Comparison of zirconia- and silica-based reversed stationary phases for separation of enkephalins

In this study, the separation of biologically active peptides on two zirconia-based phases, polybutadiene (PBD)-ZrO2 and polystyrene (PS)-ZrO2, and a silica-based phase C18 was compared. Basic differences in interactions on both types of phases led to quite different selectivity. The retention characteristics were investigated in detail using a variety of organic modifiers, buffers, and temperatures. These parameters affected retention, separation efficiency, resolution and symmetry of peaks. Separation systems consisting of Discovery PBD-Zr column and mobile phase composed of a mixture of acetonitrile and phosphate buffer, pH 2.0 (45:55, v/v) at 70 degrees C and Discovery PS-Zr with acetonitrile and phosphate buffer, pH 3.5 in the same (v/v) ratio at 40 degrees C were suitable for a good resolution of enkephalin related peptides. Mobile phase composed of acetonitrile and phosphate buffer, pH 5.0 (22:78, v/v) was appropriate for separation of enkephalins on Supelcosil C18 stationary phase.     

Use of the simplex method to optimize the mobile phase for the micellar chromatographic separation of inorganic anions

The sequential simplex strategy has been used to optimize the mobile phase used for separation of inorganic anions by micellar chromatography on a C(18)- micro Bondapak column, with absorption detection at 230 nm. The amount of acetonitrile and the concentration of phosphate buffer (pH 6.0) were chosen for optimization. The optimum mobile phase was found to be 38% acetonitrile in 18.2 mmol L(-1) phosphate buffer (pH 6.0) containing 10 mmol L(-1) cetyltrimethylammonium bromide (CTAB); this optimum was achieved within seven experiments. The separation of the five anions (nitrite, nitrate, iodide, thiocyanate, and thiosulfate) was accomplished in 18 min.     

High-Performance Liquid Chromatography Analysis of Hexamethylmelamine and its Demethylated and Hydroxylated Metabolites in Mice Plasma

Abstract

A selective assay of hexamethylmelamine and its metabolites in mice plasma has been developed. The method uses protein precipitation with acetonitrile in glass centrifuge tubes and analysis of the resulting supernatant by reverse-phase high performance liquid chromatography on a Lichrocart RP 18 column with a mobile phase acetonitrile-phosphate buffer containing n-propylamine (pH 7,5). The influence of various parameters on the separation (solvent composition, pH, n-propylamine content, temperature) has been examined.     

Effects of the operation parameters on Hydrophilic Interaction Liquid Chromatography separation of phenolic acids on zwitterionic monolithic capillary columns

Strongly polar phenolic acids are weakly retained and often poorly separated in reversed-phase (RP) liquid chromatography. We prepared zwitterionic polymethacrylate monolithic columns for micro-HPLC by in situ co-polymerization in fused-silica capillaries. The capillary monolithic columns prepared under optimized polymerization conditions show some similarities with the conventional particulate commercial ZIC-HILIC silica-based columns, however have higher retention and better separation selectivity under reversed-phase conditions, so that they can be employed for dual-mode HILIC-RP separations of phenolic acids on a single column. The capillary polymethacrylate monolithic sulfobetaine columns show excellent thermal stability and improved performance at temperatures 60–80 °C. The effects of the operation conditions on separation were investigated, including the type and the concentration of the organic solvent in the aqueous-organic mobile phase (acetonitrile and methanol), the ionic strength of the acetate buffer and temperature. While the retention in the RP mode decreases at higher temperatures in mobile phases with relatively low concentrations of acetonitrile, it is almost independent of temperature at HILIC conditions in highly organic mobile phases. The best separation efficiency can be achieved using relatively high acetate buffer ionic strength (20–30 mmol L⁻¹) and gradient elution with alternately increasing (HILIC mode) and decreasing (RP mode) concentration of aqueous buffer in aqueous acetonitrile. Applications of the monolithic sulfobetaine capillary columns in alternating HILIC-RP modes are demonstrated on the analysis of phenolic acids in a beer sample.     

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.     

