Development and validation of a simple high‐performance liquid chromatography analytical method for simultaneous determination of phytosterols,cholesterol and squalene in parenteral lipid emulsions

A simple analytical method for simultaneous determination of phytosterols, cholesterol and squalene in lipid emulsions was developed owing to increased interest in their clinical effects. Method development was based on commonly used stationary (C₁₈, C₈ and phenyl) and mobile phases (mixtures of acetonitrile, methanol and water) under isocratic conditions. Differences in stationary phases resulted in peak overlapping or coelution of different peaks. The best separation of all analyzed compounds was achieved on Zorbax Eclipse XDB C₈ (150 × 4.6 mm, 5 μm; Agilent) and ACN–H₂O–MeOH, 80:19.5:0.5 (v/v/v). In order to achieve a shorter time of analysis, the method was further optimized and gradient separation was established. The optimized analytical method was validated and tested for routine use in lipid emulsion analyses.     

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.     

Liquid chromatographic behavior of two alanine‐substituted calix[4]arene‐bonded silica gel stationary phases

Two new kinds of alanine‐substituted calix[4]arene stationary phases of 5,11,17,23‐p‐tert‐butyl‐25,27‐bis(l ‐alanine‐methylester‐N‐carbonyl‐methoxy)‐26,28‐dihyroxycalix[4]arene‐bonded silica gel stationary phase (BABS4) and 5, 11, 17, 23‐p‐tert‐butyl‐25,26,27,28‐tetra(l ‐alanine‐methylester‐N‐carbonyl‐methoxy)‐calix[4]arene‐bonded silica gel stationary phase (TABS4) were prepared and characterized in the present study. They were compared with each other and investigated in terms of their chromatographic performance by using polycyclic aromatic hydrocarbons, disubstituted benzene isomers, and mono‐substituted benzenes as solute probes. The results indicated that both BABS4 and TABS4 exhibited multiple interactions with analytes. In addition, the commonly used Tanaka characterization protocol for the evaluation of commercially available stationary phases was applied to evaluate the properties of these two new functionalized calixarene stationary phases. The Tanaka test results were compared with Zorbax Eclipse XDB C₁₈ and Kromasil phenyl columns, respectively. BABS4 has stronger hydrogen‐bonding capacity and ion‐exchange capacity than TABS4, and features weaker hydrophobicity and hydrophobic selectivity. Both of them behave similarly in stereoselectivity. Both BABS4 and TABS4 are weaker than C₁₈ and phenyl stationary phases in hydrophobicity and hydrophobic selectivity.     

Validated LC Method for Determination of Enantiomeric Purity of Apremilast Using Polysaccharide-Type Stationary Phases in Polar Organic Mode

Enantioseparation of an anti-psoriatic agent, apremilast (APR), was performed by HPLC using polysaccharide-type chiral stationary phases in polar organic mode for the first time. The separation capability of six different chiral columns (Chiralpak AD, Chiralpak IA, Chiralpak AS, Lux Amylose-2, Chiralcel OD and Chiralcel OJ-H) was investigated using neat MeOH and ACN. During the preliminary experiments the best results were obtained on Chiralpak IA column with ACN (R_s?=?5.4). The effects of binary mobile phases on the resolutions and retention factors were also investigated containing different percentages of MeOH:ACN. U-shaped retention pattern was obtained when plotting the retention factors of the APR enantiomers versus the MeOH content of the binary mobile phases on Chiralpak IA column. For further method optimizations an L25 orthogonal array table was employed altering the concentration of MeOH in ACN, column temperature, and flow rate. The best result was achieved on Chiralpak IA column with 80/20 (v/v%) MeOH/ACN with 0.7 mL min^?1 flow rate at 25 °C (R_s?=?5.4, t₂?=?7.45 min). Thermodynamic analysis revealed an enthalpy-driven enantioseparation. The developed HPLC method was validated according to the ICH guideline Q2(R1) and proved to be reliable, linear, precise and accurate for the determination of 0.1% R-enantiomer as chiral impurity in S-APR as well as quantification of the S-enantiomer.

