Analysis of bitter orange dietary supplements for natural and synthetic phenethylamines by LC-MS/MS.

Citrus aurantium, commonly known as bitter orange, is a popular dietary supplement ingredient sold worldwide. Bitter orange supplements are sold primarily as weight management and sports performance products and have gained popularity after Ephedra products were banned from the US market. Supplements containing synephrine are reported to exhibit adverse cardiovascular effects especially in the presence of caffeine. In this study, an LC-MS/MS method was established to quantify five natural amines (synephrine, octopamine, tyramine, N-methyltyramine, and hordenine) and four synthetic phenethylamines (phenylephrine, methylsynephrine, etilefrine, and isopropyloctopamine) in dietary supplements sold in the US. The method was validated and found to have acceptable performance to accurately measure analytes in complex botanical products. The average recoveries from a blank matrix were 88-125% with an RSD of 0.5-7.0%. Fifty-nine products labeled to contain bitter orange peel, extract, or its amines were purchased and their amine content was measured. Several products were found to contain higher amounts of amines than that expected from a typical bitter orange extract. Of the 23 products that made label claims for synephrine, only 5 products (22%) were within 80-120% of labeled synephrine content. The presence of synthetic amines, methylsynephrine (up to 240 mg/daily serving), and isopropyloctopamine (up to 76 mg/daily serving), whose effects in humans are not known, were detected in six products and one product, respectively. While the use of methylsynephrine and isopropyloctopamine are not permitted in dietary supplements, hordenine, N-methyltyramine, and octopamine are currently listed on the FDA's Dietary Supplement Ingredient Advisory List.

Multimodal imaging of hallucinogens 25C- and 25I-NBOMe on blotter papers.

Due to the much lower production costs but similar effects to lysergic acid diethylamide (LSD), phenethylamine derivatives are sold as a cheaper replacement or deceptively as LSD itself. These potent hallucinogenic substances can lead to severe intoxication, thus a more profound understanding of their use is required. This includes the elucidation of the manufacturing processes for the commonly used blotter papers and the assessment of the risk of overdosing because of a heterogeneous distribution on the blotter papers. Besides the rapid detection of the analytes, the manufacturing process was elucidated by three different imaging techniques and liquid chromatography-mass spectrometry (LC-MS). A blotter paper sample, containing the two hallucinogenic phenethylamine derivatives 25I-NBOMe and 25C-NBOMe, was analyzed by complementary techniques such as micro x-ray fluorescence (µXRF), laser ablation (LA)-inductively coupled plasma-optical emission spectroscopy (ICP-OES), matrix assisted laser desorption ionization (MALDI)-MS, and with LC-MS after extraction. Using the signal from chlorine and iodine within the compounds, µXRF proved to be the fastest, cheapest and easiest method for identification, requiring no sample preparation at all. LA-ICP-OES provided three-dimensional information of the elements in the blotter paper. These results helped to confirm the assumption that manufacturers spray the compounds onto the paper. Whereas µXRF and LA-ICP-OES detected signals for chlorine and iodine, MALDI-MS-imaging showed the molecular distribution of both analytes. LC-MS analyses as a complementary method support the imaging results. Quantitative results for different drug hotspots revealed a heterogeneous distribution of the drugs on the blotter paper implying an inherent risk of overdosing for consumers.

Phosphorene nanocomposite with high environmental stability and antifouling capability for simultaneous sensing of clenbuterol and ractopamine.

