Fast gradient elution reversed-phase liquid chromatography with diode-array detection as a high-throughput screening method for drugs of abuse. II. Data analysis

In Part I of this work, we developed a method for the detection of drugs of abuse in biological samples based on fast gradient elution liquid-chromatography coupled with diode array spectroscopic detection (LC-DAD). In this part of the work, we apply the chemometric method of target factor analysis (TFA) to the chromatograms. This algorithm identifies the target compounds present in chromatograms based on a spectral library, resolves nearly co-eluting components, and differentiates between drugs with similar spectra. The ability to resolve highly overlapped peaks using the spectral data afforded by the DAD is what distinguishes the present method from conventional library searching methods. Our library has a mean list length (MLL) of 1.255 and a discriminating power of 0.997 when both retention index and spectral factors are considered. The algorithm compares a library of 47 different compounds of toxicological relevance to unknown samples and identifies which compounds are present based on spectral and retention index matching. The application of a corrected retention index for identification rather than raw retention times compensates for long-term and column-to-column retention time shifts and allows for the use of a single library of spectral and retention data. Training data sets were used to establish the search and identification parameters of the method. A validation data set of 70 chromatograms was used to calculate the sensitivity (correct identification of positives) and specificity (correct identification of negatives) of the method, which were found to be 92% and 94%, respectively.     

Prediction of elution bandwidth for purine compounds by a retention model in reversed-phase HPLC with linear-gradient elution

In reversed-phase liquid chromatography, the retention mechanism of solute has been studied under linearly programmed gradient mobile-phase conditions. The separation of a mixture of four purine compounds (purine, theobromine, theophylline, and caffeine) was considered as a practical case in two binary mobile phase systems, water/methanol and water/acetonitrile. The retention model which describes how the retention factor is related to the mobile-phase composition has been developed in various mathematical forms to predict the retention time in both linear and gradient elutions. For a pulse injection of sample, two important factors, the retention time and the bandwidth of solute, might be computable to predict the elution profiles estimated by the distribution function, such as the Gaussian distribution function. In this work, a prediction method based on the analogue of the retention model was proposed to calculate the bandwidth in linear gradient elutions. Band broadening was caused by the different migration velocities of the front and rear ends of the solute band in a chromatographic column. Therefore, the migration behaviors of the front and rear ends of the solute band were explained with the same retention model which had been used to predict the retention time of solute. For the well retained solutes, theophylline and caffeine, the predicted bandwidth and experimentally obtained bandwidth showed good agreement in both isocratic and gradient elutions.     

基于深度学习的保留时间预测方法的研究进展及应用

在基于液相色谱-质谱联用的蛋白质组学研究中,肽段的保留时间作为有效区分不同肽段的特征参数,可以根据肽段自身的序列等信息对其进行预测.使用预测得到的保留时间辅助质谱数据鉴定肽段序列可以提高鉴定的准确性,因此对保留时间预测的工作一直受到领域内的广泛关注.传统的保留时间预测方法通常是根据氨基酸序列计算肽段的理化性质,进而计算...     

Dielectric spectroscopy on aqueous solutions of pyridine and its derivatives

The complex (dielectric) permittivity has been measured as a function of frequency between 1 MHz and 40 GHz for aqueous solutions of pyridine, 2- and 3-methylpyridine, as well as 2,4- and 2,6-dimethylpyridine at various temperatures and solute concentrations. Different relaxation spectral functions are used to analytically represent the data, in particular the Cole-Cole function. The solute contribution to the extrapolated static permittivity has been calculated to show that, in correspondence with other aqueous solutions of organic molecules and ions, the permittivity of the solvent seems to be enhanced with respect to the pure water value. Also in accordance with other aqueous systems it is found that the principal dielectric relaxation time for equimolar solutions of stereo isomers at the same temperature may significantly differ from one another. A further result is the finding of an unusually strong temperature dependence in the relaxation time of the 1 molar solution of 2,6-dimethylpyridine.     

