Sabrina Flor

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.

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.

Development and validation of a capillary electrophoresis method for determination of enantiomeric purity and related substances of esomeprazole in raw material and pellets

A capillary electrophoresis method using CDs for quality control of esomeprazole (ESO) in terms of enantiomeric purity and related substances in raw material and pellets was developed. ESO is the S‐enantiomer of omeprazole (OMZ). Several parameters were evaluated, including type and concentration of buffer and CD, concentration of additives and electrolyte pH. Resolution between the enantiomers of OMZ obtained for each parameter tested was calculated and the presence of the main related substance such as OMZ sulfone was carefully monitored. The optimized system consisted of 100 mM Tris‐phosphate buffer pH 2.5 with 20 mM 2‐hydroxypropyl‐β‐CD, 1 mM sodium dithionite, temperature at 15°C, voltage at 28 kV, and UV detection at 301 nm. Once optimized, the electrophoretic system was validated according to ICH guidelines. The limits of detection and quantification for R‐OMZ were 0.6 μg/mL (0.06% w/w of ESO) and 2.0 μg/mL (0.2% w/w of ESO), respectively. A mean concentration of R‐OMZ <0.2% limit established by the United States Pharmacopeia (USP) was found in the raw material and six‐pellet samples of ESO. No other impurities were found in the samples under these conditions. Therefore, the developed method was found to be appropriate not only for enantiomeric quality control of ESO but also for the analysis of ESO and the main related substance in raw material and pharmaceutical formulations as well as for stability indicating studies.

Novel nelfinavir mesylate loaded d-α-tocopheryl polyethylene glycol 1000 succinate micelles for enhanced pediatric anti HIV therapy: In vitro characterization and in vivo evaluation

Worldwide more than 35 million people are living with Human Immunodeficiency Virus (HIV) where 3.3 million are children. This translates in approximately 700 new daily infections in children only in 2012. Prolonged High Activity Antiretroviral Therapy (HAART) regimes could present low-patient compliance, especially in children, affecting therapeutic success. Nelfinavir mesylate (NFV) is a non-peptidic HIV-1 protease inhibitor (IP) which was the first IP recommended for pediatric use (>2 years-old). It exhibits pH-dependant aqueous solubility which results highly restricted at physiological pH values. The former represents a main clinical limitation due to the reduction on drug absorption along the small intestine after an oral administration, leading to unpredictable drug bioavailability. Moreover a liquid formulation of NFV is not available worldwide, preventing appropriate dose adjustment and more convenient administration. In this framework, the present investigation reports the development of a NFV highly concentrated aqueous formulation for a more appropriate management of pediatric anti-HIV therapy. The aim was to encapsulate NFV within D-α-tocopheryl polyethylene glycol 1000 succinate micelles to improve its aqueous solubility and its oral pharmacokinetic parameters. Results show that NFV aqueous solubility was increased up to 80.3 mg/mL. NFV-loaded micelles exhibited a hydrodynamic diameter of 5.6 nm and a spherical morphology as determined by dynamic light scattering and transmission electronic microscopy, respectively. In vitro NFV release profile demonstrated a cumulative drug release of 56% at 6 h. Finally, in vivo data showed a significant (p<0.01) increase of Area-Under-the-Curve between 0 and 24 h for NFV encapsulated in micelles in comparison with a NFV suspension prepared with glycerin 20% v/v and carboxymethylcellulose sodium 0.5% w/v, representing an increment on drug oral relative bioavailability of 1.71-fold. Thereby, this formulation represents an innovative nanotechnological platform to improve pediatric HIV pharmacotherapy.

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.

