Search

In the present study, an attempt was made to achieve separation of enantiomers within a minute in nano-LC and CEC. In order to achieve this goal several parameters were optimized from the viewpoint of the property of chiral analytes, concentration of the chiral selector in the packing material, capillary dimensions, and separation mode. The enantiomers of several of the applied chiral sulfoxides could be resolved with the analysis time

β-cyclodextrin derivatives, namely 6A methylamino-β-CD and hepta-methylamino-β-CD have been used as chiral selectors for the enantiomeric separation of a number of acidic and basic compounds by capillary electrophoresis employing either coated or uncoated capillaries. The effects of the CD type and concentration and the pH of the background electrolyte on the mobility and chiral resolution of the analytes have been studied. The use of monomethylamino-β-CD in a coated capillary allowed the enantiomeric resolution of phenyl lactic acid, warfarin and acenocoumarol but was not successful for tiaprofen and its 3-isomer. The heptamethylamino derivative, under the same experimental conditions, was a better chiral selector than the monosubstuted CD toward the arylpropionic acids and phenyl lactic acid while the anticoagulant drugs showed poor or no chiral resolution. Inversion of migration order was obtained for phenyl lactic acid, warfarin and acenocoumarol enantiomers. The enantiomers of basic compounds of pharmaceutical interest, namely propranolol, terbutaline, ketamine, chlorpheriramine and isoproterenol were only resolved using monomethylamino-β-CD dissolved in a phosphate buffer containing tetramethylammonium ions.

The separation of hydroquinone and some of its ether derivatives was studied by micellar electrokinetic chromatography with sodium dodecyl sulphate as an anionic surfactant in the background electrolyte. The optimized method was used for the determination of hydroquinone in a sample of skin-toning cream. On-column detection at 254 nm with caffeine as an internal standard gave good quantitative results.

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.

A high-performance liquid chromatographic method has been developed for the determination of the recent serotonin and norepinephrine reuptake inhibitor (SNRI) venlafaxine and its main active metabolite, O-desmethylvenlafaxine, in human plasma. Separation was obtained by using a reversed-phase column (C8, 150 x 4.6 mm I.D., 5 microm) and a mobile phase composed of 75% aqueous phosphate buffer containing triethylamine at pH 6.8 and 25% acetonitrile. Fluorescence detection was used, exciting at lambda=238 nm and monitoring the emission at lambda=300 nm. Citalopram was used as the internal standard. A careful pre-treatment of plasma samples was developed, using solid-phase extraction with C1 cartridges (100 mg, 1 mL). The limit of quantification (LOQ) was 1.0 ng mL(-1) and the limit of detection (LOD) was 0.3 ng mL(-1) for both analytes. The method was applied with success to plasma samples taken from patients undergoing treatment with venlafaxine. Precision data, as well as accuracy results, were satisfactory and no interference from other drugs was found. Hence, the method is suitable for therapeutic drug monitoring of venlafaxine and its main metabolite in depressed patients' plasma.

In this work, the simultaneous enantioseparation of the second-generation antidepressant drug mirtazapine and its main metabolites 8-hydroxymirtazapine and N-desmethylmirtazapine by chiral CEC is reported. The separation of all enantiomers under study was achieved employing a capillary column packed with a vancomycin-modified diol stationary phase. With the aim to optimize the separation of the three pairs of enantiomers in the same run, different experimental parameters were studied including the mobile phase composition (buffer concentration and pH, organic modifier type and ratio, and water content), stationary phase composition, and capillary temperature. A capillary column packed with vancomycin mixed with silica particles in the ratio (3:1) and a mobile phase composed of 100 mM ammonium acetate buffer (pH 6)/H(2)O/MeOH/ACN (5:15:30:50, by vol.) allowed the complete enantioresolution of each pair of enantiomers but not the simultaneous separation of all the studied compounds. For this purpose, a packing bed composed of vancomycin-CSP only was tested and the baseline resolution of the three couples of enantiomers was achieved in a single run in less than 30 min, setting the applied voltage and temperature at 25 kV and 20 degrees C, respectively. In order to show the potential applicability of the developed CEC method to biomedical analysis, a study concerning precision, sensitivity, and linearity was performed. The method was then applied to the separation of the enantiomers in a human urine sample spiked with the studied compounds after suitable SPE procedure with strong cation-exchange (SCX) cartridges.