反相高效液相三元流动相台阶梯度分离条件的快速优化方法

 根据溶质在柱内的迁移规律 ,建立了一种利用线性梯度实验快速获得溶质保留值方程系数 ,然后以串行响应函数为优化指标进行多台阶梯度分离条件优化的方法。与利用等度实验获得保留值方程的方法相比 ,该法可以大大缩短优化时间。通过该方法对芳香胺和衍生化氨基酸样品进行了分离 ,获得了满意的分离度 ,表明该方法的预测精度很好。     

The prediction of the peak width at half height in HPLC

The prediction of the peak width at half height is an important aspect in the optimization of the chromatographic operating conditions. In this paper, a linear relationship, between the peak widths at half height and the retention values with various isocratic elution is observed. In gradient elution, however, the relationship between the peak widths at half height and the so-called invented retention values that correspond to the mobile phase composition by eluting the solute from the column end is developed. We believe that there is almost the same band width at half height inside the column (in unit of length) for different solutes. The peak width at half height in the chromatogram (in unit of time) is mainly determined by the capacity factor of the solute when it is eluted from the column end. The larger the capacity factor of a solute eluted from the column end, the more slowly will be the solute eluted from the column end and the wider will be the peak width at half height. It is possible to predict the peak width at half height in various isocratic and gradient elutions by using this linear relationship.     

Analysis of the equations for the temperature dependence of the retention index. II. Physico-chemical meaning of the parameters

A general expression for the meaning of various parameters from the retention index-temperature equations was found. This is a linear combination of two solute dependent factors consisting of differences of enthalpies and respective entropies of solution, between the solute and the reference inferior n-alkane. The particular coefficients for each parameter are stationary phase dependent factors. They were calculated from methylene contributions to the thermodynamic functions of solution in SE-30 and Carbowax-20M, revealing the influence of the used temperature range. A short review of structural influences on dI/dT is included.     

Sample Solvent as Analyte in High Performance Liquid Chromatography

Abstract

Solvents vary in their behavior in high performance liquid chromatography (HPLC). Water and methanol, among others, are widely used in the mobile phase as well as solvents for the solute. Few reports indicate that the solvent used for the solute can behave as an analyte. Normally, it is generally accepted that the solute solvent, a non-constituent of the mobile phase will be the first eluent. However, a solvent which is a component of the sample can show up as an unexpected peak with its own identity. This solvent may show a similar retention time as some of the unknown components of the sample. This indicates that in some cases the quantitative results may be the sum of the absorptivity of the solute and solvent used for the sample. It is assumed that some solvents show no absorption in the ultraviolet region at which the analysis is being conducted. Depending on the mobile phase composition some solvents can be detected at the wavelengts or wavelengths used for analysis. Water, ethylacetate, and methanol showed absorption at 210 nm when present in the sample being analyzed with a mobile phase of acetonitrile-methanol using a C₁₈ column. These solvents overlapped or showed retention times the same as estriol and testosterone.     

Combined effect of temperature and organic modifier concentration on the retention under single mode gradient conditions in reversed-phase HPLC

The combined effect of temperature, T, and organic modifier concentration, phi, on the retention under gradient conditions in RPLC is studied by considering, both theoretically and experimentally gradients, of phi at constant T and gradients of T at constant phi. Two approaches are examined: in the first approach the prediction of the elution time of a sample solute is based on the isocratic/isothermal properties of this solute. The second approach is based on a direct fitting procedure of a proper retention model to 2-D isocratic/T-gradient or isothermal/phi-gradient retention data. These approaches were tested using alkylbenzes in eluting systems modified by ACN. We found that both approaches can give excellent predictions under certain prerequisites. However, the first approach exhibits the notable advantage that it can be used effectively to predict retention times under any kind of phi-gradients at constant T or T-gradients at constant phi. The second approach has the advantage that it is relatively simple but its applicability is very restricted since its predictions are satisfactory only if the gradients are of the same kind with those used in the fitting procedure and the conditions lie within those used for fitting.     

