Use of vancomycin chiral stationary phase for the enantiomeric resolution of basic and acidic compounds by nano-liquid chromatography

In this paper we studied the potentiality of nano-liquid chromatography (nano-LC) for the enantiomeric resolution of both basic and acidic compounds of pharmaceutical interest using a vancomycin modified silica stationary phase. Experiments were carried out in a fused silica capillary of 75 microm I.D. packed with chiral modified silica particles of 5 microm diameter, the detection, was done on-line at 195 nm. Enantiomeric resolution of alprenolol, atenolol, metoprolol, oxprenolol, pindolol, propranolol (basic compounds) and some acidic analytes, namely 2-[(5'-benzoyl-2'-hydroxy)phenyl]propionic acid (DF1738Y), 2-[(4'-benzoyloxy-2'-hydroxy)phenyl]propionic acid (DF1770Y), ketoprofen, indoprofen and suprofen was studied by nano-LC utilizing mobile phases containing methanol-acetonitrile-ammonium formate or acetate. The effect of mobile phase composition (buffer type and concentration, organic modifier type and concentration) on chiral resolution (Rs), retention factor (k) and retention time (tR) was also investigated. Good enantiomeric resolution was achieved for basic compounds utilizing the mobile phase containing 90% (MeCN-MeOH)/5% water/5% of 100 mM ammonium acetate pH 4.5. Acidic compounds such as DF1738Y and DF1770Y were better resolved at lower pH 3.5 while ketoprofen, indoprofen and suprofen exhibited the highest resolution at pH 4.5; in this case the mobile phase contained MeOH or MeCN (90%), 5% buffer and 5% of water. The nano-LC method was validated using R-(+)-propranolol as an internal standard finding good repeatability, detection limit, correlation coefficient and recovery and applied to the assay of a pharmaceutical formulation containing a racemic mixture of metoprolol.     

Use of vancomycin silica stationary phase in packed capillary electrochromatography: III. enantiomeric separation of basic compounds with the polar organic mobile phase

The separation of basic compounds into their enantiomers was achieved using capillary electrochromatography in 50 or 75 microm inner diameter (ID) fused-silica capillaries packed with silica a stationary phase derivatized with vancomycin and mobile phases composed of mixtures of polar organic solvents containing 13 mM ammonium acetate. Enantiomer resolution, electroosmotic flow, and the number of theoretical plates were strongly influenced by the type and concentration of the organic solvent. Mobile phases composed of 13 mM ammonium acetate dissolved in mixtures of acetonitrile/methanol, ethanol, n-propanol, or isopropanol were tested and the highest enantioresolutions were achieved using the first mobile phase, allowing the separation of almost all investigated enantiomers (9 from 11 basic compounds). The use of capillaries with different ID (50 and 75 microm ID) packed with the same chiral stationary phase revealed that a higher number of theoretical plates and higher enantioresolution was achieved with the tube with lowest ID.     

Use of vancomycin silica stationary phase in packed capillary electrochromatography I. Enantiomer separation of basic compounds

Chiral separation of basic compounds was achieved by using 75 or 100 microm ID fused-silica capillaries packed with a vanoomycin-modified diol silica stationary phase. The capillary was firstly packed for about 12 cm with a slurry mixture composed of diolsilica (3:1) then with the vancomycin modified diol-silica (3:1) (23 cm), and finally with diol-silica (3:1) for about 2 cm. Frits were prepared by a heating wire at the two ends of the capillary; the detector window was prepared at 8.5 cm from the end of the capillary where vancomycin was not present. The influence of the mobile phase composition (pH and concentration, organic modifier type and concentration) on the velocity of the electroosmotic flow, chiral resolution and enantioselectivity was studied. Good enantiomeric resolution was achieved for atenolol, oxprenolol, propranolol, and venlafaxine using a mobile phase composition of 100 mM ammonium acetate solution (pH 6)/water/acetonitrile (5:5:90 v/v/v) while for terbutaline a mixture of 5:15:80 v/v/v provided the best separations. The use of methanol instead of acetonitrile caused a general increase of enantiomer resolution of the studied compounds together with a reduction of efficiency and detector response. However, the combination of acetonitrile and methanol in the mobile phase (as, e.g., 10% methanol and 80% acetonitrile) allowed to improve the enantiomer resolution with satisfactory detector response.     

