Catherine K. Markopoulou

Validation of a novel LC-MS/MS method for the quantitation of colistin A and B in human plasma.

A liquid chromatography/tandem mass spectrometry (LC-MS/MS) method, characterized by complete automation and high-throughput, was developed for the determination of colistin A and B in human plasma. All sample preparation procedures were performed by using 2.2 mL 96-deep-well plates, whereas robotic liquid-handling workstations were utilized for all liquid transfer steps, including solid-phase extraction (SPE). The whole preparation procedure was very rapid, whereas the method had a very short chromatographic run time of just 2 min. Sample analysis was performed by reversed phase LC-MS/MS, with positive electrospray ionization, using multiple reaction monitoring. The absence of available purified colistin A and B standards led to the development of a novel LC method with evaporative light-scattering detector for the determination of their stoichiometries in the standard mixture, along with its purity. The proposed bioanalytical method was fully validated and it was proven to be selective, accurate, precise, reproducible and suitable for the determination of colistin A and B in human plasma. It was applied successfully to a pharmacokinetic study for the determination of both analytes in samples of patients.

An HPLC Method for the Separation and Simultaneous Determination of Antihistamines, Sympathomimetic Amines and Dextromethorphan in Bulk Dpug Material and Dosage Forms

Abstract A high-performance liquid chromatography (HPLC) method for the simultaneous determination of bamipine, phenylephrine or (phenylpropanolamine, ephedrine) and dextromethorphan has been developed. The method proved suitable for the analysis of the compounds in various pharmaceutical formulations. From the sample solutions no interference was observed on the chromatographs. The accuracy of the assay has been assessed. Recovery studies have been also carried out by adding known quantities of the active ingredients to placebo samples (spiking recoveries).

A Simultaneous Analysis by High Performance Liquid Chromatography of Bamipine Combined with Tricyclic Antidepressants and/or Antipsychotics in Dosage Forms

Abstract A reversed phase high-performance liquid chromatographic method is described for the simultaneous determination of antihistamines, tricyclic antidepressants and anti-psychotics in pharmaceutical formulations and in spiked placebos. The separation was performed on an octadecyl-silica column using acetonitrile: tetrahydrofuran: 0.015 M aqueous ammonium acetate (53:42: 5) as mobile phase. The presence of ammonium acetate both shortens the elution time and improves the symmetry of the chromatographic peaks. Measurements were made at 251 nm.

HPLC and Chemometrically‐Assisted Spectrophotometric Estimation of Two Binary Mixtures for Combined Hypertension Therapy

Abstract Three different methods are developed for the determination of felodipine with either (+)–metoprolol tartrate salt or ramipril. The high performance liquid chromatography (HPLC) method depends on the simultaneous separation of each drug in a reverse phase Hypersil BDS C18 3 µm (150×4.6) column at 25°C. Elution was carried out with a mobile phase consisting of 0.015 M 1‐heptanesulfonic acid sodium salt‐methanol‐acetonitrile (35:40:25, v/v/v, pH 2.5). Optimization of the separation in terms of mobile phase composition is crucial to the method development, which is discussed in detail. Quantitation was achieved with UV detection at 210 nm. Moreover, the resolution of the two binary mixtures separately, has also been accomplished by using a mobile phase consisting of 0.015 M sodium dihydrogen phosphate monohydrate‐methanol‐acetonitrile 40:30:30, v/v/v, at pH 6.5 (λ=230 nm) for the determination of felodipine with metoprolol, and at pH 2.5 (λ=210 nm) for felodipine with ramipril, respectively. The other two chemometrically‐assisted spectrophotometric methods that have been used were “Derivative‐Ratio” and “Partial Least Squares” PLS. These approaches were successfully applied to quantify each drug in the mixture using the information of the zero order UV spectra between 210–420 nm. Both methods could determine, with linearity, in the range of 1.56–15.60 µg/mL for ramipril, 4.82–80.40 µg/mL for metoprolol, 1.61–17.69 µg/mL for felodipine. They were successfully applied to the “dose uniformity” test of the two binary combinations in commercial tablets.

