RESUMEN
Reversed-phase (RP) liquid chromatography is an important tool for the characterization of materials and products in the pharmaceutical industry. Method development is still challenging in this application space, particularly when dealing with closely-related compounds. Models of chromatographic selectivity are useful for predicting which columns out of the hundreds that are available are likely to have very similar, or different, selectivity for the application at hand. The hydrophobic subtraction model (HSM1) has been widely employed for this purpose; the column database for this model currently stands at 750 columns. In previous work we explored a refinement of the original HSM1 (HSM2) and found that increasing the size of the dataset used to train the model dramatically reduced the number of gross errors in predictions of selectivity made using the model. In this paper we describe further work in this direction (HSM3), this time based on a much larger solute set (1014 solute/stationary phase combinations) containing selectivities for compounds covering a broader range of physicochemical properties compared to HSM1. The molecular weight range was doubled, and the range of the logarithm of the octanol/water partition coefficients was increased slightly. The number of active pharmaceutical ingredients and related synthetic intermediates and impurities was increased from four to 28, and ten pairs of closely related structures (e.g., geometric and cis-/trans- isomers) were included. The HSM3 model is based on retention measurements for 75 compounds using 13 RP stationary phases and a mobile phase of 40/60 acetonitrile/25 mM ammonium formate buffer at pH 3.2. This data-driven model produced predictions of ln α (chromatographic selectivity using ethylbenzene as the reference compound) with average absolute errors of approximately 0.033, which corresponds to errors in α of about 3 %. In some cases, the prediction of the trans-/cis- selectivities for positional and geometric isomers was relatively accurate, and the driving forces for the observed selectivity could be inferred by examination of the relative magnitudes of the terms in the HSM3 model. For some geometric isomer pairs the interactions mainly responsible for the observed selectivities could not be rationalized due to large uncertainties for particular terms in the model. This suggests that more work is needed in the future to explore other HSM-type models and continue expanding the training dataset in order to continue improving the predictive accuracy of these models. Additionally, we release with this paper a much larger data set (43,329 total retention measurements) at multiple mobile phase compositions, to enable other researchers to pursue their own lines of inquiry related to RP selectivity.
Asunto(s)
Cromatografía de Fase Inversa , Interacciones Hidrofóbicas e Hidrofílicas , Cromatografía de Fase Inversa/métodos , Isomerismo , Preparaciones Farmacéuticas/química , Preparaciones Farmacéuticas/análisis , Modelos Químicos , Peso Molecular , Agua/químicaRESUMEN
Method development in comprehensive two-dimensional liquid chromatography (LC×LC) is a challenging process. The interdependencies between the two dimensions and the possibility of incorporating complex gradient profiles, such as multi-segmented gradients or shifting gradients, make trial-and-error method development time-consuming and highly dependent on user experience. Retention modeling and Bayesian optimization (BO) have been proposed as solutions to mitigate these issues. However, both approaches have their strengths and weaknesses. On the one hand, retention modeling, which approximates true retention behavior, depends on effective peak tracking and accurate retention time and width predictions, which are increasingly challenging for complex samples and advanced gradient assemblies. On the other hand, Bayesian optimization may require many experiments when dealing with many adjustable parameters, as in LC×LC. Therefore, in this work, we investigate the use of multi-task Bayesian optimization (MTBO), a method that can combine information from both retention modeling and experimental measurements. The algorithm was first tested and compared with BO using a synthetic retention modeling test case, where it was shown that MTBO finds better optima with fewer method-development iterations than conventional BO. Next, the algorithm was tested on the optimization of a method for a pesticide sample and we found that the algorithm was able to improve upon the initial scanning experiments. Multi-task Bayesian optimization is a promising technique in situations where modeling retention is challenging, and the high number of adjustable parameters and/or limited optimization budget makes traditional Bayesian optimization impractical.
