New light on the use of Theobroma cacao by Late Classic Maya.

Cacao seeds, Theobroma cacao, provide the basis for a ceremonially important Mesoamerican food. Past efforts to identify cacao in ceramics focused on highly decorative vessel forms associated with elite ceremonial contexts, creating assumptions as to how cacao was distributed and who could access it. This study examines 54 archaeological ceramic sherds from El Pilar (Belize/Guatemala) of Late Classic (600 to 900 CE) residential and civic contexts representing a cross-section of ancient Maya inhabitants. Identification of cacao in ancient sherds has depended on the general presence of theobromine; we used the discrete presence of theophylline, a unique key biomarker for cacao in the region. Analysis was done by grinding off all outside surfaces to reduce contamination, pulverizing the inner clay matrix, extracting absorbed molecules, and concentrating the extractions. In order to obtain especially high selectivity and low limits of detection, our study utilized the technique of resonance-enhanced multiphoton ionization coupled with laser-desorption jet-cooling mass spectrometry. This technique isolates molecules in the cold gas phase where they can be selectively ionized through a resonant two-photon process. Of the sherds analyzed, 30 samples (56%) were found to contain significant amounts of theophylline and thus test positive for cacao. Importantly, cacao is present in all contexts, common to all Maya residents near and far from centers.

Noncompetitive Determination of Small Analytes by Sandwich-Type Lateral Flow Assay Based on an Aptamer Kissing Complex.

Here, we present the proof-of-concept of a lateral flow assay (LFA) that is capable of detecting small-molecule targets in a noncompetitive manner by deploying a sandwich-type format based on the aptamer kissing complex (AKC) strategy. A fluorescently labeled hairpin aptamer served as the signaling agent, while a specific RNA hairpin grafted onto the strip served as the capture element. The hairpin aptamer switched from an unfolded to a folded form in the presence of the target, resulting in kissing interactions between the loops of the reporter and the capture agents. This design triggered a target-dependent fluorescent signal at the test line. The AKC-based LFA was developed for the detection of adenosine, achieving a detection limit in the micromolar range. The assay revealed the presence of the same analyte in urine. The method also proved effective with another small molecule (theophylline). We believe that the AKC-based LFA approach could overcome many of the shortcomings associated with conventional signal-off methods and competitive processes.

The analysis of methylxanthine fractions obtained from Camellia sinensis cultivated in Turkey and effects on the in vitro inhibition of CYP2D6 enzyme.

Tea is a worldwide consumed herbal beverage and it was aimed in this study to reveal the major fractions of green and black tea in order to enlighten the in vitro inhibition potency on the well-known drug metabolizing enzyme CYP2D6 activity. Methylxanthine fractions were extracted from green and black tea and a yield of 0.265 g (1.06%) for 25 g of dried black tea and 0.302 g (1.2%) for 25 g of green tea was calculated. High-performance liquid chromatography analysis represented that the major components of the methylxanthine fractions were caffeine, theobromine, and theophylline. Methylxanthine content of black tea was 368.25 ± 4.6 µg/ml caffeine, 89.30 ± 2.3 µg/ml theobromine, and 3.40 ± 0.5 µg/ml theophylline, whereas that of green tea was 176.50 ± 3.7 µg/ml caffeine, 53.85 ± 1.4 µg/ml theobromine, and 2.06 ± 0.7 µg/ml theophylline. The results of concentration-dependent inhibition studies were 76% green tea, 75% black tea, and 55% caffeine at concentration of 10 mg/ml. The inhibition rates of green and black tea on CYP2D6 activity were 76% and 75%, respectively, where that of quinidine, the well-known inhibitor of CYP2D6, was 82%. Our results indicate that green and black tea is very likely to modify the CYP2D6 enzyme activity.

Electrochemical sensing platform based on covalent organic framework materials and gold nanoparticles for high sensitivity determination of theophylline and caffeine.

