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.

Analysis of diltiazem and its related substances by HPLC and HPLC/MS.

Diltiazem (DTZ) is an optically active calcium channel blocker having a benzodiazepine structure. The drug used in therapy is (+)-cis-diltiazem with configuration (2S,3S). To describe the analytical profile of DTZ different stationary phases (RP-18, RP-8, monolithic support) were tested. The best separation of DTZ from A, B, E and F was obtained using as stationary phase a RP-8 or a monolithic RP-18. The characterization of impurities was carried out using two analytical systems, HPLC and HPLC/MS.

Micellar electrokinetic chromatography for determination of drug partition in phospholipids.

The lipophilicity of pipemidic, nalidixic and oxolinic acids was determined by forming phospholipidic micelles directly in an electrophoretic capillary. Phosphatidylcholine derivatives, namely L-alpha-dilauroyl phosphatidylcholine (DLPC) or L-alpha-dimiristoyl phosphatidylcholine (DMPC), were added in the run buffer (50 mM phosphate buffer at pH 7.4). To obtain a mixed micelle, phospholipidic derivatives and sodium cholate were together added in the run buffer. Considering the increasing of migration time when phosphatidylcholine derivative is added in the run buffer, Ks can be determined and then quinolones lipophilicity.

Direct determination by capillary electrophoresis of cardiovascular drugs, previously included in liposomes.

The lipophilicity of some cardiovascular drugs was determined by capillary electrophoresis (CE). Mexiletine, amlodipine and indapamide, the drugs considered, were in contact with liposomial vescicles for 2, 4 or 6 h. After the contact time the drugs, penetrated into liposomial vesicles, were determined by CE using phosphate buffer (pH 6.3 or 7.4) or borate buffer (pH 9). The lipophilicity of three drugs was determined considering the drug percentage penetrated into liposomial vesicles. The found lipohilicity order was amlodipine > mexiletine > indapamide.

Use of a hepta-Tyr antibiotic modified silica stationary phase for the enantiomeric resolution of D,L-loxiglumide by electrochromatography and nano-liquid chromatography.

Hepta-Tyr antibiotic modified silica stationary phase was used for the chiral resolution of D,L-loxiglumide, a new drug under investigation proposed for the treatment of gastrointestinal diseases. The chiral stationary phase was packed into fused silica capillaries of 75 microm i.d. for a length of only 7 cm and used for both capillary electrochromatography (CEC) and nano-liquid chromatography (nano-LC) running the experiments with the same instrumentation; in order to increase the electroosmotic flow (EOF) the antibiotic stationary phase was mixed with amino-silica particles (3:1, w/w) generating a relatively high reversed EOF. The enantiomeric resolution of loxiglumide by CEC was strongly influenced by several experimental parameters such as applied electric field, mobile phase composition, capillary temperature, etc. Optimum experimental conditions were found applying 15 kV at 20 degrees C and eluting with acetonitrile-sodium phosphate buffer at pH 6 (1:1, v/v). The same capillary was tested for nano-LC experiments. Good chiral separation of loxiglumide was achieved selecting the appropriate mobile phase considering the type and concentration of organic modifier. The nano-LC optimised method was therefore validated and applied to the analysis of a pharmaceutical formulation declared to contain only D-loxiglumide.

Chiral investigation of midodrine, a long-acting alpha-adrenergic stimulating agent.

Midodrine hydrochloride is a peripheral alpha(1)-adrenoreceptor agonist that induces venous and arterial vasoconstriction. Midodrine, after oral or intravenous administration, undergoes enzymatic hydrolysis and releases deglymidodrine, a pharmacologically active metabolite. Midodrine and deglymidodrine have a chiral carbon in the 2-position. To investigate the bioactivity of racemates and enantiomers of the drug and metabolite, three chromatographic chiral stationary phases, Chiralcel OD-H, Chiralcel OD-R, and alpha(1)-AGP, were evaluated for enantiomeric resolution. Good enantioseparation of midodrine racemate was obtained using the Chiralcel OD-H column. This stationary phase was then used to collect separately the midodrine enantiomers. By alkaline hydrolysis of rac-midodrine and each separated enantiomer, rac-deglymidodrine and its enantiomers were prepared. The control of the enantiomeric purity was carried out by alpha(1)-AGP stationary phase, while the hydrolysis of rac-midodrine and its enantiomers was controlled by capillary electrophoresis using trimethyl-beta-cyclodextrin as chiral selector. The pharmacological activity of the two racemates and the two enantiomeric pairs was tested in vitro on a strip of rabbit descending thoracic aorta. The tests continued that the activity of the drug and metabolite is due only to the (-)-enantiomer because neither of the (+)-enantiomers is active.

