Johan Viaene

Improved sensitivity of the nano ultra-high performance liquid chromatography-tandem mass spectrometric analysis of low-concentrated neuropeptides by reducing aspecific adsorption and optimizing the injection solvent

Obtaining maximal sensitivity of nano UHPLC-MS/MS methods is primordial to quantify picomolar concentrations of neuropeptides in microdialysis samples. Since aspecific adsorption of peptides to Eppendorf tubes, pipette tips and UHPLC vials is detrimental for method sensitivity, a strategy is presented to reduce adsorption of these peptides during standard preparation. Within this respect, all procedural steps from dissolution of the lyophilized powder until the injection of the sample onto the system are investigated. Two peptides of the neuromedin family, i.e. neuromedin B and neuromedin N, and a neuromedin N-related neuropeptide, neurotensin, are evaluated. The first part of this study outlines a number of parameters which are known to affect peptide solubility. The main focus of the second part involves the optimization of the sample composition in the UHPLC vial by using design of experiments. Contradictory findings are observed concerning the influence of acetonitrile, salts and matrix components. They are found important for injection of the peptides into the system, but crucially need to be excluded from the dilution solvent. Furthermore, the type of surface material, temperature and the pipetting protocol considerably affect the adsorption phenomenon. Statistical analysis on the results of the central composite design reveals that the highest peptide responses are obtained with the injection solvent consisting of 13.1% V/V ACN and 4.4% V/V FA. This aspect of the optimization strategy can be identified as the main contributor to the gain in method sensitivity. Since the reduction of peptide adsorption and the optimization of the injection solvent resulted in a clear and quantifiable signal of the three peptides, optimization of both issues should be considered in the early stage of method development, in particular when the analysis of low-concentration peptide solutions is envisaged.

Seasonal, gender and regional variations in total phenolic, flavonoid, and condensed tannins contents and in antioxidant properties from Pistacia atlantica ssp. leaves

Abstract Context: The widespread use of Pistacia atlantica Desf. ssp. (Anacardiaceae) in traditional medicine can be partly attributed to the content of its secondary metabolites, in particular, the phenolic compounds. Objective: The effects of harvest period, growing region and gender on the phenolic compounds, flavonoids and condensed tannins contents were studied, as well as on the antioxidant activities of P. atlantica leaves in order to provide a scientific basis for optimal collection. Materials and methods: Leaves were collected monthly from April to October 2010 in two Algerian sites. The powdered leaves were used for preparing the ethyl acetate extract. Contents of total phenolics (TPC), flavonoids (FC) and condensed tannins (CTC) were determined spectrophotometrically. Antioxidant activity was evaluated through radical scavenging activity (RSA) of 2,2-diphenyl-1-picrylhydrazyl (250 μM) and the reducing power capacity (RPC) determination by K3Fe(CN)6 (1%). Results: The TPC was found to vary from 79 ± 13 to 259 ± 8 mg gallic acid equivalents/g of dry weight (DW) during the study period. The RSA and RPC varied between 262 ± 18 and 675 ± 21 mg Ascorbic Acid Equivalent (AAE)/g DW, and from 259 ± 16 to 983 ± 20 mg AAE/g DW, respectively. A seasonal pattern was observed consisting of a decrease in TPC content and RPC from spring to autumn. The FC, CTC and RSA did not show a seasonal pattern. Discussion and conclusion: Our findings showed that secondary metabolite content and antioxidant activities of P. atlantica leaves were more influenced by harvest time and growing region than by gender.

An improved microbore UHPLC method with electrochemical detection for the simultaneous determination of low monoamine levels in in vivo brain microdialysis samples.

The simultaneous determination of the monoamines dopamine (DA), noradrenaline (NA) and serotonin (5-HT) in in vivo microdialysis samples remains challenging because of the low extracellular neurotransmitter levels in different brain regions, specific sample characteristics, and the quest for high temporal resolution and a multi-target strategy in neuropharmacological research. A fast and sensitive microbore (1.0mm i.d. column) UHPLC method coupled to electrochemical detection (ECD) is developed by means of design of experiments with the emphasis on sufficient retention of NA within an acceptable total analysis time. Indeed, NA is the earliest eluting compound and often interferes with the broad solvent front originating from the sample matrix. The sensitive UHPLC-ECD assay (LLOQ of 100pM for NA and 150pM for DA and 5-HT) with an analysis time of 8min for standard solutions and 20min for in vivo microdialysis samples originating from rat hippocampus, prefrontal cortex and striatum, is validated applying accuracy profiles. The combination of in vivo microdialysis and microbore UHPLC-ECD has shown to be particularly suitable for future contributions to neuropharmacological research on the monoaminergic system.

