Evangelos Gikas

Simultaneous determination of oleuropein and its metabolites in plasma by high-performance liquid chromatography.

A method based on high-performance liquid chromatography (HPLC) with a diode array detection system was developed and validated aiming at the simultaneous determination of oleuropein (OE) and its metabolites, hydroxytyrosol (HT) and tyrosol (T), in human plasma. These phenolic components are believed to play a vital role in the prevention of coronary artery disease and atherosclerosis. The proposed method includes a clean-up solid-phase extraction procedure (using a C(18) column) with high recovery efficiency (85-100%). The statistical evaluation of the method reveals good linearity, accuracy and reproducibility for all the compounds analyzed with RSD values less than 6.5%, while the detection limit is 50 ng/ml for both OE and T and 75 ng/ml for HT. This assay can be employed in bioavailability studies of olive oil phenolic compounds, thus assisting the evaluation of their pharmacological role.

Simultaneous quantification of oleuropein and its metabolites in rat plasma by liquid chromatography electrospray ionization tandem mass spectrometry

Oleuropein (OE) is the cardinal bioactive compound derived from Olea europaea and possesses numerous beneficial properties for human health. However, despite the plethora of analytical methods that have studied the biological fate of olive oil-derived bioactive compounds, no validated methodology has been published to date for the simultaneous determination of OE, along with all its major metabolites. In this study, a liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI MS/MS) method has been developed and validated for the quantification of OE, simultaneously with its main metabolites hydroxytyrosol, 2-(3,4-dihydroxyphenyl)acetic acid, 4-(2-hydroxyethyl)-2-methoxy-phenol or homovanillyl alcohol, 2-(4-hydroxy-3-methoxyphenyl)acetic acid or homovanillic acid, and elenolic acid in rat plasma matrix. Samples were analyzed by LC-ESI MS/MS prior to and after enzymatic treatment. A solid-phase extraction step with high mean recovery for all compounds was performed as sample pretreatment. Calibration curves were linear for all bioactive compounds over the range studied, while the method exhibited good accuracy, intra- and inter-day precision. The limit of detection was in the picogram range (per milliliterof plasma) for HT and OE and in the nanogram range (per milliliter of plasma) for the other analytes, and the method was simple and rapid. The developed methodology was successfully applied for the simultaneous quantification of OE and its aforementioned metabolites in rat plasma samples, thus demonstrating its suitability for pharmacokinetics, as well as bioavailability and metabolism studies.

Simultaneous quantification of daptomycin and rifampicin in plasma by ultra performance liquid chromatography: Application to a pharmacokinetic study.

A rapid and simple method based on ultra performance liquid chromatography (UPLC) with ultra violet detection has been developed for the determination of daptomycin (DPT) and rifampicin (RFM) in rabbit plasma using 4-nitrophenol as internal standard (IS). Sample preparation involved protein precipitation with an acetonitrile:methanol mixture and centrifugation. Chromatographic separation was achieved on an Acquity BEH C18 column (100mmx2.1mm, 1.7microm) using gradient elution with methanol and 0.1% aqueous TFA. The total analysis time was 4.5min with DPT and RFM eluting at 1.9 and 2.1min, respectively. The method was fully validated with a lower limit of quantitation (LLOQ) of 2microgmL(-1) for both DPT and RFM. The intra- and inter-day precision, measured as % relative standard deviation, were less than 12.1 for DPT and 10.7 for RFM, respectively. This validated method was successfully applied to a pharmacokinetic study involving intravenous administration of 14mgkg(-1) DPT and 30mgkg(-1) RFM to rabbits.

Daptomycin use in a neonate: serum level monitoring and outcome.

We present a case of successfully treated persistent bacteremia due to Gram-positive bacteria with daptomycin in a preterm neonate. Daptomycin was given at higher doses (6 mg/kg/dose) and shorter intervals (every 12 hours) than those recommended for adults with no adverse events. Peak and trough serum concentrations of daptomycin were 27.3 and 11.6 μg/mL [DOSAGE ERROR CORRECTED] at day 4 as well as 22.9 and 7.9 μg/mL [DOSAGE ERROR CORRECTED]at day 11 after initiation of treatment, respectively.

Determination of colistin A and colistin B in human plasma by UPLC–ESI high resolution tandem MS: Application to a pharmacokinetic study