Ibuprofen quality control by electrochromatography.

The quality control of drugs is generally made by HPLC. This control could be made also by capillary electrochromatography (CEC). In this paper we report the analysis by CEC of ibuprofen, a well-known anti-inflammatory non steroidic drug, and some of its impurities. The analyses were performed in a 100-microm inner diameter (I.D.) fused silica capillary, packed with RP-18 stationary phase. The mobile phase was a mixture of 100 mM formic acid solution (pH 2.5), water and acetonitrile (ACN). The ACN percentage in the mobile phase and the applied voltage were carefully studied to well resolve the drug from each impurity. The results, obtained determining ibuprofen and related compounds by CEC, showed the selectivity and efficiency of the method.

Determination of MPTP, a toxic impurity of pethidine.

Pethidine, predominantly a mu-receptor agonist, is a phenyl-piperidinic synthetic drug. It is used in the management of moderate to several pain. A possible hydrolytic degradation of an ester group can generate a very toxic compound, the N-methyl-4-phenyl-1,2,3,6 tetrahydropyridine (MPTP) which contaminates the drug. Because of the toxicity of MPTP a suitable method for its determination must be selective and sensitive. Afterwards we propose simple methods to determine pethidine and MPTP by capillary electrophoresis (CE), MECK and RP-high performance liquid chromatography (HPLC) looking at the limit of detection obtained using these three techniques. CE was carried out using as running buffer ammonium acetate (pH 8.3). MECK was performed with a borate buffer (pH 8.3) containing sodium dodecylsulphate and trimethyl-beta-cyclodextrins. RP-HPLC was carried out on a RP18 stationary phase, using as mobile phase a mixture of phosphate buffer (pH 7) containing acetonitrile and 1% of diethylamine.

Use of short-end injection capillary packed with a glycopeptide antibiotic stationary phase in electrochromatography and capillary liquid chromatography for the enantiomeric separation of hydroxy acids.

A new chiral stationary phase (CSP) was prepared by reacting MDL 63,246 (Hepta-Tyr), a glycopeptide antibiotic belonging to the teicoplanin family, with 5-microm diol-silica particles. The CSP mixed with 5-microm amino silica particles (3:1) was packed into 75-microm fused-silica capillaries for only 6.6 cm and used for electrochromatographic experiments analyzing several hydroxy acid enantiomers. A reversed electroosmotic flow carried both analytes and mobile phase towards the anode in a short time (1-3 min), being baseline resolved all the studied analytes. In order to achieve the fastest enantiomeric resolution of the studied hydroxy acids, the effect of several experimental parameters such as mobile phase composition (organic modifier type and concentration, pH of the buffer and ionic strength), capillary temperature and applied voltage on enantioresolution factor, retention time, enantioselectivity were evaluated. The packed capillary column allowed the separation of mandelic acid enantiomers in less than 72 s with resolution factor Rs=2.18 applying a voltage of 30 kV and eluting with a mobile phase composed by 50 mM ammonium acetate (pH 6)-water-acetonitrile (1:4:5, v/v). The CSP was also tested in the capillary liquid chromatography mode resolving all the studied enantiomers applying 12 bar pressure to the mobile phase [50 mM ammonium acetate (pH 6)-water-methanol-acetonitrile, 1:4:2:3, v/v)], however, relatively long analysis times were observed (12-20 min).

Determination of losartan and hydrochlorothiazide in tablets by CE and CEC.

Capillary Electrophoresis (CE) and Capillary Electrochromatography (CEC) have been used to determine losartan and hydrochlorothiazide. The CE separation was carried out in an uncoated capillary filled with a 100 mM sodium borate pH 9 solution containing trimethyl-beta-cyclodextrins. CEC was performed using a capillary packed with a RP-18 stationary phase. The mobile phase was a mixture of 50 mM ammonium acetate pH 7, water, acetonitrile (1/1.5/7.5). By CE and CEC suitable methods to determine simultaneously losartan and hydrochlorothiazide in working standard mixture or pharmaceutical form were obtained. The proposed methods are very simple and both gave accurate and precise results.

