Polarity-based fractionation in proteomics: hydrophilic interaction vs reversed-phase liquid chromatography.

During recent decades, hydrophilic interaction liquid chromatography (HILIC) ahs been introduced to fractionate or purify especially polar solutes such as peptides and proteins while reversed-phase liquid chromatography (RPLC) is also a common strategy. RPLC is also a common dimension in multidimensional chromatography. In this study, the potential of HILIC vs RPLC chromatography was compared for proteome mapping of human peripheral blood mononuclear cell extract. In HILIC a silica-based stationary phase and for RPLC a C18 column were applied. Then separated proteins were eluted to an ion trap mass spectrometry system. Our results showed that the HILIC leads to more proteins being identified in comparison to RPLC. Among the total 181 identified proteins, 56 and 38 proteins were fractionated specifically by HILIC and RPLC, respectively. In order to demonstrate this, the physicochemical properties of identified proteins such as polarity and hydrophobicity were considered. This analysis indicated that polarity may play a major role in the HILIC separation of proteins vs RPLC. Using gene ontology enrichment analysis, it was also observed that differences in physicochemical properties conform to the cellular compartment and biological features. Finally, this study highlighted the potential of HILIC and the great orthogonality of RPLC in gel-free proteomic studies. Copyright © 2015 John Wiley & Sons, Ltd.

Freeze-dried amorphous dispersions for solubility enhancement of thermosensitive API having low molecular lipophilicity.

The present study focuses on the development of an alternative 'thermally gentle' strategy such as freeze-drying to obtain not only solubility enhanced but also physically stabilised amorphous solid dispersions of acetazolamide, which melt with decomposition (M.P.~260°C). The solid dispersions were prepared by freeze-drying an aqueous dispersion of acetazolamide containing a lyoprotectant as sugar alcohol (mannitol) in 1:0.5, 1:1 and 1:2 proportions by weight. All the proportions of solid dispersions reported a marked increase in solubility characteristics compared to those of pure drug; with outstanding performance by 1:1 ratio of about 6 folds rise in saturation solubility and 90% drug release in about initial 30 minutes. This could be attributed to the formation amorphous molecular dispersions, cosolvent effect of mannitol on dispersed acetazolamide as well as its local solubilisation effect at the diffusion layer. During stability study also, 1:1 ratio of solid dispersions reported an insignificant change in solubility characteristics subjected to an unchanged amorphous nature. Such physical stability could be attributed to decreased molecular mobility of the drug molecules in amorphous carrier because of weaker drug-carrier interactions. Thus, it was demonstrated that freeze-drying is an effective method of forming dissolution-enhanced, amorphous solid dispersions of thermally degradable APIs.

Pharmacokinetics and cardiovascular effect of etoricoxib in the absence or presence of St. John's Wort in rats.

The effect of chronic administration of etoricoxib (EXB), in the absence or presence of St. John's Wort (SJW), on its pharmacokinetic parameters and blood pressure was investigated in rats.Rats were divided into 3 groups, each group received daily different oral treatment for 3 weeks. Rats' blood pressures were monitored initially, after 1 and 3 weeks of treatment, and after 1 week of discontinuing dosing of both drugs. EXB pharmacokinetic parameters in the absence or presence of SJW were calculated after 3 weeks.SJW was significantly affected EXB pharmacokinetic parameters. The steady state peak plasma concentration and terminal half-life were reduced by 32% and 91%, respectively, due to a > 3 fold increase in its apparent clearance which is a concentration and time dependent effect. EXB was significantly increased (P<0.001) Rats' blood pressure while, co-administration of EXB and SJW was not significantly affect (P>0.05) rats' blood pressure as compared to the control.Monitoring blood pressure of patients anticipated taking EXB for extended period should be advised. The co-administration of SJW with EXB should be avoided since SJW would greatly reduce EXB concentrations by inducing its metabolism.

