A new approach to the effective preparation of plasma samples for rapid drug quantitation using on-line solid phase extraction mass spectrometry.

A procedure that permits rapid development of an optimized solid phase extraction (SPE) method for the analysis of drugs in plasma by on-line solid phase extraction-mass spectrometry (SPE-MS) has been developed. This procedure employs the concept of manipulating the pH and the percentage of organic solvent in the chromatographic mobile phase to affect the retention behaviors of both the matrix components and the analytes of interest. This resulted in the effective removal of matrix interferences from biological samples during SPE. During a the method development, only generic HPLC gradient approaches were needed, and multiple samples were pooled so that several SPE methods could be investigated at once. The analysis time per sample was 1.3 minutes. Thus, the time involved in the entire method development (analysis of a set of samples) was less than one hour. With the knowledge of the retention behaviors of the analytes with respect to the pH and the percentage of organic, it was then possible to compose an optimized SPE-MS method. This method consisted of a base/organic and then an acid/organic washing step, followed by a rapid gradient elution step. Due to the rigorous washing procedure, most matrix interferences were removed, and analytes eluted off the SPE sorbent suffered from very little matrix interference. Thus, quantitation of drugs in plasma by a single quadrupole mass spectrometer could be accomplished, something that was not possible when only a generic gradient was used for on-line SPE-MS. In addition, both external and internal calibration curves could be obtained for the concentration range from 5 to 500 ng/mL with correlation coefficients of 0.99 (using 1/x as a weighting factor) and relative standard deviations (RSDs) less than 10%. The results achieved were comparable to those obtained by the use of a triple quadrupole mass spectrometer. Moreover, the robustness of the method was tested by continuously injecting plasma samples. During 136 runs, the absolute peak area variation for these three basic drugs was less than 15% without taking the signal variation from the mass spectrometer into account. Significantly, the on-line developed method can be directly transferred to a 96-well format SPE plate.     

Theoretical background of monolithic short layer ion-exchange chromatography for separation of charged large biomolecules or bioparticles

The retention and peak spreading in linear gradient elution of charged large biomolecules were investigated by using numerical simulations. Oligo-DNA separation by monolithic anion-exchange chromatography was chosen as a model system. The peak width and the retention were well predicted by using the parameters obtained by gradient elution experiments at different gradient slopes. As the distribution coefficient at the peak retention volume K_R decreases with increasing molecular size, the peak became sharper for larger DNAs. This is due to very large effective charge (binding site) values of large DNAs (20–60). The peak width was well correlated with K_R based on the model equation developed for linear gradient elution of proteins. It was shown that the monolithic disk is best suited for very large charged biomolecule separations at high flow velocities with shallow gradients slopes.     

Surfactant-mediated hydrophobic interaction chromatography of proteins: gradient elution

Addition of 3-[(3-cholamidopropyl)dimethylammonio]-l-propanesulphonate (CHAPS) to mobile phases in gradient elution hydrophobic interaction chromatography (HIC) on SynChropak Propyl causes changes in observed elution times for nine globular proteins. The nine proteins showed different percentage reductions in capacity factor, k', demonstrating the ability of CHAPS to change the selectivity of the separations. Three basic types of gradient experiments have been explored for surfactant-mediated gradient elution HIC. Type I gradients are conducted with constant salt and variable surfactant concentration. Type II gradients with variable salt and constant surfactant concentration, and Type III gradients with variable salt and surfactant concentrations. By the criterion of a linear relationship between gradient time and retention time the linear solvent strength condition applies to Type II and Type III gradients. Type III gradients, with the fastest re-equilibration time, are preferable for repetitive analyses. Type I gradients are relatively ineffective in making use of the solvent strength of CHAPS, and Types I and II gradients require long equilibration times due to large changes in surface concentration of CHAPS which occur during elution. The presence of CHAPS had a negligible effect on peak shapes of the proteins examined, except for bovine serum albumin which yielded a narrower, less distorted peak in the presence of CHAPS.     