A high-performance liquid chromatographic method for the determination in human plasma of the recent noradrenergic and specific serotonergic antidepressant (NaSSA) mirtazapine and its two main metabolites, N -desmethylmirtazapine and 8-hydroxymirtazapine, has been developed. Fluorescence detection was used, exciting at λ = 290 nm and monitoring emission at λ = 370 nm. Separation was obtained by using a reversed-phase column (C8, 250 mm × 4.6 mm I.D., 5 μm) and a mobile phase composed of 75% aqueous phosphate buffer containing triethylamine at pH 3.0 and 25% acetonitrile. Melatonin was used as the internal standard. A careful pre-treatment of plasma samples was developed, using solid-phase extraction with phenyl cartridges (100 mg, 1 mL). The calibration curves were linear over a working range of 5–150 ng mL −1 for mirtazapine and of 2.5–75.0 ng mL −1 for N -desmethylmirtazapine and 8-hydroxymirtazapine. The limit of quantitation (LOQ) was 2.5 ng mL −1 and the limit of detection (LOD) was 1.25 ng mL −1 for all analytes. The method was applied with success to plasma samples from depressed patients undergoing treatment with mirtazapine. Precision data, as well as accuracy results, were satisfactory and no interference from other drugs was found. Hence the method is suitable for therapeutic drug monitoring of mirtazapine and its metabolites in depressed patients’ plasma.

The lipophilicity of some cardiovascular drugs was determined by capillary electrophoresis (CE). Mexiletine, amlodipine and indapamide, the drugs considered, were in contact with liposomial vescicles for 2, 4 or 6 h. After the contact time the drugs, penetrated into liposomial vesicles, were determined by CE using phosphate buffer (pH 6.3 or 7.4) or borate buffer (pH 9). The lipophilicity of three drugs was determined considering the drug percentage penetrated into liposomial vesicles. The found lipohilicity order was amlodipine > mexiletine > indapamide.

The lipophilicity of pipemidic, nalidixic and oxolinic acids was determined by forming phospholipidic micelles directly in an electrophoretic capillary. Phosphatidylcholine derivatives, namely l -α-dilauroyl phosphatidylcholine (DLPC) or l -α-dimiristoyl phosphatidylcholine (DMPC), were added in the run buffer (50 mM phosphate buffer at pH 7.4). To obtain a mixed micelle, phospholipidic derivatives and sodium cholate were together added in the run buffer. Considering the increasing of migration time when phosphatidylcholine derivative is added in the run buffer, Ks can be determined and then quinolones lipophilicity.

There is a need for fast, simple and reliable analytical methods for the analysis of fluoxetine and norfluoxetine in patients who voluntarily or involuntarily have taken an overdose of the drug. A new liquid chromatographic method with diode array detection is presented herein for the determination of fluoxetine and its main active metabolite norfluoxetine in human plasma for toxicological purposes. A mobile phase composed of acetonitrile and aqueous tetramethylammonium perchlorate allows to obtain the complete separation of the analytes on a C18 reversed phase column. The fast and accurate sample pre-treatment step is carried out by means of solid-phase extraction using hydrophilic-lipophilic balance cartridges and loading 100 µL of plasma only. This procedure gives satisfactory extraction yield values, as well as good plasma sample purification from matrix interference. Linearity was obtained in the 150-3000 ng/mL range for both analytes. Selectivity with respect to other psychotropic drugs was satisfactory. The method seems to be suitable for the analysis of fluoxetine and its metabolite in human plasma for depressed patients in overdose.

This work proposes a new capillary electrochromatography (CEC) method for determination of drug partition in membrane phospholipids. CEC experiments were carried out in a 100 μm (ID) fused-silica capillary, partially packed with a chromatographic phospholipid stationary phase, so-called Immobilized Artificial Membrane, IAM.PC.DD2. The observed retention values were corrected by both the electro-osmotic and electrophoretic mobility values, measured by capillary electrophoresis (CE) experiments, assuming the values of the logarithms of “chromatographic” affinity factors, log kCEC as indexes of affinity for phospholipids. Analogously to biochromatography, all the values were determined with a totally aqueous mobile phase, or extrapolated to 100% aqueous buffer. The analytes were 16 structurally unrelated compounds, of basic, neutral, and acidic nature. To evaluate the effectiveness of CEC data to describe partition in phospholipids, log kCEC were related to both log P and log ⁡ k w IAM values. log P are the lipophilicity values expressed as the logarithms of n-octanol/water partition coefficients and log ⁡ k w IAM are the retention data measured by High Performance Liquid Chromatography (HPLC) on an IAM.PC.DD2 column, assumed as the reference values for phospholipid affinity. Phospholipid affinity scale by CEC related to that achieved by HPLC, but only if two different subclasses were considered separately, i.e. protonated and unprotonated analytes; indeed, all the compounds protonated at the experimental pH value (7.0) were retained stronger in CEC than in HPLC. This discrepancy may be due to the use of different buffers in CEC and HPLC since, to avoid the occurrence of a high current, the eluent in CEC experiments was of different composition and lower ionic strength than in HPLC. CEC analyses were faster and required lower amounts of both solvent and stationary phase than HPLC; moreover, with the exception of only three analytes, all analyses were performed with 100% aqueous eluents avoiding time-consuming and tedious extrapolation procedures.

