Applications of Monte-Carlo simulation and chemometric techniques for development of bioanalytical liquid chromatography method for estimation of rosuvastatin calcium

In the present work, we investigated the development of a bioanalytical HPLC method of rosuvastatin (RSV) calcium as per the Quality by Design (QbD)-based systematic chemometric tools. At first, the method objectives were framed and critical analytical attributes (CAAs) were chosen. Risk assessment and factor screening was performed using Hybrid Risk Matrix and Plackett–Burman design for identifying vital factors influencing the critical method parameters (CMPs). Monte-Carlo simulation analysis was conducted which confirmed excellent process robustness (Ppk >1.33) for the studied ranges of CMPs. Furthermore, systematic method development was carried out using custom experimental design, where mobile phase ratio, pH, and injection volume were taken as CMPs at three levels. The obtained trials were evaluated for peak area, retention time, theoretical plates, and peak tailing as CAAs. Mathematical response surface modeling was carried out and optimal chromatographic solution was identified using response optimizer plots. Method transfer was made to bioanalytical scale for estimation of the analyte in rat plasma samples. Extensive method validation was performed as per the ICH Q2 guideline, which indicated validation parameters within the acceptable limits. Overall, the studies construed successful development of QbD compliant HPLC method of rosuvastatin with potential utility bioanalytical testing.     

QbD-driven development and validation of an efficient bioanalytical UPLC method for estimation of olmesartan medoxomil

The present studies describe quality by design-based development of bioanalytical ultra performance liquid chromatography method of olmesartan medoxomil. Initially, method objectives were defined and critical analytical attributes (CAAs) earmarked. Method optimization was conducted using a central composite design for optimizing mobile phase ratio and injection volume as the critical method parameters (CMPs) identified from risk assessment and factor screening studies, and evaluated for their influence on peak area, theoretical plates, and asymmetry factor as CAAs. Chromatographic separation was achieved using acetonitrile:water solvent system containing 0.1% orthophosphoric acid (54:46, v/v) as the mobile phase with UV detection at 243 nm. Further optimization of bioanalytical extraction process was accomplished using a Box–Behnken design selecting extraction time, centrifugation speed, and centrifugation time as the CMPs identified from failure mode and effect analysis, and evaluated for percent recovery, peak asymmetry, and theoretical plate count as the CAAs. Establishment of calibration curve indicated linearity between concentration range of 100 and 800 ng mL^?1, excellent accuracy and precision with limit of detection and limit of quantification as 6.2 and 19.0 ng mL^?1, respectively. Drug stability studies indicated mean percent recovery ranging between 92.4 and 97.3% under various stress conditions.     

Application of chemometric approach for development and validation of high performance liquid chromatography method for estimation of ropinirole hydrochloride

A systematic Quality by Design approach was employed for developing an isocratic reversed‐phase liquid chromatographic technique for the estimation of ropinirole hydrochloride in bulk drug and pharmaceutical formulations. LiChrospher RP 18‐5 Endcapped column (25 cm × 4.6 mm id) at ambient temperature (25 ± 2°C) was used for the chromatographic separation of the drug. The screening of factors influencing chromatographic separation of the active pharmaceutical ingredient was performed employing fractional factorial design to identify the influential factors. Optimization of the selected factors was carried out using central composite design for selecting the optimum chomatographic conditions. The mobile phase employed was constituted of Solvent A/Solvent B (65:35 v/v) (Solvent A [methanol/0.05 M ammonium acetate buffer, pH 7, 80:20 v/v] and Solvent B [high performance liquid chromatography grade water]) and used at 0.6 mL/min flow rate, while UV detection was performed at 250 nm. Linearity was achieved in the drug concentration range 5–100 µg/mL (R² = 0.9998) with limits of detection and quantification of 1.02 and 3.09 µg/mL, respectively. Method validation was performed as per ICH guidelines followed by forced degradation studies, which indicated good specificity of the developed method for detecting ropinirole hydrochloride and its possible degradation products in the bulk drug and pharmaceutical formulations.     

