Evagelos Gikas

Development and validation of a UPLC‐UV method for the determination of daptomycin in rabbit plasma

Daptomycin (DPT) is a lipopeptide antibiotic with potent bactericidal activity in vitro against Gram-positive bacteria, which has attracted the attention of the scientific community due to its unique mechanism of action and due to the immediate need for new antibiotics in the era of multidrug resistance. In order to assess its pharmacokinetics in rabbits a new analytical method has been developed and validated using ultra performance liquid chromatography in conjugation with ultraviolet detection for the quantitation of the antibiotic in rabbit plasma, using the internal standard methodology. The separation was achieved employing a C(18) column with gradient elution using 0.1% aq. trifluoroacetic acid and methanol. The total analysis time was 2.5 min. The sample pretreatment employed protein precipitation with acetonitrile-methanol mixture and centrifugation. The method was validated in terms of linearity, precision, accuracy, sensitivity, robustness, short-term and freeze-thaw stability and was applied to the quantification of DPT in plasma samples obtained from rabbits treated with 25 mg kg(-1) DPT.

Quantitation of oleuropein and related metabolites in decoctions of Olea europaea leaves from ten Greek cultivated varieties by HPLC with diode array detection (HPLC-DAD)

Abstract An extraction procedure and chromatographic methodology for the simultaneous quantitation of four major constituents in the boiling water extracts (decoctions) of Olea europaea leaves has been developed. The four studied constituents were oleuropein, elenolic acid, hydroxytyrosol, and tyrosol. The quantitation was performed using HPLC‐DAD, whereas qualitative data were acquired using LC‐MS. The developed methodology was applied in the study of ten Olea europaea varieties commonly cultivated in Greece. The chromatographic analysis revealed important differences among the varieties. The decoction of variety gaidouroelia was identified as the best source of oleuropein, but it was completely lacking of elenolic acid. The decoction of variety koronaiiki was the best source of hydroxytyrosol, whereas the variety mastoides was the best source of tyrosol and elenolic acid. In addition, the methanol and acetone extracts of one of the studied varieties (koranaiiki) were investigated, in order to compare the concentration of oleuropein in the extracts and the decoction. Interestingly, only a very low percent of the total oleuropein is present in the traditionally prepared decoction, while elenolic acid, which is a minor constituent of the extracts, was found to be one of the major constituents of the decoction.

Determination of Isoflavones in the Aerial Part of Red Clover by HPLC-Diode Array Detection

Abstract Red clover (Trifolium pratense L.) is a biennial plant which has been used as feeding material for ruminants, but also as a health food for humans due to its estrogenic, antispasmodic, and expectorant properties. Red clover contains a large number of flavones, the four most important being daidzein and genistein and their precursors formononetin and biochanin A, respectively. The purpose of the current project was to quantify these four isoflavones in the aerial part of red clover in samples collected at the flowering, vegetative, and fruiting stages, and accordingly, to determine which of the three growth stages of red clover, contains the highest isoflavone amount. Thus a method based on reversed‐phase high performance liquid chromatography (HPLC) using diode array detection has been developed and validated. The linearity, precision, accuracy, and sensitivity of the method allow for the fast and reliable determination of the aforementioned substances from the aerial part of red clover. Analysis of the plant at different growth stages showed that the highest amount of isoflavones was detected during the vegetative stage.

Use of liquid chromatography/electrospray ionization tandem mass spectrometry to study the degradation pathways of terbuthylazine (TER) by Typha latifolia in constructed wetlands: identification of a new TER metabolite.

S-Triazines are used worldwide as herbicides for agricultural and non-agricultural purposes. Although terbuthylazine (TER) is the second most frequently used S-triazine, there is limited information on its metabolism. For this reason, an analytical method based on liquid chromatography/electrospray ionization tandem mass spectrometry (LC-ESI MS/MS) has been developed aiming at the identification of TER and its five major metabolites (desisopropyl-hydroxy-atrazine, desethyl-hydroxy-terbuthylazine, desisopropyl-atrazine, hydroxy-terbuthylazine and desethyl-terbuthylazine) in constructed wetland water samples. The separation of TER and its major metabolites was performed by reversed-phase high-performance liquid chromatography (HPLC) on a C(8) column using a gradient elution of aqueous acetic acid 1% (solvent A) and acetonitrile (solvent B), followed by MS/MS analysis on a triple quadrupole mass spectrometer. The data-depended analysis (DDA) scan approach has been employed and the main degradation pathways of both hydroxyl and chloro (dealkylated and alkylated) metabolites are elucidated through the tandem mass spectral (MS/MS) interpretation of triazine fragments under CID conditions. In addition, another major metabolite of TER, namely N2-tert-butyl-N4-ethyl-6-methoxy-1,3,5-triazine-2,4-diamine, has been identified. This methodology can be further employed in biodegradation studies of TER, thus assisting the assessment of its environmental impact.

Determination of herbicide terbuthylazine and its major hydroxy and dealkylated metabolites in constructed wetland sediments using solid phase extraction and high performance liquid chromatography-diode array detection

This paper describes the development of a new analytical method for the analysis of the herbicide TER and its degradation products in sediment samples. This method, based on high-performance liquid chromatography with diode-array detection, was validated for the simultaneous determination of TER and its major metabolites, desethylterbuthylazine, desisopropyatrazine, hydroxyterbuthylazine, desethylhydroxyatrazine and desethylhydroxyterbuthylazine. This method includes a cleanup and a solid-phase extraction step, using ultra-pure water and MCX cartridges respectively, with an overall recovery efficiency ranging from 89.3 to 97.9%. The statistical evaluation demonstrates good linearity with correlation coefficients >0.999 and adequate accuracy and precision for all analytes, with% Er and RSD values up to 10.5% and 8.3% respectively. The limit of detection for all substances was found to be 3.3 ng g−1. This method can be employed in remediation studies of TER and its major metabolites in sediments of constructed wetlands leading to useful results for the degradation and dispersion of TER in the vertical profile of wetland sediment substrates.

