Multi-component calibration and analysis in liquid chromatography

Multi-wavelength detectors offer improved detection capabilities for liquid chromatographic methods, but require improvements in data analysis methodology to utilize all the available information. In this work, various methods for improving the quantitative results obtained from liquid chromatography with full spectrum fluorescence detection were studied. The availability of multi-wavelenght information allows overlapped chromatographic peaks to be resolved. Different approaches were investigated for developing calibration models that use all of the available spectral information, and are compatible with a variety of methods for quantification, including factor analysis, Kalman filtering and rank annihilation. These methods were compared for their ability to resolve overlapped chromatographic peaks and their accuracy in quantification.     

Thin Layer Chromatography-Laser Mass Spectrometry (TLC-LMS) of Triphenylmethane Dyes: Initial Results

Abstract

Laser mass spectra (LMS) of triphenylmethane dyes were obtained directly from a thin layer chromatographic (TLC) plate using the LAMMA-1000 laser microprobe. Spectra obtained from TLC plates were very similar to those obtained from standard substrates, the only difference being that at threshold power densities spectra from the TLC plates show greater fragmentation. The region below m/z 100 is mostly obscured by peaks due to the chromatographic support; above this range, peaks can be identified corresponding to the molecular cation and simple neutral losses. The inherent lateral rèsolution of the Instrument (～5 μm) was used to resolve multiple components in broad chromatographic spots. The TLC-LMS combination has the advantage that components can be identified even if they are not completely resolved chromatographically.     

Deconvolution and quantitation of unresolved mixtures in liquid chromatographic-diode array detection using evolving factor analysis

Summary A recently developed self-modeling curve resolution method based in different factor analysis techniques has been applied for the first time to the study of liquid-chromatography-diode array data under situation where the separation of two components is not achieved. Two applications are reported: the resolution and quantitation of a coeluted mixture of carbamate pesticides pirimicarb and 1-naphthol, and the estimation of the concentration profiles of the double peak obtained in the elution of the triazine metabolite chlorodiamino-s-triazine. Different methods of quantitation are compared, including Evolving Factor Analysis and Rank annihilation. Quantitation from the area of the elution profiles once the component spectra have been transformed for their area contribution to the signal, gives a relative composition for pirimicarb and naphthol pesticides which agrees with the known sample composition. In the case of the unknown triazine mixture, an approximate quantitation of the two peaks obtained for this metabolite is obtained by assuming equal signal contribution or equal maximum absorbance of the individual spectra of the two detected components.     

The Quantitation of Lecithin,Sphingomyelin and Their Ratios by Infrared Spectroscopy

Abstract

We describe a new method for measuring lecithin and its ratio to sphingomyelin with the use of infrared spectrophotometry. The method offers quantitation of lecithin, and sphingomyelin and their ratios, in approximately two hours, without the use of thin layer chromatography. Once the extraction of phospholipids is completed, an infrared spectral scan from 4000 to 400 (cm′) is obtained from which two peaks are measured and quantitated for lecithin and sphingomyelin and their ratios.     

Data handling in HPLC-diode array UV-spectrometry

Summary The mathematical resolution of overlapped chromatographic peaks obtained in HPLC with a diode array UV detector has received considerable attention recently. The purpose of the proposed methods is the quantitation and identification of unknown solutes in complex mixtures by an efficient use of all available data. Basically two approaches have been proposed: the first one resolves the concentration profiles and calculates the pure spectra by applying a minimal number of assumptions, which is denoted self-modeling-curve resolution. The second one is based on a match of pure spectra available in a spectral library with the measured mixture spectra. In this paper both approaches are evaluated with respect to their performance to provide the pure spectra and an accurate quantitation of the concentrations.     

