CBD oils on the Belgian market: A validated MRM GC-MS/MS method for routine quality control using QuEChERS sample clean up.

Quality control of CBD oils on the Belgium market showed that the CBD content not always corresponds to the label claim. There is a pressing need to develop new analytical methods specifically developed to the assay of such oily samples. Analytical issues are, however, encountered for routine analyses due to the matrix complexity, high cost of cannabinoid standards and low Δ9-THC concentrations. An oily matrix could cause technical damages to analytical instruments and reduce the lifetime of the chromatographic columns. This paper proposes a procedure combining a sample cleanup by QuEChERS, removing the oily matrix, followed by a validated MRM GC-MS/MS method for the routine analysis of CBD oil samples. Eighteen CBD samples were selected on the Belgium market for analysis. This method allows the quantification of CBD, the legality check for the Δ9-THC content by a CBN standard and the screening of seven other cannabinoids namely CBN, CBDV, CBT, CBC, Δ8-THC, THCV and CBG. The method was validated at three concentration levels (0.5-1-2% (w/v)) for CBD and (0.05-0.1-0.2% (w/v)) for CBN. The detection limits for CBT, CBD, CBC, Δ8-THC, CBN and for the other cannabinoids of interest, were 10 and 14 ng/mL respectively. The accuracy profile values for CBD and CBN showed that the ß-expectation tolerance intervals did not exceed the acceptance limits of ± 20%, meaning that 90% of future measurements will be included within this error range.

Discrimination of legal and illegal Cannabis spp. according to European legislation using near infrared spectroscopy and chemometrics.

Aerial parts containing cannabidiol can be purchased in a legal way but cannabis used as recreational drug is illegal in most European countries. Δ9-tetrahydrocannabinol is one of the main cannabinoids responsible for the psychotropic effect. European Union countries and Switzerland authorize a concentration of THC of 0.2 % and 1.0 % w/w, respectively, for smoking products and industrial hemp. Public health inspectors and law enforcement officers need to check the legality of samples. Therefore there is a need for innovative approaches, allowing quality control of these products in an easy way and preferably on site. In many countries, cultivation of industrial hemp is permitted if the THC content does not exceed 0.2 % w/w. A portable equipment could be a useful measuring tool for farmers to check for the THC content at regular time. In this work, 189 samples were analysed with a benchtop and a handheld NIR device in order to create two classification methods according to European and Swiss laws. All samples were also analysed by GC-FID to determine their THC concentration. Supervised analysis was applied in order to establish the best model. For the first classification, the accuracy was 91% for the test set with the benchtop data and 93 % for the test set with the handheld data. For the second classification, the accuracies were respectively 91 % and 95 %. The obtained models, hyphenating spectroscopic techniques and chemometrics, enable to discriminate legal and illegal cannabis samples according to European and Swiss laws.

Isoniazid Bactericidal Activity Involves Electron Transport Chain Perturbation.

Accumulating evidence suggests that the bactericidal activity of some antibiotics may not be directly initiated by target inhibition. The activity of isoniazid (INH), a key first-line bactericidal antituberculosis drug currently known to inhibit mycolic acid synthesis, becomes extremely poor under stress conditions, such as hypoxia and starvation. This suggests that the target inhibition may not fully explain the bactericidal activity of the drug. Here, we report that INH rapidly increased Mycobacterium bovis BCG cellular ATP levels and enhanced oxygen consumption. The INH-triggered ATP increase and bactericidal activity were strongly compromised by Q203 and bedaquiline, which inhibit mycobacterial cytochrome bc1 and FoF1 ATP synthase, respectively. Moreover, the antioxidant N-acetylcysteine (NAC) but not 4-hydroxy-2,2,6,6-tetramethylpiperidin-1-oxyl (TEMPOL) abrogated the INH-triggered ATP increase and killing. These results reveal a link between the energetic (ATP) perturbation and INH's killing. Furthermore, the INH-induced energetic perturbation and killing were also abrogated by chemical inhibition of NADH dehydrogenases (NDHs) and succinate dehydrogenases (SDHs), linking INH's bactericidal activity further to the electron transport chain (ETC) perturbation. This notion was also supported by the observation that INH dissipated mycobacterial membrane potential. Importantly, inhibition of cytochrome bd oxidase significantly reduced cell recovery during INH challenge in a culture settling model, suggesting that the respiratory reprogramming to the cytochrome bd oxidase contributes to the escape of INH killing. This study implicates mycobacterial ETC perturbation through NDHs, SDHs, cytochrome bc1, and FoF1 ATP synthase in INH's bactericidal activity and pinpoints the participation of the cytochrome bd oxidase in protection against this drug under stress conditions.

