Multilinear Gradient Elution Research Articles

The development of retention time prediction model using multilinear gradient profiles of seven pharmaceuticals

The ICH guidance on pharmaceutical development recommends a systematic development approach including robustness studies which assure performance of manufacturing and analytical method development of drug product. The retention model by T. Kawabe et al have an excellent correlation between observed and predicted retention time in various kinds of pharmaceutical compounds during isocratic elution by the multiple regression modeling of solvent strength parameters. However, it cannot be successfully applied to the predictability of the retention time during multilinear gradient elution and also it does not consider the instrument dependent parameters such as dwell volume. The current study demonstrated that the solution of the fundamental gradient elution equation was applied to T. Kawabe’s retention time prediction model to predict the retention time using a multilinear gradient profile with taking the delay volume of HPLC system into account. Seven pharmaceutical compounds were used for evaluation of prediction models for retention time. The predicted retention time was compared with the measured retention time obtained by several multilinear gradient using two HPLC systems with different dwell volume. The evaluated prediction error (%) was 1.10 % and 1.54 % with H-Class and Nexera XR HPLC systems, respectively. In order to evaluate the robustness of the analytical method and to set the system suitability test (SST) for proper method performance, the design space for the ACN/MeOH mixture ratio in the total organic solvent and the full width at half maximum (FWHM) relationship to the minimum resolution was simulated by the developed retention time prediction. The optimized condition of the ACN/MeOH mixture ratio, the acceptance criterion of the SST for achieving the robust separation was estimated based on the simulated design space. As a conclusion, the developed retention time prediction will be useful during analytical method transfer among different manufacturing/analytical sites of the pharmaceuticals with different HPLC systems.

Computer aided optimization of multilinear gradient elution in liquid chromatography

Analytical expressions for retention time and peak compression factor are deduced by assuming quadratic solvent strength model and multilinear gradient elution. Based on these expressions, a program for the optimization of multilinear gradient profile is written with Visual Basic for Applications in Excel using genetic algorithm. The program is applied to search for a gradient profile for the separation of twelve compounds that are degraded from lignin. It is shown that the predicted and experimental chromatograms are well consistent. A better separation of the compounds is achieved under an S-shaped multilinear gradient profile than that obtained under linear gradient profile.

Retention prediction and separation optimization under multilinear gradient elution in liquid chromatography with Microsoft Excel macros

A package of Excel VBA macros have been developed for modeling multilinear gradient retention data obtained in single or double gradient elution mode by changing organic modifier(s) content and/or eluent pH. For this purpose, ten chromatographic models were used and four methods were adopted for their application. The methods were based on (a) the analytical expression of the retention time, provided that this expression is available, (b) the retention times estimated using the Nikitas-Pappa approach, (c) the stepwise approximation, and (d) a simple numerical approximation involving the trapezoid rule for integration of the fundamental equation for gradient elution. For all these methods, Excel VBA macros have been written and implemented using two different platforms; the fitting and the optimization platform. The fitting platform calculates not only the adjustable parameters of the chromatographic models, but also the significance of these parameters and furthermore predicts the analyte elution times. The optimization platform determines the gradient conditions that lead to the optimum separation of a mixture of analytes by using the Solver evolutionary mode, provided that proper constraints are set in order to obtain the optimum gradient profile in the minimum gradient time. The performance of the two platforms was tested using experimental and artificial data. It was found that using the proposed spreadsheets, fitting, prediction, and optimization can be performed easily and effectively under all conditions. Overall, the best performance is exhibited by the analytical and Nikitas-Pappa's methods, although the former cannot be used under all circumstances.