A series of phosphorene (BP) nanocomposites was prepared to realize simultaneous electrochemical determination of clenbuterol (CLB) and ractopamine (RAC). CLB and RAC are the most commonly used ß-agonists in animal-derived food. The BP nanohybrid was obtained by co-decoration with both mono(6-mercapto-6-deoxy)-ß-cyclodextrin and poly(3,4-ethylenedioxythiophene) nanoparticles. It displays high stability, antifouling capability, a large electrochemical active surface and good electrochemical response. The electrochemical assisted antifouling strategy was selected by further eliminating the fouling of the electrode surface using continuous cyclic voltammetry. The electrode was employed for electrochemical sensing of CLB and RAC at typical peak voltages of 0.8 and 1.0 V (vs. SCE). Responses are linear in the 0.3-90 µM concentration range for CLB, and from 0.3 to 9.4 µM for RAC under optimal conditions. The limit of detection are 0.14 and 0.12 µM, respectively. The sensor was employed for simultaneous determination of CLB and RAC in (spiked) beef, feed and bovine serum samples with acceptable recoveries. Graphical abstractAn electrochemically assisted anti-fouling method for simultaneous voltammetric nanosensing of clenbuterol (CLB) and ractopamine (RAC) in edible cattle product samples using high-stable and anti-foul phosphorene (BP) co-decorated with mono(6-mercapto-6-deoxy)-ß-cyclodextrin (S-ß-CD) and poly(3,4-ethylenedioxythiophene) (PEDOTNPs).

Photoelectrochemical determination of ractopamine based on inner filter effect between gold nanoparticles and graphitic carbon nitride-copper(II) polyphthalocyanine coupled with 3D DNA stabilizer.

Copper(II) polyphthalocyanine (CuPPc) was combined with graphitic carbon nitride (g-C3N4) to form a heterojunction with enhanced photoelectrochemical (PEC) signal. A sensitive PEC method was developed for determination of ractopamine based on a PEC inner filter effect between gold nanoparticles (AuNPs) and the g-C3N4/CuPPc. A gold electrode was modified with g-C3N4/CuPPc and the DNA was linked to the AuNPs. Initially, the PEC signal is weak due to the inner filter effect between the AuNPs and g-C3N4/CuPPc. In the presence of ractopamine, it interacts with the aptamer and the complementary chain (C chain) is released. This triggers the entropy-driven cyclic amplification and results in the release of the substrate B chain (SB chain) from three-dimensional DNA stabilizer. The probe is released from the electrode due to the interaction of probe DNA and the SB chain. As a result, the PEC signal increases linearly in the 0.1 pmol·L-1 to 1000 pmol·L-1 ractopamine concentration range. The detection limit is 0.03 pM, and the relative standard deviation is 3.4% (at a 10 pmol·L-1 level; for n = 11). The method has been successfully applied to the determination of ractopamine in pork samples. Graphical abstract Schematic presentation of detection method based on PEC inner filter effect between AuNPs and the g-C3N4/CuPPc being fabricated for ractopamine. 3D DNA was used as stabilizer to decrease the PEC blank signal.

The impacts of temperature, alcoholic degree and amino acids content on biogenic amines and their precursor amino acids content in red wine.

The aim was to study how factors such as temperature, alcoholic degree, and amino acids supplementation are able to influence the content of tyramine, histamine, 2-phenylethylamine, tryptamine and their precursor amino acids in winemaking process. Biogenic amines and amino acids were quantified at the beginning, middle and end of alcoholic fermentation, and at the end of malolactic fermentation. In general, samples produced with amino acid supplementation did not show the highest concentrations of biogenic amines, except for histamine, which content increased with the addition of the four amino acids. The synthesis of tyramine was mainly affected by the temperature and alcoholic degree, the formation of phenylethylamine was largely influenced by alcoholic degree, and tryptamine synthesis principally depended on temperature. Interestingly, there was interaction between these three factors for the biogenic amines studied. In conclusion, winemaking conditions should be established depending on the biogenic amine which synthesis is required to be controlled.

Overview of regulation of dietary supplements in the USA and issues of adulteration with phenethylamines (PEAs).