The resistance of gram-negative bacteria to most available antibiotics and the lack of new antimicrobial agents have prompted the re-emergence of colistin (CS) as potent treatment against most gram-negative microorganisms. Optimal dosing with CS suffers from poor pharmacokinetic characterization mainly due to the analytical challenge of assaying CS in biological fluids and the limited information on quantitative analysis of CS in plasma using high resolution mass spectrometry (MS). Hence, 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 quadrupole time of flight (QTOF) instrument has been developed and fully validated for the quantification of CS in human plasma. After the pretreatment of plasma samples by solid phase extraction (SPE) and the addition of the internal standard (reserpine, RSP) the analytes were chromatographed on an Acquity BEH C8 column (100 mm × 2.1 mm, 1.7 μm) using gradient elution with 0.5% aqueous acetic acid (AcOH) and acetonitrile with 0.5% AcOH (with CSA and CSB eluting at 1.39 and 1.31 min, respectively). Accurate mass measurement correction was performed on line using the leukine-enkephaline standard. The method presented good fit (regression coefficient≥0.998) over the quantitation range of 0.2-300 and 0.03-4.5 μg mL(-1) with the lower limit of quantitation (LLOQ) being 0.02 and 0.03 μg mL(-1) for CSA and CSB in human plasma, respectively. The intra- and inter-day precision, measured as %relative standard deviation, was better than 10%, whereas the accuracy expressed as %relative error was also better than 10%. The short term, freeze-thaw (three cycles) and in process stability showed non-significant degradation of CS under these conditions. The validation results showed that the developed method demonstrated adequate selectivity and sensitivity. The method has been successfully applied to plasma samples from patients suffering from cystic fibrosis and treated with CS, and the pharmacokinetic profile has been calculated.

Synthesis and Pharmacological Evaluation of Novel Adenine-Hydrogen Sulfide Slow Release Hybrids Designed as Multitarget Cardioprotective Agents

This work deals with the design, synthesis, and evaluation of the cardioprotective properties of a number of novel hybrid compounds combining the adenine nucleus with a suitable H2S slow-releasing moiety, coupled via a stable ether bond. The H2S release rate of the hybrids and their ability to increase cGMP were estimated in vitro. The most promising derivatives 4 and 11, both containing 4-hydroxythiobenzamide moiety as H2S donor, were selected for further in vivo evaluation. Their ability to release H2S in vivo was recorded using a new fully validated UPLC-DAD method. Both compounds reduced significantly the infarct size when administered at the end of sustained ischemia. Mechanistic studies showed that they conferred enhanced cardioprotection compared to adenine or 4-hydroxythiobenzamide. They activate the PKG/PLN pathway in the ischemic myocardium, suggesting that the combination of both pharmacophores results in synergistic cardioprotective activity through the combination of both molecular pathways that trigger cardioprotection.

Preliminary pharmacokinetic study of the anticancer 6BIO in mice using an UHPLC-MS/MS approach

HighlightsFirst LC–MS/MS report for determination of 6BIO in mice plasma.Simple sample treatment method and rapid method for routine analysis.Full validation of the developed method using FDA, EMA and ICH procedures.Preliminary pharmacokinetics study of 6BIO in mice. Abstract Indirubins represent a group of natural and synthetic products with bio‐activities against numerous human cancer cell lines acting by inhibiting protein kinases. The natural sources of indirubins are plants of Isatis sp., Indigofera sp., and Polygonum sp., recombinant bacteria, mammalian urine and some marine mollusks. Specifically, the halogenated derivative 6‐bromo indirubin‐3′‐oxime (6BIO) possesses increased selectivity against GSK‐3. However, to our knowledge, no analytical method to determine 6BIO in biological fluids has been developed till now. Therefore, a rapid, sensitive and high throughput UHPLC‐MS/MS methods were developed and validated to evaluate the concentrations of 6BIO in mice plasma. Plasma samples were pre‐treated by protein precipation using cold mixture of methanol: acetonitrile (9:1, v/v) and separations were carried out on a Hypersil Gold C18 column (50 × 2.1 mm i.d.; 1.9 &mgr;m p.s.) using 0.1% acetic acid and methanol as mobile phase at a flow rate of 500 mL/min in a gradient mode. For quantitation, a hybrid LTQ‐Orbitrap MS equipped with an electro‐spray ionization source was used applying a selected reaction monitoring (SRM) option. The monitored transitions were m/z 354.0 → 324.0 for 6BIO and 297.1 → 282.1 for afromorsin (used as the internal standard) in the negative mode. Following the EMA, ICH and FDA guidelines for validation of analytical procedures, the assay method was fully validated in terms of selectivity, linearity, recovery, matrix effect, accuracy, precision, stability, and robustness. The validated methods were successfully applied to the pharmacokinetic studies of 6BIO following an oral administration to mice at the dose of 50 mg/kg. The results indicated that 6BIO possesses a Tmax of 30 min, a half‐life of 1 h, and low plasma bioavailability.

Mass spectrometry and the Mediterranean

paving this way. The idea for this Special Issue arose during the Mediterranean ea Region Countries Mass Spectrometry Workshop (MEDMS III) hat took place in Athens, Greece from June 28 to July 2, 2015. cientists from different disciplines across the Mediterranean and eyond, came together to discuss advances in mass spectrometry nd relevant applications. This Special Issue provides an overview f what was presented at the Workshop but also brings together ass spectrometrists and scientists from different fields who use nd develop mass spectrometry methods. The selection of the research articles and reviews presented ere, offers a taste of the plethora of contemporary mass specrometry applications around the world. Mass spectrometry-based pproaches including metabolomics, proteomics, imaging as well s novel methodologies have been implemented here to help