A. Álvarez-Lueje

How changes in the sequence of the peptide CLPFFD-NH2 can modify the conjugation and stability of gold nanoparticles and their affinity for β-amyloid fibrils

In a previous work, we studied the interaction of beta-amyloid fibrils (Abeta) with gold nanoparticles (AuNP) conjugated with the peptide CLPFFD-NH2. Here, we studied the effect of changing the residue sequence of the peptide CLPFFD-NH2 on the efficiency of conjugation to AuNP, the stability of the conjugates, and the affinity of the conjugates to the Abeta fibrils. We conjugated the AuNP with CLPFFD-NH 2 isomeric peptides (CDLPFF-NH2 and CLPDFF-NH2) and characterized the resulting conjugates with different techniques including UV-Vis, TEM, EELS, XPS, analysis of amino acids, agarose gel electrophoresis, and CD. In addition, we determined the proportion of AuNP bonded to the Abeta fibrils by ICP-MS. AuNP-CLPFFD-NH2 was the most stable of the conjugates and presented more affinity for Abeta fibrils with respect to the other conjugates and bare AuNP. These findings help to better understand the way peptide sequences affect conjugation and stability of AuNP and their interaction with Abeta fibrils. The peptide sequence, the steric effects, and the charge and disposition of hydrophilic and hydrophobic residues are crucial parameters when considering the design of AuNP peptide conjugates for biomedical applications.

A selective HPLC method for determination of lercanidipine in tablets.

An HPLC reversed phase method using both UV (356 nm) and electrochemical (1000 mV) detection was developed in order to determine lercanidipine in commercial tablets. Repeatability and reproducibility were adequate. For quantification we have used the calibration plot method for lercanidipine concentration ranging between 1 x 10(-5) and 1 x 10(-4) M. Also, the proposed method is sufficiently selective to distinguish the parent drug and the degradation products after hydrolysis, photolysis or chemical oxidation. Furthermore, the typical excipients included in the drug formulation (talc, lactose, cornstarch, microcrystalline cellulose, carboxymethylcellulose and magnesium stearate) do not interfere with the selectivity of the method. Finally, the proposed chromatographic method was successfully applied to the quantitative determination of lercanidipine in commercial tablets.

Simultaneous voltammetric determination of levodopa, carbidopa and benserazide in pharmaceuticals using multivariate calibration

An analytical methodology based on differential pulse voltammetry (DPV) on a glassy carbon electrode and the partial least-squares (PLS-1) algorithm for the simultaneous determination of levodopa, carbidopa and benserazide in pharmaceutical formulations was developed and validated. Some sources of bi-linearity deviation for electrochemical data are discussed and analyzed. The multivariate model was developed as a ternary calibration model and it was built and validated with an independent set of drug mixtures in presence of excipients, according with manufacturer specifications. The proposed method was applied to both the assay and the uniformity content of two commercial formulations containing mixtures of levodopa-carbidopa (10:1) and levodopa-benserazide (4:1). The results were satisfactory and statistically comparable to those obtained by applying the reference Pharmacopoeia method based on high performance liquid chromatography. In conclusion, the methodology proposed based on DPV data processed with the PLS-1 algorithm was able to quantify simultaneously levodopa, carbidopa and benserazide in its pharmaceuticals formulations using a ternary calibration model for these drugs in presence of excipients. Furthermore, the model appears to be successful even in the presence of slight potential shifts in the processed data, which have been taken into account by the flexible chemometric PLS-1 approach.

Electrochemical study of nisoldipine: analytical application in pharmaceutical forms and photodegradation