The multi-billion dollar dietary supplement industry is global in reach. The industry has been criticized for problems related to poor quality control, safety, misbranding, and adulteration. In this review, we describe how the US Food and Drug Administration (FDA) regulates dietary supplements within the framework of the Federal Food, Drug, and Cosmetic Act (FD&C Act). The Dietary Supplement Health and Education Act of 1994 (DSHEA), which amended the FD&C Act, gave the FDA the authority to promulgate Good Manufacturing Practices for dietary supplements and required that manufacturers provide the FDA information supporting a conclusion that the ingredients are reasonably expected to be safe if the dietary ingredients were not marketed in the USA before 15 October 1994. Recent amendments to the FD&C Act require that serious dietary-supplement-related adverse events be reported to the FDA and provide the agency with mandatory recall authority. We discuss the presence of naturally occurring (e.g. Ephedra, Citrus aurantium, Acacia) and synthetic (e.g. ß-methylphenethylamines, methylsynephrine, α-ethyl-phenethylamine) biologically active phenethylamines (PEAs) in dietary supplements and of PEA drugs (e.g. clenbuterol, fenfluramine, sibutramine, lorcaserin) in weight-loss products. Regulatory actions against manufacturers of products labelled as dietary supplements that contain the aliphatic amines 1,3-dimethylamine and 1,3-dimethylbutylamine, and PEAs such as ß-methylphenethylamine, aegeline, and Dendrobium illustrate the FDA's use of its authority under the FD&C Act to promote dietary supplement safety. Published 2016. This article is a U.S. Government work and is in the public domain in the USA.

The interaction of fiber, supplied by distillers dried grains with solubles, with an antimicrobial and a nutrient partitioning agent on nitrogen balance, water utilization, and energy digestibility in finishing pigs.

The objective of this study was to determine if a higher-fiber diet alters the response of finishing pigs to an antimicrobial (tylosin phosphate [TP]) and a nutrient partitioning agent (ractopamine HCl [RAC]) in terms of N and water utilization and energy digestibility. Seventy-two gilts (initial BW = 107.4 ± 4.2 kg) were blocked by weight and allotted to 1 of 8 dietary treatments. Treatments were arranged as a 2 × 2 × 2 factorial: distillers dried grains with solubles (DDGS; 0 vs. 30%), RAC (0 mg of RAC/kg and 0.70% standardized ileal digestible [SID] Lys vs. 5 mg of RAC/kg and 0.95% SID Lys) and TP (0 vs. 44 mg of TP/kg). Pig was the experimental unit, with 9 replications per treatment. Pigs were housed in individual metabolism crates and fed treatment diets for 17 d. Feed was provided twice daily, as much as the pigs could consume within 1 h per meal, and water was provided to the pigs between feeding periods, ad libitum. Fecal and urine collection occurred on d 7 and 8 and on d 15 and 16, for sampling periods 1 and 2, respectively. Pigs fed the DDGS diets had reduced ADG ( < 0.001) and ADFI ( < 0.0001). The apparent total tract digestibility (ATTD) of N and GE were lower for the 30% DDGS diets than the 0% DDGS diets ( < 0.0001). Ractopamine improved ADG ( < 0.0001), G:F ( < 0.0001), and N retention ( < 0.001) and tended to increase daily water intake ( < 0.10). Pigs fed RAC had higher N intake and urinary excretion and lower N retention in Period 2 than in Period 1 ( < 0.05), indicating a decline in the response to RAC over time. Tylosin phosphate did not affect ADFI or G:F but did improve ATTD of N ( < 0.05). There was a tendency for a TP × DDGS interaction ( < 0.10) for ADG, where TP tended to increase ADG in pigs fed 0% DDGS diets ( < 0.10) but not in pigs fed 30% DDGS diets ( > 0.10). Pigs fed DDGS diets had higher N intake ( < 0.01) and higher fecal ( < 0.0001) and urinary ( < 0.01) N excretion with no difference in N retention (g/d). Overall, RAC increased N retention by 33% ( < 0.0001) and the response to RAC was similar in both corn-soybean meal-based and corn-soybean meal-DDGS-based diets. Tylosin phosphate tended to improve growth performance in pigs fed corn-soybean meal-based diets but not in diets containing 30% DDGS; however, this response was not explained by changes in N balance or in energy digestibility.

Brewing effect on levels of biogenic amines in different coffee samples as determined by LC-UV.

Eight biogenic amines (spermine, spermidine, putrescine, histamine, tyramine, phenylethylamine, cadaverine and serotonin) were determined by LC-UV after derivatization with dansyl-chloride in both ground coffee and coffee beverages obtained by different methods. In ground coffee, the most relevant amine was PUT, followed by SPD, HIS, TYR, CAD, SPM, PHE, and SER, with the total BAs content decreasing as the roasting degree increased. In coffee brews, the order was PUT, SPM, TYR, CAD, SPD, PHE, HIS, and SER, but at a very low level in comparison with the amount of BAs determined in roasted ground coffee. Beverages prepared by espresso, capsule, and pod machines had the lowest BAs contents, as a result of the thermal and physical stress imposed on ground coffee by these methods, while mocha contained the highest BAs amounts owing to lower pressure and longer brewing time.