Chiral discrimination by HPLC and CE and antifungal activity of racemic fenticonazole and its enantiomers.

Fenticonazole is a chiral antifungal agent, used in therapy as the racemic mixture. The investigation on the chirality of fenticonazole is reported in this study. rac-Fenticonazole was resolved by HPLC and by capillary electrophoresis (CE). The chiral stationary phase (CSP), used in HPLC, was Daicel OD-H, a commercial phase, which allowed the separate collection of the two enantiomers. The chiral selectors used for CE were some cyclodextrin derivatives. The analysis time required from CE was about the half the HPLC enantioseparation time. The biological activity of the rac-mixture and each individual enantiomer was tested against Cryptococcus neoformans and two Aspergillus nidulans strains. The minimum inhibitory concentration (MIC) evaluation showed that the eutomer was the enantiomer chromatographically more retained and had a longer migration time in the electrophoretic enantioseparation. The CD spectrum of the eutomer showed a positive Cotton effect.

Simultaneous determination of losartan and hydrochlorothiazide in tablets by high-performance liquid chromatography.

A method for the simultaneous determination of losartan potassium and hydrochlorothiazide in tablets is described. The procedure, based on the use of reversed-phase high-performance liquid chromatography, is linear in the concentration range 3.0-7.0 microg ml(-1) for losartan and 0.5-2.0 microg ml(-1) for hydrochlorothiazide, is simple and rapid and allows accurate and precise results. The limit of detection was 0.08 microg ml(-1) for losartan and 0.05 microg ml(-1) for hydrochlorothiazide.

Enatioseparation and anti-rhinovirus activity of 3-benzylchroman-4-ones.

In a series of homo-isoflavonoids, chloro-substituted rac-3-benzylchroman-4-ones (3 d-f) showed an antiviral in vitro activity against selected picornaviruses. In order to study the anti-rhinovirus activity of each stereoisomer, racemic mixtures of 3 d and 3 e were successfully resolved by high-performance liquid chromatography, using a Whelk-O 1 column as chiral stationary phase. The CD spectra confirm that the two eluates of each compound are enantiomers but do not allow the assignment of their absolute configurations. The antiviral activity of the isomers and their racemates was tested in vitro against human rhinovirus serotype 1B and 14 infection, by means of the plaque reduction assay. All homoisoflavonoids tested exhibited an inhibitory effect on rhinovirus replication with an activity depending on virus serotype and compound. The two enantiomers of each compound and the corresponding racemate were equipotent, clearly showing that the configuration of the chiral center in position 3 does not influence the activity against both rhinovirus serotypes.

Determination of silymarine in the extract from the dried silybum marianum fruits by high performance liquid chromatography and capillary electrophoresis.

Silybine (SBN), isosilybine (ISBN), silycristine (SCN), silydianine (SDN), and taxifoline (TXF) are the main active flavanoids, generally found in the dried fruits of silybum marianum. The concentrations of these compounds, excepted TXF, are all together usually expressed as silymarine content. In this paper the determination of the silymarine titre was made by high performance liquid chromatography (HPLC), and high performance capillary electrophoresis (HPCE). Two reversed stationary phases, RP-18 and RP-8, were observed comparing the resolutions of all considered flavanoids with each stationary phases. The HPCE was carried out considering the possible improvement in the resolution of SBN, CN, SDN and TXF using, 8-cyclodextrines or organic modifier. The qualitative and quantitative data obtained by HPLC and HPCE were compared.

LTB4 as marker of 5-LO inhibitory activity of two new N-omega-ethoxycarbonyl-4-quinolones.

The supposed 5-LO inhibitory activity of two N-omega-ethoxycarbonyl-4-quinolones was tested determining leukotriene B4 (LTB4) in RBL-1 cell cultures, pretreated with the two compounds of interest. LTB4, obtained by solid-phase extraction (SPE) from cell cultures supernatants, was determined by micellar electrokinetic chromatography (MEKC). The analysis was performed using an uncoated capillary, filled with borate buffer at pH 8.3, containing 12.5 mM SDS as micelles generator. Therefore, following the decreasing of LTB4 it was possible to verify the 5-LO inhibitory activity of two quinolone derivatives. To asses the suitability of the use of LTB4 as marker of the activity of the new compounds, the analysis was repeated using quercetin, a well known 5-LO inhibitor.

