Desirability-based optimization and its sensitivity analysis for the perindopril and its impurities analysis in a microemulsion LC system

In the current paper the application of multiobjective optimization (MOOP) technique, via Derringer's desirability function, to a microemulsion liquid chromatographic (MELC) method is described. Chromatographic separation of perindopril tert-butylamine and its four impurities was selected as the case study. Central composite design (CCD) with fractional factorial design, ± 0.5 α star design and four replications in central point was applied for a response surface study, in order to examine in depth the effects of the most important factors. As factors that influence the system mostly (i) content of ethyl acetate and (ii) butyl acetate in composite internal phase, (iii) content of sodium dodecyl sulfate (surfactant) and (iv) n-butanol (co-surfactant), as well as (v) pH of the mobile phase were selected. Retention factor of (a) perindoprilat and (b) impurity Y 31 and (c) resolution factor for impurities Y 32 and 33 were chosen for simultaneous optimization. By adjustment of the importance coefficients and weights, according to defined objectives, the optimal mobile phase composition was predicted to be: 0.24% w/v butyl acetate, 0.3% w/v ethyl acetate, 2% w/v SDS, 7.75% w/v n-butanol and pH of the mobile phase 3.7. The sensitivity analysis of desirability function for these optimal conditions was conducted for the first time in LC separations, by applying a sensitivity procedure. The performed sensitivity analysis confirmed that the higher overall desirability does not necessarily mean a better solution. The accuracy of prediction might be affected if the optimal levels of input variables, achieved from several design points, end up with equal settings and different corresponding overall desirability. In our study this was not the issue, which confirmed the adequacy of predicted optimum.     

Simultaneous analysis of irbesartan and hydrochlorothiazide: an improved HPLC method with the aid of a chemometric protocol

Experimental design method was used for HPLC determination of irbesartan and hydrochlorothiazide in combined dosage forms. The traditional approach for optimization of experiments is time-consuming, involves a large number of runs and does not allow establishing the multiple interacting parameters. The main advantages of the experimental design method include the simultaneous screening of a larger number of factors affecting response and the estimation of possible interactions. On the basis of preliminary experiments, three factors-independent variables were selected as inputs (methanol content, pH of the mobile phase and temperature) and as dependent variables, five responses (resolution, symmetry of irbesartan peak, symmetry of hydrochlorothiazide peak, retention factor of irbesartan and retention factor of hydrochlorothiazide) were chosen. A full 23 factorial design, where factors were examined at two different levels ("low" and "high") was used to determine which factors had an effect on the studied response. Afterwards, experimental design was used to optimize these influent parameters in the previously selected experimental domain. The novelty of our method lies in the optimization step accomplished by Derringer's desirability function. After optimizing the experimental conditions a separation was conducted on a Supelcosil C(18) (150 mm × 4.6 mm, 5 mm particle size) column with a mobile phase consisting of methanol-tetrahydrofuran-acetate buffer 47:10:43 v/v/v, pH 6.5 and a column temperature of 25 °C. The developed method was successfully applied to the simultaneous separation of these drug-active compounds in their commercial pharmaceutical dosage forms.     

An improved HPLC method with the aid of a chemometric protocol: simultaneous analysis of amlodipine and atorvastatin in pharmaceutical formulations

Statistical experimental design and Derringer's desirability function were applied to develop an improved RP-HPLC method for the simultaneous analysis of amlodipine and atorvastatin in pharmaceutical formulations. Four independent factors were considered: acetonitrile content in the mobile phase; buffer pH; buffer concentration; and flow rate. The preliminary screening step was carried out, according to a 2(4-1) fractional factorial design, to identify the significant factors affecting the analysis time response. Then central composite design was applied for a response surface study, in order to examine in depth the effects of the most important factors. Subsequently, Derringer's desirability function was employed to simultaneously optimize the six responses: retention factor of first peak; two resolutions; and three retention times, each having a different target. This procedure allowed deduction of two separate optimum conditions, intended for the analysis of quality control and plasma samples, within the experimental domain. The predicted optimum for the quality control samples was: methanol-acetonitrile-15 mM K(2)HPO(4) buffer (pH 5.33) (10:42.08:47.92, v/v/v) as the mobile phase and 1.12 mL/min as the flow rate. The method using this optimized condition showed higher sensitivity and shorter analysis time than the previously published reports. The optimized assay condition was validated according to International Conference on Harmonization guidelines.     