Inhibition of monoamine oxidase (MAO) by α-ethylphenethylamine and N,α-diethylphenethylamine, two compounds related to dietary supplements.

Phenethylamines can interact with the metabolic enzyme monoamine oxidase (MAO), which can cause neurochemical dysfunction or changes in drug potency. A methamphetamine analog, N,α-diethylphenethylamine (N,α-DEPEA), was recently discovered in athletic performance-enhancing supplements, along with discovery of its metabolite, α-ethylphenethylamine (AEPEA). In vitro inhibition of human recombinant MAO by AEPEA and N,α-DEPEA was evaluated by measuring the fluorescence of 4-hydroxyquinoline produced from MAO substrate, kynuramine. AEPEA competitively inhibited human recombinant MAO A (Ki = 14.0 µM), which was 17-fold stronger compared to MAO B (Ki = 234 µM). Furthermore, N,α-DEPEA was a weak inhibitor of both MAO A (Ki = 251 µM) and MAO B (Ki = 159 µM). Trends regarding MAO A inhibition were explored among structural analogs, yielding the following ranking: amphetamine (Ki = 5.3 µM), AEPEA (Ki = 14.0 µM), methamphetamine (Ki = 17.2 µM), phentermine (Ki = 196 µM), and N,α-DEPEA (Ki = 251 µM). This study provides important data relating chemical structures and biochemical effects for two emerging compounds associated with dietary supplements.

LC-MS-MS analysis of dietary supplements for N-ethyl-α-ethyl-phenethylamine (ETH), N, N-diethylphenethylamine and phenethylamine.

There has been a recent rise in the number of cases of athletes being banned from competition because of positive tests for prohibited substances in their biological specimens. Most of these substances are on the World Anti-Doping Agency (WADA) prohibited list, while others are not specifically named on the list. N-Ethyl-α-ethyl-phenethylamine (ETH), a derivative of phenethylamine (PEA), is one of these unlisted substances and shares chemical and biological effects to the amphetamines, which are listed on the WADA prohibited substances list. It is classified as Category 6B stimulant on the list. This study was directed toward the development of an liquid chromatography tandem mass spectrometry (LC-MS-MS) method for the analysis of ETH in performance-enhancing dietary supplement. A standard was prepared and confirmed by spectroscopic analysis, which was then used to develop the analytical procedure. The procedure was validated and found to have an limit of detection of 2.5 ng/mL, limit of quantification of 5 ng/mL and upper limit of linearity of 500 ng/mL, with within-day variability at the 10-ng/mL level range of 3.88-7.89% (n = 6) and 1.39-3.36% (n = 6) for the 100-ng/mL level. The day-to-day variability was 9.8% for the low control and 3.1% for the high control. The method was used to analyze a variety of dietary supplements for ETH as well as PEA and its N, N-diethyl derivative (NDP).

Effects of dietary L-carnitine and ractopamine HCl on the metabolic response to handling in finishing pigs.

