-MS-MS chromatograms. Matched filtering, which works as a low-pass filter in the frequency domain, cannot effectively eliminate low frequency flicker noise contributed by these spikes. In addition, simple median filtering does not provide adequate improvement despite being able to smooth out most spikes in the chromatograms.     

DSC examination of alloys

The formation and thermal dissolution of dispersed particles was studied in aluminium alloys. It was found that only high heating rates (80°C min⁻¹) could provide DSC curves characteristic of the phase structure of the samples. The kinetic evaluation of the DSC curves was carried out by least squares curve fitting. In this way reasonable kinetic parameters and reliable peak resolution could be obtained for the overlapping peaks.     

Influence of the peak height distribution on separation performances: Discrimination factor and effective peak capacity

Summary A new index of performance of the chromatographic separation between two adjacent peaks, the discrimination factor, d₀, is defined. It is normalized between 0 and 1 and is directly and easily determined from the chromatogram. It does not depend on any assumption regarding peak shape, except that the peak profiles of individual sample components have a single mode. Its value depends on the relative heights of the two peaks as well as on their separation. The separation power of a chromatographic system is classically measured by its peak capacity, defined on the basis of constant resolution between adjacent peaks. A previously developed statistical theory of the composition of mixtures makes it possible to extend the concept of peak capacity by taking into account the peak height distribution in typical average chromatograms. A new parameter, the effective peak capacity, is defined for this purpose on the basis of a constant discrimination factor between adjacent peaks. It allows to take into account the distribution of peak heights in statistical theories of the evaluation of complex chromatograms and in the measurement of the limit of determination in quantitative analysis. The characteristics of the two new parameters, the discrimination factor and effective peak capacity, are discussed and compared with those of their classical homologs, resolution and peak capacity, in the case of gaussian component peaks of equal widths.     

Optimisation of data handling and detection conditions for automated chromatographic assay software

Summary The principle of automated chromatographic peak detection and analysis software is summarized, and critical steps are systematically studied. As the only parameter to be entered is the acquisition frequency, evaluation of its effect on software performance is discussed. In the case of relatively noisy chromatograms, it is shown experimentally that numerous points per peak have to be taken, leading to quite fast computer acquisition procedures. The use of discrete Fourier transform filtration techniques can modify peak shapes and a comparative study evaluates the relative errors induced in the shapes and characteristics of the chromatographic profiles. Optimisation of filtering conditions is achieved and it is shown that for a filter position only 2% of the Nyquist frequency no deformation occurs in the chromatographic profile. Detection of the start and finish of chromatographic peaks is optimized according to a simple four step iterative procedure. In the case of simulations, the difference between the values used to simulate peaks and those calculated by the software are less than 1%.     

小波变换用于重叠色谱峰组分信息的提取

提出了一个小波变换用于提取重叠色谱中组分信息的理论公式，并将它应用于三组分和五组分重叠色谱实验数据的解析。结果表明：在定定分离度范围内，当相邻色谱峰峰宽相近时，根据此公式的计算结果，可以将各组分的信息从重叠色谱峰信号中提取出来。洒工作对小波变换用于重叠峰的解析具有重要意义。     

Deconvolution method for accurate determination of overlapping peak areas in chromatograms.

A method is described for deconvoluting chromatograms which contain overlapping peaks. Parameters can be selected to ensure that attenuation of peak areas is uniform over any desired range of peak widths. A simple extension of the method greatly reduces the negative overshoot frequently encountered with deconvolutions. The deconvoluted chromatograms are suitable for integration by conventional methods.     

A new approach to data reduction and evaluation in high-resolution time-of-flight mass spectrometry using a time-to-digital convertor data-recording system

A new effective and robust approach to the detection of incompletely resolved peaks, and evaluation of their parameters in high-resolution time-of-flight mass spectra for time-to-digital convertor (TDC) data acquisition mode, is described. The method is based on fast construction of a smoothed continuous curve that approximates the initial data (transformed to a constant relative width of time intervals for ion counting) with respect to precision of measurements. The first derivative of this curve is used for correction of skewness of the peak shape as far as possible. A contribution of the second derivative is subtracted from the smoothed curve for better resolution of partially resolved peaks. The comparison of local maxima of this resulting final curve with those for the initial smoothed curve allows reliable detection of the peaks and to test whether or not they are spoiled by overlapping. Ion counting performed by TDC gives an opportunity to estimate standard deviations of peak locations and their intensities. These values proved to be close to theoretically minimal standard deviations for these parameters for single fully resolved peaks. Thus, estimates of the main parameters of mass peaks by the described method are close to statistically efficient estimators for these parameters.     