Multiobjective Optimization Approach in Evaluation of Chromatographic Behaviour of Zolpidem Tartrate and Its Degradation Products

Multiresponse optimization methodology in combination with experimental design was employed as a powerful technique for simultaneous optimization of input variables significant for evaluation of chromatographic behaviour of zolpidem tartrate, zolpacid, oxozolpidem, zolpyridine and zolpaldehyde towards various responses. In the first stage of the investigation fractional factorial design was used to decrease the number of variables that should be studied in detail. Among examined variables, pH of the mobile phase, percentage of organic modifier and buffer concentration showed to be statistically important and were consequently optimized with central composite design and Derringer??s desirability function. Four responses were considered, the retention factors of zolpacid and zolpaldehyde (the first and last peak) and the resolutions between critical peaks. Optimal conditions included Luna C₁₈(2) analytical column (250?mm?x?4.6?mm, 5???m particle size), mobile phase consisted of methanol?C10?mM ammonium acetate (68.4:31.6, v/v, pH 5.4) and column temperature of 35???C. The flow rate of the mobile phase was 1?mL?min^?1 and the detection was performed at 254?nm. At the end, the method was successfully validated in accordance with ICH guideline and subsequently applied to the analysis of commercially available zolpidem tartrate tablets.     

Method development and validation for optimised separation of salicylic, acetyl salicylic and ascorbic acid in pharmaceutical formulations by hydrophilic interaction chromatography and response surface methodology

This paper introduces a design of experiments (DOE) approach for method optimisation in hydrophilic interaction chromatography (HILIC). An optimisation strategy for the separation of acetylsalicylic acid, its major impurity salicylic acid and ascorbic acid in pharmaceutical formulations by HILIC is presented, with the aid of response surface methodology (RSM) and Derringer's desirability function. A Box-Behnken experimental design was used to build the mathematical models and then to choose the significant parameters for the optimisation by simultaneously taking both resolution and retention time as the responses. The refined model had a satisfactory coefficient (R2>0.92, n=27). The four independent variables studied simultaneously were: acetonitrile content of the mobile phase, pH and concentration of buffer and column temperature each at three levels. Of these, the concentration of buffer and its cross-product with pH had a significant, positive influence on all studied responses. For the test compounds, the best separation conditions were: acetonitrile/22 mM ammonium acetate, pH 4.4 (82:18, v/v) as the mobile phase and column temperature of 28°C. The methodology also captured the interaction between variables which enabled exploration of the retention mechanism involved. It would be inferred that the retention is governed by a compromise between hydrophilic partitioning and ionic interaction. The optimised method was further validated according to the ICH guidelines with respect to linearity and range, precision, accuracy, specificity and sensitivity. The robustness of the method was also determined and confirmed by overlying counter plots of responses which were derived from the experimental design utilised for method optimisation.     

Analysis of human parathyroid hormone (1-84) products. Separation of a major impurity in synthetic products by ion-pairing reversed-phase high-performance liquid chromatography

Human parathyroid hormone (1-84) is a naturally occurring polypeptide that acts as the major regulator of calcium ion homeostasis. It can be efficiently produced through both synthetic and biosynthetic routes and, as such, highly selective analytical methods are required for the detection of a wide range of impurities. Herein we report on the development of an ion-pairing reversed-phase HPLC method for the analysis of human parathyroid hormone and the separation of impurities including a major, unidentified impurity detected in synthetic preparations. This impurity could not be resolved using trifluoroacetic acid-based methods generally used for monitoring purity levels in commercial products. Separation conditions consisted of a gradient elution of 0.155 M sodium chloride containing 0.037 M sodium pentanesulfonate, pH 5.6, as mobile phase A and acetonitrile as mobile phase B. Separations were carried out on an octadecylsilyl silica column maintained at 50 degrees C. Both column temperature and pH of mobile phase A significantly affected the separation of the major impurity. The major impurity eluted after the main human parathyroid peak and was detected in the two commercial synthetic products analyzed. Several minor impurities eluting before and after the main peak were also detected. Purity levels measured by the developed HPLC method (method C) were similar to those previously measured by capillary electrophoresis. Analysis of purified recombinant human parathyroid hormone did not show the presence of this impurity. This method offers a significant advantage for the purity assessment of human parathyroid hormone.     