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Reversed-phase HPLC of peptides: Assessing column and solvent selectivity on standard, polar-embedded and polar endcapped columns

We desired to evaluate the chromatographic selectivity for peptides of silica-based RP high-performance liquid chromatography stationary phases with various modifications (polar embedding and polar endcapping on C(18) columns; ether-linked phenyl column with polar endcapping) compared with n-alkyl (C(18), C(8)) and aromatic phenylhexyl columns. Thus, we have designed and synthesized two series of synthetic peptide standards with the sequence Gly-Gly-Leu-Gly-Gly-Ala-Leu-Gly-X-Leu-Lys-Lys-amide, where the N-terminal either contains a free α-amino group (AmC series) or is N(α)-acetylated (AcC series) and where position X is substituted by Gly, Ala, Val, Ile, Phe or Tyr. These represent series of peptides with single substitutions of n-alkyl (Gly相似文献   

Improvement of the chromatographic separation performance of an imidazolium ionic liquid functionalized silica column by in situ anion‐exchange with dodecyl sulfonate and dodecylbenzene sulfonate anions

The anionic part of ionic liquids can provide additional interactions during chromatographic separations. In this work, the chromatographic separation performance of a silica column functionalized with 1‐propyl‐3‐methylimidazolium chloride ionic liquid was improved by in situ anion‐exchange from chloride anions to dodecyl sulfonate anions and dodecylbenzene sulfonate anions. The separation performances of these ionic liquid functionalized phases were investigated and compared with each other using polycyclic aromatic hydrocarbons, phthalates, parabens, and phenols as model compounds. Results indicated that the new columns presented a better chromatographic separation than the original one. This was ascribed retention mechanism from organic anions. The introduction of dodecyl sulfonate anions increased the hydrophobicity of stationary phase. Furthermore, the phenyl groups of dodecylbenzene sulfonate anions could provide an enhanced selectivity to aromatic compounds such as polycyclic aromatic hydrocarbons by π–π interactions. Analysis repeatability of the new columns was satisfactory (RSD of retention time, 0.10–0.40%; RSD of peak area, 0.66–0.84%).     

Analysis of perfluorinated chemicals in sludge: Method development and initial results

A fast, rigorous method was developed to maximize the extraction efficacy for ten perfluorocarboxylic acids and perfluorooctanesulfonate from waste-water-treatment sludge and to quantitate using liquid chromatography, tandem-mass spectrometry (LC/MS/MS). First, organic solvents were tested for extraction efficiency, including acetonitrile (ACN), methanol (MeOH), isopropanol (IPA), tetrahydrofuran (THF), and 50/50 ACN/MeOH (v/v). Among the extractants tested, 50/50 ACN/MeOH yielded the best results for our combined criteria of extraction efficacy and solvent-handling convenience. Second, chemical pretreatment prior to solvent extraction was tested with sodium hydroxide (NaOH), potassium hydroxide (KOH), hydrochloric acid (HCl), and potassium persulfate (K₂S₂O₈). Pretreatment with NaOH and HCl effectively recovered additional PFCs from the sludge, but KOH and K₂S₂O₈ digestion were less effective than no pretreatment. Third, cleanup methods were investigated with solid-phase extraction using HLB (hydrophilic–lipophilic balanced) and WAX (weak-anion exchange) stationary phases, and with ion-pairing. The HLB stationary phase yielded a slight edge over the other two cleanup strategies in terms of recoverable PFCs and chromatographic separation. Finally, the appropriateness of isotopically labeled PFCs for quantitating unlabeled PFCs using isotopic dilution in complex sludge extracts was evaluated by comparison to results obtained with the standard-addition method. A National Institute of Standards and Technology (NIST) domestic sludge (CRM 2781) was analyzed using our finalized method and compared with previously reported results.     

Comparative Chiral Separation of Thalidomide Class of Drugs Using Polysaccharide-Type Stationary Phases with Emphasis on Elution Order and Hysteresis in Polar Organic Mode

