Determination of beta-carboline alkaloids in foods and beverages by high-performance liquid chromatography with electrochemical detection at a glassy carbon electrode modified with carbon nanotubes

Simple and sensitive methods for the separation and quantification of β-carboline alkaloids in foods and beverages by HPLC with electrochemical detection at carbon nanotubes-modified glassy carbon electrodes (CNTs-GCE) are reported. Electrode modification with multi-wall CNTs produced an improved amperometric response to β-carbolines, in spite of the working medium consisting of methanol:acetonitrile: 0.05 mol L⁻¹ Na₂HPO₄ solution of pH 9.0 (20:20:60). On the contrary to that observed at a bare GCE, a good repeatability of the amperometric measurements carried out at +900 mV versus Ag/AgCl (R.S.D. of 3.2% for i_p, n = 20) was achieved at the CNTs-GCE. Using an Ultrabase C₁₈ column and isocratic elution with the above mentioned mobile phase, a complete resolution of the chromatographic peaks for harmalol, harmaline, norharmane, harmane and harmine, was achieved. Calibration graphs over the 0.25-100 μM range with detection limits ranging between 4 and 19 ng mL⁻¹, were obtained. The HPLC-ED at CNTs-GCE method was applied to the analysis of beer, coffee and cheese samples, spiked with β-carbolines at concentration levels corresponding to those may be found in the respective samples. The steps involved in sample treatment, such as extraction and clean-up, were optimized for each type of sample. Recoveries ranging between 92 and 102% for beer, 92 and 101% for coffee, and 88 and 100% for cheese, at sub-μg mL⁻¹ or g⁻¹ analytes concentration levels were achieved.     

In vitro antioxidant activities of five β-carboline alkaloids,molecular docking,and dynamic simulations

Structural Chemistry - Experimental and computational studies were performed to determine the antioxidant activities of harmine, harmaline, harmalol, harmane, and...     

An accurate and precise high-performance liquid chromatography method for the rapid quantification of the novel HIV integrase inhibitor raltegravir in human blood plasma after solid phase extraction

The quantification of the HIV integrase inhibitor raltegravir in blood plasma is described using solid phase extraction (SPE) coupled with an accurate high-performance liquid chromatography assay with ultraviolet (UV) detection. The method was validated over the range of 20-10,000 ng/mL using simple sample preparation and chromatography. The SPE method was optimized to be selective and highly efficient. The buffer’s ionic strength and pH were optimized for retaining RAL and the internal standard on the column, the percentage of methanol was optimized in the cleaning step to remove unwanted plasma contaminants, and the type and amount of acid was optimized for complete elution of the compounds. This method has no interference with other potentially co-administered antiretrovirals or common drugs. Average recoveries for the extraction method were consistently high: 90% for raltegravir and 90% for the internal standard diazepam. This method was found to be accurate and precise. Within day (n = 6) and between day (n = 18) accuracies ranged from 97.5 to 104.4%. Within-day (n = 6) and between-day (n = 18) precision ranged from 1.4 to 3.8%, and from 2.4 to 7.9%, respectively. This is the first published method to use simple UV technology and reliable SPE extraction methodology for the quantification of raltegravir in human plasma. This method can be easily implemented in most bioanalytical laboratories.     

Antioxidant and Anti-Inflammatory Effects of Peganum harmala Extracts: An In Vitro and In Vivo Study