Pharmacokinetics of ketorolac loaded to polyethylcyanoacrylate nanoparticles using UPLC MS/MS for its determination in rats.

Polyethylcyanoacrylate (PECA) nanoparticles (NPs) have been employed as biodegradable polymeric carriers for oral (PO) delivery of ketorolac. The nanoparticles were prepared by polymerization technique at room temperature in a continuous aqueous phase at pH 2.5. This polymerization technique was able to hold 76-96% of ketorolac and the drug loading was a monomer concentration dependent. The feasibility of PECA NPs as PO controlled drug delivery systems of ketorolac was investigated in two groups of rats which were given orally either ketorolac tromethamine solution (1.5 mg/kg) or the selected ketorolac NPs aqueous dispersion (1.6 mg/kg). Ketorolac plasma concentrations were measured by a new fully validated specific, precise and accurate ultra-performance liquid chromatography tandem mass spectrometry (UPLC MS/MS) assay. The detection was performed on Waters TQ detector via negative electrospray ionization in a multiple reaction monitoring mode. Linear response (r(2)> or =0.995) was observed over the range of 10-10,000 ng/ml of ketorolac, with the lower limit of quantification of 5 ng/ml with 1 microl injection volume. The intra- and inter-day precision (relative standard deviation, R.S.D.) values were <10% and the accuracy (relative error) was < or =8 for ketorolac concentrations. The drug solution is rapidly absorbed, distributed, and eliminated and shows a monophasic elimination phase. The assay was sensitive to follow pharmacokinetics of ketorolac in rats up to 24 h after a PO dose of its aqueous solution or NPs suspension. After NPs administration the mean Cmax, 5.0+/-1.3 mg/l, was attained at 1 h. The drug was successfully maintained around this elevated plasma drug concentration up to 6h (>2t(1/2)), in rats. The AUC was significantly higher after the NPs suspension than the solution of ketorolac. This present study provides evidence that the sorption of ketorolac to PECA NPs could control the drug release/elimination in rats.

Influence of water soluble polymers on hydroxypropyl-beta-cyclodextrin complexation of rofecoxib.

Rofecoxib (Rb) is a nonsteroidal anti-inflammatory drug (NSAID) with poor aqueous solubility. The present study was undertaken to investigate the influence of water-soluble polymers namely sodium carboxymethyl cellulose (Na CMC), polyvinylpyrrolidine (PVP) and polyethylene glycol (PEG-6000) on hydroxypropyl beta-cyclodextrin (HP beta-CD) complexation of Rb. The complexes were prepared by kneading, autoclaving and precipitation techniques in 1:1 and 1:2 molar ratios. The aqueous solubility enhancement of Rb by these polymers is found to be of the following order: Na CMC > PVP > PEG-6000. Complexes were characterized by Fourier transform infrared (FTIR) spectroscopy, Nuclear magnetic resonance (NMR) and X-ray diffractometry (XRD) techniques. In vitro dissolution studies were carried out on tablets formulated from molar ratios of the complexes prepared by different techniques.

Synthesis and biodistribution of 2-[123I]iodomelatonin in normal mice.

Melatonin demands that this hormone and its receptors be well understood. With this aim in mind, synthetic melatonin was radioiodinated with no-carrier-added (n.c.a.) sodium iodide-123 using in situ generated peracetic acid as oxidizing agent for electrophilic iodination at room temperature. The radiochemical yield was typically greater than 80% after 20 min reaction time especially when relatively small amounts of activities were used (

Enantiomeric separation of glutethimide derivatives using a Ceramospher RU-2 column.

