E Gikas

Oleuropein prevents doxorubicin-induced cardiomyopathy interfering with signaling molecules and cardiomyocyte metabolism

Oleuropein, a natural phenolic compound, prevents acute doxorubicin (DXR)-induced cardiotoxicity but there is no evidence regarding its role in chronic DXR-induced cardiomyopathy (DXR-CM). In the present study, we investigated the role of oleuropein in DXR-CM by addressing cardiac geometry and function (transthoracic echocardiography), cardiac histopathology, nitro-oxidative stress (MDA, PCs, NT), inflammatory cytokines (IL-6, Big ET-1), NO homeostasis (iNOS and eNOS expressions), kinases involved in apoptosis and metabolism (Akt, AMPK) and myocardial metabonomics. Rats were randomly divided into 6 groups: Control, OLEU-1 and OLEU-2 [oleuropein at 1000 and 2000 mg/kg in total, respectively, intraperitoneally (i.p.) for 14 days], DXR (18 mg/kg, i.p. divided into 6 equal doses for 2 weeks), DXR-OLEU-1 and DXR-OLEU-2 (both oleuropein and DXR as previously described). Impaired left ventricular contractility and inflammatory and degenerative pathology lesions were encountered only in the DXR group. The DXR group also had higher MDA, PCs, NT, IL-6 and Big ET-1 levels, higher iNOS and lower eNOS, Akt and AMPK activation compared to controls and the oleuropein-treated groups. Metabonomics depicted significant metabolite alterations in the DXR group suggesting perturbed energy metabolism and protein biosynthesis. The effectiveness of DXR in inhibiting cell proliferation is not compromised when oleuropein is present. We documented an imbalance between iNOS and eNOS expressions and a disturbed protein biosynthesis and metabolism in DXR-CM; these newly recognized pathways in DXR cardiotoxicity may help identifying novel therapeutic targets. Activation of AMPK and suppression of iNOS by oleuropein seem to prevent the structural, functional and histopathological cardiac effects of chronic DXR toxicity.

Development and validation of an ultra performance liquid chromatography–tandem mass spectrometry method for the quantification of daptomycin in human plasma

A rapid, simple and accurate analytical method based on ultra performance liquid chromatography (UPLC) combined with electrospray ionization (ESI) tandem mass spectrometry (MS/MS) on a hybrid q TOF instrument has been developed and fully validated for the quantification of daptomycin (DPT) in human plasma. The samples were analyzed after simple pretreatment involving protein precipitation, while chromatographic separation of DPT and the internal standard (reserpine) was achieved on an Acquity BEH C18 column (100 mm × 2.1 mm, 1.7 μm) using gradient elution with 0.1% aqueous formic acid (FA) and acetonitrile with 0.1% FA (with DPT eluting at 2.60 min). The method presented good fit (r>0.999) over the quantification range of 0.01-10 μg mL⁻¹ with the lower limit of quantitation (LLOQ) being 0.01 μg mL⁻¹ of human plasma for DPT. The intra- and inter-day precision, measured as % relative standard deviation, was less than 11% for DPT. The validation results showed that the developed method demonstrated adequate selectivity, sensitivity, precision and accuracy and therefore was successfully applied to the analysis of clinical samples following intravenous (iv) administration of 5.4 mg kg⁻¹ DPT to patients suffering from post-traumatic osteomyelitis induced by methicillin-resistant Staphylococcus aureus (MRSA). The developed methodology is the first report of an accurate mass tandem MS method for the analysis of this potent antibiotic in human plasma and can be used to further study pharmacokinetic, bioequivalence and even metabolic aspects related to this drug.