Simultaneous Determination of Clobutinol Together with Some Anti‐inflammatory Drugs in Urine by HPLC

Abstract An isocratic high performance liquid chromatography method is described for simultaneous determination of clobutinol hydrochloride together with some anti‐inflammatory drugs, such as diclofenac, meloxicam, and nimesulide in urine. For the development and optimization of the system, three different buffers containing ammonium acetate, tetraethylammonium hydrogen sulfate (THAS), and tetrabutylammonium hydrogen sulfate (THBS) were investigated, because it has been proven that different salts added in the mobile phases considerably affect solute retention and selectivity. The effect of salt content in the aqueous portion of the mobile phase together with pH over a wide range, was investigated. A response surface method based on non‐linear multiple regression analysis was employed to illustrate the changes in k′ values as a function of a range of pH values and different salts contents. On the basis of the chromatographic behavior of clobutinol together with the other drugs determined, optimum chromatographic conditions were achieved with good peak symmetry, reasonable retention time, and noticeable separation. The intra‐ and inter‐day accuracy and precision at low, medium, and high concentration(s) for the compounds were in the range %error 5.40–11.50 and %RSD 1.76–5.06, respectively.

Quantitative High Performance Liquid Chromatographic Determination of Bamipine Combined with Tricyclic Antidepressants and/or Antipsychotics in Pharmaceutical Formulations

Abstract An advanced reserved phase High Performance Liquid Chromatographic (HPLC) method using UV detection, at 251, is presented for the simultaneous determination of bamipine combined with tricyclic antidepressants and/or antipsychotics in various pharmaceutical formulations. Sample analyses were performed on a bonded reserved-phase C-2,5 μm, 250 × 4.6 mm ID (Lichrosorb RP-2)column using aqueous ammonium acetate at constant ionic strength 0.015 M and acetonitrile 35: 65 as eluent, at a flow rate of 0.9 ml/min. The pH was adjusted to 4.7 with acetic acid. The presence of acetate buffer both shortens the elution time and improves the symmetry of the chromatographic peaks. The retention time was for bamipine, prochlorperazine, trifluoperazine, thioridazine, chlorprothixene, haloperidol, clomipramine, trimipramine 8.83, 11.83, 11.50, 11.08, 10.42, 8.05, 10.25, 9.67 min respectively. Linearity and precision data have been assessed. The method also involves an investigation of the dependence of the capacity ...

Modelling by partial least squares the relationship between the HPLC mobile phases and analytes on phenyl column

Twenty-five descriptors and 61 structurally different analytes have been used on a partial least squares (PLS) to latent structure technique in order to study chromatographically their interaction mechanism on a phenyl column. According to the model, 240 different retention times of the analytes, expressed as Y variable (log k), at different % MeOH mobile-phase concentrations have been correlated with their theoretical most important structural or molecular descriptors. The goodness-of-fit was estimated by the coefficient of multiple determinations r(2) (0.919), and the root mean square error of estimation (RMSEE=0.1283) values with a predictive ability (Q(2)) of 0.901. The model was further validated using cross-validation (CV), validated by 20 response permutations r(2) (0.0, 0.0146), Q(2) (0.0, -0.136) and validated by external prediction. The contribution of certain mechanism interactions between the analytes, the mobile phase and the column, proportional or counterbalancing is also studied. Trying to evaluate the influence on Y of every variable in a PLS model, VIP (variables importance in the projection) plot provides evidence that lipophilicity (expressed as Log D, Log P), polarizability, refractivity and the eluting power of the mobile phase are dominant in the retention mechanism on a phenyl column.

