Correction of retention time shifts for chromatographic fingerprints of herbal medicines

In this study, the combination of chemometric resolution and cubic spline data interpolation was investigated as a method to correct the retention time shifts for chromatographic fingerprints of herbal medicines obtained by high-performance liquid chromatography-diode array detection (HPLC-DAD). With the help of the resolution approaches in chemometrics, it was easy to identify the purity of chromatographic peak clusters and then resolve the two-dimensional response matrix into chromatograms and spectra of pure chemical components so as to select multiple mark compounds involved in chromatographic fingerprints. With these mark components determined, the retention time shifts of chromatographic fingerprints might be then corrected effectively. After this correction, the cubic spline interpolation technique was then used to reconstruct new chromatographic fingerprints. The results in this work showed that, the purity identification of the chromatographic peak clusters together with the resolution of overlapping peaks into pure chromatograms and spectra by means of chemometric approaches could provide the sufficient chromatographic and spectral information for selecting multiple mark compounds to correct the retention time shifts. The cubic spline data interpolation technique was user-friendly to the reconstruction of new chromatographic fingerprints with correction. The successful application to the simulated and real chromatographic fingerprints of two Cortex cinnamomi, fifty Rhizoma chuanxiong, ten Radix angelicae and seventeen Herba menthae samples from different sources demonstrated the reliability and applicability of the approach investigated in this work. Pattern recognition based on principal component analysis for identifying inhomogenity in chromatographic fingerprints from real herbal medicines could further interpret it.     

Validation of a chromatography data analysis software

The performance of chromatography data analysis software packages is of cardinal importance when the precision and the accuracy of a chromatographic system are evaluated. Users cannot rely on a procedure generating chromatographic data of known accuracy. Holistic approaches cannot always be entirely trusted. We propose a new method consisting in validating a data analysis package against computer generated chromatograms of exactly known characteristics by feeding these chromatograms into the vendor supplied software and comparing the results supplied by the software and the exact answers. We simulated symmetrical and tailing chromatograms and processed these signals with the Agilent Technologies (formerly Hewlett-Packard) ChemStation software. The noise profile (i.e. the power spectrum of the baseline) was determined for a HPLC UV detector prior to the calculations, and chromatograms of different signal-to-noise ratios were used for the analysis. For every chromatogram, we simulated 25 replicates with identical signal-to-noise ratios but different noise sequences. In this manner, both the random and the systematic errors of the retention data and peak shape characteristics can be evaluated. When analyzing tailing peaks, we simulated the effects of extra-column band broadening and those of column overload. Our calculations show that the general performance of the data analysis system studied is excellent. The contribution of the random error originating from the data analysis procedure is in most cases negligible compared to the repeatability of the chromatographic measurement itself.     

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.     

An information-based computational technique for estimation of chromatographic peak purity

Assessment of the purity of chromatographic peaks is an important step in developing and validating purification procedures for complex mixtures. While curve-fitting techniques can be useful for determining the retention times and relative concentrations of the components of a chromatographic peak, their utility is limited by the lack of unambiguous criteria for determining the number of such components. In this work, we present a computational technique for analyzing chromatograms to estimate the number of components, their retention times, and their relative concentrations. In contrast to Fourier-transform-based techniques, the technique we present does not require manual peak identification. It is based on curve-fitting and uses the Akaike information criterion to estimate the number of components. Application of the technique to chromatograms obtained from size-exclusion and reverse-phase chromatography of test mixtures indicates that it is useful for the characterization of complex mixtures.     

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.     

Chemometric quality control of chromatographic purity

It is common practice in chromatographic purity analysis of pharmaceutical manufacturing processes to assess the quality of peak integration combined by visual investigation of the chromatogram. This traditional method of visual chromatographic comparison is simple, but is very subjective, laborious and seldom very quantitative. For high-purity drugs it would be particularly difficult to detect the occurrence of an unknown impurity co-eluting with the target compound, which is present in excess compared to any impurity. We hypothesize that this can be achieved through Multivariate Statistical Process Control (MSPC) based on principal component analysis (PCA) modeling. In order to obtain the lowest detection limit, different chromatographic data preprocessing methods such as time alignment, baseline correction and scaling are applied. Historical high performance liquid chromatography (HPLC) chromatograms from a biopharmaceutical in-process analysis are used to build a normal operation condition (NOC) PCA model. Chromatograms added simulated 0.1% impurities with varied resolutions are exposed to the NOC model and monitored with MSPC charts. This study demonstrates that MSPC based on PCA applied on chromatographic purity analysis is a powerful tool for monitoring subtle changes in the chromatographic pattern, providing clear diagnostics of subtly deviating chromatograms. The procedure described in this study can be implemented and operated as the HPLC analysis runs according to the process analytical technology (PAT) concept aiming for real-time release.     