Optimisation of gradient elution with serially-coupled columns Part II: Multi-linear gradients

An interpretive approach is reported for the simultaneous optimisation of resolution and analysis time in the separation of complex samples by reversed-phase liquid chromatography, using serially-coupled columns of different lengths and nature, and multi-linear gradient elution. The results complement those reported in JCA 1350 (2014) 51 for single linear gradients, using combinations of five columns: three C18 columns of different characteristics, a cyano column and a phenyl column. The relationships between the logarithm of the retention factor (lnk) and the organic solvent content (φ), and between the peak half-widths and the retention times, for each analyte, were determined using isocratic retention times. From the established correlations, the gradient retention time was predicted using the fundamental equation for gradient elution (modified to be used for serially-coupled columns), and with this information, the peak profile. Two chromatographic goals (maximal peak purity and minimal analysis time) were simultaneously evaluated using the Pareto optimality concept. The rigorous predictive system can be easily applied to columns from different manufacturers and succeeds at different levels: single and combined columns, using isocratic and gradient elution. The computation time for gradient elution optimisation was significantly reduced to practical values with the help of genetic algorithms (GAs).

Analytical separation of enantiomers by gas chromatography on chiral stationary phases.

Analytical separation of enantiomers by gas chromatography on chiral stationary phases.

A multiresidual method based on ion-exchange chromatography with conductivity detection for the determination of biogenic amines in food and beverages

In the present work a sensitive and accurate method by ion chromatography and conductimetric detection has been developed for the determination of biogenic amines in food samples at microgram per kilogram levels. The optimized extraction procedure of trimethylamine, triethylamine, putrescine, cadaverine, histamine, agmatine, spermidine, and spermine from real samples, as well as the separation conditions based on a multilinear gradient elution with methanesulfonic acid and the use of a weak ionic exchange column, have provided excellent results in terms of resolution and separation efficiency. Extended calibration curves (up to 200 mg/kg, r > 0.9995) were obtained for all the analyzed compounds. The method gave detection limits in the range 23-65 μg/kg and quantification limits in spiked blank real samples in the range 65-198 μg/kg. Recovery values ranged from 82 to 103 %, and for all amines, a good repeatability was obtained with precision levels in the range 0.03-0.32 % (n = 4). The feasibility and potential of the method were tested by the analysis of real samples, such as tinned tuna fish, anchovies, cheese, wine, olives, and salami.

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.

Multilinear gradient elution optimization in reversed-phase liquid chromatography based on logarithmic retention models: Application to separation of a set of purines, pyrimidines and nucleosides

The analytical solutions of the fundamental equation of the multilinear gradient elution are derived in two cases, when the dependence of the logarithm of the solute retention (ln k) upon the volume fraction of organic modifier ( φ) is a three-parameter logarithmic expression, and when a simple linear relationship between ln k and ln φ is adopted. The derived theoretical expressions for retention times under multilinear gradient conditions are embodied to simple algorithms for fitting gradient data and especially for resolution optimization. Their performance was examined by using a mixture of 16 model compounds chosen among purines, pyrimidine and nucleosides in eluting systems modified by acetonitrile. It was found that the accuracy of the predicted gradient retention times is very satisfactory even if the simple logarithmic expression for the retention behavior of solutes, i.e. the linear dependence of ln k upon ln φ, is used.

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.

High Throughput Screening for the Design and Optimization of Chromatographic Processes: Automated Optimization of Chromatographic Phase Systems

AbstractChromatographic processes can be optimized in various ways. However, the two most prominent approaches are either based on statistical data analysis or on experimentally validated simulation models. Both approaches heavily rely on experimental data, the generation of which usually imposes a significant bottleneck on rational process design. Hence, here a closed‐loop optimization strategy is proposed in that an automated high throughput liquid handling platform is combined with a genetic algorithm. This setup enables process optimization on the mini‐scale and thus saves time as well as material costs. The practicability and robustness of the proposed high throughput method is demonstrated with two exemplary optimization tasks: first, optimization of the buffer composition in the capture step for a binary protein mixture (lysozyme and cytochrome), and second, optimization of multilinear gradient elution for the separation of a ternary mixture (ribonuclease and cytochrome, and lysozyme).

