Database-aided ultrahigh-performance liquid chromatography Q-Exactive-Orbitrap tandem mass spectrometry putatively identifies 16 unexpected compounds and three anticounterfeiting pharmacopoeia quality markers for Perillae Fructus.

RATIONALE: Perillae Fructus (PF) is a common traditional Chinese medicine (TCM) for the treatment of asthma. It has not been effectively characterized by rosmarinic acid (RosA), which is currently designed as the sole quality indicator in the Chinese Pharmacopoeia. METHODS: This study introduced a database-aided ultrahigh-performance liquid chromatography equipped with quadrupole-Exactive-Orbitrap mass spectrometry (UHPLC/Q-Exactive-Orbitrap MS/MS) technology to putatively identify the compounds in PF, followed by literature research, quantum chemical calculation, and molecular docking to screen potential quality markers (Q-markers) of PF. RESULTS: A total of 27 compounds were putatively identified, 16 of which had not been previously found from PF. In particular, matrine, scopolamine, and RosA showed relatively high levels of content, stability, and drug-likeness. They exhibited interactions with the asthma-related target and demonstrated the TCM properties of PF. CONCLUSIONS: The database-aided UHPLC/Q-Exactive-Orbitrap MS/MS can identify at least 27 compounds in PF. Of these, 16 compounds are unexpected, and three compounds (matrine, scopolamine, and RosA) should be considered anticounterfeiting pharmacopoeia Q-markers of PF.

Assessment of tropane alkaloid levels in Brazilian buckwheat flour products: A novel LC-UHPLC-MS/MS approach using solid-liquid extraction at low temperature.

In recent years, the monitoring of tropane alkaloids, specifically hyoscyamine and scopolamine, in food has become a pressing concern. This is due to increasing reports of food contamination with these compounds worldwide, raising awareness about the potential risks associated with their consumption. A novel method is proposed here for the determination of the sum of (+)-hyoscyamine, (-)-hyoscyamine, and (-)-scopolamine in buckwheat-based matrices, using solid-liquid extraction at low temperature and quantification by bidimensional chromatography coupled to tandem mass spectrometry. The validated method presented a linear response in the concentration range of 2.5-15 µg kg-1 (r > 0.99). The precision and accuracy were in the ranges from 0.8 to 11.0 % and from 96 to 103 %, respectively. The limit of quantification (LOQ) was 2.5 µg kg-1. No contamination was found at levels above the LOQ in any of the 18 samples analyzed (buckwheat flour, grains, and gluten-free mix).

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.

Evaluation of Tropane Alkaloids in Teas and Herbal Infusions: Effect of Brewing Time and Temperature on Atropine and Scopolamine Content.

Atropine and scopolamine belong to the tropane alkaloid (TA) family of natural toxins. They can contaminate teas and herbal teas and appear in infusions. Therefore, this study focused on analyzing atropine and scopolamine in 33 samples of tea and herbal tea infusions purchased in Spain and Portugal to determine the presence of these compounds in infusions brewed at 97 °C for 5 min. A rapid microextraction technique (µSPEed®) followed by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) was used to analyze the selected TAs. The results showed that 64% of the analyzed samples were contaminated by one or both toxins. White and green teas were generally more contaminated than black and other herbal teas. Of the 21 contaminated samples, 15 had concentrations above the maximum limit for liquid herbal infusions (0.2 ng/mL) set by Commission Regulation (EU) 2021/1408. In addition, the effects of heating conditions (time and temperature) on atropine and scopolamine standards and naturally contaminated samples of white, green, and black teas were evaluated. The results showed that at the concentrations studied (0.2 and 4 ng/mL), there was no degradation in the standard solutions. Brewing with boiling water (decoction) for 5 and 10 min allowed for higher extraction of TAs from dry tea to infusion water.

Tear film instability is associated with weakened colocalization between occludin and MUC5AC in scopolamine-induced dry eye disease (DED) rats.

