An ultrasound assisted extraction-solid-phase extraction-ultra-performance liquid chromatography combined strategy for atropine determination in Atropa belladonna leaves.

Atropine is an antimuscarinic alkaloid identified in Atropa belladonna. In pharmacopeias, percolation is standardized as an extraction method for A. belladonna leaves, along with liquid-liquid extraction as a cleanup procedure and titration as an analytical method for assaying the atropine in the leaves. In this study, a faster, solvent-saving, and more reliable method for quality control of A. belladonna samples was developed. Ultrasound-assisted extraction was proposed and optimized by fractional factorial design followed by Box-Behnken design. For modeling atropine content, the following optimal conditions were established: particle size, 180 µm; percentage methanol in water, 50%; volume of solvent, 15 ml; time of extraction, 60 min; and number of extractions, two. This led to a significant improvement in atropine extraction (P < 0.001). For cleanup, solid-phase extraction was used as an alternative to liquid-liquid extraction, giving similar results, with higher reproducibility. Finally, for the atropine assay, a UPLC method was validated as a substitute for the classic titration method. Taken together, the development of an ultrasound-assisted extraction-solid-phase extraction-UPLC approach allowed the determination of atropine content in A. belladonna leaves in a time- and solvent-saving manner, with high reliability.

Development of CE-C4D Method for Determination Tropane Alkaloids.

A fast method for the determination of tropane alkaloids, using a portable CE instrument with a capacitively coupled contactless conductivity detector (CE-C4D) was developed and validated for determination of atropine and scopolamine in seeds from Solanaceae family plants. Separation was obtained within 5 min, using an optimized background electrolyte consisting of 0.5 M acetic acid with 0.25% (w/v) ß-CD. The limit of detection and quantification was 0.5 µg/mL and 1.5 µg/mL, respectively, for both atropine and scopolamine. The developed method was validated with the following parameters-precision (CV): 1.07-2.08%, accuracy of the assay (recovery, RE): 101.0-102.7% and matrix effect (ME): 92.99-94.23%. Moreover, the optimized CE-C4D method was applied to the analysis of plant extracts and pharmaceuticals, proving its applicability and accuracy.

Tale of Two Alkaloids: pH-Controlled Electrochemiluminescence for Differentiation of Structurally Similar Compounds.

Electrochemiluminescence (ECL) has increased in popularity as a result of its inherent advantages, including but not limited to portability, simplicity of use, and low reagent consumption. However, its significant advantages are often over shadowed as a result of its limited specificity. ECL emissions are intrinsically broad and lack the definition of other available analytical techniques. Furthermore, species with similar functional groups have almost identical electrochemical behavior and thus typically emit within approximately the same potential region. Within this contribution we have demonstrate the use of pH controlled ECL to prove the presence of two individual species within a mixed sample. Analysis at a single pH would not provide this information. We have illustrated the potential of this methodology to quantify scopolamine alongside sister tropane alkaloid atropine, a known ECL interferent. Previously the two alkaloids could not be distinguished from one another using a single technique which did not involve a separation strategy. pH controlled ECL is a simple approach to improve the specificity of a basic [Ru(bpy)3]2+ film based sensor. By exploiting molecular characteristics, such as pKa, we have been able to fine-tune our methodology to facilitate identification of analytes previously exhibiting indistinguishable ECL emission. Thus, by improving specificity, while maintaining operational simplicity and inexpensive design, we have been able to highlight the potential power of ECL for identification of structurally similar compounds. Further improvements of specificity, such as demonstrated within this contribution, will only further future applications of ECL sensors across a range of different fields.

Utilization of an Electrochemiluminescence Sensor for Atropine Determination in Complex Matrices.

A major challenge within forensic science is the development of accurate and robust methodologies that can be utilized on-site for detection at crime scenes and can be used for analyzing multiple sample types. The recent expansion of electrochemical sensors to tackle this hurdle requires sensors that can undergo analysis without any pretreatment. Given the vast array of samples that are submitted for forensic analysis, this can pose a major challenge for all electrochemical sensors, including electrochemiluminescent (ECL)-based sensors. Within this contribution, we demonstrate the capacity for an ECL-based sensor to address this challenge and it is potential to detect and quantify atropine from a wide range of samples directly from herbal material to spiked solutions. This portable platform demonstrates satisfactory analytical parameters with linearity across a concentration range of 0.75 to 100 µM, reproducibility of 3.0%, repeatability of 9.2%, and a detection limit of ∼0.75 µM. The sensor displays good selectivity toward alkaloid species and, in particular, the hallucinogenic tropane alkaloid functionality within complex matrices. This portable sensor provides rapid detection alongside low cost and operational simplicity, thus, providing a basis for the exploitation of ECL-based sensors within the forensic arena.

