Relationship between selectivity and average resolution in comprehensive two-dimensional separations with spectroscopic detection

The average value of the multivariate selectivity (SEL) of randomly positioned peaks in a multi-component separation is shown to equal the average fraction of peaks that are singlets, as predicted by statistical-overlap theory (SOT). This equality is the basis for proposing a useful metric, specifically the average minimum resolution of nearest-neighbor peaks, for the performance of comprehensive two-dimensional (2D) separations. Furthermore this metric was computed both without ancillary spectroscopic information and with the assistance of such help, specifically multi-wavelength UV-vis spectra, acquired during the separation. Separations are simulated with randomly positioned peaks over wide ranges of total number of peaks, first- and second-dimension peak capacity, dimensionless first-dimension sampling time, and spectral diversity. The specific version of the general multivariate selectivity concept that is used here--identified as SEL--gives the relative precision of quantification when using the PARAFAC (parallel factor analysis) method, a popular curve resolution algorithm. The SEL values of all peaks were calculated, averaged, and compared to the predictions of SOT. In the absence of auxiliary spectral data, the SEL-based average minimum resolution required to separate two peaks in a 2D separation is 0.256, compared to resolution of 0.5 if no chemometric assistance is available. This was found to be valid over a wide range of conditions and is essentially independent of peak crowding. With the assistance of the spectral data, the requisite minimum resolution substantially improves, that is, it decreases, especially when peak crowding is severe. The requisite minimum resolution decreases even further, up to a limit, as the spectral diversity is increased. In contrast, the SEL-based average under-sampling correction factor is virtually independent of the presence of the additional spectral data, and additionally is about the same as calculated with SOT from the average number of maxima in closely analogous simulations. The use of selectivity greatly increases the fraction of peaks that are singlets, relative to the number of singlet maxima, especially when spectral assistance is added. The insensitivity of the under-sampling correction factor to either the use of selectivity or added spectral data simplifies optimization of the corrected peak capacity in on-line comprehensive 2D separations.     

基于积分渐进展开的气相色谱重叠峰快速解析法

论文提出用积分渐进展开解析气相色谱重叠峰,该方法有3个主要步骤：首先将谷峰或肩峰分成两个积分区域,得到一个子区域的积分方程和一个重叠峰面积的代数方程;然后用数值积分求出这两个方程计算中所需要的峰面积,再用积分渐进公式将积分方程展开成代数方程;最后,将这两个方程与峰高约束方程联立后,得到一个非线性代数方程组,用Gauss-Seidel迭代可以快速求解方程组,方程收敛的最大迭代次数不超过20次。仿真和实验结果表明,解析的峰高和峰面积误差均很小,峰面积最大误差低于6.44%,峰高的最大误差约为6.80%。由于该算法精度高,效率高,所以这个方法可以用于气相色谱重叠峰和一般色谱峰的实时在线解析。     

基于无基底扣除的数据趋势累积谱峰检测算法

谱峰的检测分析在色谱技术研究中具有十分重要的作用,但在色谱数据采集、传输的过程中,不同程度的噪声干扰给谱峰检测带来了极大的困难.目前传统的谱峰检测算法普遍通过基底扣除的方式对谱峰的形态进行预定义,将谱峰分为单峰、重叠峰等多个种类.针对不同种类的谱峰采用不同的检测方法,这就导致了传统的谱峰检测算法具有高复杂度、低自动化程...     

分解色谱重叠峰的参数契合法

本文研究了色谱重叠峰一阶导数曲线中各极值点的相对位置与重叠子峰面积比之间的关系，提出了一种模式参数契合的色谱重叠峰分解方法，它在准确性方面优于传统的垂线法，在速度方面优于模型拟合法，可用于实时快速处理。     

Deconvolution of overlapped peaks based on the exponentially modified Gaussian model in comprehensive two-dimensional gas chromatography

Comprehensive two-dimensional gas chromatography (GC x GC) has attracted much attention for the analysis of complex samples. Even with a large peak capacity in GC x GC, peak overlapping is often met. In this paper, a new method was developed to resolve overlapped peaks based on the mass conservation and the exponentially modified Gaussian (EMG) model. Linear relationships between the calculated sigma, tau of primary peaks with the corresponding retention time (tR) were obtained, and the correlation coefficients were over 0.99. Based on such relationships, the elution profile of each compound in overlapped peaks could be simulated, even for the peak never separated on the second-dimension. The proposed method has proven to offer more accurate peak area than the general data processing method.     