A new covalent organic framework (COF) has been prepared with 1,3,6,8-tetra(4-formyl phenyl) pyrene (TFPPy) and 2,6-diaminopyridine (DP) as building units through a Schiff base reaction by a simple tube oven heating procedure and the structure of the COF has been characterized in detail. The obtained DP-Py COF is employed to fabricate a novel electrochemical sensing platform for sensitive and selective determination of theophylline (TP) and caffeine (CAF) simultaneously through compounding with AuNPs; the peak positions of TP and CAF are 0.95 V and 1.28 V, respectively. The synergistic effect between DP-Py COF and AuNPs effectively enhances the analytical sensitivity for the target analytes. Under the optimized experimental conditions, the electrochemical sensing platform shows a sensitive voltammetric response and wide linear range to both TP and CAF, and the detection limits are 0.19 µM and 0.076 µM (S/N = 3), respectively. This method has been successfully used for the determination of TP and CAF in compound paracetamol capsules and black tea samples. The recovery and relative standard deviations (RSD) of TP are 99.3~101% and 97.6~101% and 1.3~2.0% and 1.3~2.1%, respectively, and the recovery and RSD of CAF are 96.1~102% and 99.4~104% and 2.8~3.9% and 1.7~3.2%, respectively. Compared with traditional detection methods, the constructed sensing platform has better performance and is expected to be widely used also in other real sample analyses.

Electrochemical fabrication of reduced MoS2-based portable molecular imprinting nanoprobe for selective SERS determination of theophylline.

A new portable molecular imprinting polymer (MIP)-SERS nanoprobe is fabricated by a convenient electrochemical method. Single-layered MoS2 is electrochemically reduced on a screen-printed electrode as the scaffold. Functional monomers o-phenylenediamine (oPD), template theophylline (THP), and SERS-active Au nanoparticles (AuNPs) are then one-step electropolymerized on the scaffold. The morphology of the nanoprobe is found to be a three-dimensional and porous structure. The abundant AuNPs with the size of 45~50 nm are trapped within the growing MIP instead of being confined to the surface. The thickness of MIP film is calculated to 25.1 nm. The nanoprobe displays a strong SERS effect for THP using 532 nm as excitation wavelength with a detection limit (LOD) of 0.01 nM. The SERS peak intensity at 1487 cm-1 increases linearly with the concentration of THP in the range 0.1 nM to 0.1 mM. After the template is removed, the imprint-removed nanoprobe is generated for selective binding of THP. The re-binding kinetics study implies the portable MIP-SERS nanoprobe can reach the adsorption equilibrium within 8 min. This nanoprobe exhibits low SERS interference for structural analogues theobromine (THB) and caffeine (CAF). The nanoprobe was employed to THP determination in tea drink samples, with recoveries ranging from 99.0 to 102.0% and relative standard deviations of < 5.0%. Graphical abstractSchematic representation of a portable molecular imprinting SERS nanoprobe used for selective and sensitive theophylline recognition. The nanoprobe is fabricated by one-step electropolymerized o-phenylenediamine (oPD), theophylline, and electroreduced Au nanoparticles (AuNPs) on reduced MoS2 (rMoS2) modified screen-printed electrode (SPE).

Preparation of porous aromatic framework modified graphene oxide for pipette-tip solid-phase extraction of theophylline in tea.

A new material called as porous aromatic frameworks modified graphene oxide (PAFs-GO) was synthesized, and it was used as an adsorbent in pipette-tip SPE for the effective purification and enrichment of theophylline in tea sample by HPLC. The properties of PAFs-GO were characterized by field emission SEM, FTIR, thermogravimetry analysis and Brunauer Emmett Teller N2 adsorption-desorption analysis. The results of static adsorption and dynamic adsorption test showed PAFs-GO had higher adsorption ability (93.25 mg/g) than graphene oxide. The LOD and LOQ of the method were 0.0141 and 0.0471 µg/mL, respectively. The acceptable method reproducibility was found as intra- and inter-day precisions, yielding the RSDs <4.62%. By introducing PAFs as support skeleton, the specific surface area of GO was effectively increased, and the penetrability was improved. Studies showed that the proposed method had been successfully applied for purification and enrichment of theophylline in complex tea matrix.

Flow-injection chemiluminescence of the luminol-potassium periodate system enhanced by TGA-capped CdTe quantum dots for the determination of theophylline.