Electrosprayed mesoporous particles for improved aqueous solubility of a poorly water soluble anticancer agent: in vitro and ex vivo evaluation

ABSTRACT Encapsulation of poorly water‐soluble drugs into mesoporous materials (e.g. silica) has evolved as a favorable strategy to improve drug solubility and bioavailability. Several techniques (e.g. spray drying, solvent evaporation, microwave irradiation) have been utilized for the encapsulation of active pharmaceutical ingredients (APIs) into inorganic porous matrices. In the present work, a novel chalcone (KAZ3) with anticancer properties was successfully synthesized by Claisen‐Schmidt condensation. KAZ3 was loaded into mesoporous (SBA‐15 and MCM‐41) and non‐porous (fumed silica, FS) materials via two techniques; electrohydrodynamic atomization (EHDA) and solvent impregnation. The effect of both loading methods on the physicochemical properties of the particles (e.g. size, charge, entrapment efficiency, crystallinity, dissolution and permeability) was investigated. Results indicated that EHDA technique can load the active in a complete amorphous form within the pores of the silica particles. In contrast, reduced crystallinity (˜79%) was obtained for the solvent impregnated formulations. EHDA engineered formulations significantly improved drug dissolution up to 30‐fold, compared to the crystalline drug. Ex vivo studies showed EHDA formulations to exhibit higher permeability across rat intestine than their solvent impregnated counterparts. Cytocompatibility studies on Caco‐2 cells demonstrated moderate toxicity at high concentrations of the anticancer agent. The findings of the present study clearly show the immense potential of EHDA as a loading technique for mesoporous materials to produce poorly water‐soluble API carriers of high payload at ambient conditions. Furthermore, the scale up potential in EHDA technologies indicate a viable route to enhance drug encapsulation and dissolution rate of loaded porous inorganic materials.

Using the partial least squares method to model the electrospray ionization response produced by small pharmaceutical molecules in positive mode.

RATIONALE The signal intensity in electrospray ionization mass spectrometry (ESI-MS) positive mode is affected by parameters that are related to the physicochemical properties and structural features of a molecule. Accordingly, the combined interactions of an analyte and the mobile phase used is still an area that demands further clarifications since there is no general pattern regarding the nature of a molecule and the mechanism by which vapor-phase ions are produced. METHODS A multivariate analysis method, such as Partial Least Squares (PLS), provides the opportunity to correlate the effect of a large number of parameters interpreting this complex procedure with the use of appropriate mathematical algorithms. This work involves the development of models containing up to 84 X variables which characterize the analytes studied (99) focusing on their positive or negative effect on the vapor-phase ion formation process. These descriptors are correlated with the signal response of the positively charged analyte ions which corresponds to the Y variable. RESULTS The results showed that parameters referring or directly related to the ionization percentage of basic or acidic groups of an analyte can be used to determine the signal response on positive ESI-MS mode. Structural characteristics, polar surface area, lipophilicity, the ability of analytes to acts as hydrogen bond donors or acceptors, water solubility, density of a solid, surface tension of the substance and the number of free rotatable bonds are descriptors which are of secondary importance, still they cannot be considered negligible. CONCLUSIONS The models derived are proved to be reliable for the investigation of such mechanisms, with a small number of components and good linearity (R(2) >83%, Q(2) >70%).

Validation of a direct non-destructive quantitative analysis of amiodarone hydrochloride in Angoron® formulations using FT-Raman spectroscopy

Raman spectroscopy was applied for the direct non-destructive analysis of amiodarone hydrochloride (ADH), the active ingredient of the liquid formulation Angoron((R)). The FT-Raman spectra were obtained through the un-broken as-received ampoules of Angoron((R)). Using the most intense vibration of the active pharmaceutical ingredient (API) at 1568cm(-1), a calibration model, based on solutions with known concentrations, was developed. The model was applied to the Raman spectra recorded from three as-purchased commercial formulations of Angoron((R)) having nominal strength of 50mgml(-1) ADH. The average value of the API in these samples was found to be 48.56+/-0.64mgml(-1) while the detection limit of the proposed technique was found to be 2.11mgml(-1). The results were compared to those obtained from the application of HPLC using the methodology described in the European Pharmacopoeia and found to be in excellent agreement. The proposed analytical methodology was also validated by evaluating the linearity of the calibration line as well as its accuracy and precision. The main advantage of Raman spectroscopy over HPLC method during routine analysis is that it is considerably faster and no solvent consuming. Furthermore, Raman spectroscopy is non-destructive for the sample. However, the detection limit for Raman spectroscopy is much higher than the corresponding for the HPLC methodology.