DETERMINATION OF VALPROIC ACID IN HUMAN PLASMA BY HPLC WITH FLUORESCENCE DETECTION

ABSTRACT A simple and sensitive high-performance liquid chromatographic (HPLC) method has been developed for the determination of valproic acid in human plasma. The method is based on pre-column derivatization using a new fluorescent reagent, N-(7-methoxy-4-methyl-2-oxo-2H-6-chromenyl)-2-bromoacetamide. The internal standard for the assay procedure was cyclohexanecarboxylic acid. The optimum monitoring conditions and the stability of the derivatives were investigated. The derivatization reaction proceeds in acetone in the presence of potassium carbonate and the crown ether, 18-crown-6 at 30C with a reaction time of 30 min. The resulting derivatives were separated under isocratic conditions (acetonitrile–water, 60 : 40, v/v) on a LiChrospher RP-18 column (125 × 4.0 mm, i.d. 5 μm) and were detected fluorimetrically at a wavelength of 435 nm with an excitation of 345 nm. All chromatographic experiments were carried out at a flow rate 1.0 mL/min at ambient temperature. The method was fully validated and applied for the determination of valproic acid over the concentration range of 6.0–150.0 mg/mL. The detection limit for valproic acid added to plasma sample was 0.13 μg/mL plasma (3 pg on column). The recovery of valproic acid averaged 100.4%. The accuracy of the assay ranged from −0.5% to 1.3%. The method was shown to be highly reproducible and it seems to be adequate for routine therapeutic drug monitoring.

Acidic hydrolysis of bromazepam studied by high performance liquid chromatography. Isolation and identification of its degradation products

A kinetic study on the acidic hydrolysis of bromazepam was carried out in 0.01 M hydrochloric acid solution at 25 and 95 degrees C. A reversed-phase HPLC method was developed and validated for the determination of bromazepam and its degradation products. Bromazepam degraded by a consecutive reaction with a reversible first step. Two degradation products were isolated and identified by infrared, 1H and 13C nuclear magnetic resonance and mass spectroscopy. Spectroscopic data indicated that N-(4-bromo-2-(2-pyridylcarbonyl)phenyl)-2-aminoacetamide was the intermediate degradation product of this acid hydrolysis, whereas 2-amino-5-bromophenyl-2-pyridylmethanone was the final one. Therefore, the mechanism of this acid-catalysed hydrolysis involved initial cleavage of the 4,5-azomethine bond, followed by slow breakage of the 1,2-amide bond. Statistical evaluation of the HPLC method revealed its good linearity and reproducibility. Detection limits were 3.8 x 10(-7) M for bromazepam, 6.25 x 10(-7) M for the intermediate and 8.16 x 10(-7) M for the benzophenone derivative.

Kinetic study on the degradation of prazepam in acidic aqueous solutions by high-performance liquid chromatography and fourth-order derivative ultraviolet spectrophotometry

A reversed-phase HPLC method was developed for the kinetic investigation of the acidic hydrolysis of prazepam which was carried out in hydrochloric acid solutions of 0.01, 0.1 and 1.0 M. In addition, a fourth-order derivative method for monitoring the parent compound itself was proposed and evaluated. One intermediate was observed by HPLC, which should be formed from breakage of the azomethine linkage. Further slow hydrolysis of the amide bond led to the benzophenone product that was isolated and identified. The mechanism of hydrolysis was biphasic, showing a consecutive reaction with a reversible step. Relative standard deviation was less than 2% for HPLC and less than 5% for the derivative method. Detection limits were 1.2 x 10(-7) M for the former method and 6.7 x 10(-7)M for the latter. Accelerated studies at higher temperatures were employed. Results of HPLC and fourth-order derivative methods were statistically the same.

Hydroxytyrosol ameliorates metabolic, cardiovascular and liver changes in a rat model of diet-induced metabolic syndrome: pharmacological and metabolism-based investigation.

Graphical abstract Figure. No Caption available. Abstract Metabolic syndrome is a clustering of interrelated risk factors for cardiovascular disease and diabetes. The Mediterranean diet has been proposed as an important dietary pattern to confer cardioprotection by attenuating risk factors of metabolic syndrome. Hydroxytyrosol (HT) is present in olive fruit and oil, which are basic constituents of the Mediterranean diet. In this study, we have shown that treatment with HT (20 mg/kg/d for 8 weeks) decreased adiposity, improved impaired glucose and insulin tolerance, improved endothelial function with lower systolic blood pressure, decreased left ventricular fibrosis and resultant diastolic stiffness and reduced markers of liver damage in a diet‐induced rat model of metabolic syndrome. These results were accompanied by reduced infiltration of monocytes/macrophages into the heart with reduced biomarkers of oxidative stress. Furthermore, in an HRMS‐based metabolism study of HT, we have identified 24 HT phase I and II metabolites, six of them being over‐produced in high‐starch, low‐fat diet fed rats treated with HT compared to obese rats on high‐carbohydrate, high‐fat diet. These results provide direct evidence for cardioprotective effects of hydroxytyrosol by attenuation of metabolic risk factors. The implications of altered metabolism of HT in high‐carbohydrate, high‐fat diet fed obese rats warrant further investigation.