Aloe emodin is an anthraquinone compound found in Aloe vera and other species of the Asphodelaceae and the Polygonaceae families, which has recently attracted much attention as a prospective antineoplastic agent. A HPLC method with tandem UV absorption and fluorimetric detection, was developed and validated for the analysis of aloe emodin in products obtained from Aloe leaves, such as capsules, tablets, dried extracts and mother tinctures. The stationary phase was a C18 reversed-phase column and the mobile phase was composed of water and methanol (30/70, v/v). Satisfactory linearity was obtained over the 10.0-1000.0 ng/mL range for photodiode array detection (limit of detection: 3 ng/mL) and over the 2.5-1000.0 ng/mL range for fluorimetric detection (limit of detection: 0.8 ng/mL). Aloe emodin levels were determined in Aloe vera extracts and commercial formulations by both detection means, with good precision (R.S.D. < 9%) and accuracy (recovery > 85%) and consistent results. Thus, the method seems to be suitable for the analysis of aloe emodin in different herbal and commercial products; it could also be useful for the identity confirmation of formulations and extracts.

PubMed: 19266552, In this paper, the simultaneous separation of several polyphenols such as (+)-catechin, (-)-epicatechin, (-)-epigallocatechin, theophylline, caffeine in green and black teas by capillary electrochromatography (CEC) was developed. Several experimental parameters such as stationary phase type, mobile phase composition, buffer and pH, inner diameter of the columns, sample injection, were evaluated to obtain the complete separation of the analysed compounds. Baseline resolution of the studied polyphenols was achieved within 30 min by using a capillary column (id 100 µm) packed with bidentate C18 particles for 24.5 cm and a mobile phase composed of 5 mM ammonium acetate buffer pH 4 with H2O/ACN (80:20, v/v). The applied voltage and the temperature were set at 30 kV and 20°C. Precision, detection and quantification limits, linearity, and accuracy were investigated. A good linearity (R2 > 0.9992) was achieved over a concentration working range of 2-100 µg/mL for all the analytes. LOD and LOQ were 1 and 2 µg/mL, respectively, for all studied compounds. The CEC method was applied to the analysis of those polyphenols in green and black tea samples after an extraction procedure. Good recovery data from accuracy studies ranged between 90% and 112% for all analytes

The present paper deals with the development of a rapid and feasible high-performance liquid chromatographic method for the determination of trazodone and its main active metabolite 3-(1-clorophenyl)piperazine (m-CPP) in human plasma. Trazodone is a second-generation antidepressant with serotonin antagonist activity. The metabolite seems to be involved in the onset of some side effects of trazodone therapy, thus its determination is very important during therapeutic drug monitoring. Separation was achieved using a C8 reversed-phase column and a mobile phase composed of aqueous phosphate buffer (70%), containing triethylamine, at pH 3.5 and acetonitrile (30%). The UV detector was set at 255 nm and loxapine was used as the internal standard. An original pre-treatment procedure of plasma samples was developed, based on solid-phase extraction with C8 reversed phase cartridges (50 mg, 1 mL). The obtained extraction yields values were higher than 90% and precision, expressed as RSD, was lower than 5.6%. The method was successfully applied to plasma samples from depressed patients undergoing therapy with trazodone; accuracy results were satisfactory (recovery > 91%). Thus, the method seems to be suitable for the therapeutic drug monitoring of trazodone and its main active metabolite in depressed patients' plasma.

A capillary electrophoretic method has been developed for the enantioselective analysis of amisulpride in pharmaceutical formulations, using β-cyclodextrin sulfate as the chiral selector. Several parameters, such as cyclodextrin type and concentration, buffer concentration and pH and capillary temperature were investigated for method optimisation. Baseline enantioseparation of the racemic compound was achieved in less than 10 minutes using a fused silica capillary (50 μm I.D. and 33.0 cm, 8.5 cm, total and effective length, respectively), filled with a background electrolyte consisting of a 10 mM citrate buffer at pH 3.5 supplemented with 0.22% (w/v) β-cyclodextrin sulfate at 20°C and applying a voltage of +15 kV. Formulation analysis was carried out after analyte extraction by methanol. The method was fully validated, with good results in terms of precision, selectivity, accuracy and amount of drug found with respect to the label claim. Thus, the method seems to be suitable for the enantiomeric analysis of amisulpride in pharmaceutical formulations.

