Silvia Lucangioli

Bile acid profiles by capillary electrophoresis in intrahepatic cholestasis of pregnancy

ICP (intrahepatic cholestasis of pregnancy) is characterized by pruritus and biochemical cholestasis, including raised SBAs (serum bile acids) and, usually, elevated aminotransferases levels. However, AHP (asymptomatic hypercholanaemia of pregnancy) is defined as the presence of total SBA levels above the cut-off value (11 microM) in healthy pregnant women, thus elevation of total SBAs do not necessarily reflect an ICP condition. The aim of the present study was to describe clinical, obstetric, perinatal and biochemical findings, as well as the SBA profile, in pregnant women studied in the third trimester of pregnancy in order to define characteristic patterns of individual bile acids that enable women with ICP to be distinguished from AHP and healthy pregnancies. Free and conjugated ursodeoxycholic (UDCA), cholic (CA), lithocholic (LCA), deoxycholic (DCA) and chenodeoxycholic (CDCA) acids were evaluated by CE (capillary electrophoresis) in 41 patients (15 of them simultaneously by HPLC), in 30 healthy pregnant women and in 10 non-pregnant women. A highly significant correlation between CE and HPLC for total SBAs (r=0.990) and for individual SBAs was found. Normal pregnant women had higher total SBA levels than non-pregnant women (due to an increase in taurine-conjugated dihydroxy SBAs). Women with ICP had higher levels of total SBAs, the free/conjugated ratio, LCA, CA, CDCA and DCA than normal pregnant women. Newborns from women with ICP had lower birth weight and gestational age. Women with AHP had higher levels of conjugated dihydroxy SBAs than normocholanaemic patients, without any evidence of a clinical difference. In conclusion, the present study has shown a clear difference in SBA profiles between ICP and normal pregnancies (including AHP), involving a shift towards a characteristic hydrophobic composition in women with ICP.

Analysis of cis-trans isomers and enantiomers of sertraline by cyclodextrin-modified micellar electrokinetic chromatography

In this work development, optimization and validation of a cyclodextrin-modified micellar electrokinetic chromatography (CD-modified MEKC) method is proposed to resolve separation of the sertraline hydrochloride and synthesis-related substances. Sertraline hydrochloride, the cis-(1S,4S) enantiomer form, is used as an antidepressant therapeutic agent. A buffer concentration composed of 20 mM sodium borate, pH 9.0 with 50 mM sodium cholate, 15 mM sulfated beta-cyclodextrin and 5 mM hydroxypropyl-beta-cyclodextrin was found to be the most suitable background electrolyte. Quantitation of the impurities at levels of 0.1% in different samples of the bulk drug was determined. A comparison of the results with those obtained by HPLC methodology was also accomplished. The method proved appropriate for testing the purity of sertraline hydrochloride in bulk drug.

Physicochemical, biological and drug-release properties of gallium crosslinked alginate/nanoparticulate bioactive glass composite films

The aim of this work was to develop biodegradable and bioactive materials with sufficient structural integrity and prophylaxis effect against infection based on alginate-bioactive glass composite. The incorporation of bioactive glass nanoparticles (NBG) into Ga-crosslinked alginate films significantly improved their mechanical properties when compared with films fabricated with micron-sized bioactive glass particles. In addition, Ga-alginate films containing NBG induced a bacteriostatic effect in vitro towards S. aureus due to the presence of Ga ions (Ga3+), whose release is controlled solely by crosslinking the ion with alginate. Biomineralization studies in simulated body fluid suggested the deposition of hydroxyapatite on the surface of the films indicating their bioactive nature. In addition, the films were shown to feature biocompatibility toward osteoblast-like cells. Thus, it was shown that Ga-crosslinked composite films possessed relevant physicochemical, biological and controlled bacteriostatic effects which make these materials promising candidates for bone tissue engineering applications.