The chemiluminescence (CL) behaviour of the luminol-potassium periodate system enhanced by CdTe quantum dots capped with thioglycolic acid (TGA-CdTe QDs) was studied using kinetic experiments, CL spectra, UV-vis absorption spectra and fluorescence spectra. The production of oxygen-containing reactant intermediates (O2 •- and OH• ) in the present CL system was verified by CL. The possible CL mechanism was discussed in detail. Furthermore, theophylline (THP) was determined based on its enhancement of the CL intensity of the CdTe QDs-luminol-potassium periodate system coupled with a flow-injection technique. Under these optimized conditions, the linear range was found to be from 1.0 × 10-8 to 1.0 × 10-5  g/mL with a detection limit of 2.8 × 10-9  g/mL (3σ). The recoveries for the determination of THP in tablets were from 98.2 to 99.6%.

A universal aptasensing platform based on cryonase-assisted signal amplification and graphene oxide induced quenching of the fluorescence of labeled nucleic acid probes: application to the detection of theophylline and ATP.

This study describes a universal fluorometric method for sensitive detection of analytes by using aptamers. It is based on the use of graphene oxide (GO) and cryonase-assisted signal amplification. GO is a strong quencher of FAM-labeled nucleic acid probes, while cryonase digests all types of nucleic acid probes. This makes the platform widely applicable to analytes for which the corresponding aptamers are available. Theophylline and ATP were chosen as model analytes. In the absence of targets, dye-labeled aptamers are in a flexible single strand state and adsorb on the GO. As a result, the probes are non-fluorescent due to the efficient quenching of dyes by GO. Upon the addition of a specific target, the aptamer/target complex desorbed from the GO surface and the probe becomes fluorescent. The released complex will immediately become a substrate for cryonase digestion and subsequently releasing the target to bind to another aptamer to initiate the next round of cleavage. This cyclic reaction will repeat again and again until all the related-probes are consumed and all fluorophores light up, resulting in significant fluorescent signal amplification. The detection limits are 47 nM for theophylline and 22.5 nM for ATP. This is much better than that of known methods. The assay requires only mix-and-measure steps that can be accomplished rapidly. In our perception, the detection scheme holds great promise for the design enzyme-aided amplification mechanisms for use in bioanalytical methods. Graphical abstract A cryonase-assisted signal amplification (CASA) method has been developed by using graphene oxide (GO) conjugated with a fluorophore-labeled aptamer for fluorescence signal generation. It has a large scope because it may be applied to numerous analytes.

Simultaneous voltammetric determination of acetaminophen, naproxen, and theophylline using an in-situ polymerized poly(acrylic acid) nanogel covalently grafted onto a carbon black/La2O3 composite.

Lanthanum oxide nanomaterials were decorated with carbon black (CB) and grafted with a poly(acrylic acid) nanogel to obtain a composite material (CB-g-PAA/La2O3) for simultaneous determination of acetaminophen (AMP), naproxen (NPX), and theophylline (TPH). The nanogel was synthesized by in-situ free radical polymerization. The composite was dropped onto a glassy carbon electrode (GCE), and the modified GCE displays robust electrocatalytic activity towards AMP, NPX, and TPH, with voltammetric signals that are enhanced compared to a bare GCE. Features of merit for AMP, NPX, and TPH, respectively, include (a) peak potentials of 0.42, 0.85 and 0.12 V (vs. Ag/AgCl), (b) linear ranges from 0.05-887, 0.05-884, and 0.02-888 µM, and (c) detection limits of 20, 35, and 15 nM. The practical applicability of the CB-g-PAA/La2O3/GCE was illustrated by analyzing serum and urine samples. Graphical abstract Schematic presentation of simultaneous electrochemical sensing of acetaminophen (AMP), naproxen (NPX), and theophylline (TPH) in real sample analysis using poly(acrylic acid) nanogel covalently grafted onto a carbon black/La2O3 composite (CB-g-PAA/La2O3/GCE).

Validation of an LC-MS/MS Method for the Quantification of Caffeine and Theobromine Using Non-Matched Matrix Calibration Curve.

