Indirect resolution of beta-blocker agents by reversed-phase capillary electrochromatography

The indirect resolution of five beta-adrenoreceptor blocking agents (propranolol, oxprenolol, pindolol, metoprolol, and atenolol) using precolumn derivatization with (+)-1-(9-fluorenyl)ethyl chloroformate (FLEC), and capillary electrochromatography (CEC) is reported. Three octadecylsilanized (ODS) silica gel-based stationary phases, differing in particle diameter and carbon surface density for suitability of determination of the enantiomeric composition of these substances in drugs and biological fluids, were studied. Under optimum separation conditions, all the investigated compounds were baseline-resolved within acceptable analysis times (i.e., between 10-16 min). The resolution values ranged between 1.80 and 1.14, and the separation factors were in no case less than 1.07. The possible use of the developed CEC method for the determination of enantiomeric composition of beta-adrenoreceptor blocking agents in drug substances was investigated. A study, in which the enantiomers of metoprolol were examined regarding specificity, reproducibility, limit of detection, and ruggedness, was developed in accordance with some analytical procedures for method validation.     

Use of nano-liquid chromatography for the analysis of glycyrrhizin and glycyrrhetic acid in licorice roots and candies

Glycyrrhizin (G) and glycyrrhetic acid (GA) were separated by using nano-liquid chromatography (nano-LC) in a fused silica capillary packed with RP18 stationary phase (75 microm ID, effective length 33 cm, packed 23 cm) eluting at 300 nL/min in a gradient mode. The mobile phase was a mixture of water-MeOH-MeCN-acetic acid (29:35:35:1, v/v/v/v) that was delivered for one minute and after this was modified by reducing the water content (14:42.5:42.5:1, v/v/v/v). The intra-day and inter-day relative standard deviations (of retention time and peak area) were satisfactory (lower than 2.9 and 4%, respectively). The linearity of the nano-LC method was assessed in the range 0.62-5.00 microg/mL and 80-200 microg/mL for GA and G, with R2 = 0.996 and 0.995, respectively. The licorice was extracted with a mixture of ethanol-water, diluted with the mobile phase, and injected for the analysis.     

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.     

Rapid assay of vitamin E in vegetable oils by reversed-phase capillary electrochromatography

A rapid capillary electrochromatographic (CEC) method for the analysis of vitamin E in vegetable oils is reported. Vitamin E consists of a group of eight isomers, tocopherols (TOHs) and tocotrienols. The separation of four TOHs (alpha-, gamma-, delta-TOH), alpha-tocopherol acetate (alpha-TOH-Ac), and an antioxidant compound, butylated hydroxytoluene (BHT) used to prevent TOH autoxidation, was optimized. The CEC experiments were carried out in a 75 microm inner diameter (ID) fused-silica capillary, partially packed with 3 microm C(18 )stationary phase (33 cm total length, 8.4 cm and 7 cm effective and packed lengths, respectively). The optimum mobile phase was a polar organic phase composed of a mixture of methanol-acetonitrile in the ratio 50/50 v/v containing 0.01% ammonium acetate, applying a voltage and temperature set at -25 kV and 20 degrees C, respectively. The tocopherols and the BHT were successfully separated within 2.5 min using the short-end injection method. Under these experimental conditions, repeatability of retention time and peak area, analyte detection and quantitation limits, linearity, precision, and accuracy were studied. The CEC method was applied to determine the content of TOHs in different commercially available oils of virgin olive, hazelnut, sunflower, and soybean. The extraction of vitamin E isomers from oil samples was achieved using methanol and a methanol-isopropanol mixture.     

Spectrophotometric determination of hyoscine butylbromide and famciclovir in pure form and in pharmaceutical formulations

A simple, rapid, and extractive spectrophotometric method was developed for the determination of hyoscine butylbromide (HBB) and famciclovir (FCV) in pure and pharmaceutical formulations. These methods are based on the formation of yellow ion-pair complexes between the basic nitrogen of the drug and four sulphonphthalein acid dyes, namely; bromocresol green (BCG), bromothymol blue (BTB), bromocresol purple (BCP) and bromophenol blue (BPB) in phthalate buffer of pH range (3.0-3.5). The formed complexes were extracted with chloroform and measured at 420, 412, 409 and 415nm for HBB and at 418, 412, 407 and 414nm for FCV using BCG, BTB, BCP and BPB, respectively. The analytical parameters and their effects on the reported systems are investigated. Beer's law was obeyed in the range 1.0-20mugmL(-1) with correlation coefficient (n=6)>/=0.9997. The molar absorptivity, Sandell sensitivity, detection and quantification limits were also calculated. The composition of the ion pairs was found 1:1 by Job's method in all cases and the conditional stability constant (K(f)) of the complexes have been calculated. The free energy changes (DeltaG) were determined for all complexes formed. The proposed methods have been applied successfully for the analysis of the studied drugs in pure and pharmaceutical formulations with percentage recoveries ranges from 99.84 to 100.26. The results were in good agreement with those obtained by the official methods.     

