Development of a rapid extraction and high-performance liquid chromatographic separation for amitriptyline and six biological metabolites

The development of a rapid high-performance liquid chromatographic method for the determination of amitriptyline, amitriptyline-N-oxide, 10-hydroxyamitriptyline, 10-hydroxynortriptyline (E and Z isomers), nortriptyline and desmethylnortriptyline in plasma and liver tissue is described. A liquid--liquid extraction with hexane--butanol and back-extraction into phosphoric acid provides efficient extraction of amitriptyline-N-oxide along with amitriptyline and the other metabolites. A Supelcosil C8 reversed-phase column with 5-micron packing and a methanol--sodium phosphate buffer--amine modifier mobile phase was used. The combination of mobile phase pH and amine modifier concentration for the best separation within a reasonable analysis time for all seven solutes plus an internal standard was determined using a factorial design coupled with a multi-factor window diagram technique. Ultraviolet detection at 214 nm provided limits of detection of approximately 1 ng/ml.     

Separation of C1-C5 aliphatic carboxylic acids on a highly sulfonated styrene-divinylbenzene copolymer resin column with a C6 aliphatic carboxylic acid solution as the mobile phase

Simultaneous separation of C₁-C₅ aliphatic carboxylic acids (formic, acetic, propionic, iso-butyric, butyric, iso-valeric and valeric acids) on a highly sulfonated styrene-divinylbenzene copolymer resin column (TSKgel SCX,  mm i.d.) was performed with C₆ aliphatic carboxylic acids (3-methyl-n-valeric, iso-caproic and caproic acids) solutions as the mobile phases. Using 0.05 mM sulfuric acid at pH 4.0 as the mobile phase, although good separation of these C₁-C₅ acids was achieved, peaks of the C₅ acids (iso-valeric and valeric acids) with large hydrophobicity tailed strongly. In contrast, using 1 mM C₆ acids at pH ca. 4.0 as the mobile phases, although vacant peaks corresponding to the C₆ acids in the mobile phase appeared, the peak shapes of the C₅ acids were improved greatly. Excellent simultaneous separation, symmetrical peaks and relatively high-sensitivity conductimetric detection for these C₁-C₅ acids were achieved on the TSKgel SCX column in 15 min with 1 mM iso-caproic acid at pH 4.0 as the mobile phase.     

Separation of the Enantiomers of Thyroxine by HPLC with Chiral Mobile Phases

A high-performance liquid chromatographic (HPLC) method has been developed for separation of the enantiomers of thyroxine (T4). A silica gel column was used in conjunction with a chiral mobile phase (CMP) comprising 35:65 (v/v) acetonitrile–water containing 0.1 mM copper(II) acetate, 0.2 mM L-proline, and 0.5 mM triethylamine (TEA), pH 5.42. The flow rate was 1.0 mL min^?1 and the analysis temperature 40 °C. L-T4 was eluted before D-T4 by mobile phase containing L-proline copper complex. Inversion of the chirality of the mobile phase resulted in reversal of this order of elution. A racemic mobile phase containing DL-proline copper complex resulted in no separation. The enantioseparation phenomenon is discussed. When the method was used to determine the concentration of T4 enantiomers in the serum of a patients with thyroid disease, different concentrations of T4 enantiomers were found in different patents.     

Rapid and preparative separation of traditional Chinese medicine Evodia rutaecarpa employing elution-extrusion and back-extrusion counter-current chromatography: Comparative study

