Gas Chromatographic Method for Analysis of p-Synephrine in Citrus aurantium L. Products

A method has been developed and validated for the identification and quantification of p-synephrine in Citrus aurantium L. products. The approach comprises GC-FID analysis followed by GC-MS confirmation after clean-up by solid phase extraction with a strong cation-exchange phase and derivatization with cyclohexanone. Oxazolidine derivative of p-synephrine was subjected to analysis by nuclear magnetic resonance (¹H and ¹³C NMR), GC-FID and GC-MS. The GC method was validated and was found to be linear in the range of 125–500 mg%. Intra-day and inter-day precisions were 3.60 and 3.59%, respectively. Mean recovery from extract of C. aurantium was 78.1 ± 3.64%. The selectivity of the method was further improved by confirmation of oxazolidine derivative of p-synephrine by GC-MS.     

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.     

Simultaneous determination of Ephedra sinica and Citrus aurantium var. amara alkaloids by ion-pair chromatography

Ephedra sinica (Ma Huang) preparations have recently gained a lot of attention because of serious side effects associated with their prolonged consumption. Citrus aurantium var. amara is now used as an alternative, despite the fact that similar side effects are suspected. We have developed and validated the first analytical procedure for the simultaneous determination of all major alkaloids from both species. Using the ion-pairing reagent SDS, a C-18 stationary phase (3 μm material) and a pH-gradient for elution enabled the baseline separation of six alkaloids ((±)-octopamine, (±)-synephrine, tyramine, (−)-norephedrine, (+)-pseudoephedrine and (−)-ephedrine) within less than 30 min. The method is sensitive (LOD ≤ 4.6 ng and LOQ ≤ 16.2 ng on-column), selective (l-tyrosine and l-phenylalanine, two closely related amino acids did not interfere), accurate (recovery rates of spiked samples were between 97.5 and 102.0%), repeatable (σ_rel ≤ 4.6%) and precise (intra-day variation ≤7.7%, inter-day variation ≤7.0%). Without the need of a special sample treatment different matrices (plant material, commercial products) were successfully analyzed for their alkaloid content. Dominant alkaloids were (−)-ephedrine (0.9-1.6%) and/or (±)-synephrine (0.1-3.0%). Whether a product contained Ephedra-alkaloids or not could be determined in all investigated samples unambiguously.     

A Comparative Study of the Chemical Composition by SPME-GC/MS and Antiradical Activity of Less Common Citrus Species

Citrus secondary metabolites, such as terpene compounds, are very important for human health due to their bioactivity including anti-inflammatory, anti-cancer, and antioxidant effects. In this work, for the first time, the volatile chemical composition of peels and juices from four different Citrus species (C. junos, Citrus × aurantium, C. aurantium ‘Bizzarria’ and C. medica ‘Florentina’, commonly known as Yuzu jeune, Oni Yuzu, Bizzarria orange and Florence cedar, respectively) was investigated by Solid-Phase Microextraction-Gas Chromatography/Mass Spectrometry (SPME-GC/MS) technique and the antiradical activity was also examined. The results showed that limonene and γ-terpinene were the main volatile substances detected both in the juices and in the peels, followed by other minority compounds responsible for the phyto-complex of the unique aromas which characterize each individual analyzed Citrus species. Principal component analysis (PCA), performed on volatile compounds, showed both some correlation as well as a clear separation between the juice and the peel of each species. Among them, Oni Yuzu juice was found to be the richest in total polyphenols and flavonoids while its capacity to scavenge ABTS^•+ and DPPH^• radicals was similar to that of Yuzu Jeune and Bizzarria orange.     

Gas Chromatographic Method for Analysis of p-Synephrine in Citrus aurantium L. Products

A method has been developed and validated for the identification and quantification of p-synephrine in Citrus aurantium L. products. The approach comprises GC-FID analysis followed by GC-MS confirmation after clean-up by solid phase extraction with a strong cation-exchange phase and derivatization with cyclohexanone. Oxazolidine derivative of p-synephrine was subjected to analysis by nuclear magnetic resonance (¹H and ¹³C NMR), GC-FID and GC-MS. The GC method was validated and was found to be linear in the range of 125–500 mg%. Intra-day and inter-day precisions were 3.60 and 3.59%, respectively. Mean recovery from extract of C. aurantium was 78.1 ± 3.64%. The selectivity of the method was further improved by confirmation of oxazolidine derivative of p-synephrine by GC-MS.     

