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Drug-drug interactions may amplify or diminish their intended effects, or even produce entirely new effects. Multicomponent mixture HPLC analysis offers a thorough and effective method for comprehending the makeup and behavior of complicated materials, advancing research and development across a range of scientific and industrial domains. A novel experimental design-assisted HPLC methodology for the concurrent investigation of the drug-drug interaction of pholcodine, ephedrine, and guaifenesin in biological fluids has been established. Rather than the routine methodology, the application of the factorial design-HPLC method offers a powerful and efficient tool for the analysis of these compounds. Both mixed and full factorial designs were employed to assess the impact of variable factors on chromatographic results. Utilizing an isocratic elution mode on a C 18 column, the chromatographic separation was carried out. 15% Methanol, 5% acetonitrile, and 80% phosphate buffer with 0.1%(v/v) triethylamine set to pH 3 make up the mobile phase flowing at rate 1.0 mL/min. The calibration curves of the drugs show excellent linearity over a concentration ranges: 0.20-13.0 µg/mL for PHO, 0.50-20.0 µg/mL for EPH and 0.70-20.0 µg/mL for GUA with LOQ values of 0.18, 0.38 and 0.50. The fast separation and quantitation in less than 6 min is an advantage. Also, the method includes a robust sample preparation protocol for the analysis of complex biological samples, ensuring high selectivity and precision. The ease, speed and cost-effectiveness of the method are ideal for supporting in vitro studies, including drug-drug interaction investigations, especially in bioanalytical labs., Competing Interests: Declarations Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

Rationale: In recent years, ephedrine psychoactive substances have attracted much attention due to their prevalence in water bodies and potential threat to aquatic ecosystems. Psychoactive substances have been considered as a new type of environmental pollutant due to their unpredictable potential risks to the behavior and nervous system of non-target organisms. A rapid, sensitive, selective, and robust method for the quantification of three ephedrine psychoactive substances in sewage is needed., Methods: An ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed for the simultaneous determination of three ephedrine psychoactive substances in water. The optimal processing conditions were determined by optimizing the chromatography-mass spectrometry and solid-phase extraction (SPE) conditions (e.g., the SPE column, sample pH, washing, and elution), and the treatment conditions were determined; this was achieved via positive ion scanning in multiple reaction monitoring mode. Poly-Sery MCX was selected as the extraction column, with samples loaded at pH 3. And 4-mL solution of 2% formic acid (FA) aqueous solution was used as the eluent; the target compounds were eluted with 5 mL of 5% NH 4 OH in acetonitrile (ACN) solution. The best results were obtained when the residue was resolubulization in ACN after nitrogen evaporation., Results: The developed UPLC-MS/MS showed a good linear relationship in the range of 0-50.00 μg/L, with determination coefficients (R 2 ) greater than 0.9990. The detection limit and quantitation limit were 0.05-0.10 and 0.20-0.50 μg/L, respectively. Recovery rates of the target compounds in blank sewage at three different concentrations ranged from 92.37% to 106.31%, with relative standard deviations (RSDs) of 0.77%-4.83% (n = 7)., Conclusions: This method has been successfully applied to the analysis of surface water and domestic sewage, and the samples were processed stably, indicating that the method is practical for the determination of ephedrine psychoactive drugs in water bodies., (© 2024 John Wiley & Sons Ltd.)

A ternary mixture incorporating Hydroxyzine hydrochloride (HYX), Ephedrine hydrochloride (EPH) and Theophylline (THP) frequently prescribed for the treatment of respiratory diseases. Herein, two spectrophotometric methods are designated and applied to resolve these three components in their mixture. Method A is ratio-subtraction combined with derivative spectrophotometry, where THP can be determined directly at its λmax 271 nm (neither HYX or EPH interfere), then for determination of HYX and EPH, the ternary mixture was divided by 22 μg/mL of THP and after subtraction of the plateau region, HYX can be determined directly at its λmax 234.2 nm (absence of EPH intervention). Finally, the third derivative (3D) spectrophotometric approach was utilized to estimate EPH by detecting the peak amplitude at 222 nm with Δλ = 4 and a scaling factor 100. Principal Component Regression (PCR) and Partial Least Squares (PLS), two multivariate calibration approaches, were applied effectively in Method B. This method effectively quantified the mixture under investigation by using the absorption spectra obtained from suitable solutions of the three components in the 210-230 nm region. The calibration models were evaluated using cross-validation with PCR and PLS, producing statistical characteristics that demonstrate the effectiveness of the calibration models. Synthetic and pharmaceutical preparations were also used to conduct external validation. In pharmaceutical formulation, these methods were successfully applied to analyze HYX, EPH, and THP without overlap from formulation's excipients. Moreover, the study's findings were statistically contrasted with those of earlier reported HPLC method. Appraisal approaches were used to determine whether the new spectrophotometric methods had an adverse environmental impact involving the Green Analytical Procedure Index (GAPI) and the AGREE (Analytical Greenness). These evaluations delivered information about the methods' eco-friendliness and sustainability, proving that they are in line with ecologically attributed practices. Furthermore, the Blue Applicability Grade Index (BAGI) was utilized to identify and verify the feasibility and practicality of the suggested approaches., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright: © 2024 AlSalem et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Brain can be a useful specimen for toxicology testing as it is a protected and isolated organ with lower metabolic activity than other tissues, but there is currently no published data supporting the stability of stimulant drugs in prepared brain homogenates. Brain homogenates were evaluated to determine the stability of the following stimulant drugs: amphetamine, benzoylecgonine, bupropion, cocaethylene, cocaine, ephedrine, methylenedioxyamphetamine, methylenedioxymethamphetamine, methamphetamine, and phentermine. Four different homogenates were prepared at a 1:4 dilution with deionized water and fortified at 500 ng/mL of: cocaine without sodium fluoride, cocaine with 1% sodium fluoride, stimulant drugs other than cocaine without sodium fluoride, and stimulant drugs other than cocaine with 1% sodium fluoride. The fortified homogenates were aliquoted into 13 × 100-mm screw cap tubes and stored at room temperature (∼20°C), refrigerated (2-8°C), or frozen (<-5°C) and analyzed in triplicate on Days 0, 1, 3, 7, 14, 30, 60, and 90. Analytes were considered stable as long as the difference in analyte/internal standard response ratio from Day 0 was less than 20% and the peaks met qualitative acceptance criteria. All analytes were stable for up to 90 days when stored frozen with or without sodium fluoride and had variable stability at all other evaluated conditions., (© The Author(s) 2024. Published by Oxford University Press. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site–for further information please contact journals.permissions@oup.com.)

Background and Aims: The use and manufacture of methamphetamine has increased in Afghanistan in recent years. Recent research and reports have pointed to the ephedra plant, which grows wildly, as a key source of ephedrine used in the manufacture of methamphetamine. This paper aimed to estimate the relative efficiencies and scale of inputs required to manufacture methamphetamine in Afghanistan., Methods: Monte Carlo simulations model of the amount of ephedra or cold medications needed to render a pure kilogram of methamphetamine in Afghanistan, accounting for uncertainty in ranges of key parameters informed from the literature and elsewhere. Final estimates were extrapolated to recent seizure totals., Results: For dried ephedra, the median estimate is 196.8 kg (25th-75th percentiles 119.3-346.6 kg) needed to produce 1 kg of methamphetamine compared with 27.9 kg (25th-75th percentiles 21.9-36.8 kg) for cold medications. Nearly 2.7 t of methamphetamine were seized in Afghanistan in 2021. Assuming a purity range of 50%-90%, some 266-478 t of dried ephedra or 38-68 t of cold medication would need to have been processed., Conclusion: Simulated estimates show that considerable amounts of either ephedra or cold medication are needed to produce 1 kg of methamphetamine in Afghanistan. This raises questions about the plausibility of ephedra as the dominant source of Afghanistan's methamphetamine., (© 2024 Society for the Study of Addiction.)