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Algoritmos , Teorema de Bayes , Cromatografía Liquida/métodos , Plaguicidas/aislamiento & purificación , Plaguicidas/análisisRESUMEN
Hydroxypropyl methyl cellulose (HPMC) is a type of cellulose derivative with properties that render it useful in e.g. food, cosmetics, and pharmaceutical industry. The substitution degree and composition of the ß-glucose subunits of HPMC affect its physical and functional properties, but HPMC characterization is challenging due to its high structural heterogeneity, including many isomers. In this study, comprehensive two-dimensional liquid chromatography-mass spectrometry was used to examine substituted glucose monomers originating from complete acid hydrolysis of HPMC. Resolution between the different monomers was achieved using a C18 and cyano column in the first and second LC dimension, respectively. The data analysis process was structured to obtain fingerprints of the monomers of interest. The results revealed that isomers of the respective monomers could be selectively separated based on the position of substituents. The examination of two industrial HPMC products revealed differences in overall monomer composition. While both products contained monomers with a similar degree of substitution, they exhibited distinct regioselectivity.
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Derivados de la Hipromelosa , Glucosa/química , Glucosa/análisis , Hidrólisis , Derivados de la Hipromelosa/química , Isomerismo , Cromatografía Líquida con Espectrometría de MasasRESUMEN
High-throughput screening (HTS) workflows are revolutionizing many fields, including drug discovery, reaction discovery and optimization, diagnostics, sensing, and enzyme engineering. Liquid chromatography (LC) is commonly deployed during HTS to reduce matrix effects, distinguish isomers, and preconcentrate prior to detection, but LC separation time often limits throughput. Although subsecond LC separations have been demonstrated, they are rarely utilized during HTS due to limitations associated with the speed of common autosamplers. In this work, these limits are overcome by utilizing droplet microfluidics for sample introduction. In the method, a train of samples segmented by air are continuously pumped into the inlet of an LC injection valve that is actuated once each sample fills the sample loop. Coupled with 2.1 mm diameter × 5 mm long columns packed with 2.7 µm superficially porous C18 particles operated at 5 mL/min, the injector enabled separation of 3 components at 1 s/sample and analysis of a 96-well plate in 1.6 min with <2% peak area relative standard deviation. Analyte-dependent carryover was minimized by including wash droplets composed of organic solvent in between sample droplets. High-throughput LC coupled with mass spectrometric detection using the segmented flow injector was applied to a screen of inhibitors of a cytochrome P450-catalyzed hydroxylation reaction. Measurements of the reaction substrate and product concentrations made using fast LC with the segmented flow injector correlated well with measurements made using a more conventional, 3 min LC method. These results demonstrate the potential for droplet microfluidics to be used for sample introduction during high-throughput LC analysis.
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Microfluídica , Cromatografía Liquida/métodos , Espectrometría de Masas/métodosRESUMEN
The present work describes a re-parameterization of the Neue Kuss (NK) model for describing retention in liquid chromatography, and this re-parameterized model is used to fit a large set of isocratic retention measurements with improved convergence properties relative to the original parameterization of the model. Next, an experimental design for retention measurements using mobile phase gradient elution conditions is proposed for the purpose of obtaining accurate and precise NK parameters. Simulated retention data for mobile phase gradient elution conditions with two different levels of noise, as well as an essentially zero noise level were fit with the re-parameterized model. The results showed that the re-parameterized fits yielded average (absolute value) prediction errors for the parameters at the highest noise level of 7.2 % for S1,ref, 18 % for S2,ref and 6.2 % for kref (the re-parameterized NK model parameters). These errors were significantly smaller than those for the original parameterization of the NK model, where the errors were 23 % for S1, 25 % for S2 and 160 % for kw (the original NK model parameters). Furthermore, isocratic retention factors predicted using these model parameters were found to have an average magnitude of error of 0.51 % for the re-parameterized model, as opposed to 6800 % for the model with the original parameterization. A further test of this approach was carried out for independent experimental measurements for five solutes on a C18 column. The average magnitude of error of the isocratic retention factors predicted from parameters obtained from fits of gradient data was 1.6 %, provided that the range of organic solvent compositions that the solute sampled in the mobile phase gradient experiments was consistent with the isocratic experiments. These results indicate that the re-parameterization of the NK model allows for significant improvements in the fitting process, and that the proposed experimental design allows for NK parameters to be extracted from mobile phase gradient experiments, with prediction accuracies of isocratic retention factors on the order of 1-2 %.