Response surface optimization of ultrasound‐assisted flavonoids extraction from the flower of Citrus aurantium L. var. amara Engl

Citrus aurantium L. var. amara Engl is a member of genus Citrus (Rutaceae) and has been used in Chinese medicine with the effectiveness of digestant and expectorant. Ultrasonic‐assisted extraction process for maximum flavonoids from the flower of Citrus aurantium L. var. amara Engl was investigated by response surface methodology. Through single factor experiment, ranges of the main variables (including ethanol concentration, solid/liquid ratio, extraction time and temperature) affecting the extraction yield of flavonoids were confirmed. Box–Behnken central composite design consisting of 24 experimental runs and 5 replicates at zero point was then applied and a regress model was obtained to predict the optimal extraction yield. The ANOVA indicated that the regression equation fits very well with the actual situation, reflecting the relationship between the extraction yield of flavonoids and extraction conditions. The optimal conditions were as follows: extraction temperature 72.11°C, time 51.89 min, ethanol concentration 51.19% and liquid/solid ratio of 40:10. Under the optimal conditions, the maximum response value of yield (1.88%) was consistent with the experimental value (1.87%), indicating the feasibility and validation of response surface methodology in optimizing the extraction of flavonoids from the flower of Citrus aurantium L. var. amara Engl.     

Simultaneous quantification of adrenergic amines and flavonoids in C. aurantium, various Citrus species, and dietary supplements by liquid chromatography

An analytical method was developed for the simultaneous quantitative analysis of 6 amines and 20 flavonoids in fruits and extracts of 30 Citrus species, including C. aurantium, near-Citrus relatives, and dietary supplements by liquid chromatography with photodiode array detection. The separation was achieved with a Phenomenex Synergi Hydro reversed-phase column using gradient mobile phase of sodium acetate buffer (pH 5.5) and acetonitrile. Elution was run at a flow rate of 1.0 mL/min and UV at 254, 280, and 330 nm. Among the amines analyzed, synephrine, the main component, was present in the levels from 0.11 to 2.0 mg/g dry weight in 21 Citrus species and 0.07 to 18.62% in dietary supplements claiming to contain C. aurantium. The flavanones and flavones were analyzed in the same Citrus samples and were species-specific. The levels of flavones were very low compared with those of flavanones. The method facilitated the simultaneous quantification of 6 amines and 20 flavonoids in various Citrus species, the distinction between the different Citrus species, and the analysis of dietary supplements containing C. aurantium.     

Recent studies on selected botanical dietary supplement ingredients

The market for botanical dietary supplements in the US has grown rapidly during the last 15 years. Use of newly introduced botanical ingredients has often outpaced an adequate scientific understanding of the ingredients themselves. This may lead to problems, including misidentification, mislabeling, adulteration, and toxicity related to the intended ingredient or one substituted for it. This article reviews recent work with several botanical ingredients (Ephedra, Citrus species, Hoodia gordonii, Teucrium, isoflavones) that illustrates the complexity of the current situation and approaches that contribute to ensuring the quality of botanical ingredients. Recent work with contamination of botanical products by mycotoxins is also reviewed. The need for tools for botanical authentication and methods for reproducible extraction of bioactive constituents is critical. Such tools, and improved analytical techniques for identifying potentially bioactive constituents in fresh plant material and in concentrated extracts and for detection of hazardous contaminants, are expected to improve the overall quality and safety of botanical dietary supplement ingredients.     

Citrus aurantium L. Active Constituents,Biological Effects and Extraction Methods. An Updated Review

Citrus genus is a prominent staple crop globally. Long-term breeding and much hybridization engendered a myriad of species, each characterized by a specific metabolism generating different secondary metabolites. Citrus aurantium L., commonly recognized as sour or bitter orange, can exceptionally be distinguished from other Citrus species by unique characteristics. It is a fruit with distinctive flavor, rich in nutrients and phytochemicals which possess different health benefits. This paper presents an overview of the most recent studies done on the matter. It intends to provide an in-depth understanding of the biological activities and medicinal uses of active constituents existing in C. aurantium. Every plant part is first discussed separately with regards to its content in active constituents. All extraction methods, their concepts and yields, used to recover these valuable molecules from their original plant matrix are thoroughly reported.     

