Malva sylvestris L. extract suppresses desferrioxamine‐induced PGE2 and PGD2 release in differentiated U937 cells: the development and validation of an LC‐MS/MS method for prostaglandin quantification

Malva sylvestris is a species used worldwide as an alternative to anti‐inflammatory therapies; however, its mechanism of action remains unknown. In this paper, the anti‐inflammatory effects of M. sylvestris alcoholic extracts were evaluated by measuring the pro‐inflammatory mediators PGE₂ and PGD₂ in desferrioxamine‐stimulated phorbol 12‐myristate 13‐acetate‐differentiated U937 cells. An HPLC‐DAD fingerprint of the M. sylvestris extract was performed and caffeic acid, ferulic acid and scopoletin were identified and quantified. An HPLC‐MS/MS method was developed and validated to separate and measure the prostaglandins. The lower limits of detection (~0.5 ng/mL for PGE₂ and PGD₂) and quantification (1.0 ng/mL for PGE₂ and PGD₂) indicated that the method is highly sensitive. The calibration curves showed excellent coefficients of correlation (r > 0.99) over the range of 1.0–500.0 ng/mL, and at different levels, the accuracy ranged from 96.4 to 106.4% with an RSD < 10.0% for the precision study. This method was successfully applied using U937‐d cells. A significant dose‐dependent reduction of PGE₂ and PGD₂ levels occurred using 10 µg/mL (10.74 ± 2.86 and 9.60 ± 6.89%) and 50 µg/mL of extract (48.37 ± 3.24 and 53.06 ± 6.15%), suggesting that the anti‐inflammatory mechanisms evoked by M. sylvestris may be related to modulation of these mediators. Copyright © 2014 John Wiley & Sons, Ltd.     

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.     

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.     

Characterisation of ultra-thin films of oxidised bacterial cellulose for enhanced anchoring and build-up of polyelectrolyte multilayers

The process of catalytic oxidation of bacterial cellulose (BC) ultra-thin films with 2,2,6,6-tetramethyl-1-piperidinyloxy was investigated along with their capability to adsorb oppositely charged polyelectrolytes of chitosan and alginate. The time-dependent oxidation of BC films was analysed by X-ray photoelectron spectroscopy and quartz crystal microbalance (QCM) experiments. A negatively charged surface was achieved by inserting carboxylic groups, which was augmented by prolonged media exposure (17.9 %), compared with a fast oxidation process (9.1 %). Polyelectrolyte deposition was followed by QCM, which indicated that BC oxidation increased the first layer uptake of chitosan 17-fold (?105.0?±?1.5 Hz) in comparison with unoxidised BC (?6.0?±?0.2 Hz), confirming the capability of oxidised BC surfaces to exhibit strong electrostatic interactions and to support the build-up of a thicker multilayer system. These findings indicate that oxidised BC surfaces are capable of immobilising and detecting several charged species.     

Development and validation of two methods based on high-performance liquid chromatography-tandem mass spectrometry for determining 1,2-benzopyrone, dihydrocoumarin, o-coumaric acid, syringaldehyde and kaurenoic acid in guaco extracts and pharmaceutical preparations

In this study, two HPLC-ESI-MS/MS methods were developed and validated for the determination of 1,2-benzopyrone (COU), o-coumaric acid (OCA), kaurenoic acid (KAU), syringaldehyde (SYR), and dihydrocoumarin (DIH) in guaco extracts and pharmaceutical preparations (syrup and oral solution). The chromatographic separation was achieved using a C18 XBridge 150×2.1-mm (5-μm particle size) column maintained at 25°C. The mobile phases consisted of a gradient of water and acetonitrile containing 0.05% formic acid or 5 mM ammonium formate for the positive and negative ion modes, respectively. All of the calibration curves showed excellent coefficients of correlation (r≥0.9970) over the ranges of 1.25-400 ng/mL for coumarin, 10-600 ng/mL for dihydrocoumarin, 5-250 ng/mL for KAU, and 25-500 ng/mL for o-coumaric acid and syringaldehyde. The range of recovery was 96.3-103% with an RSD% of <4.85% for intraday and interday precision. The results indicate that the developed methods are fast, efficient, and sensitive for the quantification of the guaco metabolites in extracts and pharmaceutical forms while avoiding purification and derivatization steps.     

LC–QToF–MS method for quantification of ethambutol,isoniazid, pyrazinamide and rifampicin in human plasma and its application

In this research, we developed and validated a liquid chromatography coupled to mass spectrometry (LC–QToF–MS) method for simultaneous quantification of the anti-tuberculosis drugs ethambutol, isoniazid, pyrazinamide and rifampicin in human plasma. Plasma samples spiked with cimetidine (internal standard) were extracted using protein precipitation with acetonitrile containing 1% formic acid. Separation was performed using a C₁₈ column under flow gradient conditions with water and acetonitrile, both containing 5 mm ammonium formate and 0.1% formic acid. The method was validated according to the ANVISA and US Food and Drug Administration guidelines for bioanalytical method validation. The calibration curve was linear over a concentration range of 0.2–5 μg ml⁻¹ for ethambutol, 0.2–7.5 μg ml⁻¹ for isoniazid, 1–40 μg ml⁻¹ for pyrazinamide and 0.25–2 μg ml⁻¹ for rifampicin, all with adequate precision and accuracy. The method was reproducible, selective and free of carryover and matrix effects. The validated LC–QToF–MS method was successfully applied to real samples and shown to be applicable to future therapeutic and pharmacokinetic monitoring studies.     