An integrated approach to study novel properties of a MALDI matrix (4-maleicanhydridoproton sponge) for MS imaging analyses.

The chemical properties accounting for the operation of a valuable matrix used in matrix-assisted laser desorption ionization (MALDI) to perform mass spectrometry imaging (MSI), namely 3-(4,5-bis(dimethylamino)napthalen-1-yl)furan-2,5-dione (4-maleicanhydridoproton sponge, MAPS), have been elucidated also by comparison with the parent molecule 1,8-bis(dimethylamino) naphthalene (so-called proton sponge, PS). Both compounds present the bis(dimethylamino) groups, apt to efficiently trap a proton imparting positive charge. Only MAPS, though, owns the maleicanhydrido function acting as electrophile and yielding covalently bound adducts with a variety of analytes. In this way, MAPS performs as "carrier" for the analyte (A) of interest, at the same time minimizing the presence of useless, background ions. The covalent character of the adducts, [MAPS+H + A]+, is testified by their collision-induced dissociation pattern, quite distinct from the one displayed by [PS + H]+, while PS does not form any [PS + H + A]+, thus confirming the key role of the maleicanhydrido functionality of MAPS. Vibrational spectroscopy of [MAPS+H + A]+ adducts (A = H2O, NH3) provided further structural evidence. The presence of a mobile proton on A was found to be a requisite for adduct formation by electrospray ionization of acetonitrile solutions, pointing to a possible role of MAPS in discriminating competing analytes based on molecular features. The performance of MAPS has been verified in MALDI-MSI of Atropa belladonna berries, exploiting MAPS binding to atropine. Graphical abstract ᅟ.

Delivering Inorganic and Organic Reagents and Enzymes from Zein and Developing Optical Sensors.

Nowadays, interest in using environmentally friendly materials is increasing in many fields. However, the rational design of sensors with biodegradable materials is a challenge. The main aim of this work is to show the possibility of using zein, a protein from corn, as a biodegradable and low-cost material for immobilizing, stabilizing, and delivering different kind of reagents for developing optical sensors. Enzymes, metallic salts, and aromatic and small organic compounds were tested. In addition, different techniques of immobilization, entrapment and adsorption, were used, and different formats, such as solid devices and also multiwell platforms, were proposed. The capacity of zein for immobilizing two reagents together, enzyme and substrate, into a multianalysis format was also shown. Two applications were developed as examples: a colorimetric assay based on a ferric hydroxamate reaction for ester drugs, which was applied in atropine determination in pills, and a fluorimetric enzymatic multiwell-plate biodevice applied in phosphate determination in human serum and urine. Zein demonstrated being not only a green alternative but also a versatile polymer for developing sensors from reagents with different natures in different formats and matrices, thereby resulting in different applications.

Simultaneous quantification of atropine and scopolamine in infusions of herbal tea and Solanaceae plant material by matrix-assisted laser desorption/ionization time-of-flight (tandem) mass spectrometry.