The enantiomeric resolution of p-acetylaminoglutethimide and p-nitroglutethimide was achieved on a Ceramospher RU-2 column using methanol as the mobile phase. The flow rates of the mobile phase were 1.0 and 0.5 mL/min for p-acetylaminoglutethimide and p-nitroglutethimide, respectively, with UV detection at 254 nm. The values of alpha of the resolved enantiomers of p-acetylaminoglutethimide and p-nitroglutethimide were 1.63 and 1.24 while the values of Rs were 1.44 and 0.86 respectively. The possible chiral mechanism was the formation of transient diastereomeric intermediates between the enantiomers and the chiral selector (1,10-phenanthroline) ruthenium II complex which was stabilized by pi-pi interactions.

Continuous wavelet transformation applied to the simultaneous quantitative analysis of two-component mixtures.

In this paper we developed a graphical method based on Haar (HA) and Mexican (MEX) one-dimensional continuous wavelet transforms and we applied it to a mixture of hydrochlorothiazide (HCT) and spironolactone (SP) in the presence of strongly overlapping signals. Keeping in mind to obtain an appropriately transformed spectrum, we tested several values of the scaling parameter a and the point number of the analysed spectrum in the concentration range of 2-22 microg/ml for both active compounds. The optimal values of the scale parameters and the corresponding frequencies were found to be a = 32 and 0.031 for HA and a = 30 and 0.008 for MEX corresponding to 400 points. HA and MEX methods based on a zero crossing technique were applied to the analysed signal and their regression lines at the selected points were obtained. The validation of the above methods was carried out by analysing different synthetic mixtures containing HCT and SP. MATLAB 6.5 software was used for one-dimensional wavelet analysis and the basic concepts about wavelet method were briefly explained. The method developed in this paper is rapid, easy to apply, inexpensive and is suitable for analysing the overlapping signals of compounds in their mixtures without any chemical pre-treatment.

Determination of nabumetone in pharmaceutical formulation by flow injection analysis (FIA) with UV-detection.

A precise and accurate FIA method for the quantification of nabumetone (NAB) in pharmaceuticals is described. The best suitable carrier solvent system consisted of ethanol: water (30:70 v/v). Sample solution (4.7 x 10(-6) M NAB) was prepared in this solvent and injected to the instrumental system at a flow rate of 1.2 ml x min(-1). The signals were detected by a UV detector at 228.8 nm. The calibration curves of NAB was linear in the concentration range of 1.4 x 10(-6) M-2.8 x 10(-5) M. The intra- and inter-assay precision were less than 2.6%. The method exhibited a good linearity with the correlation coefficients. The LOD and LOQ values were found to be 4.4 x 10(-7) and 1.3 x 10(-6) M, respectively. The effects of the tablet excipients were insignificant at the 95% probability level. The calculated tablet content was 99% which is agreement with the ranges stated by pharmacopoeias.

Thermodynamic study of the enantiomeric resolution of flurbiprofen by HPLC using Chiralpak AD-RH column.

The chiral resolution of (+/-)-flurbiprofen was achieved using water-acetonitrile (60:40, v/v) containing 0.1% acetic acid on a Chiralpak AD-RH column at 20 degrees C. The enantio-resolution was studied with different percentages of acetonitrile. Thermodynamic parameters (enthalpy, entropy and free energy) were calculated by carrying out the enantio-resolution experiments at 0 to 60 degrees C. The enantio-resolution was found to be exothermic in nature. Attempts have been made to explain the mechanism of chiral resolution of flurbiprofen on the Chiralpak AD-RH column.

Direct enantiomeric resolution of some cardiovascular agents using synthetic polymers imprinted with (-)-S-timolol as chiral stationary phase by thin layer chromatography.

