Roberto Gotti

Capillary electrophoresis of phytochemical substances in herbal drugs and medicinal plants

This paper reviews the applications of electromigration methods in analysis of phytochemical substances in herbal drugs and medicinal plants. A short description of the basic principles of capillary electrophoretic techniques is firstly given, then the overview deals with the applications of selected methods published in the period 2005-2010. The phytochemical substances have been classified according to their chemical nature (e.g. alkaloids, polyphenols, carbohydrates, lipids, terpenes) and the applied CE approaches, namely CZE, NACE, MEKC, MEEKC and CEC, together with the different detection methods, are critically discussed for each of the considered classes of natural compounds.

Differentiation of green tea samples by chiral CD-MEKC analysis of catechins content.

A chiral CD‐MEKC method, enantioselective for catechin and gallocatechin, was developed, validated and applied to the analysis of tea samples. The method was addressed to the fast and simultaneous quantitation of the most represented and biologically important green tea catechins and methylxanthines. The CD‐MEKC was based on SDS as surfactant (90 mM) and hydroxypropyl‐β‐CD (25 mM) as chiral selector, under acidic conditions (25 mM borate–phosphate buffer, pH 2.5). The method was first applied to study the thermal epimerisation of epi‐structured catechins, (−)‐epicatechin and (−)‐epigallocatechin, to non‐epi‐structured (−)‐catechin and (−)‐gallocatechin. The latter compounds, being non‐native molecules, were for the first time regarded as useful phytomarkers of tea samples subjected to thermal treatment. The proposed method was applied to the analysis of more than twenty tea samples of different geographical origins (China, Japan, Ceylon), having undergone different storage conditions and manufacturing processes. Finally, factor analysis was used to visualise the useful information contained in the data set, showing that it was possible to distinguish tea samples on the basis of their different contents of native and non‐native catechins.

Cyclodextrin-MEEKC for the analysis of oxybutynin and its impurities

The development of a cyclodextrin-MEEKC method for the analysis of oxybutynin and five related impurities is described. Experimental design strategies were applied in order to reach baseline separation of the compounds in a short analysis time. Mixture design made it possible to find the best composition for the microemulsion acting as pseudostationary phase, which was constituted by 89.1% 10 mM borate buffer pH 9.2, 1.7% n-heptane, 9.2% SDS/n-butanol in 1:2 ratio. The addition of (2-hydroxypropyl)-beta-cyclodextrin to the background electrolyte was found to improve analysis performance. A Doehlert design, for the factors cyclodextrin concentration and voltage, was carried out and Derringer desirability function led to the identification of 18 mM and 29 kV as the optimal values. Applying the optimum conditions, separation of all the compounds, including the enantiomers of impurity 1, was obtained in less than 12 min. The method was validated according to ICH guidelines for drug assay and determination of impurities and was applied to oxybutynin tablet analysis.

Mitochondrial Pathway Mediates the Antileukemic Effects of Hemidesmus Indicus, a Promising Botanical Drug

Background Although cancers are characterized by the deregulation of multiple signalling pathways, most current anticancer therapies involve the modulation of a single target. Because of the enormous biological diversity of cancer, strategic combination of agents targeted against the most critical of those alterations is needed. Due to their complex nature, plant products interact with numerous targets and influence several biochemical and molecular cascades. The interest in further development of botanical drugs has been increasing steadily and the FDA recently approved the first new botanical prescription drug. The present study is designed to explore the potential antileukemic properties of Hemidesmus indicus with a view to contributing to further development of botanical drugs. Hemidesmus was submitted to an extensive in vitro preclinical evaluation. Methodology/Principal Findings A variety of cellular assays and flow cytometry, as well as a phytochemical screening, were performed on different leukemic cell lines. We have demonstrated that Hemidesmus modulated many components of intracellular signaling pathways involved in cell viability and proliferation and altered the protein expression, eventually leading to tumor cell death, mediated by a loss of mitochondrial transmembrane potential and increased Bax/Bcl-2 ratio. ADP, adenine nucleotide translocator and mitochondrial permeability transition pore inhibitors did not reverse Hemidesmus-induced mitochondrial depolarization. Hemidesmus induced a significant [Ca2+]i raise through the mobilization of intracellular Ca2+ stores. Moreover, Hemidesmus significantly enhanced the antitumor activity of three commonly used chemotherapeutic drugs (methotrexate, 6-thioguanine, cytarabine). A clinically relevant observation is that its cytotoxic activity was also recorded in primary cells from acute myeloid leukemic patients. Conclusions/Significance These results indicate the molecular basis of the antileukemic effects of Hemidesmus and identify the mitochondrial pathways and [Ca2+]i as crucial actors in its anticancer activity. On these bases, we conclude that Hemidesmus can represent a valuable tool in the anticancer pharmacology, and should be considered for further investigations.