Quantitation and identification of polynuclear aromatic hydrocarbons by liquid chromatography and multiwavelength absorption spectrometry

This paper describes the combined use of liquid chromatographic separation with multiwavelength absorption detection for the quantitation and identification of polynuclear aromatic hydrocarbons. Quantiative results are presented for three- and four-component mixtures with complete and partial separation. Qualitative data are presented for a twelve-component synthetic mixture and for extracts from a filter used to collect particulate emissions from a biomass-gasifier system. It is shown that multiwavelength data-processing methods commonly applied to absorption spectra can be applied with significant advantage to second-derivative spectra. Results indicate that significant improvements can be achieved by the use of different wavelength ranges for different components in mixtures. Detection limits for single-component samples varied between 3 and 11 μg l^?1 for eleven hydrocarbons and the scatter about calibration plots for mixtures was in the range of 0.05 to 0.4 μg ml^?1. The identities of all twelve components in the synthetic mixture were confirmed and nineteen components were identified in the sample from the biomass gasifier filter.     

High Performance Liquid Chromatographic analysis of the Biological Response Modifier Synthetic Double-Stranded RNA (dsRNA,Ampligen)

Abstract

A high performance liquid chromatographic method was developed for analysis of the antitumor agent synthetic, double-stranded RNA (dsRNA, Ampligen) in plasma. In this procedure the agent was extracted from plasma by ion-exchange chromatography and then degraded to its primary components, inosine and cytidine, by treatment with nuclease and alkaline phosphatase. Inosine and cytidine derived from degradation of ampligen were quantified by reversed phase HPLC. Standard curves for quantitation of inosine derived from ampligen were generated by addition of various amounts of ampligen to plasma. Utilizing this method, extraction of drug from the plasma was approximately 70–80%. Standard curves were linear over the concentration range of 0.25 to 100 ug/ml plasma. There were no peaks from plasma which interfere with quantitation of inosine. The approximate lower limit of detection of drug by this method was 0.25 ug/ml plasma. Interassay and intra-assay mean variability of standards was 9.6 and 3.2% respectively. Analysis of plasma samples obtained from one patient after infusion of ampligen (640 mg/m²) show that this method is sensitive enough to monitor plasma for clinical pharmacology studies of ampligen.     

断层扫描分析法用于联用色谱二维数据的解析Ⅰ定性分析:原理与应用

提出一种对色谱-光谱二维数据进行信息处理的全新手段——断层扫描分析法。它以逐层“剥离”的方式对二维数据进行“断层扫描”,并结合褶合曲线：兮析理论进行数据挖掘,可实现色谱峰纯度检验和归属判定、重叠色谱峰解折与定量分析等功能,并最终获得分析体系的全部定性定量信息。本文介绍了断层扫描分析法用于定性分析的原理及其在实验体系中的应用情况。结果表明：断层扫描分析法用于定性分析可实现色谱峰定性(归属)鉴别、纯度检验以及选择性区域的识别等,在中药等复杂体系的分析中具有重要的研究意义和良好的应用前景。     

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.     

Component reconstruction in the primary space of spectra and concentrations. Alternating regression and related direct methods

A set of methods that extract the spectral components in a chromatographic run is considered. The methods do not need libraries of previously known spectra or retention times. The methods have been developed for two-dimensional spectra but they can also be used for chromatographic analyses with a single-channel detector. The methods are direct; they do not use principal components as the starting point. Alternating regression (AR) remains in the primary space of spectra and concentrations during the calculations. Random numbers are used as the starting spectra. Regression is used to solve first for the concentrations, then for the spectra. The method uses two kinds of constraints: all spectra and concentrations are forced to be positive; and all concentration profiles are forced to a unimodal shape with a single local maximum. It is assumed that all observations are a linear sum of components. Compact alternating regression (CAR) is a new variant of the basic AR. The idea is to replace multiplication of a large matrix by two multiplications of smaller matrices. This typically speeds up the iterations by a factor of ten. AR and CAR have been successfully used with combined techniques such as gas chromatography—mass spectrometry and liquid chromatography with UV—visible detection. The reliability of the solution is checked by repeatedly injecting noise and performing the analysis several times. This produces estimates of confidence intervals. AR and CAR have recently been extended to handle single-dimensional signals. Examples are single-channel detectors such as the flame ionization detector in gas or liquid chromatography with a fixed-wavelength UV detector. A batch of samples is used as the observation matrix. As a result, one obtains both the concentrations and the elution shapes of individual chromatographic peaks.     