Reversed-phase capillary electrochromatography for the simultaneous determination of acetylsalicylic acid, paracetamol, and caffeine in analgesic tablets

The separation and simultaneous determination of caffeine, paracetamol, and acetylsalicylic acid in two analgesic tablet formulations was investigated by capillary electrochromatography (CEC). The effect of mobile phase composition on the separation and peak efficiency of the three analytes was studied and evaluated; in particular, the influence of buffer type, buffer pH, and acetonitrile content of the mobile phase was investigated. The analyses were carried out under optimized separation conditions, using a full-packed silica capillary (75 microm ID; 30.0 cm and 21.5 cm total and effective lengths, respectively) with a 5 microm C8 stationary phase. A mixture of 25 mM ammonium formate at pH 3.0 and acetonitrile (30:70 v/v) was used as the mobile phase. UV detection was at 210 nm. Good linearity was found in the range of 50-200, 20-160, and 4-20 microg/mL for acetylsalicylic acid (r2=0.9988), paracetamol (r2=0.9990) and caffeine (r2=0.9990), respectively. Intermediate precision (RSD interday) as low as 0.1-0.8% was found for retention times, while the RSD values for the peak area ratios (Aanalyte/AIS) were in the range of 1.9-2.9%. The optimized CEC method was applied to the analysis of the studied compounds present in commercial tablets.     

Chiral separation of beta-adrenergic antagonists, profen non-steroidal anti-inflammatory drugs and chlorophenoxypropionic acid herbicides using teicoplanin as the chiral selector in capillary liquid chromatography

Three groups of structurally diverse chiral compounds were used to study the interaction mechanism responsible for stereoselective recognition with teicoplanin as chiral selector in capillary liquid chromatography. Teicoplanin-based chiral stationary phase (CSP) was used. The effect of the variation of mobile phase composition on retention and enantioselective separation was studied. The mobile phase composition suitable for enantioresolution of the various chiral compounds differed according to the interaction forces needed for chiral recognition. Mobile phases with high buffer portion (70-90 vol.%) were preferred for separation of enantiomers of profen non-steroidal anti-inflammatory drugs and chlorophenoxypropionic acid herbicides that require hydrophobic interactions, inclusion and pi-pi interactions for stereoselective recognition with teicoplanin. Higher concentration triethylamine in the buffer (0.5-1.0%) increased resolution of these acids. On the other hand, H-bonding and electrostatic interactions are important in stereoselective interaction mechanism of beta-adrenergic antagonists with teicoplanin. These interaction types predominate in the reversed phase separation mode with high organic modifier content (95% methanol) and in polar organic mobile phases. For this reason beta-adrenergic antagonists were best enantioresolved in the polar organic mode. The mobile phase composed of methanol/acetic acid/triethylamine, 100/0.01/0.01 (v/v/v), provided enantioresolution values of all the studied beta-adrenergic antagonists in the range 1.1-1.9. Addition of teicoplanin to the mobile phase, which was suitable for enantioseparation of certain compounds on the CSP, was also investigated. This system was used to dispose of nonstereoselective interactions of analytes with silica gel support that often participate in the interaction with CSPs. Very low concentration of teicoplanin in the mobile phase (0.1 mM) resulted in enantioselective separation of 2,2- and 2,4-chlorophenoxypropionic acids.     

Separation of enantiomers by nanoliquid chromatography and capillary electrochromatography using a bonded cellulose trisphenylcarbamate stationary phase

A cellulose trisphenylcarbamate-bonded chiral stationary phase was applied to nano-liquid chromatography (nano-LC) and capillary electrochromatography (CEC) with nonaqueous and aqueous solutions as the mobile phases. Several chiral compounds were successfully resolved on the prepared phase by nano-LC. The applicability of nonaqueous CEC on a cellulose derivative stationary phase was investigated with the organic solvents methanol, hexane, 2-propanol, and tetrahydrofuran (THF) containing acetic acid, as well as triethylamine as the mobile phases. Enantiomers of warfarin and praziquantel were baseline-resolved with plate numbers of 82,300 and 38,800 plates/m, respectively, for the first eluting enantiomer. The influence of applied voltage, concentration of nonpolar solvent, apparent pH, and buffer concentration in the mobile phase on the electroosmotic flow (EOF) and the mobility of the enantiomers was evaluated. Enantioseparations of trans-stilbene oxide and praziquantel were also achieved in aqueous CEC with plate numbers of 111,100 and 107,400 plates/m, respectively, for the first eluting enantiomer. A comparison between nonaqueous CEC and aqueous CEC based on a cellulose trisphenylcarbamate stationary phase was discussed. Pressure-assisted CEC was examined for the chiral separation of praziquantel and faster analysis with high enantioselectivity was acquired with the proper pressurization of the inlet vial.     