Determination of Three Main Chlorogenic Acids in Water Extracts of Coffee Leaves by Liquid Chromatography Coupled to an Electrochemical Detector.

Coffee is a beverage widely consumed in the world. The coffee species most commercialized worldwide are Arabica (Coffea arabica) and Robusta (Coffea canephora). Roasted coffee beans are the most used, but coffee leaves are also consumed as infusion in several countries for traditional medicinal purposes. They contain several interesting phenolic antioxidant compounds mainly belonging to chlorogenic acids (CGAs). In the present work, a liquid chromatography-electrochemical detection (LC-EC) method was developed for the determination of three main chlorogenic acid isomers, namely 3-, 4-, and 5-caffeoylquinic acids (CQA), in coffee leaves aqueous extracts. Samples from eight coffee species, namely; Coffea arabica, Coffea canephora, Coffea liberica, Coffea humilis, Coffea mannii, Coffea charrieriana, Coffea anthonyi, and Coffea liberica var. liberica, were grown and collected in tropical greenhouses. Linearity of the calibration graphs was observed in the range from the limit of quantification to 1.0 × 10-5 M, with R² equal to 99.9% in all cases. High sensitivity was achieved with a limit of detection of 1.0 × 10-8 M for 3-CQA and 5-CQA (i.e., 3.5 µg/L) and 2.0 × 10-8 M for 4-CQA (i.e., 7.1 µg/L). The chromatographic profile of the samples harvested for each Coffea species was studied comparatively. Obtained raw data were pretreated for baseline variations and shifts in retention times between the chromatographic profiles. Principal Component Analysis (PCA) was applied to the pretreated data. According to the results, three clusters of Coffea species were found. In the water sample extracts, 5-CQA appeared to be the major isomer, and some species contained a very low amount of CQAs. Fluctuations were observed depending on the Coffea species and harvesting period. Significant differences between January and July were noticed regarding CQAs content. The species with the best CQAs/caffeine ratio was identified. The LC-EC data were validated by liquid chromatography-high resolution mass spectrometry (LC-HRMS).

Metabolomics fingerprint of coffee species determined by untargeted-profiling study using LC-HRMS.

Coffee bean extracts are consumed all over the world as beverage and there is a growing interest in coffee leaf extracts as food supplements. The wild diversity in Coffea (Rubiaceae) genus is large and could offer new opportunities and challenges. In the present work, a metabolomics approach was implemented to examine leaf chemical composition of 9 Coffea species grown in the same environmental conditions. Leaves were analyzed by LC-HRMS and a comprehensive statistical workflow was designed. It served for univariate hypothesis testing and multivariate modeling by PCA and partial PLS-DA on the Workflow4Metabolomics infrastructure. The first two axes of PCA and PLS-DA describes more than 40% of variances with good values of explained variances. This strategy permitted to investigate the metabolomics data and their relation with botanic and genetic informations. Finally, the identification of several key metabolites for the discrimination between species was further characterized.

Electrochemical Detectors in Liquid Chromatography: Recent Trends in Pharmaceutical and Biomedical Analysis.

Liquid chromatography (LC) coupled to an electrochemical (EC) detector is a complementary analytical tool compared to LC coupled with optical or mass spectrometry detectors (LC-MS). LC-EC can be applied to the determination of molecules difficult to be analyzed by other commercially available detectors. New EC detector design and new working electrode material have extended the scope of application in the field of pharmaceutical compounds analysis. Combining EC with LC-MS offers additional advantages compared to optical detectors in terms of drug stability and drug metabolism mimicry studies. Selected literature devoted to pharmacologically active compounds in their dosage forms, herbal drugs in natural products, drug residues in feed and/or in biological samples are reported in this review.

Liquid chromatography-electrochemical detection for the determination of ethoxyquin and its dimer in pear skin and salmon samples.

Ethoxyquin (EQ) is widely used as a synthetic antioxidant in animal feed, an antiscalding agent in apples and pears and as a color preservative in some spices. Since the presence of EQ in food products could cause negative health effects it is necessary to develop reliable analytical methods to evaluate the risk of human exposure. In this work, a sensitive, selective and accurate method based on solid-liquid extraction followed by clean-up with solid sorbent and liquid chromatography-electrochemical detection analysis with boron doped diamond electrode (LC-EC) for the determination of ethoxyquin and its dimer (EQDM) in pear skin and salmon samples, was developed. The method was validated according to the European Commission guidelines. The main variables of extraction were accurately optimized. The amounts of solid sorbents for clean-up procedure were optimized by using experimental design. A Box-Behnken design to obtain the optimum conditions was applied. For validation, a matrix-matched calibration was established and a recovery assay with spiked samples was carried out. The limits of detection (LODs) found were 0.05 and 0.1mgkg-1 for EQ and its dimer, respectively. The precision (as relative standard deviation, RSD) was lower than 15% with recoveries of compounds close to 100% in spiked samples.