电荷耦合器件检测器同时测定多组分荧光物质

用电荷耦合器件检测器组装的荧光检测装置，检测荧光混合物的荧光光谱，借助卡尔曼滤波处理测量数据，进行多组分荧光物质同时测定，获得了满意结果。     

A one-dimensional kalman filter for peak resolution

A one-dimensional Kalman filter algorithm is presented that resolves several overlapped liquid chromatographic peaks without algebraic operations of matrices. The resolving powers or filtering reliability of the algorithm is independent of the number of peaks, but depends on both the peak overlap (resolution value R_s) and the signal-to-noise ratio more significantly than the usual multi-dimensional Kalman filter. The reliability is shown to be similar to that of the multi-dimensional filter for the resolution of overlapped Gaussian peaks with limited R_s and S/N values.     

Application of Computer-Aided Spectral Deconvolution Technique in Validation of High Performance Liquid Chromatographic Peaks

Abstract

Reliable analysis with high performance liquid chromatography (HPLC) requires purity of the eluting peak. The present work has combined the advantages of the availability of full spectral data from HPLC photodiode array UV detector and computer algorithms to perform chromatographic peak purity check. A deconvolution technique based on multicomponent analysis has been applied to the UV spectra of co-eluting components. This method employs residual error (Relative-fit-error, RFE) between predicted spectrum and analyte's spectrum to detect presence of other component or contaminant. Typical RFE values for uncontaminated chromatographic peaks of norethisterone and ethynyloestradiol range between 1 and 3, while contaminated peaks have RFE values as large as 145. A systematic increase in ‘relative-fit error’ from 1.10 to 145 was observed for peaks of norethisterone when contaminated to varied extent with ethynyloestradiol. Extent of peak overlap in chromatogram was also mapped out with this technique. The co-prescribed oral contraceptive, norethisterone and ethynyloestradiol were used as model in this work. An advantage of the method is its applicability when the contaminant's spectrum is unavailable. The method, unlike several earlier techniques, is also applicable to chromatograms with concidental elution time for the components.     

Simultaneous quantitative analysis of isobars by tandem mass spectrometry from unresolved chromatographic peaks

A method was developed for the simultaneous quantitation of isobars from unresolved chromatographic peaks. The method is based on differences in branching ratios of ion abundances in their tandem mass spectra and an assumption that the product ion mass spectra of a mixture can be considered as a linear combination of the spectra of individual constituents. We present analytical equations and a matrix-based approach for deconvoluting the concentration of individual components from the total peak intensity for two and three isobars and also a matrix-based generalization to any number of compounds. The feasibility of the simultaneous analysis of mixtures containing two compounds was assessed. The approach was evaluated for the analysis of structural isomers of methylmalonic and succinic acids in human plasma and urine samples for a group of 270 samples. The linear regression equation, standard error and correlation coefficient for the agreement with a traditional method utilizing chromatographic separation of the isomers were y = 0.999x - 0.005, 0.024 micro mol l(-1), and 0.985, respectively. The utility of a spectral contrast angle as a predictor of analysis feasibility was evaluated.     

Capillary SFC-FTIR – effects of the flow-cell on chromatographic resolution

The quantitative effects on chromatographic resolution of FTIR flow-cells for detection in capillary SFC have been determined. A suitably designed cell with a volume of 500 nl was shown to cause only modest broadening of chromatographic peaks obtained from the surfactant mixture Triton X-100, showing that subsequent detection methods can therefore be used in a multi-hyphenated chromatographic ensemble. A larger-volume (980 nl) cell was found to give satisfactory results with 100 μ columns, but not with 50 μm columns. The practicability of a multi-hyphenated system was illustrated by a preliminary capillary SFC-UV-FTIR-FID study on a plant extract. Good infrared spectra were obtained, together with well-resolved UV and (subsequent) FID chromatograms.     