正交投影用于多波长色谱重叠峰分析

 将正交投影分辨 (OPR)技术用于多波长色谱重叠峰分辨 ,当色谱峰中最大重叠度小于或等于波长数时 ,用这一方法能从多波长色谱重叠峰中获得完全真解。基于双波长色谱分析 ,提出了一种新的色谱重叠峰中背景校正、组分数和纯组分信号区确定以及各组分重叠情况的分析方法 ,即双波长特征信息分析 (DWCI)。该法被成功的用于三组分双峰和双组分单峰重叠色谱的分析。     

基于分形理论分辨重叠峰的新算法

将分形理论用于仪器分析信号的解析,提出一种面向分析谱图中重叠信号处理的分形分辨算法.通过对信号进行分形分析,采用分形维数可有效地反映信号的特征,准确地得到谱峰个数和位置的信息,避免人为判断的误差,实现重叠复合信号的分辨.实验表明,这种新的重叠谱峰分辨法能用于光谱、电化学、色谱等仪器分析数据的处理     

Molecular level comparison of water extractives of maple and oak with negative and positive ion ESI FT‐ICR mass spectrometry

Soluble extractives in wood function to protect living trees from destructive agents and also contribute to wood color and fragrance. Some extractive components have biological activities with medical applications. They also play important roles in wood processing and related applications. To increase the knowledge of wood chemistry, maple and oak were extracted by water. Ultraviolet/visible (UV/vis) spectroscopy indicated the presence of a phenolic compound, resorcinol, in maple extractives having higher molecular mass and more aromatic components than oak extractives. Negative and positive electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI FT‐ICR‐MS) identified thousands of formulas in the two samples in the m/z range of 200 to 800. They mainly fall into the lignin‐like, carbohydrate‐like, and tannin‐like compound categories. The top 25 peaks (ie, formulas) with the highest relative magnitude in negative ESI represented nearly 50% of the summed total spectral magnitude of all formulas assigned in the maple and oak extractives. Furthermore, the base peak (ie, most abundant peak) accounted for about 14% of the total abundance in each wood sample. Literature comparisons identified 17 of 20 formulas in the top five peaks of the four spectra as specific bioactive compounds in trees and other plants, implying the potential to explore utilization of maple and oak extractives for functional and medicinal applications. The various profiling of the top 25 peaks from the two samples also suggested the possible application of FT‐ICR‐MS for detecting chemical markers useful in profiling and identification of wood types and sources.     

Peak area measurement of severely overlapped pairs

Summary Based on the ratio of two apparent heights and an empirical correction factor, a method is presented for quantitation of peak areas of severely overlapped pairs. This method can be applied to a wide range of area ratios and peak asymmetries, provided there exists a clear and precise valley-except for shoulder peaks. The relative errors of the first peak are usually within ±3% and theoretical relative error limits are −7.0% to +5.5%. Peak asymmetry of a severely overlapped pair can be determined using the ratio of the front half-width to the rear half-width at 10% peak height of an overlapping profile. The asymmetry so determined is an apparent one and the relative errors are −4% to +5.3% for peaks with 90% relative valley, depending on area ratio, degree of overlap and asymmetry. An empirical area equation for the first peak involving area ratio, asymmetry, resolution and the area measured by a perpendicular drop algorithm is also developed.     