Application of Multilinear Gradient Elution for Optimization of Separation of Chlorophenols Using Derringer’s Desirability Function

Multilinear gradient elution was applied for the simultaneous optimization of resolution and analysis times of nine chlorophenols separated by HPLC. The first relationship of ln k versus φ was determined using the isocratic retention time for each analyte. For prediction of gradient retention times of analytes, the fundamental equation of gradient elution was numerically solved. Then a grid search program was used to simulate chromatograms under each new condition. Two different chromatographic goals, analysis time and minimum distance between adjacent peaks, were evaluated simultaneously using Derringer’s desirability function for each chromatogram. The sigmoid function was used to transform the optimization criteria to desirability values. Under optimal conditions, a good agreement was observed between predicted and experimental values of the chromatographic response function when analysis time was less than 40 min.

Optimisation of multilinear gradient elutions in reversed-phase liquid chromatography using ternary solvent mixtures

The multilinear gradient elution theory for binary mobile phases in reversed-phase liquid chromatography presented in [P. Nikitas, A. Pappa-Louisi, A. Papageorgiou, J. Chromatogr. A 1157 (2007) 178] is extended to ternary gradients. For the evaluation of this theory and the performance of the various fitting and optimisation algorithms we used 13 o-phthalaldehyde (OPA) derivatives of amino acids with mobile phases modified by acetonitrile and methanol. It is shown that the theory can lead to high quality predictions of the retention times under gradients elutions and optimisation of ternary gradients provided that we use a six-parameter expression for the logarithm of the retention factor, lnk, and the adjustable parameters of this expression are determined from ternary isocratic data.

Simple algorithms for fitting and optimisation for multilinear gradient elution in reversed-phase liquid chromatography

The theory of the multilinear gradient elution in reversed-phase liquid chromatography (RP-HPLC) presented in [P. Nikitas, A. Pappa-Louisi, P. Agrafiotou, J. Chromatogr. A 1120 (2006) 299] is modified to increase its flexibility. In addition, it is embodied to simple algorithms for fitting gradient data and especially for resolution optimisation under multilinear gradient conditions. In particular, two new algorithms for fitting and one for optimisation are tested and compared with conventional algorithms. Their performance was examined using 13 o-phthalaldehyde (OPA) derivatives of amino acids with mobile phases modified by acetonitrile. It was found that the new proposed algorithms, a repeated application of the Levenberg–Marquardt (LM) method for fitting (R_LM) and a modified descent algorithm for optimisation (RND_D), in combination with the modified theory of the multilinear gradient elution can lead to high quality predictions of the retention times and optimisation results.

IMPROVED MICRO-METHOD FOR THE HPLC ANALYSIS OF CAFFEINE AND ITS DEMETHYLATED METABOLITES IN HUMAN BIOLOGICAL FLUIDS AFTER SPE

An improved high performance liquid chromatographic-photodiode array method, using a multi-linear gradient elution, is described for the simultaneous determination of caffeine and its eight primary metabolites: xanthine (XA), 7-methylxanthine (7-MX), 3-methylxanthine (3-MX), 1-methylxanthine (1-MX), isocaffeine (IC), theobromine (TB), paraxanthine (PA) and theophylline (TP). The separation method Is based on mobile phase optimization and off-line solid-phase extraction (SPE) from human biological fluids: blood serum and urine. The eluting system consisted of ammonium acetate 0.05 M (pH=7) and methanol (90:10 v/v) changing to (40:60 v/v) over a period of 30 min. Identification of metabolites was achieved by photodiode array detector at 270 nm resulting in 2 ng limit of detection, while linearity held up to 20 ng/μL for each compound. Lamotrigine was used as internal standard at a concentration of 10.0 ng/μL. The statistical evaluation of the method was examined performing intra-day (n=8) and inter-day (n=8) and was found to be satisfactory with high accuracy and precision results. High extraction recoveries were achieved from blood serum and Furine using Merck C8 400 mg and Oasis HLB cartridges respectively, requiring small volumes, 40 μL of blood and 100 μL of urine. The separation was achieved on octylsilica, using a Silasorb C8, 10 μm, 250 × 4.6 mm analytical column at ambient temperature and proved to be highly selective, sensitive, reproducible and rapid regarding the nine compounds.