Human alpha1-glycoprotein acid as chiral selector in the enantioseparation of midodrine and deglymidodrine racemates by HPLC.

Human alpha1-acid glycoprotein (alpha1-AGP) has been used as a chiral stationary phase (CSP) for the enantioseparation of midodrine and deglymidodrine racemates in the same HPLC run. The imobilized AGP resulted as the best chiral selector for the enantioresolution of two compounds. Due to the modification of alpha1-AGP characters as a result of changing the composition of the mobile phase, an attempt study of the watery mobile phase (ionic strength and pH of the buffer, nature and concentration of the organic modifier) allowed for an increase in the enantioselectivity of the chromatographic system and an optimization of the resolution base-line of both enantiomeric pairs.

The indirect UV detection in the analysis of ursodeoxycholic acid and related compounds by HPCE.

A high-performance capillary zone electrophoretic (HPCE) assay has been developed for the determination of ursodeoxycholic acid (UDCA) and its usual impurities. Considering the low molecular absorptivity of UDCA and its related compounds indirect UV detection was used. The electrophoretic capillary was filled with a background electrolyte (BGE) containing an UV absorbing ion: benzoic acid (BA) or 5,5-diethylbarbituric acid (DBA). To enhance the selectivity of the assay diimethyl-beta-cyclodextrines (D-beta-CDs) or trimethyl-beta-cyclodextrines (T-beta-CDs) have been added to the running buffer together with methylcellulose or urea. All considered impurities were well resolved with two buffers studied, with the exception of methylursodehoxycholate, a neutral compound.

Determination of the binding of a beta 2-blocker drug, frusemide and ceftriaxone to serum proteins by capillary zone electrophoresis.

A modified Hummel-Dreyer method was used to study the binding of drugs with serum proteins by high performance capillary electrophoresis. The study was carried out to check the possible interaction between serum proteins and a highly selective beta 2-blocker, ICI 118551 (ICI). To prove the suitability of the method the protein binding of frusemide and ceftriaxone, drugs previously investigated, was also studied. The analyses were carried out by injecting a solution of s alpha(1)-acidic glycoprotein (alpha(1)-AGP) or human serum albumin in 70 mM NaH2PO4/Na2HPO4(pH 7.4) buffer into an uncoated fused silica capillary filled with the same buffer. In the capillary, maintained at a working temperature of 35 degrees C, a known amount of the ICI, frusemide or ceftriaxone was added. The method allows the bound drug to be determined directly.

HPTLC and reflectance mode densitometry of anthocyanins in Malva silvestris L.: a comparison with gradient-elution reversed-phase HPLC.

Aqueous alcoholic mallow flower extracts were analyzed both by HPTLC-densitometry in the reflectance mode at 530 nm and by reversed-phase HPLC with gradient elution. For the mallow flower anthocyanins the best chromatographic resolution was obtained by HPLC, which revealed only two main compounds, confirmed by FAB-MS: malvidin 3,5-O-diglucoside (malvin) and malvidin 3-O-(6"-O-malonylglucoside)-5-O-glucoside. The HPTLC densitometric method on cellulose plates provides accuracy, reproducibility and selectivity for the quantitative analysis of the anthocyanins and this method was shown to be much more sensitive than the HPLC-DAD system, at 530 nm. Both methods give comparable quantitative results for total anthocyanins when applied to mallow flowers from two different sources: Italy and Albania.

Analysis of non-benzodiazepinic anxiolytic agents by capillary zone electrophoresis.

A simple capillary electrophoretic method was developed for the analysis of a new generation of serotonergic anxiolytics and their related substances: zalospirone, gepirone, ipsapirone and buspirone. All compounds run in a Tris/phosphate buffer at pH 3 as cations and the experimental conditions allowed good resolution of four drugs and their principal impurities. The analyses were made using two different kinds of capillary. The suitability of CZE and HPLC methods for the analysis of these non-benzodiazepinic anxiolytic agents and their impurities was compared.