Application of new methodologies based on design of experiments, independent component analysis and design space for robust optimization in liquid chromatography

HPLC separations of an unknown sample mixture and a pharmaceutical formulation have been optimized using a recently developed chemometric methodology proposed by W. Dewé et al. in 2004 and improved by P. Lebrun et al. in 2008. This methodology is based on experimental designs which are used to model retention times of compounds of interest. Then, the prediction accuracy and the optimal separation robustness, including the uncertainty study, were evaluated. Finally, the design space (ICH Q8(R1) guideline) was computed as the probability for a criterion to lie in a selected range of acceptance. Furthermore, the chromatograms were automatically read. Peak detection and peak matching were carried out with a previously developed methodology using independent component analysis published by B. Debrus et al. in 2009. The present successful applications strengthen the high potential of these methodologies for the automated development of chromatographic methods.     

Direct injection determination of benzoylecgonine, heroin, 6-monoacetylmorphine and morphine in serum by MLC

A simple and sensitive direct injection chromatographic procedure is developed for the determination of heroin, two of its metabolites (morphine and 6-monoacetylmorphine (6-MAM)), and benzoylecgonine (a metabolite of cocaine) in serum samples. The proper resolution of the four substances is obtained with a chemometrics approach, where the retention is modelled as a first step using the retention factors obtained in a limited number of mobile phases. Afterwards, an optimisation criterion that takes into account the position and shape of the chromatographic peaks is applied. The mobile phase selected to carry out the analysis was 0.1 mol L(-1) SDS-4% (v/v) butanol buffered at pH 7, in which the separation is performed in less than 18 min. The limits of quantification were in the 17-36 ng mL(-1) range. Intra- and inter-day assay accuracy and precision (below 3%) were obtained following ICH guidelines. The method developed was applied to the determination of the drugs studied in serum samples with good recoveries (90-104%). Serum samples from subjects that have been ingested cocaine and heroin were also analysed. The samples were injected directly in the chromatographic system without any pretreatment.     

Development of a generic micellar electrokinetic chromatography method for the separation of 15 antimalarial drugs as a tool to detect medicine counterfeiting

Since antimalarial drugs counterfeiting is dramatically present on the African market, the development of simple analytical methods for their quality control is of great importance. This work consists in the CE analysis of 15 antimalarials (artesunate, artemether, amodiaquine, chloroquine, piperaquine, primaquine, quinine, cinchonine, mefloquine, halofantrine, sulfadoxine, sulfalen, atovaquone, proguanil, and pyrimethamine). Since all these molecules cannot be ionized at the same pH, MEKC was preferred because it also allows separation of neutral compounds. Preliminary experiments were first carried out to select the most crucial factors affecting the antimalarials separation. Several conditions were tested and four parameters as well as their investigation domain were chosen: pH (5-10), SDS concentration (20-90 mM), ACN proportion (10-40%), and temperature (20-35°C). Then, the experimental design methodology was used and a central composite design was selected. Mathematical modeling of the migration times allowed the prediction of optimal conditions (29°C, pH 6.6, 29 mM SDS, 36% ACN) regarding analyte separation. The prediction at this optimum was verified experimentally and led to the separation of 13 compounds within 8 min. Finally, the method was successfully applied to the quality control of African antimalarial medicines for their qualitative and quantitative content.     

Integrated quality by design (QbD) and design of experiments (DoE) approach for UFLC determination of telaprevir in rat serum

An ultrafast liquid chromatographic bioanalytical method was developed and validated for the determination of telaprevir in Wistar albino rat serum. Principles of quality by design (QbD) were implemented for enhancing the bioanalytical liquid–liquid extraction of telaprevir from rat serum. A Box–Behnken design was utilized in the studies by selecting extraction time, centrifugation speed, and vortex time as the critical method variables for evaluating their effect on the critical analytical attribute, i.e., %recovery of telaprevir. Chromatographic separation was achieved within a run time of 10?min using a C-18 column and mobile phase comprising of methanol:borate buffer of pH 9 (90:10 v/v) flowing at 1.2?mL/min. Photodiode array detection was performed at 270?nm. Results of validation studies were satisfactory. The method was linear over a concentration of 25–10,000?ng/mL. Limit of detection for the developed method was 10?ng/mL. Further, design of experiments (DoE) used for inter-day accuracy and precision study suggested superior method reliability. This integrated QbD- and DoE-based approach ensured the development of a validated and reliable analytical method for optimum bioanalysis of telaprevir in biological matrix.     