Background: Spectrophotometric resolution of a mixture of several drugs is considered a cheaper, simpler, and more versatile alternative compared to costly chromatographic instruments., Objective: The work aims to resolve the interfering spectra of ephedrine hydrochloride, naphazoline nitrate, and methylparaben in nasal preparations using smart spectrophotometric methods., Method: In our work, derivative and dual-wavelength methods were combined to eliminate this interference, under the name of derivative dual-wavelength method. Other methods, namely successive derivative subtraction and chemometric analysis, were also able to eliminate this interference. The methods have proven their applicability as they follow the International Conference on Harmonization (ICH) requirements regarding repeatability, precision, accuracy, selectivity, and linearity. Eco-scale, GAPI, and AGREE tools were used to estimate the possible environmental effects of the methods., Results: Acceptable results for repeatability, precision, accuracy, selectivity, and linearity were obtained. Limit of detection (LOD) values were 2.2 for ephedrine and 0.3 for naphazoline. The correlation coefficients were above 0.999. The methods were proven to be safe for application., Conclusions: The introduced methods are cheap and easily implemented compared to chromatographic techniques. They can be used in purity-checking of raw material and estimation of concentrations in market formulations. The replacement of the published chromatographic techniques with our developed methods is useful when needing to save money, effort, and time., Highlights: The three components of a decongestant nasal preparation were determined using cheap, green, and versatile spectrophotometric methods that keep the advantages of chromatographic techniques, including accuracy, reproducibility, and selectivity., (© The Author(s) 2023. Published by Oxford University Press on behalf of AOAC INTERNATIONAL. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

This study focused on the separation, characterization, content determination, and antiviral efficacy research on colloidal particles with different sizes in Maxing Shigan Decoction(MXSG). The mixed colloidal phase of MXSG was initially separated into small colloidal particle segment(S), medium colloidal particle segment(M), and big colloidal particle segment(B) using ultrafiltration. Further fine separation was performed using size-exclusion chromatography. Dynamic light scattering(DLS) and transmission electron microscopy(TEM) were employed to characterize the size and morphology of the separated colloidal particles. UPLC-MS/MS was used to determine the content of ephedrine, amygdalin, glycyrrhizic acid, and the EDTA complexometric titration was used to measure the calcium(Ca~(2+)) content in different colloidal phases. Finally, a respiratory syncytial virus(RSV) infection mouse model was established using intranasal administration. The experimental groups included a blank group, a model group, a ribavirin group, an MXSG group, an S group, an M group, and a B group. Oral administration was given for treatment, and pathological changes in mouse lung tissue and organ indices were evaluated. The results of the study showed that the distribution of ephedrine, amygdalin, glycyrrhizic acid, and Ca~(2+) content was not uniform among different colloidal segments. Among them, the B segment had the highest proportions of the three components, except for Ca~(2+), accounting for 46.35%, 53.72%, and 92.36%, respectively. Size-exclusion chromatography separated colloidal particles with uniform morphology in the size range of 100-500 nm. Compared to the S and M segments, the B segment showed an increased lung index inhibition rate(38.31%), spleen index, and thymus index in RSV-infected mice, and it improved the infiltration of inflammatory cells and lung injury in the lung tissue of mice. The complex components in MXSG form colloidal particles of various sizes and morphologies through heating, and small-molecule active components such as ephedrine, amygdalin, glycyrrhizic acid, and Ca~(2+) participate in the assembly to varying degrees. The main material basis for the antiviral effect of MXSG is the colloidal particles with certain particle sizes formed by the assembly of active components during the heating process.

A previous 1 H-NMR method allowed the quantification of ephedrine alkaloids; however, there were some disadvantages. The cyclized derivatives resulted from the impurities of diethyl ether were identified and benzene was selected as the better extraction solvent. The locations of ephedrine alkaloids were confirmed with 2D NMR. Therefore, a specific 1 H-NMR method has been modified for the quantification of ephedrine alkaloids. Accordingly, twenty Ephedrae Herba samples could be classified into three classes: (I) E. sinica -like species; (II) E. intermedia -like species; (III) others (lower alkaloid contents). The results indicated that ephedrine and pseudoephedrine are the major alkaloids in Ephedra plants, but the concentrations vary greatly determined by the plant species and the collection locations.

A total of 147 oral Kampo prescriptions, which are used clinically in Japan, were evaluated for their anti-glycation activity. Kakkonto demonstrated significant anti-glycation activity, prompting further analysis of its chemical constituents using LC-MS, which revealed the presence of two alkaloids, fourteen flavonoids, two but-2-enolides, five monoterpenoids, and four triterpenoid glycosides. To identify the components responsible for its anti-glycation activity, the Kakkonto extract was reacted with glyceraldehyde (GA) or methylglyoxal (MGO) and analyzed using LC-MS. In LC-MS analysis of Kakkonto reacted with GA, the peak intensity of ephedrine was attenuated, and three products from ephedrine-scavenging GA were detected. Similarly, LC-MS analysis of Kakkonto reacted with MGO revealed two products from ephedrine reacting with MGO. These results indicated that ephedrine was responsible for the observed anti-glycation activity of Kakkonto. Ephedrae herba extract, which contains ephedrine, also showed strong anti-glycation activity, further supporting ephedrine's contribution to Kakkonto's reactive carbonyl species' scavenging ability and anti-glycation activity.

Ephedra herba is a conventional Chinese medicine to treat cold, fever, asthma, edema, and lung diseases in the clinic. At present, most pharmacokinetic studies focus on the pharmacokinetic process of alkaloids in normal animals. However, the non-alkaloid components are also active. In addition, the pharmacokinetic studies under pathological state make more sense for clarifying the material basis of efficacy. In this study, a sensitive and rapid ultra-high-performance-tandem mass spectrometry method was developed and applied to determine nine bioactive components (ephedrine, pseudoephedrine, methylephedrine, (+)-catechin, epicatechin, vitexin, vicenin-2, cinnamic acid, and ferulic acid) in normal, common cold and nephrotic syndrome rats after the oral administration of Ephedra herba. Compared to the normal group, except for ferulic acid, the exposure levels of the other eight components were significantly increased and the plasma clearance clearly declined in common cold rats. Similarly, the exposure levels of seven components other than cinnamic acid and ferulic acid were also significantly augmented and the plasma clearance decreased significantly in nephrotic syndrome rats. In brief, the pathological conditions of the common cold and nephrotic syndrome could lead to alterations in the pharmacokinetics profiles of the nine components, which provide a reference for further exploration of the pharmacodynamics basis of Ephedra herba., (© 2023 Wiley-VCH GmbH.)

Amphetamine and cathinone derivatives are abused recreationally due to the sense of euphoria they provide to the user. Methodologies for the rapid detection of the drug derivative present in a seized sample, or an indication of the drug class, are beneficial to law enforcement and healthcare providers. Identifying the drug class is prudent because derivatisation of these drugs, to produce regioisomers, for example, occurs frequently to circumvent global and local drug laws. Thus, newly encountered derivatives might not be present in a spectral library. Employment of benchtop nuclear magnetic resonance (NMR) could be used to provide rapid analysis of seized samples as well as identifying the class of drug present. Discrimination of individual amphetamine-, methcathinone-, N-ethylcathinone and nor-ephedrine-derived fluorinated and methylated regioisomers is achieved herein using qualitative automated 1 H NMR analysis and compared to gas chromatography-mass spectrometry (GC-MS) data. Two seized drug samples, SS1 and SS2, were identified to contain 4-fluoroamphetamine by 1 H NMR (match score median = 0.9933) and GC-MS (RR t  = 5.42-5.43 min). The amount of 4-fluoroamphetamine present was 42.8%-43.4% w/w and 48.7%-49.2% w/w for SS1 and SS2, respectively, from quantitative 19 F NMR analysis, which is in agreement with the amount determined by GC-MS (39.9%-41.4% w/w and 49.0%-49.3% w/w). The total time for the qualitative 1 H NMR and quantitative 19 F NMR analysis is ~10 min. This contrasts to ~40 min for the GC-MS method. The NMR method also benefits from minimal sample preparation. Thus, benchtop NMR affords rapid, and discriminatory, analysis of the drug present in a seized sample., (© 2021 John Wiley & Sons, Ltd.)

