Manuela Martinefski

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.

MINIATURIZED HPLC-UV METHOD FOR ANALYSIS OF COENZYME Q10 IN HUMAN PLASMA

Coenzyme Q10 (CoQ10) is a cofactor in the respiratory chain and a potent endogenous antioxidant. Abnormally low levels of CoQ10 in the circulatory system are often involved with pathological states such as heart failure, mitochondrial and muscular diseases, and cancer. The development of simple, rapid, and highly sensitive methods capable of quantifying low CoQ10 levels in plasma is, therefore, increasingly required. In this work, we developed a miniaturized HPLC-UV system for the determination CoQ10 in human plasma using an XTerra microcolumn (50 mm × 2.1 mm i.d., 3.5 µm particle size), methanol:water (98:2, v/v) as mobile phase, 275 nm, flow rate of 0.3 mL/min, 30 °C column temperature, and 2 µL injection volume. The extraction procedure consists of a plasma precipitation with 1-propanol and evaporation under nitrogen to allow a 1.5-fold enrichment. The chromatographic analysis was accomplished in 7 min. As a result, it was possible to quantitate CoQ10 in small sample volumes down to 0.07 µM and detect as low as 0.02 µM in real plasma, with good accuracy and precision, even in pathological conditions. The proposed method was found to be suitable for routine CoQ10 determination in clinical laboratories.

Aerobic degradation of ibuprofen in batch and continuous reactors by an indigenous bacterial community

ABSTRACT Water from six points from the Riachuelo-Matanza basin was analyzed in order to assess ibuprofen biodegradability. In four of them biodegradation of ibuprofen was proved and degrading bacterial communities were isolated. Biodegradation in each point could not be correlated with sewage pollution. The indigenous bacterial community isolated from the point localized in the La Noria Bridge showed the highest degradative capacity and was selected to perform batch and continuous degradation assays. The partial 16S rRNA gene sequence showed that the community consisted of Comamonas aquatica and Bacillus sp. In batch assays the community was capable of degrading 100 mg L−1 of ibuprofen in 33 h, with a specific growth rate (μ) of 0.21 h−1. The removal of the compound, as determined by High performance liquid chromatography (HPLC), exceeded 99% of the initial concentration, with a 92.3% removal of Chemical Oxygen Demand (COD). In a down-flow fixed-bed continuous reactor, the community shows a removal efficiency of 95.9% of ibuprofen and 92.3% of COD for an average inlet concentration of 110.4 mg. The reactor was kept in operation for 70 days. The maximal removal rate for the compound was 17.4 g m−3 d−1. Scanning electron microscopy was employed to observe biofilm development in the reactor. The ability of the isolated indigenous community can be exploited to improve the treatment of wastewaters containing ibuprofen.

New analytical strategies applied to the determination of Coenzyme Q10 in biological matrix.

In the last few years the importance of Coenzyme Q10 (CoQ10) determination has gained clinical relevance. CoQ10 is a redox-active, lipophilic substance integrated in the mitochondrial respiratory chain which acts as an electron carrier for the production of cellular energy. In addition, it is recognized as a primary regenerating antioxidant playing an intrinsic role against oxidative damage. There are some reports of low CoQ10 levels in a number of disorders, such as cancer, muscular, neurodegenerative, cardiological, and reproductive diseases. Therefore, it is a priority to develop analytical methodologies for evaluating CoQ10 in matrices of greater importance for the correct diagnosis of diseases, simple enough to be used in routine clinical laboratories. In this chapter two recently developed techniques, capillary electrophoresis and microHPLC, for the analysis of CoQ10 in biological matrices, are studied.

A novel non-invasive sampling method using buccal mucosa cells for determination of coenzyme Q10

Coenzyme Q10 (CoQ10) is an important cofactor in the mitochondrial respiratory chain and a potent endogenous antioxidant. CoQ10 deficiency is often associated with numerous diseases, and patients can benefit from CoQ10 supplementation, being more effective when diagnosed and treated early. Due to the increased interest in CoQ10 deficiency, several methods for CoQ10 analysis from plasmatic, muscular, fibroblast, and platelet matrices have been developed. These sampling techniques are not only highly invasive but also too traumatic for periodic clinical monitoring. In the present work, we describe the development and validation of a novel non-invasive sampling method for quantification of CoQ10 in buccal mucosa cells (BMCs) by microHPLC. This method is suitable for using in a routine laboratory and useful for sampling patients in pediatry. CoQ10 correlation was demonstrated between BMCs and plasma levels (Spearman r, 0.4540; p < 0.001). The proposed method is amenable to be applied in the post treatment monitoring, especially in pediatric patients as a non-invasive sample collection. More studies are needed to assess whether this determination could be used for diagnosis and if this matrix could replace the traditional ones.