Enantiomeric analysis of drugs in water samples by using liquid–liquid microextraction and nano-liquid chromatography

The nano-LC technique is increasingly used for both fast studies on enantiomeric analysis and test beds of novel stationary phases due to the small volumes involved and the short conditioning and analysis times. In this study, the enantioseparation of 10 drugs from different families was carried out by nano-LC, utilizing silica with immobilized amylose tris(3-chloro-5-methylphenylcarbamate) column. The effect on chiral separation caused by the addition of different salts to the mobile phase was evaluated. To simultaneously separate as many enantiomers as possible, the effect of buffer concentration in the mobile phase was studied, and, to increase the sensitivity, a liquid–liquid microextraction based on the use of isoamyl acetate as sustainable extraction solvent was applied to pre-concentrate four chiral drugs from tap and environmental waters, achieving satisfactory recoveries (>70%).     

Optical isomer separation of flavanones and flavanone glycosides by nano-liquid chromatography using a phenyl-carbamate-propyl-β-cyclodextrin chiral stationary phase

In this paper a phenyl-carbamate-propyl-β-cyclodextrin stationary phase was employed for the enantioseparation of several flavonoids, including flavanones and methoxyflavanones by using nano-liquid chromatography (nano-LC). The same stationary phase was also used for the diastereoisomeric separation of two flavanone glycosides. The compounds: flavanone, 2′-hydroxyflavanone, 4′-hydroxyflavanone, 6-hydroxyflavanone, 7-hydroxyflavanone, 4′-methoxyflavanone, 6-methoxyflavanone, 7-methoxyflavanone, hesperetin, hesperidin, naringenin and naringin were studied using reversed, polar organic and normal elution modes. The effect of the nature and composition of the mobile phase (organic modifier type, buffer and water content in the reversed phase mode) on the enantioresolution (R_s), retention factor (k) and enantioselectivity (α) were investigated. Baseline resolution of all studied flavonoids, with the exception of 2′-hydroxyflavanone and naringin, was achieved in reversed phase mode using a mixture of MeOH/H₂O at different ratios as mobile phase. Good results, in terms of peak efficiency and short analysis time, were obtained adding 1% triethylammonium acetate pH 4.5 buffer to MeOH/H₂O mixture. The separation of the studied compounds was also performed in polar organic mode. By using 100% of MeOH as mobile phase, the resolution was achieved for the studied analytes, except for 7-hydroxyflavanone, 2′-hydroxyflavanone, naringenin, hesperidin and naringin. Normal mode was tested employing a mixture of EtOH/hexane/TFA as mobile phase achieving the enantiomeric and diastereomeric separation of only hesperetin and hesperidin, respectively. The use of nano-LC technique for the resolution of flavanones optical isomers allowed to achieve good resolutions in shorter analysis time compared to the results reported in literature with conventional HPLC.     

Two fluoroalcohols as components of basic buffers for liquid chromatography electrospray ionization mass spectrometric determination of antibiotic residues

Two fluoroalcohols--1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) and 1,1,1,3,3,3-hexafluoro-2-methyl-2-propanol (HFTB)--were evaluated for the first time as volatile buffer acids in the basic mobile phase for reversed-phase chromatography with electrospray ionization-mass spectrometric (LC-ESI-MS) detection of five antibiotics. Chromatographic separation as well as positive and negative ion ESI-MS intensities using these novel buffer components were compared to traditional buffer systems. Overall, the highest signal intensities and best chromatographic separation for the five antibiotics (ciprofloxacin, norfloxacin, ofloxacin, sulfadimethoxine and sulfamethoxazole) were achieved using 5 mM HFIP as the buffer acid to methanol : water mobile phase (pH of the aqueous component adjusted to 9.0 with ammonium hydroxide). Comparable results were achieved using 5 mM HFTB (pH adjusted to 9.0 with ammonium hydroxide). The suitability of HFIP for analysis of antibiotic residues in lettuce is demonstrated.     