An HPLC method for determination of azadirachtin residues in bovine muscle

A high-performance liquid chromatography (HPLC) method for the determination of azadirachtin (A and B) residues in bovine muscle has been developed. Azadirachtin is a neutral triterpene and chemotherapeutic agent effective in controlling some pest flies in horses, stables, horns and fruit. The actual HPLC method uses an isocratic elution and UV detection. Liquid-liquid extraction and solid-phase purification was used for the clean-up of the biological matrix. The chromatographic determination of these components is achieved using a C18 analytical column with water-acetonitrile mixture (27.5:72.5, v/v) as mobile phase, 1 mL/min as flow rate, 45 °C column temperature and UV detector at 215 nm. The azadirachtin peaks are well resolved and free of interference from matrix components. The extraction and analytical method developed in this work allows the quantitation of azadirachtin with precision and accuracy, establishing a lower limit of quantitation of azadirachtin, extracted from the biological matrix.     

Background subtraction for fluorescence detection in thin-layer chromatography with derivative spectrometry and the adaptive Kalman filter

Background signals must be removed from analyte responses before reliable qualitative and quantitative results can be obtained. For cases in which a reliable estimate for the background response cannot be obtained, the combination of two mathematical approaches yields accurate quantitative results. First-derivative spectrometric methods, in conjunction with a curve-resolution approach based on the adaptive Kalman filter, are used to compensate for difficulties caused by background responses which are not reproducible. Derivative spectrometric techniques reduce the relative magnitude of low-frequency systematic deviations in the spectra. In this case, this has the effect of localizing model errors to restricted regions of the spectra, which in turn meets the major requirement for successful utilization of the adaptive Kalman filter. The approach is applied to fluorimetric detection for thin-layer chromatography in the quantification of polynuclear atomatic hydrocarbon compounds. Results are given which demonstrate that this combined approach yields accurate estimates for concentrations of components in overlapped chromatographic zones. A derivative spectrometric approach in conjunction with a regular Kalman-filter fit gives less accurate results, and an adaptive Kalman filter used to fit the raw spectral data fails to give any reliable quantitative information. The combined approach using derivative spectrometry anal the adaptive Kalman filter is shown to give 8-fold lower detection limits for anthracene when compared to traditional background-subtraction methods.     

Kalman filter deconvolution after cubic splines background removal in the HPLC determination of Cr(III) and Al(III) as 8-hydroxyquinolines

Summary A procedure for the simultaneous quantitation of Al(III) and Cr(III) ions by reversed-phase HPLC, after pre-column complexation with 8-hydroxyquinoline, is described. The deconvolution of the partially overlapped peaks was by the Kalman filter method which yielded accurate and precise results. Background removal from the chromatograms was by a new approach employing cubic splines as interpolators between the peak valleys. Finally, it is shown that the Kalman filter deconvolution, after subtraction of the background by cubic spline interpolation, allowed quantitation of Al(III) and Cr(III) down to 25 ppb for each metal. These concentrations were not detectable by conventional integration methods due to a very low signal-to-noise ratio.     

HELP法在中草药分析中的应用研究-冬虫夏草的化学成分测定

基于直观推导式演进特征投影（ＨＥＬＰ）法,对冬虫夏草子座和虫体分别进行了多组分同时定性定量测定．结果表明,ＨＥＬＰ法能减少样本提取分离的步骤,降低色谱分离条件的要求,提高检测准确度．联用色谱检测与化学计量学解析法相结合将为复杂中草药分析提供一种全新手段     

中药川芎和赤芍配伍规律的方法研究

提出应用HPLC-PAD-MS研究中药配伍多种化学成分的基本思路,指出应用色谱保留时间、紫外光谱及质谱等多信息、综合对照的方法确定合煎液中各峰的归属,从而进一步研究了中药的配伍规律.首先建立了一套稳定、重复而可靠的液相色谱分析方法进行分离,再用统一的分析方法对分离的色谱图进行数据处理.在HPLC-PAD-MS分析中可得到各成分的保留时间、紫外光谱和分子量信息.通过比较合煎液和单煎液中各成分的保留时间及分子量信息可确定合煎液各峰的归属,最后比较了单煎液和合煎液的化学成分变化.结果表明,川芎和赤芍配伍后有一新峰产生,同时有两个成分含量发生变化.