Enantioseparation and determination of asenapine in biological fluid micromatrices by HPLC with diode array detection.

Asenapine is a recent drug approved in the European Union for the treatment of bipolar disorder. An original approach has been developed for asenapine analysis in patients treated with the drug, including miniaturized microsampling procedures, separation and quantitation of drug enantiomers. An original enantioselective method based on high-performance liquid chromatography with diode array detection was developed and applied to the determination of asenapine enantiomer levels in innovative haematic samples: four micromatrices have been tested, two based on dried matrix spots (dried blood spots and dried plasma spots) and two based on volumetric absorptive microsampling (from blood and plasma). Chiral separation was achieved on a cellulose-tris(3,5 dimethylphenylcarbamate) column, with a mobile phase containing bicarbonate buffer and acetonitrile. The method was validated with satisfactory results of linearity and precision on all matrices that showed also a significant performance in terms of stability, feasibility and reliability, when compared to fluid plasma sampling, handling and processing. Among micromatrices, both volumetric absorptive microsampling types were superior to dried matrix spots in terms of data reproducibility and correspondence with plasma levels. The bioanalytical approach proposed herein provides for the first time a chiral high-performance liquid chromatographic method for the determination of asenapine enantiomers, coupled to a very effective microsampling strategy.

Quantitative evaluation of auraptene and umbelliferone, chemopreventive coumarins in citrus fruits, by HPLC-UV-FL-MS.

An analytical strategy, based on the development of two HPLC methods with spectrophotometric (UV), spectrofluorometric (FL), and mass spectrometric (MS) detection, has been developed to investigate the presence of and to quantitate two important chemopreventive coumarins, auraptene and umbelliferone, in foodstuffs. The analytes were determined in fruits, and fruit parts, of plants belonging to the Citrus , Poncirus , and Fortunella genera, to test their nutraceutical potential. The method validation has been carried out according to international guidelines, with good results in terms of precision (RSD < 6.9%) and extraction yields (>91%). Application to the quantitative analysis of auraptene and umbelliferone in several kinds of citrus fruits was successful, providing reliable and consistent data. Exploiting three different kinds of detection, the analytical methodology proposed herein has been demonstrated to be sound but versatile, as well as reliable. Performances and results were compared and always found in good agreement among themselves. Thus, this approach is suitable for the identification and simultaneous quantitation of auraptene and umbelliferone in citrus fruits, with the aim of evaluating their nutraceutical potential.

A novel test using dried blood spots for the chromatographic assay of methadone.

A novel test has been developed for the analysis of methadone in dried blood spot specimens from patients undergoing methadone maintenance treatment. An isocratic reversed-phase high-performance liquid chromatography method with coulometric detection has been optimized for the determination of methadone. The clean-up of dried blood spots was performed by means of an original microextraction by packed sorbent procedure after microwave-assisted extraction of the drug with a suitable solvent. Extraction yields were satisfactory, always being higher than 90.0 %. The calibration curve was linear over the 4-500 ng mL(-1) concentration range. The method had satisfactory sensitivity (limit of quantitation of 4 ng mL(-1)), precision (relative standard deviation less than 5.8 %), selectivity and accuracy (recovery greater than 87.0 %). It was successfully applied to dried blood spot samples collected from heroin-addicted patients undergoing methadone maintenance therapy at dosages between 40 and 240 mg day(-1). The statistical analysis (Bland-Altman plot) showed that the results were in good agreement with those found from the analysis of plasma samples obtained from the same patients. Thus, the method has proved to be suitable for the monitoring of methadone by means of dried blood spots.

Analysis of Aloe-based phytotherapeutic products by using nano-LC-MS.