Two experiments (384 pigs; C22 × L326; PIC) were conducted to determine the interactive effect of dietary L-carnitine and ractopamine HCl (RAC) on the metabolic response of pigs to handling. Experiments were arranged as split-split plots with handling as the main plot and diets as subplots (4 pens per treatment). Dietary L-carnitine (0 or 50 mg/kg) was fed from 36.0 kg to the end of the experiments (118 kg), and RAC (0 or 20 mg/kg) was fed the last 4 wk of each experiment. At the end of each experiment, 4 pigs per pen were assigned to 1 of 2 handling treatments. Gently handled pigs were moved at a moderate walking pace 3 times through a 50-m course and up and down a 15° loading ramp. Aggressively handled pigs were moved as fast as possible 3 times through the same course, but up and down a 30° ramp, and shocked 3 times with an electrical prod. Blood was collected immediately before and after handling in Exp. 1 and immediately after and 1 h after handling in Exp. 2. Feeding RAC increased (P < 0.01) ADG and G:F, but there was no effect (P > 0.10) of L-carnitine on growth performance. In Exp. 1 and 2, aggressive handling increased (P < 0.01) blood lactate dehydrogenase (LDH), lactate, cortisol, and rectal temperature and decreased blood pH. In Exp. 1, there was a RAC × handling interaction (P < 0.06) for the difference in pre- and posthandling blood pH and rectal temperature. Aggressively handled pigs fed RAC had decreased blood pH and increased rectal temperature compared with gently handled pigs, demonstrating the validity of the handling model. Pigs fed RAC had increased (P < 0.01) LDH compared with pigs not fed RAC. Pigs fed L-carnitine had increased (P < 0.03) lactate compared with pigs not fed L-carnitine. In Exp. 2, pigs fed RAC had lower (P < 0.02) blood pH immediately after handling, but pH returned to control levels by 1 h posthandling. Lactate, LDH, cortisol, and rectal temperature changes from immediately posthandling to 1 h posthandling were not different (P > 0.10) between pigs fed L-carnitine and those fed RAC, indicating that L-carnitine did not decrease recovery time of pigs subjected to aggressive handling. These results suggest that pigs fed 20 mg/kg of RAC are more susceptible to stress when handled aggressively compared with pigs not fed RAC. Dietary L-carnitine fed in combination with RAC did not alleviate the effects of stress. This research emphasizes the importance of using proper animal handling techniques when marketing finishing pigs fed RAC.

Monitoring of ractopamine concentration in the mixture of this feed additive with vitamin mineral complex and with swine feed by HPLC.

Ractopamine (RAC) analysis at all stages in the feed chain until its final mixing into swine feed is necessary to ensure the safety of all meat consumers and to decrease waste and the cost of supplementation of feed. Two suitable HPLC methods were developed and validated for RAC determination in vitamin mineral complex (VMC) and in swine feed. Both methods employed reverse-phase (C18 column at 40°C) and isocratic elution, but with some modifications to the methods. Validation parameters, such as selectivity, linearity, precision, trueness and robustness, were shown to be within the acceptable range. Therefore, the developed methods can be successfully applied for the monitoring of RAC concentrations in samples of VMC and swine feed ensuring economy to producers and security to consumers of swine meat.

"Bath salts" and "plant food" products: the experience of one regional US poison center.

Abuse of psychogenic substances sold as "bath salts" and "plant food" has escalated in recent years in the United States (USA). Previous reports suggest regional differences in the primary active ß-keto phenylalkylamines found in these products and the corresponding signs and symptoms reported after exposure. Currently, there are only limited studies describing the clinical effects associated with reported "bath salts" exposure in the USA. This study describes the clinical effects associated with "bath salt" and "plant food" exposures as reported to the poison center serving the state of North Carolina (Carolinas Poison Center). We performed a retrospective review of the Carolinas Poison Center database for all cases of reported human exposure to "bath salt" and "plant food" products from 2010 to 2011 with specific attention to clinical effects and routes of exposure. Additionally, we reviewed therapies used, trended the volume of exposure cases reported over the study period, and evaluated the distribution of calls within state counties using descriptive statistics. Carolinas Poison Center received 485 total calls and 409 reported exposure calls regarding "bath salt" or "plant food" products between January of 2010 and December of 2011. The peak of reported exposures occurred in May of 2011. Clinical effects commonly reported in the exposure cases generated from these calls included tachycardia (53.3 %, n = 218), agitated/irritable (50.4 %, n = 206), hallucination/delusions (26.7 %, n = 109), and hypertension (25.2 %, n = 103). In addition to intravenous fluids, common therapies included benzodiazepines (46.0 %, n = 188), sedation (13.4 %, n = 55), alkalinization (3.90 %, n = 16), antihistamine (4.16 %, n = 17), and intubation (3.67 %, n = 15). Haloperidol was the antipsychotic agent used most often to treat agitation (n = 40). Serious complications associated with reported exposure to "bath salt" and "plant food" products included rhabdomyolysis, renal failure, excited delirium syndrome, and death. While treatments have not been empirically determined, sedation with benzodiazepines, aggressive cooling for hyperthermic patients, and use of small doses of antipsychotics for choreoathetoid movements not controlled with benzodiazepines are not likely to be harmful.