RATIONALE: Atropine (Atr) and scopolamine (Scp) are toxic secondary plant metabolites of species within the Solanaceae genus that can accidentally or intentionally reach the food store chain by inaccurate harvesting of any plant material, e.g. for herbal tea infusions. Ingestion may cause severe anticholinergic poisoning thus requiring risk-oriented determination in food and beverages. The suitability of matrix-assisted laser desorption/ionization time-of-flight (tandem) mass spectrometry, MALDI-TOF MS(/MS), should be characterized for simultaneous analysis. METHODS: We herein present the first MALDI-TOF MS(/MS) procedure for quantitative determination of both alkaloids in herbal tea infusions and Solanaceae plant material. A standard additions procedure using triply deuterated Atr as internal standard was developed and validated. RESULTS: Tropane alkaloids were detected without interferences and the standard additions procedure allowed reliable quantification. Intraday and interday precision were less than 17% and corresponding accuracies were between 77% and 112%. Limits of detection in the spotting solution were found at 5 ng/mL (Atr) and 0.5 ng/mL (Scp). The assay was applied to diverse tea infusions as well as to berries and leaves of deadly nightshade and angel's trumpet. CONCLUSIONS: The usefulness of MALDI-TOF MS(/MS) for investigations of plant-derived samples to prove contaminations by small basic compounds was demonstrated. The elaborate procedure is reliable but quite laborious to obtain quantitative results, but MALDI-TOF MS(/MS) was also shown to be a valuable tool for rapid qualitative screening for Atr and Scp in plant extracts.

Simultaneous analysis of tropane alkaloids in teas and herbal teas by liquid chromatography coupled to high-resolution mass spectrometry (Orbitrap).

A new method has been developed for the simultaneous determination of 13 tropane alkaloids in tea and herbal teas using high-performance liquid chromatography coupled to an Exactive-Orbitrap analyzer. A mixture of methanol, water, and formic acid was used for the extraction of the target compounds followed by a solid-phase extraction step. The validated method provided recoveries from 75 to 128% with intra- and interday precision lower than or equal to 24% (except for apoatropine). Limits of quantification ranged from 5 to 20 µg/kg. Eleven tea and herbal tea samples and two contaminated samples with Datura stramonium seeds were analyzed. Tropane alkaloids were detected in six samples with concentrations from 5 (apoatropine) to 4340 µg/kg (sum of physoperuvine, pseudotropine, and tropine), whereas concentrations from 5 (apoatropine) to 1725 µg/kg (sum of physoperuvine, pseudotropine, and tropine) were found in the contaminated samples.

[Doping relevant substances in horse feed]. / Dopingrelevante Substanzen in Futtermitteln für Pferde.

INTRODUCTION: Horse feed material of Swiss and foreign production available on the Swiss market was tested with regard to possible contamination with 7 different alkaloids, atropine and colchicine. Twenty-eight feed samples as well as 2 poppy seed samples were analyzed. Out of 28 feed samples 18 were positive for prohibited substances in Equestrian sports. The concentration of prohibited substances was in none of the samples high enough to cause an effect on the body of the horse. The poppy seed sample, which was obtained by conventional harvesting and cleaning methods, contained a very high Morphine concentration compared to the sample which was harvested by hand. Despite high quality standards a contamination with prohibited substances in horse feed cannot be excluded.

INTRODUCTION: Divers aliments pour chevaux de provenance indigène et étrangère disponibles sur le marché suisse ont été examinés quant à une éventuelle contamination par 7 alcaloïdes différents ainsi que par l'atropine et la colchicine. On a analysé 28 échantillons de fourrages ainsi que 2 échantillons de graines de pavot provenant de cultures suisses. Dix-huit des vingt-huit échantillons contenaient des substances pouvant avoir une importance en matière de dopage dans les sports équestres. La concentration des substances recherchées n'était, dans aucun des échantillons de fourrages, assez élevée pour qu'on puisse tabler avec un effet sur le corps du cheval. L'échantillon de graines de pavot produites avec les techniques usuelles de récolte et de nettoyage présentait, par rapport à celui composé de graines récoltées à la main, un taux de morphine inhabituellement élevé. La présente étude de 28 échantillons d'aliments démontre qu'une contamination avec des substances ayant une importance en matière de dopage ne peut pas être exclue, même avec des standards de qualité élevés.

Selective Determination of Atropine Using poly Dopamine-Coated Molecularly Imprinted Mn-Doped ZnS Quantum Dots.

In this study, a selective method for the determination of atropine in pharmaceutical formulations was proposed. L-cysteine capped Mn-doped ZnS quantum dots (QDs) were prepared in an in-situ method using sodium thiosulfate precursor and characterized by spectrofluorometer, Fourier transform infrared spectroscopy (FT-IR), transmission electron microscope (TEM) and X-ray diffractometer (XRD). Dopamine hydrochloride was used as a precursor for preparation of poly dopamine-coated molecularly imprinted Mn-doped ZnS quantum dots. Finally, these prepared molecularly imprinted Mn-doped ZnS quantum dots were used for determination of atropine in pharmaceutical formulations. The obtained linear range for determination of atropine was in the range of 2 × 10(-8) - 7 × 10(-6) M, with a correlation coefficient (R(2)) of 0.9889; and the detection limit (S/N = 3) was 3.2 nM.