Lithocholic acid as a biomarker of intrahepatic cholestasis of pregnancy during ursodeoxycholic acid treatment

Background The diagnosis and treatment of intrahepatic cholestasis of pregnancy (ICP) has important implications on fetal health. The biochemical parameter commonly used in the diagnosis of ICP is the determination of the concentration of total serum bile acids (TSBA). However, bile acid profile, especially lithocholic acid (LCA) analysis is a more sensitive and specific biomarker for differential diagnosis of this pathology and also could be an alternative to evaluate the efficiency of ursodeoxycholic acid (UDCA) for ICP treatment. Methods Serum bile acid (SBA) profiles including LCA determination, were studied in 28 ICP patients using a capillary electrophoresis method. The effects of UDCA treatment on bile acid profile, were analysed in 23 out of 28 ICP patients and the two samples obtained before and 15 days after treatment were compared. Two samples taken as controls were also obtained from each of five patients without therapy. Results A dramatic decrease in LCA concentrations and maintenance of TSBA concentrations were found in all patients after UDCA therapy, whereas SBA profiles together with LCA values did not change in patients without therapy. Conclusion We propose LCA as an alternative biomarker and a more sensitive parameter than TSBA to evaluate the effectiveness of UDCA treatment, at least in ICP patients from Argentina.

A capillary electrophoretic system based on a novel microemulsion for the analysis of coenzyme Q10 in human plasma by electrokinetic chromatography

A new analytical method for determination of coenzyme Q10 (2,3‐dimethoxy‐5‐methyl‐6‐decaprenyl‐1,4‐benzoquinone, CoQ10) in human plasma was developed based on CE using a double tensioactive microemulsion. CoQ10 was quantitatively extracted into 1‐propanol/hexane and quantified by MEEKC. The microemulsion was prepared by mixing 1.4% w/w sodium bis(2‐ethylhexyl) sulfosuccinate, 4% w/w cholic acid, 1% w/w octane, 8.5% w/w butanol, 0.1% w/w PVA and 85% w/w 10 mM Tris buffer at pH 9.0. The optimized electrophoretic conditions included the use of an uncoated silica capillary of 60 cm total length and 75 μm id, an applied voltage of 20 kV, room temperature and 214 nm ultraviolet detection. Selectivity, linearity, LOD, LOQ, precision and accuracy were evaluated as the parameters of validation. Owing to its simplicity and reliability, the proposed method can be an advantageous alternative to the traditional methodology for the quantitation of CoQ10 in human plasma with good accuracy and precision.

Determination of bile acids in pharmaceutical formulations using micellar electrokinetic chromatography

A micellar electrokinetic chromatography method (MEKC) has been developed and validated for the determination of bile acids (BA) such as ursodeoxycholic acid (UDCA), dehydrocholic acid (DHCA) and deoxycholic acid (DCA) in pharmaceuticals for quality control purpose. The background electrolyte consisted of 20 mM borate-phosphate buffer containing 50 mM sodium dodecylsulfate (SDS), and acetonitrile as additive. UV detection was set at 185 nm. Selectivity, linearity, range, repeatability, intermediate precision and accuracy showed good results. Comparison of the values obtained by MEKC and HPLC methods were in close agreement.

Simultaneous determination of nine endogenous steroids in human urine by polymeric-mixed micelle capillary electrophoresis.

A new CE system based on the use of polymeric‐mixed micelles (cholic acid, SDS and the poloxamine Tetronic® 1107) was developed for the simultaneous determination of nine steroids in human urine. This method allows the baseline separation and quantitation of cortisol, androstenedione, estriol, dehydroepiandrosterone sulfate, testosterone, dehydroepiandrosterone, estrone, progesterone and estradiol in less than 25 min showing to be sensitive enough to detect low concentrations of these steroids in urine samples (5–45 ng/mL). The optimized electrophoretic conditions were performed using a 50 cm×75 μm capillary, 18 kV, 25°C, with 44 mM cholic acid, 10 mM SDS, 0.05% w/v tetronic® 1107, 2.5% v/v methanol, 2.5% v/v tetrahydrofuran in 5 mM borate – 5 mM phosphate buffer (pH=8.0) as a background electrolyte and a dual 210/254 UV‐detection. The method can simultaneously determine 0.1–120 μg/mL, which corresponds to 5–6000 ng/mL of steroids in 2 mL urine. The recoveries ranged between 82.4 and 101.5%. Due to its simplicity, speed, accuracy and reliability, the proposed method could be a potential alternative to the traditional methodologies used with clinical purposes.