The study objective was to analyse the phytochemical constituents in aerial extracts of these plants using an HPTLC method and optimization by quality by design. Qualitative analysis of ephedrine in hydro-alcoholic extract was done via HPTLC, using a mobile phase of toluene-ethyl acetate-chloroform-formic acid in the ratio of 1:0.5:0.5:01 and the peaks were monitored at 366 nm. In the hydro-alcoholic aerial part extract, ephedrine was identified using the HPTLC method and the retardation factor (Rf) value was found to be 0.69 ± 0.01 and 0.69 ± 0.01, as compared with the standard sample. The extraction of plant materials was done using different concentration of water and alcohol solvents and quality by design was applied to optimize the extraction process and to find out the best extraction in an 80:20 ratio of hydro-alcoholic extract. In the hydro-alcoholic extract, the ephedrine was characterized using the HPTLC method and compared with the standard solution, and this method was used in herbal as well as academic research for the identification of ephedrine in poly herbal formulations as well as the ephedrine present in different plant extracts. Response surface methodology software was utilized to predict the path or choose the best extraction method. Sida rhombifolia and Sida cordifolia can be used as substitutes for Ephedra gerardiana based on the HPTLC profile., (© 2022 John Wiley & Sons Ltd.)

Ephedrae Herba is one of the most commonly used herbal medicines, and it has been shown that most of the clinical efficacy for cold and asthma is exerted by its alkaloidal components. A simple and sensitive high-performance liquid chromatography method was developed using a perfluorooctyl column for the simultaneous determination of five alkaloids (norephedrine, norpseudoephedrine, ephedrine, pseudoephedrine, and methylephedrine) in Ephedrae Herba. The mobile phase comprising acetonitrile and 15 mM ammonium trifluoroacetate was used to elute the targets in isocratic elution mode. The method was validated for linearity (R 2  > 0.999), repeatability, intraday and interday precision, recoveries with trueness (93.87-110.99%), limits of detection (5.35-5.76 µg/mL), and limits of quantification (20 µg/mL). The quantitative results revealed that the developed method was precise and accurate. Then it was successfully applied to determine the difference in the contents of three batches of Ephedrae Herba from three pharmaceutical companies., (© 2022 Wiley-VCH GmbH.)

Background: Due to the strong association between the chemistry of medicinal plants and their biological properties, it is important to determine their phytochemical composition to justify experimental tests., Objective: The aim of this study was to evaluate the in vitro antioxidant and the enzyme inhibitory properties and to identify the bioactive compounds present in the extracts of Ephedra nebrodensis growing in Algeria., Methods: Total phenolic and flavonoids content in these extracts were quantified by Folin- Ciocalteu and aluminum chloride methods. The antioxidant capacity was assessed using DPPH, ABTS, β-carotene/linoleic acid, CUPRAC and FRAP assays, and in vitro cholinesterase activity against acetylcholinesterase and butyrylcholinesterase were evaluated. The chemical constituents of the extracts were analyzed by high-performance liquid chromatography coupled with mass spectrometric detection and gas chromatography. For the acute toxicity study, extracts were administered to mice at single dose of 2 g/kg and 5 g/kg by gavage., Results: Plant extracts were rich in phenolic compounds. Ethyl acetate extract presented the highest phenolic (238.44 ± 1.50 μg GAE /mg of extract) and flavonoid (21.12 ± 0.00 μg QE /mg of extract) contents. Likewise, ethyl acetate extract showed potent radical scavenging and reducing properties. Ethanol-acetone extract showed inhibitory activity against acetylcholinesterase, and was a potent inhibitor of butyrylcholinesterase. In all extracts, flavonoids were the most abundant compounds. The phytochemical investigation showed the presence of alkaloids (ephedrine and pseudo-ephedrine). In the acute toxicity, the LD 50 was superior to 5 g/kg body weight. There were no alterations in the histology of the liver and kidneys., Conclusion: This study demonstrated a good antioxidant potential and anticholinesterase activity of aerial parts of E. nebrodensis., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Background: Weight loss and sports supplements containing deterenol have been associated with serious adverse events including cardiac arrest., Objective: To determine the presence and quantity of experimental stimulants in dietary supplements labeled as containing deterenol sold in the United States., Methods: Dietary supplements available for sale in the US and labeled as containing deterenol or one of its synonyms (e.g., isopropylnorsynephrine and isopropyloctopamine) were purchased online. For each brand, one container or subsample was analyzed by NSF International (Ann Arbor, MI) and one container or subsample by the Netherland's National Institute for Public Health and the Environment (RIVM, Bilthoven, The Netherlands). When differences existed between the two containers or subsamples of the same brand, both products were reanalyzed by Sciensano (Brussels, Belgium). NSF International carried out qualitative and quantitative analyses using ultra-high-performance liquid chromatography (UHPLC) quadrupole-Orbitrap mass spectrometry. RIVM performed qualitative and quantitative analysis using UHPLC quadrupole time-of-flight mass spectrometry. Sciensano carried out qualitative analysis using UHPLC quadrupole-Orbitrap mass spectrometry., Results: Seventeen brands of supplements were analyzed. Many brands included more than one prohibited stimulant in the same product: 4 brands (24%, 4/17) included 2 stimulants, 2 (12%, 2/17) combined 3 stimulants, and 2 (12%, 2/17) combined 4 stimulants. The range of quantities per recommended serving size of the 9 stimulants detected were 2.7 mg to 17 mg of deterenol; 1.3 mg to 20 mg of phenpromethamine (Vonedrine); 5.7 mg to 92 mg of beta-methylphenylethylamine (BMPEA); 18 mg to 73 mg of octodrine; 18 mg to 55 mg of oxilofrine; 48 mg of higenamine; 17 mg of 1,3-dimethylamylamine (1,3-DMAA); 1.8 mg to 6.6 mg of 1,3-dimethylbutylamine (1,3-DMBA); and 5.3 mg of 1,4-dimethylamylamine (1,4-DMAA)., Conclusion: Weight loss and sports supplements listing deterenol as an ingredient contained 9 prohibited stimulants and 8 different mixtures of stimulants, with as many as 4 experimental stimulants per product. These cocktails of stimulants have never been tested in humans and their safety is unknown.

A highly specific and sensitive proton nuclear magnetic resonance ( 1 H-NMR) method has been developed for the quantification of ephedrine alkaloid derivatives in Ephedra herbal commercial prescriptions. At the region of δ 4.0 to 5.0 ppm in the 1 H NMR spectrum, the characteristic signals are separated well from each other, and six analogues in total, methylephedrine (ME), ephedrine (EP), norephedrine (NE), norpseudoephedrine (NP), pseudoephedrine (PE), and methylpseudoephedrine (MP) could be identified. The quantities of these compounds are calculated by the relative ratio of the integral values of the target peak for each compound to the known concentrations of the internal standard anthracene. The present method allows for a rapid and simple quantification of ephedrine alkaloid derivatives in Ephedra -related commercial prescriptions without any preliminary purification steps and standard compounds, and accordingly it can be a powerful tool to verify different Ephedra species. In comparison to conventional chromatographic methods, the advantages of this method include the fact that no standard compounds are required, the quantification can be directly performed on the crude extracts, a better selectivity for various ephedrine alkaloid derivatives, and the fact that a very significant time-gain may be achieved.

