Radu Oprean

The development of spectrophotometric and electroanalytical methods for ascorbic acid and acetaminophen and their applications in the analysis of effervescent dosage forms

The electroanalytical study of ascorbic acid, acetaminophen and of several mixtures of these compounds in different ratios has been made by using a carbon paste electrode (CPE-graphite:solid paraffin 2:1) as working electrode and an Ag/AgCl reference electrode. The potential curves were recorded using different concentrations of ascorbic acid and acetaminophen by measuring samples between 10 and 50 microl. The oxidation reactions were studied in a potential range from -0.1 to +1.3 V with different sweep rates, at different current sensitivities, in stationary working conditions and stirring before each replicate. The oxidation of ascorbic acid occurs at +0.31 +/- 0.02 V and the oxidation of acetaminophen at +0.60 +/- 0.05 V; meanwhile, the current has a linear variation for the following concentration ranges: 10(-3)-10(-2) M for the ascorbic acid and 3 x 10(-6)-7.5 x 10(-3) M for acetaminophen (r2 = 0.999 for both ascorbic acid and acetaminophen). The mixtures of ascorbic acid and acetaminophen were made as follows: 1:1, 1:2, 1:3, 2:1, and 3:1. The studies revealed the alteration of the voltammograms processed according to the validation methodology. The best potential variation range for different current sensitivities, the influence of the sweep rate, of the solvent volume and of the pH were studied. The mutual interferences of the compounds in the mixtures and the electroactive compounds in the pharmaceutical dosage forms, especially effervescent ones, also made the object of the research. The same mixtures were studied using the direct spectrophotometric method that revealed a lot of spectral interferences. In order to solve this problem, an appropriate separation or an indirect spectrophotometric method (the apparent content curves method) were used. The spectrophotometric and voltammetric methods developed were used to determine ascorbic acid and acetaminophen in different dosage forms (vials, tablets, suppositories and effervescent dosage forms). The results were compared with those obtained by other techniques.

Application of new methodologies based on design of experiments, independent component analysis and design space for robust optimization in liquid chromatography

HPLC separations of an unknown sample mixture and a pharmaceutical formulation have been optimized using a recently developed chemometric methodology proposed by W. Dewé et al. in 2004 and improved by P. Lebrun et al. in 2008. This methodology is based on experimental designs which are used to model retention times of compounds of interest. Then, the prediction accuracy and the optimal separation robustness, including the uncertainty study, were evaluated. Finally, the design space (ICH Q8(R1) guideline) was computed as the probability for a criterion to lie in a selected range of acceptance. Furthermore, the chromatograms were automatically read. Peak detection and peak matching were carried out with a previously developed methodology using independent component analysis published by B. Debrus et al. in 2009. The present successful applications strengthen the high potential of these methodologies for the automated development of chromatographic methods.

Polyphenolic content, antioxidant and antimicrobial activities of Lycium barbarum L. and Lycium chinense Mill. leaves.

This study was performed to evaluate the in vitro antioxidant and antimicrobial activities and the polyphenolic content of Lycium barbarum L. and L. chinense Mill. leaves. The different leave extracts contain important amounts of flavonoids (43.73 ± 1.43 and 61.65 ± 0.95 mg/g, respectively) and showed relevant antioxidant activity, as witnessed by the quoted methods. Qualitative and quantitative analyses of target phenolic compounds were achieved using a HPLC-UV-MS method. Rutin was the dominant flavonoid in both analysed species, the highest amount being registered for L. chinense. An important amount of chlorogenic acid was determined in L. chinense and L. barbarum extracts, being more than twice as high in L. chinense than in L. barbarum. Gentisic and caffeic acids were identified only in L. barbarum, whereas kaempferol was only detected in L. chinense. The antioxidant activity was evaluated by DPPH, TEAC, hemoglobin ascorbate peroxidase activity inhibition (HAPX) and inhibition of lipid peroxidation catalyzed by cytochrome c assays revealing a better antioxidant activity for the L. chinense extract. Results obtained in the antimicrobial tests revealed that L. chinense extract was more active than L. barbarum against both Gram-positive and Gram-negative bacterial strains. The results suggest that these species are valuable sources of flavonoids with relevant antioxidant and antimicrobial activities.