Development of new extraction method based on liquid–liquid–liquid extraction followed by dispersive liquid–liquid microextraction for extraction of three tricyclic antidepressants in plasma samples

In the present study, a new extraction method based on a three–phase system, liquid–liquid–liquid extraction, followed by dispersive liquid–liquid microextraction has been developed and validated for the extraction and preconcentration of three commonly prescribed tricyclic antidepressant drugs – amitriptyline, imipramine, and clomipramine – in human plasma prior to their analysis by gas chromatography–flame ionization detection. The three phases were an aqueous phase (plasma), acetonitrile and n–hexane. The extraction mechanism was based on the different affinities of components of the biological sample (lipids, fatty acids, pharmaceuticals, inorganic ions, etc.) toward each of the phases. This provided high selectivity toward the analytes since most interferences were transferred into n–hexane. In this procedure, a homogeneous solution of the aqueous phase (plasma) and acetonitrile (water–soluble extraction solvent) was broken by adding sodium sulfate (as a phase separating agent) and the analytes were extracted into the fine droplets of the formed acetonitrile. Next, acetonitrile phase was mixed with 1,2–dibromoethane (as a preconcentration solvent at microliter level) and then the microextraction procedure mentioned above was performed for further enrichment of the analytes. Under the optimum extraction conditions, limits of detection and lower limits of quantification for the analytes were obtained in the ranges of 0.001–0.003 and 0.003–0.010 μg mL⁻¹, respectively. The obtained extraction recoveries were in the range of 79–98%. Intra– and inter–day precisions were < 7.5%. The validated method was successfully applied for determination of the selected drugs in human plasma samples obtained from the patients who received them.     

Chaotropic chromatography method development for the determination of aripiprazole and its impurities following analytical quality by design principles

In this paper, development of robust and reliable chaotropic chromatography method for the determination of aripiprazole and its impurities, following Analytical Quality by Design principles is presented. The efficient baseline separation and accurate determination of aripiprazole and its four impurities from tablets were set as Analytical Target Profile. In line with it, the influence of Critical Method Parameters (acetonitrile content, concentration of perchloric acid in water phase, and column temperature) on predefined Critical Method Attributes (separation of the critical pair of peaks, retention of the first and last eluting peak) was investigated with aid of the Central Composite Design. Further on Design Space, where Critical Method Parameters meet predefined acceptance limits with a high level of probability (π ≥ 85%), was computed as a result of performed Monte Carlo simulations. A normal operating conditions corresponding to 34% of acetonitrile, 66% of 42.5 mM perchloric acid, and column temperature at 35°C were selected from created Design Space. Robustness testing of the quantitative performances of the developed method was conducted combining Plackett–Burman design with alias matrix approach. Through the additional validation testing, reliability of the developed method for the use in the routine practice was completely confirmed.     

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.     

Air‐assisted liquid–liquid extraction coupled with dispersive liquid–liquid microextraction and a drying step for extraction and preconcentration of some phthalate esters from edible oils prior to their determination by GC

In this work, a new, cheap, simple, fast, and low organic solvent consuming procedure is proposed for isolation, enrichment, and gas chromatographic determination of some phthalate esters in edible oils. The method is based on a combination of air‐assisted liquid–liquid extraction and dispersive liquid–liquid microextraction followed by a drying step under N₂ gas. Several experimental parameters affecting both extraction and preconcentration steps were investigated and optimized. Under the optimum conditions for the proposed method, wide linear ranges (0.05–800 μg/L) and low detection limits (0.007–0.023 μg/L) were observed. The ranges of enrichment factors and extraction recoveries were 68–340 and 14–68%, respectively. Eventually, the target analytes were successfully determined in different edible oils using the proposed method.     

QbD-based development of an extraction procedure for simultaneous quantification of telmisartan,amlodipine besylate and chlorthalidone in combination complex matrix formulation

The main objective of this study was to establish an efficient extraction procedure for the estimation of telmisartan, amlodipine and chlorthalidone from their combination in sample matrix using an analytical quality by design approach. Initial screening studies were performed for optimization of a suitable diluent to extract active components from sample matrix. Further, the same study was extended for the identification of critical method attributes and the factors affecting the analytical target profile. This study also explains the rugged and robust quantitative determination of combinations drugs with a shorter run time. The design of experimental studies confirms that the current center point parameters are well suited to recoveries. The chromatographic separation was achieved with an X-Terra RP8, 150 × 4.6 mm, 3.5 μm column with an isocratic mobile phase (mixture of 20 mm aqueous ammonium acetate and acetonitrile). To demonstrate the stability-indicating nature of the optimized method, forced degradation studies were conducted and proved. The optimized method was validated according to International Conference on Harmonization guidelines.     