Fast HPLC method for the determination of glimepiride, glibenclamide, and related substances using monolithic column and flow program

This work presents a fast method for the simultaneous separation and determination of glimepiride, glibenclamide, and two related substances by RP LC. The separation was performed on a Chromolith Performance (RP-18e, 100 mm x 4.6 mm) column. As mobile phase, a mixture of phosphate buffer pH 3, 7.4 mM, and ACN (55:45 v/v) was used. Column oven temperature was set to 30 degrees C. The total chromatographic run time was 80 s. This was achieved using a flow program from 5 to 9.9 mL/min. Precisions of the interday and the intraday assay for both retention times and peak areas for the four analyzed compounds were less than 1.2%. The method showed good linearity and recovery. The short analysis time makes the method very valuable for quality control and stability testing of drugs and their pharmaceutical preparations.     

Design of experiment techniques for the optimization of chromatographic analysis conditions: A review

Design of experiment (DoE) techniques have been widely used in the field of chromatographic parameters optimization as a valuable tool. A systematic literature review of the available DoE techniques applied to the development of a chromatographic analysis method is presented in this paper. First, the most common available designs and the implementation steps of DoE are comprehensively introduced. Then the studies in recent 10 years for the application of DoE techniques in various chromatographic techniques are discussed, such as capillary electrophoresis, liquid chromatography, gas chromatography, thin-layer chromatography, and high-speed countercurrent chromatography. Current problems and future outlooks are finally given to provide a certain inspiration of research in the application of DoE techniques to the different chromatographic techniques field. This review contributes to a better understanding of the DoE techniques for the efficient optimization of chromatographic analysis conditions, especially for the analysis of complex systems, such as multicomponent drugs and natural products.     

An experimental design based strategy to optimize a capillary electrophoresis method for the separation of 19 polycyclic aromatic hydrocarbons

Because of their high toxicity, international regulatory institutions recommend monitoring specific polycyclic aromatic hydrocarbons (PAHs) in environmental and food samples. A fast, selective and sensitive method is therefore required for their quantitation in such complex samples. This article deals with the optimization, based on an experimental design strategy, of a cyclodextrin (CD) modified capillary zone electrophoresis separation method for the simultaneous separation of 19 PAHs listed as priority pollutants. First, using a central composite design, the normalized peak-start and peak-end times were modelled as functions of the factors that most affect PAH electrophoretic behavior: the concentrations of the anionic sulfobutylether-β-CD and neutral methyl-β-CD, and the percentage of MeOH in the background electrolyte. Then, to circumvent computational difficulties resulting from the changes in migration order likely to occur while varying experimental conditions, an original approach based on the systematic evaluation of the time intervals between all the possible pairs of peaks was used. Finally, a desirability analysis based on the smallest time interval between two consecutive peaks and on the overall analysis time, allowed us to achieve, for the first time in CE, full resolution of all 19 PAHs in less than 18 min. Using this optimized capillary electrophoresis method, a vegetable oil was successfully analyzed, proving its suitability for real complex sample analysis.     

Chromatographic elution process design space development for the purification of saponins in Panax notoginseng extract using a probability‐based approach

A Monte Carlo method was used to develop the design space of a chromatographic elution process for the purification of saponins in Panax notoginseng extract. During this process, saponin recovery ratios, saponin purity, and elution productivity are determined as process critical quality attributes, and ethanol concentration, elution rate, and elution volume are identified as critical process parameters. Quadratic equations between process critical quality attributes and critical process parameters were established using response surface methodology. Then probability‐based design space was computed by calculating the prediction errors using Monte Carlo simulations. The influences of calculation parameters on computation results were investigated. The optimized calculation condition was as follows: calculation step length of 0.02, simulation times of 10 000, and a significance level value of 0.15 for adding or removing terms in a stepwise regression. Recommended normal operation region is located in ethanol concentration of 65.0–70.0%, elution rate of 1.7–2.0 bed volumes (BV)/h and elution volume of 3.0–3.6 BV. Verification experiments were carried out and the experimental values were in a good agreement with the predicted values. The application of present method is promising to develop a probability‐based design space for other botanical drug manufacturing process.     