Identification of coffee leaves using FT-NIR spectroscopy and SIMCA.

Abundant literature has been devoted to coffee beans (green or roasted) chemical description but relatively few studies have been devoted to coffee leaves. Given the fact that coffee leaves are used for food and medicinal consumption, it was of interest to develop a rapid screening method in order to identify coffee leaves taxa. Investigation by Fourier - Transform near infrared spectroscopy (FT-NIRS) was performed on nine Coffea taxa leaves harvested over one year in a tropical greenhouse of the Botanic Garden Meise (Belgium). The only process after leaves harvesting was an effective drying and a homogeneous leaves grinding. FT-NIRS with SIMCA analysis allowed to discriminate the spectral profiles across taxon, aging stage (mature and senescence coffee leaves) and harvest period. This study showed that it was possible (i) to classify the different taxa, (ii) to identify their aging stage and (iii) to identify the harvest period for the mature stage with a correct classification rate of 99%, 100% and 90%, respectively.

Determination of groundwater mercury (II) content using a disposable gold modified screen printed carbon electrode.

Mercury (II) measurements were performed thanks to a newly developed electrochemical method using a disposable gold modified screen printed carbon electrode. The method has a wide dynamic range (1-100 µg/L), a good accuracy and a limit of detection in compliance with WHO standards. The application of the method to several groundwater samples made it possible to identify, for the first time, mercury content higher than the recommended WHO standard value in a gold mining activity area in the northern part of Burkina Faso. The accuracy of the assay was checked by ICP/MS.

An experimental design approach to optimize an amperometric immunoassay on a screen printed electrode for Clostridium tetani antibody determination.

An immunoassay for the determination of anti-tetani antibodies has been developed using a screen printed electrode (SPE) as solid support for toxoid (antigen) immobilization. The assay was performed in guinea pig serum. The immunoreaction and the subsequent amperometric detection occurred directly onto the SPE surface. The assay consisted of spiking the anti-tetani sample directly onto the toxoid modified SPE, and then a second antibody, i.e. a HRP-labeled anti-immunoglobulin G, was deposited onto the biosensor. Subsequent amperometric detection was realized by spiking 10 µL of a hydroquinone (HQ) solution into 40 µL of buffer solution containing hydrogen peroxide. An experimental design approach was implemented for the optimization of the immunoassay. The variables of interest, such as bovine serum albumin (BSA) concentration, incubation times and labeled antibody dilution, were optimized with the aid of the response surface methodology using a circumscribed central composite design (CCCD). It was observed that two factors exhibited the greatest impact on the response, i.e. the anti-tetani incubation time and the dilution factor of the labeled antibody. It was discovered that in order to maximize the response, the dilution factor should be small, while the anti-tetani antibody incubation time should be long. The BSA concentration and the HRP-anti-IgG incubation had very limited influence. Under the optimized conditions, the immunoassay had a limit of detection of 0.011 IU/mL and a limit of quantification of 0.012 IU/mL. These values were below the protective human antibody limit of 0.06 IU/mL.

Contribution of Electrochemistry to the Biomedical and Pharmaceutical Analytical Sciences.

All analytical techniques have experienced major progress since the last ten years and electroanalysis is also involved in this trend. The unique characteristics of phenomena occurring at the electrode-solution interface along with the variety of electrochemical methods currently available allow for a broad spectrum of applications. Potentiometric, conductometric, voltammetric and amperometric methods are briefly reviewed with a critical view in terms of performance of the developed instrumentation with special emphasis on pharmaceutical and biomedical applications.

Liquid Chromatography with Amperometric Detection at a Silver Based Detector for the Determination of Thiocompounds: Application to the Assay of Thiopurine Antimetabolites in Urine.