Asunto(s)
Cromatografía de Fase Inversa , Proyectos de Investigación , Cromatografía Liquida/métodos , Solventes/química , Cromatografía Líquida de Alta Presión/métodosRESUMEN
Method development in comprehensive two-dimensional liquid chromatography (LC × LC) is a complicated endeavor. The dependency between the two dimensions and the possibility of incorporating complex gradient profiles, such as multi-segmented gradients or shifting gradients, renders method development by "trial-and-error" time-consuming and highly dependent on user experience. In this work, an open-source algorithm for the automated and interpretive method development of complex gradients in LC × LC-mass spectrometry (MS) was developed. A workflow was designed to operate within a closed-loop that allowed direct interaction between the LC × LC-MS system and a data-processing computer which ran in an unsupervised and automated fashion. Obtaining accurate retention models in LC × LC is difficult due to the challenges associated with the exact determination of retention times, curve fitting because of the use of gradient elution, and gradient deformation. Thus, retention models were compared in terms of repeatability of determination. Additionally, the design of shifting gradients in the second dimension and the prediction of peak widths were investigated. The algorithm was tested on separations of a tryptic digest of a monoclonal antibody using an objective function that included the sum of resolutions and analysis time as quality descriptors. The algorithm was able to improve the separation relative to a generic starting method using these complex gradient profiles after only four method-development iterations (i.e., sets of chromatographic conditions). Further iterations improved retention time and peak width predictions and thus the accuracy in the separations predicted by the algorithm.
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Algoritmos , Anticuerpos Monoclonales , Computadores , Espectrometría de Masas , Cromatografía LiquidaRESUMEN
Analytical data processing often requires the comparison of data, i.e. finding similarities and differences within separations. In this context, a peak-tracking algorithm was developed to compare multiple datasets in one-dimensional (1D) and two-dimensional (2D) chromatography. Two application strategies were investigated: i) data processing where all chromatograms are produced in one sequence and processed simultaneously, and ii) method optimization where chromatograms are produced and processed cumulatively. The first strategy was tested on data from comprehensive 2D liquid chromatography and comprehensive 2D gas chromatography separations of academic and industrial samples of varying compound classes (monoclonal-antibody digest, wine volatiles, polymer granulate headspace, and mayonnaise). Peaks were tracked in up to 29 chromatograms at once, but this could be upscaled when necessary. However, the peak-tracking algorithm performed less accurate for trace analytes, since, peaks that are difficult to detect are also difficult to track. The second strategy was tested with 1D liquid chromatography separations, that were optimized using automated method-development. The strategy for method optimization was quicker to detect peaks that were still poorly separated in earlier chromatograms compared to assigning a target chromatogram, to which all other chromatograms are compared. Rendering it a useful tool for automated method optimization.