Peganum harmala (P. harmala) belongs to the family Zygophyllaceae, and is utilized in the traditional medicinal systems of Pakistan, China, Morocco, Algeria, and Spain to treat several chronic health disorders. The aim of the present study was to identify the chemical constituents and to evaluate the antioxidant, anti-inflammatory, and toxicity effects of P. harmala extracts both in vitro and in vivo. Sequential crude extracts including 100% dichloromethane, 100% methanol, and 70% aqueous methanol were obtained and their antioxidant and anti-inflammatory effects evaluated both in vitro and in vivo. The anti-inflammatory effect of the extract was investigated using the carrageenan-induced paw edema method in mice, whereas the toxicity of the most active extract was evaluated using an acute and subacute toxicity rat model. In addition, we have used the bioassay-guided approach to obtain potent fractions, using solvent–solvent partitioning and reversed phase high performance liquid chromatography from active crude extracts; identification and quantification of compounds from the active fractions was achieved using electrospray ionization mass spectrometry and high performance liquid chromatography techniques. Results revealed that the 100% methanol extract of P. harmala exhibits significant in vitro antioxidant activity in DPPH assay with an IC₅₀ of 49 µg/mL as compared to the standard quercetin with an IC₅₀ of 25.4 µg/mL. The same extract exhibited 63.0% inhibition against serum albumin denaturation as compared to 97% inhibition by the standard diclofenac sodium in an in vitro anti-inflammatory assay, and in vivo anti-inflammatory against carrageenan-induced paw edema (75.14% inhibition) as compared to 86.1% inhibition caused by the standard indomethacin. Furthermore, this extract was not toxic during a 14 day trial of acute toxicity when given at a dose of 3 g/kg, indicating that the lethal dose (LD₅₀) of P. harmala methanol extract was greater than 3 g/kg. P. harmala methanolic fraction 2 obtained using bioassay-guided fractionation showed the presence of quinic acid, peganine, harmol, harmaline, and harmine, confirmed by electrospray ionization mass spectrometry and quantified using external standards on high performance liquid chromatography. Taken all together, the current investigation further confirms the antioxidant, anti-inflammatory, and safety aspects of P. harmala, which justifies its use in folk medicine.     

Determination of oleic acid in sunflower seeds by infrared spectroscopy and multivariate calibration method

A method for the determination of oleic acid in sunflower seeds is proposed. One hundred samples of sunflower seeds were analyzed by near-infrared diffuse reflectance spectroscopy (NIRDRS). The direct measures were realized in ground and sifted seeds. The PLS multivariate calibration model was obtained using first derivate absorbance values as response matrix, while the oleic acid concentration matrix was obtained analyzing the seed samples by gas chromatography with a flame ionization detector (GC-FID). The NIRDRS-PLS model was validated externally using unknown samples of sunflower seeds. The accuracy and precision of the method was evaluated using GC as reference method. The following figures of merit (FOM) were obtained: LOD = 3.4% (w/w); LOQ = 11.3% (w/w); SEN = 8 × 10⁻⁵; SEL = 0.15; analytical sensibility (γ) = 1.5 and linear range (LR) = 18.1-89.2% (w/w). This method is useful for the fast determination of oleic acid in sunflower seeds and for quality control of raw materials.     

Analysis of cytokinin nucleotides by capillary zone electrophoresis with diode array and mass spectrometric detection in a recombinant enzyme in vitro reaction

A capillary zone electrophoresis (CZE) method for separation of adenosine and N⁶-isopentenyladenosine (cytokinin) nucleotides was developed, optimized and validated. Aqueous solutions of several amino acids were evaluated as the background electrolyte constituents. Separation of six nucleotides in less than 20 min with high theoretical plate number (up to 400 000 for isopentenyladenosine triphosphate) was achieved using a 100 mM sarcosine/ammonia buffer at pH 10.0. The detection limits of the CZE-UV method are in the low micromolar range (0.69–1.27 μmol L⁻¹). Good repeatability of migration times (within 1.3%), peak areas (within 1.8%) and linearity (R² > 0.999) was achieved over the concentration range 5–1000 μmol L⁻¹. The method was used to assay the activity of the recombinant Arabidopsis thaliana isopentenyltransferase 1 (AtIPT1). Baseline separation of isopentenylated nucleotides by CE–ESI-MS using a volatile buffer (30 mM ammonium formate; pH 10.0) was accomplished. The identities of the reaction products – isopentenyladenosine di- and triphosphate were confirmed by HPLC-QqTOF-MS. Dephosphorylation of ATP was observed as a parallel reaction.     