Objective: The electrochemical analysis of ephedrine which is a sympathometric drug has been studied using poly(Nile blue A) modified glassy carbon electrodes, cyclic voltammetry, differential pulse voltammetry and square wave voltammetry., Methods: The modified electrodes were prepared by potential cycling electropolymerization of Nile blue A in 0.1 M phosphate buffer solution at pH 6.0. The redox behavior of ephedrine was investigated in different buffer solutions at pH values between 5.5 and 9.0., Results: Scan rate studies showed that the electron transfer reaction of ephedrine was diffusion controlled. A linear response was obtained between the peak current and the ephedrine concentration in the range of 0.6 to 100 μM with a limit of detection of 2.91×10 -3 μM for differential pulse voltammetry in Britton-Robinson buffer solution at pH 9.0. The linearity range of ephedrine in human urine was between 1.0 and 100 μM with a detection limit of 8.16 nM., Conclusion: The recovery studies in both pharmaceutical dosage forms and urine showed that the proposed method ensured good selectivity, precision and accuracy without any interference from inactive excipients., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Dried terrestrial stems of Ephedra sinica are known as 'Ephedra herb.' The pharmacological effects are mainly related to two major ingredients, (-)-ephedrine and (+)-pseudoephedrine (total alkaloids which are defined in Japanese Pharmacopoeia, TA). In this study, in order to aid in cultivation and breeding, the stability of TA content and stem dry weight of 46 E. sinica genets was evaluated from the first year of transplantation to the sixth year. TA content and composition ratio of these genets were stable after the second year, and dry weight was stable after the fourth year. These traits showed high inter-genet variability but low annual variability for each genet. Additionally, rank correlation coefficients of each trait among the genets were high. There was no significant correlation between these traits. Furthermore, to assess the reproducibility of these traits in clones, we evaluated TA content and dry weight of three clonal lines with high TA contents. TA content and composition ratio of the clonal lines were also stable after the second year of transplantation, and dry weight of the clonal lines was also stable after the fourth year. Moreover, TA content and composition ratio in each clonal line were comparable with those of each original genet after the second year. These results suggested that ephedrine alkaloids content and dry weight of E. sinica plants are stable, and that these traits are highly reproducible in clones. Therefore, selection breeding of E. sinica using vegetative propagation can be effective for high and stable quality of Ephedra herb.

In forensic science, drug profiling is clarifying the identity of seized drugs of abuse based on their physicochemical properties and it is applied to various drugs, including crystalline methamphetamine. Impurity analysis is particularly important in drug profiling because the impurities can be a measure for speculating how the methamphetamine was synthesized in the clandestine laboratories. However, developments in scientific techniques have allowed the synthesis of high-purity, homogeneous crystalline methamphetamine, and thus new techniques to characterize methamphetamine are needed. In this study, we developed a method for chiral separation of ephedrine and its stereoisomers by supercritical fluid chromatography. Ephedrine is a common starting compound for methamphetamine synthesis. It possesses two chiral center carbon atoms and has four stereoisomers, (1R,2S)-(-)-ephedrine, (1S,2R)-(+)-ephedrine, (1S,2S)-(+)-pseudoephedrine, and (1R,2R)-(-)-pseudoephedrine. Because the stereostructure of ephedrines contained in methamphetamine seizure reflects the starting materials and the synthetic pathways, the stereoisomer ratio will provide additional information for drug profiling. The developed method achieved rapid separation of four isomers in about 11min with low limits of detection (1pg on column). Due to a switching valve connecting a chromatograph to a mass spectrometer, dense methamphetamine sample solutions containing small amount of ephedrines could be analyzed directly with a simple pre-treatment. Using multivariate analysis, 44 real samples were objectively grouped based on stereoisomer ratio. Our method is expected to improve the profiling of crystalline methamphetamine., Competing Interests: Declaration of Competing Interest The authors report no declarations of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Bioanalysis of polar analytes using liquid chromatography-tandem mass spectrometry (LC-MS/MS) remains a significant challenge because of their poor chromatographic retention on the commonly used reversed-phase LC columns and the resulting severe ionization suppression from coeluting matrix components. Here we present a novel approach to perform ultrahigh-throughput and chromatography-free bioanalysis of polar compounds using a prototype acoustic ejection mass spectrometer (AEMS) platform. Previously developed for direct analysis of solid or liquid samples by MS, the open port interface (OPI) has recently been modified and coupled to an acoustic nanoliter dispenser to enable high-speed direct MS analysis from 384-well plates with a reported speed as fast as 0.5 s/sample. Ionization suppression was reduced due to the >1000 fold dilution of the original sample by the carrier solvent in the AE-OPI-MS operation. Taking full advantage of the chromatography-free and suppression-reducing features of this prototype instrument, we successfully demonstrated the ultrahigh-throughput bioanalysis of metformin, a small polar substrate commonly used in high-throughput in vitro transporter inhibition assays in the early ADME profiling space in drug discovery. The AEMS platform achieved a speed of 2.2 s/sample using only 10 nL of sample volume. Similar bioanalytical and biological results from actual assay samples were obtained by AEMS when compared to those obtained by the fastest LC-MS/MS method previously reported, along with a 15-fold speed advantage and ∼500-fold less sample consumption to enable future assay miniaturization. The general applicability of this novel approach to bioanalysis of several classes of polar analytes including ethambutol, isoniazid, ephedrine, and gemcitabine in biological matrices was further demonstrated.

Interpretation of amphetamine-type stimulant (ATS) findings in urine samples can be challenging without chiral information. We present a sensitive enantioselective high-performance liquid chromatography-tandem mass spectrometry method for the quantification of (R)-amphetamine, (S)-amphetamine, (R)-methamphetamine, (S)-methamphetamine, (1R,2R)-pseudoephedrine, (1S,2S)-pseudoephedrine, (1R,2S)-ephedrine, (1S,2R)-ephedrine, (1R,2S)-norephedrine, (1S,2R)-norephedrine, (R)-cathinone, (S)-cathinone, and (1S,2S)-norpseudoephedrine (cathine) in urine. The method was successfully applied to more than 100 authentic urine samples from forensic casework. In addition, samples from a controlled self-administration of (1S,2S)-pseudoephedrine (Rinoral, 1200 mg within 6 days) were analyzed. The results strengthen the hypothesis that (1R,2S)-norephedrine is a minor metabolite of amphetamine and methamphetamine. We suggest cathine and (1S,2R)-norephedrine as minor metabolites of amphetamine racemate in humans. Small methamphetamine concentrations detected in samples with high concentrations of amphetamine could result from a metabolic formation by methylation of amphetamine although in samples with an (R)/(S) ratio for methamphetamine < 1 an additional (previous) (S)-methamphetamine consumption seems likely. Our data suggest that even amphetamine concentrations exceeding methamphetamine concentrations in urine can be caused by the biotransformation of methamphetamine to amphetamine as long as no (R)-amphetamine is detected. However, without chiral information, such findings might be (falsely) assumed as a co-consumption of both substances. Cathinone enantiomers detected in urine samples with high amphetamine concentrations can be interpreted as metabolites of amphetamine. In addition, the results of the self-administration study revealed that both cathinone enantiomers are minor metabolites of (1S,2S)-pseudoephedrine, which is the active ingredient of various medicines used for cold. The enantioselective analysis is a powerful tool to avoid the misinterpretation of ATS findings in urine samples., (© 2020 The Authors. Drug Testing and Analysis published by John Wiley & Sons Ltd.)

The emergence of chemically masked illicit drugs represents a challenge to global initiatives that are working to prevent their manufacture and distribution. Targeted analytical techniques currently used by law enforcement to identify unknown materials rely on spectroscopic and spectrophotometric databases that do not currently include some of these compounds, making their identification challenging. This study aimed to update compound spectral libraries to aid in the rapid detection and identification of these masked drugs, as well as to provide insight into their synthetic procedures. Five commonly employed protecting groups, acetyl, p-tosyl, methoxycarbonyl, Fmoc, and t-Boc, were appended to pseudoephedrine, ephedrine, methamphetamine, and MDMA. Characterization was carried out using NMR, GC-MS, FTIR, high-resolution LC-MS/MS, and common screening color tests. Some of the methoxycarbonyl and t-Boc derivatives and all of the Fmoc derivatives showed partial or full thermal degradation or rearrangement during GC-MS analysis, while LC-MS/MS analysis did not always show characteristic fragmentation that would allow unambiguous assignment of the structure. Restricted rotation in some of the derivatives meant that NMR assignments could only be made using NMR spectra acquired at elevated temperature. Therefore, GC-MS and LC-MS/MS analyses serve complementary roles for these derivatives, with NMR providing confirmation of structure for the pure materials if necessary., (© 2020 John Wiley & Sons, Ltd.)