Comparative Studies on Polyphenolic Composition, Antioxidant and Antimicrobial Activities of Schisandra chinensis Leaves and Fruits

The aim of this paper was to evaluate the antioxidant and antimicrobial activities and the polyphenolic content of Schisandra chinensis (Turcz.) Baill. leaves and fruits. The leaves are an important source of flavonoids (35.10 ± 1.23 mg RE/g plant material). Qualitative and quantitative analyses of the polyphenolic compounds were achieved using a HPLC-UV-MS method. The main flavonoid from the leaves was isoquercitrin (2486.18 ± 5.72 μg/g plant material), followed by quercitrin (1645.14 ± 2.12 μg/g plant material). Regarding the fruit composition, the dominant compound there was rutin (13.02 ± 0.21 μg/g plant material), but comparing with the leaves, fruits can be considered a poor source of phenolic compounds. The antioxidant activity was evaluated by DPPH, TEAC, hemoglobin ascorbate peroxidase activity inhibition (HAPX), inhibition of lipid peroxidation catalyzed by cytochrome c and EPR spectroscopic assays, revealing a better antioxidant activity for the S. chinensis leaves extract. In the antimicrobial assay, S. chinensis leaves extract showed efficient activities against the targeted bacteria, being more active than the fruits extract. The results suggest the leaves of S. chinensis as a valuable source of antioxidant compounds with significant antioxidant activity.

Essential oils analysis. II. Mass spectra identification of terpene and phenylpropane derivatives

Mass spectra are widely used in order to identify the peaks resulting from a chromatographic separation. The most common approach to solve the problem for unknowns on whom very little other structural information is available is the use of a retrieval algorithm and a reference mass spectra database. The wide variety of mass spectra recorded with different instruments under various experimental conditions can lead to erroneous results. In order to improve the accuracy of the results, we proposed earlier an identification algorithm, which combines the information obtained from both GC and MS fingerprints. This paper presents a new algorithm based on the comparison of the unknown mass spectra with several libraries (including Wiley and NIST) by using reverse and direct search algorithms respectively. The results of the comparisons were quantified with respect to the match quality and the interference compounds. A global match index for the comparison using all the above information was computed and the results were presented as the match probability. This index expresses more accurately the matches between unknown and all the available libraries mass spectra. In order to verify our algorithm, we tried to identify the compounds separated by GC-MSD from different species of Acorus calamus L. (Araceae) essential oils. The probability of the matches increases compared with the quality of matches resulting from Wiley and NIST libraries.

Comparison of GC-MS and TLC techniques for asarone isomers determination

Two chromatographic methods (GC-MS and TLC) have been developed for separation and determination of alpha and beta asarone from essential oils and alcoholic extracts. The study has been performed on the Acorus calamus (I) and Asarum europaeum (II) essential oils of Romanian origin and the alcoholic extract of Acorus calcamus L (III) and it is a consequence of the International Boards exigency regarding the presence of beta asarone in food, beverages and pharmaceuticals. The isomers were determined using both internal and external standard methods. Both SIM and SCAN techniques were used and the results were compared regarding the chromatographic resolution and interference compounds. The method exhibits good repeatability and low detection limit but is expensive and time consuming. The two isomers concentrations are 5.2- 6.7 microg ml(-1) (I), 460-510 microg ml(-1) (II) and 2.7 5.7 microg ml (III) for alpha asarone and 91-98 microg ml(-1) (I), 24-29 microg ml(-1) (II) and 88 97 microg ml(-1) (III) for beta asarone. The TLC method was developed as an alternative for the GC method. The separation was performed on silica gel plates using toluene: ethyl acetate 8:2 as mobile phase. The evaluation of the chromatograms was made by densitometry using multiple wavelength. The sum of the two isomers are between 80-120 microg ml(-1) (I) and 127-145 microg ml(-1) (III) using spectrophotometric detection and between 73-93 microg ml(-1) (I) and 99-105 microg ml(-1) (III) using fluorimetric detection. The results of the two chromatographic methods were compared. Even the GC is more sensitive, mathematical computations for spots optimization and interference elimination could improves the TLC quality results.