Liquid chromatographic retention and separation of phenols and related aromatic compounds on reversed phase columns

Summary Isocratic column liquid chromatographic systems with UV absorbance detection at 280 nm have been developed for the separation of 29 phenolics and related compounds.The selectivity was investigated on silica-, carbon- and polymer-based separation columns for the separation of phenolic type of components. The effects of various acetonitrile/buffer mixtures, and pH of the mobile phase, and their impact on the retention of the phenols was assessed. Tables of retention times on the four columns for the 29 phenols with two different acetonitrile/buffer mixtures, together with the retention times at three pHs from 6.5 to 2.3 with varying levels of organic modifier on the LiChrospher RP 18 column are presented.As an application, the analysis of real river water samples from the Ebro river is described using a solid phase extraction step prior to injection into the chromatographic system.     

Determination of water-soluble vitamins in vitamin premixes,bioacitve dietary supplements,and pharmaceutical preparations using high-efficiency liquid chromatography with gradient elution

The influence of the pH and temperature of the mobile phase on the retention and separation of 14 vitamins and vitameric forms has been studied for four different stationary phases. The chromatographic conditions allowing the separation of 12 water-soluble vitamins and vitameric forms have been proposed. It has been established that the best separation of water-soluble vitamins can be achieved by employing gradient elution. The mobile phase A, (0.6% phosphoric acid) pH 1.5–1.8; mobile phase B, acetonitrile. For the separation of nicotinic and ascorbic acid it is preferable to use stationary phases Luna C18(2) and Gemini C18, while for the separation of riboflavin and riboflavin-5-phosphate the Synergy Hydro RP and Zorbaz SB-C18 stationary phases should be used. The selected conditions have been used for the determination of vitamins in commercial samples of vitamin preparations. The results are in good accordance with the producer data.     

Comparative Analysis of Acyclovir Esters Stability in Solutions: The Influence of the Substituent Structure,Kinetics, and Steric Effects

Reversed‐phase high‐performance liquid chromatography has been applied to the determination of acyclovir (ACV) esters such as acetate, isobutyrate, pivalate, ethoxycarbonate, and nicotinate. All analyses were carried out at laboratory temperature using a column LiChrospher RP‐18 (250 × 4 mm, 5 µm) and a proper mobile phase consisting of acetonitrile and phosphate buffer (pH 6 or 6.7) or acetonitrile and potassium dihydrogen phosphate, and acetic acid. The methods were validated by the determination of the following parameters: selectivity, precision, accuracy, and linearity. Kinetic studies on the hydrolysis were investigated in solutions at 310 K over the pH range 0.42–1.38. The pH‐profiles indicated specific acid‐catalyzed and spontaneous water‐catalyzed degradation. The stability of the studied ACV esters were determined not only by steric factors. In the case of ethoxycarbonyl ester of ACV, the hydrolysis was a two‐step reaction.     

Comparison of various reversed-phase columns for the simultaneous determination of ephedrines in urine by high-performance liquid chromatography

Different reversed-phase columns for basic analytes were compared for the simultaneous determination of ephedrines in urine, such as LiChrospher 60 RP-Select B, LiChrospher 100 RP18, Hypersil BDS-C18, Inertsil ODS-2, Spherisorb ODS-B and Symmetry Shield RP8. Symmetry Shield was the only column which did not require the use of high concentrations of buffer and triethylamine. With this column, a good separation of the six ephedrines and the internal standard was achieved using 50 mM phosphate buffer–25 mM triethylamine as a mobile phase. Linearity, precision and accuracy were satisfactory for the levels usually found in urine. Due to these all parameters the developed analytical method was found to be suitable for the application in the doping field.     