Determination of pharmacological levels of harmane, harmine and harmaline in mammalian brain tissue, cerebrospinal fluid and plasma by high-performance liquid chromatography with fluorimetric detection

Increased blood aldehyde levels, as occur in alcohol intoxication, could lead to the formation of beta-carbolines such as harmane by condensation with indoleamines. Endogenous beta-carbolines, therefore, should occur in specific brain areas where indoleamine concentrations are high, whilst exogenous beta-carbolines should exhibit an even distribution. The author presents direct and sensitive methods for assaying the beta-carbolines harmane, harmine and harmaline in brain tissue, cerebrospinal fluid and plasma at picogram sample concentrations using reversed-phase high-performance liquid chromatography with fluorimetric detection and minimal sample preparation. Using these assay methods, it was found that the distribution of beta-carbolines from a source exogenous to the brain results in a relatively even distribution within the brain tissue.     

Development and validation of an HPLC method for the determination of process-related impurities in pridinol mesylate, employing experimental designs

A simple high performance liquid chromatographic method for the determination of process-related impurities in bulk drug of the central anticholinergic compound pridinol mesylate, has been developed and validated. Spectroscopically characterized synthetic impurities were used as standards. The chromatographic separation was optimized employing an experimental design strategy, and was achieved on a C₁₈ column with a mobile phase containing 50 mM potassium phosphate buffer (pH 6.4), MeOH and 2-propanol (20:69:11, v/v/v), delivered at a flow rate of 1.0 mL min⁻¹. UV detection was performed at 245 nm. The optimized method was thoroughly validated, demonstrating to be selective, when the chromatogram was recorded with a diode-array detector and peak purities were evaluated (>0.9995). The method is robust and linear (r² > 0.99) over the range 0.05-2.5% (5-250% with regards to the 1% specification limit for both process-related impurities); it is also precise, regarding repeatability (RSD ≤ 1.5% for all of the analytes) and intermediate precision aspects and LOQ values for the impurities are below 0.01%. Method accuracy, evidenced by low bias of the results and analyte recoveries in the range of 99.1-102.7%, was assessed at five analyte concentration levels. The usefulness of the determination was also demonstrated through the analysis of different lots of pridinol mesylate bulk substance. The results indicate that the method is suitable for the quality control of the bulk manufacturing of pridinol mesylate drug substance.     

Separation of Tic-hydantoin enantiomers,potent sigma-1 agonists,by high performance liquid chromatography and capillary electrophoresis

Stereospecific separations of seven Tic-hydantoin sigma-1 agonists were performed by both HPLC method using derivatized cellulose and amylose chiral stationary phases and capillary electrophoresis (CE) method using neutral and anionic cyclodextrins added in the background electrolyte (BGE). An optimal baseline separation (R_s > 3.3 with analysis times < 25 min) was readily obtained with all silica-based celluloses and amyloses using a normal-phase methodology. CE was used as an alternative technique to HPLC for the Tic-hydantoin derivatives separation. The enantiomers were fully resolved with highly sulfated β-cyclodextrins at pH 2.5 (R_s > 1.5 with analysis times <11 min). Both methods were validated in terms of linearity, detection and quantification limits. They were used to check the enantiomeric purity of the enantiomers.     

An on-line method for pressurized hot water extraction and enzymatic hydrolysis of quercetin glucosides from onions