Stable isotope-free quantitative shotgun proteomics combined with sample pattern recognition for rapid diagnostics

Mass spectrometry (MS) has become a powerful tool for the quantitative analysis of complex protein samples. A high-throughput strategy for the comparative analysis of multiple protein samples with high complexity becomes more and more important. Two strategies, spectral count and peak intensity, for label-free MS analysis of prefractionated complex mixtures have been described recently to be useful for quantitation. Here we compare both strategies for rapid and quantitative 1-D shotgun LC/MS/MS analyses of highly complex protein mixtures using silica-based monolithic columns. First, we validated linearity and sensitivity of these methods by spiking varying amounts of an internal standard protein in a complex plant protein extract. Secondly, quantitative data of proteins of Medicago truncatula nodules were visualized with independent components analysis using data either obtained from spectral count or peak integration performed with commercial software. Spectral count showed apparent advantages over peak integration because several peptides per protein are automatically averaged, the linear dynamic range of quantitation increases in complex matrices and the number of quantified proteins surpasses the number of proteins using peak integration. Thus, for the need of rapid comparative analysis of highly complex protein samples, spectral count enables sample pattern recognition and identification of biomarkers in nongel based proteomic studies.     

Quantitative analysis of 17 amino acids in tobacco leaves using an amino acid analyzer and chemometric resolution

A method was developed for quantifying 17 amino acids in tobacco leaves by using an A300 amino acid analyzer and chemometric resolution. In the method, amino acids were eluted by the buffer solution on an ion‐exchange column. After reacting with ninhydrin, the derivatives of amino acids were detected by ultraviolet detection. Most amino acids are separated by the elution program. However, five peaks of the derivatives are still overlapping. A non‐negative immune algorithm was employed to extract the profiles of the derivatives from the overlapping signals, and then peak areas were adopted for quantitative analysis of the amino acids. The method was validated by the determination of amino acids in tobacco leaves. The relative standard deviations (n = 5) are all less than 2.54% and the recoveries of the spiked samples are in a range of 94.62–108.21%. The feasibility of the method was proved by analyzing the 17 amino acids in 30 tobacco leaf samples.     

改进遗传算法用于未知组分数的重叠色谱峰的解析

按照指数修正的Gauss卷积色谱峰模型，构造了结合模拟退火及变长染色体的改进遗传算法，并结合爬山法，可以方便快捷地进行数目未知的重叠色谱峰的精确解析；此法成功地应用于大鼠脑微透析液氨基酸定量分析中，解析结果良好。     

三元重叠色谱峰面积的定量方法

本从色谱峰的ＥＭＧ模型出发，通过对重叠色谱峰的模拟和回归分析，提出了一种三元重叠色谱峰的面积的定量方法，三元重叠色谱峰的每一个峰面积可以由峰面积比和总面积求得，此法所需的数据都由实验色谱图上测得，峰面积计算结果的相对误差小于±５％，适用于相对峰谷为５０％－９５％的三元重叠色谱峰面积的定量。     

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.     

Unravelling the composition of very complex samples by comprehensive gas chromatography coupled to time-of-flight mass spectrometry. Cigarette smoke

The potential and current limitations of comprehensive two-dimensional gas chromatography coupled to time-of-flight mass spectrometry (GC x GC-TOF-MS) for the analysis of very complex samples were studied with the separation of cigarette smoke as an example. Because of the large number of peaks in such a GC x GC chromatogram it was not possible to perform manual data processing. Instead, the GC-TOF-MS software was used to perform peak finding, deconvolution and library search in an automated fashion; this resulted in a peak table containing some 30000 peaks. Mass spectral match factors were used to evaluate the library search results. The additional use of retention indices and information from second-dimension retention times can substantially improve the identification. The combined separation power of the GC x GC-TOF-MS system and the deconvolution algorithm provide a system with a most impressive separation power.     

Spectral correlation of high-performance liquid chromatography-diode array detection data from two independent chromatographic runs peak tracking in pharmaceutical impurity profiling

A novel qualitative analytical method for peak tracking in impurity profiling control by the correlation of spectra was established. Two-dimensional (2D) standard spectrochromatographic data produced by high-performance liquid chromatography with diode array detection (HPLC-DAD) were compared with sample data to develop two-dimensional chromatographic spectral correlative maps. Taking full advantage of separation efficiency of HPLC and spectral specificity of the analytes, the method was successfully used to recognize impurities in quinolone antibacterials, when in combination with relative retention times (RRTs). For the comparison of spectra was expanded to three-dimensional space, simultaneous identification of the chromatographic peaks can be obtained rapidly without preparation and injection of a reference solution, even when the mobile phase changed or the peaks of multi-component samples overlapped.     