Background: Allergic rhinitis, acute nasal congestion and sinusitis are one of the most common health problems and have a major effect on the quality of life. Several medications are used to improve the symptoms of such diseases in humans. Pharmaceutical pomade form containing Ephedrine (EPD) HCl, Naphazoline (NPZ) HCl, Antazoline (ANT) HCl, and Chlorobutanol (CLO) is one of them., Objective: For these reasons, this study includes the development of spectrophotometric and chromatographic methods for the determination of EPD HCl, NPZ HCl, ANT HCl, and CLO active agents in the pharmaceutical pomade., Method: In the spectrophotometric method, third-order derivative of the amplitudes at 218 nm n=5 and the first-order derivative of the amplitudes 254 nm n=13 was selected for the determination of EPD, ANT, respectively while NPZ was determined by the second derivative at 234 nm and n=21. Colorimetric detection was applied for assay analysis of CLO at 540 nm. Furthermore, a reverse phase high performance liquid chromatographic (RP- HPLC) method has been developed and optimized by using Agilent Zorbax Eclipse XDB C18 (75 mm x 3.0 mm, 3.5μm) column. The column temperature was 40°C, binary gradient elution was used and the mobile phase consisted of 15 mM phosphate buffer in distilled water (pH 3.0) and methanol, and the flow rate was 0.6 mL min -1 and the UV detector was detected at 210 nm. The linear operating range was obtained as 11.97-70, 0.59-3, 2.79-30, and 2.92-200 μg mL -1 for EPD HCl, NPZ HCl, ANT HCl, and CLO respectively., Results: The LOD values were found to be 3.95, 0.19, 0.92 and 0.96 μg mL -1 for EPD HCl, NPZ HCl, ANT HCl, and CLO in the spectrophotometric method, respectively. The linear ranges in the RP-HPLC method were 8.2-24.36 μg mL -1 , 0.083 - 0.75 μg/mL, 2.01-7.5 μg mL -1 and 2.89-24.4 μg mL -1 for EPD HCl, NPZ HCl, ANT HCl, and CLO, respectively. The LOD values in the validation studies were 2.7, 0.025, 0.66 and 0.86 μg mL -1 for EPD HCl, NPZ HCl, ANT HCl, and CLO in RP-HPLC method respectively., Conclusion: The results of the spectrophotometric and chromatographic methods were compared and no differences were found between the two methods., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

This work presents a strategy based on the in-line coupling of SPE and CE for the chiral determination of cathinones (R,S-mephedrone, R,S-4-methylephedrine, and R,S- methylenedioxypyrovalerone) in urine samples, using a sample pretreatment based on liquid-liquid extraction. The chiral separation of the compounds is achieved by adding a mixture of 8 mM 2-hydroxypropil β-CD and 5 mM β-CD to the BGE, which consists of 70 mM of monosodium phosphate aqueous solution at pH 2.5. Oasis HLB was the selected sorbent for the in-line SPE device, and to reduce analysis time and LODs, several parameters affecting the in-line SPE system were evaluated, such as pressure and time of sample injection and dimensions of the SPE device. The highest preconcentration factors were achieved by using 3 bar of injection pressure for 20 min with an in-line SPE device of 2 mm length and 150 µm of i.d. The developed method was applied to determine the presence of the compounds in spiked urine samples. The LODs obtained were between 3 and 8 ng/mL, and these levels were below the usual concentrations at which these drugs are present in urine from cathinone abusers. Thus, the optimized method has the potential to be applied for toxicological and forensic purposes., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Here, we describe a study of screen characterization of aptamers targeting the chiral drug ephedrine using the non-immobilized graphene oxide (GO) SELEX. The improved method of long and short chains was here used to prepare the ssDNA library. The Resonance Rayleigh Scattering (RRS) method was first used to monitor the screening process. Through high-throughput sequencing, the genetic sequence data of 90,487 aptamers were obtained. Through the analysis of the parameters of free energy value and secondary structure prediction model of high repeatability sequence, the 10 candidate sequences were identified. Finally, a best-fit aptamer named EP08 was identified by combining the dissociation experiment. The binding affinity and binding mechanism of the aptamer and target were analyzed using an isothermal titration colorimetry (ITC) experiment and circular dichromatic (CD) experiment. The binding affinity (Kd) of the EP08 aptamer to ephedrine is approximately 2.86 ± 0.24 μM. This novel DNA aptamer will help in the future development of a new method for the identification and detection of chiral drug ephedrine., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Field surveys of Ephedra plants were conducted in the Zaravshan Mountains of Tajikistan. E. equisetina, E. intermedia, and their putative hybrids were collected. They were identified based on their phenotypes and their sequences of nuclear ribosomal DNA internal transcribed spacer 1 (ITS1) region. Sequencing and species-specific PCR analyses of their ITS1 sequences revealed six putative hybrids of E. equisetina and E. intermedia. The total ephedrine and pseudoephedrine content of most of the Ephedra samples collected in Tajikistan were higher than the 0.7% lower limit prescribed by the Japanese pharmacopoeia, 17th edition (JP17), and varied from 0.34 to 3.21% by dry weight. The total alkaloid level of E. intermedia (11E08-1) cultivated in Japan varied from 1.77 to 2.30% by dry weight, which was much higher than the 0.7% lower limit prescribed by JP17.

The use of stable isotope ratio mass spectrometry (IRMS) as a profiling tool for methylamphetamine has evolved over the last decade. Stable isotope ratios of carbon (δ 13 C), nitrogen (δ 15 N), and hydrogen (δ 2 H) of methylamphetamine are useful in determining the precursor used to manufacture methylamphetamine, and in many cases the synthetic origin of the methylamphetamine precursor. More recently, samples of seized methylamphetamine show that a resolution step is being employed in the manufacturing process. We sought to determine whether the δ 13 C, δ 15 N, and δ 2 H values were affected by either a resolution performed on racemic methylamphetamine or a resolution on racemic ephedrine, a commonly used precursor to methylamphetamine. We found that for the types of resolution studied, IRMS is still able to provide useful information on the provenance of a methylamphetamine sample., (© 2018 John Wiley & Sons, Ltd.)

Sleeping aids are often abused in the commission of drug-facilitated crimes. Generally, there is little evidence that a victim ingested a spiked drink unknowingly because the unconscious victim cannot report the situation to the police immediately after the crime occurred. Although conventional segmental hair analysis can estimate the number of months since a targeted drug was ingested, this analysis cannot determine the specific day of ingestion. We recently developed a method of micro-segmental hair analysis using internal temporal markers (ITMs) to estimate the day of drug ingestion. This method was based on volunteer ingestion of ITMs to determine a timescale within individual hair strands, by segmenting a single hair strand at 0.4-mm intervals, corresponding to daily hair growth. This study assessed the ability of this method to estimate the day of ingestion of an over-the-counter sleeping aid, diphenhydramine, which can be easily abused. To model ingestion of a diphenhydramine-spiked drink unknowingly, each subject ingested a dose of diphenhydramine, followed by ingestion of two doses of the ITM, chlorpheniramine, 14days apart. Several hair strands were collected from each subject's scalp several weeks after the second ITM ingestion. Diphenhydramine and ITM were detected at specific regions within individual hair strands. The day of diphenhydramine ingestion was estimated from the distances between the regions and the days of ITM ingestion. The error between estimated and actual ingestion day ranged from -0.1 to 1.9days regardless of subjects and hair collection times. The total time required for micro-segmental analysis of 96 hair segments (hair length: 3.84cm) was approximately 2days and the cost was almost the same as in general drug analysis. This procedure may be applicable to the investigation of crimes facilitated by various drugs., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The presence of coloring matters in syrups usually interferes with the spectrophotometric determination of active pharmaceutical ingredients. A novel approach was introduced to eliminate the interference of sunset yellow (coloring matter) in Cyrinol syrup. Smart, simple, accurate, and selective spectrophotometric methods were developed and validated for the simultaneous determination of a ternary mixture of carbinoxamine maleate, pholcodine, and ephedrine hydrochloride in syrup. Four of the applied methods used ratio spectra: successive derivative subtraction coupled with constant multiplication, successive derivative of ratio spectra, ratio subtraction coupled with ratio difference, and ratio spectra continuous wavelet transforms zero-crossing. In addition, a method that was based on the presence of an isosbestic point, the amplitude summation method, was also established. A major advantage of the proposed methods is the simultaneous determination of the mentioned drugs without prior separation steps. These methods were successfully applied for the determination of laboratory-prepared mixtures and a commercial pharmaceutical preparation without interference from additives, thus proving the selectivity of the methods. No significant difference regarding both accuracy and precision was observed upon statistical comparison of the results obtained by the proposed methods with each other and with those of official or reported ones.