This article proposes a chromatographic method for the analysis of extracts of Aloe plants. The method was developed with a laboratory assembled nano-LC system coupled with a UV detector, followed by an IT-mass spectrometer. With a step gradient mode of ACN/H(2)O mixtures and employing a capillary column packed with C(18) (100 µm id), a complete separation of the following anthrones was achieved: aloin (in its two isomeric forms A and B), 5-hydroxyaloin and 7-hydroxyaloin (in its two isomeric forms A and B). The optimized nano-LC-MS method was validated for the quantification of aloin, the main component of Aloe with known pharmacological activities. RSD values obtained for retention time and peak areas were 1.3 and 12.1%, respectively. LOD and LOQ values of 0.4 and 1.5 µg/mL were obtained for each aloin isomer. The method was applied to the analysis of Aloe vera and A. ferox extracts in order to acquire a fingerprint, characteristic for each plant. Several phenolic compounds were detected by UV and identified by MS. A. vera and A. ferox showed different profiles and it was possible to discriminate them. Several commercial formulations, declared to contain Aloe extracts, were analyzed. Comparing their chromatograms with those obtained from A. vera and A. ferox, it was possible to recognize the Aloe species and to determine aloin.

Fast CE analysis of adrenergic amines in different parts of Citrus aurantium fruit and dietary supplements.

A CE method has been developed for the simultaneous analysis of the adrenergic amines synephrine, octopamine and tyramine in Citrus aurantium (bitter orange) fruit extracts and in dietary supplements. The analytes were separated on a fused silica capillary (50 microm id, 40.0 cm effective length, 48.5 cm total length) using a BGE composed of phosphate buffer (pH 2.5, 50 mM) and applying a 30 kV potential. The samples were injected hydrodynamically at 50 mbar for 25 s. The use of photodiode array detection (lambda=195 nm) allowed the quantification of the analytes and the control of peak purity. The method has been fully validated, obtaining satisfactory values of precision and extraction yield. The analytes are extracted with water from the dried whole fruits or fruit parts (endocarp, mesocarp and exocarp) or from the commercial formulations and directly injected into the CE apparatus. The results obtained were satisfactory in terms of precision (RSD < 5.7%) and accuracy (recovery > 89%). Thus, the method has demonstrated to be suitable for the qualitative and quantitative determination of synephrine, octopamine and tyramine in C. aurantium extracts, for dietary supplement quality control and for food adulteration identification.

HPLC-F analysis of melatonin and resveratrol isomers in wine using an SPE procedure.

An original analytical method has been developed for the determination of the antioxidants trans-resveratrol (t-RSV) and cis-resveratrol (c-RSV) and of melatonin (MLT) in red and white wine. The method is based on HPLC coupled to fluorescence detection. Separation was obtained by using a RP column (C8, 150 mm x 4.6 mm id, 5 mum) and a mobile phase composed of 79% aqueous phosphate buffer at pH 3.0 and 21% ACN. Fluorescence intensity was monitored at lambda = 386 nm while exciting at lambda = 298 nm, mirtazapine was used as the internal standard. A careful pretreatment of wine samples was developed, using SPE with C18 cartridges (100 mg, 1 mL). The calibration curves were linear over the following concentration ranges: 0.03-5.00 ng/mL for MLT, 3-500 ng/mL for t-RSV and 1-150 ng/mL for c-RSV. The LOD values were 0.01 ng/mL for MLT, 1 ng/mL for t-RSV and 0.3 ng/mL for c-RSV. Precision data, as well as extraction yield and sample purification results, were satisfactory. Thus, the method seems to be suitable for the analysis of MLT and resveratrol isomers in wine samples. Moreover, wine total polyphenol content and antioxidant activity were evaluated.

Determination of the antidepressant paroxetine and its three main metabolites in human plasma by liquid chromatography with fluorescence detection.

A high-performance liquid chromatographic method has been developed for the determination in human plasma of the specific serotonin reuptake inhibitor (SSRI) antidepressant paroxetine and its three main metabolites (M1, M2, M3). Fluorescence detection was used, exciting at lambda = 294 nm and monitoring emission at lambda = 330 nm for paroxetine (lambda(exc) = 280 nm, lambda(em) = 330 nm for M1 and M2; lambda(exc) = 268 nm, lambda(em) = 290 nm for M3). Separation was obtained on a reversed-phase C18 column using a mobile phase composed of 66.7% aqueous phosphate at pH 2.5 and 33.3% acetonitrile. Imipramine (lambda(exc) = 252 nm, lambda(em) = 390 nm) was used as the internal standard. A careful pre-treatment of plasma samples was developed, using solid-phase extraction with C8 cartridges (50 mg, 1 mL). The calibration curves were linear over a working range of 2.5-100 ng mL(-1) for paroxetine and of 5-100 ng mL(-1) for all metabolites. The limit of detection (LOD) was 1.2 ng mL(-1) for PRX and 2.0 ng mL(-1) for the metabolites. The method was applied with success to plasma samples from depressed patients undergoing treatment with paroxetine. Hence, the method seems to be suitable for the therapeutic drug monitoring of paroxetine and its main metabolites in depressed patients' plasma.

