Optimization via experimental design of an SPE-HPLC-UV-fluorescence method for the determination of valsartan and its metabolite in human plasma samples

A chemometric approach was applied for the optimization of the extraction and separation of the antihypertensive drug valsartan and its metabolite valeryl-4-hydroxy-valsartan from human plasma samples. Due to the high number of experimental and response variables to be studied, fractional factorial design (FFD) and central composite design (CCD) were used to optimize the HPLC-UV-fluorescence method. First, the significant variables were chosen with the help of FFD; then, a CCD was run to obtain the optimal values for the significant variables. The measured responses were the corrected areas of the two analytes and the resolution between the chromatographic peaks. Separation of valsartan, its metabolite valeryl-4-hydroxy-valsartan and candesartan M1, used as internal standard, was made using an Atlantis dC18 100 mm x 3.9 mm id, 100 angstroms, 3 microm chromatographic column. The mobile phase was run in gradient elution mode and consisted of ACN with 0.025% TFA and a 5 mM phosphate buffer with 0.025% TFA at pH 2.5. The initial percentage of ACN was 32% with a stepness of 4.5%/min to reach the 50%. A flow rate of 1.30 mL/min was applied throughout the chromatographic run, and the column temperature was kept to 40+/-0.2 degrees C. In the SPE procedure, experimental design was also used in order at achieve a maximum recovery percentage and extracts free from plasma interferences. The extraction procedure for spiked human plasma samples was carried out using C8 cartridges, phosphate buffer (pH 2, 60 mM) as conditioning agent, a washing step with methanol-phosphate buffer (40:60 v/v), a drying step of 8 min, and diethyl ether as eluent. The SPE-HPLC-UV-fluorescence method developed allowed the separation and quantitation of valsartan and its metabolite from human plasma samples with an adequate resolution and a total analysis time of 1 h.     

Optimization of the separation of a group of triazine herbicides by micellar capillary electrophoresis using experimental design and artificial neural networks

The micellar electrokinetic chromatography separation of a group of triazine compounds was optimized using a combination of experimental design (ED) and artificial neural network (ANN). Different variables affecting separation were selected and used as input in the ANN. A chromatographic exponential function (CEF) combining resolution and separation time was used as output to obtain optimal separation conditions. An optimized buffer (19.3 mM sodium borate, 15.4 mM disodium hydrogen phosphate, 28.4 mM SDS, pH 9.45, and 7.5% 1-propanol) provides the best separation with regard to resolution and separation time. Besides, an analysis of variance (ANOVA) approach of the MEKC separation, using the same variables, was developed, and the best capability of the combination of ED-ANN for the optimization of the analytical methodology was demonstrated by comparing the results obtained from both approaches. In order to validate the proposed method, the different analytical parameters as repeatability and day-to-day precision were calculated. Finally, the optimized method was applied to the determination of these compounds in spiked and nonspiked ground water samples.     

Determination of hydroxy acids in cosmetics by chemometric experimental design and cyclodextrin‐modified capillary electrophoresis

A CD‐modified CE method was established for quantitative determination of seven hydroxy acids in cosmetic products. This method involved chemometric experimental design aspects, including fractional factorial design and central composite design. Chemometric experimental design was used to enhance the method's separation capability and to explore the interactions between parameters. Compared to the traditional investigation that uses multiple parameters, the method that used chemometric experimental design was less time‐consuming and lower in cost. In this study, the influences of three experimental variables (phosphate concentration, surfactant concentration, and methanol percentage) on the experimental response were investigated by applying a chromatographic resolution statistic function. The optimized conditions were as follows: a running buffer of 150 mM phosphate solution (pH 7) containing 0.5 mM CTAB, 3 mM γ‐CD, and 25% methanol; 20 s sample injection at 0.5 psi; a separation voltage of ?15 kV; temperature was set at 25°C; and UV detection at 200 nm. The seven hydroxy acids were well separated in less than 10 min. The LOD (S/N = 3) was 625 nM for both salicylic acid and mandelic acid. The correlation coefficient of the regression curve was greater than 0.998. The RSD and relative error values were all less than 9.21%. After optimization and validation, this simple and rapid analysis method was considered to be established and was successfully applied to several commercial cosmetic products.     