A silver amperometric detector coupled to liquid chromatography (LC) was used for the determination of 6-thioguanine (6-TG) and two of its metabolites, thiouric acid (TU) and 2-amino-6-mercaptopurine riboside (6-TGR). The silver detector coupled to LC operated at a low applied potential (0.08 V vs Ag/AgCl) and offered a chromatogram with peak responses corresponding to molecules interacting with silver, namely, chloride ions and small soluble biothiols in addition to the organothiol drug compounds investigated. Online electrochemical surface cleaning permitted the improvement of the repeatability and peak shape of the recorded signal compared to direct current amperometric detection (AD) when operating in chloride containing media. The studied molecules were eluted isocratically within 5 min on a reversed-phase C18 column without interference from endogenous biothiols present in urine samples. Diluted urine samples (1:1) were directly injected in the LC setup; a linear calibration curve was obtained between peak area and analyte concentration between 0.1 and 10 µM for all the studied molecules. Limits of detection (LODs) were 0.03, 0.008, and 0.01 µM, and the limits of quantification (LOQs) were 0.1, 0.02, and 0.03 µM for TU, 6-TG, and 6-TGR, respectively. Within-day RSDs were 2%, 0.8%, and 1% and between-day RSDs were 2%, 0.9%, 2% for TU, 6-TG, and 6-TGR, respectively. Recoveries in spiked urine were 99.8%, 99.9%, and 99.0% for TU, 6-TG, and 6-TGR, respectively.

Flow-through enzyme immobilized amperometric detector for the rapid screening of acetylcholinesterase inhibitors by flow injection analysis.

A commercially available thin-layer flow-through amperometric detector, with the sensing block customized in an original design, was applied to the screening of drug compounds known as acetylcholinesterase (AChE) inhibitors. AChE from electric eel was covalently immobilized onto a cysteamine modified gold disk adjacent to a silver disk working electrode. On-line studies were performed by flow injection analysis (FIA) in PBS buffer pH 7.4. Seven commercially available AChE inhibitors used in the medical field, namely neostigmine, eserine, tacrine, donepezil, rivastigmine, pyridostigmine and galantamine as well as two natural compounds, quercetin and berberine, were investigated. The same trend of inhibitory potency as described in the literature was observed. Of particular interest and in addition to the determination of the IC50 values, this flow-through system allowed the study of both, the stability of the enzyme-inhibitor complex and the kinetic of the enzyme activity recovery.

Nanoimmunoassay onto a screen printed electrode for HER2 breast cancer biomarker determination.

A chip format sandwich-type immunoassay based on Nanobodies(®) (Nbs) with the Human Epidermal Growth Factor Receptor (HER2) extracellular domain as antigen model has been developed. The HER2 is considered as an important biomarker because its overexpression causes an aggressive type of breast cancer. Nbs are single domain antigen-binding fragments derived from camelid heavy-chain antibodies. The strategy of the presently developed sandwich immunoassay takes advantage of the small size of Nbs for the detection of the electroactive redox tracer onto the screen printed electrode (SPE). A capture anti HER2 Nb was covalently immobilized onto the SPE, and the detection Nb, raised against another epitope of HER2, was labeled with horseradish peroxidase (HRP). The biosensor signal corresponded to the electroreduction of para-quinone generated at the SPE by the HRP in the presence of hydroquinone and hydrogen peroxide. The best performing and optimized immunoassay conditions consisted of 2 and 20 min for the first and the second incubation times, respectively. The amperometric signal obtained was proportional to the logarithm of HER2 concentration between 1 and 200 µg/mL and the modified SPE storage stability lasted for at least three weeks. Determination of HER2 in human cells has been realized.

Inhibition of myeloperoxidase activity by the alkaloids of Peganum harmala L. (Zygophyllaceae).