Automated mass analysis of low-molecular-mass bacterial proteome by liquid chromatography-electrospray ionization mass spectrometry

Due to the presence of a large number of proteins in cell extracts, ion chromatograms of cell extracts obtained by electrospray ionization mass spectrometry (ESI-MS) can be quite complicated. It is found that the elevated baseline in an ion chromatogram contains many protein signals. One deficiency of current commercially available LC-ESI-MS data interpretation software is found to be the lack of functional operation that allows automated mass spectral integration and interpretation over signals hidden in the baseline. This current limitation can be overcome by a technique that involves the introduction of artificial pulses to an ion chromatogram by removing the solvent mixer in the HPLC pump. These artificial pulses are treated as chromatographic peaks by the software, thereby allowing automated spectral integration over the duration of a pulse. The reliability of mass analysis from the integrated spectra is shown to be dependent on spectral interpretation parameters such as mass spectral baseline threshold. The application of this method is demonstrated for rapid detection and mass analysis of low-molecular-mass proteins from cell extracts of Escherichia coli or Bacillus globigii.     

Three-dimensional ion mass chromatograms of oil hydrocarbon and heteroatomic compound types

The type composition of oil and oil products is usually determined by either the summation of all individuals of this compound type found from GC or GC/MS data or using appropriate generalized analytical features specific for a compound type as a whole. The specific representation of mass spectra of a complex mixture as a table of 14 homological series allows the analyst to visualize characteristic ion clusters specific for the compound types. These ion clusters form a “type mass spectrum” for each compound type. In the mass chromatograms of ions of a homologous ion series, these ion clusters form peculiar three-dimensional chromatographic peaks, whose width along the retention time axis corresponds to the isomer distribution for the homologue, molecular mass distribution (if molecular ions are considered), or structural features of the system of fused rings (for fragment ions) and “volume,” the concentration of the compound type. Three-dimensional chromatographic peaks for compound types are similar to usual peaks for individual compounds in ion mass chromatograms.     

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.     

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.     

Real-time target selection optimization to enhance alignment of gas chromatograms

An improved method for real-time selection of the target for the alignment of gas chromatographic data is described. Further outlined is a simple method to determine the accuracy of the alignment procedure. The target selection method proposed uses a moving window of aligned chromatograms to generate a target, herein referred to as the window target method (WTM). The WTM was initially tested using a series of 100 simulated chromatograms, and additionally evaluated using a series of 55 diesel fuel gas chromatograms obtained with four fuel samples. The WTM was evaluated via a comparison to a related method (the nearest neighbor method (NNM)). The results using the WTM with simulated chromatograms showed a significant improvement in the correlation coefficient and the accuracy of alignment when compared to the alignments performed using the NNM. A significant improvement in real-time alignment accuracy, as assessed by a correlation coefficient metric, was achieved with the WTM (starting at ∼1.0 and declining to only ∼0.985 for the 100th sample), relative to the NNM (starting at ∼1.0 and declining to ∼0.4 for the 100th sample) for the simulated chromatogram study. The results determined when using the WTM with the diesel fuels also showed an improvement in correlation coefficient and accuracy of the within-class alignments as compared to the results obtained from the NNM. In practice, the WTM could be applied to the real-time analysis of process and feedstock industrial streams to enable real-time decision making from the more precisely aligned chromatographic data.     