Elution parameters in constant-pressure, single-ramp temperature-programmed gas chromatography

The dependence of the degree of interaction of a solute with the stationary phase at the time of its elution from the column in temperature-programmed GC is best described by interaction level of the solute. The latter represents the fraction of a solute residing in the stationary phase relative to the total amount of the solute. A simple approach to the evaluation of interaction levels of eluting solutes in a single-ramp temperature program is proposed. In a single-ramp temperature program having no preceding temperature plateau, all solutes that elute at temperatures that are about 60 degrees C higher than the initial temperature of the heating ramp elute with nearly the same interaction levels that can be found as exp(-r), where r is dimensionless heating rate. A specially designed temperature plateau preceding the ramp causes all solutes eluting during the entire time of the ramp to elute with nearly the same interaction levels equal to exp(-r). A transformation of the interaction level of a solute into its retention factor or mobility factor (a fraction of a solute in a mobile phase in relation to the total amount of the solute) and vice versa is also described.     

Multilinear gradient elution optimisation in reversed-phase liquid chromatography using genetic algorithms

The treatment presented in a recent paper [P. Nikitas, A. Pappa-Louisi, J. Chromatogr. A, 1068 (2005) 279] is extended to multilinear gradients, i.e. continuous gradients consisting of a certain number of linear portions. Thus, the experimental lnk versus phi curve, where k is the retention factor of a sample solute under isocratic conditions and phi is the volume fraction of the organic modifier in the water-organic mobile phase, is subdivided into a finite number of linear portions resulting in simple analytical expressions for the solute gradient retention time. These expressions of the retention time are directly used in an optimisation technique based on genetic algorithms. This technique involves first the determination of the theoretical dependence of k upon phi by means of gradient measurements, which in turn is used by the genetic algorithm for the prediction of the best gradient profile. The validity of the analytical expressions and the effectiveness of the optimisation technique were tested using fifteen underivatized amino acids and related compounds with mobile phases modified by acetonitrile. It was found that the adopted methodology exhibits significant advantages and it can lead to high quality predictions of the gradient retention times and optimisation results.     

Rapid identification of fatty acid methyl esters using a multidimensional gas chromatography-mass spectrometry database

A multidimensional approach for the identification of fatty acid methyl esters (FAME) based on GC/MS analysis is described. Mass spectra and retention data of more than 130 FAME from various sources (chain lengths in the range from 4 to 24 carbon atoms) were collected in a database. Hints for the interpretation of FAME mass spectra are given and relevant diagnostic marker ions are deduced indicating specific groups of fatty acids. To verify the identity of single species and to ensure an optimized chromatographic resolution, the database was compiled with retention data libraries acquired on columns of different polarity (HP-5, DB-23, and HP-88). For a combined use of mass spectra and retention data standardized methods of measurement for each of these columns are required. Such master methods were developed and always applied under the conditions of retention time locking (RTL) which allowed an excellent reproducibility and comparability of absolute retention times. Moreover, as a relative retention index system, equivalent chain lengths (ECL) of FAME were determined by linear interpolation. To compare and to predict ECL values by means of structural features, fractional chain lengths (FCL) were calculated and fitted as well. As shown in an example, the use of retention data and mass spectral information together in a database search leads to an improved and reliable identification of FAME (including positional and geometrical isomers) without further derivatizations.     

Compilation of an ESI-MS Library of β-Lactam Antibiotics for Rapid Identification of Drugs

β-Lactam antibiotics are among the most frequently used in clinical therapy. Thirty-three cephalosporin and eleven penicillin antibiotics frequently used in China have been used to create an ESI-MS library. A single-quadrupole mass analyzer was used for rapid identification. The product ions of these 44 β-lactam antibiotics were assigned to establish the fragmentation patterns and a standard ESI-MS library was established. Along with the specificity that product ion spectral information provides, the library provides another means to help identify β-lactam antibiotics. This is an important complement to the retention time from LC separations, and a valuable tool for confirming the identity of counterfeit drugs.     