Molecular imprinted polymers (MIPs) of S-timolol were prepared as chiral stationary phases (CSPs) in thin layer chromatography (TLC). The resolution of the enantiomers of some cardiovascular drugs, including propranolol, atenolol, timolol, nadolol, nifedipine and verapamil were investigated on these CSPs. A mobile phase system of either methanol or acetonitrile was used and the effects of acetic acid content of the mobile phases were also investigated. The best resolution was achieved for enantioseparation of propranolol, timolol and atenolol on plates based on MIP of (-)-S-timolol using methacrylic acid as functional monomer (alpha = 1.52, 1.6, 1.59) respectively, using acetonitrile containing 5% acetic acid and (alpha = 1.47, 1.52, 1.5) in methanol containing 1% acetic acid as mobile phases. The results obtained show that TLC based on MIPs could be applied in the direct separation of several beta-adrenergic drugs. As the side chains on beta-blockers are similar, it is possible that this method could also be used for the resolution of other racemates in this family of drugs. Racemic drugs structurally related to print molecule, were completely resolved into two spots with the MIP plates. In general the retention of (+)-R-isomers was greater than that of (-)-S-isomers, indicating lower stereoselectivity of the MIPs to the dextrorotatory isomers. The method offers a rapid, sensitive and reliable method for quality control for these drugs.

Distribution and muscle-sparing effects of clenbuterol in hindlimb-suspended rats.

Based on their anabolic properties in skeletal muscles, beta-adrenergic agonists are of interest as potential countermeasures to microgravity-induced skeletal muscle atrophy. The levels of clenbuterol (Cb), a beta(2)-adrenergic agonist, in both plasma and skeletal muscle were higher in hindlimb-suspended rats than in their nonsuspended Cb-treated controls. Cb treatment was shown to help maintain the body weight in suspended rats, while reducing the amount of mesenteric fat. However, hindlimb suspension attenuated Cb's lipolytic effects. In skeletal muscle, the magnitude of response to unloading and Cb treatment followed a general regional pattern and was muscle and type specific. The highest magnitude of response to unloading was in predominantly slow-twitch muscles, and the least responsive were the predominately fast-twitch muscles.

The effect of oral absorption enhancers on the in vivo performance of insulin-loaded poly(ethylcyanoacrylate) nanospheres in diabetic rats.

Poly(ethylcyanoacrylate) (PECA) nanospheres have been employed as biodegradable polymeric carriers for oral (po) delivery of insulin. The main goal of this investigation was to screen various absorption enhancers, which were used to protect insulin-loaded PECA, by following their in vivo performance after oral administrations to streptozotocin-induced diabetic rats. The nanospheres were prepared by polymerization in a continuous aqueous phase at pH 2.5 and in the presence of Pluronic 68 (0.5%). This polymerization technique was able to hold 85 +/- 7.5% of insulin added, 30 min after initiation of polymerization. The drug loading was evaluated by HPLC. Insulin absorption after oral administration was evaluated by its hypoglycemic effect. The addition of protease inhibitor to insulin-loaded PECA nanospheres significantly reduced the blood glucose level after po administrations. Capric acid (0.5%) showed the maximum reduction in blood glucose level (bG max = 34.4 +/- 4.1 mg%), while cholic acid (0.5%) showed the fastest reduction in blood glucose level (Tmax, G = 6.0 +/- 0.2 h) among the tested oral absorption enhancers. Insulin associated with PECA nanospheres retained its biological activity up to 12 days in 50% of the rats in the presence of glycyrrhizic acid (1%). The per cent pharmacological availabilities (PA%) were in the order of capric acid (0.5%) > glycyrrhizic acid (1%) > deoxycholic acid (0.5%) > hydroxypropyl-beta-cyclodextrin (HPbetaCD) (1%) > cholic acid (0.5%). There was no significant difference in the PA% between capric acid (0.5%), glycyrrhizic acid (1%) and deoxycholic acid (0.5%). Although sodium cholate (0.5%) showed the lowest increase in PA% (3.1 +/- 3.11%), its Tmax G was the shortest (6 +/- 0.2 h) among the tested absorption enhancers. The reduction in blood glucose levels was maintained < 200 mg/dL in the order of glycyrrhizic acid (166 +/- 56 h) > capric acid (72 h) >; deoxycholic acid (66 +/- 6 h) > no enhancer (64 +/- 16 h) > HPbetaCI) (31 +/- 17.5) > cholic acid (22 +/- 5.7 h) > aprotenin (20 +/- 3.5 h). Therefore, capric acid and glycyrrhizic acid could be successfully used as oral absorption enhancers.