Prediction of protein retention at gradient elution conditions in ion-exchange chromatography.

This work presents a prediction procedure for protein retention in ion-exchange chromatography, where two linear gradient experiments of different length give the protein retention time at other linear gradients. The procedure predicts the retention time of early and late eluting proteins with similar precision and predictions by extrapolation deviate approximately 3% or less from the experimental retention times. By using the ionic strength, this procedure predicts protein retention times obtained with divalent ions in the eluent more accurately than a well-established procedure that uses the protein co-ion concentration.     

Modeling of salt and pH gradient elution in ion‐exchange chromatography

The separation of proteins by internally and externally generated pH gradients in chromatofocusing on ion‐exchange columns is a well‐established analytical method with a large number of applications. In this work, a stoichiometric displacement model was used to describe the retention behavior of lysozyme on SP Sepharose FF and a monoclonal antibody on Fractogel SO₃ (S) in linear salt and pH gradient elution. The pH dependence of the binding charge B in the linear gradient elution model is introduced using a protein net charge model, while the pH dependence of the equilibrium constant is based on a thermodynamic approach. The model parameter and pH dependences are calculated from linear salt gradient elutions at different pH values as well as from linear pH gradient elutions at different fixed salt concentrations. The application of the model for the well‐characterized protein lysozyme resulted in almost identical model parameters based on either linear salt or pH gradient elution data. For the antibody, only the approach based on linear pH gradients is feasible because of the limited pH range useful for salt gradient elution. The application of the model for the separation of an acid variant of the antibody from the major monomeric form is discussed.     

Prediction of peptide retention times in normal-phase liquid chromatography with only a single gradient run.

Previous studies of peptide separation by normal-phase liquid chromatography have shown a linear relationship between the logarithm of the capacity factor and the logarithm of the volume fraction of modifier in the mobile phase. This permitted the use of a model to predict isocratic and gradient retention times based on data obtained by two initial gradient runs. In the present study, chromatographic behavior of 25 peptides in normal-phase liquid chromatography with isocratic elution have been studied and a linear relationship between the slope (S) and intercept [log k(0)] was obtained. This relationship was combined with the algorithm of prediction reported in the previous paper. The prediction of peptide retention times with only a single experimental gradient retention data was investigated.     

Retention prediction of a set of amino acids under gradient elution conditions in hydrophilic interaction liquid chromatography

The analysis of amino acids presents significant challenges to contemporary analytical separations. The present paper investigates the possibility of retention prediction in hydrophilic interaction chromatography (HILIC) gradient elution based on the analytical solution of the fundamental equation of the multilinear gradient elution derived for reversed‐phase systems. A simple linear dependence of the logarithm of the solute retention (ln k) upon the volume fraction of organic modifier (φ) in a binary aqueous‐organic mobile is adopted. Utility of the developed methodology was tested on the separation of a mixture of 21 amino acids carried out with 14 different gradient elution programs (from simple linear to multilinear and curved shaped) using ternary eluents in which a mixture of methanol and water (1:1, v/v) was the strong eluting member and acetonitrile was the weak solvent. Starting from at least two gradient runs, the prediction of solute retention obtained under all the rest gradients was excellent, even when curved gradient profiles were used. Development of such methodologies can be of great interest for a wide range of applications.     

梯度液相色谱中任意等度、线性和阶梯梯度组合下的保留时间公式

基于线性溶剂强度模型,应用特征线分析的方法求解梯度洗脱模式下的理想液相色谱模型。在考虑到梯度延迟时间会对溶质的保留时间造成影响的情况下,得到适合于梯度液相色谱中任意等度、线性和阶梯梯度组合条件下的保留时间推导公式。应用这些公式计算任意的梯度条件下的保留时间,并将得到的结果与数值计f算的结果进行比较,二者完全一致,从而验证了推导得到的保留时间公式的正确性。由于这些公式具有形式简单、适用范围广等优点,因此可方便地应用于实际应用中,具有较高的实用价值。     