Study on the racemization of synephrine by off-column chiral high-performance liquid chromatography

In this study, the racemization kinetic parameters of R-(−)-synephrine, the active phenethylamine alkaloid of Citrus aurantium L., were determined by means of an off-column HPLC method. Enantioseparation was carried out in different buffer solutions and solvents on a chiral stationary phase (CSP) with cellobiohydrolase as the chiral selector (Chiral-CBH, 100 mm × 4.0 mm i.d., 5 μm). The mobile phase was 10 mM sodium phosphate buffer (pH 6.0)-2-propanol (95:5, w/w), with 50 μM disodium EDTA, at 0.8 mL/min. The column was thermostatted at 20 °C and detection was set at 225 nm. The influence of pH value, ionic strength, temperature and addition of organic modifier on the rate constant, the half-life of racemization and the free energy barrier of racemization of R-(−)-synephrine were determined. Among the different chemical and physical parameters evaluated as affecting the racemization of naturally occurring R-(−)-synephrine, pH, temperature and addition of an organic co-solvent appear to have the strongest effect, while ionic strength does not exert a significant influence on the racemization rate. The results of the present study indicated that synephrine racemization is possible at high temperature at both acidic and basic pH values; therefore, the extraction procedure of R-(−)-synephrine from the plant material should be carried out under specific conditions to preserve the stereochemical integrity and the biological activity of this secondary metabolite.     

Rapid Chiral Separation by Flow-Through Chromatography with a Biporous Stationary Phase

Enantiomer separation is an area of increasing importance in chemistry and in the pharmaceutical industry. Although liquid chromatography is one of the most practical processes for chiral resolution, the high mass-transfer resistance of most commercially available packings makes chiral resolution time-consuming. In this work, a novel chiral stationary phase (CSP) with wide pores was prepared by coupling bovine serum albumin to a biporous resin with triazine as a linker. The rigid biporous medium was fabricated by radical suspension copolymerization with solid granules and solvents as porogenic agents. Studies by scanning electron microscopy and mercury intrusion porosimetry revealed the matrix contained two types of pore—micropores smaller than 180 nm and macropores of 500–7300 nm. Because the macropores provide convective flow channels for the mobile phase, the chromatographic process can be operated at high flow rate with low back-pressure. The biporous CSP was applied to the resolution of d, l-tryptophan (Trp). When the flow velocity was as high as 1800 cm h^–1, d, l-Trp could still be separated. By comparison of the resolution of d, l-Trp on this CSP at high flow velocities with that predicted theoretically for conventional supports we concluded that the increased flow velocity had little effect on the resolution of enantiomers with biporous packings. The results indicate that the protein biporous CSP is promising for high-speed resolution of enantiomers.Revised: 8 October 2004 and 3 January 2005     

Determination of Bitter Orange alkaloids in dietary supplements standard reference materials by liquid chromatography with ultraviolet absorbance and fluorescence detection

Four adrenergic amines [synephrine, octopamine, tyramine, and n-methyltyramine] were determined in a variety of Bitter Orange containing dietary supplements. Two extraction techniques were evaluated in detail: Soxhlet extraction and sonication extraction. A liquid chromatographic separation using a reversed-phase C(18) stationary phase and the ion-pairing reagent sodium dodecyl sulfate was developed to separate the Bitter Orange alkaloids. Ultraviolet absorbance detection at 220 nm and fluorescence detection with excitation at 273 nm and emission at 304 nm were used for the alkaloid detection. The method described was used for the assignment of the levels of the predominant alkaloids in three candidate standard reference materials containing Bitter Orange.     

Production of <Emphasis Type="SmallCaps">d</Emphasis>-tagatose,a Functional Sweetener,Utilizing Alginate Immobilized <Emphasis Type="Italic">Lactobacillus fermentum</Emphasis> CGMCC2921 Cells