Enantiomer separation of flavour and fragrance compounds by liquid chromatography using novel urea-covalent bonded methylated beta-cyclodextrins on silica

A novel methylated beta-cyclodextrin chiral stationary phase (CSP-ME), which was chemically immobilised onto porous silica via multiple urea-linkages was synthesised. The CSP-ME chiral stationary phase depicted good enantiomer separation abilities for some well-known flavour as well as fragrance compounds using high-performance liquid chromatography under reverse phase conditions. The optimum resolution for alpha-ionone, 3-methyl-alpha-ionone, flavanone, 5-methoxyflavanone, 6-methoxyflavanone, 7-methoxyflavanone, hesperetin, naringenin and taxifolin was achieved using a mobile phase composition consisting of 1 wt.% triethylammonium acetate buffer (pH 4.68)-methanol. The effects of pH of triethylammonium acetate buffer and the methanol-acetonitrile content of the mobile phase composition on their retention time and resolution were examined to optimise the separation conditions.     

Investigation of polar stationary phases for the separation of sympathomimetic drugs with nano-liquid chromatography in hydrophilic interaction liquid chromatography mode

In this study, the retention and selectivity of a mixture of basic polar drugs were investigated in hydrophilic interaction chromatographic conditions (HILIC) using nano-liquid chromatography (nano-LC). Six sympathomimetic drugs including ephedrine, norephedrine, synephrine, epinephrine, norepinephrine and norphenylephrine were separated by changing experimental parameters such as stationary phase, acetonitrile (ACN) content, buffer pH and concentration, column temperature. Four polar stationary phases (i.e. cyano-, diol-, aminopropyl-silica and Luna HILIC, a cross-linked diol phase) were selected and packed into fused silica capillary columns of 100 μm internal diameter (i.d.). Among the four stationary phases investigated a complete separation of the all studied compounds was achieved with aminopropyl silica and Luna HILIC stationary phases only. Best chromatographic results were obtained employing a mobile phase composed by ACN/water (92/8, v/v) containing 10 mM ammonium formate buffer pH 3. The influence of the capillary temperature on the resolution of the polar basic drugs was investigated in the range between 10 and 50 °C. Linear correlation of ln k vs. 1/T was observed for all the columns; ΔH° values were negative with Luna HILIC and positive with aminopropyl- and diol-silica stationary phases, demonstrating that different mechanisms were involved in the separation.To compare the chromatographic performance of the different columns, Van Deemter curves were also investigated.     

Preparation and enantiomer separation behavior of selectively methylated β-cyclodextrin-bonded stationary phases for high performance chromatography

Native and three selectively methylated β-cyclodextrin (β-CD)-bonded stationary phases without an unreacted spacer arm for liquid chromatography were prepared, where heptakis(2-O-methyl)-β-CD, heptakis(3-O-methyl)-β-CD and heptakis(2,3-di-O-methyl)-β-CD were used as the methylated β-CDs. The enantiomer separation abilities of the resulting β-CD stationary phases for 12 pairs of dansylamino acid enantiomers and six pairs of N-3,5-dinitrobenzoyl amino acid methyl esters as model solutes were investigated. The effects of pH and methanol content of the mobile phase on the retention and resolution were examined to optimize the mobile phase conditions. The optimum resolution for the dansylamino acids was achieved using a mobile phase consisting of 1.0% triethylammonium acetate buffer (pH 5.0)–methanol (v/v 4/6) on the β-CD stationary phase. Heptakis(3-O-methyl)- and heptakis(2,3-di-O-methyl)-β-CD-bonded stationary phases showed little enantiomer separation abilities for the dansylamino acids. The heptakis(2-O-methyl)-β-CD-bonded stationary phase exhibited no enantioselectivities for those solutes.

For the N-3,5-dinitrobenzoyl amino acid methyl esters, the optimum resolution was achieved using a mobile phase consisting of 1.0% triethylammonium acetate buffer (pH 5.0)–methanol (v/v 9/1) on a heptakis(2-O-methyl)-β-CD stationary phase. The heptakis(2,3-di-O-methyl)-β-CD-bonded stationary phases exhibited no enantioselectivities for the N-3,5-dinitrobenzoyl amino acid methyl esters. β-CD and heptakis(3-O-methyl)-β-CD-bonded stationary phases had no enantiomer separation abilities for those solutes except for the N-3,5-dinitrobenzoyl phenylalanine methyl ester.     