High‐performance liquid chromatographic determination of multi‐mycotoxin in cereals and bean foodstuffs using interference‐removal solid‐phase extraction combined with optimized dispersive liquid–liquid microextraction

A novel pre‐treatment was proposed for the simultaneous determination of aflatoxins, ochratoxin A and zearalenone in foodstuffs using high‐performance liquid chromatography with fluorescence detection. The analytical procedure was based on a first step using a quick, easy, cheap, effective, rugged, and safe based extraction procedure, followed by salting out and purification with a C₁₈ solid‐phase extraction column as interference removal clean‐up. Subsequently, collected supernatant was subjected to dispersive liquid–liquid microextraction. Response surface methodology based on central composite design was employed to optimize conditions in the microextraction procedure. Under the optimum conditions, satisfactory analytical performance with recoveries ranging from 63.22 to 107.6% were achieved in different types of cereals and beans, as well as desirable precisions (0.81–8.13%). Limits of detections and quantifications for these six mycotoxins ranging from 0.03 to 13 μg/kg and 0.22 to 44 μg/kg, respectively, were obtained. Finally, the established method was successfully validated by four certified reference materials (P  = 0.897 > 0.05) and applied to 79 samples from local markets.     

Development and validation of a reversed‐phase high‐performance liquid chromatographic method with solid‐phase extraction for the quantification of hydrochlorothiazide in ex vivo permeation studies

Hydrochlorothiazide (HCT) is a diuretic used to treat hypertension. In order to study its intestinal permeation behavior applying an ex vivo methodology, a rapid, sensitive and selective reversed‐phase liquid chromatography (RP‐HPLC) method coupled with UV detection (RP‐HPLC UV) was developed for the analysis of HCT in TC199 culture medium used as mucosal and serosal solutions in the everted rat intestinal sac model. Also, analytical procedures for the quantification of HCT by RP‐HPLC with UV detection required a sample preparation step by solid‐phase extraction. The method was validated in the concentration range of 8.05 × 10⁻⁷ to 3.22 × 10⁻⁵ m for HCT. Chromatographic parameters, namely carry‐over, lower limit of quantification (1.4491 × 10⁻⁷ m ), limit of detection (3.8325 × 10⁻⁸ m ), selectivity, inter‐ and intraday precision and extraction recovery, were determined and found to be adequate for the intended purposes. The validated method was successfully used for permeability assays across rat intestinal epithelium applying the ex vivo everted rat gut sac methodology to study the permeation behavior of HCT.     

High‐performance liquid chromatographic determination of anti‐ hypertensive drugs on dried blood spots using a fluorescence detector – method development and validation

A selective and sensitive high‐performance liquid chromatography method with fluorescence detection for simultaneous determination of irbesartan, losartan and valsartan on dried blood spots (DBS) has been developed and validated. It involves solvent extraction of a punch of DBS followed by reversed‐phase liquid chromatography on a Lichrospher^® 100 RP‐18e column. Fluorescence detection was performed at 259 and 385 nm as excitation and emission wavelengths, respectively. The detection limits of irbesartan, losartan and valsartan were 1.8, 3.6 and 1.8 ng/mL respectively. The mean recoveries of irbesartan, losartan and valsartan were 98.68, 98.42 and 97.81%, respectively. The mean inter‐day and intra‐day precisions of irbesartan, losartan and valsartan were 2.07 and 1.34%, 1.42 and 1.48%, and 3.20 and 2.15% respectively. The proposed method was simple and rapid. Design of experiments was used to evaluate the robustness of the method. Copyright © 2011 John Wiley & Sons, Ltd.     

Developing an alcoholic‐assisted dispersive liquid–liquid microextraction for extraction of pentachlorophenol in water

Optimization of alcoholic‐assisted dispersive liquid–liquid microextraction of pentachlorophenol (PCP) and determination of it with high‐performance liquid chromatography (UV‐Vis detection) was investigated. A Plackett‐Burman design and a central composite design were applied to evaluate the alcoholic‐assisted dispersive liquid–liquid microextraction procedure. The effect of seven parameters on extraction efficiency was investigated. The factor studied were type and volume of extraction and dispersive solvents, amount of salt, and agitation time. According to Plackett‐Burman design results, the effective parameters were type and volume of extraction solvent and agitation time. Next, a central composite design was applied to obtain optimal condition. The optimized conditions were obtained at 170‐μL 1‐octanol and 5‐min agitation time. The enrichment factor of PCP was 242 with limits of detection of 0.04 μg L^?1. The linearity was 0.1–100 μg L^?1 and the extraction recovery was 92.7%. RSD for intra and inter day of extraction of PCP were 4.2% and 7.8%, respectively for five measurements. The developed method was successfully applied for the determination of PCP in environmental water samples.     