Quality by Design enabled enhanced bioanalytical extraction and UFLC determination of vilazodone from rat serum

An ultrafast liquid chromatographic bioanalytical method was developed and validated for the determination of vilazodone in Wistar rat serum. Principles of quality by design were implemented for enhancing the bioanalytical liquid–liquid extraction of vilazodone 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 analytical attribute, i.e., %recovery of vilazodone. Chromatographic separation was achieved within a run time of 10?min using a C-18 column and mobile phase comprising of methanol:phosphate buffer of pH 7 (85:15 v/v) flowing at 1.5?mL/min. Photodiode array detection was performed at 242?nm. Results of validation studies were satisfactory. The method was linear over a concentration of 100–2,000?ng/mL with acceptable accuracy and precision. Limits of detection and quantitation for the developed method were 50 and 100?ng/mL, respectively. This QbD-based approach was found suitable for routine bioanalysis of vilazodone in the biological matrix.     

Computer-assisted method development and optimization in high-performance liquid chromatography

This paper reports the use of DryLab, a computer simulation software package, to assist in the development and optimization of a reversed-phase high-performance liquid chromatographic (HPLC) method for the separation of a model drug candidate and its degradation products. Prior to the optimization process, columns with various bonded phases are evaluated for their chromatographic performance using the sample of interest. Simultaneous optimization of two separation variables and the use of resolution maps to predict the optimal conditions are illustrated. Options to optimize column conditions (column length and flow-rate) to further reduce run time are briefly discussed. The accuracy of DryLab-predicted retention times and resolution is compared with experimental values. The DryLab software used in this study provided satisfactory predictions for the selected model, with average errors of less than 3.5 and 11.8% for retention time and resolution, respectively.     

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.     

Rational experimental design for bioanalytical methods validation illustration using an assay method for total captopril in plasma

Generally, bioanalytical chromatographic methods are validated according to a predefined programme and distinguish a pre-validation phase, a main validation phase and a follow-up validation phase. In this paper, a rational, total performance evaluation programme for chromatographic methods is presented. The design was developed in particular for the pre-validation and main validation phases. The entire experimental design can be performed within six analytical runs. The first run (pre-validation phase) is used to assess the validity of the expected concentration-response relationship (lack of fit, goodness of fit), to assess the specificity of the method and to assess the stability of processed samples in the autosampler for 30 h (benchtop stability). The latter experiment is performed to justify overnight analyses. Following approval of the method after the pre-validation phase, the next five runs (main validation phase) are performed to evaluate method precision and accuracy, recovery, freezing and thawing stability and over-curve control /dilution. The design is nested, i.e., many experimental results are used for the evaluation of several performance characteristics. Analysis of variance (ANOVA) is used for the evaluation of lack of fit and goodness of fit, precision and accuracy, freezing and thawing stability and over-curve control/ dilution. Regression analysis is used to evaluate benchtop stability. For over-curve control/ dilution, additional to ANOVA, also a paired comparison is applied. As a consequence, the recommended design combines the performance of as few independent validation experiments as possible with modern statistical methods, resulting in optimum use of information. A demonstration of the entire validation programme is given for an HPLC method for the determination of total captopril in human plasma.     

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.     

Simultaneous determination of ciprofloxacin hydrochloride and hydrocortisone in ear drops by high performance liquid chromatography

The aim of the study was to design and validate a reversed phase high performance liquid chromatography method for the separation and quantification of two active pharmaceutical ingredients (ciprofloxacin hydrochloride, hydrocortisone) and a preservative (benzyl alcohol) in ear drops. Effective separation of the examined compounds was achieved on a GraceSmart? RP 18 column (150 mm × 4.6 mm, 5 μm) with gradient elution and a diode array detector. The total assay run time was 25 min. Analytical method validation assays were performed. Validation parameters used for the evaluation were: specificity, linearity, trueness, precision (repeatability and reproducibility), limit of detection and limit of quantitation. Results of the validation procedure (high recoveries, good standard deviations, no interfering peaks at the retention times corresponding to the analytes) confirm that the developed chromatographic method can be applied for routine analysis of ear drops.     