PURPOSE: Dry eye disease (DED) is a disease with tear film instability because of multiple factors. This study was conducted to explore roles of occludin and MUC5AC in tear film instability in DED rat model. METHODS: A total of 20 SD rats were divided into DED group (n = 10) and normal control (NC) group (n = 10). DED rat model was established by subcutaneously injecting with scopolamine hydrobromide. Clinical examinations, including tear breakup time (tBUT), Schirmer's test and corneal fluorescein staining, were conducted to determine corneal functions. Transmission electron microscopy was used to measure the ultrastructures of corneal epithelial cells. Western blotting assay was used to identify occludin expression in corneal tissues of DED rats. Real-time PCR (RT-PCR) was performed to verify gene transcription of occludin and MUC5AC. Colocalization between occludin and MUC5AC was identified with confocal fluorescence microscopy. RESULTS: Tear breakup time was significantly shorter, and corneal fluorescein staining score was predominantly higher in DED rats compared to those in normal rats (P < 0.05). Normal rats showed a steady tear secretion throughout the whole experiments, while DED rats showed a dramatic reduction on day 14. DED rats demonstrated ultrastructural damage of Golgi apparatus and endoplasmic reticulum in corneal epithelial cells. Occludin and MUC5AC expressions were significantly downregulated in corneal tissue of DED rats compared with those of normal rats (P < 0.05). Percentage of occludin-MUC5AC-colocalized corneal epithelial cells in DED rats was significantly less compared with those in normal rats (P < 0.01). CONCLUSIONS: Tear film stability was damaged in scopolamine-induced DED rats because of the weakened colocalization between occludin and MUC5AC molecule. This study would provide a potential clue for the pathogenesis and a promising theoretical basis for clinical work of DED.

Improved Analytical Approach for Determination of Tropane Alkaloids in Leafy Vegetables Based on µ-QuEChERS Combined with HPLC-MS/MS.

This work presents an optimized methodology based on the miniaturization of the original QuEChERS (µ-QuEChERS) followed by liquid chromatography coupled to mass spectrometry (HPLC-MS/MS) for the determination of tropane alkaloids (TAs), atropine, and scopolamine in leafy vegetable samples. The analytical methodology was successfully validated, demonstrating quantitation limits (MQL) ≤ 2.3 ng/g, good accuracy, and precision, with recoveries between 90-100% and RSD ≤ 13% for both analytes. The method was applied to the analysis of TA-producing plants (Brugmansia versicolor, Solandra maxima, and Convolvulus arvensis). High concentrations of scopolamine were found in flowers (1771 mg/kg) and leaves (297 mg/kg) of B. versicolor. The highest concentration of atropine was found in flowers of S. maxima (10.4 mg/kg). Commercial mixed leafy vegetables contaminated with B. versicolor and S. maxima were analysed to verify the efficacy of the method, showing recoveries between 82 and 110% for both analytes. Finally, the method was applied to the analysis of eighteen samples of leafy vegetables, finding atropine in three samples of mixed leafy vegetables, with concentrations of 2.7, 3.2, and 3.4 ng/g, and in nine samples with concentrations ≤MQL. In turn, scopolamine was only found in a sample of chopped Swiss chard with a concentration ≤MQL.

Atropine and Scopolamine in Maize Products from the Retail Stores in the Republic of Serbia.