A novel environmentally sound continuous-flow hot water extraction and enzymatic hydrolysis method for determination of quercetin in onion raw materials was successfully constructed using a stepwise optimization approach. In the first step, enzymatic hydrolysis of quercetin-3,4′-diglucoside to quercetin was optimized using a three level central composite design considering temperature (75–95 °C), pH (3–6) and volume concentration of ethanol (5–15%). The enzyme used was a thermostable β-glucosidase variant (termed TnBgl1A_N221S/P342L) covalently immobilized on either of two acrylic support-materials (Eupergit^® C 250L or monolithic cryogel). Optimal reaction conditions were irrespective of support 84 °C, 5% ethanol and pH 5.5, and at these conditions, no significant loss of enzyme activity was observed during 72 h of use. In a second step, hot water extractions from chopped yellow onions, run at the optimal temperature for hydrolysis, were optimized in a two level design with respect to pH (2.6 and 5.5), ethanol concentration (0 and 5%) and flow rate (1 and 3 mL min⁻¹) Obtained results showed that the total quercetin extraction yield was 1.7 times higher using a flow rate of 3 mL min⁻¹ (extraction time 90 min), compared to a flow rate of 1 mL min⁻¹ (extraction time 240 min). Presence of 5% ethanol was favorable for the extraction yield, while a further decrease in pH was not, not even for the extraction step alone. Finally, the complete continuous flow method (84 °C, 5% ethanol, pH 5.5, 3 mL min⁻¹) was used to extract quercetin from yellow, red and shallot onions and resulted in higher or similar yield (e.g. 8.4 ± 0.7 μmol g⁻¹ fresh weight yellow onion) compared to a conventional batch extraction method using methanol as extraction solvent.     

Determination of four major saponins in the seeds of Aesculus chinensis Bunge using accelerated solvent extraction followed by high-performance liquid chromatography and electrospray-time of flight mass spectrometry

A new method based on accelerated solvent extraction (ASE) followed by a reliable high-performance liquid chromatography-diode array detector (HPLC-DAD) and positive ion electrospray-time of flight mass spectrometry (ESI-TOF/MS) analysis has been developed for the characterization and quantification of four major saponins in extracts of the seeds of Aesculus chinensis Bunge (Semen Aesculi). The saponins escin Ia, escin Ib, isoescin Ia and isoescin Ib were extracted from seeds of A. chinesis Bunge via ASE, and the operational parameters of ASE were optimized, such as extraction solvent, extraction temperature, static extraction time and extraction cycles. The optimized procedure employed 70% MeOH as extraction solvent, 120 °C of extraction temperature, 7 min of static extraction time, 60% flush volume and the extraction recoveries of the four compounds were nearly to 100% for two cycles. The HPLC conditions are as follows: SinoChrom ODS BP C₁₈ (4.6 mm × 200 mm, 5 μm) column, acetonitrile and 0.10% phosphoric acid solution as mobile phase, flow rate is 1.0 mL min⁻¹, detection length of UV is 203 nm, injection volume is 10 μL. The results indicated that the developed HPLC method is simple, sensitive and reliable for the determination of four major saponins in seeds of A. chinesis Bunge with a good linearity (r² > 0.9994), precision (relative standard deviation (R.S.D.) <1.5%) and the recovery ranges of 95.2-97.3%. The limits of detection (LOD) of the four compounds were in the range of 0.40-0.75 μg mL⁻¹. This assay can be readily utilized as a quality control method for Semen Aesculi and other related medicinal plants.     

High-performance thin-layer chromatography densitometric method for the quantification of harmine, harmaline, vasicine, and vasicinone in Peganum harmala

Peganum harmala Linn. seed is a reputed drug of the Indian systems of medicine. We report a simple high-performance thin-layer chromatography (HPTLC) densitometric method for the quantification of 4 alkaloids, viz., harmine, harmaline, vasicine, and vasicinone from P. harmala seed. The 4 compounds were resolved in ethyl acetate-methanol-ammonia (7 + 1 + 0.3, v/v/v) mobile phase. The method was validated for precision, repeatability, and accuracy. Instrumental precision was 0.27, 1.53, 0.39, and 1.15% [relative standard deviation (RSD)] and repeatability of the method was 1.01, 0.79, 0.13, and 1.62% RSD for harmine, harmaline, vasicine, and vasicinone, respectively. Accuracy of the method was checked by a recovery study conducted at 3 different levels, and the average recovery was 97.88% for harmine, 97.69% for harmaline, 98.38% for vasicine, and 98.28% for vasicinone. The 4 compounds were quantified in P. harmala seed using the method, and it was found to contain 0.44, 0.096, 0.25, and 0.0007% (w/w) of each, respectively. The proposed HPTLC densitometric method for the quantification of the 4 compounds was found to be simple, precise, specific, sensitive, and accurate. It can be used for routine quality control of P. harmala seed and has the ability to quantify the 4 marker compounds in the formulations containing P. harmala. It also can be used to quantify any of these marker compounds in other herbal drugs.     