Nano-liquid chromatography analysis of dansylated biogenic amines in wines

In this work, the simultaneous analysis of 10 biogenic amines (ethanolamine, methylamine, tryptamine, 2-phenylethylamine, putrescine, cadaverine, histamine, tyramine, spermidine and spermine) in wines by nano-liquid chromatography (nano-LC) using UV detection and a capillary bidentate C(18) column of 100 microm I.D. is proposed. The 10 selected amines, which are the most important to be determined in wine samples, were derivatized with dansyl-chloride (Dns-Cl) previous to their nano-LC determination. Excess of the derivatizing agent as well as other components of the samples were eliminated by the use of an on-line cleaning step employing a C(18) trapping column which also provided a pre-concentration effect. The mobile phase composed of acetonitrile, water, acetic acid and triethylamine (TEA) mixture was pumped at a low flow rate (634 nL/min). Limits of detection (LODs) achieved ranged between 18.3 and 48.3 ng/mL; while calibration curves showed good linearity (R(2)>0.9924). The method was applied to the analysis of this group of amines in white and red wine samples after suitable treatment with polyvinylpyrrolidone (PVP) and extraction with C(18) cartridges.     

Separation of basic compounds of pharmaceutical interest by using nano-liquid chromatography coupled with mass spectrometry

Nano-liquid chromatography-mass spectrometry (nano-LC-MS) was evaluated for the separation of basic compounds of pharmaceutical interest. The separation of selected beta-blockers, namely nadolol, oxprenolol, alprenolol and propranolol in the presence of terbutaline was performed using two 75 microm I.D. capillaries packed with two different RP18 stationary phases (SP). The best results concerning resolution and efficiency were achieved using the SP where free silanol groups were not present. As expected, this latter SP proved to be very efficient and symmetry factors were observed mainly in the case of the more retained analytes. Baseline resolution of all studied basic compounds was achieved with the Cogent bidentate C18 silica phase (CBC18) eluting analytes at 800 nL/min with a mobile phase containing 500 mM ammonium acetate pH 4.5-water-methanol (1:8:91, v/v/v). The separated basic compounds were revealed using on-column UV detector at 205 nm and electrospray-ion-trap mass spectrometer (ESI-MS). The packed capillary was connected to the MS through a commercial sheath liquid interface or a sheathless nano-spray interface and in both cases the sensitivity was studied and the results compared. Limit of detection (LOD) as low as 0.1 ng/mL was measured for nadolol using the sheathless nano-spray interface and the capillary column packed with the CBC18 stationary phase.     

Determination of key flavonoid aglycones by means of nano‐LC for the analysis of dietary supplements and food matrices

A method for the analysis of flavonoids (myricetin, quercetin, naringenin, hesperitin, and kaempferol), with interesting bioactivity, has been developed and validated utilizing nano‐LC technique. In order to find optimal conditions, capillary columns (75 μm id × 10 cm) packed with different types of stationary phases, Kinetex® C18 core–shell (2.6 μm particle size), Hydride‐based RP‐C18 (sub‐2 μm particle size), and LiChrospher® 100 RP‐18 endcapped (5 μm particle size) were evaluated. The method was validated using Hydride‐based RP‐C18 stationary phase, with sub‐2 μm particle size. A good chromatographic performance, expressed in terms of repeatability (RSD, in the range 1.63–4.68% for peak area), column‐to‐column reproducibility (RSD not higher than 8.01% for peak area), good linearity and sensitivity was obtained. In particular limit of detection values between 0.07 and 0.31 μg/mL were achieved with on column focusing technique. The method was applied to the determination of studied flavonoids in dietary supplements as well as in food matrices. The amount of quercetin found in the first analyzed dietary supplement, was in agreement to the labeled content. In the other samples, where the content of flavonoids was not labeled, most of the studied flavonoids were determined in amounts somewhere comparable to those reported in literature.     

Impact of Spray Cone Angle on the Performances of Methane/Diesel RCCI Engine Combustion under Low Load Operating Conditions

The behaviors of spray, in Reactivity Controlled Combustion Ignition (RCCI) dual fuel engine and subsequent emissions formation, are numerically addressed. Five spray cone angles ranging between 5° and 25° with an advanced injection timing of 22° Before Top Dead Center (BTDC) are considered. The objective of this paper is twofold: (a) to enhance engine behaviors in terms of performances and consequent emissions by adjusting spray cone angle and (b) to outcome the exergy efficiency for each case. The simulations are conducted using the Ansys-forte tool. The turbulence model is the Renormalization Group (RNG) K-epsilon, which is selected for its effectiveness in strongly sheared flows. The spray breakup is governed by the hybrid model Kelvin–Helmholtz and Rayleigh–Taylor spray models. A surrogate of n-heptane, which contains 425 species and 3128 reactions, is used for diesel combustion modeling. The obtained results for methane/diesel engine combustion, under low load operating conditions, include the distribution of heat transfer flux, pressure, temperature, Heat Release Rate (HRR), and Sauter Mean Diameter (SMD). An exergy balance analysis is conducted to quantify the engine performances. Output emissions at the outlet of the combustion chamber are also monitored in this work. Investigations show a pressure decrease for a cone angle θ = 5° of roughly 8%, compared to experimental measurement (θ = 10°). A broader cone angle produces a higher mass of NO_x. The optimum spray cone angle, in terms of exergy efficiency, performance, and consequent emissions is found to lie at 15° ≤ θ ≤ 20°.     

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.