Comparison of the performance of different reversed-phase columns for liquid chromatography separation of 11 pollutant phenols

A systematic optimization of the HPLC separation of a mixture containing 11 pollutant phenols (PPs) using a Hypersil ODS (250 mm x 4.6 mm, 5 microm) column and UV-DAD detection has been carried out. The binary mobile phases used were obtained by mixing 50 mM phosphate (pH = 3.0) and methanol, ACN, or THF as organic modifiers. After selecting ACN as an organic modifier, the effects of pH and temperature on PPs separation were studied. A mobile phase of 50 mM acetate (pH = 5.0)-ACN (60:40 v/v) at 50 degrees C allowed the separation of 11 phenols but not to baseline in 17 min. To improve the performance of this separation, the following RP columns were tested: Luna C18 (2), Purospher C18, Synergi C12, Synergi Fusion C18, Gemini C18, Luna Cyano, Lichrospher C8, and Envirosep-PP (polymeric). In all the cases, the performance (analysis time, retention, selectivity, resolution, asymmetry factors, and efficiency) was evaluated. A further reoptimization of the mobile phase was carried out for all the columns by studying the ACN content and pH, with the aim of improving the above-mentioned separations and selecting the most suitable one for PPs analysis.     

Analysis of phenolic acids in fruits by HPLC with monolithic columns

Reversed-phase HPLC was optimised for simultaneous determination of several derivatives of benzoic and hydroxycinnamic acids (so-called phenolic acids) in plums using a commercially available monolithic column. Mobile phase pH and concentration of organic modifier (methanol and acetonitrile) were tested in order to obtain the best resolution. Satisfactory separation was achieved in gradient mode with a mobile phase consisting of 50 mM phosphate buffer at pH 2.2 (solvent A) and acetonitrile (solvent B). The limits of detection for a UV detector ranged between 0.098 and 2.04 microg/mL for vanillic acid and p-hydroxyphenylacetic acid, respectively. The developed method was used for monitoring the content of polyphenolic acids in plums during their ripening process. The presence of these constituents was confirmed by checking their MS spectra.     

Determination of Antiviral Drugs and Their Metabolites Using Micro-Solid Phase Extraction and UHPLC-MS/MS in Reversed-Phase and Hydrophilic Interaction Chromatography Modes

Two new ultra-high performance liquid chromatography (UHPLC) methods for analyzing 21 selected antivirals and their metabolites were optimized, including sample preparation step, LC separation conditions, and tandem mass spectrometry detection. Micro-solid phase extraction in pipette tips was used to extract antivirals from the biological material of Hanks balanced salt medium of pH 7.4 and 6.5. These media were used in experiments to evaluate the membrane transport of antiviral drugs. Challenging diversity of physicochemical properties was overcome using combined sorbent composed of C₁₈ and ion exchange moiety, which finally allowed to cover the whole range of tested antivirals. For separation, reversed-phase (RP) chromatography and hydrophilic interaction liquid chromatography (HILIC), were optimized using extensive screening of stationary and mobile phase combinations. Optimized RP-UHPLC separation was carried out using BEH Shield RP18 stationary phase and gradient elution with 25 mmol/L formic acid in acetonitrile and in water. HILIC separation was accomplished with a Cortecs HILIC column and gradient elution with 25 mmol/L ammonium formate pH 3 and acetonitrile. Tandem mass spectrometry (MS/MS) conditions were optimized in both chromatographic modes, but obtained results revealed only a little difference in parameters of capillary voltage and cone voltage. While RP-UHPLC-MS/MS exhibited superior separation selectivity, HILIC-UHPLC-MS/MS has shown substantially higher sensitivity of two orders of magnitude for many compounds. Method validation results indicated that HILIC mode was more suitable for multianalyte methods. Despite better separation selectivity achieved in RP-UHPLC-MS/MS, the matrix effects were noticed while using both chromatographic modes leading to signal enhancement in RP and signal suppression in HILIC.     

Liquid Chromatographic Separation of Some Common Benzodiazepines and their Metabolites

Abstract

A series of benzodiazepines commonly encountered in forensic samples were separated using isocratic reversed-phase liquid chromatography. These compounds display a wide range of capacity factors on a C₁₈ stationary phase in a pH 8 phosphate buffer and methanol mobile phase. Clorazepate must be analyzed under basic mobile phase conditions to prevent its decomposition to N-desmethyldiazepam. The separation of common parent benzodiazepines such as chlordiazepoxide, diazepam and flurazepam from their corresponding metabolites was achieved under a variety of reversed-phase conditions.     