HPLC-PDA analysis of ACE-inhibitors,hydrochlorothiazide and indapamide utilizing design of experiments

A simple, rapid, precise, accurate and sensitive high performance liquid chromatographic method has been developed for simultaneous determination of ACE inhibitors with hydrochlorothiazide and indapamide in pharmaceutical formulations. ‘Design of Experiments’ (DoE) using ‘central composite design’ (CCD) was applied to facilitate method development and optimization. Mobile phase was optimized utilizing response surface methodology using Design Expert software. Chromatographic separation was achieved on Hypersil®-Gold C₁₈ (100 × 4.6 mm, 3 μm, Thermo Fisher Scientific, USA), column at 25 °C. The mobile phase was 58% buffer (5 mM KH₂PO₄, containing triethylamine 0.25 ml/L), 25% acetonitrile and 17% methanol (pH adjusted to 2.8 ± 0.1). The analysis was performed at 215 nm. The mobile phase flow rate was 1.0 ml/min and injection volume 10 μl. The method was validated for linearity, limits of quantitation and detection, accuracy, precision, ruggedness and robustness as per the International Conference on Harmonization (ICH) guidelines. Calibration curves (for lisinopril, hydrochlorothiazide, captopril, imidapril, perindopril, indapamide and trandolapril) were linear in the concentration range of 5–35 μg/ml. The limit of detection and limit of quantitation for experimental drugs ranged from 0.03 to 0.61 and 0.08–1.84 μg/ml respectively.     

Multi-objective optimisation strategy based on desirability functions used for chromatographic separation and quantification of l-proline and organic acids in vinegar

An optimisation strategy based on desirability functions together with experimental design has been used to optimise a chromatographic method applied to the separation and quantification of l-proline and seven organic acids in vinegar samples. Chromatographic problems often force to reach a compromise between different experimental variables in order to achieve the best chromatographic separation. The importance of the use of multi-objective optimisation methods lies in the ability to cope with this kind of problems. The quality of the multicriteria optimisation method was tested through the validation of the analytical parameters of the final chromatographic method developed. The versatility of this methodology allows to use it in other chromatographic applications resulting in a suitable adaptive procedure to solve new analytical problems. Furthermore, the determination of l-proline and organic acids in vinegar is useful for several industrial goals such as the correct monitoring of fermentation, for the study of nutrient needs at all times during the twofold fermentation process and for the detection of possible adulterations in the final product.     

Modelling of GC and HPLC separations of simazin and atrazin by experimental design methodology

Summary Experimental Design methodology allows the modelling and optimization of the chromatographic separation of similar pesticides (triazine family) by GC and HPLC. The GC separation of simazin and atrazin is well modelled by a first degree equation, involving injected volume, carrier gas pressure and rising oven temperature. The LC is modelled by a second degree equation, depending on injected volume, eluent flow and composition. These calculated models allow easy optimization of the separations, using isoresponse curves.     

应用质量源于设计的理念优化大鼠血浆中大黄蒽醌的分析方法

应用质量源于设计理念建立一种高效液相色谱-荧光检测法(HPLC-FLD)用于测定大鼠血浆中5种大黄蒽醌。用Plackett-Burman设计考察流动相中甲醇含量、pH值、流速、柱温和进样体积对色谱峰的分离度、理论塔板数、最末洗脱峰的保留时间和拖尾因子的影响,结果显示流动相中甲醇含量、流速和柱温对色谱系统的影响显著(p<0.05)。继而采用Box-Behnken设计结合响应面法考察上述三因素对分离度、保留时间和理论塔板数的影响。用Derringer渴求函数评价了响应值的综合作用。得出最优色谱条件为:以甲醇-0.1%(v/v)磷酸水溶液(81.4:18.6, v/v)为流动相等度洗脱,流速1.1 mL/min,柱温31℃,荧光检测激发波长为440 nm,发射波长为540 nm。建立的模型显示良好的预测性。结果表明:质量源于设计的理念可有效地应用于优化高效液相色谱分析方法。     

Computational chemical analysis of the retention of acidic drugs on a pentyl-bonded silica gel in reversed-phase liquid chromatography

A fast method to obtain a quantitative structure-retention relationship is required in chromatography for the rapid optimization of chromatographic separation conditions. Chromatographic data of acidic drugs are analyzed by a computational chemical method to simulate chromatographic simulation. The direct interaction between a model phase and a drug is calculated as an energy value using the molecular mechanics calculation of CAChe. Computational chemistry using a model adsorbent is a new method for quantitative analysis of retention in reversed-phase liquid chromatography. The correlation coefficient is 0.878 (n = 19) between the retention factors of acidic drugs and interaction energy values of the final structure (DeltaFS) between an acidic drug and model pentyl-bonded phase.     

