Fotini N. Bazoti

Study of the interaction between the amyloid beta peptide (1–40) and antioxidant compounds by nuclear magnetic resonance spectroscopy

Amyloid beta peptide (Abeta) aggregation leads to the senile plaque formation, a process that is strongly influenced by oxidative stress and is considered as the molecular basis of various neurodegenerative diseases, such as Alzheimers disease (AD). Endogenous antioxidants or dietary derived compounds may down-regulate this process. In this study, the interaction of two antioxidants, oleuropein (OE) and melatonin (M), with Abeta is monitored through nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry. The concerted application of these two analytical techniques provides new experimental evidence and residue-specific insights into the interacting Abeta peptide amino acids that are implicated in this process. Both antioxidant compounds interact in a similar way with the peptide and cause chemical shift variations. The most pronounced resonance changes have been observed for the 1H-15N signals of N-terminal region and Leu17-Phe20 residues, as monitored by NMR titration studies.

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.

Localization of the noncovalent binding site between amyloid-β-peptide and oleuropein using electrospray ionization FT-ICR mass spectrometry

Abnormal accumulation and aggregation of amyloid-β-peptide (Aβ) eventually lead to the formation and cerebral deposition of amyloid plaques, the major pathological hallmark in Alzheimer’s disease (AD). Oleuropein (OE), an Olea europaea L. derived polyphenol, exhibits a broad range of pharmacological properties, such as antioxidant, anti-inflammatory, and antiatherogenic, which could serve as combative mechanisms against several reported pathways involved in the pathophysiology of AD. The reported noncovalent interaction between Aβ and OE could imply a potential antiamyloidogenic role of the latter on the former via stabilization of its structure and prevention of the adaptation of a toxic β-sheet conformation. The established β-sheet conformation of the Aβ hydrophobic carboxy-terminal region and the dependence of its toxicity and aggregational propensity on its secondary structure make the determination of the binding site between Aβ and OE highly important for assessing the role of the interaction. In this study, two different proteolytic digestion protocols, in conjunction with high-sensitivity electrospray ionization mass spectrometric analysis of the resulting peptide fragments, were used to determine the noncovalent binding site of OE on Aβ and revealed the critical regions for the interaction.

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.

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.

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.

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.

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.

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.

Post-Translationally Modified Proteins: Glycosylation and Disulfide Bond Formation

Even though most medicines have historically been small molecules, many newly approved drugs over the last two decades have been derived from proteins. For the past few years, protein therapeutics have been enjoying the fastest growth within the global pharmaceutical industry.