The use of chemometrics to study multifunctional indole alkaloids from Psychotria nemorosa (Palicourea comb. nov.). Part I: Extraction and fractionation optimization based on metabolic profiling.

Extraction methods evaluation to access plants metabolome is usually performed visually, lacking a truthful method of data handling. In the present study the major aim was developing reliable time- and solvent-saving extraction and fractionation methods to access alkaloid profiling of Psychotria nemorosa leaves. Ultrasound assisted extraction was selected as extraction method. Determined from a Fractional Factorial Design (FFD) approach, yield, sum of peak areas, and peak numbers were rather meaningless responses. However, Euclidean distance calculations between the UPLC-DAD metabolic profiles and the blank injection evidenced the extracts are highly diverse. Coupled with the calculation and plotting of effects per time point, it was possible to indicate thermolabile peaks. After screening, time and temperature were selected for optimization, while plant:solvent ratio was set at 1:50 (m/v), number of extractions at one and particle size at ≤180μm. From Central Composite Design (CCD) results modeling heights of important peaks, previously indicated by the FFD metabolic profile analysis, time was set at 65min and temperature at 45°C, thus avoiding degradation. For the fractionation step, a solid phase extraction method was optimized by a Box-Behnken Design (BBD) approach using the sum of peak areas as response. Sample concentration was consequently set at 150mg/mL, % acetonitrile in dichloromethane at 40% as eluting solvent, and eluting volume at 30mL. Summarized, the Euclidean distance and the metabolite profiles provided significant responses for accessing P. nemorosa alkaloids, allowing developing reliable extraction and fractionation methods, avoiding degradation and decreasing the required time and solvent volume.

The use of chemometrics to study multifunctional indole alkaloids from Psychotria nemorosa (Palicourea comb. nov.). Part II: Indication of peaks related to the inhibition of butyrylcholinesterase and monoamine oxidase-A.

Psychotria nemorosa is chemically characterized by indole alkaloids and displays significant inhibitory activity on butyrylcholinesterase (BChE) and monoamine oxidase-A (MAO-A), both enzymes related to neurodegenerative disorders. In the present study, 43 samples of P. nemorosa leaves were extracted and fractionated in accordance to previously optimized methods (see Part I). These fractions were analyzed by means of UPLC-DAD and assayed for their BChE and MAO-A inhibitory potencies. The chromatographic fingerprint data was first aligned using correlation optimized warping and Principal Component Analysis to explore the data structure was performed. Multivariate calibration techniques, namely Partial Least Squares (PLS1), PLS2 and Orthogonal Projections to Latent Structure (O-PLS1), were evaluated for modelling the activities as a function of the fingerprints. Since the best results were obtained with O-PLS1 model (RMSECV=9.3 and 3.3 for BChE and MAO-A, respectively), the regression coefficients of the model were analyzed and plotted relative to the original fingerprints. Four peaks were indicated as multifunctional compounds, with the capacity to impair both BChE and MAO-A activities. In order to confirm these results, a semi-prep HPLC technique was used and a fraction containing the four peaks was purified and evaluated in vitro. It was observed that the fraction exhibited an IC50 of 2.12μgmL(-1) for BChE and 1.07μgmL(-1) for MAO-A. These results reinforce the prediction obtained by O-PLS1 modelling.

