Florentin Tache

HPLC-DAD determination of Metformin in human plasma using derivatization with p-nitrobenzoyl chloride in a biphasic system

An optimized method for determination of Metformin at ppb level in human plasma is described. Derivatization of Metformin with p-nitrobenzoyl chloride was carried out in a biphasic system. The derivative extracted in the organic layer (dichloromethane) was concentrated and analyzed by HPLC-DAD, using an isocratic elution (water/methanol mixture 65/35), monitored at 280 nm. Good selectivity against co-extracted matrix components was observed. A detection limit of 10 ppb Metformin in human plasma was reached.

Validation of a LC-fluorescence method for determination of free captopril in human plasma, using a pre-column derivatization reaction with monobromobimane

Both derivatization of free captopril in human plasma samples using monobromobimane as fluorescent label and the corresponding HPLC-fluorescence detection (FLD) method were validated. Calibration curve for the fluorescent captopril derivative in plasma samples is linear in the ppb-ppm range with a detection limit of 4 ppb and an identification limit of 10 ppb (P%: 90; nu > or = 5). These methods were successfully applied on bioequivalence studies carried out on some marketed pharmaceutical formulations.

Large-volume injection of sample diluents not miscible with the mobile phase as an alternative approach in sample preparation for bioanalysis: an application for fenspiride bioequivalence

BACKGROUND Liquid-liquid extraction of target compounds from biological matrices followed by the injection of a large volume from the organic layer into the chromatographic column operated under reversed-phase (RP) conditions would successfully combine the selectivity and the straightforward character of the procedure in order to enhance sensitivity, compared with the usual approach of involving solvent evaporation and residue re-dissolution. Large-volume injection of samples in diluents that are not miscible with the mobile phase was recently introduced in chromatographic practice. The risk of random errors produced during the manipulation of samples is also substantially reduced. RESULTS A bioanalytical method designed for the bioequivalence of fenspiride containing pharmaceutical formulations was based on a sample preparation procedure involving extraction of the target analyte and the internal standard (trimetazidine) from alkalinized plasma samples in 1-octanol. A volume of 75 µl from the octanol layer was directly injected on a Zorbax SB C18 Rapid Resolution, 50 mm length × 4.6 mm internal diameter × 1.8 µm particle size column, with the RP separation being carried out under gradient elution conditions. Detection was made through positive ESI and MS/MS. Aspects related to method development and validation are discussed. CONCLUSIONS The bioanalytical method was successfully applied to assess bioequivalence of a modified release pharmaceutical formulation containing 80 mg fenspiride hydrochloride during two different studies carried out as single-dose administration under fasting and fed conditions (four arms), and multiple doses administration, respectively. The quality attributes assigned to the bioanalytical method, as resulting from its application to the bioequivalence studies, are highlighted and fully demonstrate that sample preparation based on large-volume injection of immiscible diluents has an increased potential for application in bioanalysis.

Estimation of the lipophilic character of flavonoids from the retention behavior in reversed phase liquid chromatography on different stationary phases: A comparative study

The retention behavior of some flavonoids in reversed phase liquid chromatography (RPLC) was investigated using different chemistries of the modified silicagel based stationary phases. Highly end-capped octadecyl silicagel (ODS), polar embedded linker octadecyl silicagel (SB-18 Aqua), phenyl silicagel and pentafluorophenyl modified silicagel (PFP) were used as stationary phases. The mobile phase consisted in acetonitrile/acidified water mixtures, at different fractions of volume. The lipophilicity was estimated through different chromatographic descriptors, as it follows: log k(w), m log k, S, φ(0) and PC1/log k. The chromatographic behavior observed on the mentioned stationary phases was evaluated by means of various graphical profiles and correlation matrices. Additionally, new information about the characteristics of the stationary phases and their (dis)-similarities were provided through lipophilicity charts and by scatterplots of loadings obtained by applying principal component analysis (PCA) to retention data. Furthermore, the experimental lipophilicity indices estimated from retention data were correlated with the computed descriptors, at a high level of statistical significance.

Solvent and salting effects on sample preparation for the determination of fenofibric acid in human plasma by HPLC-DAD

Abstract The solvent and salting effects induced on the sample preparation procedure applied to plasma samples containing fenofibric acid and 4-chlorophenyl-4′-hydroxyphenyl methanone (internal standard) are evaluated. Sodium chloride addition during a deproteinization step using both methanol and phosphoric acid influences the recovery of the analytes as well as the selectivity of the process. The chromatographic method allows high sample volume injection (500 μl) with the focusing of both analytes in the stationary phase. The synthesized high porosity octadecylsilica material allows a fast elution gradient at 4 ml/min flow-rate and a complete analysis within 7 min. UV-detection is made at 295 nm and quantitation limit in the 20 ng/ml concentration level can be achieved. The method can be successfully applied for bioequivalence studies on fenofibrate, administrated as prodrug (fenofibric acid represents its main active metabolite) in pharmaceutical formulations. The main parameters used in studying the retention behavior of the internal standard and FEFA were also estimated.

Lipophilicity indices derived from the liquid chromatographic behavior observed under bimodal retention conditions (reversed phase/hydrophilic interaction): application to a representative set of pyridinium oximes.