The enantioseparation of four phthalimide derivatives (thalidomide, pomalidomide, lenalidomide and apremilast) was investigated on five different polysaccharide-type stationary phases (Chiralpak AD, Chiralpak AS, Lux Amylose-2, Chiralcel OD and Chiralcel OJ-H) using neat methanol (MeOH), ethanol (EtOH), 1-propanol (PROP), 2-propanol (IPA) and acetonitrile (ACN) as polar organic mobile phases and also in combination. Along with the separation capacity of the applied systems, our study also focuses on the elution sequences, the effect of mobile phase mixtures and the hysteresis of retention and selectivity. Although on several cases extremely high resolutions (R_s > 10) were observed for certain compounds, among the tested conditions only Chiralcel OJ-H column with MeOH was successful for baseline-separation of all investigated drugs. Chiral selector- and mobile-phase-dependent reversals of elution order were observed. Reversal of elution order and hysteresis of retention and enantioselectivity were further investigated using different eluent mixtures on Chiralpak AD, Chiralcel OD and Lux Amylose-2 column. In an IPA/MeOH mixture, enantiomer elution-order reversal was observed depending on the eluent composition. Furthermore, in eluent mixtures, enantioselectivity depends on the direction from which the composition of the eluent is approached, regardless of the eluent pair used on amylose-based columns. Using a mixture of polar alcohols not only the selectivities but the enantiomer elution order can also be fine-tuned on Chiralpak AD column, which opens up the possibility of a new type of chiral screening strategy.     

Preparation of high‐selective HPLC packing materials based on alternating copolymer‐grafted silica

Three alternating copolymer‐grafted silica stationary phases for use in high‐selective RP‐HPLC were prepared from two vinyl monomers selected from styrene, N‐methylmaleimide, N‐octadecylmaleimide, and octadecyl acrylate; they were characterized by elemental analyses, thermogravimetric analysis, diffuse reflectance infrared Fourier transform spectroscopy, and ¹³C cross‐polarization magic‐angle spinning NMR spectroscopy. Aspects of molecular‐shape selectivity were evaluated for three different columns using Standard Reference Material 869b, Column Selectivity Test Mixture for Liquid Chromatography. The best selectivity for isomer separations was obtained for the stationary phase prepared with a copolymer of octadecyl acrylate and N‐octadecylmaleimide, which was able to separate 16 polycyclic aromatic hydrocarbons (Standard Reference Material 1647e) in an isocratic elution. In this paper, the effectiveness of this phase is also demonstrated by separation of tocopherol isomers.     

Enantiomeric separation of racemic sulphoxides on chiral stationary phases by gas and liquid chromatography

Summary The enantiomeric resolution of seven racemic sulphoxides on chiral stationary phases has been investigated by gas and liquid chromatography. In gas chromatography the separations were performed on octakis-(2,6-di-O-pentyl-3-O-butyryl)-γ-cyclodextrin (FS Lipodex-E) and heptakis-(2,6-di-O-methyl-3-O-pentyl)-β-cyclodextrin (DMP-β-CD). Both stationary phases were suitable for separation of the enantiomers of the sulphoxides. With one exception for each series all racemetes could be resolved on both stationary phases; FS Lipodex-E was more enantioselective than DMP-β-CD, whereas the latter seemed more generally applicable. Liquid chromatographic separations with Chiralcel-OB as stationary phase were significantly improved by optimization of mobile phase composition and temperature. Resolution factors up to R_s=6 were achieved indicating that the improved separations could now be easily used for preparative purposes.     

Selectivity comparison of acetonitrile-methanol-water ternary mobile phases on an octadecylsiloxane-bonded silica stationary phase

The solvation parameter model was used in this study to investigate various intermolecular interactions that influence retention on the standard C18 stationary phase for the solvent system acetonitrile:methanol (ACN:MeOH, 1:1). In comparison to the organic mobile phase modifiers acetonitrile, acetone, methanol, 2-propanol, and tetrahydrofuran, the solvent strength for the ACN:MeOH (1:1) solvent system was evaluated. To facilitate the interpretation of various intermolecular interactions that contribute to retention on a standard C18 stationary phase for the solvent system ACN:MeOH (1:1), system maps were constructed and compared with those of acetone, tetrahydrofuran, acetonitrile, 2-propanol, and methanol. The solvation parameter models were constructed for the ternary solvent system ACN:MeOH (1:1)-water, and in the models constructed, the coefficient of determination values were from 0.998 to 0.999, the Fisher statistic values for the models were from 1687 to 4015, and the standard error of the estimate values ranged from 0.022 to 0.029. The solvent system ACN:MeOH (1:1) has retention properties more similar to methanol than acetonitrile, indicating methanol's influence is more dominant.     