A quantitative HPLC-MS/MS method for studying internal concentrations and toxicokinetics of 34 polar analytes in zebrafish (Danio rerio) embryos.

An analytical method using high-performance liquid chromatography-tandem mass spectrometry was developed to determine internal concentrations of 34 test compounds such as pharmaceuticals and pesticides in zebrafish embryos (ZFE), among them, cimetidine, 2,4-dichlorophenoxyacetic acid, metoprolol, atropine and phenytoin. For qualification and quantification, multiple reaction monitoring mode was used. The linear range extends from 0.075 ng/mL for thiacloprid and metazachlor and 7.5 ng/mL for coniine and clofibrate to 250 ng/mL for many of the test compounds. Matrix effects were strongest for nicotine, but never exceeded ±20 % for any of the developmental stages of the ZFE. Method recoveries ranged from 90 to 110 % from an analysis of nine pooled ZFE. These findings together with the simple sample preparation mean this approach is suitable for the determination of internal concentrations from only nine individual ZFE in all life stages up to 96 h post-fertilization. Exemplarily, the time course of the internal concentrations of clofibric acid, metribuzin and benzocaine in ZFE was studied over 96 h, and three different patterns were distinguished, on the basis of the speed and extent of uptake and whether or not a steady state was reached. Decreasing internal concentrations may be due to metabolism in the ZFE.

A two-electrode system-based electrochemiluminescence detection for microfluidic capillary electrophoresis and its application in pharmaceutical analysis.

A two-electrode configuration powered by batteries was designed for a microchip capillary electrophoresis-electrochemiluminescence system. A home-made working electrode for end-column mode detection and wall-jet configuration was made up of a platinum wire (0.3 mm diameter) and a quartz capillary (320 µm internal diameter). The platinum wire served as a pseudoreference electrode. The configuration of the detection power supply comprised two D-size batteries (connected in series), a switch, and an adjustable resistor. The microchip consisted of two layers: the bottom layer was a glass sheet containing injection and separation channels; the upper layer was polydimethylsiloxane block. In order to reduce the loss of electrochemiluminescence signal, a coverslip (0.17 mm thickness) was used as the floor of the detection reservoir. The performance of the system was demonstrated by separation and detection of atropine, anisodamine and proline. The linear response for proline ranged from 5 µM to 100 µM (r = 0.9968), and the limit of detection was 1.0 µM (S/N = 3). The system was further applied to the measurement of atropine in atropine sulfate injection solutions with the limit of detection 2.3 µM. This new system is a potential tool in pharmaceutical analysis.

Determination of Atropine by HPLC in Plant of Datura by Liquid-Liquid Extraction and Magnet Solid-Phase Extraction.

Atropine is a tropane alkaloid found in abundance in Datura plant. We used two liquid-liquid extraction methods and magnet solid-phase extraction to compare the amount of atropine in these two types of plants (Datura innoxia and Datura stramonium). The surface magnetic nanoparticle Fe3O4 correction with an amine and dextrin, and finally, magnetic solid-phase extraction Fe3O4@SiO2-NH2-dextrin (MNPs-dextrin), was prepared. We determined the effect of significant parameters in the removal step and optimization of atropine measurements with half-fractional factorial design (25-1) and response surface methodology via central composite design. The optimum conditions are for desorption solvent = 0.5 mL methanol and desorption time of 5 min. We obtained an extraction recovery of 87.63% with a relative standard deviation of 4.73% via six frequented measurements on a 1 µg L-1 atropine standard solution based on the optimum condition. Preconcentration factors for MNPs are 81, limit of detection = 0.76 µg L-1 and limit of quantitation = 2.5 µg L-1.

Effect of pH and temperature on tropane alkaloids within a processing strategy to provide safe infant cereal-based food.