The anodic and cathodic behavior of nisoldipine, 3-isobutyl-5-methyl-1,4-dihydro-2,6-dimethyl-4-(2-nitrophenyl)pyridine-3 ,5-dicarboxylate, are reported. This drug belongs to the nitroaryl-1,4-dihydropyridine family, known as calcium channel antagonist and employed in therapeuticalls as peripheral and cerebral vasodilators, in the treatment of the arterial hypertension. The cathodic response corresponds to the reduction of the nitroaromatic group to generate the hydroxylamine derivative. The study by dc and d.p.p. reveals the appearance of four signals depending on pH: Signal I (pH 1-11.5) R - NO2 + 4H+ + 4e- --> R - NHOH + H2O; Signal II (pH 1-5) R - N+H2OH + 2H+ + 2e- --> RN+H3 + H2O; Signal III (pH > 11.5) R - NO2 + e- <--> R - NO2.-; Signal IV (pH > 11.5) R - NO2.- + 3e- + 4H+ --> R - NHOH. In contrast, the anodic response corresponds to the oxidation of the 1,4-dihydropyridine ring to generate the corresponding pyridine derivative. Both, cathodic (d.p.p.) and anodic signals (d.p.v.) were employed to develop analytical methodology for the determination of the drug. The repeatability of the measurements for both methods was adequate with R.S.D. of 1.4% (n = 10) and 2.1% (n = 10) for d.p.p. and d.p.v., respectively. Also recovery studies, 103.8% (R.S.D. 2.65%) by d.p.p. and 98.7% (R.S.D. 2.1%) by d.p.v. show that the accuracy and precision of the developed methods were adequate. The analytical methods were successfully applied to the determination of nisoldipine in both tablets and capsules. In addition, a preliminary study of the photostability of nisoldipine (using both UV and artificial day light) was completed. The identity of the main electroactive photodegradation products by GC with spectrometry detection is provided.

Electrochemical Study of Flutamide, an Anticancer Drug, and Its Polarographic, UV Spectrophotometric and HPLC Determination in Tablets

Flutamide, 4-nitro-3-trifluoromethyl-isobutylanilide, a synthetic antiandrogenic with therapeutic use in prostatic cancer, is electrochemically studied to propose a new electroanalytical alternative for its quantitative determination in pharmaceutical forms. Flutamide is shown to be electrochemically reducible in aqueous and mixed media, due to the presence of the nitroaromatic moiety in the molecule. From the mixed media studies, a nitro radical anion derivative from flutamide is described. From the pH dependence of the polarographic peak an apparent pKa of 4.8 was obtained and corroborated by means of UV spectroscopy. Based on the linear relation between the peak current and the flutamide concentrations a differential pulse polarographic method was developed. For comparative purposes spectrophotometric and HPLCUV methodologies were developed. Furthermore the developed methods were checked for selectivity using hydrolysis, photolysis, thermolysis and chemical oxidation.

Electrochemical Oxidation of 4‐Methyl‐1,4‐dihydropyridines in Protic and Aprotic Media. Spin Trapping Studies

This work reports the electrochemical oxidation of a new series of 4‐methyl‐1,4‐dihydropyridine derivatives with platelet activation factor antagonistic activity. Differential pulse and cyclic voltammetry studies on a glassy carbon electrode showed an irreversible single peak due to the oxidation of the dihydropyridine ring via two electrons. Rotating disk electrode studies show a linear dependence between the current and the rotating rate, indicating that the oxidation process is diffusion‐controlled. Calculated diffusion coefficients did not exhibit significant differences between the derivatives. Voltammetric apparent values for the protonation‐deprotonation equilibrium of the N‐heterocyclic nitrogen are compared with 4‐phenyl substituted 4‐aryl‐1,4‐dihydropyridine (1,4-DHP) derivatives. Based on voltammetric experiments on glassy carbon electrodes, it is seen that the potential peak values are directly related to the electron density on the dihydropyridine ring, wherein C-5 substituent with electron‐withdrawing character produces the most oxidizable 1,4-DHP derivative. The oxidation mechanism follows the general pathway: electron, , electron, , with formation of an unstable cation radical in the first step. A one‐electron oxidation intermediate was confirmed with controlled potential electrolysis and electron spin resonance experiments. On applying N‐benzilydene‐t‐butylamine‐N‐oxide as the spin trap, the unstable radical intermediates from the oxidation of 4‐methyl‐1,4‐dihydropyridine derivatives were intercepted. Comparison of the electron spin resonance spectra of the nitroxide spin adducts revealed no significant differences in the splitting constants of the radicals.

Ionic liquids for improving the extraction of NSAIDs in water samples using dispersive liquid-liquid microextraction by high performance liquid chromatography-diode array-fluorescence detection.