Live retention database for identification of compounds in capillary gas chromatographic analysis --Principle and structure

A live retention database for compound identification in isothermal and any step temperature programmed capillary gas chromatography has been developed. The database utilizes the Kovats retention indices of compounds on a given stationary phase and the retention time of n-alkanes measured at isothermal conditions on the column to be used, together with the programming parameters. Identification is performed by search operation that compares the calculated results with the retention values of unknown peaks. Cross-reference of the search results of different operating conditions is performed automatically by the database in order to increase the reliability of the identification. The error of the database conversion is ≤± 0.5 index unit, or ≤± 1% on retention time. This paper describes the principle and the structure of the database in detail. The experimental results for different calsses of compounds tested at divers operating conditions will be presented in Part Ⅱ.     

梯度液相色谱保留时间公式在梯形梯度洗脱研究中的应用

根据前期得到的梯度液相色谱保留时间计算公式,在不指定溶剂强度模型形式的前提下,探讨了梯形梯度洗脱的一些特点。对于溶质在梯形梯度坡度上流出时的情形,推导得到溶质流出色谱柱所对应的流动相组成(φ_R)随梯度斜率(B)变化的表达式。该公式表明,在该情形中φ_R将会随着B值的增加而增加。对于溶质在梯形梯度最后一个等度区间流出时的情形,如果初始和终止流动相组成保持不变而仅有梯度的斜率发生变化时,从理论上证明了溶质保留时间(t_R)与梯度斜率的倒数(1/B)之间呈线性关系。实验中以C18色谱柱为固定相,甲醇-水为流动相,联苯为样品,测定了不同流动相组成以及梯形梯度条件下的保留时间,所得到的实验值与理论值吻合,从而验证了理论方法的正确性。     

Method to predict the bandwidth of elution profile under the linear gradient elution in reversed‐phase HPLC

Solute migration in a chromatographic column is an important consideration when designing batch or continuous chromatographic separation processes. Most design methods for the chromatographic processes are based on the equilibrium theory which concerns only the migration velocity of the solute. However, in real cases, it is important to predict the zone spreading which occurs by axial dispersion and mass transfer resistance. To predict the actual solute profiles in the column or effluent stream, numerical methods to solve nonlinear partial differential equations have been used. However, these methods involve much time and expense. In this work, two different rate factors are considered to predict the characteristics of the solute profiles. The first is solute migration velocity and the second is the zone spreading rate. The zone spreading rate can be estimated by the apparent axial dispersion coefficient which is obtained from the height of the equivalent theoretical plate in particular. Four benzene derivatives (benzene, toluene, p‐xylene, and acetophenone) were used as model solutes, and two mobile phase systems, water/methanol and water/ACN, were used in RP‐HPLC. The bandwidths and retention times of the solutes were predicted under several linear gradient conditions. The predicted and experimental bandwidths and retention times showed good agreement.     

Micellar enhanced ultrafiltration of phenolic derivatives from their mixtures

Micellar enhanced ultrafiltration (MEUF) of different phenolic derivatives including meta-nitrophenol (MNP), catechol (CC), para-nitrophenol (PNP), and beta-napthol (BN) in their binary mixture has been studied. A 1:1 ratio of the mixture of (i) MNP with CC and (ii) PNP with BN is taken for the MEUF experiments using a cationic surfactant, namely, cetyl(hexadecyl)pyridinium chloride (CPC). An organic polyamide membrane with molecular weight cutoff of 1000 is used. Experiments are conducted using an unstirred batch cell and a continuous cross-flow cell. The effects of various operating conditions, e.g., concentrations of surfactant and solute in the feed, transmembrane pressure drop, and cross-flow rate (for cross-flow experiments) on the permeate flux and the observed retention of each solute have been studied in detail. The retention of solutes without using the surfactant varies from 3 to 15% only at a typical feed solute concentration of 0.09 kg/m3, whereas, under the same operating pressure (345 kPa), retention at the end of the experiment increases to about 66 to 99.8% depending on the nature of solute in the batch cell using surfactant micelles (10 kg/m3). Retention of solutes is less in the case of the two-component feed solution compared to the single-component feed solution. An increase in flux to the range of 9 to 16% is realized in cross-flow experiments compared to batch cell flux after one hour of operation.