Chiral capillary liquid chromatography based on penicillin G acylase immobilized on monolithic epoxy silica column

An epoxy derivatized monolithic silica capillary column (100 μm i.d.) was used as a support for immobilization of penicillin G acylase (PGA), an enzyme used in the production of semisynthetic antibiotics. In order to allow for sensitive UV detection, the PGA-based monolithic capillary column was coupled to an open fused-silica capillary via a TFE (Teflon®) shrink tube sleeve (1 cm long, 300 μm i.d.), which proved to be a robust, dead-volume free and easily replaceable connector. This configuration resulted in a duplex fritless column for capillary liquid chromatography (CLC) and electrically assisted CLC (eCLC). In particular, using the driving pressure (2-12 bar) supplied by the commercial CE instruments, CLC separations could be obtained in short time due to the low column backpressure of the monolith. In particular, the developed stationary phase characterized by the chiral recognition ability of PGA, was successfully applied in enantioseparation of arylpropionic acids of pharmaceutical interest (i.e., profens). As an example, by using a 7 cm long monolith capillary column, the enantioresolution (Rs>3.0) of rac-ketoprofen was achieved in less than 2 min (pressure 12 bar) with a minimum plate height in the order of 20 μm and using as a mobile phase a 50 mM phosphate buffer pH 7.0. Validation data such as repeatability of retention time (intraday<0.62, n=6; interday<1.62, n=9; and column-to-column<10.5, n=2), linearity (r²=0.999), and sensitivity (LOQ 0.25% (w/w) of (R)-ketoprofen with respect to (S)-ketoprofen) showed good method performance. The method was successfully applied to the determination of (S)-ketoprofen in pharmaceutical samples (tablets).

Development of a capillary electrophoresis method for the assay of ramipril and its impurities: An issue of cis–trans isomerization

The development of a rapid and selective capillary electrophoresis method for the quantitation of ramipril and its eight main impurities in pharmaceutical dosage form is described. Ramipril and three of its impurities contain a proline-similar moiety which causes in solution the presence of interconverting cis-trans isomers with respect to the amide bond. The interplay between electrophoretic migration and isomerization may yield the presence of an undesired interconversion zone between the two isomer peaks in the electropherogram, depending on the experimental conditions. Different capillary electrophoresis operative modes and pseudostationary phases were evaluated, both in normal and reverse polarity, in order to find the essential analytical parameters which could make it possible to overcome this issue and thus accurately quantify the analytes. The best results were obtained by using microemulsion electrokinetic chromatography in reverse polarity, where all the compounds which undergo cis-trans interconversion migrate as a single narrow peak. Experimental design led to identification of the following optimised conditions: background electrolyte, microemulsion made by 88.95% of 90 mM phosphate pH 2.5, 1.05% of n-heptane and 10.00% of SDS/n-butanol in 1:2 ratio; voltage, -26 kV; temperature, 17°C. Applying these conditions, the baseline separation of the analytes was obtained in about 10 min. Validation of the method following ICH guidelines was carried out and the procedure was applied to a real sample of ramipril tablets.

Cytotoxic activity of guaiazulene on gingival fibroblasts and the influence of light exposure on guaiazulene-induced cell death.

Guaiazulene (GA) is widely used as a natural ingredient in many health care products and solutions. Although it has been reported to have interesting biological effects, GA and azulene derivatives have been proven to be cytotoxic against normal human cells and human tumor cells; moreover, guaiazulene has shown photomutagenic properties on bacterial strains. Therefore, we evaluated and compared the cytotoxicity of GA at different concentrations on human gingival fibroblast (HGF) cell cultures under normal conditions and under UV irradiation (UV-A dose: 6.4 J/cm(2)). The compound tested was found to significantly reduce cell viability (dose-dependent trend, IC(50) 72.1 μM), decrease protein procollagen α1 type I synthesis, a marker for HGF protein, and COL1A1 mRNA expression. The cytotoxic effects were accompanied by activation of an intrinsic apoptotic pathway, studied using transmission electron microscopy (TEM) and caspase-3 activation. The light exposure of the cell culture treated decreased GA-induced cell death (IC(50) 128.9 μM), suggesting a photoprotective effect due to the photodegradation of the toxic agent, guaiazulene. Furthermore, the products of the photodegradation reaction of GA proved not to be toxic against HGFs.

Determination of dermatan sulfate and chondroitin sulfate as related substances in heparin by capillary electrophoresis

Capillary electrophoresis (CE) was applied to the quantitation of dermatan sulfate (DS) and chondroitin sulfate (CS) as related substances in sodium heparin. The method is based on the selective digestion of either CS and DS contained in the main drug heparin, by using chondroitinase ABC (specific for both DS and CS) and chondroitinase AC (specific for only CS). The unsaturated disaccharides released after exhaustive digestion, can be separated by CE using a 110mM phosphate buffer, pH 3.5 as the background electrolyte in a fused silica capillary (64.5cmx50mum i.d.) at 40 degrees C and -30kV. Since the level of each disaccharide released upon enzymatic digestion corresponds to its content in the native glycosaminoglycan, the amount of CS and DS was determined by proportion with the released disaccharides. In particular, DeltaUA-->GalNAc-4S Na(2) and DeltaUA-->GalNAc-6S Na(2) were selected for quantitation of CS and DS because of their significant response and short migration time (less than 7min).The method was validated for linearity, accuracy, precision and it showed to be able in detecting selectively, DS and CS at impurity level (LOD 0.01%, w/w). The proposed CE approach was finally applied to real samples. The results obtained were found in excellent correlation with those achieved by the analysis of the same samples using the official USP method based on high performance anion exchange chromatography (HPAEC) with pulsed amperometric detector.