The cereal grains, which represent the cultivated grasses fruits, supply almost half of the total caloric requirements for humans and provide more nourishment compared with any other class of the food. Out of many cereals used for food, maize, rice, and wheat are the most important food resources for humans, representing 94% of the total cereals consumption. According to the data of the Republic Institute of Statistics for the year 2018, the harvested areas of corn amount to 906,753 hectares. The production of about 7 million tons was achieved with an average yield of 7.7 t/ha according to the Ministry of Agriculture of the Republic of Serbia. Serbia is still among the ten largest exporters of wheat and corn in the world for the period of 2014/15-2017/18. More precisely, it ranks seventh in the export of corn. Utilization of maize products for food animal nutrition (1000 t) is 491,48, and for industrial processing (1000 t) 278,862 expressed as the total consumption (1000 t) is 769,910. Therefore, a total of 103 samples of maize products were analyzed for the presence of toxins, i.e., tropane alkaloids (TAs). The samples were collected from the retail stores in the Republic of Serbia in 2021 and analyzed for the presence of atropine and scopolamine (33 corn grits, 39 polenta, and 31 semolina samples). Therefore, the Recommendation 2015/976/EU on the monitoring of TAs in food was adopted by the EU Commission to obtain more occurrence data on TAs in food. The monitoring extent, however, is restricted because reliable analytical methods and appropriate sensitivity are limited. There was a limit of 1 g/kg for each atropine and scopolamine in cereals containing millet, sorghum, buckwheat, or their derivatives. All the samples were analyzed by the LC-MS/MS. The LOQ was set at 1.0 µg/kg. Out of the total 103 tested samples, 32 samples (31.1%) were contaminated with atropine and scopolamine in concentrations above the LOQ. The highest concentrations of the studied TAs were observed in a semolina sample-atropine: 58.80 µg/kg, scopolamine: 10.20 µg/kg. The obtained results indicate that the TAs concentrations are above the LOQ which can be considered potential human and animal health hazards.

Rapid Detection and Determination of Scopolamine in the Leaf Extract of Black Henbane (Hyoscyamus niger L.) Plants Using a Novel Nanosensor.

BACKGROUND: Scopolamine is among the most essential tropane alkaloids used to remedy various nervous system disorders such as urinary incontinence, motion sickness, and spasmodic movements because of its anticholinergic and antispasmodic effects. OBJECTIVE: In this study, an optical nanosensor was fabricated using nano-Dragendorff's reagent to detect and determine scopolamine in different plant parts at different stages of growth. METHOD: For fabrication of the sensing phase, GO-g-PCA/DR was synthesized by encapsulation of Dragendorff's reagent (DR) on the graphene oxide grafted with poly citric acid (GO-g-PCA) with ultrasonication for 15 min and stirred for 80 min at room temperature, and then it was immobilized on a triacetyl cellulose membrane. The kinetic absorption profiles were recorded at 360 nm, which is concerned with the reaction between immobilized GO-g-PCA/DR and different concentrations of scopolamine. RESULTS: The nanosensor showed a rapid, strong, and stable response to the scopolamine solution with changing the absorption spectrum at 360 nm. The reaction was completed in a period of 300 s. The SEM, AFM, and FT-IR analysis of nanocomposites and nanosensors show the successful synthesis of GO-g-PCA/DR and the reaction between nanosensor and scopolamine. All experiments were performed at the wavelength of 360 nm, room temperature, pH 7 (the scopolamine solution pH), and 300 s. The nanosensor had a linear range of 0.65 to 19.63 µg/mL and 0.19 ± 0.025 µg/mL as the limit of detection for scopolamine determination. In order to reuse the designed nanosensor, it was recovered with ethanol, and the color ultimately returned to its original state. CONCLUSIONS: This in situ nanosensor can determine the scopolamine in real samples with easy reversibility, extended lifetime, and reproducibility of the sensing phase response. HIGHLIGHTS: A sensitive, precise, and fast response optical nanosensor is designed for in situ determination of scopolamine in real samples.

Neuroprotective Effect of Yucca schidigera Roezl ex Ortgies Bark Phenolic Fractions, Yuccaol B and Gloriosaol A on Scopolamine-Induced Memory Deficits in Zebrafish.

Y. schidigera contains a number of unusual polyphenols, derivatives of resveratrol and naringenin, called spiro-flavostilbenoids, which have potent in vitro anti-inflammatory, antioxidant, and moderate cholinesterase inhibitory activities. To date, these compounds have not been tested in vivo for the treatment of neurodegenerative diseases. The aim of the present study was to evaluate the effects of both single spiro-flavostilbenoids (yuccaol B and gloriosaol A) and phenolic fractions derived from Y. schidigera bark on scopolamine-induced anxiety and memory process deterioration using a Danio rerio model. Detailed phytochemical analysis of the studied fractions was carried out using different chromatographic techniques and Nuclear Magnetic Resonance (NMR). The novel tank diving test was used as a method to measure zebrafish anxiety, whereas spatial working memory function was assessed in Y-maze. In addition, acetylcholinesterase/butyrylcholinesterase (AChE/BChE) and 15-lipooxygenase (15-LOX) inhibition tests were performed in vitro. All pure compounds and fractions under study exerted anxiolytic and procognitive action. Moreover, strong anti-oxidant capacity was observed, whereas weak inhibition towards cholinesterases was found. Thus, we may conclude that the observed behavioral effects are complex and result rather from inhibition of oxidative stress processes and influence on cholinergic muscarinic receptors (both 15-LOX and scopolamine assays) than effects on cholinesterases. Y. schidigera is a source of substances with desirable properties in the prevention and treatment of neurodegenerative diseases.