Analysis of aromatic and terpenic constituents of pepper extracts by capillary electrochromatography.

An original method based on CEC has been developed for the determination of aromatic and terpenic compounds in extracts of spices obtained from Piper nigrum. The method is based on the use of a fused silica capillary (effective length: 23.5 cm, internal diameter: 100 microm) packed with a C18 sorbent (packing length: 23 cm, particle size: 5 microm). The mobile phase is a 50 mM, pH 6.0 ammonium acetate/ACN (10:90 v/v) mixture. Applying a 30 kV voltage, the following 11 compounds were separated and analysed: terpinen-4-ol, caryophyllene oxide, limonene, alpha-pinene, 3-carene, beta-pinene, alpha-humulene, beta-caryophyllene, alpha-phellandrene, eugenol and piperine. Compound determination is carried out using a diode-array detector set at 265 and 338 nm for alpha-phellandrene and piperine, respectively, and at 210 nm (reference subtraction at 282 nm) for all the other analytes. The optimised method has been validated with good results in terms of linearity, limits of quantitation, detection and precision. The CEC method was successfully applied to the analysis of essential oils and methanolic extracts of 'black', 'white' and 'green' pepper.

Simultaneous determination of aromatic and terpenic constituents of cloves by means of HPLC with diode array detection.

An HPLC method has been developed for the simultaneous determination of aromatic and terpenic constituents of cloves on a C8 RP column, with the mobile phase consisting of a pH 3.5 phosphate buffer-triethylamine (30%) and acetonitrile (70%); a flow rate of 1.2 mL/min and a diode-array detector were used. Complete separation of all analytes (eugenol (EUG), eugenol acetate (AEUG), beta-caryophyllene, a-humulene and caryophyllene oxide) was achieved within 7 min. Good linearity was found in the range 0.125-40.0 microg/mL for EUG and AEUG and in the range 0.250-20.0 microg/mL for the terpenic compounds. After validation, the method was successfully applied to the analysis of clove oil and clove extract samples. The results obtained indicate good accuracy (recovery percentage mean value corresponding to 99.9%) and satisfactory precision.

Simultaneous HPLC analysis, with isocratic elution, of glycyrrhizin and glycyrrhetic acid in liquorice roots and confectionery products.

Glycyrrhizin (1), the main active principle of Glycyrrhiza glabra (liquorice) roots, is extensively used in herbal medicines, in pharmaceutical preparations and confectionery products. A feasible and reliable method which allows the simultaneous analysis of 1 and its aglycone, 18beta-glycyrrhetic acid (2), by means of an isocratic HPLC procedure is described. The system uses a C8 column as the stationary phase, and a mixture of acetonitrile, methanol, water and glacial acetic acid as the mobile phase. Good linearity was found in the concentration ranges 1-50 and 0.05-2.50 microg/mL for 1 and 2, respectively. A simple and rapid sample pre-treatment, based on the extraction of the two analytes with a mixture of water and ethanol, was developed for the examination of liquorice confectionery products and root samples. The HPLC method was shown to be appropriate, in terms of precision and feasibility, for the quality control of the analytes in these matrices.

Separation and analysis of glycyrrhizin, 18beta-glycyrrhetic acid and 18alpha-glycyrrhetic acid in liquorice roots by means of capillary zone electrophoresis.