Tropane alkaloids (TAs) are secondary metabolites from weeds that can contaminate cereals and vegetables during harvest. Due to their toxicity, the Regulation (EC) 2023/915 sets maximum levels for atropine and scopolamine in cereal-based foods for infants containing millet, sorghum, buckwheat or their derived products. The aim of this study was to evaluate the effect of pH and temperature on the stability of TAs, as possible parameters in thermal processing to mitigate this chemical hazard in cereal-based infant food. The effect of pH (4 and 7) and temperature (80 °C and 100 °C) was assessed in buffer solutions. Also, treatment at 180 °C was performed in spiked and naturally incurred millet flour to assess the effect of high temperature, simulating cooking or drying, on the stability of TAs in the cereal matrix. The fate of 24 TAs was assessed by UHPLC-MS/MS. TAs showed high thermostability, although it was variable depending on the specific compound, pH, temperature and treatment time. In buffer solutions, higher degradation was found at 100 °C and pH 7. In spiked millet flour at 180 °C for 10 min, scopolamine and atropine contents decreased by 25 % and 22 %, similarly to other TAs which also showed a slow thermal degradation. Atropine, scopolamine, anisodamine, norscopolamine, scopine and scopoline were found in naturally contaminated millet flour. Interestingly, naturally incurred atropine was more thermostable than when spiked, showing a protective effect of the cereal matrix on TAs degradation. The present results highlight the need for an accurate monitorization of TAs in raw materials, as this chemical hazard may remain in infant cereal-based food even after intense thermal processing.

Forensic electrochemistry: sensing the molecule of murder atropine.

We present the electroanalytical sensing of atropine using disposable and economic screen printed graphite sensors. The electroanalytical determination of atropine is found to be possible over the concentration range of 5 µM to 50 µM with a detection limit of 3.9 µM (based on 3-sigma) found to be possible. We demonstrate proof-of-concept that this approach provides a rapid and inexpensive sensing strategy for determining the molecule of murder atropine in diet Coca-Cola samples.

Studies on prokinetic, laxative and spasmodic activities of Phyllanthus emblica in experimental animals.

This study was aimed to provide pharmacological basis for the medicinal use of Phyllanthus emblica fruit in indigestion and constipation using the in-vivo and in-vitro assays. The crude extract of the dried fruits of Phyllanthus emblica (Pe.Cr) and its fractions were tested positive for alkaloids, saponins, tannins, terpenes, flavonoids, sterols and coumarins. Pe.Cr at the doses of 100 and 300 mg/kg exhibited the prokinetic and laxative activities in mice, which were found partially sensitive to atropine. In isolated guinea-pig ileum and rabbit jejunum, the crude extract and its aqueous fraction (Pe.Aq) caused concentration-dependent and partially atropine-sensitive stimulatory effects followed by relaxation at higher tested concentrations, being more efficacious in guinea pig, while more potent in rabbit tissues. The petroleum fraction (0.003-0.1 mg/mL) exhibited fully atropine-sensitive contractions in both guinea-pig and rabbit tissues. However, the ethyl acetate and chloroform fractions (0.003-1.0 mg/mL) showed only spasmolytic activity when studied in spontaneously contracting rabbit jejunum. This study showed that the Phyllanthus emblica possesses prokinetic and laxative activities in mice along with spasmodic effect in the isolated tissues of guinea pig and rabbit, mediated partially through activation of muscarinic receptors; thus, this study provides a rationale for the medicinal use of Phyllanthus emblica fruits in indigestion and constipation.

[Simultaneous determination of scopolamine hydrobromide, atropine sulfate, ephedrine hydrochloride and pseudoephedrine hydrochloride in Zhichuanling oral liquid with HPLC].

