Optimising reversed-phase liquid chromatographic separation of an acidic mixture on a monolithic stationary phase with the aid of response surface methodology and experimental design

An optimization strategy for the separation of an acidic mixture by employing a monolithic stationary phase is presented, with the aid of experimental design and response surface methodology (RSM). An orthogonal array design (OAD) OA(16) (2(15)) was used to choose the significant parameters for the optimization. The significant factors were optimized by using a central composite design (CCD) and the quadratic models between the dependent and the independent parameters were built. The mathematical models were tested on a number of simulated data set and had a coefficient of R(2) > 0.97 (n = 16). On applying the optimization strategy, the factor effects were visualized as three-dimensional (3D) response surfaces and contour plots. The optimal condition was achieved in less than 40 min by using the monolithic packing with the mobile phase of methanol/20 mM phosphate buffer pH 2.7 (25.5/74.5, v/v). The method showed good agreement between the experimental data and predictive value throughout the studied parameter space and were suitable for optimization studies on the monolithic stationary phase for acidic compounds.     

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.     

Combined column–mobile phase mixture statistical design optimization of high-performance liquid chromatographic analysis of multicomponent systems

A statistical approach for the simultaneous optimization of the mobile and stationary phases used in reversed-phase liquid chromatography is presented. Mixture designs using aqueous mixtures of acetonitrile (ACN), methanol (MeOH) and tetrahydrofuran (THF) organic modifiers were performed simultaneously with column type optimization, according to a split-plot design, to achieve the best separation of compounds in two sample sets: one containing 10 neutral compounds with similar retention factors and another containing 11 pesticides. Combined models were obtained by multiplying a linear model for column type, C8 or C18, by quadratic or special cubic mixture models. Instead of using an objective response function, combined models were built for elementary chromatographic criteria (retention factors, resolution and relative retention) of each solute or pair of solutes and, after their validation, the global separation was accomplished by means of Derringer's desirability functions. For neutral compounds a 37:12:8:43 (v/v/v/v) percentage mixture of ACN:MeOH:THF:H₂O with the C18 column and for pesticides a 15:15:70 (v/v/v) ACN:THF:H₂O mixture with the C8 column provide excellent resolution of all peaks.     

Determination of textile dyes by means of non-aqueous capillary electrophoresis with electrochemical detection

Eight textile dye compounds including five cationic dyes, namely, basic blue 41, basic blue 9, basic green 4, basic violet 16 and basic violet 3, and three anionic dyes, acid green 25, acid red 1 and acid blue 324, were separated and detected by non-aqueous capillary electrophoresis (NACE) with electrochemical detection. Simultaneous separations of acid and basic dyes were performed using an acetonitrile-based buffer. Particular attention was paid to the determination of basic textile dyes. The optimized electrophoresis buffer for the separation of basic dyes was a solvent mixture of acetonitrile/methanol (75:25, v/v) containing 1 M acetic acid and 10 mM sodium acetate. The limits of detection for the basic dyes were in the range of 0.1–0.7 μg mL⁻¹. An appropriate solid-phase extraction procedure was developed for the pre-treatment of aqueous samples with different matrices. This analytical approach was successfully applied to various water samples including river and lake water which were spiked with textile dyes.     

Chromatographic methods for the separation of biocompatible iron chelators from their synthetic precursors and iron chelates