Sensitive and Selective Fluorescence Detection of Phenoxyacid Herbicides by Liquid Chromatography with On-Line Ion-Pair Extraction

Abstract

Fluorescent probes, especially a newly synthesized N-substituted 1-cyanobenz[f]-isoindole quaternary ammonium fluorophore, were used as counter ions in a reaction detector for on-line ion-pair extraction of phenoxyacid herbicides. The probe was used in an on-line post-column set-up coupled to a reversed-phase chromatographic system. After separation on an C-8-bonded silica column using an aqueous methanol (pH 2.5) mobile phase, the herbicides were on-line deprotonated by post-column addition of a 10mM sodium phosphate buffer (pH 8.0), in which the probe was dissolved. Subsequently, the ion-pairs were extracted on-line with chloroform-1-butanol (80:20, v/v) and were monitored by fluorescence detection. Using this system, at least seven herbicides could be separated. The detection limits of 2,4-dichlorophe xyacetic acid and 2,4,5-trichlorophenoxyacetic acid were 400 pg (S/N = 3). The repeatability, based on peak height measurements, for 100ng injections was about 0.5%. Calibration curves were linear over the investigated range of 1–100ng, with correlation coefficients of 0.999 for the two analytes. Application to a drinking water sample is presented.     

高效液相色谱β-环糊精键合固定相直接拆分硫代和硒代缩水甘油醚对映异构体

 用两种不同的方法合成了β-环糊精键合固定相。通过对硝基苯胺异物体的拆分,对键合相进行了评价。拆分了3种硫代缩水甘油醚和2种未见文献报道的硒代缩水甘油醚。     

The effect of stationary phase on lipophilicity determination of beta-blockers using reverse-phase chromatographic systems

Evaluation of lipophilicity parameters for basic compounds using different chromatographic stationary phases is presented. An HPLC method for determination of lipophilic molecule-stationary phase interactions was based on gradient analysis. Differences in correlation between the lipophilicity of compounds and experimental chromatographic results obtained in pseudo-membrane systems showed a strong influence of stationary phase structure and physico-chemical properties. beta-Blocker drugs with varying lipophilicity and bio-activity were chosen as test compounds. The stationary phases used for the study were monolithic rod-structure C18 and silica gel octadecyl phase SG-C18 as reference material. The second group was silica gel-based polar-embedded alkylamide and cholesterolic phases. The mobile phase was composed of acetonitrile or methanol with ammonium acetate, and a linear gradient of methanol and acetonitrile in mobile phase was performed. A linear correlation of plots of log k(g) = f(log P) was observed, especially for polar-embedded phases, and this allowed log P(HPLC) to be calculated. The behavior of stationary phases in methanol and acetonitrile buffer showed differences between obtained log P(HPLC) values.     

核苷与碱基的苯胺甲基键合硅胶固定相高效液相色谱分离

建立苯胺甲基键合硅胶固定相(PAMS)高效液相色谱分离核苷与碱基的方法；研究流动相有机溶剂浓度、磷酸缓冲液pH值、离子强度对核苷和碱基在该键合固定相上的色谱保留及分离选择性的影响，用磷酸缓冲液(pH＝4)为流动相快速分离了部分核苷与碱基。     