Phenylethylamines from Browningia candelaris (Cactaceae) / Feniletilaminas de Browningia candelaris (Cactaceae)

The analysis by gas chromatography coupled with mass spectrometry (GC-MS) of the alkaloidal extract of Browningia candelaris (Cactaceae) showed the presence of N-acetyl-3,4-dimethoxyphenylethylamine; N,N-dimethyl-3,4-dimethoxyphenylethylamine; N,N-dimethyl-4-methoxyphenylethylamine; and 4-methoxyamphetamine. The presence of these psychoactive compounds is discussed in terms of their possible ritual use in Andean cultures of Northern Chile.

El análisis por medio de cromatografía de gases acoplada a espectrometría de masas (CG-EM) del extracto alcaloidal de Browningia candelaris (Cactaceae) mostró la presencia de N-acetil-3,4-dimetoxifeniletilamina; N,N-dimetil-3,4-dimetoxifeniletilamina; N,N-dimetil-4-metoxifeniletilamina y 4-metoxianfetamina. La presencia de estos compuestos psicoactivos se discute en términos de su posible utilización en ceremonias mágico-religiosas por culturas andinas del norte de Chile.

Development and validation of a pre-column reversed phase liquid chromatographic method with fluorescence detection for the determination of primary phenethylamines in dietary supplements and phytoextracts.

A sensitive and selective reversed-phase liquid chromatographic (RP-LC) method was developed and validated to determine octopamine, tyramine and Tyrosine (Tyr) in complex matrices as formulations and phytoextracts (Citrus aurantium), after pre-column derivatization with o-phthaldialdehyde (OPA) reagent. The chromatographic separations were performed at room temperature on a Phenomenex Luna C18 column using methanol and sodium acetate buffer (pH 5.5) by varying composition gradient elution as mobile phase and detected flurometrically at λ(em)=455 nm with λ(ex)=340 nm. The results obtained by the proposed method were compared with those achieved by a validated direct RP-LC method with fluorescence detection at λ(em)=310 nm with λ(ex)=275 nm, as reference method, using a Phenomenex Gemini C18 column under isocratic elution conditions with acetonitrile and sodium 1-heptanesulphonate (pH 3), as mobile phase. The higher sensitivity of the derivatization method (detection limit about 0.06 pmol) allowed the sure determination of octopamine present in traces in the examined samples. The repeatability of method (RSD) was ≤1.90% and there was no significant difference between repeatability and intermediate precision data. Recovery studies showed good results 99.5-101.3% with RSD ranging from 0.8 to 1.2%. All analyses were performed by mild conditions in absence of preliminary difficult extraction methodologies or laborious step of sample pre-treatment.

Determination of Citrus aurantium protoalkaloids using HPLC with acidic potassium permanganate chemiluminescence detection.

Acidic potassium permanganate chemiluminescence was explored as a sensitive and selective mode of detection for phenolic phenethylamines (adrenergic amines) in consumer products containing Citrus aurantium extracts. Nine commercially available weight-loss products were analysed using rapid reversed-phase chromatography with a monolithic column (separation time of 4 min). The results were in good agreement with package labelling, with some notable exceptions. The products contained a wide concentration range of synephrine and total adrenergic amines, and the difference in consumer intake was even greater when the manufacturers' recommended daily consumption was considered. The quantity of the extract, often specified on the packaging as equivalent grams of dry C. aurantium fruit, was a poor indicator of the concentration of the active ingredients. Methionine, a thioether amino acid contained in some weight-loss products, was identified as a potential interferent for this mode of detection.

Fast high-performance liquid chromatography analysis of phenethylamine alkaloids in Citrus natural products on a pentafluorophenylpropyl stationary phase.