Chromatographic methods have been developed for the separation of the three novel biocompatible iron chelators pyridoxal isonicotinoyl hydrazone (PIH), salicylaldehyde isonicotinoyl hydrazone (SIH), and pyridoxal 2-chlorobenzoyl hydrazone (o-108) from their synthetic precursors and iron chelates. The chromatographic analyses were achieved using analytical columns packed with 5 microm Nucleosil 120-5 C18. For the evaluation of all chelators in the presence of the synthetic precursors, EDTA was added to the mobile phase at a concentration of 2 mM. The best separation of PIH and its synthetic precursors was achieved using a mixture of phosphate buffer (0.01 M NaH2PO4, 5 mM 1-heptanesulfonic acid sodium salt; pH 3.0) and methanol (55:45, v/v). For separation of SIH and its synthetic precursors, the mobile phase was composed of 0.01 M phosphate buffer (pH 6.0) and methanol (60:40, v/v). o-108 was analyzed employing a mixture of 0.01 M phosphate buffer (pH 7.0), methanol, and acetonitrile (60:20:20, v/v/v). These mobile phases were slightly modified to separate each chelator from its iron chelate. Furthermore, a RP-TLC method has also been developed for fast separation of all compounds. The chromatographic methods described herein could be applied in the evaluation of purity and stability of these drug candidates.     

Experimental design optimization of a capillary zone electrophoresis method for the screening of several diuretics and ACE inhibitors

Experimental design methodologies are applied to the development of a capillary zone electrophoretic method for the separation of the angiotensin-converting enzyme inhibitor enalapril and its derivative enalaprilat and the diuretics xipamide and hydrochlorothiazide. The effects of pH, buffer concentration, proportion of boric acid in the mixed boric acid-potassium dihydrogen phosphate background electrolyte, temperature, applied voltage, and percentage of organic modifier are studied. Critical factors are identified in a screening design (a 2(6-2) fractional factorial design), and afterwards, optimal conditions for the separation are reached by means of an optimization design (a 2(2) + 2 x 2 + k central composite design). The studied response is the resolution between peaks. The four studied compounds can be separated in less than 3.5 min using an electrolyte of 20mM boric acid-potassium dihydrogen phosphate (75:25, v/v) with 5% MeOH adjusted to pH 8.0 with KOH, at a potential of 30 kV. The detection wavelength and temperature are 206 nm and 35 degrees C, respectively.     

CEC separation of insect oostatic peptides using a strong-cation-exchange stationary phase

The separation of several insect oostatic peptides (IOPs) was achieved by using CEC with a strong-cation-exchange (SCX) stationary phase in the fused-silica capillary column of 75 microm id. The effect of organic modifier, ionic strength, buffer pH, applied voltage, and temperature on peptides' resolution was evaluated. Baseline separation of the studied IOPs was achieved using a mobile phase containing 100 mM pH 2.3 sodium phosphate buffer/water/ACN (10:20:70 v/v/v). In order to reduce the analysis time, experiments were performed in the short side mode where the stationary phase was packed for 7 cm only. The selection of the experimental parameters strongly influenced the retention time, resolution, and retention factor. An acidic pH was selected in order to positively charge the analyzed peptides, the pI's of which are about 3 in water buffer solutions. A good selectivity and resolution was achieved at pH <2.8; at higher pH the three parameters decreased due to reduced or even zero charge of peptides. The increase in the ionic strength of the buffer present in the mobile phase caused a decrease in retention factor for all the studied compounds due to the decreased interaction between analytes and stationary phase. Raising the ACN concentration in the mobile phase in the range 40-80% v/v caused an increase in both retention factor, retention time, and resolution due to the hydrophilic interactions of IOPs with free silanols and sulfonic groups of the stationary phase.     

卤代乙酸及其结构相近化合物的高效毛细管电泳分离

氟、氯、溴等卤代乙酸是结构非常相近的离子型化合物,对它们的分离测定比较困难。用高效毛细管电泳法在碱性或酸性缓冲液条件下可将它们分离。在酸性缓冲液条件下,可提高有机酸分离的选择性。较低的操作电压有利于提高阴离子的分离度,而改变温度对分离度的影响不大。     

Strategy for the chiral separation of non-acidic pharmaceuticals using capillary electrochromatography