A Validated RP–LC Method for Simultaneous Determination of Losartan Potassium and Amlodipine Besilate in Pharmaceutical Preparations

A simple RP–LC method for simultaneous quantification of losartan and amlodipine and separation of their degradation products has been developed. For this purpose we tested appropriated mobile phase pH range, flow rate, temperature and different columns. The method was validated with an ODS column. A gradient of acetonitrile and phosphate pH 3.0 buffer was utilized as mobile phase. The linearity was determined at 50–150% level. Individual recoveries at 70–130% level ranged from 98.8 to 100.5% for losartan and 96.4–101.2% for amlodipine. The robustness was also evaluated. Although losartan has much higher quantities than amlodipine in commercial tablets, this method allowed simultaneous quantification for both drugs.     

Influence of mobile phase composition on electroosmotic flow velocity, solute retention and column efficiency in open-tubular reversed-phase capillary electrochromatography

The effects of some experimental parameters, such as the volume fraction and type of organic modifier in the mobile phase, and the concentration, type and pH of the buffer on the electroosmotic flow velocity, the retention behavior of test solutes, and the column efficiency have been investigated in capillary electrochromatography (CEC) using an open-tubular column of 9.60 microm I.D. with a porous silica layer chemically modified with C18 as stationary phase. The retention of a group of polycyclic aromatic hydrocarbons (PAHs) used as a test mixture varied significantly by changing the organic modifier content in the hydroorganic mobile phase according to the reversed-phase-like selectivity of the stationary phase. In addition, an increase in the percentage of organic modifier resulted in a slight increase in the linear velocity of the EOF. On the other hand, when the phosphate buffer concentration was increased over the range 1-50 mM, the electroosmotic mobility fell dramatically, the retention of the solutes decreased steadily, and the plate height showed a significant increase. The results obtained with phosphate, trishydroxymethylaminomethane or 2-morpholinoethanesulfonic acid as buffers were similar when pH remained constant. Optimization in CEC was essential to achieve further enhancement of separation performance, because the analysis time and separation resolution are essentially affected when varying operating parameters. Separations of seven PAHs with more than 100000 plates are presented within 4 min analysis time.     

A Rapid High Performance Liquid Chromatographic Method for the Simultaneous Measurement of Six Tricyclic Antidepressants

Abstract

A reversed-phase high pressure liquid chromatographic procedure has been developed for the quantitation of the concentration of six different tricyclic antidepressants in the plasma of patients undergoing routine drug therapy. Plasma samples were extracted into a 97:3 hexane:isoamyl alcohol solution and then extracted back into dilute acid. A Supelcosil C-8 column with 5 micron packing was employed in combination with an acetonitrile/ phosphate buffer/diethylamine mobile phase. At an optimized mobile phase pH of 7.22, baseline separation of all six tricyclic antidepressants plus the internal standard was achieved within 8 minutes. UV detection at 254 nm resulted in limits of detection of 2.5 μg/L for each drug. The potential interferences from 13 different benzodiazepines and neuroleptics was investigated. Five of the 13 parent drugs and three metabolites were found to interfere with this tricyclic antidepressant assay.     

A Simple Isocratic High-Performance Liquid Chromatographic (HPLC) Determination of Naproxen in Human Plasma using a Microbore Column Technique

Abstract

A simple and sensitive HPLC method was developed for the determination of naproxen in human plasma. The assay employs a microbore column packed with a C18 reversed-phase material (5 μm ODS Hypersil) with an isocratic mixture of acetonitrile and 10 mM phosphate buffer, pH 2.5 (40:60, v/v) as the mobile phase. The mobile phase was pumped at a flow rate of 0.5 ml/min. For sample analysis 200 μl of acetonitrile containing internal standard (flurbiprofen) was added to 100 μl of plasma. After centrifugation 10 mM phosphate buffer, pH 7.4 (200 μl) was added to the tube, then vortexed and centrifuged. The supernatant (20 μl) was injected onto the HPLC column. The chromatographic separation was monitored by a fluorescence detector at an emission wavelength of 350 nm with an excitation wavelength of 225 nm. The direct precipitation of plasma protein using acetonitrile gave a good recovery for both naproxen and the internal standard. The detection limit was 0.1 μg/ml for naproxen. The intra- and inter-assay coefficients of variation at different concentrations evaluated were less than 10%.     