ETHNOPHARMACOLOGICAL RELEVANCE: Seeds and aerial parts of Peganum harmala L. are widely used in Algeria as anti-inflammatory remedies. Evaluation of Peganum harmala total alkaloids extracts and pure ß-carboline compounds as an anti-inflammatory treatment by the inhibition of an enzyme key of inflammatory, myeloperoxidase (MPO) and HPLC quantification of the alkaloids from the different parts of plant. MATERIALS AND METHODS: MPO inhibition was tested using taurine chloramine test. The inhibition of LDL oxidation induced by MPO was carried out. The molecular docking analysis of Peganum harmala alkaloids on MPO was performed using the Glide XP docking protocol and scoring function and the redox potential of alkaloids was determined using an Epsilon potentiostat. The concentration of harmala alkaloids was determined using HPLC analysis. RESULTS: The HPLC profiling of the active total alkaloids indicates that ß-carboline e.g. harmine, harmaline, harmane, harmol and harmalol are major components. As ß-carbolines resemble tryptamine, of which derivatives are efficient inhibitors of MPO, the harmala alkaloids were tested for their activity on this enzyme. Total alkaloids of the seeds and of the aerial parts strongly inhibited MPO at 20µg/mL (97±5% and 43±4%, respectively) whereas, at the same concentration, those of the roots showed very low inhibition (15±6%). Harmine, harmaline and harmane demonstrated a significant inhibition of MPO at IC50 of 0.26, 0.08 and 0.72µM respectively. These alkaloids exerted a similar inhibition effects on MPO-induced LDL oxidation. Molecular docking analysis of Peganum harmala alkaloids on MPO showed that all active Peganum harmala alkaloids have a high affinity on the active site of MPO (predicted free energies of binding up to -3.1kcal/mol). Measurement of redox potentials versus the normal hydrogen electrode clearly differentiated (i) the high MPO inhibitory activity of harmine, harmaline and harmane (+1014, 1014 and 1003mV, respectively); and (ii) the low activity of harmalol and harmol (+629/778 and 532/644mV, respectively). A reverse phase HPLC method has been developed to determine simultaneously five alkaloids of Peganum harmala. Seeds contained all five ß-carboline derivatives with the main active alkaloids, harmaline and harmine, being up to 3.8% and 2.9%, respectively. Up to 3.2% of harmine was determined in the roots. The four ß-carboline derivatives, harmine, harmaline, harmane and harmalol were identified in the aerial parts. The highest inhibitory effect observed in seeds and the moderate effect of aerial parts could be explained by their harmine and harmaline content. In contrast, the very weak inhibition of the root extract, despite the presence of harmine, may tentatively be explained by the high concentration of harmol which can reduce Compound II of MPO to the native form. CONCLUSION: The inhibition of MPO by Peganum harmala ß-carboline alkaloids, herein reported for the first time, may explain the anti-inflammatory effect traditionally attributed to its herbal medicine.

A new chemical criteria for white wine: the glutathione equivalent capacity.

The present work aimed at showing the interest in applying liquid chromatography with amperometric detection at a silver electrode (LC-EC-Ag) in order to record a chromatogram highly selective to non volatile aminothiols present in white wines. By integrating and summing the peak area of the aminothiols and by normalizing with respect to the peak area of an injected standard solution of glutathione (1 µM) a new quantitative criteria for white wine characterization is proposed namely, the glutathione equivalent capacity (GEC). The LC setup uses a C18 column in isocratic mode and the analysis takes less than 4 min. The wine sample needs no sample treatment other than dilution with the mobile phase. This new methodology and concept is illustrated by the LC-EC-Ag analysis of several white wines of different origins especially Alsace Riesling wines and Riesling grape juice. It is anticipated that in addition to the determination of the GEC, the developed method may be of interest for establishing a white wine "signature" based on the chromatographic profile.

Determination of aminothiols by liquid chromatography with amperometric detection at a silver electrode: application to white wines.

Liquid chromatography coupled to a silver electrode based flow-through amperometric detector (LC-EC-Ag) was developed for the determination of aminothiols in white wines. The C18 reversed phase LC system operated in the isocratic mode at 0.7 mL min(-1) and used an acidic mobile phase composed of formic acid, EDTA, sodium nitrate, sodium hydroxide, and methanol 1% (v/v) at pH 4.5. The working electrode operated at 0.08 V vs Ag/AgCl, 3M KCl and its manual cleaning was realized once a month by smoothing on a polishing cloth. The analyzed aminothiols were resolved and eluted within 4 min, and all standard curves were linear in the range 2×10(-7)-2×10(-5) M. The analyzed wine samples needed no preparation other than dilution with the mobile phase. The concentration of cysteine (CYS), homocysteine (HCYS), glutathione (GSH) and N-acetylcysteine (NAC) in bottled white wines, determined by the method of standard addition, was found to be in the low µM range (0.2-2 mg L(-1)) depending on the wine type and its age.

Development of an acetylcholinesterase immobilized flow through amperometric detector based on thiocholine detection at a silver electrode.

This paper describes the use of a commercially available thin-layer flow through a detector with the sensing block customized in an original design for acetylcholinesterase (AChE) immobilization and suitable for inhibition studies by flow injection analysis (FI). AChE was chemically linked onto a gold disk substrate adjacent to a silver disk electrode. The downstream positioned silver electrode, poised at 0.08 V vs. Ag/AgCl, KCl 3M, permitted the sensitive amperometric detection of liberated thiocholine (TCh) using acetylthiocholine (ATCh) as enzyme substrate. A typical Michaelis-Menten curve was obtained for ATCh within the concentration range 1×10(-5)-1×10(-2)M with a Kmapp of 5.93×10(-4)M. ATCh quantification was achieved with a limit of detection (LOD) of 5.3×10(-6)M. The utility of the developed FI setup was demonstrated for AChE inhibition studies using neostigmine as model compound. The IC50 for neostigmine was obtained to be 1.45×10(-7)M.