In completion of an earlier defined generic chiral screening approach, a generic separation strategy for basic, bifunctional, and neutral compounds was proposed and evaluated. This strategy adds to a previously defined strategy for acidic compounds. The screening experiment of the actual strategy used a mobile phase of 5 mM phosphate buffer pH 11.5/ACN (30/70 v/v), a temperature of 25 degrees C, and a voltage of 15 kV. The selected chiral stationary phases were Chiralpak AD-RH, Chiralcel OD-RH, Chiralcel OJ-RH, and Chiralpak AS-RH, all based on polysaccharide selectors. It was seen that 31 out of 48 test compounds were partially or baseline-resolved under screening conditions. After execution of the optimization steps of the strategy, this number increased to 41, with a total of 21 baseline-separated compounds. Combined with the results obtained from the acidic test set examined in the earlier defined strategy, of all tested compounds 82.5% showed enantioselectivity and 49.2% could be baseline-separated.     

Separation of alpha-, beta-, gamma-, delta-tocopherols and alpha-tocopherol acetate on a pentaerythritol diacrylate monostearate-ethylene dimethacrylate monolith by capillary electrochromatography

This work reports the first use of a monolith with method development for the separation of tocopherol (TOH) compounds by CEC with UV detection. A pentaerythritol diacrylate monostearate-ethylene dimethacrylate (PEDAS-EDMA) monolithic column has been investigated for an optimised condition to separate alpha-, beta-, gamma- and delta-TOHs, and alpha-tocopherol acetate (TAc). The PEDAS-EDMA monolith showed a remarkably good selectivity for separation of the TOH isomers including the beta- and gamma-isomers which are not easily separated by standard C8 or C18 particle-packed columns. Retention studies indicated that an RP mechanism was involved in the separation on the PEDAS-EDMA column, but polar interactions with the underlying ester and hydroxyl groups enhanced the separation of the problematic beta- and gamma-isomers. Separation of all the compounds was achieved within 25 min using 3:10:87 v/v/v 100 mM Tris buffer (pH 9.3)/methanol/ACN as the mobile phase. The method was successfully applied to a pharmaceutical sample with recoveries from 93 to 99%. Intraday and interday precisions (%RSD) for peak area and retention time were less than 2.3. LODs for all four TOHs and TAc were below 1 ppm.     

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.     

Cationic double-chained surfactant as pseudostationary phase in micellar electrokinetic capillary chromatography for drug separations

The cationic double-chained surfactant didodecyldimethylammonium bromide (DDAB) was used as pseudostationary phase (PSP) in micellar electrokinetic capillary chromatography (MEKC). Its performance on the three kinds of drugs, i.e., basic, acidic, and neutral drugs, was systematically investigated. Nicotine, cotinine, caffeine, lidocaine, and procaine were selected as the model basic drugs. Good baseline separation and high efficiency were obtained under the optimal separation condition that consisted of 50mM phosphate (pH 4.0) and 0.08 mM DDAB. Three basic phenylenediamine isomers can also be well separated with DDAB in buffer. In addition, DDAB can form cationic bilayer on the capillary wall, thus the wall adsorption of basic analytes was greatly suppressed. Compared with commonly used CTAB, the separation of basic drugs was significantly improved with a much lower amount of DDAB present in the buffer. The DDAB-involved MEKC also showed superiority to CTAB upon the separation of acidic drugs, amoxicillin and ampicillin. In the case of neutral compounds, a good separation of resorcinol, 1-naphthol and 2-naphthol was achieved with 0.1mM DDAB and 30% (v/v) acetonitrile (ACN) present in buffer. Hence, it was concluded that the double-chained cationic surfactant DDAB can be a good substitute for traditional single-chained surfactant CTAB in MEKC.     