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。     

Development and validation of spectrophotometric, TLC and HPLC methods for the determination of lamotrigine in presence of its impurity

Three reliable, rapid and selective methods have been developed and validated for the determination of lamotrigine in the presence of its impurity, 2,3-dichlorobenzoic acid. The first method is spectrophotometric method using p-chloranilic acid forming a colored product with lambda(max) 519+/-2 nm. All variables affecting the reaction have been investigated and the conditions were optimized. Beer's law was obeyed over a concentration range of 10-200 microg ml(-1) with mean accuracy 100.13+/-0.44%. The molar ratio of the formed ion-association complex is found to be 1 : 1 as deduced by Job's method. The conditional stability constant (K(f)), standard free energy (DeltaG), molar absorptivity(epsilon), and sensitivity index were evaluated. The second method is based on TLC separation of the cited drug (Rf=0.75+/-0.01) from its impurity (Rf=0.23+/-0.01) followed by densitometric measurement of the intact drug spots at 275 nm. The separation was carried on silica gel plates using ethyl acetate : methanol : ammonia 35% (17 : 2 : 1 v/v/v) as a mobile phase. The linearity range was 0.5-10 microg/spot with mean accuracy 99.99+/-1.33%. The third method is accurate and sensitive stability-indicating HPLC method based on separation of lamotrigine from its impurity on a reversed phase C(18) column, using a mobile phase of acetonitrile : methanol : 0.01 M potassium orthophosphate (pH 6.7+/-0.1) (30 : 20 : 50 v/v/v) at ambient temperature 25+/-5 degrees C and UV detection at 275 nm in an overall analysis time of about 6 min., based on peak area. The injection repeatability, intraday and interday repeatability were calculated. The procedure provided a linear response over the concentration range 1-12 microg ml(-1) with mean accuracy of 99.50+/-1.30%. The proposed methods were successfully applied for the determination of lamotrigine in bulk powder, in dosage form and in presence of its impurity. The results obtained were analyzed by ANOVA to assess that no significant difference between each of the three methods and the reported one. The validation was performed according to USP guidelines.     

Use of an immiscible diluent in ionic liquid / ion pair LC for the assay of an injectable analgesic

Injectable solutions used in treatment of intense pain are based on combinations of active ingredients such as metamizole sodium (MTZ), pitofenone hydrochloride (PTF) and fenpiverine bromide (FPB). The simultaneous chromatographic assay of such combinations poses difficulties due to their structural variety, highly polar character, and wide concentration ranges (500 mg mL⁻¹ for MTZ, 2 mg mL⁻¹ for PTF and 0.02 mg mL⁻¹ for FPB). Fast hydrolysis of MTZ on aqueous dilution causes additional problems due to impurity (MTC) formation. Sodium hexane sulphonate (10 mM) was used as ion pairing agent for PTF, FPB and MTC in a mobile phase consisting of 48/52 (v/v) methanol and aqueous 0.2% triethylamine at pH=3. The ionic liquid 1-butyl-1-methyl-pyrrolidinium tetrafluoroborate (10 mM) was used as mobile phase additive to preserve the MTZ peak symmetry. The minor active ingredient FPB was selectively extracted into 1-octanol by ion pair formation with picric acid. A 20 μL aliquot of the organic layer was directly injected into the column.      