Quantitation of the Flavonols Quercetin and Kaempferol in the Leaves of Trigonella foenum-graecum by High-Performance Liquid Chromatography – Diode Array Detection

Trigonella foenum-graecum is known for the strong hypoglycemic effects of the seed extract and for its significant allelopathic effects. The leaf extract however has not been studied thoroughly up-to-date and no data are available regarding the quantitative content of the plants leaves. The purpose of this study was to quantify the flavonoid content and to assess the flavonoid seasonal variation in the leaves of the plant during the three developmental stages, vegetative, flowering, and fruiting. An HPLC method has been developed and validated employing a reversed phase C18 column and Diode Array Detection. Sample pre-treatment was performed by methanol:water (80:20 v:v) extraction and glucoside hydrolysis with HCl. The method allowed the reliable determination of the aforementioned substances in the Trigonellas leaves revealing the highest amount of flavonoids during the flowering stage.

A New Coumarin Based Fluorogenic Derivatization Reagent for Labelling Free Carboxyl Groups (Br‐MOZC)

Abstract A new coumarin based bromomethyl fluorescent probe 4‐bromomethyl coumarin[a,b‐c,d]‐oxazine‐3‐one was synthesized as a fluorescent carboxylic acid derivatizing agent. The synthetic route is described, as well as its application for the determination of the model compound n‐butyric acid. The derivatization reaction was conducted in acetone using 18‐crown‐6 as catalyst. The derivatization solution was separated by high performance liquid chromatography on a reversed‐phase ODS C‐18 column (150 × 4.6 mm i.d., 5 µm) with a mobile phase of acetonitrile and water (70:30, v/v) at a flow rate of 1.0 mL/min. The excitation and emission wavelengths were set to 435 and 345 nm, respectively. The detection limit was determined to be below 10 pg of the derivatized acid on the column. The probe has been used for the simultaneous determination of six aliphatic fatty acids.

Simultaneous Determination of Herbicide Terbuthylazine and Its Major Hydroxy and Dealkylated Metabolites in Typha latifolia L. Wetland Plant Using SPE and HPLC-DAD

Abstract The worldwide application of s-Triazines as herbicides for agricultural and nonagricultural purposes, results in a significant environmental pollution. It is necessary to develop sustainable and environmental friendly techniques in order to remediate surface water from the aforementioned organic substances. Phytoremediation with the wetland plant Typha latifolia L. is a technique that could potentially aid the restoration of polluted surface water. However, there is no analytical method for the determination of terbuthylazine (TER) and its metabolites in Typha latifolia L. and the assessment of the effectiveness of such a procedure. For this reason a method based on high performance liquid chromatography with diode array detection was developed and validated for the simultaneous determination of terbuthylazine (TER) and its major degradation products, desethyl-terbuthylazine, desisopropy-atrazine, hydroxy-terbuthylazine, desethyl-hydroxy-atrazine, and desethyl-hydroxy-terbuthylazine. This method includes both a cleanup and a solid phase extraction step (using Florisil and MCX cartridges, respectively) with adequate overall recovery efficiency (71–96%). The statistical evaluation of the method reveals good linearity, accuracy, and precision for the compounds determined, with RSD values not exceeding 10.5%, while the limit of detection for all analytes was found to be 17 ng g−1. This method can be employed in phytoremediation studies of TER by Typha latifolia L. in constructed wetlands.

Transport and dissipation study of the herbicide terbuthylazine and its major metabolites in wetland sediment substrates planted with Typha latifolia L

Abstract It is widely recognized that the organic micropollutants, coming from the intensive agricultural use of land, are the major thread against surface and ground water. However, they are an environmental engineering challenge in order to encounter the pollution by the use of constructed wetlands. The aim of this work is the study of the potential transport and dissipation of the herbicide terbuthylazine (TER) and its major hydroxy and dealkylated metabolites at the vertical profile of a constructed wetland sediment substrate, planted with Typha latifolia L., in order to determine the processes and study the possible remediation mechanisms for wetland ecosystems contaminated by the aforementioned substances. The results show that the dissipation of TER exhibits a gradient behavior through depth of the sediment substrate of wetlands and its major degradation products follow the effect of biotic and abiotic mechanisms of degradation in the bioreactor substrate. Moreover, the greater recovery of the herb...

MOZPhCSE, a New Coumarin Based Fluorescent Derivatization Reagent

Abstract The synthesis of a new fluorescent probe 2‐(3,7‐dioxo‐2‐phenyl‐3,7‐dihydrochromeno[7,6‐b][1,4]oxazin‐9‐yl)acetic acid succinimidyl ester was described. The reagent reacts easily with primary amines and forms highly fluorescent derivatives that can be separated and detected with an HPLC‐fluorometric detector. The new reagent was evaluated using pentylamine as a model compound. The separation was achieved on a C18 column using a mixture of acetonitrile:ammonium acetate buffer as the mobile phase. Statistical evaluation of the methodology described reveals good linearity, precision and accuracy, and high sensitivity. Application of the methodology can be used for the separation of four aliphatic amines (pentylamine, hexylamine, octylamine, and decylamine).