A rapid, sensitive and efficient analytical method based on the use of ionic liquids for determination of non-steroidal anti-inflammatory drugs (NSAIDs) in water samples was developed. High-performance liquid chromatography equipped with a diode array and fluorescence detector was used for quantification of ketoprofen, ibuprofen and diclofenac in tap and river water samples. This new method relies on the use of two ionic liquids with multiple functionalities: one functions as an extraction solvent (1-butyl-3-methylimidazolium hexafluorophosphate ([BMIM][PF6]), and the other changes the polarity in the aqueous medium (1-butyl-3-methylimidazolium tetrafluoroborate, ([BMIM][BF4]). Factors such as the type and volume of the ILs and dispersive solvent, sample volume, and centrifugation time were investigated and optimized. The optimized method exhibited good precision, with relative standard deviation values between 2% and 3%, for the three NSAIDs. Limits of detection achieved for all of the analytes were between 17 and 95 ng mL(-1), and the recoveries ranged from 89% to 103%. Furthermore, the enrichment factors ranged from 49 to 57. The proposed method was successfully applied to the analysis of NSAIDs in tap and river water samples.

Voltammetric behaviour of bromhexine and its determination in pharmaceuticals.

A complete electrochemical study and a novel electroanalytical procedure for bromhexine quantitation are described. Bromhexine in methanol/0.1molL(-1) Britton-Robinson buffer solution (2.5/97.5) shows an anodic response on glassy carbon electrode between pH 2 and 7.5. By DPV and CV, both peak potential and current peak values were pH-dependent in all the pH range studied. A break at pH 5.5 in E(P) versus pH plot revealing a protonation-deprotonation (pK(a)) equilibrium of bromhexine was observed. Spectrophotometrically, an apparent pK(a) value of 4.3 was also determined. An electrodic mechanism involving the oxidation of bromhexine via two-electrons and two-protons was proposed. Controlled potential electrolysis followed by HPLC-UV and GC-MS permitted the identification of three oxidation products: N-methylcyclohexanamine, 2-amino-3,5-dibromobenzaldehyde and 2,4,8,10-tetrabromo dibenzo[b,f][1,5] diazocine. DPV at pH 2 was selected as optimal pH for analytical purposes. Repeatability, reproducibility and selectivity parameters were adequate to quantify bromhexine in pharmaceutical forms. The recovery was 94.50+/-2.03% and the detection and quantitation limits were 1.4x10(-5) and 1.6x10(-5)molL(-1), respectively. Furthermore, the DPV method was applied successfully to individual tablet assay in order to verify the uniformity content of bromhexine. No special treatment of sample were required due to excipients do not interfered with the analytical signal. Finally the method was not time-consuming and less expensive than the HPLC one.

Electrochemistry of interaction of 2-(2-nitrophenyl)-benzimidazole derivatives with DNA

In this study the interaction between new benzimidazole molecules, 2-(2-nitrophenyl)-1H-benzimidazole (NB) and N-benzoyl-2-(2-nitrophenyl)-benzimidazole (BNB), with dsDNA and ssDNA was assessed at pH 7.4. Using differential pulse voltammetry at glassy carbon electrode, both molecules were electrochemically reduced due to the presence of a nitro group in their structures. When DNA was added to the solution, the electrochemical signal of NB and BNB decreased and shifted to more negative potentials. The interaction mode was electrostatic when ionic strength was low. Under this condition DNA-nitro complexes were characterized and binding constant values of 8.22 x 10(4)M(-1) and 3.08 x 10(6)M(-1) for NB and BNB with dsDNA were determined. On the other hand, only NB was able to interact when a high concentration of NaCl was used. Finally, a glassy carbon electrode modified with carbon nanotubes and DNA was tested in order to determine the nitrocompound in solution. The electrochemical reduction of the nitrocompound adsorbed on GCE/CHIT-CNT/DNA was used as an analytical signal. Using 10 min as accumulation time, a linear dependence was observed between 20 and 80 microM nitrocompound concentrations and the electrode response. Detection and quantification limits in the range of microM were determined.

HPLC Determination of Nimesulide in Tablets by Electrochemical Detection

Abstract An analytical chromatographic method for the determination of nimesulide in pharmaceutical forms has been developed. The method is based on high performance liquid chromatographic (HPLC) and electrochemical detection. Chromatography was performed on a μBondapak/μPorasil C-18 column (150 mmx 3.9 mm I.D.). The mobile phase consisted of pH 3 buffer phosphate-methanol (40:60, v/v) at a flow-rate of 1 mL/min. The analytes were detected by electrochemical detection in the pulse mode at 1200 mV. For comparative purposes both an HPLC with diode array detector and a spectrophotometric method were employed. The results of the recovery study (mean 101.07, rsd 1.44) show that HPLC with an electrochemical detection method is adequately precise and accurate and can be recommended for the determination of nimesulide in tablets.