Current measurement of nonaqueous solvents: Applicatons to capillry electrophoresis and electrochromatography

The solvents acetonitrile, methanol, N, N-dimethylformamide, dimethyl sulfoxide, formamide, and deionized water were investigated for their ability to support current flow without added electrolyte. Using open tubular capillary electrophoresis, currents were measured to be in the nanoampere (10^?9 A) range for all solvents but formamide (10^?6 A). Comparisons with flow data showed no clear relationship between current and electroosmotic flow. Packed capillary columns (3-μm ODS) were used for separations using both pure solvent and hydrophobic dyes showed mild retention in pure ACN. A 16 polynuclear aromatic hydrocarbon (PAH) standard soluton was separated in 80/20 ACN/H₂O with reduced plate heights (h) between 2.8 and 3.1 for retained species. A separation of nine androstenediones was achieved using a 70/30 MeOH/H₂O mobile phase.     

Separation of polycyclic aromatic hydrocarbons with a C60 bonded silica phase in microcolumn liquid chromatography

A chemically bonded C₆₀ silica phase was synthesized as a stationary phase for liquid chromatography (LC) and its retention behavior evaluated for various polycyclic aromatic hydrocarbons (PAHs) using microcolumn LC. The results indicate that the C₆₀ bonded phase offers selectivity different from that of octadecylsilica (ODS) bonded phases in the separation of isomeric PAHs. With the C₆₀ phase, PAH molecules having a partial structure similar to that of the C₆₀ molecule, e.g. triphenylene and perylene, were retained longer than with ordinary ODS stationary phases. The results also show that good correlation exists between the retention data with this C₆₀ bonded phase and with C₆₀ itself as the stationary phase.     

Laterally attached liquid-crystalline polymers as stationary phases. Effect of temperature in RP HPLC and comparison with a commercial C18 stationary phase

Summary Specific side-on-fixed liquid-crystalline polymers (SOLCP) have been synthesized for use in silica-modified stationary phases in high-performance liquid chromatography (HPLC). The mesogenic side group of the SOLCP is composed of three benzoate-type phenyl rings with terminal alkoxy chains and is laterally linked to a polysiloxane backbone via an alkyl ester spacer arm. The dependence of the logarithm of the retention factor on the reciprocal temperature showed that the liquid-crystalline anisotropic order was conserved in the small pores (200 ? diameter) of the silica gel. The first-order nematic-isotropic transition is lost and probably becomes second-order. Adsorption enthalpies for the liquid-crystalline stationary phases have been measurement for three polycyclic aromatic hydrocarbon isomers (ortho-terphenyl, triphenylene, and chrysene) and compared with those for a commercial C₁₈ phase. The adsorption enthalpies never exceeded 30 kJ mol⁻¹, i.e. ten times the thermal agitation energy,RT. They were always less on the SOLCP stationary phase than on the C₁₈ column, emphasizing the more rigid structure of the liquid crystalline phase and its mechanism based upon adsorption. Better separation of steroids, pesticides and amino acids were obtained with the LCP-coated silica than the commercial bonded C₁₈ column. Four small peptides were successfully separated by using pure water as mobile phase.     

Prediction of retention times of polycyclic aromatic hydrocarbons and <Emphasis Type="Italic">n</Emphasis>-alkanes in temperature-programmed gas chromatography

We have developed an iterative procedure for predicting the retention times of polycyclic aromatic hydrocarbons (PAHs) and n-alkanes during separations by temperature-programmed gas chromatography. The procedure is based on estimates of two thermodynamic properties for each analyte (the differences in enthalpy and entropy associated with movements between the stationary and mobile phases) derived from data acquired experimentally in separations under isothermal conditions at temperatures spanning the range covered by the temperature programs in ten-degree increments. The columns used for this purpose were capillary columns containing polydimethylsiloxane-based stationary phases with three degrees of phenyl substitution (0%, 5%, and 50%). Predicted values were mostly within 1% of experimentally determined values, implying that the method is stable and precise. Figure Predicted values were mostly within 1 % of experimentally determined values, thus implying that the method is stable and precise     

Polarizable biphenyl polysiloxane stationary phase for capillary column gas chromatography

A crosslinkable biphenylmethylpolysiloxane stationary phase was synthesized for capillary column gas chromatography and compared with methyl, phenyl, and cyanopropyl polysiloxane stationary phases for the separation of isomeric polycyclic aromatic compounds. While the new phase gave similar separations of nonpolar isomers when compared to the nonpolar phases, separations of polar isomers were greatly improved because of the induced polarity of the biphenyl group of the stationary phase by the solute molecules. This polarizable stationary phase offers a unique selectivity which is not available in other stationary phases.     