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Algoritmos , Análisis de Datos , Cromatografía Liquida/métodos , Cromatografía de Gases/métodosRESUMEN
The importance of therapeutic peptides continues to increase in the marketplace for treating a range of diseases including diabetes and obesity. Quality control analyses for these pharmaceutical ingredients usually depends on reversed-phase liquid chromatography, and it is critically important to ensure that no impurities coelute with the target peptide at levels that would compromise the safety or effectiveness of the drug products. This can be challenging due to the broad range of properties of impurities that can be present on one hand (e.g., amino acid substitutions, chain cleavages, etc.), and the similarity of other impurities on the other hand (e.g., d-/l-isomers). Two-dimensional liquid chromatography (2D-LC) is a powerful analytical tool that is well suited to address this particular problem; the first dimension can be used to detect impurities over a broad range in properties, while the second dimension can be used to focus specifically on those species that might coelute with the target peptide in the first dimension. While hundreds of papers have been published on the use of 2D-LC for proteomics applications, there are very few papers that have focused on its use for characterisation of therapeutic peptides. This paper is the second in a two-part series. In Part I of the series, we studied several different column / mobile phase combinations that could be useful in 2D-LC separations of therapeutic peptides, with a focus on selectivity, peak shape, and complementarity to other combinations, particularly for isomeric peptides under mass spectrometry-friendly conditions (i.e., volatile buffers). In this second part in the series, we describe a strategy to derive second-dimension (2D) gradient conditions that both, ensure elution from the 2D column, and increase the likelihood of resolving peptides with very similar properties. We find that a two-step process yields conditions that place the target peptide in the middle of the 2D chromatogram. This process begins with two scouting gradient elution conditions in the second dimension of a 2D-LC system, followed by building and refining a retention model for the target peptide using a third separation. The process is shown to be generically useful by developing methods for four model peptides, and application to a sample of degraded model peptide to demonstrate its utility for resolving impurities in a real sample.
Asunto(s)
Cromatografía de Fase Inversa , Péptidos , Cromatografía de Fase Inversa/métodos , Cromatografía Liquida/métodos , Espectrometría de Masas/métodos , Péptidos/análisis , Preparaciones Farmacéuticas , Cromatografía Líquida de Alta Presión/métodosRESUMEN
The liquid chromatography (LC) analysis of small molecule pharmaceutical compounds and related impurities is crucial in the development of new drug substances, but developing these separations is usually challenging due to analyte structural similarities. Tandem-column LC (TC-LC) has emerged as a powerful approach to achieve alternative separation selectivity compared to conventional single column separations. However, one of the bottlenecks associated with use of tandem column approaches is time-consuming column pair screening and selection. Herein, we compared critical resolution (Rc) in single column vs. TC-LC separations for a given set of small molecule pharmaceutical compounds and developed a column selection workflow that uses separation simulations based on parameters from the hydrophobic subtraction model (HSM) of reversed-phase selectivity. In this study, HSM solute parameters were experimentally determined for a small molecule pharmaceutical (Linrodostat) and ten of its related impurities using multiple linear regression of their retentions on 16 selected RPLC columns against in-house determined HSM column parameters. Rc values were calculated based on HSM database column parameters for a pool of about 200 available stationary phases in both single-phase column (2.1 mm i.d. × 100 mm) or tandem column paired (two 2.1 mm i.d. × 50 mm) formats. Four column configurations (two single and two tandem) were predicted to achieve successful separations under isocratic HSM separation conditions, with a fifth tandem pair predicted to have a single co-elution. Of these five potential candidates, one tandem pair yielded compete baseline resolution of the 11-component mixture in an experimental separation. In this specific case, the tandem column pairs outperformed single-phase columns, with better predicted and experimental Rc values for the Linrodostat mixture under the HSM separation conditions. The results reported in this study demonstrated the enormous selectivity potential of TC-LC in pharmaceutical compound separations and are consistent with our previous study that examined the potential of tandem column approaches using purely computational means, though there is room for substantial improvement in the prediction accuracy. The proposed workflow can be used to prioritize a small number of column combinations by computational means before any experiments are conducted. This is highly attractive from the point of view of time and resource savings considering over 200,000 different tandem column pairings are possible using columns for which there are data in the HSM database.