Green extraction approach based on µSPEed® followed by HPLC-MS/MS for the determination of atropine and scopolamine in tea and herbal tea infusions.

A high throught methododology based on a green extraction technique, µSPEed®, followed by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) has been proposed for the analysis of atropine and scopolamine in tea and herbal tea infusions. For this, a digiVOL® Digital Syringe was used with different sorbents and working conditions to obtain a fast and efficient µSPEed® extraction. The best performance was achieved with a PS/DVB sorbent phase, sample loading of 5 × 500 µL and elution with 2 × 100 µL aliquots of methanol. The strategy based on µSPEed® followed by HPLC-MS/MS was validated, attaining quantitation limits lower than 0.15 ng mL-1 and recoveries between 94 and 106% for both analytes and applied to seventeen tea and herbal tea infusions. Fourteen infusions showed contamination with one or both analytes above the maximum content legislated (sum of atropine and scopolamine < 0.2 ng mL-1).

Scopolamine and atropine in feeds - determination with liquid chromatography mass spectrometry.

Tropane alkaloids (TAs) are naturally occurring plant toxins. Due to the fact that TA-producing plants can enter the food chain, they pose a risk for animals and human health. Therefore, sensitive analytical methods need to be developed to provide an adequate safety of feed and food. The presented method is based on liquid chromatography-mass spectrometry detection and enables the determination of scopolamine and atropine in compound feeds at a low level of contamination. Limits of quantification for scopolamine and atropine were 0.92 and 0.93 µg kg-1, respectively. Scopolamine-D3 and atropine-D3 were used for quantification. The method was successfully validated and applied to the analysis of 42 feed samples. Among investigated feeds, 67% contained at least one of the monitored alkaloids. Soybean meals were the feed materials contaminated most often, also with the highest determined concentrations of TAs, which reached 147.9 µg kg-1.

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.

Determination of atropine and scopolamine in spinach-based products contaminated with genus Datura by UHPLC-MS/MS.

Datura species are well known because of their high concentration of tropane alkaloids, which has led to poisoning episodes when Datura is accidentally mixed with edible crops. Therefore, the European Union has set a maximum level in cereal-based infant food products of 1 µg kg-1 for atropine and scopolamine. However, the occurrence of these compounds in other commodities has become a global concern. Spinach and derived products can be contaminated with Datura innoxia leaves. In this study, we tested frozen spinachs and spinach-based infant food products. The determination was carried out by UHPLC-MS/MS after applying the QuEChERS method as sample treatment. The LOQs were below 0.016 µg kg-1, achieving satisfactory results in terms of precision, accuracy, and matrix effects. The obtained results (ranging between 0.02 and 8.19 µg kg-1) were close to the maximum level set by the European Union for 24% of the samples tested.

Indirect competitive enzyme-linked immunosorbent assay based on a broad-spectrum monoclonal antibody for tropane alkaloids detection in pig urine, pork and cereal flours.

In this study, an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) based on a broad-spectrum monoclonal antibody for tropane alkaloids (TAs) was established for the rapid screening of atropine, scopolamine, homatropine, apoatropine, anisodamine, anisodine and L-hyoscyamine residues in pig urine, pork and cereal flour samples through a simple sample preparation procedure. The half inhibitory concentrations of atropine, homatropine, L-hyoscyamine, apoatropine, scopolamine, anisodamine and anisodine were 0.05, 0.07, 0.14, 0.14, 0.24, 5.30 and 10.15 ng mL-1, respectivelyThe detection and quantitative limits of this method for TAs in samples were 0.18-73.18 and 0.44-74.77 µg kg-1. The spiked recoveries ranged from 69.88% to 147.93%, and the coefficient of variations were less than 14%. Good correlation (R2 = 0.9929) between the results of the ic-ELISA and the high performance liquid chromatography-tandem mass spectrometry support the reliability of the developed ic-ELISA method.