Application of extraction disks in dissolution tests of clenbuterol and levothyroxine tablets by capillary electrophoresis

Sample preparation procedures using octadecyl (C18) extraction disks were developed to obtain accurate and reproducible results for determinations of clenbuterol(20 micrograms per dose) and levothyroxine (100 micrograms per dose) in dissolution media of solid oral dosage forms. Preconcentration of samples allowed final concentrations of 1.1 micrograms/ml of clenbuterol and 4.0 micrograms/ml of levothyroxine to be reached prior to CE analysis. The results obtained by CE were in good agreement with those of HPLC. The precision of the migration time, peak area, peak height and accuracy were determined in both intra-day (n = 6) and inter-day (n = 18) assays. Linearity was demonstrated over the ranges 0.5-80.0 micrograms/ml of clenbuterol and 1.0-30.0 micrograms/ml of levothyroxine. The mean recoveries were higher than 94.0%, ranging from 50 to 125% levels with respect to dose potencies. The proposed methodology may be generally applied to determine drugs at ng/ml concentrations.

Simultaneous determination of free and conjugated bile acids in serum by cyclodextrin-modified micellar electrokinetic chromatography

A simultaneous determination of 15 free and most conjugated forms of bile acids (BA) in serum using capillary electrophoresis is described. The optimized and validated method proposed in this work is straightforward and rapid, employing affordable equipment. A background electrolyte of 5 mM beta-cyclodextrin, 5 mM 2-hydroxypropyl-beta-cyclodextrin, 50 mM SDS and sodium borate-dihydrogen phosphate pH 7.0 with 10% of acetonitrile was used. The complete separation of 15 BA, not easily achievable with other methods, is performed in less than 12 min using a UV detector with good precision and accuracy. BA were extracted from pretreated serum samples using a C(18)-solid-phase extraction and the recovery values ranged from 65 to 107.8%. Limits of quantitation were between 0.58 and 3.2 microM. This method proved to be suitable to determine individual BA profiles which are more useful than total serum bile acids as indicators of metabolic disorders and hepatobiliary diseases.

The use of coenzyme Q0 as a template in the development of a molecularly imprinted polymer for the selective recognition of coenzyme Q10.

In this work, a novel molecularly imprinted polymer (MIP) for use as a solid phase extraction sorbent was developed for the determination of coenzyme Q10 (CoQ10) in liver extract. CoQ10 is an essential cofactor in mitochondrial oxidative phosphorylation and a powerful antioxidant agent found in low concentrations in biological samples. This fact and its high hydrophobicity make the analysis of CoQ10 technically challenging. Accordingly, a MIP was synthesised using coenzyme Q0 as the template, methacrylic acid as the functional monomer, acetonitrile as the porogen, ethylene glycol dimethacrylate as the crosslinker and benzoyl peroxide as the initiator. Various parameters affecting the polymer preparation and extraction efficiency were evaluated. Morphological characterisation of the MIP and its proper comparison with C18 as a sorbent in solid phase extraction were performed. The optimal conditions for the molecularly imprinted solid phase extraction (MISPE) consisted of 400 μL of sample mixed with 30 mg of MIP and 600 μL of water to reach the optimum solution loading. The loading was followed by a washing step consisting of 1 mL of a 1-propanol solution (1-propanol:water, 30:70,v/v) and elution with 1 mL of 1-propanol. After clean-up, the CoQ10 in the samples was analysed by high performance liquid chromatography. The extraction recoveries were higher than 73.7% with good precision (3.6-8.3%). The limits of detection and quantification were 2.4 and 7.5 μg g(-1), respectively, and a linear range between 7.5 and 150 μg g(-1) of tissue was achieved. The new MISPE procedure provided a successful clean-up for the determination of CoQ10 in a complex matrix.