Use of different stationary phases for separation of isoniazid, its metabolites and vitamin B6 forms

The ability of different stationary phases developed for the analysis of polar compounds (ZIC-HILIC, ZIC-pHILIC and Zorbax SB-Aq) to separate isoniazid, its metabolites (acetylisonazid, pyridoxal isonicotinoyl hydrazone, pyridoxal isonicotinoyl hydrazone 5-phosphate), pyridoxine, pyridoxal and pyridoxal 5-phosphate under MS compatible conditions was systematically investigated using HPLC-UV. The mobile phase strength, pH and buffer concentration were modified to assess their impact on the retention of these compounds. The best available separation of the compounds was achieved using 1 mM ammonium formate (pH≈6) and ACN (20:80, v/v) on ZIC-HILIC and employing 5 mM ammonium formate (pH 3.0) and ACN (40:60, v/v) on ZIC-pHILIC. A gradient profile using 0.5 mM ammonium formate (pH≈6) and MeOH (0-12 min: 10% MeOH, 12-15 min: 10-50% MeOH, 15-35 min: 50% MeOH, 35.0-35.2 min: 50-10% MeOH, 35.2-45.0 min: 10% MeOH) provided the best separation of the compounds on Zorbax SB-Aq. Subsequent LC-MS analysis demonstrated that ZIC-HILIC is useful for the analysis of pyridoxine, pyridoxal and pyridoxal isonicotinoyl hydrazone. However, the chromatographic conditions developed for the analysis of the compounds on Zorbax SB-Aq are capable of achieving the best separation of all compounds in this study with the higher sensitivity for most of the analytes.     

一种混合模式反相弱阳离子交换色谱填料的制备及其保留机理

采用“点击化学”的合成方法制备了一种混合配体的辛基-羧基共同键合硅胶（OCS）材料,经元素分析和红外光谱表征,证明了辛基和羧基官能团均已成功键合到硅胶表面。将其作为混合模式反相弱阳离子交换（RP/WCX）的固定相填料,以3种同系物阳离子表面活性剂作为探针分子,定量研究了该固定相的混合模式保留机理,考察了溶质保留因子与盐浓度和溶质亚甲基数目的关系,验证了混合模式固定相的单点和两点保留机理的数学模型,并进一步将其应用于一系列标准碱性混合物的分离。结果表明：两点保留机理更加符合实验的结果;根据混合模式两点保留机理的数学方程,可以得到单一的疏水或离子交换作用力对总保留的影响,对混合模式色谱的实际分离应用提供了有价值的参考。本文建立了反相弱阳离子交换混合模式OCS固定相的保留机理的定量模型,并证明了该固定相在碱性化合物的分离中具有很大的应用前景。     

Characterization of pharmaceutical drugs by a modified nonaqueous capillary electrophoresis--mass spectrometry method

A simple method for the separation and characterization of a group of nine basic compounds, comprising seven tricyclic antidepressant and two bronchodilator drugs, by nonaqueous capillary electrophoresis (NACE) employing ultraviolet and mass spectrometry detection is described. After optimization of the electrophoresis separation conditions, including the compositions of the electrolyte and the organic solvent, a reliable separation of all nine basic analytes was achieved in 80 mM ammonium formate dissolved in a methanol-acetonitrite (80:20 v/v) mixture, having an apparent pH of 8.7. The volatile nonaqueous electrolyte system used with a normal electroosmotic flow polarity also provided an optimal separation condition for the characterization of the analytes by mass spectrometry. When results were compared with reversed-phase gradient and isocratic high-performance liquid chromatography (HPLC) methods, the NACE method provided greater efficiency, achieving baseline resolution for all nine basic compounds in less than 30 min. The NACE method is suitable for use as a routine procedure for the rapid separation and characterization of basic compounds and is a viable alternative to HPLC for the separation of a wide range of pharmaceutical drugs.     

Application of a multivariate approach for analyte focusing by micelle collapse‐micellar electrokinetic chromatography for analyzing sunscreen agents in cosmetics