Application of a streamlined LC-MS/MS methodology for the determination of atropine and scopolamine in cereals from Asian and African countries.

Tropane alkaloids are toxic secondary metabolites produced by a wide variety of plants that can be present in edible materials or animal feed. Several human poisoning cases through consumption of cereals were reported over the last years and highlighted the need for reliable and robust analytical methodologies for safety control. To rationalize analyses in high-throughput laboratory environments dealing with shorter and shorter turn-around-around time, the scope of our multi mycotoxins method was extended to the analysis of two regulated tropane alkaloids, namely atropine and scopolamine. Extraction procedure is based on the QuEChERS (Quick, Easy, Cheap, Efficient, Rugged, and Safe) approach followed by liquid chromatography tandem mass spectrometry (LC-MS/MS) detection. Quantification is performed by the isotopic dilution approach using labelled isotopomers as internal standard. The procedure was validated at two fortification levels (0.5 µg/kg and 10 µg/kg) on different cereal-based products according to the European SANTE/12682/2019 document and performance parameters such as precision (RSD(r) ≤ 6%, RSD(iR) ≤ 6%) and recovery (82-114%) fulfilled its requirements. The limit of quantification (0.5 µg/kg) is low enough to ensure compliance with existing regulations. The method was further applied on 95 cereals and cereal-based products collected from Asian and African countries. All samples were found free of the two targeted TAs, with the exception of a rice-based product in which both atropine and scopolamine were quantified at 9.6 µg/kg and 2.6 µg/kg, respectively. A total of 29 cereals samples, shown to be free of both atropine and scopolamine were also analysed for mycotoxins. Aflatoxins, fumonisins, and deoxynivalenol were sporadically detected at levels below the maximum levels defined by the European Union legislation often considered as the most stringent regulation.

Determination of tropane alkaloids in cereals, tea and herbal infusions: Exploiting proficiency testing data as a basis to derive interlaboratory performance characteristics of an improved LC-MS/MS method.

Monitoring of tropane alkaloids is regulated in the European Union in cereal-based foods for infants and young children, tea and herbal infusions. The European Commission's Joint Research Centre (JRC) developed an improved LC-MS/MS analytical method using a pentafluorophenyl column, validated it and conducted two proficiency tests targeting these food categories. A subset of the data gathered from laboratories that used the JRC method was additionally exploited to derive interlaboratory performance characteristics. The method showed fit-for-purpose figures of merit. The LOQs for atropine and scopolamine were around 0.4 and 1.2 µg/kg in cereal products, and in tea and herbal infusions, respectively. Uncertainties varied from 15 to 25%. The reproducibility varied from 11 to 38% for scopolamine and from 17 to 44% for atropine at levels ranging from 0.18 to 18.8 and 1.2-54.0 µg/kg, respectively. Recoveries ranged from 71 to 96%. These performance parameters render the method a good candidate for standardisation.

Electrochemiluminescent sensors as a screening strategy for psychoactive substances within biological matrices.

With the rapid growth and appearance of novel psychoactive substances (NPS) onto the global drug market, the need for alternative screening methodologies for implementation within clinical environments is substantial. The immunoassay methods currently in use are inadequate for this new drug trend with the potential for misdiagnosis and subsequent administration of incorrect patient treatment increased. This contribution illustrates a strong proof-of-concept for the use of electrochemiluminescence (ECL) as a screening methodology for NPS within biological fluids, using the hallucinogen scopolamine as a model compound. A low cost, easy-to-use and portable sensor has been developed and successfully employed for the detection of scopolamine at clinically relevant concentrations within a variety of biological matrices, including human pooled serum, urine, artificial saliva and sweat, without any prior sample preparation required. Moreover, assessment of the sensor's potential as a point-of-care wearable device was performed with sample collection from the surface of skin, demonstrating its capability for the qualitative identification of scopolamine despite collection of only minimal volumes off the skins surface. The developed sensor described herein exhibits a strong proof-of-concept for the employment of such ECL sensors as point-of-care devices, where the sensors ease of use and removal of time-consuming and complex sample preparation methods will ultimately increase its usability by physicians, widening the avenues where ECL sensors could be employed.