FAST AND SENSITIVE NEW HPLC-UV METHOD FOR DETERMINATION OF OMEPRAZOLE AND MAJOR RELATED SUBSTANCES IN PHARMACEUTICAL FORMULATION

A simple, fast, and sensitive HPLC method with UV detection has been developed for the quantitation of omeprazole (OMZ) and major related substances in raw material and pharmaceutical formulation (paste) using a column of reduced length (50 mm) and diameter (2.1 mm) packed with hybrid particles. Chromatographic conditions were: 25°C, 1 µl injection volume, and UV detection at WV of 280 nm. The flow rate was 0.3 mL/min using methanol-phosphate buffer (pH 7.6) (40:60) as the mobile phase. Chromatographic purity was also determined with the same chromatographic conditions. The method was validated according to international guidelines (ICH guidelines) for specificity, linearity, LOD, LOQ, precision, accuracy, and robustness. The HPLC-UV method was found to be suitable for the quality control and stability studies of OMZ in a pharmaceutical formulation.

Silica core–shell particles for the dual delivery of gentamicin and rifamycin antibiotics

Increasing bacterial resistance calls for the simultaneous delivery of multiple antibiotics. One strategy is to design a unique pharmaceutical carrier that is able to incorporate several drugs with different physico-chemical properties. This is highly challenging as it may require the development of compartmentalization approaches. Here we have prepared core-shell silica particles allowing for the dual delivery of gentamicin and rifamycin. The effect of silica particle surface functionalization on antibiotic sorption was first studied, enlightening the role of electrostatic and hydrophobic interactions. This in turn dictates the chemical conditions for shell deposition and further sorption of these antibiotics. In particular, the silica shell deposition was favored by the positively charged layer of gentamicin coating on the core particle surface. Shell modification by thiol groups finally allowed for rifamycin sorption. The antibacterial activity of the core-shell particles against Staphylococcus aureus and Pseudomonas aeruginosa demonstrated the dual release and action of the two antibiotics.

Simple, Highly Sensitive Micro HPLC Method for the Determination of Coenzyme Q10 and its Major Related Substances

Abstract A simiple, rapid, and highly sensitive fast microbore HPLC method with UV detection has been developed for the quantitation of coenzyme Q10 (Co-Q10) and related substances in raw material, pharmaceutical, and cosmetic formulations using a column with hybrid particles. Chromatographic conditions were: 30ºC column temperature, 1 µL injection volume at 275 nm. The Co-Q10 analysis was performed within 2.5 minutes with a flow rate of 0.3 mL/min. with a mobile phase consisted of 100% methanol, and the related substances were separated with a gradient elution based on methanol-water. The developed method was validated for selectivity, linearity, precision, accuracy, and robutness. The method was found to be suitable for the quality control of Co-Q10 in pharmaceutical and cosmetic products, as well as the stability indicating studies.

Novel and highly sensitive mixed-polymeric electrokinetic chromatography system for determination of contaminants and impurities of heparin samples

A mixed‐polymeric electrokinetic chromatography system has been developed for the simultaneous determination of a contaminant like oversulfated condroitin sulfate (OSCS) and impurities expressed as dermatan (Der) in heparin (Hep) samples. The EKC system consisted of 0.5% w/v polymeric β‐CD, 0.4% w/v tetronic® 1107 and 400 mM tris‐phosphate buffer at pH 3.5. The optimized electrophoretic conditions included the use of an uncoated‐silica capillary of 50 cm of total length and 75 μm id, an applied voltage of −7 kV, a temperature of 30°C and 200 nm UV‐detection. The highly sensitive method developed showed low values of LOD, 0.07% w/w (0.07 μg/mL) (OSCS) and 0.1% w/w (0.1 μg/mL) (Der), and values of LOQ 0.2% w/w (0.2 μg/mL) (OSCS) and 0.3% w/w (0.3 μg/mL) (Der) with a concentration level of Hep sample as low as 0.1 mg/mL. Additional parameters of validation such as specificity, linearity, accuracy, and robustness were evaluated according to international guidelines. Owing to its simplicity, high sensitivity, and reliability, the proposed method can be an advantageous alternative to the traditional methodologies for the analysis of Hep in raw material and specially in finished products because of the low amounts of Hep sample required.

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.