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Cromatografía Líquida de Alta Presión , Cromatografía Líquida de Alta Presión/métodos , Cromatografía Liquida/métodos , Soluciones , Interacciones Hidrofóbicas e HidrofílicasRESUMEN
The current study describes the development of a 2D-LC-MS-based strategy for assessing main peak purity in the analysis of pharmaceutical peptides. The focus is on 2D-LC using reversed-phase (RP) separations in both dimensions, and particularly peptide isomer selectivity, since compounds with the same mass to charge ratio are not readily differentiated by mass spectrometry and therefore must be separated chromatographically. Initially, 30 column / mobile phase combinations were evaluated for both general separation performance (i.e., selectivity and peak shape) and isomer selectivity using forcibly degraded peptide samples and mixtures of synthetic diastereomers. A ranking of more than 300 UV and MS chromatograms suggests that when developing a new method, screening a set of four columns and four volatile mobile phases with differing characteristics should be adequate to both cover the selectivity space, and yield good separation performance. When 2D-LC-MS is to be used to evaluate peak purity for a new method, our results show that a second-dimension separation comprising a C8/C18 column possessing no ionic functionality, and an acetic acid / ammonium acetate mobile phase buffered at pH 5, provides good selectivity at 25 °C for peptide isomers with a MW <10 kDa. Retention data for 29 diverse peptides (1 < MW < 14 kDa, 3.7 < pI < 12.5) measured in this study using a variety of column and mobile phase conditions (i.e., 30 in total) are consistent with the classification of these various chromatographic conditions using the previously reported Peptide RPC Column Characterisation Protocol. For the investigated peptides trifluoroacetic acid was found to reduce selectivity differences between columns of diverse properties, probably due to its potential to form ion-pairs with peptides. Trifluoroacetic acid often improves peak shape for very large peptides (i.e. MW > 10 kDa). In the current dataset which also contain smaller peptides it received the highest ranking for 40% of the column and mobile phase combinations due to better selectivity and/or peak shape. The reported work here constitutes part one of a series of two papers. The second paper focuses on the use of retention modelling for rapid and accurate selection of the shallow gradients (i.e., << 1% ACN/min) required to obtain sufficient peptide isomer retention and separation in the second dimension. The overall results presented in this series of papers provides the guidance needed to develop a 2D-LC-MS method from start to finish for the analysis of main peak purity of therapeutic peptides.
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Cromatografía de Fase Inversa , Péptidos , Cromatografía de Fase Inversa/métodos , Ácido Trifluoroacético , Cromatografía Liquida/métodos , Espectrometría de Masas/métodos , Péptidos/análisis , Preparaciones Farmacéuticas , Cromatografía Líquida de Alta Presión/métodosRESUMEN
The majority of liquid chromatography (LC) methods are still developed in a conventional manner, that is, by analysts who rely on their knowledge and experience to make method development decisions. In this work, a novel, open-source algorithm was developed for automated and interpretive method development of LC(-mass spectrometry) separations ("AutoLC"). A closed-loop workflow was constructed that interacted directly with the LC system and ran unsupervised in an automated fashion. To achieve this, several challenges related to peak tracking, retention modeling, the automated design of candidate gradient profiles, and the simulation of chromatograms were investigated. The algorithm was tested using two newly designed method development strategies. The first utilized retention modeling, whereas the second used a Bayesian-optimization machine learning approach. In both cases, the algorithm could arrive within 4-10 iterations (i.e., sets of method parameters) at an optimum of the objective function, which included resolution and analysis time as measures of performance. Retention modeling was found to be more efficient while depending on peak tracking, whereas Bayesian optimization was more flexible but limited in scalability. We have deliberately designed the algorithm to be modular to facilitate compatibility with previous and future work (e.g., previously published data handling algorithms).
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Algoritmos , Quimiometría , Teorema de Bayes , Cromatografía Liquida/métodos , Espectrometría de Masas/métodosRESUMEN
Multi-dimensional liquid chromatography techniques play an important role in the analysis of complex mixtures. The keys to maximizing peak capacity in these methods are fast sampling rates and sufficient complementarity between the first- (1D) and second- (2D) dimension separations. One way that these criteria have been met is by using 2D parallel column arrays. This review covers demonstrations of this approach in the literature that have been published over the past three decades. Two or more identical 2D columns can be operated in a sequential order to permit increased separation times and higher peak capacities in the second dimension without the concomitant decrease in sampling rate. The parallel column arrays can also be operated simultaneously to reduce total analysis time. Columns with different stationary phase chemistries can be used in the 2D column array to increase complementarity by utilizing specific stationary phases for various first dimension fractions. More recently, this type of platform has been used to automate the development of two-dimensional (2D) achiral-chiral LC methods. These strategies, as well as recent efforts toward the development of integrated, spatial multi-dimensional LC devices that include parallel column arrays, are discussed here.