[Nitrogen metabolism and secondary metabolism regulation of Atropa belladonna by exogenous NO under NaCl stress].

Atropa belladonna seedlings were used as experimental materials and cultivated by soil culture method. Different concentrations(0,0.05,0.1,0.2,0.5 mmol·L~(-1))of NO donor sodium nitroprusside(SNP) were sprayed on the leaves. The effects of different concentrations of SNP and different treatment time(4,8,12,16 d) on nitrogen metabolism, secondary metabolite content, precursor content of tropane alkaloid synthesis pathway and expression of key enzyme genes under 100 mmol·L~(-1) NaCl stress were studied. The results showed that with the prolongation of salt stress, the nitrogen metabolism and the accumulation of secondary metabolites of A. belladonna were inhibited to some extent. After treatment with different concentrations of exogenous SNP, the ammonium nitrogen content decreased dramatically, and the contents of nitrate nitrogen, free amino acid, soluble protein and the activities of key enzymes of nitrogen metabolism(NR, GS, GDH) were all greatly improved; the contents of precursor amino acids(ornithine, arginine) and polyamines(Put, Spd, Spm) in the secondary metabolic pathway have increased to varying degrees. The qRT-PCR analysis showed that exogenous SNP treatment can effectively promote the high expression of key enzyme genes PMT, TRⅠ and H6H in the secondary metabolic pathway of A. belladonna, and the production of hyoscyamine and scopolamine were increased notably. In summary, the application of appropriate concentration of SNP can effectively alleviate the inhibition of salt stress on the nitrogen metabolism and secondary metabolism of Atropa belladonna, and enhance its salt tolerance. Overall, 0.1 mmol·L~(-1) and 0.2 mmol·L~(-1) SNP treatment achieved the most remarkable effect.

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.

Nitrogen metabolism and secondary metabolism regulation of Atropa belladonna by exogenous NO under NaCl stress / 中国中药杂志

Atropa belladonna seedlings were used as experimental materials and cultivated by soil culture method. Different concentrations(0,0.05,0.1,0.2,0.5 mmol·L~(-1))of NO donor sodium nitroprusside(SNP) were sprayed on the leaves. The effects of different concentrations of SNP and different treatment time(4,8,12,16 d) on nitrogen metabolism, secondary metabolite content, precursor content of tropane alkaloid synthesis pathway and expression of key enzyme genes under 100 mmol·L~(-1) NaCl stress were studied. The results showed that with the prolongation of salt stress, the nitrogen metabolism and the accumulation of secondary metabolites of A. belladonna were inhibited to some extent. After treatment with different concentrations of exogenous SNP, the ammonium nitrogen content decreased dramatically, and the contents of nitrate nitrogen, free amino acid, soluble protein and the activities of key enzymes of nitrogen metabolism(NR, GS, GDH) were all greatly improved; the contents of precursor amino acids(ornithine, arginine) and polyamines(Put, Spd, Spm) in the secondary metabolic pathway have increased to varying degrees. The qRT-PCR analysis showed that exogenous SNP treatment can effectively promote the high expression of key enzyme genes PMT, TRⅠ and H6H in the secondary metabolic pathway of A. belladonna, and the production of hyoscyamine and scopolamine were increased notably. In summary, the application of appropriate concentration of SNP can effectively alleviate the inhibition of salt stress on the nitrogen metabolism and secondary metabolism of Atropa belladonna, and enhance its salt tolerance. Overall, 0.1 mmol·L~(-1) and 0.2 mmol·L~(-1) SNP treatment achieved the most remarkable effect.