Consumption of Ephedra alkaloids is prohibited in-competition by the World Anti-Doping Agency (WADA). In Taiwan, colds are often treated with Chinese herbal formulae containing Herba Ephedrae. We screened products sold in Taiwan and preliminarily assessed their relationships with WADA threshold violations. Fifty-six concentrated powder products, including 19 Chinese herbal formulae that contained Herba Ephedrae, were collected. The content of Ephedra alkaloids, namely ephedrine (E), methylephedrine (ME), norpseudoephedrine (NPE; cathine), pseudoephedrine (PE), and norephedrine (NE; phenylpropanolamine), was determined using a validated high-performance liquid chromatography method. The results revealed that the phenotypic indicators of the collected products, E/PE and E/total ratios, were 1.52-4.70 and 0.49-0.72, respectively, indicating that the Herba Ephedrae species in these products was probably E. sinica or E. equisetina, but not E. intermedia. The contents of E, ME, NPE, PE, and NE and the total alkaloid contents in the daily doses of the products were 0.45-34.97, 0.05-4.87, 0.04-3.61, 0.15-12.09, and 0.01-2.00 mg and 0.68-53.64 mg, respectively. The alkaloid contents followed a relatively consistent order (E > PE > ME ≈ NPE > NE), even for products from different manufacturers. We calculated that single doses of 50.0% and 3.6% of the products would result in the WADA thresholds of E and NPE being exceeded, respectively. Our data provide critical information for athletes and medical personnel, who should be wary of using complex Chinese herbal formulae in addition to over-the-counter products., (Copyright © 2017 John Wiley & Sons, Ltd.)

As part of our continuing study of ephedrine alkaloids-free Ephedra Herb extract (EFE) in pursuit of its approval as a crude drug preparation, we identified two quantitative markers for the quality control of the manufacturing process of EFE and sought to establish cost-effective and simple methods for quantitative analyses. We analysed Ephedra Herb extracts grown in different habitats and collection years by liquid chromatography/high-resolution mass spectrometry (LC/HRMS) and detected two notable peaks common to each extract. These peaks were identified as vicenin-2 (1) and isovitexin 2″-O-rhamnoside (2). Quantitative analyses using the isocratic condition of LC/MS showed that the content percentages of 1 and 2 in EFE were 0.140-0.146% and 0.350-0.411%, respectively. We concluded that 1 and 2 were adequate quality control markers for quantitative analysis of EFE. Furthermore, we quantitatively analysed apigenin (3), an aglycon common to 1 and 2, and found that the conversion factors of 1 to 3 and 2 to 3 were 1.3 and 1.5, respectively. Therefore, we concluded that 3 was a secondary standard for quantifying the contents of 1 and 2 in EFE. A series of results obtained from this study will be valuable for the quality control of EFE.

Para-methoxymethamphetamine (PMMA) has caused numerous fatal poisonings worldwide and appears to be more toxic than other ring-substituted amphetamines. Systemic metabolism is suggested to be important for PMMA neurotoxicity, possibly through activation of minor catechol metabolites to neurotoxic conjugates. The aim of this study was to examine the metabolism of PMMA in humans; for this purpose, we used human liver microsomes (HLMs) and blood samples from three cases of fatal PMMA intoxication. We also examined the impact of CYP2D6 genetics on PMMA metabolism by using genotyped HLMs isolated from CYP2D6 poor, population-average, and ultrarapid metabolizers. In HLMs, PMMA was metabolized mainly to 4-hydroxymethamphetamine (OH-MA), whereas low concentrations of para-methoxyamphetamine (PMA), 4-hydroxyamphetamine (OH-A), dihydroxymethamphetamine (di-OH-MA), and oxilofrine were formed. The metabolite profile in the fatal PMMA intoxications were in accordance with the HLM study, with OH-MA and PMA being the major metabolites, whereas OH-A, oxilofrine, HM-MA and HM-A were detected in low concentrations. A significant influence of CYP2D6 genetics on PMMA metabolism in HLMs was found. The catechol metabolite di-OH-MA has previously been suggested to be involved in PMMA toxicity. Our studies show that the formation of di-OH-MA from PMMA was two to seven times lower than from an equimolar dose of the less toxic drug MDMA, and do not support the hypothesis of catechol metabolites as major determinants of fatal PMMA toxicity. The present study revealed the metabolite pattern of PMMA in humans and demonstrated a great impact of CYP2D6 genetics on human PMMA metabolism., (Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics.)

In January 2017, counterfeits of the hepatitis C drug 'HARVONI ® Combination Tablets' (HARVONI ® ) were found at a pharmacy chain through unlicensed suppliers in Japan. A total of five lots of counterfeit HARVONI ® (samples 1-5) bottles were found, and the ingredients of the bottles were all in tablet form. Among them, two differently shaped tablets were present in two of the bottles (categorized as samples 2A, 2B, 4A, and 4B). We analyzed the total of seven samples by high-resolution LC-MS, GC-MS and NMR. In samples 2A, 3 and 4B, sofosbuvir, the active component of another hepatitis C drug, SOVALDI ® Tablets 400 mg (SOVALDI ® ), was detected. In sample 4A, sofosbuvir and ledipasvir, the active components of HARVONI ® , were found. A direct comparison of the four samples and genuine products showed that three samples (2A, 3, 4B) are apparently SOVALDI ® and that sample 2A is HARVONI ® . In samples 1 and 5, several vitamins but none of the active compounds usually found in HARVONI ® (i.e., sofosbuvir and ledipasvir) were detected. Our additional investigation indicates that these two samples are likely to be a commercial vitamin supplement distributed in Japan. Sample 2B, looked entirely different from HARVONI ® and contained several herbal constitutents (such as ephedrine and glycyrrhizin) that are used in Japanese Kampo formulations. A further analysis indicated that sample 2B is likely to be a Kampo extract tablet of Shoseiryuto which is distributed in Japan. Considering this case, it is important to be vigilant to prevent a recurrence of distribution of counterfeit drugs.

Maoto, a traditional Japanese Kampo medicine, has been used to treat various respiratory diseases, including respiratory infections and influenza. Ephedrine (EPD), the main ingredient in maoto, is also clinically used to treat respiratory diseases. However, the pharmacokinetics and distribution of EPD in the lungs after the administration of maoto have not been demonstrated. This study aimed to determine the concentrations, distribution, and pharmacokinetics of EPD and its precursor methylephedrine (MEPD) in the lungs of rats orally administered maoto (1 and 4 g/kg). We used liquid chromatography-electrospray ionization-tandem mass spectrometry to measure the ingredient concentrations. Both ingredients were detected in maoto-treated lung homogenates. Next, we examined the distribution of both ingredients in lung sections by using matrix-assisted laser desorption/ionization-mass spectrometry imaging, a powerful tool for the visualization of the distribution of biological molecules. The mass spectrometry imaging analysis detected only EPD and provided the first visual demonstration that EPD is distributed in the alveoli, bronchi, and bronchioles in the lungs of rats orally administered maoto (4 g/kg, three times at 2-h intervals). These results suggest that the pharmacological efficacy of maoto for the amelioration of respiratory symptoms is related to the distribution of EPD in the lung.

To establish the quality control methods for the standard decoction of Ephedrae Herba, and provide the reference for quality evaluation method of all Chinese herbal medicine decoction.Standard decoction of Ephedrae Herba was prepared, and UPLC-UV fingerprint was established to determine the total contents of ephedrine and pseudoephedrine. Then UPLC-QTOF/MS was used to confirm the major common peaks in the fingerprint to clarify the main chemical constituents in the decoction. In addition, the stability of the process was evaluated by calculating the parameters such as the extraction ratio, transfer rate of the index components and the pH values.In the decoction of Ephedrae Herba, the total average concentration of ephedrine and pseudoephedrine was (2.11±0.70) g•L⁻¹; the similarities of all the fingerprints were more than 0.85; there were 10 major common peaks in the fingerprint, including alkaloids, flavonoids and organic acids; the extraction ratio was (17±3.2)%, and the overall transfer rate of ephedrine and pseudoephedrine was (32.4±8.1)%.The method for evaluating the quality of standard decoction of Ephedrae Herba was established in this article, providing reference for the quality control of products which were stemmed from the water extract of Ephedrae Herba., Competing Interests: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose., (Copyright© by the Chinese Pharmaceutical Association.)