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Mezclas Complejas , Cromatografía Liquida/métodosRESUMEN
Efforts to model and simulate various aspects of liquid chromatography (LC) separations (e.g., retention, selectivity, peak capacity, injection breakthrough) depend on experimental retention measurements to use as the basis for the models and simulations. Often these modeling and simulation efforts are limited by datasets that are too small because of the cost (time and money) associated with making the measurements. Other groups have demonstrated improvements in throughput of LC separations by focusing on "overhead" associated with the instrument itself - for example, between-analysis software processing time, and autosampler motions. In this paper we explore the possibility of using columns with small volumes (i.e., 5 mm x 2.1 mm i.d.) compared to conventional columns (e.g., 100 mm x 2.1 mm i.d.) that are typically used for retention measurements. We find that isocratic retention factors calculated for columns with these dimensions are different by about 20%; we attribute this difference - which we interpret as an error in measurements based on data from the 5 mm column - to extra-column volume associated with inlet and outlet frits. Since retention factor is a thermodynamic property of the mobile/stationary phase system under study, it should be independent of the dimensions of the column that is used for the measurement. We propose using ratios of retention factors (i.e., selectivities) to translate retention measurements between columns of different dimensions, so that measurements made using small columns can be used to make predictions for separations that involve conventional columns. We find that this approach reduces the difference in retention factors (5 mm compared to 100 mm columns) from an average of 18% to an average absolute difference of 1.7% (all errors less than 8%). This approach will significantly increase the rate at which high quality retention data can be collected to thousands of measurements per instrument per day, which in turn will likely have a profound impact on the quality of models and simulations that can be developed for many aspects of LC separations.
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Programas Informáticos , Cromatografía Líquida de Alta Presión/métodos , Cromatografía Liquida/métodos , Simulación por Computador , Indicadores y ReactivosRESUMEN
The use of chemometric methods based on the analysis of variances (ANOVA) allows evaluation of the statistical significance of the experimental factors used in a study. However, classical multivariate ANOVA (MANOVA) has a number of requirements that make it impractical for dealing with metabolomics data. For this reason, in recent years, different options have appeared that overcome these limitations. In this work, we evaluate the performance of three of these multivariate ANOVA-based methods (ANOVA simultaneous component analysis-ASCA, regularized MANOVA-rMANOVA, and Group-wise ANOVA-simultaneous component analysis-GASCA) in the framework of metabolomics studies. Our main goals are to compare these various ANOVA-based approaches and evaluate their performance on experimentally designed metabolomic studies to find the significant factors and identify the most relevant variables (potential markers) from the obtained results. Two experimental data sets were generated employing liquid chromatography coupled to mass spectrometry (LC-MS) with different complexity in the design to evaluate the performance of the statistical approaches. Results show that the three considered ANOVA-based methods have a similar performance in detecting statistically significant factors. However, relevant variables pointed by GASCA seem to be more reliable as there is a strong similarity with those variables detected by the widely used partial least squares discriminant analysis (PLS-DA) method.