Profiling propolis flavonoids by means of micellar electrokinetic capillary chromatography, capillary gas chromatography and bactericidal action

Summary There is no proportional correlation between increasing organic solvent polarity from hexane to methanol and the extractability of propolis solids by the solvents or the bactericidal action of the propolis extracts obtained. Hexane (17.7%) and chloroform (62.1%) were the poorest and the best extractants, respectively, for propolis solids. The antibiotic activity of the extracts againstStaphylococcus strains decreased in the order hexane≥ethanol>methanol. Different capillary GC profiles were obtained for persilyl derivatives of propolis extracted with hexane and methanol and for propolis collected in different Brazilian provinces, suggesting the influence of flora variability on propolis composition. Wavelength-selected DAD detection revealed MEKC to be an innovative and sensitive method for monitoring the occurrence in propolis of the flavonoids and phenolcarboxylic acids thought to be responsible for the antimicrobial activity of propolis.     

A new HPLC–MS/MS method for the simultaneous quantification of SGLT2 inhibitors and metformin in plasma and its application to a pharmacokinetic study in healthy volunteers

Monitoring the plasma concentrations of metformin and sodium‐glucose cotransporter‐2 inhibitors (canagliflozin, dapagliflozin and empagliflozin) is essential for pharmacokinetic and bioequivalence studies and therapeutic monitoring. The present work therefore aimed to develop and validate a high‐performance liquid chromatography coupled to tandem mass spectrometry (HPLC–MS/MS) method for the simultaneous quantification of these drugs in human plasma. The analyses were performed using an Agilent 1200 HPLC system coupled to an Applied Biosystems API 3200 triple quadrupole MS/MS with electrospray ionization in positive ion mode. After one‐step protein precipitation of plasma with acetonitrile containing 0.1% formic acid, chromatographic separation was achieved on an Xbridge C₁₈ column, with a mobile phase consisting of a gradient of water and acetonitrile, both containing 1 mm ammonium formate and 0.1% formic acid. Quantification was performed in multiple reaction monitoring mode using m/z 130.1 → 71.1 for metformin, m/z 462.0 → 191.2 for canagliflozin, m/z 426.1 → 167.1 for dapagliflozin and m/z 468.0 → 354.9 for empagliflozin. The proposed method was validated and demonstrated to be adequate for the quantification of metformin, canagliflozin, dapagliflozin and empagliflozin for clinical monitoring, pharmacokinetics and bioequivalence studies.     

1,4-Dihydropyridine/BF3OEt2 for the reduction of imines: Influences of the amount of added BF3OEt2 and the substitution at N-1 and C-4 of the dihydropyridine ring

We have evaluated four 1,4-dihydropyridines (DHPs 1a, 1b, 1c and 1d) as reducing agents, which presented free (hydrogenated) or phenyl-substituted N-1 and C-4 positions of the DHP ring. Reactions combining each of the DHP and different amounts of BF₃OEt₂ were evaluated for the reduction of imine 2a (N-benzylideneaniline). DHP simultaneously substituted at N-1 and C-4 (1a), and DHP substituted at C-4 (1b) gave lower yields for reduction of 2a in comparison with DHPs 1c and 1d (both unsubstituted at the C-4 position). By evaluating the amount of added BF₃OEt₂ to the reaction mixture, we have found that DHP 1c (substituted at N-1) provided its best yield for amine 3a (82%) when associated with stoichiometric amounts BF₃OEt₂, while DHP 1d (N-1- and C-4-unsubstituted derivative) was more effective (90% yield) with catalytic quantities of the Lewis acid. The reaction system using DHP 1c under stoichiometric BF₃OEt₂ could also be successfully applied with additional imine examples and under reductive amination conditions.     

Simultaneous spectrophotometric determination of pyrantel pamoate and febantel in pharmaceutical preparations using partial least-squares regression

The multivariate spectroscopic determination of the combination of pyrantel pamoate (PP) and febantel (FB) in pharmaceutical preparations was carried out by partial least-squares regression. The UV absorbance spectra of standard mixtures of PP and FB were measured at concentrations ranging from 5.76–11.52 and 6–12 μg/mL, respectively, in a diluent solution of methanol and acetonitrile. The best model was selected with variable selection (280–320, 380–400 nm), 2 latent variables and mean centered. External validation was performed using 13 different mixtures, which provided prediction errors lower than 1%. The method was validated in accordance with international and Brazilian guidelines. This method was successfully employed to quantify the drug content in two different pharmaceutical formulations (solid — capsules, liquid — oral suspensions) with good prediction capacity. Furthermore, its advantages include the use of simple and accessible reagents and equipment, and a low operating cost.