Characterization of silicones by direct-deposition GC/FT-IR

Materials used in the manufacture of silicone breast prostheses were analyzed by supercritical fluid chromatography (SFC) and high temperature gas chromatography (GC) coupled with direct deposition FT-IR detection. The chromatographic resolution and deposition quality were found to be unsatisfactory for SFC/FT-IR; however, high temperature GC/FT-IR produced excellent results. The chromatograms and spectra obtained allowed for the identification of poly-dimethyl silicones and mono-, di-, and tri-diphenylsubstituted poly-dimethyl silicones. An automated method of baseline correction was developed that allowed functional group chromatograms due to molecules containing Si-phenyl bonds to be constructed without significant interference from Si-methyl bonds despite the fact that the Si-C stretching mode of Si-phenyl bands overlapped significantly with the strong bands due to Si-CH₃ groups.     

Peak alignment using wavelet pattern matching and differential evolution

Retention time shifts badly impair qualitative or quantitative results of chemometric analyses when entire chromatographic data are used. Hence, chromatograms should be aligned to perform further analysis. Being inspired and motivated by this purpose, a practical and handy peak alignment method (alignDE) is proposed, implemented in this research for one-way chromatograms, which basically consists of five steps: (1) chromatogram lengths equalization using linear interpolation; (2) accurate peak pattern matching by continuous wavelet transform (CWT) with the Mexican Hat and Haar wavelets as its mother wavelets; (3) flexible baseline fitting utilizing penalized least squares; (4) peak clustering when gap of two peaks is smaller than a certain threshold; (5) peak alignment using differential evolution (DE) to maximize linear correlation coefficient between reference signal and signal to be aligned. This method is demonstrated with both simulated chromatograms and real chromatograms, for example, chromatograms of fungal extracts and Red Peony Root obtained by HPLC-DAD. It is implemented in R language and available as open source software to a broad range of chromatograph users (http://code.google.com/p/alignde).     

Novel inverse liquid chromatography detector configuration for studying solid-liquid adsorption

Inverse liquid chromatography (ILC) is a physicochemical chromatographic technique for studying solid-liquid interactions and surface properties of unknown solid materials. However, such studies are dependent on the ability to self pack the sample powder into a LC column in a reliable and robust manner. In practise, this is often difficult and anomalous chromatograms can be encountered quite frequently. Results obtained with a novel LC system configuration utilising both pre and post-column UV-vis detectors for ILC studies are reported here which greatly assist in our attempts to interpret such chromatograms. The operational issues for using a two detector system are discussed including the use of both detectors in the linear concentration regime; with the choice of an optimal detector wavelength being the most critical factor. The two detector system was demonstrated to be capable of verifying reversible adsorption processes, as well as assisting in the interpretation of complex or problematic chromatograms. This paper demonstrates that the two detector ILC system can offer a number of practical benefits in the study of complex chromatographic phenomena.     

A gradient anion exchange chromatographic method for the speciation of arsenic in lobster tissue extracts

A gradient anion exchange chromatographic technique was developed for the separation of arsenobetaine (AsB), arsenocholine (AsC), arsenite (As(III)), arsenate (As(V)), monomethylarsonic acid (MMAA) and dimethylarsinic acid (DMAA) in one chromatographic run. This technique used low residue ammonium carbonate buffer and the inductively coupled plasma-mass spectrometry (ICP-MS) chromatograms showed little baseline drift. Gradient elution improved peak shape and peak separation. The separation was completed in approximately 27 min with low detection limits (0.017-0.029 mug As kg(-1)). Baseline resolution of all the arsenic species evaluated was achieved when the concentration of AsC was less than approximately 12.5 mug As kg(-1). This technique was successfully applied to different extracts of a standard reference material, TORT-2, and lobster tissue. AsB was found to be the major arsenic species present. AsC, DMAA, MMAA and As(V) were also found, although MMAA was not detected in all of the TORT-2 extracts. Two unknown peaks found may be due to the presence of arsenosugars or other arsenic species. Discrepancy between extraction recoveries previously determined using flow injection-ICP-MS and the high-performance liquid chromatography-ICP-MS was observed in some cases. The differences may be due to the extraction technique and/or conditions at which the extractions were performed.     

High-performance liquid chromatographic method with quantitative comparisons of whole chromatograms of raw and steamed Panax notoginseng

A high-performance liquid chromatographic (HPLC) method coupled with chromatographic pattern matching was developed to differentiate whole chromatograms of raw and steamed Panax notoginseng objectively and quantitatively. The major peaks differentiating chromatograms of raw and steamed samples were also identified for the first time in this herb. The raw and steamed P. notoginseng roots and its products were successfully differentiated. The quantitative differences between the chromatograms were correlated to the duration of steaming. Chromatographic pattern matching allows rapid, simple, automated, and quantitative comparisons of complex chromatograms. It is a useful tool in ensuring safety and quality of herbal products.     