Characterization and prediction of retention in gradient-elution normal phase HPLC with ternary gradients

Summary Chromatographic behaviour of phenylurea herbicides and phenols on a silica gel column with ternary gradient elution using mobile phases containing dry 2-propanol, n-heptane and dioxane was investigated. With dried solvents and the temperature controlled to ±0.1 °C, the data from repeated experiments measured on the same column after ten months of use were reproducible. Algorithms were suggested for characterization and prediction of retention in ternary gradients at a constant ratio of the two polar solvents (elution strength gradients) and at a constant sum of their concentrations (selectivity gradients). These were based on two- and three-parameter equations describing the dependence of the sample retention factors (isocratic) on the concentration of the polar solvent in binary mobile phases. Presented at the 21^st ISC held in Stuttgart, Germany, 15^th–20^th September, 1996     

Multi-linear gradient elution optimization for separation of phenylthiohydantoin amino acids using pareto optimality method

Multi-linear gradient elution was applied for simultaneous optimization of resolution and analysis times for ten phenylthiohydantoin amino acids (PTH-AAs) in liquid chromatography. Relation of lnK upon φ for each analyte was determined using isocratic retention time data, and gradient retention time of analytes was predicted using fundamental equation of gradient elution. Then a grid search program was used to predict retention time of solutes in variable space. Two different chromatographic goals-analysis time and minimum difference between adjacent peaks- were simultaneously evaluated using Pareto optimality method. Gradient program in optimum condition was: initially 24% CH₃OH/Water for 10 min, linear ramp to 34% over 5 min, to 29% over 5 min, and to 70% over 20 min. The average of calculated relative error in the prediction of the retention time in optimal conditions was -1.67% that shows a good agreement between predicted and experimental values of the chromatographic retention time in optimal condition.     

Steady-migration retention characteristics of peptides under gradient elution: application towards a dynamic separation method for minor-adjustments of the retention of peptides in RPLC

Minor-adjustment of the retention of peptides, induced by varying the mobile phase flow-rate(MPF-R), is a new dynamic separation method for simultaneously and rapidly identifying and improving the selectivity of hidden and overlapping peptide peaks. It can also-stabilize the reverse elution order of some pair-peaks under gradient elution in reverse phase liquid chromatography. The retention characteristics of peptides under gradient elution in RPLC was firstly found to be dominated by two variables of the steady region(SR) and migration region(MR). The changes in peptide retention induced by varying the MPF-R can be attributed to changes in the rate of bond breaking of multiple molecular interactions of peptides from the SR and of the mass transfer of peptides from the stationary phase to the mobile phase in the MR. The two dynamic variables were also found to independently depend on the type of peptide. Desirable results were obtained using six standard oligopeptides and a real sample of trypsin-digested lysozyme.It is expected that the quality control of peptide drugs, high dispersion of peptide peaks in peptide mapping and "bottom-up MS"in proteomics will be improved by this method, even enabling peptide purification on a preparative scale in industry.     

高效液相色谱法测定非天然氨基酸的光学纯度

 以（1- 氟-2,4-二硝基苯基）-5-L-缬氨 酰胺为手性试剂、反相高效液相色谱法测定非天然氨基酸的光学 纯度。梯度洗脱,流动相A：含体积分数为0.1％的三氟乙酸的乙腈,流动相B：体积分 数为0.1％的三氟乙酸水溶液。L-和D-对映体得到良好分离。准确测定了25种非天然 氨基酸L-和D-对映体的光学纯度。     

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。     

Evaluation of the retention dependence on the physicochemical properties of solutes in reversed-phase liquid chromatographic linear gradient elution based on linear solvation energy relationships