In this study, the chromatographic performance of a pentafluorophenylpropyl (PFPP) stationary phase was evaluated for the rapid separation of phenethylamine alkaloids (i.e. (+/-)-octopamine, (+/-)-synephrine, tyramine, N-methyltyramine and hordenine) in Citrus aurantium plant material (fruits and peel), various Citrus species, extracts and dietary supplements claiming to contain C. aurantium. The problems of phenethylamine alkaloid separation, such as peak tailing, low retention and low resolution, were successfully solved with this stationary phase. The parameters used for the method optimization included the mobile phase counter ion concentration and column temperature. A Discovery HS F5 column (150 mm x 4.6 mm i.d., 5 microm) was used, with an isocratic mobile phase composed of 10 mM ammonium acetate in 90:10 ACN-H(2)O (v/v), at a flow rate of 1.0 mL/min. The column temperature was set at 20 degrees C. The photodiode array detector monitored the eluent at 225 nm. The total analysis time was 10 min. The validation parameters, such as linearity, sensitivity, accuracy, precision and specificity, were found to be highly satisfactory. With a simple sample preparation procedure, different matrices were successfully analyzed for their alkaloid content. The results indicated that the products on sale, labeled as dietary supplements, vary widely in the quantitative composition of the active constituents: the amount of (+/-)-synephrine, the major alkaloid, in such products ranged from 0.65 to 27.41 mg/g. The other compounds were either not detected or were present at low levels. The developed method can be considered suitable for the quality control of Citrus plant material and commercial products.

Development and validation of HPLC methods for the analysis of phenethylamine and indoloquinazoline alkaloids in Evodia species.

The aim of this study was to evaluate the chromatographic performance of a PEG stationary phase, in comparison with those of C18 columns, for the HPLC analysis of phenethylamine ((+/-)-synephrine) and indoloquinazoline (rutaecarpine and evodiamine) alkaloids in methanolic extracts of fruits of Evodia rutaecarpa (Juss.) Benth. and E. rutaecarpa (Juss.) Benth. var. officinalis (Dode) Huang (i.e., E. officinalis Dode) (Rutaceae family). The method was validated and showed good linearity, precision, accuracy, sensitivity, and specificity. The highest content of both phenethylamine and indoloquinazoline alkaloids was found in methanolic fruit extracts of E. rutaecarpa, and it was closely related to the degree of maturity. E. officinalis fruits displayed low amounts of both types of alkaloids. Furthermore, an enantioselective HPLC method for the enantioseparation of (+/-)-synephrine from Evodia fruits was applied, by using a protein-based chiral stationary phase with cellobiohydrolase (CBH) as the chiral selector (Chiral-CBH). Isolation of synephrine from Evodia aqueous fruit extracts was carried out by strong cation-exchange SPE. The results of the application of the method to the analysis of Evodia samples showed that (-)-synephrine was the main component while (+)-synephrine was present in low concentration.

Behavioral and neurochemical effects of deprenyl and beta-phenylethylamine in Wistar rats.

The effects of 1-deprenyl (1-16 mg kg-1, 3.5 hr) on brain levels of endogenous beta-phenylethylamine were assessed in animals under three conditions: (1) experience of lateral hypothalamic self-stimulation; (2) electrode implantation but no self-stimulation experience; (3) no surgical intervention. The increase in striatal levels of beta-phenylethylamine with 1-deprenyl treatment was attenuated in the self-stimulation condition relative to conditions (2) and (3). This differential effect of 1-deprenyl was not observed at the level of the hypothalamus. Administration of 1-deprenyl did not affect self-stimulation behavior. Equivalent analysis of beta-phenylethylamine levels was carried out using animals injected with beta-phenylethylamine (0.5-4 mg kg-1, 0.5 hr 1P and 1-deprenyl (4 mg kg-1, 3.5 hr sc). Injected beta-phenylethylamine with deprenyl pretreatment increased self-stimulation rates; concomitant striatal levels of approximately 190 ng g-1 of beta-phenylethylamine were observed and were associated with increased brainstem 5-HIAA but no change in striatal HVA, indicating possible involvement of 5-HT in this response to beta-phenylethylamine. It is proposed that experience of electrical hypothalamic stimulation may alter endogenous striatal beta-phenylethylamine metabolism, possibly via an alteration of mechanisms governing synthesis and/or catabolism.