Traditional Chinese medicines (TCMs) have attracted much attention in recent years. Elution-extrusion and/or back-extrusion counter-current chromatography (EECCC/BECCC) both take full advantage of the liquid nature of the stationary phase. They effectively extend the solute hydrophobicity window that can be studied and rendered the CCC technique particularly suitable for rapid analysis of complex samples. In this paper, a popular traditional Chinese medicine, Evodia rutaecarpa, was used as the target complex mixture for extrusion CCC separations. With a carefully selected biphasic liquid system (n-hexane/ethyl acetate/methanol/water, 3/2/3/2, v/v) and optimized conditions (V_CM = V_C, mobile phase flow rate: 3 mL/min in descending mode, sample loading: 100 mg), five fractions could be obtained in only 100 min on a 140-mL capacity CCC instrument using both elution- and back-extrusion methods. Each fraction was analyzed and identified compared with the data of major standards using LC/MS. Moreover, the performance of both extrusion protocols was systematically compared and summarized. EECCC could be operated continuously and was found extremely suitable for high-throughput separation; however, post-column addition of a clarifying reagent is recommended to smooth the UV-signal during the extrusion process. Considering BECCC, the practical operation is very simple by just switching a 4-port valve to change the flow direction. The change of flowing direction should be done after a sufficient amount of mobile phase has flushed the column in the classical mode so that solutes with small and medium distribution constants have been eluted. Otherwise, a significant portion of the solutes will stay in the mobile phase inside the column, mix together and produce a broad peak showing in the mobile phase eluting after the stationary phase extrusion. Compared with classical CCC or other preparative separation tools, extrusion CCC approaches exhibit distinguished superiority in the modernization process of traditional Chinese medicines.     

Simultaneous Estimation of Ceftazidime and Ceftizoxime in Pharmaceutical Formulations by HPLC Method

The aim of the present study was to develop a fast, sensitive and reliable method for rapid screening of cephalosporin injectable dosage forms namely ceftazidime and ceftizoxime to the detection of counterfeit and substandard drugs that might be illegally commercialized. Ceftazidime, ceftizoxime and cefixime (IS) were separated in a X-Terra RP-18 column (250 × 4.60 mm ID × 5 ??) and DAD detector set at 290 and 260 nm. The mobile phase consisted of a mixture of methanol:water 20:80 (v/v) at a flow rate of 1.0 mL min^?1. Additionally, in order to find the optimum pH value of separation the pK _a values of studied compounds were determined by using two different methodologies. Aqueous pK _a values of studied compounds have been determined by UV-spectrophotometry and liquid chromatography were used for the determination and direct characterization of the dissociation constants by using the dependence of the capacity factor on the pH of the mobile phase in 20% (v/v) methanol?Cwater binary mixture in which separation was performed. The pH of the mobile phase was adjusted with 25 mM H₃PO₄ to 3.2. The method was shown to be linear, sensible, accurate, and reproducible over the range of analysis and it can be used to pharmaceutical formulations containing a single active ingredient within a short analysis time.     

Preparation and ion chromatographic properties of a new core-shell chromatographic support Al2O3/SiO2-10

A new stationary phase Al₂O₃/SiO₂-10 was prepared and characterized by XPS, XRD, SEM and surface analysis. The anion exchanger properties of this new stationary phase were investigated by the separation of inorganic anions in ion chromatography (IC). pH of the mobile phase, concentration and strength of the Lewis base of the elute, and the organic modifier of the mobile phase strongly affect the separation of inorganic anions, and anion exchange selectivities of the analyte on the new support are significantly different from quaternary ammonium styrene based anion exchangers. The result of separation of inorganic anions shows that the new stationary phase provides excellent column efficiency, well-defined chromatographic peaks and favorable retention times.     

Electrochemically modulated liquid chromatographic separation of triazines and the effect of pH on retention

Electrochemically modulated liquid chromatography (EMLC) manipulates analyte retention by changing the potential applied (E_app) to a conductive stationary phase. This paper applies EMLC to the separation of a set of seven triazines which are commonly used but environmentally hazardous herbicides. Experiments herein examine the influence of E_app and the pH of the mobile phase on triazine retention. The results are discussed in terms of: (1) retention of triazines of dissimilar acid strengths and by correlations with the pH of the mobile phase; (2) how changes in E_app and acid–base equilibria modulate elution; (3) qualitative insights into EMLC-based retention; and (4) potential merits of EMLC in realizing the rapid separation of the seven-component triazine mixture.     