An assessment of the retention behaviour of polycyclic aromatic hydrocarbons on reversed phase stationary phases--thermodynamic behaviour on C18 and phenyl-type surfaces

The thermodynamic retention behaviour of a linear series of polycyclic aromatic hydrocarbons (PAHs) was investigated on C18 and selected phenyl-type reversed-phase stationary phases, namely C18, C18 Aqua, Propyl-phenyl and Synergi polar-RP stationary phases, using methanol mobile phases. The Propyl-phenyl stationary phase, despite having the lowest surface coverage, was found to exhibit significantly larger enthalpic interactions to the other Phenyl-type phase (Synergi polar-RP) even though this had a much higher surface coverage. This indicated that stronger interactions between the PAHs and the stationary phase ligands were occurring on the Propyl-phenyl phase. Evaluation of the elution band profile of the PAHs in the aqueous methanol mobile phase revealed fairly symmetrical bands for the C18, C18 Aqua and Synergi polar-RP, but severe peak tailing on the Propyl-phenyl phase. A change in mobile phase from methanol to acetonitrile improved the peak shape of the PAHs on the Propyl-phenyl phase, leading to the assumption that unfavourable pi-pi interactions were occurring between the electron-rich PAHs and the electron-rich phenyl rings of the Propyl-phenyl phase.     

Cyclodextrins as a chiral mobile phase additive in nano-liquid chromatography: comparison of reversed-phase silica monolithic and particulate capillary columns

Enantioseparations of racemic nonsteroidal anti-inflammatory drugs (naproxen, ibuprofen, ketoprofen, flurbiprofen, suprofen, indoprofen, cicloprofen, and carprofen) were performed by nano-liquid chromatography, employing achiral capillary columns and heptakis(2,3,6-tri-O-methyl)-β-cyclodextrin (TM-β-CD) or hydroxylpropyl-β-cyclodextrin (HP-β-CD) as a chiral mobile phase additive (CMPA). Working under the same experimental conditions (in terms of mobile phase and linear velocity), the performance of a RP-C18 monolithic column was compared with that of a RP-C18 packed column of the same dimensions (100 μm i.d. × 10 cm). Utilizing a mobile phase composed of 30% ACN (v/v) buffered with 50 mM sodium acetate at pH 3, and containing 30 mM TM-β-CD, the monolithic column provided faster analysis but lower resolution than the packed column. This behavior was ascribed to the high permeability of the monolithic column, as well as to its minor selectivity. HP-β-CD was chosen as an alternative to TM-β-CD. Employing the monolithic column, the effects of different parameters such as HP-β-CD concentration, mobile phase composition, and pH on the retention factor and the chiral resolution of the analytes were studied. For the most of the analytes, enantioresolution (which ranged from R _s = 1.80 for naproxen to R _s = 0.86 for flurbiprofen) was obtained with a mobile phase consisting of sodium acetate buffer (25 mM, pH 3), 10% MeOH, and 15 mM HP-β-CD. When the same experimental conditions were used with the packed column, no compound eluted within 1 h. Upon increasing the percentage of organic modifier to favor analyte elution, only suprofen eluted within 30 min, with an R _s value of 1.14 (20% MeOH). Replacing MeOH with ACN resulted in a loss of enantioresolution, except for naproxen (R _s = 0.89).     

Rapid preliminary separation of petroleum hydrocarbons by solid-phase extraction cartridges

Small, prepacked columns are tested for fractionating petroleum hydrocarbons from crude oil and products oils. Both normal-phase (silica) and reversed-phase (C₁₈) stationary phases yielded fast, easy, and reproducible separations, which facilitated further analysis of the oils by gas or liquid chromatography and mass spectrometry. The different separation characteristics of the stationary phases offer great flexibility in fractionating oil, enriching special compound classes, or separating hydrocarbons from environmental matrices.