Monolithic silica columns for high-efficiency chromatographic separations

A deconvolution methodology for overlapped chromatographic signals is proposed. Several single-wavelength chromatograms of binary mixtures, obtained in different runs at diverse concentration ratios of the individual components, were simultaneously processed (multi-batch approach), after being arranged as two-way data. The chromatograms were modelled as linear combinations of forced peak profiles according to a polynomially modified Gaussian equation. The fitting was performed with a previously reported hybrid genetic algorithm with local search, leaving all model parameters free. The approach yielded more accurate solutions than those found when each experimental chromatogram was fitted independently to the peak model (single-batch approach). The improvement was especially significant for those chromatograms where the peaks were severely affected by the tails of the preceding compounds. Peak shifts among chromatograms, which are a usual source of non-bilinearity, were modelled in a continuous domain instead of in a discrete way, which avoided some drawbacks associated with latent variable methods. An experimental design involving simulated chromatograms was applied to check the method performance. Five main factors affecting the deconvolution were examined: concentration pattern, chromatographic resolution, number of batches and replicates, and noise level, which were evaluated using first- and second-order figures of merit. The method was also tested on three real samples containing compounds showing different overlap. Four multi-batch deconvolution methods were considered differing in the nature of the processed information and kind of peak matching among chromatograms. In all cases, the multi-batch deconvolution yielded better performance than the single-batch approach.     

Qualitative evaluation of chromatographic data from quality control schemes using a support vector machine

The qualitative evaluation of chromatographic data in the framework of external quality assurance schemes is considered in this paper. The homogeneity in the evaluation of chromatographic data among human experts in samples with analytes close to the limit of detection of analytical methods was examined and also a Support Vector Machine (SVM) was developed as an alternative to experts for a more homogeneous and automatic evaluation. A set of 105 ion chromatograms obtained by anti-doping control laboratories was used in this study. The quality of the ion chromatograms was evaluated qualitatively by nine independent experts (associating a score from 0 to 4) and also more objectively taking into account chromatographic parameters (peak width, asymmetry, resolution and S/N ratio). Results obtained showed a high degree of variability among experts when judging ion chromatograms. Experts applying extremely outlying evaluation criteria were identified and excluded from the data used to develop the SVM. This machine was built providing the system with qualitative information (scores assigned by experts) and with objective data (parameters) of the ion chromatograms. A seven-fold cross-validation approach was used to train and to evaluate the predictive ability of the machine. According to the results obtained, the SVM developed was found to be close to the reasoning process followed by the homogeneous human expert group. This machine also could provide a scoring system to sort laboratories according to the quality of their results. The qualitative evaluation of analytical records using a scoring system allowed the identification of the main factors affecting the quality of chromatographic analytical data, such as the specific analytical technique applied and the adherence to guidelines for reporting positive results.     

Assessment of the results from data processing systems using a digital chromatogram simulator

A digital chromatogram simulator has been used to evaluate the performance of data handling systems. The simulator can synthesize chromatograms with any desired shape from a library of peaks based on real peak shapes. Once generated, the chromatograms can be reproduced exactly any number of times. The algorithms used in data handling systems have a significant effect on the results they produce. An object of this work was to examine how well the true size of a peak was reported when the peak was detected overlapping with other peaks. A series of experiments was therefore performed in which peaks were overlapped by different amounts and in different situations. The reported results from the data systems were compared with the results which would have been reported had the peaks been fully resolved. A significant difference was found between the overlap results and the fully resolved results, which did not have a linear relationship with the parameters tested. The chromatogram simulator was found to be a powerful tool in assessing the performance of the data systems. It offers a valuable means of evaluating the quality of the results produced by these systems.     

Resolution of Overlapping Chromatographic Peaks Using a Genetic Algorithm

ABSTRACT

A genetic algorithm for resolution of overlapping chromatographic peaks (GAROCP) using real-number coding, non-uniform mutation and arithmetical crossover methods is described in this paper. It was applied to resolution of highly overlapped multicomponent high-performance liquid chromatographic peaks by fitting experimental chromatogram to the exponentially modified Gaussian (EMG) model. The genetic algorithm was used to find the minimum of fitting error to optimize the parameters in the EMG functions which determine the shape and area of each peak. The applicability of the method was investigated with both simulated signals calculated by EMG functions and experimental multicomponent overlapping chromatograms.