d-tagatose is a ketohexose that can be used as a novel functional sweetener in foods, beverages, and dietary supplements. This study was aimed at developing a high-yielding d-tagatose production process using alginate immobilized Lactobacillus fermentum CGMCC2921 cells. For the isomerization from d-galactose into d-tagatose, the immobilized cells showed optimum temperature and pH at 65 °C and 6.5, respectively. The alginate beads exhibited a good stability after glutaraldehyde treatment and retained 90% of the enzyme activity after eight cycles (192 h at 65 °C) of batch conversion. The addition of borate with a molar ratio of 1.0 to d-galactose led to a significant enhancement in the d-tagatose yield. Using commercial β-galactosidase and immobilized L. fermentum cells, d-tagatose was successfully obtained from lactose after a two-step biotransformation. The relatively high conversion rate and productivity from d-galactose to d-tagatose of 60% and 11.1 g l⁻¹ h⁻¹ were achieved in a packed-bed bioreactor. Moreover, lactobacilli have been approved as generally recognized as safe organisms, which makes this L. fermentum strain an attracting substitute for recombinant Escherichia coli cells among d-tagatose production progresses.     

Glycoconjugates of amino acids. Preparation throughN-alkylation of amino acids withN-chloroacetyl-β-glycopyranosylamines

Monoalkylation of amino acids of different structural types withN-chloroacetyl-glycosylamines was shown to be applicable for the preparation of glycoconjugates containing β-d-galactose,N-acetyl-β-d-glucosamine, β-d-mannose, and lactose residues. The glycoconjugates were synthesized from amino acids with secondary (sarcosine,l-proline) or primary (l-2- and 4-aminobutyric acids,l-tryptophan) amino groups as well as from various amino dicarboxylic acids (N-methyl-dl-aspartic,dl-aspartic,l-glutamic, anddl-2-aminoadipic acids). The derivatives obtained may be of interest for glycotargeting of physiologically active compounds of this series. Translated fromIzvestiya Akademii Nauk. Seriya Khimicheskaya, No. 7, pp. 1377–1380, July, 1999.     

Chromatographic and electrophoretic methods for the analysis of phenethylamine [corrected] alkaloids in Citrus aurantium

Citrus aurantium (bitter orange) is a plant belonging to the family Rutaceae, whose fruit extracts have been used recently for the treatment of obesity. The most important biologically active constituents of the C. aurantium fruits are phenethylamine alkaloids (i.e. octopamine, synephrine, tyramine, N-methyltyramine and hordenine). Synephrine is a primary synthesis compound with pharmacological activities such as vasoconstriction, elevation of blood pressure and relaxation of bronchial muscle. Synephrine is present in the peel and the edible part of Citrus fruit. Of the adrenergic amines of natural origin, synephrine has been found to be the main constituent of C. aurantium fruits and extracts; the other alkaloids are either absent or present in only low concentrations. It is known that synephrine and the other amines found in C. aurantium have adverse effects on the cardiovascular system, owing to adrenergic stimulation. In light of the great commercial proliferation of C. aurantium herbal medicines in recent years, this review provides an overview of various extraction, separation and detection techniques employed for the qualitative and quantitative determination of the alkaloids in C. aurantium and related species. The application of chromatographic and electrophoretic methods for the separation and determination of these active components in C. aurantium plant material and derivatives are described. Since synephrine is a chiral compound, enantioselective chromatographic and electrophoretic techniques for the analysis of synephrine enantiomers in natural products are presented. Furthermore, examples of identification of these active compounds in complex matrices by hyphenated methods, such as gas chromatography-mass spectrometry and high-performance liquid chromatography-mass spectrometry, are described. The advantages and limitations of these separation and identification methods are assessed and discussed.     

Use of TLC and UV–Visible Spectrometry for Fingerprinting of Dietary Supplements

We have studied dietary supplements containing sea-buckthorn (Hippophae rhamnoides), bilberry (Vaccinium myrtillus), and cranberry (Vaccinium vitis-idaea) by use of thin-layer chromatography (TLC), image analysis, and UV–visible spectroscopy. The results were subjected to multivariate techniques, for example cluster analysis (CA), principal-components analysis (PCA), and linear discriminant analysis (LDA). Evaluation of the discriminating power of TLC coupled with digital image analysis for characterization of the supplements was performed by use of chemometrics. Zero-order, first-order, and normalized zero-order UV–visible spectra of extracts of fruit and dietary supplements were digitized and used in the chemometric analysis. To increase the relevance of the study, the dietary supplement extracts were compared with extracts of the natural fruit. Results obtained by application of CA, PCA, and PCA–LDA to the chromatograms suggest the possibility of discrimination among dietary supplements on the basis of the raw material used for their preparation.