Two-dimensional correlation spectroscopy (2DCOS) was employed for the identification of ephedrine (Ep) and pseudoephedrine (Ps) present in illegally adulterated slimming herbal products (SHPs). Second derivative (SD) spectral pretreatment was used prior to 2DCOS analysis to highlight specific features not readily observable by Fourier transform infrared spectroscopy (FTIR), SD-FTIR, or original 2DCOS, leading to enhanced resolution and a reduced lower limit of detection (<1% in this study). After examining the power spectra of suspicious SHPs, bands containing characteristic peaks for Ep (701, 747, 1042, 1363, 1375, 1451, 1478 cm -1 etc) and/or Ps (703, 767, 1037, 1375, 1428, 1455, 1590 cm - 1, etc.) were selected to construct synchronous and asynchronous maps for further analysis, while the latter was applied to discriminate positive SHPs adulterated simultaneously with Ep and Ps. The proposed method is simple and economical and has the potential to identify other chemicals in illegally adulterated herbal products. Copyright © 2016 John Wiley & Sons, Ltd., (Copyright © 2016 John Wiley & Sons, Ltd.)

Ephedrine and pseudoephedrine are stimulant drugs whose use is prohibited in athletic competition by the World Anti-Doping Agency (WADA) at very different threshold doping violation concentrations. We use a recently developed universal approach that integrates UV photofragmentation spectroscopy of cold ions with Orbitrap mass spectrometry (MS) for highly selective and highly sensitive identification of these diastereomers. Both species can be selectively detected at a solution concentration of a few tens of ng/mL, which is almost 3 orders of magnitude lower than the threshold concentration required by WADA. Relative concentrations of the isomers in solutions have been determined with the standard deviation of 3.1%, when the ions were cooled in an ion trap maintained at T = 6 K. Considering practical implementation of the method, we evaluated its performance for a simplified instrumentation. At an affordable elevated temperature of ∼70 K and with a low-maintenance midbandwidth optical parametric oscillator, a few second measurement should yield nearly the same selectivity and only ten times lower sensitivity than with the current research grade instrument.

Oxilofrine (4-[1-hydroxy-2-(methylamino)propyl]phenol) is a pharmaceutical stimulant prescribed in dosages of 16 to 40 mg to stimulate the heart and increase blood pressure. It has never been approved for use in the USA as a prescription drug or as a dietary supplement. Several athletes, however, have been banned from sport for testing positive for oxilofrine and have claimed that they inadvertently consumed oxilofrine in sports supplements. Consumption of supplements containing oxilofrine may also pose serious health risks. For example, one brand of supplements containing oxilofrine has been linked to serious adverse events including vomiting, agitation, and cardiac arrest. We designed our study to determine the presence and quantity of oxilofrine in dietary supplements sold in the USA. A validated ultra-high performance liquid chromatography-quadrupole time of flight-mass spectrometry method was developed for the identification and quantification of oxilofrine. The separation was achieved using a reversed phase column, mass spectrometry detection, and a water/acetonitrile gradient as the mobile phase. The presence of oxilofrine was confirmed using a reference standard. We analyzed 27 brands of supplements labelled as containing a synonym of oxilofrine ('methylsynephrine') and found that oxilofrine was present in 14 different brands (52%) at dosages ranging from 0.0003 to 75 mg per individual serving. Of the supplements containing oxilofrine, 43% (6/14) contained pharmaceutical or greater dosages of oxilofrine. Following instructions on the label, consumers could ingest as much as 250 mg of oxilofrine per day. The drug oxilofrine was found in pharmacological and greater dosages in supplements labelled as containing methylsynephrine. Copyright © 2016 John Wiley & Sons, Ltd., (Copyright © 2016 John Wiley & Sons, Ltd.)

The Ephedra herb, which has been used in Kampo medicines, originates from terrestrial stems of Ephedra species. It is important to establish cultivation methods and cultivars to secure a stable supply of the Ephedra herb that would meet the quality standards for the ephedrine alkaloids content. In this study, we first grew Ephedra sinica plants derived from seeds in the field for 5 years. Then, for selective breeding of cultivars that could meet the quality standards for the ephedrine alkaloids content, we measured the content of total alkaloids (TAs), ephedrine (Eph), and pseudoephedrine (PEph) in individual plants derived from seedlings and grown for 4 years in the field. The range of the TA content in each individual plant was narrower than that among individual plants grown in the field. Therefore, individual plants were selected according to their TA content, Eph/PEph ratio, and stolon-formation capability. The selected individuals were propagated using stolons, and their TA content was studied for 2 years. In the second year, the TA content in terrestrial stems derived from stolons of the selected individuals was as high as that of their parents. Therefore, it was confirmed that the selected individuals that were propagated using stolons could produce TA reproducibly. This study suggested that selective breeding using stolon propagation is effective for stabilizing Ephedra herb TA content.

Ephedrines in dietary supplements can arise from herbs or illegal adulteration so a liquid chromatography tandem mass spectrometry (LC-MS/MS) method was developed for separation and quantification of ephedrine, pseudoephedrine, norephedrine, norepseudoephedrine, and methyl-ephedrine, some of which are isomer pairs of pseudo-structures. This method includes liquid-liquid extraction of ephedrines from three typical dietary supplement matrices-solid, liquid, and oil-as well as liquid chromatographic separation. After liquid chromatographic separation, ephedrines are qualitatively and quantitatively analyzed using triple quadrupole mass spectrometry with positive electrospray ionization in multi-reaction monitoring (MRM) mode. Ephedrine recoveries in a solid matrix ranged from 53.3-91.5%, in a liquid matrix from 56.4-102.3%, and in an oil matrix from 51.7-01.2%. Linearity ranges were 10-1000ng/g in solid and oil matrix and 1-100ng/ml in liquid matrix. Accuracy was -11.5-16.3%. Intra-day and inter-day variation are less than 5.9% and 7.3%, respectively. Expanded uncertainty of quantification is less than 0.123ng/g in a solid matrix, less than 0.139ng/ml in a liquid matrix, and less than 0.158ng/g in an oil matrix. Data collected for more than 500 routine samples are presented and discussed., (Copyright © 2016 Elsevier B.V. All rights reserved.)

In healthcare settings drug diversion and impairment of physicians are major concerns requiring a rapid and efficient method for surveillance and detection. A Direct Analysis in Real Time ion source coupled to a JEOL AccuTOF TM time-of-flight mass spectrometer (DART-MS) method was developed to screen parenteral pharmaceutical formulations for potential drug diversion. Parenteral pharmaceutical formulations are also known as injectable formulations and are used with intravenous, subcutaneous, intramuscular and intra-articular administration. A library was created using the mass spectra data collected by a DART-MS operated in switching mode at 20, 60 and 90 V settings. This library contained 17 commonly encountered drugs in parenteral pharmaceutical formulations that included the surgical analgesic: fentanyl, hydromorphone and morphine; anesthetic: baclofen, bupivacaine, ketamine, midazolam, ropivacaine and succinylcholine; and a mixture of other drug classes: caffeine, clonidine, dexamethasone, ephedrine, heparin, methadone, oxytocin and phenylephrine. Randomly selected 200 de-identified parenteral pharmaceutical formulations containing one or more drugs were submitted for analysis to the FIRM Toxicology Laboratory at Virginia Commonwealth University Health and were screened using the DART-MS. The drug contents of the de-identified formulations were previously confirmed by a published high performance liquid chromatography (HPLC) method. The drugs in the formulations were rapidly and successfully identified using the generated library. The DART-MS and HPLC results were in complete agreement for all 200 parenteral pharmaceutical formulations., (© The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Many studies have found that some dietary supplement product labels do not accurately reflect the actual ingredients. However, studies have not been performed to determine if ingredients in the same dietary supplement product vary over time. The objective of this study was to assess the consistency of stimulant ingredients in popular sports supplements sold in the United States over a 9-month period. Three samples of nine popular sports supplements were purchased over the 9-month period. The 27 samples were analyzed for caffeine and several other stimulants (including adulterants). The identity and quantity of stimulants were compared with stimulants listed on the label and stimulants found at earlier time points to determine the variability in individual products over the 9-month period. The primary outcome measure was the variability of stimulant amounts in the products examined. Many supplements did not contain the same number and quantity of stimulants at all time points over the 9-month period. Caffeine content varied widely in five of the six caffeinated supplements compared with the initial measurement (-7% to +266%). In addition, the stimulants-synephrine, octopamine, cathine, ephedrine, pseudoephedrine, strychnine, and methylephedrine-occurred in variable amounts in eight of the nine products. The significance of these findings is uncertain: the sample size was insufficient to support statistical analysis. In our sample of nine popular sports supplements, the presence and quantity of stimulants varied over a 9-month period. However, future studies are warranted to determine if the variability found is significant and generalizable to other supplements.