Glycyrrhizin is the main active compound of Glycyrrhiza glabra root extracts; according to recent studies, glycyrrhizin and its aglycon, glycyrrhetic acid, have interesting therapeutic properties. A new capillary electrophoretic method has been developed for the separation and quantification of glycyrrhizin, beta-glycyrrhetic acid and its isomer a-glycyrrhetic acid. Separation of the analytes was achieved in less than 3 min on a fused silica capillary, by injecting the samples at the short end of the capillary (effective length: 8.5 cm). The background electrolyte was composed of pH 10.0 carbonate buffer, methanol and ethylene glycol (80/10/10) and contained 0.4% beta-cyclodextrin; indomethacin was used as the internal standard. Diode array detection was used, with quantitative assays carried out at 254 nm. Linearity was found over the 5-200 and 2.5-100 microg mL(-1) concentration ranges for glycyrrhizin and glycyrrhetic acid, respectively. This method has been applied to the determination of the analytes in different matrices (liquorice roots and commercial confectionery products), and to the purity control of beta-glycyrrhetic acid obtained from the hydrolysis of glycyrrhizin. When analysing beta-glycyrrhetic acid and its epimer in roots, the samples were purified by means of a suitable solid-phase extraction (SPE) procedure with Oasis HLB cartridges, which granted good selectivity, eliminating matrix interference.

Separation and analysis of the major constituents of cloves by micellar electrokinetic chromatography.

A micellar electrokinetic chromatographic method was developed and applied for the first time to the separation and quantification of the main components of cloves, namely eugenol and beta-caryophyllene. The analytes were separated in less than ten minutes in an unmodified fused silica capillary (effective length: 40.0 cm) using a background electrolyte composed of 25 mM carbonate buffer (pH 10.0) and 60 mM sodium dodecylsulfate. Analyte concentrations were determined in clove oil and in methanolic clove extracts after simple sequential dilution with methanol and background electrolyte, and the resulting electropherograms showed no interference due to other components. Precision assays gave satisfactory results, with mean standard deviation of peak areas lower than 4.7% (lower than 1.5% for migration times). The eugenol content of a commercial clove oil was about 70% (RSD% = 3.2), while that of beta-caryophyllene was about 15% (RSD% = 4.2); in clove extracts, on the other hand, eugenol was approximately 15% (RSD% = 4.7) and beta-caryophyllene 4% (RSD% = 5.1). Accuracy was also good (mean recovery >98%).

High-performance liquid chromatographic determination of Levetiracetam in human plasma: comparison of different sample clean-up procedures.

An accurate and precise high-performance liquid chromatographic method using diode array detection for the determination of the novel antiepileptic, Levetiracetam, has been developed. Three clean-up procedures for the analysis of Levetiracetam in human plasma were implemented and evaluated, namely solid-phase extraction, deproteinization by addition of organic solvents and formation of insoluble salts. Adenosine was used as the internal standard for all three sample pretreatment procedures. Among the several cartridges used for solid-phase extraction, the hydrophilic-lypophilic balance (Oasis) HLB) phase provides the best extraction yield of Levetiracetam, together with high precision. With the two other clean-up procedures involving plasma deproteinization by addition of methanol or zinc sulphate, lower sensitivity and precision of the assays were obtained. However, they are cheaper and faster when compared with the solid-phase extraction procedure.

Quality control of commercial tablets containing the novel antipsychotic quetiapine.

Quetiapine (bis [2-(2-[4-(dibenzo[b,f][1,4]thiazepin-11-yl]ethoxy)ethanol]fumarate) is the most recent agent introduced on the drug market for the treatment of psychotic disorders. Two different analytical methods for the quality control of quetiapine in commercial formulations have been developed and compared: a spectrophotometric method and a capillary zone electrophoretic (CZE) method. The spectrophotometric assay was carried out measuring the absorbance at a wavelength of 246 nm. The CZE method used an uncoated fused-silica capillary and a pH 2.5, 50 mM phosphate buffer as the background electrolyte. The detection wavelength was 205 nm, the separation voltage was 15 kV, and a complete electrophoretic run lasts less than 2.5 min. Extraction of quetiapine from the commercial tablets consisted of a simple one-step treatment with a pH 2.5, 50 mM phosphate buffer. Linearity was observed in the 5-25 microg ml(-1) concentration range of quetiapine for the spectrophotometric method, and in the 5-50 microg ml(-1) concentration range for the electrophoretic method. Both methods gave satisfactory results in terms of repeatability and intermediate precision (RSD<1.9%). Also accuracy values were very good for both methods, the recovery being between 98.2 and 100.5%.