Quality by Design: Design of Experiments Approach Prior to the Validation of a Stability-Indicating HPLC Method for Montelukast

This paper describes a systematic design of experiments (DoE) approach by applying the principle of quality by design (QbD) to determine the design space for a stability-indicating HPLC method prior to validation. By employing DoE, a simultaneous multivariate approach was carried out for mobile phase pH, flow rate, percentage of organic content and column temperature. A two-level fractional factorial design (2^4?1 + 2 center points = 10 experiments) was employed and statistical analysis of the experimental data uncovered the significant influential chromatographic factors. The experimental data for USP tailing and resolution were analyzed statistically to screen the chromatographic factors. This approach determined the most influential chromatographic factors. During this process, inferences were evaluated from various data tables, for example, analysis of variance, summary of fit, lack of fit, and parameter estimates. The study also explained various plots such as actual vs. predicted plot, Pareto plot, and prediction profiler. The acceptable range of the chromatographic factors was displayed as a Contour plot defining the ‘design space’ of the method. The range of operating conditions that guarantee a satisfactory QbD was deduced to finalize the method prior to validation. The method is simple, rapid, and robust for the determination of montelukast in montelukast sodium oral granules dosage form. The method was validated according to ICH guidelines for accuracy, precision, linearity, range, specificity, ruggedness and robustness (one factor varied at a time). The method has been successfully transferred to the quality control department for quality analysis of manufactured batches and stability samples.     

Simple and Efficient Solution for Robustness Testing in Gradient Elution Liquid Chromatographic Methods

In this paper, an efficient way for robustness testing of gradient elution liquid chromatographic methods is proposed and tested on model mixtures comprising cilazapril, hydrochlorothiazide, and their degradation products, solutes that differ not only in polarities, but also in solubility and absorption characteristics. In general, the robustness could be tested with respect to various responses: resolution, retention factor, selectivity factor, change of detector response, etc. In chromatographic methods, the separation of the adjacent peaks is mandatory, and, consequently, the resolution is usually used as response. In isocratic elution methods, the resolution threshold depends on many factors, such as sizes of adjacent peaks, peak shapes, and asymmetry factor. At the same time, the situation is even more complex in gradient elution methods, because separation depends on a larger number of parameters, such as gradient profile, column geometry, mobile phase flow rate, column equilibration between gradient runs, etc. To ensure baseline separation, the authors propose application of separation criterion (s) as response and indirect modeling in the robustness evaluation. Examined response in this approach is represented by the difference between the retention time of the beginning of the peak and the retention time of the end of the previously eluting peak of the critical pair. Moreover, the proposed methodology included reusing experiments from the optimization phase to define a robust chromatographic region without increasing the number of experiments. It was shown that method robustness can be easily and efficiently evaluated by this methodology.     

Optimization by orthogonal array design of ion-pair HPLC separation of the enzymatic hydrolysis products of yeast RNA

<正>The separation of enzymatic hydrolysis products of yeast RNA by ion-pair HPLC was studied.A modified chromatographic response function(MCRF) was proposed to appraise the effectiveness of chromatographic separation.This function takes the number of peaks,resolution and the retention time of the last peak into consideration.It shows advantages for optimization of HPLC separation of complex mixtures.An orthogonal array design was used to separate the hydrolysate of yeast RNA and the optimal chromatographic conditions were obtained.     

Integration of high speed microwave chemistry and a statistical ‘design of experiment’ approach for the synthesis of the mitotic kinesin Eg5 inhibitor monastrol

The rapid preparation of the mitotic kinesin Eg5 inhibitor monastrol has been performed using multicomponent chemistry and combining microwave-assisted synthesis and a statistical design of experiments (DoEs) approach. By variation of the solvent, catalyst type and concentration, reaction time, and temperature a matrix of experiments for the DoE method was derived. As a result of 29 experiments with reaction times ranging from 10 to 30 min, an optimized procedure was derived that allowed the preparation of monastrol in 82%.     