In Search of an Accurate Evaluation of Intrahepatic Cholestasis of Pregnancy

Until now, biochemical parameter for diagnosis of intrahepatic cholestasis of pregnancy (ICP) mostly used is the rise of total serum bile acids (TSBA) above the upper normal limit of 11 μM. However, differential diagnosis is very difficult since overlapped values calculated on bile acids determinations, are observed in different conditions of pregnancy including the benign condition of pruritus gravidarum. The aim of this work was to determine the better markers in ICP for a precise diagnosis together with parameters associated with severity of symptoms and treatment evaluation. Serum bile acid profiles were evaluated using capillary electrophoresis in 38 healthy pregnant women and 32 ICP patients and it was calculated the sensitivity, specificity, accuracy, predictive values and the relationships of certain individual bile acids in pregnant women in order to replace TSBA determinations. The evaluation of the results shows that LCA and UDCA/LCA ratio provided information for a more complete and accurate diagnosis and evaluation of ICP than calculation of solely TSBA levels in pregnant women.

Relative bioavailability of coenzyme Q10 formulation for paediatric individualized therapy

Conduct a preliminary comparison of the bioavailability between two formulations: commercial grade coenzyme Q10 (CoQ10) powder (solid formulation) and a new oil‐in‐water liquid emulsion and their effect on other antioxidants.

Coenzyme Q in pregnant women and rats with intrahepatic cholestasis

Intrahepatic cholestasis of pregnancy is a high‐risk liver disease given the eventual deleterious consequences that may occur in the foetus. It is accepted that the abnormal accumulation of hydrophobic bile acids in maternal serum are responsible for the disease development. Hydrophobic bile acids induce oxidative stress and apoptosis leading to the damage of the hepatic parenchyma and eventually extrahepatic tissues. As coenzyme Q (CoQ) is considered an early marker of oxidative stress in this study, we sought to assess CoQ levels, bile acid profile and oxidative stress status in intrahepatic cholestasis.

A simple microHPLC-UV method for the simultaneous determination of retinol and α-tocopherol in human plasma. Application to intrahepatic cholestasis of pregnancy

(a) Objectives: the development of a simple, rapid, sensitive, and accurate HPLC-UV method for the simultaneous determination of retinol and α-tocopherol in human plasma, suitable for the evaluation of these vitamins in pregnant women suffering from intrahepatic cholestasis of pregnancy (ICP). (b) Design and methods: chromatographic separation of vitamins was achieved with a microcolumn BDS HYPERSIL C18 (100 mm × 2.1 mm, 2.4 μm) using a C18 guard column. The developed and validated HPLC method proposed in this study was applied in the assessment of plasma concentrations of retinol and α-tocopherol in normal pregnancy (n = 37) and in pregnancy complicated with ICP (n = 19). (c) Results: the validation of the method showed excellent linearity for retinol and α-tocopherol (r2: 0.998 and 0.997), high sensitivity (LOD: 0.02 and 0.11 μM, LOQ: 0.07 and 0.36 μM), and high recovery (96.0–104.0% and 99.3–102.0%), respectively. The method requires a low sample volume and all procedures of sample preparation and analytical determination take less than 20 min to be completed. The evaluation of the results in ICP patients showed that α-tocopherol significantly diminished compared to normal pregnant women (13.1 ± 2.9 vs. 22.4 ± 2.2 μM, p < 0.05) whereas retinol remains unchanged (1.27 ± 0.21 vs. 1.25 ± 0.10 μM). (d) Conclusions: a simple, accurate, reliable, selective, highly sensitive and cost effective method suitable for routine determination of retinol and α-tocopherol in biological samples is proposed. It was possible to determine the decrease of α-tocopherol in ICP patients by this method.

The importance of analytical methodology in accurate diagnosis and monitoring of intrahepatic cholestasis of pregnancy

Fil: Martinefski, Manuela Romina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquimica. Departamento de Quimica Analitica y Fisicoquimica. Catedra de Quimica Analitica; Argentina;