MultiSimplex and experimental design as chemometric tools to optimize a SPE-HPLC-UV method for the determination of eprosartan in human plasma samples

A chemometric approach was applied for the optimization of the extraction and separation of the antihypertensive drug eprosartan from human plasma samples. MultiSimplex program was used to optimize the HPLC-UV method due to the number of experimental and response variables to be studied. The measured responses were the corrected area, the separation of eprosartan chromatographic peak from plasma interferences peaks and the retention time of the analyte.The use of an Atlantis dC18, 100 mm × 3.9 mm i.d. chromatographic column with a 0.026% trifluoroacetic acid (TFA) in the organic phase and 0.031% TFA in the aqueous phase, an initial composition of 80% aqueous phase in the mobile phase, a stepness of acetonitrile of 3% during the gradient elution mode with a flow rate of 1.25 mL/min and a column temperature of 35 ± 0.2 °C allowed the separation of eprosartan and irbesartan used as internal standard from plasma endogenous compounds. In the solid phase extraction procedure, experimental design was used in order to achieve a maximum recovery percentage. Firstly, the significant variables were chosen by way of fractional factorial design; then, a central composite design was run to obtain the more adequate values of the significant variables. Thus, the extraction procedure for spiked human plasma samples was carried out using C8 cartridges, phosphate buffer pH 2 as conditioning agent, a drying step of 10 min, a washing step with methanol-phosphate buffer (20:80, v/v) and methanol as eluent liquid. The SPE-HPLC-UV developed method allowed the separation and quantitation of eprosartan from human plasma samples with an adequate resolution and a total analysis time of 1 h.     

Oil-in-Water Microemulsion High Performance Liquid Chromatographic Analysis of Pharmaceuticals

Efficient and novel oil-in-water microemulsion HPLC separations of a range of solutes have been achieved using isocratic elution and a conventional reversed-phase HPLC column. A single set of isocratic oil-in-water microemulsion HPLC conditions are shown to be successful for the resolution of a wide range of basic, neutral and acidic drugs and excipients. A robust separation method was developed for the quantitative analysis of Naproxen in a tablet formulation. The method offered a similar run time and improved chromatography when compared to conventional HPLC modes, demonstrating its potential for routine use. In developing the separations the effect on the chromatography of varying the operating parameters was studied. While generally the method was robust to changes in some variables, others markedly changed the separation selectivity, solute retention, peak-peak resolution or resulted in poor chromatographic performance. The method was found to be compatible with very low UV working detection wavelengths. As a final stage to the experimental programme, a microemulsion method was developed with the capability of analysing a range of water-insoluble drugs.     

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.

     

Improvement of the chromatographic separation of several 1,4-dihydropyridines calcium channel antagonist drugs by experimental design

A high-performance liquid chromatographic method with diode array detection has been developed and optimized for the separation of five calcium channel blockers belonging to the 1,4-dihydropyridine subgroup (nifedipine and related drugs). The possibility of the simultaneous drug analysis allows a decrease of time during the assay as well as a saving of reagents and solvents. In this work, the effect of four experimental parameters (organic modifier percentage, pH value, concentration of the buffer in the mobile phase, and column temperature) on the chromatographic resolution are investigated by experimental design in order to optimize the chromatographic separation of five 1,4-dihydropyridines (amlodipine, nitrendipine, felodipine, lacidipine, and lercanidipine). Fractional factorial design, central composite design, and finally the Multisimplex program are used to establish the optimal conditions in terms of resolution and minimum analysis time. Optimal separation of the five compounds under study is achieved in less than 12 min using a Sulpecosil LC-ABZ+Plus C18 column, a composition of mobile phase of acetonitrile-10mM acetic acid acetate buffer pH 5 (72:28, v/v) at a flow rate of 1 mL/min, a column temperature of 30 degrees C +/- 0.1 degrees C, and a detection wavelength of 238 nm.     