Capillary electrochromatography-mass spectrometry of cationic surfactants

The CEC-MS of alkyltrimethylammonium (ATMA+) ions with chain lengths ranging from C1-C18 is optimized using an internally tapered column packed with mixed mode reversed phase/strong cation exchange stationary phase. A systematic study of the CEC separation parameters is conducted followed by evaluation of the ESI-MS sheath liquid and spray chamber settings. First, the optimization of CEC separation parameters are performed including the ACN concentration, triethylamine (TEA) content, buffer pH and ammonium acetate concentration. Using 90% v/v ACN with 0.04% v/v TEA as mobile phase, the separation of longer chain C6-C18-TMA+ surfactants could be achieved in 15 min. Lowering the ACN concentration to 70% v/v provided resolution of shorter chain C1, C2-TMA+ from C6-TMA+ although the total analysis time increased to 40 min. Furthermore, variation of both the ACN and TEA content as well as ionic strength has found to significantly influence the retention of longer chain surfactants as compared to shorter chains. The optimum CEC conditions are 70% v/v ACN, 0.04% v/v TEA, pH 3.0 and 15 mM ammonium acetate. Next, the optimization of the ESI-MS sheath liquid composition is conducted comparing methanol to isopropanol followed by the use of experimental design for analysis of spray chamber parameters. Overall, the developed CEC-ESI-MS method allows quantitative and sensitive monitoring of ATMA+ from < or =10 microg/mL down to 10 ng/mL. Utilizing the optimized CEC-ESI-MS protocol, the challenging analysis of commercial sample Arquad S-50 ATMA+ containing cis-trans unsaturated and saturated soyabean fatty acid derivatives is demonstrated.     

Separation of catechins and methylxanthines in tea samples by capillary electrochromatography

In this paper, the simultaneous separation of several polyphenols such as (+)‐catechin, (–)‐epicatechin, (–)‐epigallocatechin, theophylline, caffeine in green and black teas by capillary electrochromatography (CEC) was developed. Several experimental parameters such as stationary phase type, mobile phase composition, buffer and pH, inner diameter of the columns, sample injection, were evaluated to obtain the complete separation of the analysed compounds. Baseline resolution of the studied polyphenols was achieved within 30 min by using a capillary column (id 100 μm) packed with bidentate C₁₈ particles for 24.5 cm and a mobile phase composed of 5 mM ammonium acetate buffer pH 4 with H₂O/ACN (80:20, v/v). The applied voltage and the temperature were set at 30 kV and 20°C. Precision, detection and quantification limits, linearity, and accuracy were investigated. A good linearity (R² > 0.9992) was achieved over a concentration working range of 2–100 μg/mL for all the analytes. LOD and LOQ were 1 and 2 μg/mL, respectively, for all studied compounds. The CEC method was applied to the analysis of those polyphenols in green and black tea samples after an extraction procedure. Good recovery data from accuracy studies ranged between 90% and 112% for all analytes.     

Rapid separation of barbiturates and benzodiazepines by capillary electrochromatography with 3-(1,8-naphthalimido)propyl-modified silyl silica gel

A capillary electrochromatographic (CEC) method was applied to the simultaneous separation of barbiturates (barbital, phenobarbital, secobarbital and thiopental) and benzodiazepines (nitrazepam, diazepam and triazolam). The separation was performed in a 75 microm i.d. capillary, packed with 3-(1,8-naphthalimido)propyl-modified silyl silica gel (NAIP), studying the effects of buffer pH and mobile phase composition. Using an applied voltage of 20 kV and the short-end injection method (9 cm capillary effective length), the mobile phase of 1.0 mM citrate buffer (pH 5.0) containing 45% methanol provided the baseline separation of seven toxic drugs in less than 9 min. In CEC with NAIP, the benzodiazepines were separated by the combination of hydrophobic and pi-pi interactions, whereas the separation of the barbiturates was based on the hydrophobic interaction.     

Simultaneous determination of ampicillin, cefoperazone, and sulbactam in pharmaceutical formulations by HPLC with beta-cyclodextrin stationary phase

An accurate and reproducible method for the simultaneous determination of ampicillin (AMP), sulbactam (SUL), and cefoperazone (CFP) in pharmaceutical formulations by using HPLC with beta-CD stationary phase was developed. It involved the use of the added tetraethylammonium acetate (TEAA) reagent, pH, and methanol as the significant parameters to find the optimum separation condition. A high resolution and selectivity of analytes was obtained by running the mobile phase in methanol-5 mM TEAA buffer = 35:65 (v/v, pH 4.5) at 280 nm. The mean recoveries ranged from 96.6 to 103.3% for AMP in the synthetic mixture, 97.6 to 103.0% for SUL, and 97.0 to 104.0% for CFP. The low LOD (<1.8 microg/mL) and low CV (<0.9%) assured that this method was sensitive and reproducible. The assay of analytes in commercial products exhibited that it was convenient and reproducible for routine analyses of these components in sterilized H(2)O, saline, or 5% dextrose injection solutions.