Retention models for ionizable compounds in reversed-phase liquid chromatography: Effect of variation of mobile phase composition and temperature

General models in reversed-phase liquid chromatography that have been extended to relate retention of ionizable compounds to mobile phase composition, pH and/or temperature are reviewed. In particular, the fundamentals and applications of the solvation parameter model, the polarity parameter model and several classical models based on empirical equations are presented and compared. A main parameter in all these models is the degree of ionization of the acid–base compound, which depends on both the pH of the mobile phase and the acid–base constant of the compound. Thus, on one hand, the different procedures for pH measurement in the mobile phase and their influence on the performance of the models are outlined. On the other hand, equations that relate the variation of the pH of the buffer and the pK_a of the compound with the mobile phase composition and/or temperature are reviewed and their applicability to the retention models critically discussed.     

Tetrazole-Functionalized Silica for Hydrophilic Interaction Chromatography of Polar Solutes

In this work, tetrazole-functionalized stationary phase was prepared with nitrile-modified silica by an ammonium-catalyzed (3 + 2) azide-nitrile cycloaddition reaction. The prepared stationary phase was used for separation of nucleobases and nucleosides by hydrophilic interaction chromatography (HILIC) mode. A typical HILIC mechanism was observed at higher content of acetonitrile (>85%, v/v) in the mobile phase. The retention mechanism of the column was investigated by the models used for describing partitioning and surface adsorption through adjustment ratio of water in the mobile phase, and by the influence of salt concentration, buffer pH, and temperature on the retention of solutes. The results illustrated that the surface adsorption through hydrogen bonding dominated the retention behavior of nucleobases/nucleosides under HILIC mode. From the separation ability, the tetrazole-functionalized stationary phase could become a valuable alternative for the separation of the compounds concerned.     

Optimisation of mobile phase pH in liquid chromatography. Application to fluorimetric detection of series of quinolones

Summary The dissociation constants of several quinolones in a 7% acetonitrile-water mixture are determined. A model describing the effect of pH on the retention of quinolones by an octadecylsilica stationary phase with a 7% acetonitrile-water mobile phase is proposed. The model uses pH values in the mobile phase instead of pH values in water and takes into account the effect of activity coefficients. Fluorescence of quinolones in the mobile phase is studied, and a chromatographic separation with fluorimetric detection is established. Detection limits for the proposed method range from 3 to 12 μg·L⁻¹.     

Simultaneous determination of fangchinoline and tetrandrine in Stephania tetrandra S. Moore by using 1-alkyl-3-methylimidazolium-based ionic liquids as the RP-HPLC mobile phase additives

A reversed phase high performance liquid chromatography (RP-HPLC) method for simultaneous determination of fangchinoline (FAN) and tetrandrine (TET) in Stephania tetrandra S. Moore was established by using 1-hexyl-3-methylimidazolium tetrafluoroborate as the mobile phase additives in this paper. Four types of 1-alkyl-3-methylimidazolium-based ionic liquids (ILs) were used as additives of the mobile phase to separate FAN and TET by RP-HPLC. The effects of the length of the alkyl group on the imidazolium ring and its counterion, the concentrations of IL and the pH of the mobile phase, which influenced the chromatographic behaviors of FAN and TET, were investigated in detail. The linearity, sensitivity, accuracy and repeatability of the proposed method were also investigated. The probable mechanism of the separation with ILs as the mobile phase additives was explored and discussed.     