This paper describes the results of the evaluation of retention dependence on the physicochemical properties of solutes in linear gradient elution by reversed-phase liquid chromatography (RPLC) based on linear solvation energy relationships (LSERs). Retention time data on Inertsil ODS(3) column by linear gradient elution were collected for both acetonitrile-water and methanol-water binary mobile phases under various gradient steepness. Based on the LSERs, the retention times were linearly correlated with the physicochemical properties (size, dipolarity, and hydrogen bond donor-acceptor acidity and basicity) of solutes. As predicted by LSERs, very acceptable linear relationships are observed for both mobile phases. While the magnitudes of the coefficients are modified by the gradient steepness, their signs are consistent with those obtained by isocratic elution. As obtained for isocratic elution, the dominant factors to retention in linear gradient elution of RPLC are the solutes' size and hydrogen bond acceptor basicity. The conclusions of the study allow us to predict retention in chromatographic method development by gradient elution.     

HPLC of nucleotides. II. General methods and their development for analysis and preparative separation. An approach to selectivity control

In order to develop efficient separation methods for nucleotides according to their size and heterocyclic composition, the application of ion-exchange, reverse-phase, and normal-phase adsorption HPLC has been studied. The comparative investigation of retention power and selectivity of various packings (non-polar bonded-phase and amino silicas) in relation to nucleotide length and composition yields data which enable suitable packings to be selected and a method of preparing the new packing for a particular separation problem to be formulated. Thus a new anion exchanger with high selectivity and dynamic mass transfer has been prepared for fractionation of large oligonucleotides. The effect of the eluent pH and composition (organic modifier, salt) on retention, selectivity, and resolution in ion-exchange and reverse-phase HPLC has been studied. The optimum separation conditions comprise elution with oppositely directed gradients of the salt and the modifier, use of a precolumn packing that provides the best protection for the main column without loss of its efficiency, and the optimum gradient program for the desired retention of the component of interest. The relation between loading and sample concentration has been studied and the system for gradient elution improved. Our work shows that two-dimensional separation is the most reliable and informative method for preparation of homogeneous oligonucleotides. The hydrophobic-pair ion-exchange mechanism is proposed for ion-pair chromatography. Protected and partially deblocked oligonucleotides, chemically synthesized for genetic engineering studies, have been separated with high selectivity by adsorption (normal-phase) HPLC which is efficient for gradient elution with isohydric eluents. The analysis of a monomeric composition of nano-(pico-) molar amounts of oligonucleotides has been developed; the procedure involves microcolumn digestion of the oligonucleotides with immobilized enzymes followed by microcolumn separation of the nucleoside-mononucleotide mixture. Also, a new slurry method for packing stable HPLC columns with a tightly consolidated, nonshrinkable bed of particles has been developed.     

HPLC Analysis of complex mixtures of triglycerides using gradient elutions and an ultraviolet detector

Summary A method of reserved-phase HPLC analysis for mixtures of triglycerides (TG's) that provides good resolution at acceptable analysis times for high-ECN TG's has been developed. An elution gradient of methyltert-butyl-ether (MTBE) in acetonitrile (ACN) was used with an ultraviolet detector operated at 215nm. The effect of the proportion of MTBE in the mobile phase, gradient time, temperature and sample solvent on TG retention and resolution was studied. Linear relationships were derived between the logarithm of the capacity factor (log k'), and the logarithm of selectivity (log α) and the above-mentioned chromatographic factors. The conditions selected were: an elution gradient of from 23 to 30% MTBE, an elution gradient time of 25 minutes, and a temperature of 30°C, which provided good resolution of soybean oil TG's in less than 30 minutes.     