Separation of Felted Explosives by Pressurized Microemulsion Electrokinetic Chromatography

A new MEEKC method assisted with pressure-driven mobile phase was presented for the separation of felted explosives. Microemulsion solution was composed of 80 mmol/L heptane-120 mmol/L SDS (sodium dodecyl sulphate)-900 mmol/L butanol-10 mmol/L borate at pH 9.4 and a pressure-driven flow of 0.020 mL/min under 1.3 MPa was employed to manipulate the separation. Explosives HMX (1,3,5,7-tetranitro-1,3,5,7-tetrazacyclooctane) and TATB(triamino-trinitrobenzene), which were felted on fluorine rubber F2311 (polytrifluorochlorethylene and polyvinylidene fluoride 1/1 co-polymerization) and F2314 (polytrifluorochlorethylene and polyvinylidene fluoride 4/1 co-polymerization) were well separated with very good peak shapes.     

Effects of amine modifiers on retention and peak shape in reversed-phase high-performance liquid chromatography

A systematic evaluation of the effects of 15 different amine modifiers on the retention and peak symmetry of three solutes, a primary, a secondary, and a tertiary amine, is presented. Using automated experimentation, mobile phase combinations for each modifier over a pH range of 2.5 to 8 were investigated. The effect of changing the sodium ion concentration of the mobile phase was also examined. The importance of hydrophobic, ion exchange, and hydrogen bonding interactions as mechanisms for retention and peak symmetry of positively charged solutes is discussed.     

Determination of N-methylcarbamate insecticides in water samples using dispersive liquid–liquid microextraction and HPLC with the aid of experimental design and desirability function

A rapid and simple method for the extraction and preconcentration of N-methylcarbamates (NMCs) (carbofuran, carbaryl and promecarb) in water samples using dispersive liquid–liquid microextraction (DLLME) using chemometrics was developed. Influence variables such as volume of extracting (CHCl₃) and dispersing solvents (ACN), pH and ionic strength, extraction time and centrifugation time and speed were screened in a 2^7–4 Plackett–Burman design was investigated. The significant variables were optimized by using a central composite design (CCD) combined with desirability function (DF). At optimum conditions values of variables set as 126 μL chloroform, 1.5 mL acetonitrile, 1 min extraction time, 10 min centrifugation at 4000 rpm min⁻¹, natural pH, 4.7% (w/v) NaCl, the separation was reached in less than 14 min using a C₁₈ column and an isocratic binary mobile phase (acetonitrile: water (50:50, v/v)) with flow rate of 1.0 mL min⁻¹. At optimum conditions method has linear response over 0.001–10 μg mL⁻¹ with detection limit between 0.0001 and 0.0005 μg mL⁻¹ with relative standard deviations (RSDs) in the range 2.18–5.06% (n = 6).     

Development and evaluation of new zwitterionic hydrophilic interaction liquid chromatography stationary phases based on 3-P,P-diphenylphosphonium-propylsulfonate

New zwitterionic stationary phases were synthesized by covalently bonding 3-P,P-diphenylphosphonium-propylsulfonate to silica gel. The resulting materials possess both a negatively charged sulfonate group and a positively charged quaternary phosphonium group, which means that there is no net charge over a wide pH range. The retention mechanism and chromatographic behavior of polar solutes under HILIC conditions were studied on these zwitterionic phases. Compared to the commercial ZIC-HILIC column and a bare silica gel stationary phase, the newly synthesized zwitterionic stationary phases provided greater retention, higher peak efficiency and better peak symmetry in the HILIC mode. The analytes examined included: β-blockers, nucleic acid bases and nucleosides, salicylic acid and its analogues, and water soluble vitamins. Factors, such as the type of organic modifiers, solvent composition, pH and the buffer concentration of the mobile phase, have been considered as potential variables for controlling the chromatographic retention of polar analytes.     