Phase Ratio and Equilibrium Constant in RP-HPLC Obtained from Octanol/Water Partition Constant Through Solvophobic Theory

The experimentally known dependence in RP-HPLC of the retention factor k′ on octanol/water partition coefficient (K _ow) has been examined based on solvophobic theory. The result showed that the dependence provides a means for the evaluation of phase ratio (Φ) of RP-HPLC columns, and of the equilibrium constant for a given compound and mobile phase. Using this theory, the phase ratio was evaluated for a set of seven different C18 columns (five having fully porous particles and two core–shell particles), and the equilibrium constants were calculated for four homologous series of compounds in two mobile phase systems. One mobile phase was methanol/aqueous solution of 0.1% H₃PO₄, and the other was acetonitrile/aqueous solution of 0.1% H₃PO₄. Besides providing the values for Φ for the evaluated columns, the results of the study indicated that for a specific composition of the mobile phase and for a given compound displaying only hydrophobic interactions, the equilibrium constant K(X) for different C-18 columns is basically the same. The data were further used to provide guidance in the selection of a chromatographic column for a specific separation based on K _ow values and chemical structure of the analytes. The study indicated that the separation of compounds with identical polar groups (or no polar groups) and with very close values for the K _ow cannot be achieved based only on hydrophobic interactions that dominate the separation on RP-type columns. Only column that displays polar interactions may provide a solution to such separations. For hydrocarbons with close K _ow values, the separation cannot be achieved even on columns with some polarity. On the other hand, even compounds with equal K _ow values, but with different functionalities can be separated on RP-HPLC columns without involving polar interactions. The compounds with different K _ow values are expected to be easily separated on RP-HPLC columns.     

Separation behavior of basic compounds on unbonded silicon oxynitride and silica high‐performance liquid chromatography stationary phases with reversed‐phase eluents

Unbonded silicon oxynitride and silica high‐performance liquid chromatography stationary phases have been evaluated and compared for the separation of basic compounds of differing molecular weight, pK_a, and log D using aqueous/organic mobile phases. The influences of percentage of organic modifier, buffer pH, and concentration in the mobile phase on base retention were investigated on unbonded silicon oxynitride and silica phases. The results confirmed that unbonded silicon oxynitride and silica phases demonstrated excellent separation performance for model basic compounds and both the unbonded phases examined possessed a hydrophobic/adsorption and ion‐exchange character. The silicon oxynitride stationary phase exhibited high hydrophilicity compared with silica with a reversed‐phase mobile phase. An ion‐exclusion‐type mechanism becomes predominant for the separation of three aimed bases on the silicon oxynitride column at pH 2.8. Different from silicon oxynitride stationary phase, no obvious change for the retention time of three model bases on silica stationary phase at pH 2.8 can be observed.     

Separation of Selected Beta Lactam Antibiotic Epimers on Gamma Cyclodextrin,Ion Exchange Ethylvinylbenzene/Divinylbenzene/Copolymer and Poly(Styrene-Divinylbenzene) Copolymer Stationary Phases

Abstract

High performance liquid chromatography (HPLC) stationary phases of gamma cyclodextrin, ion exchange ethylvinylbenzene/divinylbenzene (EVB/DVB) copolymer and poly (Styrene-divinylbenzene) (PRP-1) copolymer were investigated for the separation of beta lactam antibiotic epimers of cephalexin, moxalactam, ticarcillin, and carbenicillin. A combination of ion pair chromatography and inclusion complex formation improved the selectivity of moxalactam epimers on gamma cyclodextrin but had no effect on cephalexin epimers. A 10% increase in resolution was obtained for the moxalactam epimers on gamma cyclodextrin when 3mM tetrapropylammonium bromide was present in the mobile phase. Ion-exchange and reverse phase properties of the ion-exchange EVB/DVB phase coupled with perchlorate or sodium pentane sulfonate ion pair chromatography were also successful in separating some of the epimers. Retention and separation behavior of the analytes could not be easily predicted using this multiphase system. The PRP-1 phase was capable of easily resolving the epimers studied with minor adjustments in the mobile phase pH and organic modifier concentration. The PRP-1 phase would be highly recommended for the separation of antibiotic epimers based on the model compounds studied.     