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.     

Decomposition of overlapping plasmagram peaks by spectral subtraction

Low field atmospheric pressure Ion Mobility Spectroscopy (IMS) involves the careful analysis of plasmagrams with multiple peaks which can mask one another when they are closely spaced in drift time or corresponding reduced mobility. A typical signal processing approach to decomposing overlapped peaks would be to use an orthogonal decomposition technique, but unfortunately Gaussian-like functions are not orthogonal, so no unique decomposition can be guaranteed. However, each ion species in the drift tube will arrive at the Faraday plate with a known statistical distribution determined by the IMS instrument’s drift tube design, electric field strength, reagent gas flow and other instrument-specific factors such as the ion gate function. This paper presents a straightforward algorithm for decomposing plasmagrams into distinct peaks using a subtractive technique that independently estimates the statistical parameters of each peak, rejecting spurious peaks and electrical noise. The results show that for relatively short gate times, the plasmagram peaks are nearly Gaussian-shaped, but slightly fatter and asymmetric. We show that including of the gate rise and fall times is also significant in matching the plasmagram peak shape. We also show that the diffusion effects on resolution can be attributed to combinations of non-uniform ion distributions in the reaction chamber as well as detritus effects in the drift tube. Given the known peaks statistical parameters, one can then separate overlapping peaks using a straightforward spectral subtractive technique.     

用EM算法改进鸟枪法蛋白质鉴定中的无标记定量方法

蛋白质定量是探索疾病发生发展状况和寻找新药靶标的重要手段。在shotgun蛋白组学中,目前常用定量方法包括综合同位素标记后的质谱峰强度方法和无标记定量方法。根据数据类型无标记定量方法可以分为两类:基于鉴定蛋白的质谱数的方法和基于质谱峰强度的方法。本研究主要用EM算法改进基于鉴定蛋白质谱数的定量方法,并用免疫印迹实验获得的酵母全蛋白的丰度来验证EM算法改进后定量的有效性结果表明,改进后的质谱数和蛋白丰度的相关性比改进前有一定的提高。同时,利用这些数据对主要的几种基于鉴定蛋白的质谱数的模型进行了比较,发现PAI模型最好,SpS模型次之,emPAI模型最不适合于蛋白质定量。     

Spectral counting robust on high mass accuracy mass spectrometers

Mass spectrometry is central to shotgun proteomics, an application that seeks to quantify as much of the total protein complement of a biological sample as possible. The high mass accuracy, resolution, capacity and scan rate of modern mass spectrometers have greatly facilitated this endeavor. The sum of MS to MS/MS transitions in tandem mass spectrometry, the spectral count (SC), of a peptide has been shown to be a reliable estimate of its relative abundance. However, when using SCs, optimal MS configurations are crucial in order to maximize the number of low abundant proteins quantified while keeping the estimates for the highly abundant proteins within the linear dynamic range. In this study, LC/MS/MS analysis was performed using an LTQ‐OrbiTrap on a sample containing many highly abundant proteins. Tuning the LTQ‐OrbiTrap mass spectrometer to minimize redundant MS/MS acquisition and to maximize resolution of the proteome by accurately measured m/z ratios resulted in an appreciable increase in quantified low abundant proteins. An exclusion duration of 90 s and an exclusion width of 10 ppm were found best of those tested. The spectral count of individual proteins was found to be highly reproducible and protein abundance ratios were not affected by the different settings that were applied. We conclude that on a high mass accuracy instrument spectral counting is a robust measure of protein abundance even for samples containing many highly abundant proteins and that tuning dynamic exclusion parameters appreciably improves the number of proteins that can be reliably quantified. Copyright © 2010 John Wiley & Sons, Ltd.