Establishing the chromatographic fingerprint of traditional Chinese medicine standard decoction based on quality by design approach: A case study of Licorice

A novel analytical quality by design approach for developing a chromatographic fingerprint was established for analyzing complex traditional Chinese medicine, using a licorice standard decoction as an example. Considering the characteristics of integrity and ambiguity, the resolution of eight common peaks, total peak number, capacity factor distributions, and peak purity were selected as potential critical method attributes for assessing the quality of the chromatographic fingerprint. A central composite design was used to evaluate the relationship between critical method attributes and critical method parameters, including column temperature, wavelength, flow rate, formic‐acid concentration, and gradient parameters. A standard probability method was employed to calculate the design space of the fingerprint analysis parameters and evaluate the robustness of the methodology. The optimized high‐performance liquid chromatography fingerprint conditions were acetonitrile and 0.1% formic acid water gradient elution (0‐5 min, 5–19% A; 5–10 min, 19% A; 10–50 min, 19–42% A; 50–54 min, 42–100% A; 54–60 min, 100% A), column temperature 25±5°C, detection wavelength 265 nm. The design space of fingerprint analytical method based on the analytical quality by design approach not only met the requirements of the fingerprint analysis, but also improved the robustness and applicability of the fingerprint method.     

Quality by Design: Design of Experiments Approach Prior to the Validation of a Stability-Indicating HPLC Method for Montelukast

This paper describes a systematic design of experiments (DoE) approach by applying the principle of quality by design (QbD) to determine the design space for a stability-indicating HPLC method prior to validation. By employing DoE, a simultaneous multivariate approach was carried out for mobile phase pH, flow rate, percentage of organic content and column temperature. A two-level fractional factorial design (2⁴⁻¹ + 2 center points = 10 experiments) was employed and statistical analysis of the experimental data uncovered the significant influential chromatographic factors. The experimental data for USP tailing and resolution were analyzed statistically to screen the chromatographic factors. This approach determined the most influential chromatographic factors. During this process, inferences were evaluated from various data tables, for example, analysis of variance, summary of fit, lack of fit, and parameter estimates. The study also explained various plots such as actual vs. predicted plot, Pareto plot, and prediction profiler. The acceptable range of the chromatographic factors was displayed as a Contour plot defining the ‘design space’ of the method. The range of operating conditions that guarantee a satisfactory QbD was deduced to finalize the method prior to validation. The method is simple, rapid, and robust for the determination of montelukast in montelukast sodium oral granules dosage form. The method was validated according to ICH guidelines for accuracy, precision, linearity, range, specificity, ruggedness and robustness (one factor varied at a time). The method has been successfully transferred to the quality control department for quality analysis of manufactured batches and stability samples.

     

Classification of high-speed gas chromatography-mass spectrometry data by principal component analysis coupled with piecewise alignment and feature selection

A useful methodology is introduced for the analysis of data obtained via gas chromatography with mass spectrometry (GC-MS) utilizing a complete mass spectrum at each retention time interval in which a mass spectrum was collected. Principal component analysis (PCA) with preprocessing by both piecewise retention time alignment and analysis of variance (ANOVA) feature selection is applied to all mass channels collected. The methodology involves concatenating all concurrently measured individual m/z chromatograms from m/z 20 to 120 for each GC-MS separation into a row vector. All of the sample row vectors are incorporated into a matrix where each row is a sample vector. This matrix is piecewise aligned and reduced by ANOVA feature selection. Application of the preprocessing steps (retention time alignment and feature selection) to all mass channels collected during the chromatographic separation allows considerably more selective chemical information to be incorporated in the PCA classification, and is the primary novelty of the report. This methodology is objective and requires no knowledge of the specific analytes of interest, as in selective ion monitoring (SIM), and does not restrict the mass spectral data used, as in both SIM and total ion current (TIC) methods. Significantly, the methodology allows for the classification of data with low resolution in the chromatographic dimension because of the added selectivity from the complete mass spectral dimension. This allows for the successful classification of data over significantly decreased chromatographic separation times, since high-speed separations can be employed. The methodology is demonstrated through the analysis of a set of four differing gasoline samples that serve as model complex samples. For comparison, the gasoline samples are analyzed by GC-MS over both 10-min and 10-s separation times. The successfully classified 10-min GC-MS TIC data served as the benchmark analysis to compare to the 10-s data. When only alignment and feature selection was applied to the 10-s gasoline separations using GC-MS TIC data, PCA failed. PCA was successful for 10-s gasoline separations when the methodology was applied with all the m/z information. With ANOVA feature selection, chromatographic regions with Fisher ratios greater than 1500 were retained in a new matrix and subjected to PCA yielding successful classification for the 10-s separations.