Caffeine is one of the most widely consumed psycho-stimulants. The study of the beneficial effects of caffeine consumption to decrease the risk of developing several neuropsychiatric pathologies is receiving increasing attention. Thus, accurate and sensitive methods have been developed, mainly by LC-MS/MS, in order to quantify caffeine and its metabolites. These quantifications of caffeine and its metabolites by LC-MS/MS require a considerable effort to select or find a surrogate matrix, without the compounds of interest, to be used in the calibration curves. Thus, we evaluated the possibility of using calibration curves prepared in solvent instead of calibration curves prepared in human plasma. Results show that the calibration curves prepared in solvent and in human plasma were similar by comparing their slopes and interceptions, and the accuracy and precision were within the limits of acceptance for both calibration curves. This work demonstrates that, by using internal standards, it is possible to use a calibration curve in solvent instead of a calibration curve in plasma to perform an accurate and precise quantification of caffeine and theobromine.

A Simple Colorimetric System for Detecting Target Antigens by a Three-Stage Signal Transformation-Amplification Strategy.

Inexpensive, straightforward, and rapid medical diagnostics are becoming increasingly important for disease identification in time- and resource-limited settings. Previous attempts to link oligonucleotide-based aptamers and hammerhead ribozymes to form ligand-induced ribozymes have been successful in identifying a variety of small molecule and protein targets. Isothermal exponential amplification reactions (EXPAR) amplify minute amounts of nucleic acid templates without requiring special instrumentation. We introduce a colorimetric assay that we engineered using an aptamer, hammerhead ribozyme, EXPAR, and peroxidase activity in conjunction with a 3,3',5,5'-tetramethylbenzidine (TMB) substrate. This is a modular signal enhancer system that can be easily modified to detect virtually any chosen analyte target within 5-10 min with minimal technical requirements. Ligand-aptamer binding causes the ribozyme to change conformation and self-cleave. The cleaved ribozyme triggers exponential amplification of a reporter sequence during EXPAR. The amplification products fold into single-stranded DNA guanine quadruplexes that exhibit peroxidase-like activity and can oxidize a colorless TMB substrate into a colored reaction product for visual detection. As a proof of concept, we examined the bronchodilator theophylline versus its chemical analogue, caffeine. We demonstrate linear changes in absorption readout across a wide range of target concentrations (0.5-1000 µM) and the ability to visually detect theophylline at 0.5 µM with an approximately 35-fold increased specificity versus that of caffeine. This three-stage detection system is a versatile platform that has the potential to improve the rapid identification of target analytes.

Ternary deep eutectic solvent magnetic molecularly imprinted polymers for the dispersive magnetic solid-phase microextraction of green tea.

Ternary deep eutectic solvent magnetic molecularly imprinted polymers grafted on silica were developed for the selective recognition and separation of theophylline, theobromine, (+)-catechin hydrate, and caffeic acid from green tea through dispersive magnetic solid-phase microextraction. A new ternary deep eutectic solvent was adopted as a functional monomer. The materials obtained were characterized by FTIR spectroscopy, field emission scanning electron microscopy, transmission electron microscopy, NMR spectroscopy, and powder X-ray diffraction. The practical recovery of the theophylline, theobromine, (+)-catechin hydrate, and caffeic acid isolated with ternary deep eutectic solvent magnetic molecularly imprinted polymers in green tea were 91.82, 92.13, 89.96, and 90.73%, respectively, and the actual amounts extracted were 5.82, 4.32, 18.36, and 3.69 mg/g, respectively. The new method involving the novel material coupled with dispersive magnetic solid-phase microextraction showed outstanding recognition, selectivity and excellent magnetism, providing a new perspective for the separation of bioactive compounds.

Suberin Fatty Acids from Outer Birch Bark: Isolation and Physical Material Characterization.

The isolation and physical material properties of suberin fatty acids (SFAs) were investigated with special reference to their potential applications as novel pharmaceutical excipients. SFAs were isolated from outer birch bark (OBB) with a new extractive hydrolysis method. The present simplified isolation process resulted in a moderate batch yield and chemical purity of SFAs, but further development is needed for establishing batch-to-batch variation. Cryogenic milling was the method of choice for the particle size reduction of SFAs powder. The cryogenically milled SFAs powder exhibited a semicrystalline structure with apparent microcrystalline domains within an amorphous fatty acids matrix. The thermogravimetric analysis (TGA) of SFAs samples showed a good thermal stability up to 200 °C, followed by a progressive weight loss, reaching a plateau at about 95% volatilization at about 470 °C. The binary blends of SFAs and microcrystalline cellulose (MCC; Avicel PH 101) in a ratio of 25:75 (w/w) displayed good powder flow and tablet compression properties. The corresponding theophylline-containing tablets showed sustained or prolonged-release characteristics. The physicochemical and bulk powder properties of SFAs isolated from OBB are auspicious in terms of potential pharmaceutical excipient applications.