Macrocyclic polyamine‐functionalized silica as a solid‐phase extraction material coupled with ionic liquid dispersive liquid–liquid extraction for the enrichment of polycyclic aromatic hydrocarbons

In this study, silica modified with a 30‐membered macrocyclic polyamine was synthesized and first used as an adsorbent material in SPE. The SPE was further combined with ionic liquid (IL) dispersive liquid–liquid microextraction (DLLME). Five polycyclic aromatic hydrocarbons were employed as model analytes to evaluate the extraction procedure and were determined by HPLC combined with UV/Vis detection. Acetone was used as the elution solvent in SPE as well as the dispersive solvent in DLLME. The enrichment of analytes was achieved using the 1,3‐dibutylimidazolium bis[(trifluoromethyl)sulfonyl]imide IL/acetone/water system. Experimental conditions for the overall macrocycle‐SPE–IL‐DLLME method, such as the amount of adsorbent, sample solution volume, sample solution pH, type of elution solvent as well as addition of salt, were studied and optimized. The developed method could be successfully applied to the analysis of four real water samples. The macrocyclic polyamine offered higher extraction efficiency for analytes compared with commercially available C₁₈ cartridge, and the developed method provided higher enrichment factors (2768–5409) for model analytes compared with the single DLLME. Good linearity with the correlation coefficients ranging from 0.9983 to 0.9999 and LODs as low as 0.002 μg/L were obtained in the proposed method.     

Ionic‐liquid‐impregnated resin for the microwave‐assisted solid–liquid extraction of triazine herbicides in honey

Microwave‐assisted ionic‐liquid‐impregnated resin solid–liquid extraction was developed for the extraction of triazine herbicides, including cyanazine, metribuzin, desmetryn, secbumeton, terbumeton, terbuthylazine, dimethametryn, and dipropetryn in honey samples. The ionic‐liquid‐impregnated resin was prepared by immobilizing 1‐hexyl‐3‐methylimidazolium hexafluorophosphate in the microspores of resin. The resin was used as the extraction adsorbent. The extraction and enrichment of analytes were performed in a single step. The extraction time can be shortened greatly with the help of microwave. The effects of experimental parameters including type of resin, type of ionic liquid, mass ratio of resin to ionic liquid, extraction time, amount of the impregnated resin, extraction temperature, salt concentration, and desorption conditions on the extraction efficiency, were investigated. A Box–Behnken design was applied to the selection of the experimental parameters. The recoveries were in the range of 80.1 to 103.4% and the relative standard deviations were lower than 6.8%. The present method was applied to the analysis of honey samples.     

Combination of dispersive solid‐phase extraction and salting‐out homogeneous liquid–liquid extraction for the determination of organophosphorus pesticides in cereal grains

A new analytical method for the determination of organophosphorus pesticides in cereal samples was developed by combining dispersive SPE (d‐SPE) and salting‐out homogeneous liquid–liquid extraction (SHLLE). The pesticides were first extracted from cereal grains with acetonitrile, followed by d‐SPE cleanup. A 2 mL aliquot of the extract was then added to a centrifuge tube containing 9.2 mL water and 3.3 g NaCl for SHLLE. Analysis of the extract was carried out by gas chromatography coupled with flame photometric detection. The d‐SPE procedure effectively provides the necessary cleanup of the extract while SHLLE is used as an efficient concentration technique. Experimental parameters influencing the extraction efficiency including amounts of added water and salt were investigated. Recovery studies were carried out at three fortification levels, yielding recoveries in the range of 57.7–98.1% with the RSD from 3.7 to 10.9%. The reported limits of determination obtained from this study were 1 μg/kg, which is better than the conventional methods. In the analysis of 40 wheat and corn samples taken from Beijing suburbs, only two wheat samples have chlorpyrifos residue over the limits of determination.     