SIMULTANEOUS DETERMINATION OF METHYLURIC ACIDS IN BIOLOGICAL FLUIDS BY RP-HPLC ANALYSIS AFTER SOLID PHASE EXTRACTION

An automated reversed phase high performance liquid chromatographic (RP-HPLC) method, using a multi-linear gradient elution, is described for the simultaneous analysis of five methyluric acids: 7-methyluric acid (7-MU), 1-methyluric acid (1-MU), 1,3-di-methyluric acid (1,3-DMU), 1,7-di-methyluric acid (1,7-DMU), and 1,3,7-tri-methyluric acid (1,3,7-TMU). The separation method was based on mobile-phase optimization and off-line solid-phase extraction (SPE) from human biological fluids: blood serum and urine. The analytical column used was octylsilica, MZ-Kromasil, 5 μm, 250 × 4 mm i.d. Elution was performed at ambient temperature with a mixture of acetate buffer (pH = 3.5) and methanol at a volume ratio 95:5, changing to 30:70 v/v, over a period of 15 min. Isocaffeine was used as internal standard at a concentration of about 8 ng/μL. Detection was performed with a variable wavelength UV-visible detector at 280 nm, resulting in detection limits of 0.1 ng per 10 μL injection, while linearity held up to 5 ng/μL for each compound. The statistical evaluation of the method was examined performing intra day (n=8) and inter day calibration (n=8) and was found to be satisfactory, with high accuracy and precision results. High extraction recoveries, ranging from 89% to 106%, were achieved using Oasis HLB cartridges with methanol as eluent, requiring small volumes, 40 mL of blood serum and 100 mL of urine.

Solid-Phase Extraction Study and Photodiode Array RP-HPLC Analysis of Xanthine Derivatives in Human Biological Fluids

Abstract An automated reverse phase high performance liquid chromatography (HPLC)—photodiode array method using a multi linear gradient elution is described for the simultaneous analysis of nine xanthines: xanthine, 7-methylxanthine, 3-methylxanthine, 1-methylxanthine, isocaffeine, theobromine, paraxanthine, theophylline and caffeine. The separation method development was based on mobile-phase optimisation and off-line solid-phase extraction (SPE) from human biological fluids: blood serum and urine. Eluent consisted of 0.05 M CH3COONH4 and methanol (90:10 v/v) changing to (70:30 v/v) over a period of 20 min. Identification of xanthines was achieved by photodiode—array detector and quantitation was performed at 270 nm. Isocaffeine was used as internal standard at a concentration of 3.06 ng/μL. High extraction recoveries were achieved from Merck RP-18 cartridges using 1% hydrochloric acid as eluent, requiring small volumes, 40 μL of blood serum and 100 μL of urine. The separation of xanthines was achieved o...

Disappearance of Heme Metabolites Following Chelation Therapy with Meso 2,3 - Dimercaptosuccinic Acid (DMSA)

A lead poisoned adult (blood lead level 384 micrograms/dL) had a specific urinary porphyrin profile of elevated porphyrins including coproporphyrin III and 5-carboxylic acid derivatives. A multilinear gradient elution, modified with an ion-pair and a reversed phase column were employed for the separation of the diagnostically important porphyrin isomers. Fluorescence detection enhanced both sensitivity and selectivity. Both compounds were restored to normal levels following two courses of meso 2,3-dimercaptosuccinic acid: 90 mg/kg/d x 5 d at one month intervals. The decrease of clinical symptoms was associated with increase of delta aminolevulinic acid dehydratase from 53 to 230 U/mL blood and hemoglobin from 8.4 to 12.7 g/dL. Blood lead decreased from 384 to 24 micrograms/dL, urine lead from 1286 to 188 micrograms/L and urine coproporphyrin III from 5712 to 25 micrograms/L.