Monitoring of fosinopril sodium impurities by liquid chromatography-mass spectrometry including the neural networks in method evaluation

In this paper, the mass spectrometry (MS) detection has been applied for screening of fosinopril sodium impurities which arise during forced stress study. Before MS analysis, liquid chromatographic method with suitable mobile phase composition was developed. The separation was done on SunFire 100 mm x 4.6 mm 3.5 microm particle size column. The mobile phases which consisted of methanol-ammonium acetate buffer-acetic acid, in different ratios, were used in a preliminary study. Flow rate was 0.3 mL min(-1). Under these conditions, percent of methanol, concentration of ammonium acetate buffer and acetic acid content were tested simultaneously applying central composite design (CCD) and artificial neural network (ANN). The combinations of experimental design (ED) and ANN present powerful technique in method optimization. Input and output variables from CCD were used for network training, verification and testing. Multiple layer perceptron (MLP) with back propagation (BP) algorithm was chosen for network training. When the optimal neural topology was selected, network was trained by adjusting strength of connections between neurons in order to adapt the outputs of whole network to be closer to the desired outputs, or to minimize the sum of the squared errors. From the method optimization the following mobile phase composition was selected as appropriate: methanol-10 mM ammonium acetate buffer-acidic acid (80:19.5:0.5 v/v/v). This mobile phase was used as inlet for MS. According to molecular structure and literature data, electrospray positive ion mode was applied for analysis of fosinopril sodium and its impurities. The proposed method could be used for screening of fosinopril sodium impurities in bulk and pharmaceuticals, as well as for tracking the degradation under stress conditions.     

Simultaneous Determination of Dextromethorphan Hydrobromide, Pyrilamine Maleate and Sodium Benzoate in a Cough Cold Syrup by LC

A liquid chromatographic method for the simultaneous determination of dextromethorphan hydrobromide, pyrilamine maleate and sodium benzoate in cough cold syrup has been developed. The method was based on replacing heptane sulfonate by sodium chloride as ion pairing agent. The addition of sodium chloride to the mobile phase has changed the retention behaviour of the basic drugs. The separation of these compounds was achieved in less than 8 min with an isocratic mobile phase consisting of acetonitrile/0.1 M dihydrogenphosphate buffer containing 0.1 M sodium chloride (29:71 v/v) at pH 2.5 and using a Kromasil C18 column. The analysis was performed at a flow rate of 1 mL min^?1 and at a detection wavelength of 220 nm. The selectivity, linearity of calibration, accuracy, within and between days precision, limit of detection and quantification, recovery were examined as parts of the method validation. Calibration curves were linear in the range 1–140 μg mL^?1 with a regression coefficient (R ²) better than 0.999. The results of the method repeatability (intra-day) and reproducibility (inter-day) were all less than 2% (n = 6). The lowest detectable concentration of dextromethorphan hydrobromide and pyrilamine maleate varied between 0.10 and 0.12 μg mL^?1. The proposed liquid chromatographic method was satisfactorily applied for the routine quality control of dextromethorphan hydrobromide, pyrilamine maleate and sodium benzoate in cough cold syrup formulations.     

Forced Degradation Study to Develop and Validate Stability-Indicating RP-LC for Quantification of Ropinirole Hydrochloride in Its Modified Release Tablets

A forced degradation study on ropinirole hydrochloride in bulk and in its modified release tablets was conducted under the conditions of hydrolysis, oxidation and photolysis in order to develop an isocratic stability-indicating LC-UV method for quantification of the drug in tablets. An impurity peak in standard solution was found to increase under acidic and neutral hydrolytic conditions while another degradation product was formed under alkaline condition. The drug and its degradation products were optimally resolved on a Hypersil C₁₈ column with mobile phase composed of diammonium hydrogen orthophosphate (0.05 M; pH 7.2), tetrahydrofuran and methanol (80:15:5% v/v) at a flow rate of 1.0 mL min^?1 at 30 °C using 250 nm as detection wavelength. The method was linear in the range of 0.05–50 μg mL^?1 drug concentrations. The %RSD of inter- and intra-day precision studies was <1. The system suitability parameters remained unaffected during quantification of the drug on three different LC systems. Excellent recoveries (101.59–102.28%) proved that the method was sufficiently accurate. The LOD and LOQ were found to be 0.012 and 0.040 μg mL^?1, respectively. Degradation behaviour of the drug in both bulk and tablets was similar. The drug was very unstable to hydrolytic conditions but stable to oxidative and photolytic conditions. The method can be used for rapid and accurate quantification of ropinirole hydrochloride in tablets during stability testing. Based on chemical reactivity of ropinirole in different media, the degradation products were suspected to be different from the known impurities of the drug.     