The operating parameters that affect the performance of the online preconcentration technique “analyte focusing by micelle collapse‐MEKC (AFMC‐MEKC)” were examined using a multivariate approach involving experimental design to determine the sunscreen agents in cosmetics. Compared to the single‐variable approach, the advantage of the multivariate approach was that many factors could be investigated simultaneously to obtain the best separation condition. A fractional factorial design was used to identify the fewest significant factors in the central composite design (cCD). The cCD was adopted for evaluating the location of the minimum or maximum response in this study. The influences of the experimental variables on the response were investigated by applying a chromatographic exponential function. The optimized condition and the relationship between the experimental variables were acquired using the JMP software. The ANOVA analysis indicated that the Tris pH value, SDS concentration, and ethanol percentage influenced the separation quality and significantly contributed to the model. The optimized condition of the running buffer was 10 mM Tris buffer (pH 9.5) containing 60 mM SDS, 7 mM γ‐CD, and 20% v/v ethanol. The sample was prepared in 100 mM Tris buffer (pH 9.0) containing 7.5 mM SDS and 20% v/v ethanol. The SDS concentration in the sample matrix was slightly greater than the CMC value that makes the micelle be easily collapsed and the analytes be accumulated in the capillary. In addition, sunscreen agents in cosmetics after 1000‐fold dilution were successfully determined by AFMC‐MEKC.     

Separation of basic compounds of pharmaceutical interest by using nano-liquid chromatography coupled with mass spectrometry

Nano-liquid chromatography-mass spectrometry (nano-LC-MS) was evaluated for the separation of basic compounds of pharmaceutical interest. The separation of selected beta-blockers, namely nadolol, oxprenolol, alprenolol and propranolol in the presence of terbutaline was performed using two 75 microm I.D. capillaries packed with two different RP18 stationary phases (SP). The best results concerning resolution and efficiency were achieved using the SP where free silanol groups were not present. As expected, this latter SP proved to be very efficient and symmetry factors were observed mainly in the case of the more retained analytes. Baseline resolution of all studied basic compounds was achieved with the Cogent bidentate C18 silica phase (CBC18) eluting analytes at 800 nL/min with a mobile phase containing 500 mM ammonium acetate pH 4.5-water-methanol (1:8:91, v/v/v). The separated basic compounds were revealed using on-column UV detector at 205 nm and electrospray-ion-trap mass spectrometer (ESI-MS). The packed capillary was connected to the MS through a commercial sheath liquid interface or a sheathless nano-spray interface and in both cases the sensitivity was studied and the results compared. Limit of detection (LOD) as low as 0.1 ng/mL was measured for nadolol using the sheathless nano-spray interface and the capillary column packed with the CBC18 stationary phase.     

Capillary electrochromatography of 1-phenyl-3-methyl-5-pyrazolone derivatives of some mono-and disaccharides

Summary A packing procedure was adopted for capillary electrochromatography (CEC) that produces capillary columns with high separation efficiencies. The columns were fully packed, 50 cm long, with UV detection being performed through the packed section 30 cm from the inlet end. The CEC experiments were run at ambient pressure, with no additional pressure applied to the ends of the column. The stationary phase (octadecyl silica (ODS), 5 μm) promoted a high velocity electroosmotic flow (EOF), enabling rapid and efficient separation of a hydrocarbon test mixture. Some 1-phenyl-3-methyl-5-pyrazolone (PMP) derivatives of mono- and disaccharides were baseline separated, using a 5 mM NaH₂PO₄ in 80% acetonitrile and 20% water (v/v) buffer solution. CEC shows promise for future applications in carbohydrate analysis. Presented at Balaton Symposium on High Performance Separation Methods, Siófok, Hungary, September 1–3, 1999     

Native and stearic acid modified ceria-zirconia supports in normal and reversed-phase HPLC

Porous ceria-zirconia composite with narrow particle size distribution and large specific surface area was synthesized by a sol-gel process. Chromatographic properties of the native supports was investigated in normal phase mode for the separation of test mixtures of basic, neutral and acidic compounds. The new packing material exhibited polar and basic properties, which are suitable for the separation of basic compounds. Lypophilic packing has been obtained by the modification of the ceria-zirconia with stearic acid, which exhibited strong hydrophobicity relative to the native packing. Therefore, the modified ceria-zirconia behaves as a reversed-phase packing material. Different selectivity towards basic compounds was observed on the new packing compared to the native ceria-zirconia and conventional ODS stationary phase.