Determination of L-pipecolic acid in plasma using chiral liquid chromatography-electrospray tandem mass spectrometry.

BACKGROUND: L-pipecolic acid (L-PA), an important biochemical marker for the diagnosis of peroxisomal disorders, is usually determined as the racemate. We developed a chiral liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the analysis of L-PA in plasma. METHODS: We used a narrow bore chiral macrocyclic glycopeptide teicoplanin column for the enantioseparation of D-pipecolic acid (D-PA) and L-PA and interfaced the column directly to the electrospray source of a tandem mass spectrometer. We used phenylalanine-d5 as internal standard added to 50 microL of plasma followed by deproteinization, evaporation, and injection. The analysis was performed in the selected-reaction monitoring mode using two transitions: m/z 130-->m/z 84 for PA, and m/z 171-->m/z 125 for phenylalanine-d5. L-PA eluted at 7 min, and D-PA eluted at 11.7 min, whereas phenylalanine-d5 eluted at 6 min. The turnaround time for the assay was 20 min. RESULTS: Linear calibration curves were obtained in the range of 0.5-80 micromol/L. At a plasma concentration of 1.0 micromol/L, the signal-to-noise ratio was 50:1. The intra- and interassay variations were 3.1-7.9% and 5.7-13%, respectively, at concentrations of 1-50 micromol/L. Mean recoveries of L-PA added to plasma were 95% (5 micromol/L) and 102% (50 micromol/L). The method clearly distinguished between healthy individuals and peroxisomal disease patients. CONCLUSIONS: The novel LC-MS/MS method is simple, rapid, and stereoselective, and uses only 50 microL of plasma, no derivatizing reagents, and a commercially available internal standard. Sample preparation is not complex and is faster than for all other methods.

Optimization of the chiral separation of some 2-arylpropionic acids on an avidin column by modeling a combined response.

The enantiomeric separation of some nonsteroidal antiinflammatory drugs was investigated on an avidin column. An experimental design approach (central composite design) was used to evaluate the effects of three method parameters (pH, concentration of organic modifier, and buffer concentration) on the analysis time and the resolution, as well as to model these responses. This revealed that the organic modifier concentration and sometimes the pH are significant parameters to control because of their influence on both analysis time and resolution. Furthermore, the central composite design results were combined in a multicriteria decision-making approach in order to obtain a set of optimal experimental conditions leading to the most desirable compromise between resolution and analysis time.

HPLC analysis of S-adenosyl-L-methionine in pharmaceutical formulations.

A validated analytical method of S-adenosyl-L-methionine (SAM) in pharmaceutical preparations by reversed phase high performance liquid chromatography (RP-HPLC) is described. The compound is separated by a 2.1 mm x 15 cm, 5 microns--Discovery C18 column with isocratic elution. The effect of different anionic surface active agents with different molarity on the separation was studied. A direct relationship between the molarity of the surface active agents and the capacity factor (k') was found. The limit of detection was 0.49 mmol/ml and the linearity was r = 0.999 in the concentration range 20-100 micrograms/ml. Inter- and intra-assay variation was determined for three selected concentrations (20, 60, 100 micrograms/ml) by calculating the analytical recoveries with a range of 97.0-99.9%. The procedure was also suitable to check the stability of S-adenosyl-L-methionine in solution at room temperature.

Comparison of the chiral resolution of econazole, miconazole, and sulconazole by HPLC using normal-phase amylose CSPs.