Reversed-phase ion-pair separation of several weak organic acids found in urine

Retention data are presented for homovanillic acid, 5-hydroxyindoleacetic acid, p-hydroxyphenylacetic acid, vanillic acid and hippuric acid on reversed-phase C-18 columns with aqueous and 10% (v/v) methanolic acetic acid buffers. These data are fitted to a model and window diagrams are constructed from calculated model parameters. The optimum pH for separation of these acids is 4.2–4.4.     

LC Enantioseparation of Aryl-Substituted β-Lactams Using Variable-Temperature Conditions

Direct reversed-phase high-performance liquid chromatographic methods were developed for the separation of enantiomers of β-lactams. The enantiomers of 7 aryl-substituted β-lactams were separated on chiral stationary phases containing the macrocyclic glycopeptide antibiotic teicoplanin (Chirobiotic T) and teicoplanin aglycone (Chirobiotic TAG) at 10-°C increments in the range 5–45 °C, using different compositions of 0.1% aqueous triethylammonium acetate (pH 4.1)/methanol (v/v) as mobile phase. The mobile phase composition and temperature were varied to achieve baseline resolutions in a single chromatographic run. The dependence of the natural logarithms of the selectivity factors ln α on the inverse of temperature, 1/T, was used to determine the thermodynamic data on the enantiomers. The thermodynamic data revealed that all the compounds in this study undergo separation via the same enthalpy-driven chiral recognition mechanism. The different methods were compared in systematic chromatographic examinations. The effects of the organic modifier, the mobile phase composition and the temperature on the separation were investigated.     

Preparation and Evaluation of 1,3-alternate 25,27-Bis-[p-nitrobenzyloxy]-26,28-bis-[3-propyloxy]-calix[4]arene-bonded Silica Gel Stationary Phase for LC

A novel 1,3-alternate 25,27-bis-[p-nitrobenzyloxy]-26,28-bis-[3-propyloxy]-calix[4]arene-bonded silica gel stationary phase has been prepared and used for separating various selected analytes by HPLC. The effect of organic modifier and pH of the mobile phase on retention and selectivity were studied using aromatic positional isomers as an example. Application examples have been provided for separation of alkylbenzenes, PAHs, xanthine derivatives, and purine and pyrimidine bases. A selectivity comparison of the novel phase versus CalixBz and Backerbond PhenylEthyl phases has been performed.     

Preparation of Cyclodextrin Type Stationary Phase Based on Graphene Oxide and Its Application in Enantioseparation and Hydrophilic Interaction Chromatography

Graphene oxide (GO) was covalently coupled to the surface of amino silica gel by amide bond. β-cyclodextrin (β-CD) was further chemically bonded with GO to prepare a novel chiral stationary phase. The resulting material was characterized by Fourier transform-infrared (FT-IR) spectra, scanning electron microscopy (SEM), transmission electron microscopy (TEM), elemental analysis and thermogravimetric analysis (TGA). The separation of seven enantiomers was improved in varying degrees. Meanwhile, the stationary phase showed typical characteristics of hydrophilic interaction chromatography (HILIC), and four small nucleoside molecules were separated with the mobile phase of methanol-acetonitrile-water (45:45:10, V/V) in the HILIC mode. In addition, the separation mechanism of the stationary phase was further explored by studying the effects of mobile phase composition, temperature and pH value on the analyte retention. The low temperature was conducive to the separation of analytes at 20–60 °C. The addition of protonated solvent methanol significantly decreased the retention time of the four analytes. The change of pH affected the degree of protonation of the analyte, the interaction between analytes and the stationary phase, and retention time of analytes. The results showed that GO and β-CD played a synergistic effect in the chiral resolution of the chromatographic stationary phase. The retention of analytes in HILIC was attributed to their mixed-mode retention mechanisms including hydrophilic interaction, electrostatic interaction, hydrogen bonding, π-π stacking and so on.