Fast HPLC method using ion-pair and hydrophilic interaction liquid chromatography for determination of phenylephrine in pharmaceutical formulations

A rapid procedure based on a direct extraction and HPLC determination with fluorescence detection of phenylephrine in pharmaceutical sachets that include a large excess of paracetamol (65 + 1, w/w), ascorbic acid (5 + 1, w/w), and other excipients (aspartame and sucrose) was developed and validated. The final optimized chromatographic method for ion-pair chromatography used an XTerra RP18 column, 3 microm particle size, 50 x 3.0 mm id. The mobile phase consisted of a mixture of acetonitrile and buffer (10 mM sodium octane-1-sulfonate, adjusted with H3PO4 to pH 2.2; 200 + 800, v/v), with a constant flow rate of 0.3 mL/min. The separation was carried out at 30 degrees C, and the injection volume was 3 microL. Fluorescence detection was performed at excitation and emission wavelengths of 275 and 310 nm, respectively. The mobile phase parameters, such as the organic solvent fraction (acetonitrile) in mobile phase as an organic modifier, the concentration of sodium octane-1-sulfonate as a counter-ion, temperature, and pH of mobile phase, were studied. As an alternative to ion-pair chromatography, hydrophilic interaction liquid chromatography (HILIC) was investigated using a Luna HILIC column, 3 microm, 100 x 4.6 mm id. The mobile phase consisted of acetonitrile and buffer (5 mM potassium dihydrogen phosphate, adjusted with H3PO4 to pH 2.5; 750 + 250, v/v) at a flow rate of 0.8 mL/min. The separation was carried out at 25 degrees C, and the injection volume was 5 microL. The proposed method has an advantage of a very simple sample pretreatment, and is much faster than the currently utilized HPLC methods using gradient elution and UV detection. Commercial samples of sachets were successfully analyzed by the proposed HPLC method.     

Optimization of a liquid-liquid extraction method for HPLC-DAD determination of penicillin-V in human plasma

Optimization of a liquid-liquid extraction procedure used for isolation of penicillin-V in human plasma samples and the subsequent HPLC-DAD analysis are reported. Some aspects related to the stability of penicillin-V in plasma and according to pH are also given. From seven tested solvents, the highest extraction yield was obtained with methyl-t-buthyl ether. Influence of the extraction parameters, such as pH of aqueous phase, sample/solvent volumetric ratio, and extraction duration, is discussed. HPLC separation was achieved on an Eclipse XDB-C8 column, using a mobile phase composed by aqueous phosphate buffer (pH=7.3), methanol and acetonitrile in the ratio 8:1:1. Quantitation limits of 50 ng/ml were obtained. The optimal sample preparation method and HPLC-DAD separations were used for a bioequivalence study, made on 36 volunteers.     

Retention mechanisms and selectivity in internal-surface reversed-phase liquid chromatography. Studies with cyanobacterial peptide toxins

Summary Microcystins-LA,-LR,-RR,-YR and nodularin, cyanobacterial peptide toxins, were separated by internal-surface reversed-phase (ISRP), high-performance liquid chromatography. The capacity factors of the toxins were measured in the range pH 2–8 using acetonitrile, isopropanol or tetrahydrofuran in potassium dihydrogenphosphate mobile phase. The main retention mechanism of the ISRP column was reversed-phase interaction but cation-exchange offered additional selectivity at neutral and slightly acidic pH. At neutral pH (10% modifier, 0.1 M buffer) the elution order was microcystin-LA (two nonpolar residues leucine and alanine as the variable amino acids), nodularin, microcystin-LR,-YR and-RR (two basic arginines as the variable amino acids). The retention times of all toxins except microcystin-RR were substantially longer at acidic pH. At pH 2 (10% modifier, 0.1 M buffer) where the cation-exchange mechanism was inoperative the elution order was changed to microcystin-RR, nodularin, microcystin-LR,-YR and-LA. The best separation was achieved at pH 2 where even two desmethylated microcystin-RR analogs could be separated from microcystin-RR.