QbD‐oriented development and validation of a bioanalytical method for nevirapine with enhanced liquid–liquid extraction and chromatographic separation

The present studies describe the systematic quality by design (QbD)‐oriented development and validation of a simple, rapid, sensitive and cost‐effective reversed‐phase HPLC bioanalytical method for nevirapine in rat plasma. Chromatographic separation was carried out on a C₁₈ column using isocratic 68:9:23% v/v elution of methanol, acetonitrile and water (pH 3, adjusted by orthophosphoric acid) at a flow rate of 1.0 mL/min using UV detection at 230 nm. A Box–Behnken design was applied for chromatographic method optimization taking mobile phase ratio, pH and flow rate as the critical method parameters (CMPs) from screening studies. Peak area, retention time, theoretical plates and peak tailing were measured as the critical analytical attributes (CAAs). Further, the bioanalytical liquid–liquid extraction process was optimized using an optimal design by selecting extraction time, centrifugation speed and temperature as the CMPs for percentage recovery of nevirapine as the CAA. The search for an optimum chromatographic solution was conducted through numerical desirability function. Validation studies performed as per the US Food and Drug Administration requirements revealed results within the acceptance limit. In a nutshell, the studies successfully demonstrate the utility of analytical QbD approach for the rational development of a bioanalytical method with enhanced chromatographic separation and recovery of nevirapine in rat plasma. Copyright © 2015 John Wiley & Sons, Ltd.     

Separation of rutin nona(H-) and deca(H-) sulfonate sodium by ion-pairing reversed-phase liquid chromatography

Ion-pairing reversed-phase liquid chromatography (RPLC) was used to separate two polysulfonates, rutin nona(H-) sulfonate sodium and rutin deca(H-) sulfonate sodium, which have very similar chemical structures. The final product always contained both of them when one of the compounds was synthesized. Baseline separation was achieved on a C8-bonded silica column at ambient temperature. The eluent was acetonitrile-15 mM phosphate buffer solution containing 20 mM TBA (pH 6.0) (46:54, v/v). The calibration plot was linear in the concentration range 0.5-200 microg ml(-1) for both analytes. The limits of detection (LODs; 254 nm) were 0.03 microg ml(1-) for rutin nona(H-) sulfonate sodium and 0.04 microg ml(-1) for rutin deca(H-) sulfonate sodium. Three batches of rutin deca(H-) sulfonate sodium were analyzed using the assay; the results showed that the analytical performance is really satisfactory.     

Effect of chromatographic conditions on the separation and system efficiency for HPLC of selected alkaloids on different stationary phases

Retention parameters of alkaloid standards were determined on different stationary phases, i.e., octadecyl silica, base-deactivated octadecyl silica, cyanopropyl silica, preconditioned cyanopropyl silica, and pentafluorophenyl, using different aqueous eluant systems: acetonitrile-water mixtures; buffered aqueous mobile phases at pH 3 or 7.8; and aqueous eluants containing ion-pairing reagents (octane-1-sulfonic acid sodium salt and pentane-1-sulfonic acid sodium salt) or silanol blockers (tetrabutyl ammonium chloride and diethylamine). Improved peak symmetry and separation selectivity for basic solutes was observed when basic buffer, ion-pairing reagents, and, especially, silanol blockers as mobile phase additives were applied. The best separation selectivity and most symmetric peaks for the investigated alkaloids were obtained in systems containing diethylamine in the mobile phase. The influence of acetonitrile concentration and kind and concentration of ion-pairing reagents or silanol blockers on retention, peak symmetry, and system efficiency was also examined. The most efficient and selective systems were used for separation of the investigated alkaloids and analysis of Fumaria officinalis and Glaucium flavum plant extracts.