Antiplasmodial Activity, Cytotoxicity and Structure-Activity Relationship Study of Cyclopeptide Alkaloids

Cyclopeptide alkaloids are polyamidic, macrocyclic compounds, containing a 13-, 14-, or 15-membered ring. The ring system consists of a hydroxystyrylamine moiety, an amino acid, and a β-hydroxy amino acid; attached to the ring is a side chain, comprised of one or two more amino acid moieties. In vitro antiplasmodial activity was shown before for several compounds belonging to this class, and in this paper the antiplasmodial and cytotoxic activities of ten more cyclopeptide alkaloids are reported. Combining these results and the IC50 values that were reported by our group previously, a library consisting of 19 cyclopeptide alkaloids was created. A qualitative SAR (structure-activity relationship) study indicated that a 13-membered macrocyclic ring is preferable over a 14-membered one. Furthermore, the presence of a β-hydroxy proline moiety could correlate with higher antiplasmodial activity, and methoxylation (or, to a lesser extent, hydroxylation) of the styrylamine moiety could be important for displaying antiplasmodial activity. In addition, QSAR (quantitative structure-activity relationship) models were developed, using PLS (partial least squares regression) and MLR (multiple linear regression). On the one hand, these models allow for the indication of the most important descriptors (molecular properties) responsible for the antiplasmodial activity. Additionally, predictions made for interesting structures did not contradict the expectations raised in the qualitative SAR study.

Batch-to-batch N-glycosylation study of infliximab, trastuzumab and bevacizumab, and stability study of bevacizumab

Objectives Infliximab, trastuzumab and bevacizumab are among the most frequently prescribed therapeutic proteins, and like most other therapeutic proteins, are glycosylated. As differences in glycosylation may significantly change the safety and efficacy of therapeutic glycoproteins, it is extremely important to control N-glycosylation consistency. In the first part of this study, the batch-to-batch consistency of the N-glycosylation of infliximab, trastuzumab and bevacizumab was analysed. In the second part, the consistency of the N-glycosylation of bevacizumab stored in polycarbonate syringes (for off-label drug use) for 3 months was examined. Methods N-glycans were (i) enzymatically released using peptide-N-glycosidase F, (ii) reduced, and (iii) analysed using hydrophilic interaction liquid chromatography coupled with high-resolution mass spectrometry. Mass spectrometry data were interpreted using principal component analysis combined with two-way analysis of variance and Tukey post hoc tests. The biological activity of infliximab and trastuzumab was examined using enzyme-linked immunosorbent assays. Results The results of both studies make important contributions to the field of hospital pharmacy. All batches of the studied therapeutic glycoproteins (infliximab, trastuzumab and bevacizumab) varied considerably (especially in galactosylation), while the N-glycosylation of bevacizumab remained unchanged during 3-month storage. Conclusions Threshold values for batch-to-batch N-glycosylation variations should be established and batch-to-batch glycosylation consistency should be regularly tested. In our study, samples with significantly different N-glycosylation profiles showed no significant variations in biological activity, suggesting that the differences are probably not therapeutically significant.

Comparison of a triple-quadrupole and a quadrupole time-of-flight mass analyzer to quantify 16 opioids in human plasma

The aim of this work is to study whether a quadrupole time-of-flight (QToF) mass analyzer, coupled to an ultra high performance liquid chromatography (UHPLC) system, can be a valuable alternative for a triple-quadrupole (QqQ) mass analyzer, for quantitative toxicological purposes. The case study considered was the quantification of 16 opioids (6-monoacetylmorphine, buprenorphine, codeine, dihydrocodeine, ethylmorphine, fentanyl, hydrocodone, hydromorphone, morphine, norbuprenorphine, norcodeine, norfentanyl, oxycodone, oxymorphone, pholcodine and tilidine) in human plasma. Both methods were validated in parallel in terms of selectivity, matrix effects, extraction recovery, carry-over, bias, precision and sensitivity. Accuracy-profile methodology was used to determine the optimal calibration model, and to estimate bias, repeatability, intermediate precision and total error. Selectivity was demonstrated for all opioids and deuterated analogues, except for codeine-d3 on the UHPLC-QTOF. For most compounds, extraction recoveries were in the range 60 to 80% on both systems, except for the synthetic analogues, buprenorphine, fentanyl and tilidine, where large variability is observed. Carry-over was negligible on both systems. For different opioids, the optimal calibration model was different between the systems. The accuracy profiles of the majority of the opioids indicated that, over the entire tested concentration range, for more than 5% of the future measurements, total errors are expected to exceed the a priori defined 15% acceptance limit. For some exceptions, however, the measurements even suffer from total errors above 30%, which can be attributed to the solid phase extraction procedure that was applied as sample pretreatment technique. Sensitivity was generally tenfold better on the LC-QToF system, probably due to the difference in ion choice for quantification between both systems. In conclusion, the best performing system seemed to depend on the compound, on the parameter and even on the concentration. Accuracy profiles clearly provided valuable information complementary to that obtained in classical validation tests, and therefore preferably are taken into account when deciding on a methods performance.