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Metabolómica , Análisis de Varianza , Cromatografía Liquida/métodos , Espectrometría de Masas/métodos , Metabolómica/métodos , Análisis MultivarianteRESUMEN
The continuous interest in discovering new bioactive molecules derived from natural products (NP) has stimulated the development of improved screening assays to help overcome challenges in NP-based drug discovery. Here, we describe a unique platform for the online screening of acetylcholinesterase inhibitors without the need for pre-treating the sample. In the current study, we have demonstrated the ability to combine reversed-phase separation with a capillary immobilized enzyme reactor (cIMER) in two-dimensional liquid chromatography system coupled with mass spectrometry detection. We systematically investigated the effects of method parameters that are of practical significance and are known to affect the enzyme assay and interfere in the analysis such as: bioreactor dimensions, loop sizes, amount of immobilized enzyme, second dimension flow rates, reaction time, substrate concentration, presence of organic modifier, limit of detection and signal suppression. The performance of this new platform was evaluated using a mixture containing three known AChE inhibitors (tacrine, galanthamine and donepezil) and an ethanolic extract obtained from the dry bulbs of Hippeastrum calyptratum (Amaryllidaceae) was investigated to provide a proof of concept of the applicability of the platform for the analysis of complex mixtures such as those derived from NPs.
RESUMEN
At the forefront of chemistry and biology research, development timelines are fast-paced and large quantities of pure targets are rarely available. Herein, we introduce a new framework, which is built upon an automated, online trapping-enrichment multi-dimensional liquid chromatography platform (TE-Dt-mDLC) that enables: 1)â highly efficient separation of complex mixtures in a first dimension (1 D-UV); 2)â automated peak trapping-enrichment and buffer removal achieved through a sequence of H2 O and D2 O washes using an independent pump setup; and 3)â a second dimension separation (2 D-UV-MS) with fully deuterated mobile phases and fraction collection to minimize protic residues for immediate NMR analysis while bypassing tedious drying processes and minimizing analyte degradation. Diverse examples of target isolation and characterization from organic synthesis and natural product chemistry laboratories are illustrated, demonstrating recoveries above 90 % using as little as a few micrograms of material.
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Productos Biológicos , Cromatografía Liquida , Espectroscopía de Resonancia Magnética , SolventesRESUMEN
Continued adoption of two-dimensional liquid chromatography (2D-LC) in industrial laboratories will depend on the development of approaches to make method development for 2D-LC more systematic, less tedious, and less reliant on user expertise. In this paper, we build on previous efforts in these directions by describing the use of multifactorial modeling software that can help streamline and simplify the method development process for 2D-LC. Specifically, we have focused on building retention models for second dimension (2D) separations involving variables including gradient time, temperature, organic modifier blending, and buffer concentration using LC simulator (ACD/Labs) software. Multifactorial retention modeling outcomes are illustrated as resolution map planes or cubes that enable straightforward location of 2D conditions that maximize resolution while minimizing analysis time. We also illustrate the practicality of this approach by identifying conditions that yield baseline separation of all compounds co-eluting from a first dimension (1D) separation using a single combination of 2D stationary phase and elution conditions. The multifactorial retention models were found to be very accurate for both the 1D and 2D separations, with differences between experimental and simulated retention times of less than 0.5%. Pharmaceutical applications of this approach for multiple heartcutting 2D-LC were demonstrated using IEC-IEC or achiral RPLC-chiral RPLC for 2D separations of multicomponent mixtures. The framework outlined here should help make 2D-LC method development more systematic and streamline development and optimization for a variety of 2D-LC applications in both industry and academia.
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Cromatografía Liquida , Simulación por ComputadorRESUMEN
Closed form expressions for the prediction of retention times and peak widths for gradient liquid chromatography are particularly useful in understanding, rationalizing and optimizing separations. These expressions are obtained by integrating differential equations, in conjunction with a model of the variation of the retention factor as a function of mobile phase composition. Two of these models, the linear solvent strength (LSS) model and the Neue-Kuss (NK) model are explored in the present work. Here, we expand on these closed form expressions to account for effects of sample volume overload and a mismatch between the sample solvent and the initial mobile phase composition for the gradient. We show that there have been errors in expressions reported in the literature, and we have evaluated the accuracy of the predictions from the closed form expressions reported here using a recently developed liquid chromatography simulator. The expressions assume a constant plate height and consider elution across four zones of the gradient profile - elution in the sample solvent, elution in the initial (isocratic) mobile phase caused by the gradient delay volume, elution during a linear gradient, and elution post-gradient at the final (isocratic) mobile phase composition. The expressions generally give reasonably accurate predictions for retention times and peak widths, except for cases where the solute elutes during transitions between the different zones. The average magnitude of the prediction errors for retention time and peak width relative to simulation were 0.093% and 0.40% for the LSS expressions for ten amphetamine solutes at 36 different separation conditions, and 0.22% and 1.8% for the NK expressions for eight alkylbenzene solutes at 36 different separation conditions, respectively.