     

Rapid Discrimination of Citrus reticulata ‘Chachi’ by Electrospray Ionization–Ion Mobility–High-Resolution Mass Spectrometry

A common idea is that some dishonest businessmen often disguise Citrus reticulata Blanco varieties as Citrus reticulata ‘Chachi’, which places consumers at risk of economic losses. In this work, we combined high-resolution ion mobility (U-shaped mobility analyzer) with high-resolution mass spectrometry to rapidly distinguish Citrus reticulata ‘Chachi’ from other Citrus species. The samples were analyzed directly through simple extraction and the analytes were separated in one second. It only took about 1 min to perform a cycle of sample analysis and data acquisition. The results showed that polymethoxylated flavones and their isomers were separated easily by the ion mobility analyzer and preliminarily identified according to the accurate mass. Moreover, the collision cross-section values of all analytes, which could be used as auxiliary parameters to characterize and identify the compounds in the samples, were measured. Twenty-four samples were grouped as two clusters by multivariate analysis, which meant that Citrus reticulata ‘Chachi’ could be effectively differentiated. It was confirmed that the developed method had the potential to rapidly separate polymethoxylated flavones and distinguish between Citrus reticulata ‘Chachi’ and other Citrus reticulata Blanco varieties.     

Fast-scan cyclic voltammetry for the detection of tyramine and octopamine

Tyramine and octopamine are biogenic amine neurotransmitters in invertebrates that have functions analogous to those of the adrenergic system in vertebrates. Trace amounts of these neurotransmitters have also been identified in mammals. The purpose of this study was to develop an electrochemical method using fast-scan cyclic voltammetry at carbon-fiber microelectrodes to detect fast changes in tyramine and octopamine. Because tyramine is known to polymerize and passivate electrode surfaces, waveform parameters were optimized to prevent passivation. No fouling was observed for octopamine when the electrode was scanned from 0.1 to 1.3 V and back at 600 V/s, while a small decrease of less than 10% of the signal was seen for 15 repeated exposures to tyramine. The technique has limits of detection of 18 nM for tyramine and 30 nM for octopamine, much lower than expected levels in insects and lower than basal levels in some brain regions of mammals. Current was linear with concentration up to 5 μM. This voltammetry technique should be useful for measuring tyramine and octopamine changes in insects, such as the fruit fly, Drosophila melanogaster.

Liquid Chromatography for Separation and Quantitative Determination of Adrenergic Amines and Flavonoids from Poncirus trifoliatus Raf. Fruits at Different Stages of Growth

The fruits of Poncirus trifoliatus Raf. (Rutaceae) have been used against allergic diseases for generations and still occupy an important place in traditional oriental medicine. They have also been used to treat gastric and duodenal ulcers. Chemical analysis of extracts of Poncirus trifoliatus Raf. fruit at different stages of maturation revealed variations in the concentrations of flavonoids present. Fourteen flavonoids (neoeriocitrin, narirutin, naringin, hesperidin, neohesperidin, neoponcirin, poncirin, naringenin, hesperetin, sinensetin, nobiletin, heptamethoxyflavone, 5-O-demethylnobiletin, and tangeretin) and five amines (synephrine, octopamine, N-methyltyramine, hordenine, and tyramine) in extracts from the fruit of Poncirus trifoliatus Raf. were analyzed by HPLC with a C₁₈ reversed-phase column. The concentrations of the four flavonoids naringin, poncirin, narirutin, and neoponcirin was maximum during the first stage of growth and gradually decreased until the fruits were fully developed. The paper also discusses the anatomical variations observed at different stages of development of the fruit.     

Glycoconjugates of amines: alkylation of primary and secondary amines withN-chloroacetyl-β-glycopyranosylamines

Efficient monoalkylation of a series of primary and secondary amines was demonstrated with the use ofN-chloroacetylglycosylamines derived fromd-glucose,d-galactose,d-mannose,N-acetyl-d-glucosamine, and lactose. The reaction was shown to be useful for incorporation of carbohydrate residues into physiologically active compounds. Glycoconjugates of some derivatives of piperazine, 2-phenylethylamine, tryptamine, and important biogenic amines (norephedrine, octopamine, dopamine) were prepared. Translated fromIzvestiya Akademii Nauk. Seriya Khimicheskaya. No. 6, pp. 1244–1247, June, 1998.