Probing the Distribution of Water in a Multi-Component System by Solid-State NMR Spectroscopy.

PURPOSE: To characterize the distribution of water among various components in a powder blend using solid-state NMR spectroscopy. METHODS: Water sorption behavior of theophylline anhydrate and excipients was determined by dynamic vapor sorption (DVS) and Karl Fischer Titration (KFT) after storing them in humidity chambers for 1 week at room temperature (RT) and calibration curves were generated for water content vs. (1)H T 1 relaxation times. Powder blends (either with microcrystalline cellulose or lactose as diluent) were stored at different relative humidity (RH) conditions and analyzed periodically using solid-state NMR, powder X-ray diffraction, and KFT. RESULTS: Anhydrous theophylline converted to the hydrate at ≥ 84% RH. Based on the calibration curves of water content vs. relaxation times, the distribution of water in the powder blends was estimated. The total water content calculated using ssNMR was in good agreement with values measured using KFT. In blends stored at 90% RH, theophylline anhydrate-to-hydrate conversion did not occur in 1 week. CONCLUSIONS: The distribution of water in multi-component powder blends was successfully determined using correlation between (1)H T 1 relaxation times and total water content. Excipient water sorption inhibited hydrate formation in theophylline at 90% RH. Water distribution was affected by excipient type. The extent of water sorbed by excipients in blends was found to be different than their standalone equilibrium water content.

Biofunction-assisted aptasensors based on ligand-dependent 3' processing of a suppressor tRNA in a wheat germ extract.

We have developed a novel type of biofunction-assisted aptasensor that harnesses ligand-dependent 3' processing of a premature amber suppressor tRNA and the subsequent amber suppression of a reporter gene in a wheat germ extract.

Simultaneous multiplexed quantification of caffeine and its major metabolites theobromine and paraxanthine using surface-enhanced Raman scattering.

Accurate quantitative measurement of drugs and their metabolites is important as this can be used to establish long-term abuse of illicit materials as well as establish accurate drug dosing for legal therapeutics. However, the levels of drugs and xenometabolites found in human body fluids necessitate methods that are highly sensitive as well as reproducible with the potential for portability. Raman spectroscopy does offer excellent reproducibility, portability and chemical specificity, but unfortunately, the Raman effect is generally too weak unless it is enhanced. We therefore developed surface-enhanced Raman scattering (SERS) and combined it with the powerful machine learning technique of artificial neural networks to enable rapid quantification of caffeine and its two major metabolites theobromine and paraxanthine. We established a three-way mixture analysis from 10(-5) to 10(-7) mol/dm(3), and excellent predictions were generated for all three analytes in tertiary mixtures. The range we selected reflects the levels found in human body fluids, and the typical errors for our portable SERS analysis were 1.7 × 10(-6) mol/dm(3) for caffeine, 8.8 × 10(-7) mol/dm(3) for theobromine and 9.6 × 10(-7) mol/dm(3) for paraxanthine. We believe this demonstrates the exciting prospect of using SERS for the quantitative analysis of multiple analytes simultaneously without recourse to lengthy and time-consuming chromatography, a method that often has to be combined with mass spectrometry.

Quantitative on-line concentration for capillary electrophoresis with inkjet sample introduction technique.

A quantitative sample introduction method based upon inkjet injection was applied to capillary electrophoresis coupled with stacking and sweeping on-line concentration techniques. Methylxanthines were used as model compounds for the proof-of-concept of the method. The volume of injected sample could be easily manipulated by controlling the number of ejected droplets in the injection procedure. Under optimized conditions, a linear relationship between the ejected droplet number and peak area was obtained when the droplet number introduced into the capillary was less than 100. Under optimized quantitative on-line concentration conditions, the limits of detection for theobromine, caffeine, and theophylline were 1.0, 2.0, and 1.0 µM, respectively. The inkjet injection system was evaluated by comparing it with conventional injection methods. The electropherogram of the inkjet injection mode was the same as that for hydrodynamic injection mode, and no sample discrimination was observed compared with the electrokinetic injection mode. The established method was applied to the determination of methylxanthines in bottled green tea. The recoveries of theobromine, caffeine, and theophylline were 94.1, 110.6, and 86.8%, respectively. We conclude that proposed method can be used for quantitative concentration for capillary electrophoresis, thus resulting in an improved accuracy.