OBJECTIVE: To establish an HPLC method for determining the contents of scopolamine hydrobromide, atropine sulfate, ephedrine hydrochloride and pseudoephedrine hydrochloride in Zhichuanling oral liquid. METHOD: Agela Durashell RP-C18 (4. 6 mm x250 mm, 5 microm) was adopted, with acetonitrile-sodium phosphate buffer solution (0. 07 mol L-1 sodium phosphate solution with 17.5 mmol L-1 sodium dodecylsulfate adjusted to pH 6.0 with phosphoric acid solution) (30:70) as the mobile phase. The flow rate was 0. 9 mL min -1, the detection wavelength was 207 nm, and the column temperature was 25 degree C. RESULT: Scopolamine hydrobromide, atropine sulfate, ephedrine hlvdrochloride and pseudoephedrine hydrochloride showed good linear relations with peak areas within the concentration range of 0. 021 21-1. 060 5 pg (r =0. 999 3) , 0. 011 14-0. 557 microg (r = 0. 999 6) , 0. 200 56-10. 028 microg (r =0. 999 7) and 0.070 33-3. 516 5 gg (r =0. 999 6), respectively, with the average recoveries of 101.9% , 99. 80%, 100. 3%, 100. 2% (n=6). CONCLUSION: The method was so quick, simple, highly reproducible and specific that it could be used as one of quality control methods of Zhichuanling oral liquid.

Development, validation, and application of a multimatrix UHPLC-MS/MS method for quantification of Datura-type alkaloids in food.

A quantitative ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method was developed and validated for the determination of tropane alkaloids (TAs), atropine and scopolamine, in a variety of food products. The sample preparation of cereal-based food, oilseeds, honey, and pulses consisted of a solid-liquid extraction with an acidified mixture of methanol and water, while an additional step of solid-phase extraction on a cation-exchange sorbent was introduced in the treatment of teas and herbal infusions, aromatic herbs, spices and food supplements. The limits of quantification of the method varied from 0.5 to 2.5 µg kg-1. Apparent recovery was in the range of 70-120%, and repeatability and intermediate precision were below 20%. The method was successfully applied in a proficiency testing exercise as well as in the analysis of various commercial foods. Only 26% of the analysed food samples contained one or both TAs. The mean concentrations for atropine and scopolamine amounted to 21.9 and 6.5 µg kg-1, respectively, while the maximum concentrations were 523.3 and 131.4 µg kg-1, respectively. Overall, the highest levels of TA sum were found in an herbal infusion of fennel and a spice mix containing fennel and anise seeds.

A non-aqueous solid phase extraction method for alkaloid enrichment and its application in the determination of hyoscyamine and scopolamine.

A non-aqueous solid phase extraction (SPE) method utilizing silica based strong cation exchange (SCX) was developed and optimized for the enrichment of alkaloids. In this method, silica based SCX SPE columns were used for the elimination of non-alkaloid compounds and the preconcentration of alkaloids from the extracts. Mass spectrometry was employed to analyze the alkaloid-enriched fraction, and results showed that the SPE method developed in this study was effective for the removal of non-alkaloids. Then, this pretreatment method was combined with high performance liquid chromatography for the quantification of scopolamine and hyoscyamine from Scopolia tangutica Maxim. The recoveries of scopolamine and (-)-hyoscyamine were 98.51% and 91.12%, respectively. Relative standard deviation values were 1.4% for scopolamine and 1.6% for (-)-hyoscyamine. The linearity was good in the 0.01-0.8 mg mL(-1) range for hyoscyamine and 0.01-0.4 mg mL(-1) range for scopolamine.

Biochemical, microbiological and phytochemical studies on aqueous-fermented extracts from Atropa belladonna L. Part 2--Phytochemistry.

Extraction methods of fresh plants into aqueous-fermented extracts according to German Homoeopathic Pharmacopoeia (HAB), regulation nos. 33 and 34 were evaluated. In the course of production, the extraction is accompanied by fermentation and the resulting preparation is stored for at least 6 months until further processing. In part 1 of the work, the biochemical reactions proceeding in aqueous-fermented extracts from the fresh flowering herb of Atropa belladonna var. belladonna (L.) were studied and the responsible microorganisms were identified. This second part aimed at shedding light on the phytochemical changes upon manufacture and storage. Additionally, questions were addressed at the robustness of the method by varying production conditions, its reproducibility and the comparison with ethanolic extraction. Studying 110 extracts produced from 2006 to 2009 as well as model experiments on isolated lactic acid bacteria in atropine solutions proved that the active substance atropine was stable under regular pH conditions. Interestingly, no difference between aqueous-fermented and ethanolic extracts could be found with respect to atropine concentration. In contrast, the amounts of scopoletin and kaempferol glycosides from Atropa belladonna differed depending on the extraction procedure.