Development of a flow system for the determination of low concentrations of silver using Moringa oleifera seeds as biosorbent and flame atomic absorption spectrometry

In this study a method for the determination of low concentrations of silver in waters using solid-phase extraction with a flow injection analysis system and detection by flame atomic absorption spectrometry (FAAS) was developed. Moringa oleifera seeds were used as a biosorbent material. Chemical and flow variables of the on-line preconcentration system such as sample pH and flow rate, preconcentration time, eluent concentration and sorbent mass were studied. The optimum preconcentration conditions were obtained using sample pH in the range of 6.0-8.0, preconcentration time of 4 min at a flow rate of 3.5 mL min^− 1, 0.5 mol L^− 1 HNO₃ eluent at a flow rate of 4.5 mL min^− 1 and 35 mg of sorbent mass. With the optimized conditions, the preconcentration factor, precision, detection limit and sample throughput were estimated as 35 (for preconcentration of 14 mL sample), 3.8% (5.0 μg L^− 1, n = 7), 0.22 μg L^− 1 and 12 samples per hour, respectively. The developed method was successfully applied to mineral water and tap water, and accuracy was assessed through analysis of a certified reference material for water (APS-1071 NIST) and recovery tests, with recovery ranging from 94 to 101%.     

Optimization of headspace solid-phase microextraction for analysis of β-caryophyllene in a nanoemulsion dosage form prepared with copaiba (Copaifera multijuga Hayne) oil

Recent studies have shown the anti-inflammatory activity of Copaiba oils may be addressed to the high content of β-caryophyllene, the most common sesquiterpene detected, especially in the Copaifera multijuga Hayne species. In the present study, nanoemulsions were proposed as a delivery system for copaiba oil in view to treat locally inflamed skin. This article describes the optimization and validation of a stability-indicating SPME-GC method, for β-caryophyllene analysis in the nanoemulsions produced by high pressure homogenization. SPME methods are performed with PDMS (polydimethylsiloxane) fiber (100 μm). Three SPME parameters were evaluated by a three-level-three-factor Box–Behnken factorial design as potentially affecting the technique efficiency. According to the results obtained, the best conditions to extract β-caryophyllene were: (i) sampling temperature of 45 °C, (ii) sampling time of 20 min and (iii) no NaCl addition. Results coming from the forced degradation tests showed a reduction of β-caryophyllene peak area when both caryophyllene methanolic solution and nanoemulsions were exposed to acid hydrolysis, UV-A irradiation, oxidative (H₂O₂) and thermolitic (60 °C) conditions. Such reduction occurred in lower extent in the nanoemulsions, suggesting a protective effect of the formulation to β-caryophyllene content. Since no degradation products were detected in the same retention time of β-caryophyllene, the specificity of the method was demonstrated. The method was linear in the range of 0.14–0.68 μg mL⁻¹ of β-caryophyllene (r² > 0.999), and was also validated for precision (R.S.D. ≤ 5.0%), accuracy (97.85–101.87%) and robustness. Finally, the method was applied to quantification of β-caryophyllene content in the developed formulations.     