Determination of optimal extraction conditions for phenolic compounds from Pistacia atlantica leaves using the response surface methodology

The response surface methodology in combination with a Box–Behnken experimental design was performed to optimize the extraction conditions, resulting in a maximum yield of the total phenolic content (TPC) from the leaves of Pistacia atlantica. The ranges of the examined independent variables (factors), i.e. extraction time (24–72 hours), liquid-to-solid ratio (30 : 1–50 : 1 ml solvent per g dry leaf) and extraction temperature (35–55 °C), were identified by preliminary experiments. Quadratic polynomial regression models were fitted through the experimental results. They showed acceptable coefficients of multiple determinations. From the models, the liquid-to-solid ratio was found to have the most influence on the extraction of TPC. The optimum extraction conditions were found to be 72 h extraction time and 50 : 1 ml g−1 liquid-to-solid ratio. For the extraction temperature, rather high values (about 50 °C) were found to be the best. Using the optimized conditions, the TPC varied from 256 to 306 mg gallic acid equivalents per g dry leaf in different sample types.

Potentially antidiabetic and antihypertensive compounds identified from Pistacia atlantica leaf extracts by LC fingerprinting

Graphical abstract Figure. No caption available. HighlightsIn vitro evaluation of antidiabetic and antihypertensive activities of Pistacia atlantica.Effect of the growing region and gender on the &agr;‐amylase, &agr;‐glucosidase and ACE‐I inhibitory activities of P. atlantica leave extracts.Potentially antidiabetic and antihypertensive compounds indicated from fingerprints using PLS.Identify contributions of the individual phenolic compounds in P. atlantica leaves to the &agr;‐amylase, &agr;‐glucosidase and ACE‐I inhibitory activities. ABSTRACT The objective of this paper is to evaluate the variations in the ability of Pistacia atlantica leaves to inhibit enzymes linked to type 2 diabetes (&agr;‐amylase and &agr;‐glucosidase) and to hypertension (angiotensin converting enzyme‐I (ACE‐I)), depending on harvesting month, gender and growing region, as well as to identify the peaks in chromatographic fingerprints that potentially correspond to components with enzymatic inhibitory activities. In this study, LC fingerprints of P. atlantica leave extracts were developed. Peaks which were probably responsible for the anti‐amylase, anti‐glucosidase and anti‐ACE‐I activities were assigned. For the latter purpose, the relevant information was extracted, linking the chromatographic fingerprints with the activities using a linear multivariate calibration technique, i.e., Partial Least Squares (PLS) regression. Prior to the construction of the models, the fingerprints are aligned using a warping method, called Correlation Optimized Warping (COW). Besides COW, different other data pretreatment methods were applied and compared. Our findings revealed that the influence of the growing region and gender on the &agr;‐amylase, &agr;‐glucosidase and ACE‐I inhibitory activities of P. atlantica leaves was less important than the harvest time. Thirteen common peaks were selected from the chromatograms and used as a dataset to model the biological activities. The peaks potentially responsible for the biological activity of the samples were indicated by studying the regression coefficients of the models. Seven peaks corresponding to possibly anti‐amylase compounds were found, while 6 peaks were considered important for inhibiting the &agr;‐glucosidase activity. Furthermore, the regression coefficients of the hypertension model indicated eight peaks as being important for inhibiting the ACE‐I activity. The contributions of individual phenolic compounds of P. atlantica leaves to the &agr;‐amylase, &agr;‐glucosidase and ACE‐I inhibitory activities were also identified. This investigation showed that the extract of P. atlantica leaves provides a rational basis for the isolation and development of antidiabetic and antihypertensive agents.