The liquid chromatographic behavior observed under bimodal retention conditions (reversed phase and hydrophilic interaction) offers a new basis for the determination of some derived lipophilicity indices. The experiments were carried out on a representative group (30 compounds) of pyridinium oximes, therapeutically tested in acetylcholinesterase reactivation, covering a large range of lipophilic character. The chromatographic behavior was observed on a mixed mode acting stationary phase, resulting from covalent functionalization of high purity spherical silica with long chain alkyl groups terminated by a polar environment created through the vicinal diol substitution at the lasting carbon atoms (Acclaim Mixed Mode HILIC 1 column). Elution was achieved by combining different proportions of 5 mM ammonium formiate solutions in water and acetonitrile. The derived lipophilicity indices were compared with logP values resulting from different computational algorithms. The correlations between experimental and computed data sets are significant. To obtain a better insight on the transition from reversed phase to hydrophilic interaction retention mechanisms, the variation of the thermodynamic parameters determined through the van׳t Hoff approach was also discussed.

On-Line SPE on Restricted Access Adsorbent for HPLC-MS/MS Analysis of Felodipine in Plasma Samples

Direct plasma loading on a LiChrospher ADS C18 cartridge on-line coupled to an analytical Zorbax XDB C18 column was used to analyze felodipine by means of positive atmospheric pressure chemical ionization (APCI) tandem mass spectrometric (MS/MS) detection. Appropriate sensitivity, accuracy, and precision were obtained using an ion trap mass analyzer operated in a multiple reaction monitoring (MRM) mode. Diethyl-4-(2,3-dichlorophenyl)-2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylate) was used as an internal standard. The method was fully validated. The method was successfully applied to a pilot bioequivalence study made on extended release oral dosage forms containing 10 mg of felodipine, under fed and fasting conditions.

Experimental variability and data pre-processing as factors affecting the discrimination power of some chemometric approaches (PCA, CA and a new algorithm based on linear regression) applied to (+/-)ESI/MS and RPLC/UV data: Application on green tea extracts.

The influence of the experimental variability (instrumental repeatability, instrumental intermediate precision and sample preparation variability) and data pre-processing (normalization, peak alignment, background subtraction) on the discrimination power of multivariate data analysis methods (Principal Component Analysis -PCA- and Cluster Analysis -CA-) as well as a new algorithm based on linear regression was studied. Data used in the study were obtained through positive or negative ion monitoring electrospray mass spectrometry (+/-ESI/MS) and reversed phase liquid chromatography/UV spectrometric detection (RPLC/UV) applied to green tea extracts. Extractions in ethanol and heated water infusion were used as sample preparation procedures. The multivariate methods were directly applied to mass spectra and chromatograms, involving strictly a holistic comparison of shapes, without assignment of any structural identity to compounds. An alternative data interpretation based on linear regression analysis mutually applied to data series is also discussed. Slopes, intercepts and correlation coefficients produced by the linear regression analysis applied on pairs of very large experimental data series successfully retain information resulting from high frequency instrumental acquisition rates, obviously better defining the profiles being compared. Consequently, each type of sample or comparison between samples produces in the Cartesian space an ellipsoidal volume defined by the normal variation intervals of the slope, intercept and correlation coefficient. Distances between volumes graphically illustrates (dis)similarities between compared data. The instrumental intermediate precision had the major effect on the discrimination power of the multivariate data analysis methods. Mass spectra produced through ionization from liquid state in atmospheric pressure conditions of bulk complex mixtures resulting from extracted materials of natural origins provided an excellent data basis for multivariate analysis methods, equivalent to data resulting from chromatographic separations. The alternative evaluation of very large data series based on linear regression analysis produced information equivalent to results obtained through application of PCA an CA.

Ethyl Lactate as a Greener Alternative to Acetonitrile in RPLC: A Realistic Appraisal

Appropriate substitution of acetonitrile (ACN) in mobile phases for reversed-phase liquid chromatography (RPLC) by low toxicity, ecologically friendly alternative solvents emerges as a greener approach in separation sciences. Ethyl lactate is considered as a green solvent in organic synthesis, industrial extraction processes and many other applicative fields. Its ability to substitute ACN in mobile phases for RPLC applications was barely investigated. The feasibility of such a replacement was tested for the separation of the mixture of 16 polycyclic aromatic hydrocarbons listed by the Environmental Protection Agency. The analytical approach was found to be achievable, with some compromises in terms of elution order, peak efficiency and UV detectability. Thermodynamic aspects of the chromatographic process were also comparatively assessed. Correlations between the elution order and some molecular descriptors were also discussed.

LC–MS/MS approaches for the assay of bis-quaternary pyridinium oximes used as AChE reactivators in biological matrices

BACKGROUND Extreme efforts are made for the structural diversification of oximes used as AChE reactivators. Co-administration of different oximes should also be considered as a solution in therapy. Consequently, development of selective assays of oximes in biological matrices is of major importance. RESULTS Three chromatographic separation mechanisms were evaluated: hydrophilic-interaction LC; mixed reversed-phase/cation exchange (DUET); and reversed-phase ion pairing based on per-fluorinated agents. MS was used to identify and quantify oximes. Alternative preparation of whole blood and plasma samples were used based on protein precipitation through addition of acetonitrile or ionic liquids. Quality characteristics of the proposed analytical approaches are discussed. CONCLUSION The reversed-phase ion pairing based on per-fluorinated agents chromatographic separation mechanism and positive ESI-MS/MS detection produced the best results for the assay of bis-quaternary pyridinium oximes. LLOQ in the tenths of nanogram per milliliter range are achievable.