Optimization of a pressurized liquid extraction method by experimental design methodologies for the determination of fluoroquinolone residues in infant foods by liquid chromatography

In the present study, we have developed a method based on pressurized liquid extraction (PLE) and liquid chromatography with fluorescence detection (LC-FLD) for the determination of residues of fluoroquinolones (FQs) in infant food products. PLE extraction has been optimized by the application of experimental design methodologies. Initially, a fractional factorial design (FFD) was used to screen the significance of four extraction parameters: solvent composition, temperature, pressure and number of cycles. The most significant factors, identified by ANOVA analysis, were the solvent composition, temperature and pressure, which were further optimized with the aid of a face centred design (FCD) and the desirability function. The optimized operating PLE conditions were as follows: ACN/o-phosphoric acid 50 mM pH 3.0 (80:20, v/v), 80 °C, 2000 psi and three extraction cycles of 5 min. Under these conditions, recoveries of the target FQs varied between 69% and 107% with RSDs below 9%. The whole method was validated according to the Commission Decision 2002/657/EC guidelines. The proposed method has been successfully applied to the analysis of different infant food products bought in local supermarkets and pharmacies. The results showed the presence of residues of enrofloxacin in a non-compliant baby food sample corresponding to a chicken-based formulation, which were also confirmed and quantified by LC–MS/MS analysis.     

Hyphenation of optimized microfluidic sample preparation with nano liquid chromatography for faster and greener alkaloid analysis

A glass liquid–liquid extraction (LLE) microchip with three parallel 3.5 cm long and 100 μm wide interconnecting channels was optimized in terms of more environmentally friendly (greener) solvents and extraction efficiency. In addition, the optimized chip was successfully hyphenated with nano-liquid chromatography with ultraviolet and mass spectrometric detection (nanoLC–UV–MS) for on-line analysis. In this system, sample pretreatment, separation and detection are integrated, which significantly shortens the analysis time, saves labor and drastically reduces solvent consumption. Strychnine was used as model analyte to determine the extraction efficiency of the optimized 3-phase chip. Influence of organic solvent, pH of feed phase, type of alkaloid, and flow rates were investigated. The results demonstrated that the 3-phase chip nanoLC–UV/MS hyphenation combines rapid (∼25 s) and efficient (extraction efficiency >90%) sample prep, with automated alkaloid analyses. The method was applied to real samples including Strychnos nux-vomica seeds, Cephaelis ipecacuanha roots, Atropa belladonna leaves, and Vinca minor leaves.     

Optimization and validation of a low temperature microwave-assisted extraction method for analysis of polycyclic aromatic hydrocarbons in airborne particulate matter

A low temperature microwave-assisted extraction method (MAE) is reported for the analysis of polycyclic aromatic hydrocarbons (PAHs) in airborne particulate matter (PM). The main parameters affecting the extraction efficiency (choice of extractants, microwave power, and extraction time) were investigated and optimized. The optimized procedure requires a 20 ml mixture of acetone:n-hexane (1:1) for extraction of PAHs in PM at 150 W of microwave energy (20 min extraction time). Clean-up of MAE extracts was not found to be necessary. The optimized method was validated using two different SRM (1648-urban particulate matter and 1649a, urban dust). The results obtained are in good agreement with certified values. PAHs recoveries for both reference materials were between 79 and 122% with relative standard deviation ranging from 3 to 21%. Detection limits were determined based on blank determination using two kinds of quartz filter substrates (n = 10), which ranged from 0.001 (0.03) ng m⁻³ (pg/μg) for B(k)Ft to 1.119 (37.3) for Naph in ng m⁻³ (pg/μg), respectively. The repeatability and day-to-day reproducibility obtained in this study were in the range of 4-16 and 3-25% for spiked standards and SRM 1649, respectively. The optimized and validated MAE technique was applied to the extraction of PAHs from a set of real world PM samples collected in Singapore. The sum of particulate-bound PAHs in outdoor PM ranged from 1.05 to 3.45 ng m⁻³ while that in indoor PM (cooking emissions) ranged from 27.6 to 75.7 ng m⁻³, respectively.     