TLC-UV densitometric and GC-MSD methods for simultaneous quantification of morphine and codeine in poppy capsules

Thin-layer chromatographic (TLC)-UV densitometric and gas-chromatographic-mass spectrometric detection (GC-MSD) methods were developed for simultaneous quantification of morphine and codeine in poppy capsules (Papaver somniferum). Morphine and codeine were isolated by extraction with chloroform: isopropanol (3:1, v/v) at pH = 8.5 and by solid-phase extraction on Snap-Cap cartridges at pH = 8.5. The TLC-UV densitometric quantification was performed by external standard method on silica gel plates using ethyl acetate: toluene: methanol: ammonia (68:17:10:5, v/v) as developing solvent and UV detection at 275 nm. For the GC-MSD analysis, the drugs were derivatized with acetic anhydride: pyridine (1:1, v/v) and separated on a 30 m HP5 capillary column. The quantification was performed using nalorphine as internal standard.

Implementation of a design space approach for enantiomeric separations in polar organic solvent chromatography

This paper focuses on implementing a design space approach and on the critical process parameters (CPPs) to consider when applying the Quality by Design (QbD) concepts outlined in ICH Q8(R2), Q9 and Q10 to analytical method development and optimization for three chiral compounds developed as modulators of small conductance calcium-activated potassium (SK) channels. In this sense, an HPLC method using a polysaccharide-based stationary phase containing a cellulose tris (4-chloro-3-methylphenylcarbamate) chiral selector in polar organic solvent chromatography mode was considered. The effects of trifluoroacetic acid (TFA) and n-hexane concentration in an acetonitrile (MeCN) mobile phase were investigated under a wide range of column temperatures. Good correlations were found between the observed data obtained after using a central composite design and the expected chromatographic behaviours predicted by applying the design of experiments-design space (DoE-DS) methodology. The critical quality attribute represented here by the separation criterion (S(crit)) allowed assessing the quality of the enantioseparation. Baseline separation for the compounds of interest in an analysis time of less than 20 min was possible due to the original and powerful tools applied which facilitated an enhanced method comprehension. Finally, the advantage of the DoE-DS approach resides in granting the possibility to concurrently assess robustness and identify the optimal conditions which are compound dependent.

Spectrophotometric determination of fluoride in dosage forms and dental preparations

The method is based upon the reaction between fluoride ions and the coloured complex of Fe(III) with methyl salicylate to form the stable, colourless hexaflouride complex of iron. The conditions of the method (pH, time and combination ratio) were studied and a standard curve was obtained for 0.01-0.08 mg NaF ml-1, at 525 nm. A study was conducted on interference with complexing anions of Fe(III), cations that react with fluoride ions and with common ingredients of dosage forms and dental preparations. The method was validated and the results showed good precision (100.16 +/- = 2.33%) comparable with that of other analytical methods. Good results were obtained in the spectrophotometric determination of fluoride ions in a stomatological gel and in a toothpaste.

Simultaneous Enantiospecific Recognition of Several β-Blocker Enantiomers Using Molecularly Imprinted Polymer-Based Electrochemical Sensor

The development of a chiral electrochemical sensor using an electrogenerated molecularly imprinted polymer (MIP)-based ultrathin film using R(+)-atenolol (ATNL) as a template was reported. The proposed sensor exhibited distinctive enantiospecific oxidation peaks toward the R-antipodes of four β-blocker representatives and additional oxidation peaks common to both enantiomers of each studied β-blocker, allowing thus the simultaneous analysis of all of their enantiomers in a single analysis. The specific preconditioning of the polymer by alternative exposure to aqueous and nonaqueous medium was proven to be essential for the chiral recognition ability of the obtained sensor. The rebinding property of the MIP film was studied by using a well-known redox probe, a change in the morphology and diffusive permeability of the thin polymeric layer in the presence of its template being observed. The applicability of the optimized analytical procedure was demonstrated by the analysis of ATNLs enantiomers in the range of 1.88 × 10(-7)-1.88 × 10(-5) mol/L. The developed polymeric interface is the first reported transductor of a chiral electrochemical sensor able to exhibit simultaneous enantiospecificity toward several β-blocker representatives extensively used in the pharmaceutical and biomedical fields, offering good prospects in the simple, cost-effective, and fast assessment of their enantiomeric ratio and total concentration.