Validation of an analytical method to determine sulfamides in kidney by HPLC-DAD and PARAFAC2 with first-order derivative chromatograms

Six sulfamides were extracted from kidney and analysed by high-performance liquid chromatography with diode array detection (HPLC-DAD): sulfadiazine, sulfamethazine, sulfamethoxypyridazine, sulfamethoxazole, sulfadimethoxine and sulfaquinoxaline.Two main difficulties arose in identifying and quantifying the analytes. Firstly, the chromatographic peaks of the matrix interferences overlapped with those of the analytes. The uniqueness property of PARAFAC2 solved this problem. Secondly, the gradient elution caused a baseline drift. The first-order derivative of the chromatograms minimized its effect.The analytical method was validated. As the performance criteria detailed in the European Decision 2002/657/EC are based on specific signals, this paper generalizes those criteria for higher-order and non-specific signals. In this sense the proposed methodology is general and can be applied to any chromatographic method (HPLC or GC) with a detector that provide a multivariate signal (MS, DAD, EC, etc.).     

Chemometric analysis of complex hyphenated data. Improvements of the component detection algorithm

Component detection algorithm (CODA) is a method to quickly extract the high-quality mass chromatograms from complex liquid chromatography/electrospray ionization mass spectrometry (LC/MS) data sets, saving operators hours of analysis time. It appeared, however, that mass chromatograms with a limited baseline problem were ignored. This paper describes several methods to increase the tolerance for mass chromatograms with a baseline.     

Supercritical fluid chromatography/Fourier transform infrared microspectrometry

The feasibility of supercritical fluid chromatography/Fourier transform-infrared (SFC/FT-IR) microspectrometry is presented. In this approach to SFC/FT-IR, the chromatographic eluates are aspirated from the restrictor directly onto the surface of a moving window which then passes into the beam focus of an infrared microscope. Because the mobile phase is gaseous at ambient conditions, elimination of the mobile phase is easily accomplished. Detection limits in all interfaces between a chromatograph and an FT-IR spectrometer in which the mobile phase is eliminated are determined in large part by the area over which the sample is deposited. We have shown that SFC eluates can be condensed at ambient temperature into spots of 100 to 200μm in diameter. The microscope interface therefore serves to increase the sensitivity of the SFC/FT-IR measurements of these spots and detection limits in the low nanogram range are possible. Preliminary results obtained before any real attempts were made to optimize the deposition process indicate that identifiable spectra can be obtained in real time at the 50 ng level for chromatographic separations performed with a 100μm i.d. wall coated open tubular column. Reproducible reconstructed chromatograms are obtained as each deposited eluate travels through the beam focus of the microscope. The concentration profile of deposited peaks was determined by IR measurements performed at 50 μm increments over the width of the peak to ascertain the deposition size. The results described in this paper, while not yet optimized, indicate the great potential of SFC/FT-IR microspectrometry.     

Nonlinear alignment of chromatograms by means of moving window fast Fourier transfrom cross‐correlation

The preprocessing of chromatograms is essential to modern chromatography for further qualitative and quantitative analysis, especially when chromatographic instruments are used for herb products analysis involving large number of samples. To accurately compare and analyze the obtained chromatograms, it is necessary to preprocess, especially align retention time shifts. Here moving window fast Fourier transform (FFT) cross‐correlation is introduced to perform nonlinear alignment of high‐throughput chromatograms. Since elution characteristics of chromatograms will produce local similarity in retention time shifts, moving window procedure seems to be a better substitute of segmentation steps. The retention time shifts can be calculated and accelerated by FFT cross‐correlation. The artifacts can be detected and eliminated from the retention time shifts profile since the continuity of moving window procedure. The proposed method is demonstrated in comparison with recursive alignment by FFT on chromatographic datasets from herb products analysis. It is shown that the proposed method can address nonlinear retention time shift problem in chromatograms with the simple moving window procedure, which will not introduce segments size optimization problem. In additional, the parameters are intuitive and easy to adjust, which makes it off‐the‐shelf toolbox for alignment of chromatograms.