The enantioseparation of reboxetine by HPLC was investigated using chiral stationary phases (CSPs) con- taining cellulose Tris(3,5-dimethylphenyl)carbamate on silica gel (Chiralcel OD column) as the chiral selector. Reversed phase HPLC was the technique of choice for the analytical enantioseparation of rebox- etine, while the chiral semipreparative separation was obtained with the same CSP, but in normal phase conditions. The effects of the mobile phase pH and composition on analytical retention, enantioselectivity and resolution were investigated. The best performance was obtained using a mobile phase composed of 0.5 M sodium perchlorate at pH 6 and acetonitrile in the 60/40 (v/v) ratio. The semipreparative separation has allowed obtaining pure enantiomers, but required the preparation of reboxetine free base. Different n-hexane/alcohol mixtures were tested as mobile phases, varying both the nature of the alcohol and its percentage in the mobile phase. Different n-hexane/alcohol mixtures were tested as mobile phase and the best results were obtained by using a mobile phase composed of n-hexane and 2-propanol (80:20, v/v).

An original method based on capillary electrochromatography (CEC) has been developed for the identification and 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 µm) packed with a C18 sorbent (packing length: 23 cm, particle size: 5 µm). The mobile phase is a 50 mM, pH 6.0 ammonium acetate / acetonitrile (10/90, v/v) mixture. Applying a 30 kV voltage, within a 22-min electrochromatographic run the following eleven compounds are separated and analysed: the terpenes terpinen-4-ol, caryophyllene oxide, limonene, beta-pinene, 3-carene, alpha-pinene, alpha-humulene, beta-caryophyllene and alpha-phellandrene and the aromatic 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 other analytes. The optimised method has been validated with good results in terms of linearity, limits of quantitation and detection and precision. The CEC method was successfully applied to the analysis of essential oils and methanolic extracts of “black”, “white” and “green” pepper. The results show that the samples can be analysed without any pre-treatment, in fact a simple dilution with the mobile phase allows good analyte separation to be obtained without any significant interference from the oil or methanolic matrices.

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.

Glycyrrhizin (G) and glycyrrhetic acid (GA) were separated by using nano-liquid chromatography(nano-LC) in a fused silica capillary packed with RP18 stationary phase (75 micron ID, effective length 33 cm, packed 23 cm) eluting at 300 nL/min in a gradient mode. The mobile phase was a mixture of water-MeOH-MeCN-acetic acid (29:35:35:1, v/v/v/v) that was delivered for one minute and after this was modified by reducing the water content (14 :42.5 :42.5 :1, v/v/v/v). The intra-day and inter-day relative standard deviations (of retention time and peak area) were satisfactory (lower than 2.9 and 4%, respectively). The linearity of the nano-LC method was assessed in the range 0.62–5.00 microg/mL and 80–200 microg/mL for GA and G, with R2 = 0.996 and 0.995, respectively. The licorice was extracted with a mixture of ethanol-water, diluted with the mobile phase, and injected for the analysis.

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%).

Mirtazapine is a recent noradrenergic and specific serotonergic antidepressant drug. A capillary electrophoretic method has been developed for the enantioseparation and analysis of mirtazapine and its main active metabolite, N-desmethylmirtazapine, in human plasma. For method optimisation several experimental parameters were investigated, such as type and concentration of the chiral selector, buffer pH and capillary temperature. Baseline enantioseparation of the analytes was achieved in 2.5 minutes in a fused silica capillary (50 μm I.D.; 48.5 cm total length; 8.5 cm effective length) using carboxymethyl-beta-cyclodextrin, dissolved in a background electrolyte consisting of 50 mM phosphate buffer at pH 2.5, as the chiral selector. UV detection was set at 205 nm. A careful pre-treatment of plasma samples was developed, using solid-phase extraction with hydrophilic-lipophilic balance cartridges (60 mg, 3 mL), eluting the sample with methanol, then concentrating it 37.5 times before injection. Extraction yield values are very satisfactory, being the average 89% for mirtazapine and 73% for N-desmethylmirtazapine. Application of the method to some human plasma samples has given satisfactory results.

The separation and simultaneous determination of caffeine, paracetamol, and acetylsalicylic acid in two analgesic tablet formulations was investigated by capillary electrochromatography (CEC). The effect of mobile phase composition on the separation and peak efficiency of the three analytes was studied and evaluated; in particular, the influence of buffer type, buffer pH, and acetonitrile content of the mobile phase was investigated. The analyses were carried out under optimized separation conditions, using a full-packed silica capillary (75 microm ID; 30.0 cm and 21.5 cm total and effective lengths, respectively) with a 5 microm C8 stationary phase. A mixture of 25 mM ammonium formate at pH 3.0 and acetonitrile (30:70 v/v) was used as the mobile phase. UV detection was at 210 nm. Good linearity was found in the range of 50-200, 20-160, and 4-20 microg/mL for acetylsalicylic acid (r2=0.9988), paracetamol (r2=0.9990) and caffeine (r2=0.9990), respectively. Intermediate precision (RSD interday) as low as 0.1-0.8% was found for retention times, while the RSD values for the peak area ratios (Aanalyte/AIS) were in the range of 1.9-2.9%. The optimized CEC method was applied to the analysis of the studied compounds present in commercial tablets.