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Cromatografía Liquida , Simulación por Computador , Solventes , Cromatografía Liquida/métodos , Indicadores y Reactivos , Modelos Lineales , Solventes/químicaRESUMEN
A peak-tracking algorithm was developed for use in comprehensive two-dimensional liquid chromatography coupled to mass spectrometry. Chromatographic peaks were tracked across two different chromatograms, utilizing the available spectral information, the statistical moments of the peaks and the relative retention times in both dimensions. The algorithm consists of three branches. In the pre-processing branch, system peaks are removed based on mass spectra compared to low intensity regions and search windows are applied, relative to the retention times in each dimension, to reduce the required computational power by elimination unlikely pairs. In the comparison branch, similarity between the spectral information and statistical moments of peaks within the search windows is calculated. Lastly, in the evaluation branch extracted-ion-current chromatograms are utilized to assess the validity of the pairing results. The algorithm was applied to peptide retention data recorded under varying chromatographic conditions for use in retention modelling as part of method optimization tools. Moreover, the algorithm was applied to complex peptide mixtures obtained from enzymatic digestion of monoclonal antibodies. The algorithm yielded no false positives. However, due to limitations in the peak-detection algorithm, cross-pairing within the same peaks occurred and six trace compounds remained falsely unpaired.
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Algoritmos , Anticuerpos Monoclonales/análisis , Cromatografía Liquida/métodos , Péptidos/análisis , Espectrometría de Masas/métodos , Reconocimiento de Normas Patrones Automatizadas , Estándares de ReferenciaRESUMEN
It is common practice in liquid chromatography to split the flow of the effluent exiting the analytical column into two or more parts, either to enable parallel detection (e.g., coupling the separation to two destructive detectors such as light scattering and mass spectrometry (MS)), or to accommodate flow rate limitations of a detector (e.g., electrospray ionization mass spectrometry). In these instances the user must make choices about split ratio and dimensions of connecting tubing that is used between the split point and the detector, however these details are frequently not mentioned in the literature, and rarely justified. In our own work we often split the effluent following the second dimension (2D) column in two-dimensional liquid chromatography systems coupled to MS detection, and we have frequently observed post 2D column peak broadening that is larger than we would expect to result from dispersion in the MS ionization source itself. For the present paper we describe a series of experiments aimed at understanding the impact of the split ratio and post-split connecting tubing dimensions on dispersion of peaks exiting an analytical column. We start with the simple idea - based on the principle of conservation of mass - that analyte peaks entering the split point are split into two parts such that the analyte mass (and thus peak volume) entering and exiting the split point is conserved, and directly related to the ratio of flow rates entering and exiting the split point. Measurements of peak width and variance after the split point show that this simple view of the splitting process - along with estimates of additional dispersion in the post-split tubing - is sufficient to predict peak variances at the detector with accuracy that is sufficient to guide experimental work (median error of about 10% over a wide range of conditions). We feel it is most impactful to recognize that flow splitting impacts apparent post-column dispersion not because anything unexpected happens in the splitting process, but because the split dramatically reduces the volume of the analyte peak, which then is more susceptible to dispersion in connecting tubing that would not cause significant dispersion under conditions where splitting is not implemented. These results will provide practitioners with a solid basis on which rational decisions about split ratios and dimensions of post-split tubing can be made.