Analytical QbD-based systematic bioanalytical HPLC method development for estimation of quercetin dihydrate

The current work entails development of rapid, sensitive, and inexpensive high-performance liquid chromatographic method of quercetin dihydrate using the quality by design approach. Quality target method profile was defined and critical analytical attributes (CAAs) were earmarked. Chromatographic separation was accomplished on a C₁₈ column using acetonitrile and ammonium acetate buffer (35:65) %v/v (containing 0.1% acetic acid, pH 3.5) as mobile phase at 0.7?mL/min flow rate with UV detector at 237?nm. Screening studies using fractional factorial design revealed that organic modifier, injection volume, column temperature, and buffer strength have significant influence on method CAAs, namely, peak area, retention time, and peak tailing. The critical method parameters were systematically optimized using Box–Behnken design. Response surface mapping was used along with numerical optimization and desirability function for identifying the optimal chromatographic conditions. Linearity was observed in the drug concentration ranging between 2 and 50?µg/mL. Accuracy analysis revealed mean % recovery between 93.6 and 96.2%, while precision study revealed mean % recovery between 93.7 and 96.5%. Limits of detection and quantification of the developed method were found to be 12.1 and 36.6?ng/mL. Overall, the studies construed successful development of chromatographic method of quercetin with enhanced method performance.     

Optimized determination of polybrominated diphenyl ethers by ultrasound‐assisted liquid–liquid extraction and high‐performance liquid chromatography

A method based on ultrasound‐assisted liquid–liquid extraction and high‐performance liquid chromatography has been optimized for the determination of six polybrominated diphenyl ether congeners. The optimal condition relevant to the extraction was first investigated, more than 98.7 ± 0.7% recovery was achieved with dichloromethane as extractant, 5 min extraction time, and three cycles of ultrasound‐assisted liquid–liquid extraction. Then multiple function was employed to optimize polybrominated diphenyl ether detection conditions with overall resolution and chromatography signal area as the responses. The condition chosen in this experiment was methanol/water 93:7 v/v, flow rate 0.80 mL/min, column temperature 30.0°C. The optimized technique revealed good linearity (R² > 0.9962 over a concentration range of 1–100 μg/L) and repeatability (relative standard deviation < 6.3%). Furthermore, the detection limit (S/N = 3) of the method were ranged from 0.02 to 0.13 μg/L and the quantification limit (S/N = 10) ranged from 0.07 to 0.35 μg/L. Finally, the proposed method was applied to spiked samples and satisfactory results were achieved. These results indicate that ultrasound‐assisted liquid–liquid extraction coupled with high‐performance liquid chromatography was effective to identify and quantify the complex polybrominated diphenyl ethers in effluent samples.     

Vortex‐assisted liquid–liquid micro‐extraction and high‐performance liquid chromatography for a higher sensitivity methyl methacrylate determination in biological matrices

A vortex‐assisted liquid–liquid micro‐extraction coupled with high‐performance liquid chromatography, with UV–vis, is proposed to pre‐concentrate methyl methacrylate and to improve separation in biological matrices. The use of 1‐octanol as extracting phase, its volume, the need for a dispersant agent, the agitation conditions and the cooling time before phase separation were evaluated. In optimum conditions, enrichment factors of 20 (±0.5) and enrichment recovery of 99% were obtained. The straightforward association of this extraction process with the HPLC method, previously regulated by the International Organization for Standardization, afforded a detection limit of 122 ng/mL and a quantification limit of 370 ng/mL. The within‐batch precision, relative standard deviation, was 3% for a sample with 1.49 µg/mL and 4% for a sample with 13.4 µg/mL. The results showed a between batch‐precision of 21% for experiments performed on five different days, for a sample with a concentration of 1.10 µg/mL in methyl methacrylate. Copyright © 2013 John Wiley & Sons, Ltd.     

Deep eutectic solvent based homogeneous liquid–liquid extraction coupled with in‐syringe dispersive liquid–liquid microextraction performed in narrow tube; application in extraction and preconcentration of some herbicides from tea

A homogeneous liquid‐liquid extraction performed in narrow tube coupled to in–syringe‐dispersive liquid‐liquid microextraction based on deep eutectic solvent has been developed for the extraction of six herbicides from tea samples. In this method, sodium chloride as a separation agent is filled into the narrow tube and the tea sample is placed on top of the salt. Then a mixture of deionized water and deep eutectic solvent (water miscible) is passed through the tube. In this procedure, the deep eutectic solvent is realized as tiny droplets in contact with salt. By passing the droplets from the tea layer placed on the salt layer, the analytes are extracted into them. After collecting the solvent as separated layer, it is mixed with another deep eutectic solvent (choline chloride/butyric acid) and the mixture is dispersed into deionized water placed in a syringe. After adding acetonitrile to break up the cloudy state, the collected organic phase is injected into gas chromatography‐mass spectrometry. Under optimal conditions, limits of detection and quantification in the ranges of 2.6–8.4 and 9.7–29 ng/kg, respectively, were obtained. The extraction recoveries and enrichment factors in the ranges of 70–89% and 350–445 were obtained, respectively.