Application of artificial neural networks for gradient elution retention modelling in ion chromatography

Gradient elution in ion chromatography (IC) offers several advantages: total analysis time can be significantly reduced, overall resolution of a mixture can be increased, peak shape can be improved (less tailing) and effective sensitivity can be increased (because there is little variation in peak shape). More importantly, it provides the maximum resolution per time unit. The aim of this work was the development of a suitable artificial neural network (ANN) gradient elution retention model that can be used in a variety of applications for method development and retention modelling of inorganic anions in IC. Multilayer perceptron ANNs were used to model the retention behaviour of fluoride, chloride, nitrite, sulphate, bromide, nitrate and phosphate in relation to the starting time of gradient elution and the slope of the linear gradient elution curve. The advantage of the developed model is the application of an optimized two-phase training algorithm that enables the researcher to make use of the advantages of first- and second-order training algorithms in one training procedure. This results in better predictive ability, with less time required for the calculations. The number of hidden layer neurons and experimental data points used for the training set were optimized in terms of obtaining a precise and accurate retention model with respect to minimization of unnecessary experimentation and time needed for the calculation procedures. This study shows that developed, ANNs are the method of first choice for retention modelling of inorganic anions in IC.     

Computer-assisted transfer of programmed elutions in reversed-phase high-performance liquid chromatography

Computer-assisted procedures were used to simulate modifications in chromatograms caused by the transfer of elution programmes between instruments with significantly different dwell volumes. Moreover, for the first time the same approach was used to modify the elution programmes to match the chromatograms produced in the different instruments. The process may consist of making minor modifications to gradient programmes or transforming the original gradient programme into a stepwise gradient profile and/or the simultaneous programming of flow and solvent composition. The combination of these approaches has been shown to have an enormous potential for producing matched chromatograms in instrumental systems with dwell volumes that differ by several millilitres. The efficiency and robustness of the proposed procedure is demonstrated with a variety of compounds (two different mixtures of 10 and 11 analytes), mobile phases (methanol and acetonitrile gradients), flow rates (0.5-1.5 mL/min range), temperatures (35-45 degrees C interval) and gradient profiles (linear, multilinear, curved and stepwise).     

New uniform algorithm to predict reversed phase retention values under different gradient conditions

A new numerical emulation algorithm was established to calculate retention parameters in RP-HPLC with several retention times under different linear or nonlinear binary gradient elution conditions and further predict the retention time under any other binary gradient conditions. A program was written according to this algorithm and nine solutes were used to test the program. The prediction results were excellent. The maximum relative error of predicted retention time was less than 0.45%.     

Biomarkers of alcohol abuse in racehorses by liquid chromatography/tandem mass spectrometry

A rapid and sensitive method was developed for the screening, quantification and confirmation of ethyl glucuronide (EG) and ethyl sulfate (ES) as biomarkers for alcohol administration to racehorses using liquid chromatography coupled on-line with triple quadrupole tandem mass spectrometry. Urine sample aliquots (0.1 mL) were pre-treated by protein precipitation. Separation of EG and ES was achieved on an Ultra PFP column. Isocratic elution with a flush step was performed using 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B). Analysis was performed by negative electrospray ionization in multiple reaction monitoring mode. The retention times for EG and ES were 1.7 +/- 0.30 and 3.4 +/- 0.30 min, respectively. The internal standard used was d(5)-ethyl glucuronide with a retention time of 1.7 +/- 0.30 min. The entire separation was completed in <5 min. The limit of detection (LOD) and of quantification (LOQ) for both analytes were 100 ng/mL (S/N > or =3) and 500 ng/mL, respectively. The limit of confirmations (LOC) for EG and ES were 500 ng/mL and 1.0 microg/mL, respectively. The assay was linear over a concentration range of 0.5-100 microg/mL (r(2) > 0.995). Intra- and inter-day accuracy and precision were less than 15%. The analytes were stable in urine for 24 h at room temperature, 10 days at 4 degrees C and 21 days at -20 degrees C and -70 degrees C. Ion suppression or enhancement due to matrix effect was negligible. The measurement uncertainty was <14% for EG and <25% for ES. This method was successfully used for the quantification of EG and ES in urine samples following alcohol administration to research horses and for screening and confirmation of EG and ES in urine samples obtained from racehorses post-competition. The method is simple, rapid, inexpensive, and reliably reproducible.