Resolution of the enantiomers of (+/-)-econazole, (+/-)-miconazole, and (+/-)-sulconazole has been achieved on different normal-phase chiral amylose columns, Chi-ralpak AD, AS, and AR. The mobile phase used was hexane-2-propanol-diethylamine, 400:99:1 (v/v). The flow rates of the mobile phase used were 0.50 and 1.00 mL min(-1). The alpha values for the resolved enantiomers of econazole, miconazole, and sulconazole on the chiral phases were in the range 1.63 to 1.04; the Rs values varied from 5.68 to 0.32.

Validated analysis of fluvastatin in a pharmaceutical capsule formulation and serum by capillary electrophoresis.

The capillary electrophoretic behavior and the determination of fluvastatin (FLU) in capsule and serum is described in this study. Method development was conducted in a fused-silica capillary (L = 86 cm, L(eff) = 58 cm and 75 microm i.d.) and a background electrolyte consisting of 10 mM borate at pH 8 was used. The separation was performed by current-controlled system applying 41 microA, detecting at 239 nm and injecting 0.5 s vacuum injection. A good electropherogram and excellent repeatability was obtained. FLU and phenobarbital sodium (internal standard) migrated (with RSD%) at 4.8 (0.3) min and 5.2 (0.6) min, respectively. Limit of detection (LOD) and limit of quantitation (LOQ) values were found to be 1 x 10(-6) M and 2.89 x 10(-6) M, respectively. Linearity in the range of 1.03 x 10(-5) -5.15 x 10(-5) M was examined employing intra-day and inter-day studies and well-correlated calibration equations were obtained. FLU in a capsule (Lescol 40 mg declared) was found to be 41.9 +/- 0.4 mg. Furthermore, FLU was determined in serum applying standard addition technique. Good repeatability and no interference were observed. The method proposed is simple, sensitive, precise and easy to use for the determination of FLU in capsule and serum.

A comparative study of the enantiomeric resolution of several tetralone derivatives on macrocyclic antibiotic chiral stationary phases using HPLC under normal phase mode.

The enantiomeric resolution of five substituted 2-(4-pyridylalkyl)-1-tetralone derivatives has been achieved on three macrocyclic glycopeptide antibiotic chiral stationary phases namely, Chirobiotic R, T, and V columns. The mobile phase used was hexane-ethanol-triethylamine (12:8:0.01, v/v/v). The flow rates were 1 mL/min for Chirobiotic T and 2 mL/min for Chirobiotic R and V respectively. The UV detection was carried out at 254 nm. The values of alpha of the resolved enantiomers of the reported tetralone derivatives were in the range of 1.32 to 2.51 on Chirobiotic R, 2.02 to 2.88 on Chirobiotic T and 1.55 to 2.54 on Chirobiotic V respectively while the values of Rs were in the range of 1.00 to 2.50 on Chirobiotic R, 1.00 to 1.95 on Chirobiotic T and 1.00 to 1.60 on Chirobiotic V respectively. The best resolution was achieved on Chirobiotic R column.

A rapid, sensitive high performance liquid chromatographic method for the determination of meropenem in pharmaceutical dosage form, human serum and urine.

A new, simple, precise and rapid high performance liquid chromatographic method was developed for the determination of meropenem in human serum, urine and pharmaceutical dosage forms. Chromatography was carried out on an LC(18) column using a mixture of 15 mM KH(2)PO(4):acetonitrile:methanol (84:12:4; v/v/v), adjusted to pH 2.8 with H(3)PO(4). The proposed method was conducted using a reversed-phase technique, UV monitoring at 307.6 nm and cefepime as an internal standard. The retention times were 5.98 and 7.47 min for cefepime and meropenem, respectively. The detector response was linear over the concentration range of 50-10,000 ng/mL. The detection limit of the procedure was found to be 22 ng/mL. The detection limit for meropenem in human plasma was 108.4 ng/mL and the corresponding value in human urine was 179.3 ng/mL. No interference from endogenous substances in human serum, urine and pharmaceutical preparation was observed. The proposed method is sufficiently sensitive for determination of the concentrations of meropenem and may have clinical application for its monitoring in patients receiving the drug.