Determination of cadmium in alcohol fuel using Moringa oleifera seeds as a biosorbent in an on-line system coupled to FAAS

In this study a new method for determination of cadmium in alcohol fuel using Moringa oleifera seeds as a biosorbent in an on-line preconcentration system coupled to flame atomic absorption spectrometry (FAAS) was developed. Flow and chemical variables of the proposed system were optimized through multivariate designs. The limit of detection for cadmium was 5.50 μg L⁻¹ and the precision was below 2.3% (35.0 μg L⁻¹, n = 9). The analytical curve was linear from 5 to 150 μg L⁻¹, with a correlation coefficient of 0.9993. The developed method was successfully applied to spiked alcohol fuel, and accuracy was assessed through recovery tests, with recovery ranging from 97.50 to 100%.     

Analysis of β-agonists and β-blockers in urine using hollow fibre-protected liquid-phase microextraction with in situ derivatization followed by gas chromatography/mass spectrometry

A method using hollow fibre-protected liquid-phase microextraction (HF-LPME) with in situ derivatization followed by gas chromatography/mass spectrometry (GC/MS) was established for the analysis of β-agonists and β-blockers in urine. Because it can simultaneously extract and derivatize compounds of interest by methylbenzol and N-methyl-N-(trimethylsilyl) trifluoroacetamide (MSTFA) in HF-LPME, the approach overcomes the drawbacks of considerable time-consuming and tedious operation, meanwhile improves enrichment multiple. The optimized conditions were extraction for 20 min at 35 °C with 5.0 μL of mixed extraction solvent (methylbenzol/MSTFA = 1:1, v/v) with stirring speed of 925 rpm in 5.0 mL sample under pH 12.0 and 14% (w/v) NaCl. The method provided very wide linear ranges (0.25–400 ng mL⁻¹) and low detection limits in the range of 0.08–0.10 ng mL⁻¹ for clenbuterol, metoprolol and propranolol while enrichment factors reached up to 256. The analytes could be determined in spiked urine by the method with high extraction efficacy (93.79–109.04% recoveries) and precision (<9.70% RSD). It has a satisfactory result for metoprolol in practical human urine samples for a single-dose administration of 50 mg after 36 h. The proposed method only needs few microliters of organic solvent and derivatizing agent; the operation is simple, convenient and rapid for the trace analysis of β-agonists and β-blockers in biological fluids; it can be readily generalized for high sample throughput. So, it is hopeful that the study will facilitate the monitoring of β-agonists and β-blockers in the competition sports.     

Spectrofluorimetric determination of stoichiometry and association constants of the complexes of harmane and harmine with beta-cyclodextrin and chemically modified beta-cyclodextrins

The association characteristics of the inclusion complexes of the beta-carboline alkaloids harmane and harmine with beta-cyclodextrin (beta-CD) and chemically modified beta-cyclodextrins such as hydroxypropyl-beta-cyclodextrin (HPbeta-CD), 2,3-di-O-methyl-beta-cyclodextrin (DMbeta-CD) and 2,3,6-tri-O-methyl-beta-cyclodextrin (TMbeta-CD) are described. The association constants vary from 112 for harmine/DMbeta-CD to 418 for harmane/HPbeta-CD. The magnitude of the interactions between the host and the guest molecules depends on the chemical and geometrical characteristics of the guest molecules and therefore the association constants vary for the different cyclodextrin complexes. The steric hindrance is higher in the case of harmine due to the presence of methoxy group on the beta-carboline ring. The association obtained for the harmane complexes is stronger than the one observed for harmine complexes except in the case of harmine/TMbeta-CD. Important differences in the association constants were observed depending on the experimental variable used in the calculations (absolute value of fluorescence intensity or the ratio between the fluorescence intensities corresponding to the neutral and cationic forms). When fluorescence intensity values were considered, the association constants were higher than when the ratio of the emission intensity for the cationic and neutral species was used. These differences are a consequence of the co-existence of acid-base equilibria in the ground and in excited states together with the complexation equilibria. The existence of a proton transfer reaction in the excited states of harmane or harmine implies the need for the experimental dialysis procedure for separation of the complexes from free harmane or harmine. Such methodology allows quantitative results for stoichiometry determinations to be obtained, which show the existence of both 1:1 and 1:2 beta-carboline alkaloid:CD complexes with different solubility properties.