Film-coated matrix mini-tablets for the extended release of a water-soluble drug.

Extended release (ER) of water-soluble drugs from hydroxypropylmethylcellulose (HPMC) matrix mini-tablets (mini-matrices) is difficult to achieve due to the large surface area to volume ratio of the mini matrices. Therefore, the aims of this study were to control the release of a water-soluble drug (theophylline) from mini-matrices by applying ER ethylcellulose film coating (Surelease®), and to assess the effects of Surelease®:pore former (Opadry®) ratio and coating load on release rates. Mini-matrices containing 40%w/w HPMC K100M CR were coated with 100:0, 85:15, 80:20, 75:25 or 70:30 Surelease®:Opadry® to different coating weight gains (6-20%). Non-matrix mini-tablets were also produced and coated with 80:20 Surelease®:Opadry® to different coating weight gains. At low coating weight gains, nonmatrix mini-tablets released the entire drug within 0.5 h, while at high coating weight gains only a very small amount (<5%) of drug was released after 12 h. The gel formation of HPMC prevented disintegration of mini-matrices at low coating weight gains but contributed to rupture of the film even at high coating weight gains. As a result, drug release from mini-matrices was slower than that from nonmatrix mini-tablets at low coating weight gains, yet faster at high coating weight gains. An increase in the lag time of drug release from mini-matrices was observed as the concentration of Opadry® reduced or the coating weight gain increased. This study has demonstrated the possibility of extending the release of a water-soluble drug from HPMC mini-matrices by applying ER film coating with appropriate levels of pore former and coating weight gains to tailor the release rate.

Alkaloids and athlete immune function: caffeine, theophylline, gingerol, ephedrine, and their congeners.

Plant alkaloids are found in foods, beverages, and supplements consumed by athletes for daily nutrition, performance enhancement, and immune function improvement. This paper examined possible immunomodulatory roles of alkaloids in exercise contexts, with a focus on human studies. Four representative groups were scrutinized: (a) caffeine (guaranine, mateine); (b) theophylline and its isomers, theobromine and paraxanthine; (c) ginger alkaloids including gingerols and shogaol; and (d) ephedra alkaloids such as ephedrine and pseudoephedrine. Emerging or prospective alkaloid sources (Goji berry, Noni berry, and bloodroot) were also considered. Human in vitro and in vivo studies on alkaloids and immune function were often conflicting. Caffeine may be immunomodulatory in vivo depending on subject characteristics, exercise characteristics, and immune parameters measured. Caffeine may exhibit antioxidant capacities. Ginger may exert in vivo anti-inflammatory effects in certain populations, but it is unclear whether these effects are due to alkaloids or other biochemicals. Evidence for an immunomodulatory role of alkaloids in energy drinks, cocoa, or ephedra products in vivo is weak to nonexistent. For alkaloid sources derived from plants, variability in the reviewed studies may be due to the presence of unrecognized alkaloids or non-alkaloid compounds (which may themselves be immunomodulatory), and pre-experimental factors such as agricultural or manufacturing differences. Athletes should not look to alkaloids or alkaloid-rich sources as a means of improving immune function given their inconsistent activities, safety concerns, and lack of commercial regulation.

Detection of theophylline utilising portable electrochemical sensors.

The electrochemical oxidation of theophylline (TP) is investigated utilising screen-printed electrodes. Through thorough investigation of pH, we propose a reaction mechanism, finding that the oxidation of TP is stable over a wide pH range, in particular under acidic conditions. Conversely under alkaline conditions, theophylline fouls the electrode surface. The screen-printed carbon sensors are applied towards the electroanalytical sensing of TP with a remarkable amount of success in aqueous solution at physiological pH. The screen-printed sensors have been shown to be applicable to the detection of TP at unharmful, medicinally relevant (55-110 µM), and toxic concentrations in aqueous media at physiological pH. Thus this work presents a proof-of-concept approach towards TP detection utilising sensors commonly implemented in point-of-care applications.