A NEW FLUOROGENIC REAGENT FOR LABELLING CARBOXYLIC ACIDS IN HPLC

A new fluorescent reagent for carboxylic acids, N-(4-bromomethyl-7-hydroxy-2-oxo-2H-6-chromenyl) bromoacetamide (Br-MAMC) was synthesized. The fluorescence properties of Br-MAMC were monitored for a series of polar and non-polar solvents. Its applicability as a precolumn derivatization reagent in high-performance liquid chromatography was examined using undecylenic acid as a model compound. The derivatization reaction was performed in acetone in the presence of potassium carbonate and the crown ether, 18-crown-6 at 30°C for 20 min. The reaction solution was subjected to a high-performance liquid chromatographic procedure on a reversed-phase ODS C-18 column (150×4.6 mm i.d., 5 μm) with a mobile phase of acetonitrile and water (75 : 25, v/v) at a flow rate of 1.0 mL/min. The derivative of undecylenic acid was detected fluorometrically; excitation and emission wavelengths were set at 345 and 435 nm, respectively. The detection limit of the method was determined to be 12.5 pg (signal to noise ratio = 3) of the derivatized undecylenic acid on column. The derivatives of butyric, hexanoic, octanoic, decanoic, and dodecanoic acid with Br-MAMC were also well resolved with a mobile phase of acetonitrile and water (60 : 40, v/v).

Quantitation of Crocins and Picrocrocin in Saffron by HPLC: Application to Quality Control and Phytochemical Differentiation from Other Crocus Taxa

A chromatographic method was developed and fully validated for the determination of the major saffron constituents, i.e., picrocrocin and five major crocins. Dried samples (styles of Crocus sativus and other Crocus taxa) were extracted with MeOH : water (1 : 1, v/v), and chromatographic separation of the analytes was achieved by reversed-phase chromatography using a gradient elution. Full validation was performed using spiked samples with analytes, which were isolated, purified, and characterized by MS due to a lack of commercial standards. The method showed a good fit (r2 > 0.999) for all analytes with limit of quantitation values in the range of 1-15 µg/mL, and demonstrated adequate intra- and inter-precision (< 15 % RSD) and accuracy (< 7 % RE). The method was applied to the analysis of various commercial saffron samples and of indigenous Crocus taxa and allowed for the first time the absolute quantitation of several Crocus components.

Kinetic study on the acidic hydrolysis of lorazepam by a zero-crossing first-order derivative UV-spectrophotometric technique.

A zero-crossing first-order derivative UV-spectrophotometric technique for monitoring the main degradation product, 6-chloro-4-(2-chlorophenyl)-2-quinazoline carboxaldehyde, was developed to study the acidic hydrolysis of lorazepam in hydrochloric acid solutions of 0.1 M. Due to the complete overlap of the spectral bands of the parent drug and the hydrolysis product (the range between their spectral maxima was only 3 nm), the graphical methods of derivative spectrophotometry were not efficient. The relative standard deviation of the proposed technique was less than 2.4% and the detection limit was 6.6x10(-8) M. Accelerated studies at higher temperatures have been employed that enable rapid prediction of the long-term stability of this drug. Pseudo-first order reaction kinetics was observed. Kinetic parameters, k(obs) and t(1/2), were calculated, which were similar to those estimated by an HPLC method developed in our laboratory.

Kinetic Investigation on the Degradation of Lorazepam in Acidic Aqueous Solutions by High Performance Liquid Chromatography

Abstract A high performance liquid chromatographic method was developed for the kinetic investigation on the acidic hydrolysis of lorazepam in 0.01, 0.1, and 1.0 M hydrochloric acid solutions. In this study, the simultaneous determination of lorazepam and its main degradation product was performed on a reversed phase BDS C-8 column. The mobile phase consisted of a mixture of methanol: acetonitrile: buffer solution containing 0.005 M KH