A generally applicable high-performance liquid chromatographic method for the qualitative and quantitative determination of pharmaceutical preparations containing phenylephrine hydrochloride, paracetamol, ephedrine hydrochloride, guaifenesin, doxylamine succinate, and dextromethorphan hydrobromide is developed. Optimization of chromatographic conditions was performed for the gradient elution using different buffer pH values, flow rates and two C18 stationary phases. The method was developed using a Kinetex® C18 column as a core-shell stationary phase with a gradient profile using buffer pH 5.0 and acetonitrile at 2.0 mL/min flow rate. Detection was carried out at 220 nm and linear calibrations were obtained for all components within the studied ranges. The method was fully validated in agreement with ICH guidelines. The proposed method is specific, accurate and precise (RSD% < 3%). Limits of detection are lower than 2.0 μg/mL. Qualitative and quantitative responses were evaluated using experimental design to assist the method robustness. The method was proved to be highly robust against 10% change in buffer pH and flow rate (RSD% < 10%), however, the flow rate may significantly influence the quantitative responses of phenylephrine, paracetamol, and doxylamine (RSD% > 10%). Satisfactory results were obtained for commercial combinations analyses. Statistical comparison between the proposed chromatographic and official methods revealed no significant difference., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The stimulating herbal drug khat (cathine, cathinone) and its analog methcathinone are common substances of abuse in most countries. A GC-MS method was developed and validated for the detection and quantification of cathine, cathinone, methcathinone and ephedrine in oral fluid specimens. The analytes and internal standard (amphetamine-d5) were extracted from 0.5 mL oral fluids by ethyl acetate, and then the dried extracts were derivatized with heptafluorobutyric anhydride at 70°C for 30 min. The MS was used in selected ion monitoring mode. Ions monitored were m/z 117, 240 and 330 for cathine, m/z 77, 105 and 240 for cathinone, m/z 105, 210 and 254 for methcathinone, m/z 210, 254 and 344 for ephedrine and m/z 244 and 336 for amphetamine-d5 The calibration curves were linear (r(2)> 0.98) in the concentration range 20-2,000 ng/mL for all analytes. The intra- and inter-assay imprecisions were within (1.6-12.5%) and (1.5-9.5%), respectively, for all analytes. Intra-assay accuracies were between -5.9 and 6.7% for all analytes. The method was successfully applied to detect and quantify the target analytes from oral fluid specimens collected from Khat and methcathinone users., (© The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Ephedrine is one of the main precursor compounds used in the illegal production of amphetamines and related drugs. Actually, conventional analytical methods such as high-performance liquid chromatography (HPLC), capillary electrophoresis (CE), and gas chromatography-mass spectrometry (GC-MS) are used for the detection of ephedrine; sadly, these methods require qualified personnel and are time-consuming and expensive. In order to overcome these problems, in recent years, different methods have been developed based on the surface plasmon resonance (SPR) and electrochemical method. In this work, we present a simple, rapid, and effective method to detect the presence of ephedrine in solution, based on competitive fluorescence resonance energy transfer (FRET) assay. The antibody anti-ephedrine and ephedrine derivative were produced and labeled respectively, with two different fluorescent probes (donor and acceptor). The change in FRET signal intensity between donor and acceptor ephedrine compounds gives the possibility of detecting ephedrine traces of at least 0.81 ± 0.04 ppm (LOD). Graphical abstract A new Time-resolved Fluorescence Resonance Energy Transfer (FRET) assay for ephedrine detection.

Ephedrine alkaloids (EAs) have been considered the main pharmacologically active substances in Ephedra Herb (, Mao; EH) since they were first identified by Prof. N. Nagai, and are known to induce palpitation, hypertension, insomnia, and dysuria as side effects. Therefore, the administration of drugs containing EH to patients with cardiovascular-related diseases is severely contraindicated. While our previous studies suggest that some of the effects of EH may not be due to EAs, considering their side effects would be expedient to develop a new EAs-free EH extract (EFE). Here, we established a preparation method for EFE and revealed its chemical composition, including the content of herbacetin, a flavonoid aglycon present in EH and a potential putative marker for EFE quality control. In addition, we showed the antiproliferative effects of EFE against the H1975 non-small cell lung cancer (NSCLC) cell line. EFE was prepared from EH extract using the ion exchange resin SK-1B. LC/Orbitrap MS analysis revealed the removal of EAs, 6-methoxykynurenic acid, and 6-hydroxykynurenic acid from the original extract. Quantitative analysis of herbacetin using LC/MS in acid-hydrolyzed EFE showed that its content was 0.104 %. Although several alkaloidal constituents were removed from EH extract, the antiproliferative effect of EFE against H1975 cells was comparable to that of EH extract. These results indicate that EFE retained the anticancer effect of EH and demonstrated its potential for future development as a new herbal medicine with reduced side effects.

It is generally accepted that the primary pharmacological activities and adverse effects of Ephedra Herb are caused by ephedrine alkaloids. Interestingly, our research shows that Ephedra Herb also has ephedrine alkaloid-independent pharmacological actions, such as c-MET inhibitory activity. This study describes the preparation of an ephedrine alkaloids-free Ephedra Herb extract (EFE) by ion-exchange column chromatography, as well as in vitro and in vivo evaluation of its pharmacological actions and toxicity. We confirmed that EFE suppressed hepatocyte growth factor (HGF)-induced cancer cell motility by preventing both HGF-induced phosphorylation of c-Met and its tyrosine kinase activity. We also investigated the analgesic effect of EFE. Although the analgesic effect of Ephedra Herb has traditionally been attributed to pseudoephedrine, oral administration of EFE reduced formalin-induced pain in a dose-dependent manner in mice. Furthermore, we confirmed the anti-influenza virus activity of EFE by showing inhibition of MDCK cell infection in a concentration-dependent manner. All assessments of toxicity, even after repeated oral administration, suggest that EFE would be a safer alternative to Ephedra Herb. The findings described here suggest that EFE has c-Met inhibitory action, analgesic effect, and anti-influenza activity, and that it is safer than Ephedra Herb extract itself. Therefore, EFE could be a useful pharmacological agent.

The utility of matrix solid phase dispersion (MSPD) for the direct analysis of amphetamines in hair samples has been evaluated, using liquid chromatography (LC) with fluorescence detection and precolumn derivatization. The proposed approach is based on the employment of MSPD for matrix disruption and clean-up, followed by the derivatization of the analytes onto the dispersant-sample blend. The fluorogenic reagent 9-fluorenylmethyl chloroformate (FMOC) has been used for derivatization. Different conditions for MSPD, analyte purification and solid phase derivatization have been tested, using amphetamine (AMP), methamphetamine (MET), ephedrine (EPE) and 3,4-methylenedioxymethamphetamine (MDMA) as model compounds. The results have been compared with those achieved by using ultrasound-assisted alkaline digestion and by MSPD combined with conventional solution derivatization. On the basis of the results obtained, a methodology is proposed for the analysis of amphetamines in hair which integrates sample disruption, clean-up and derivatization using a C18 phase. Improved sensitivity is achieved with respect to that obtained by the alkaline digestion or by the MSPD followed by solution derivatization methods. The method can be used for the quantification of the tested amphetamines within the 2.0-20.0ng/mg concentration interval, with limits of detection (LODs) of 0.25-0.75ng/mg. The methodology is very simple and rapid (the preparation of the sample takes less than 15min)., (Copyright © 2016 Elsevier B.V. All rights reserved.)