Analysis of Cyclosporin A and Main Degradation Impurities by Cyclodextrin–Modified Micellar Electrokinetic Chromatography

Separation of the immunosuppressive drug cyclosporin A (CyA) from the closely related degradation impurities cyclosporin H (CyH, a CyA diastereomer) and isocyclosporin A (IsoCyA) was accomplished by means of cyclodextrin-modified micellar electrokinetic chromatography (CD-MEKC). Heptakis (2,3,6-tri-O-methyl)-β-cyclodextrin (TM-βCD) showed to be an effective modifiers of the sodium dodecyl sulfate (SDS) micellar system, allowing for the CD-MEKC separation of CyA from CyH and IsoCyA. By means of electric current measurements carried out using the capillary electrophoretic apparatus, the critical micelle concentration of SDS in the presence of different neutral cyclodextrins was estimated. Interestingly, it was found that TM-βCD strongly inhibited the micellization of SDS compared to β-, and hydroxypropyl-β-cyclodextrin. This unusual behavior was considered to be a key factor of the specific ability of the methylated cyclodextrin in providing CD-MEKC separation of hydrophobic cyclosporins. The optimized method consisted of: 50 mM sodium dodecyl sulfate (SDS) in 50 mM tetraborate buffer (pH 9.2) supplemented with 15 mM TM-βCD. The electrophoretic runs were performed at 30°C under the application of 22 kV in a fused-silica capillary (50 µm id, 64.5 cm total length, 56 cm length to the detector). The method was validated for linearity of CyA (within 0.25–5.00 mg/mL) and CyH and IsoCyA (within 0.25–5.0%, w/w with respect to CyA), sensitivity (LOQ 5.0 µg/mL), accuracy, and precision. The applicability of the method was proved by analysis of a commercially available pharmaceutical (gelatin capsules).

Micellar complexes of all-trans retinoic acid with polyvinylalcohol-nicotinoyl esters as new parenteral formulations in neuroblastoma

All-trans-retinoic acid (ATRA) is now included in many antitumor therapeutic schemes for the treatment of acute promyelocytic leukemia, Kaposi’s sarcoma, head and neck squamous cell carcinoma, ovarian carcinoma, bladder cancer, and neuroblastoma. Unfortunately, its poor aqueous solubility hampers its parenteral formulation, whereas oral administration of ATRA is associated with progressively diminishing drug levels in plasma, which is related to induction of retinoic acid-binding proteins and increased drug catabolism by cytochrome P450-mediated reactions. An ATRA formulation, obtained by complexation of the drug into polymeric micelles, might be suitable for parenteral administration overcoming these unwanted effects. To this purpose, amphiphilic polymers were prepared by polyvinylalcohol (PVA) partial esterification with nicotinoyl moieties and their functional properties evaluated with regard to ATRA complexation. The physicochemical characteristics of the polymers and the complexes were analyzed by 1H-NMR, Dynamic Light Scattering (DLS), Capillary Electophoresis (CE), and were correlated with the complex ability to improve the drug solubilization and release the free drug in an aqueous environment. Subsequently, the best complex, providing the highest ATRA solubilization and release, was evaluated in vitro to test its citotoxicity towards neuroblastoma cell lines. The PVA substitution degree calculated from 1H-NMR was found to be 5.0%, 8.2%, 15.3% (nicotinoyl moiety:PVA monomer molar ratio), while capillary electrophoresis analysis on the complexes revealed that the drug loadings were 0.95%, 1.20%, 4.76% (ATRA:polymer w:w) for PVA substitution degrees of 5.0%, 8.2%, and 15.3%, respectively. Complexation strongly increased ATRA aqueous solubility, which reached 1.20 ± 0.25 mg/mL. The DLS measurements of the polymers and the complexes in aqueous solutions revealed mean sizes always below 400 nm, low polydispersity (min 0.202 ± 0.013, max 0.450 ± 0.032), and size almost unaffected by concentration. Drug fractional release did not exceed 8% after 48 h. The cytotoxicity studies against neuroblastoma cell lines outlined a significant growth inhibition effect of complexed ATRA with respect to free ATRA. These data suggest that the systems analyzed may be suitable carriers for parenteral administration of ATRA and other hydrophobic antitumor drugs, where the carriers are required to improve drug aqueous solubility and delay drug release almost after their accumulation in solid tumors.