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.     

Separation and identification of different therapeutic pharmaceutical drugs on humic acid-based HPLC column: Method optimization using central composite design

Multimode chromatographic separations are highly desirable in pharmaceutical and environmental sciences. Current study deals with the application of newly developed mixed-mode end capped-immobilized humic acid onto an aminopropyl silica based chromatographic column for separation and identification of six drugs belonging to different therapeutic groups for its applicability in pharmaceutical industries. For this, central composite design was used to evaluate the separation and resolution by optimization of three most effective parameters (acetonitrile%, flow rate, and pH of mobile phase). Second-order quadratic model was used to evaluate their effect on resolution of peaks; the probability value (<0.05) obtained from analysis of variance suggested the best applicability of the model. Desirability function was applied to calculate optimum conditions (44.8% acetonitrile, 1.75 mL/min of flow rate, and 7.5 pH) required to achieve maximum separation with good resolution within 11 min. The method was validated for linearity, precision accuracy, selectivity, and sensitivity. The results revealed a highly precise (coefficient of variance > 1%), linear (R² = 0.99), and highly selective method. Moreover, the limit of detection/quantification values revealed acceptable sensitivity of the method. The developed column was compared for its efficiency with a commercially available column and found to be highly applicable for industrial applications.     

Optimization of the simultaneous determination of imatinib and its major metabolite, CGP74588, in human plasma by a rapid HPLC method using D-optimal experimental design

A simple, rapid and specific HPLC method has been developed and validated for the simultaneous determination of imatinib, a tyrosine kinase inhibitor, and its major metabolite, CGP74588, in human plasma. The optimization of the HPLC procedure involved several variables, of which the influences of each was studied. After a series of preliminary-screening experiments, the composition of the mobile phase and the pH of the added buffer solution were set as the investigated variables, while the resolution between imatinib and CGP74588 peaks, the retention time and the imatinib peak width were chosen as the dependent variables. Applying D-optimal design, the optimal chromatographic conditions for the separation were defined. The method proved to show good agreement between the experimental data and predictive values throughout the studied parameter range.The optimum assay conditions were achieved with a Chromolith™ Performance RP-8e 100 mm × 4.6 mm column and a mixture of methanol/acetonitrile/triethylamine/diammonium hydrogen phosphate (pH 6.25, 0.048 mol L⁻¹) (20:20:0.1:59.9, v/v/v/v) as the mobile phase at a flow rate of 2 mL min⁻¹ and detection wavelength of 261 nm. The run time was less than 5 min, which is much shorter than the previously optimized methods. The optimized method was validated according to FDA guidelines to confirm specificity, linearity, accuracy and precision.     

A simple and rapid high performance liquid chromatographic method with fluorescence detection for the estimation of fexofenadine in rat plasma--application to preclinical pharmacokinetics

A sensitive high performance liquid chromatographic (HPLC) method involving fluorescence detection was developed for the determination of fexofenadine (FEX), known to have low oral bioavailability, in rat plasma. In order to understand the effect of various chromatographic factors on the separation of analytes and to simultaneously optimize the resolution and analysis run time, a response surface method was used. The chromatographic separation was achieved using a Supelco C(18)-DB (250 mm x 4.6mm I.D./5 microm particle size) column with mobile phase comprising of ammonium acetate buffer and acetonitrile (63:37, v/v), delivered isocratically at a flow rate of 1.0 mL min(-1). Diphenhydramine was used as an internal standard (I.S.). The statistical evaluation of the method was examined and the method was found to be precise and accurate with a linearity range of 1-500 ng mL(-1) (r>0.9980). The intra- and inter-day precision studies showed good reproducibility with coefficients of variation (C.V.) less than 12.26%. The advantages of our method are small sample volume (100 microL), short time of analysis (13 min) and a simple sample extraction and clean-up as compared to the previously published methods. The established method provides a reliable bioanalytical methodology to carry out FEX pharmacokinetics in rat plasma.