High-performance liquid chromatographic method with amperometric detection employing boron-doped diamond electrode for the determination of sildenafil, vardenafil and their main metabolites in plasma

A simple, fast and sensitive HPLC method with electrochemical detection employing boron-doped diamond electrode (BDD) for the determination of sildenafil (Viagra™), vardenafil (Levitra™) and their main metabolites, N-desmethyl sildenafil and N-desethyl vardenafil in human plasma is presented. The assay involved drug extraction by tert-butyl methyl ether and isocratic reversed-phase liquid chromatography with amperometric detection. Complete separation of all analytes was achieved within 12 min. The mobile phase consisted of 20 mM sodium dihydrogen phosphate with 40 mM sodium perchlorate/acetonitrile (70:30, v/v), pH 3.5. The electrode working potential was +1520 mV (vs. Pd/H₂). Calibration curves were linear over the concentration range of 10–400 ng mL⁻¹. Phloretin was used as an internal standard. The limits of detection (LOD) and quantification (LOQ) for the studied analytes were within the range of 2–4 ng mL⁻¹ and 7.0–13.4 ng mL⁻¹, respectively. The developed method was applied to human plasma samples spiked with analytes at therapeutic concentrations. The study confirms the method's suitability for both pharmacokinetic studies and therapeutic monitoring.     

Titania immobilized polypropylene hollow fiber as a disposable coating for stir bar sorptive extraction-high performance liquid chromatography-inductively coupled plasma mass spectrometry speciation of arsenic in chicken tissues

The bottleneck of applying stir bar sorptive extraction (SBSE) to elemental speciation analysis is lack of suitable extraction phases with good affinities to different elemental species. In this paper, a newly high polar extraction phase of titania immobilized polypropylene hollow fiber (TiO₂-PPHF) was prepared by sol–gel immersion and low temperature hydrothermal process and the obtained TiO₂-PPHF inherits the adsorption properties of TiO₂ and the toughness of PPHF. With a suitable size of stainless steel magnetic bar inserted into the prepared TiO₂-PPHF, a disposable TiO₂-PPHF coating stir bar was obtained. The prepared TiO₂-PPHF was characterized by X-ray diffraction spectrometry and scanning electron microscopy and the significant parameters affecting the extraction efficiency of different arsenic species were studied. Based on the above facts, a new method of SBSE combined with high performance liquid chromatography (HPLC)–inductively coupled plasma mass spectrometry (ICP-MS) was developed for the speciation of phenyl arsenic compounds and their possible transformation products in chicken tissues. Under the optimal conditions, limits of detection (LODs) of the developed method for eight target arsenic species were in the range of 11.4–64.6 ng L⁻¹ with enrichment factors of 8.5–22.3 (theory enrichment factor was 50), and the relative standard deviations (RSDs) were varying from 6.3 to 12.6% (c_AsIII/V = 5 μg L⁻¹, c_{MMA,DMA,p-ASA,4-OH,3-NHPAA,PA,4-NPAA} = 10 μg L⁻¹, n = 7). The proposed method was successfully applied to the speciation of arsenic in chicken meat/liver samples and the recoveries for the spiked samples were in the range of 78.5–120.4%. In order to validate the accuracy of the proposed method, a certified reference material of BCR-627 tuna fish tissue was analyzed and the determined values were in good agreement with the certified values. The TiO₂-PPHF was demonstrated to be a highly selective coating for the target arsenic species, and could be easily prepared in batches with low cost. In addition, with the disposable coating, the carry-over effect commonly encountered in conventional SBSE was avoided.     

Trace analysis of fluoxetine and its metabolite norfluoxetine. Part I: development of a chiral liquid chromatography-tandem mass spectrometry method for wastewater samples

An enantioselective method for the determination of fluoxetine (a selective serotonin reuptake inhibitor) and its pharmacologically active metabolite norfluoxetine has been developed for raw and treated wastewater samples. The stable isotope-labeled fluoxetine and norfluoxetine were used in an extended way for extraction recovery calculations at trace level concentrations in wastewater. Wastewater samples were enriched by solid phase extraction (SPE) with Evolute CX-50 extraction cartridges. The obtained extraction recoveries ranged between 65 and 82% in raw and treated wastewater at a trace level concentration of 50 pM (15-16 ng L⁻¹). The target compounds were identified by the use of chiral liquid chromatography tandem mass spectrometry (LC-MS/MS) in selected reaction monitoring (SRM) mode. The enantiomers were successfully resolved on a chiral α₁-acid glycoprotein column (chiral AGP) with acetonitrile and 10 mM ammonium acetate buffer at pH 4.4 (3/97, v/v) as the mobile phase. The effects of pH, amount of organic modifier and buffer concentration in the mobile phase were investigated on the enantiomeric resolution (R_s) of the target compounds. Enantiomeric R_s-values above 2.0 (1.03 RSD%, n = 3) were achieved for the enantiomers of fluoxetine and norfluoxetine in all mobile phases investigated. The method was validated by assessing parameters such as cross-contamination and carryover during SPE and during LC analysis. Cross-talk effects were examined during the detection of the analytes in SRM mode. In addition, the isotopic purity of fluoxetine-d₅ and norfluoxetine-d₅ were assessed to exclude the possibility of self-contamination. The interassay precision of the chromatographic separation was excellent, with relative standard deviations (RSD) equal to or lower than 0.56 and 0.81% in raw and treated wastewaters, respectively. The method detection and quantification limits (respectively, MDL and MQL) were determined by the use of fluoxetine-d₅ and norfluoxetine-d₅. The MQL for the single enantiomers ranged from 12 to 14 pM (3.6-4.3 ng L⁻¹) in raw wastewater and from 3 to 4 pM (0.9-1 ng L⁻¹) in treated wastewater. The developed method has been employed for the quantification of (R)-fluoxetine, (S)-fluoxetine and the enantiomers of norfluoxetine in raw and treated wastewater samples to be presented in Part II of this study.     

Simultaneous extraction and clean-up of polybrominated diphenyl ethers and polychlorinated biphenyls from sheep liver tissue by selective pressurized liquid extraction and analysis by gas chromatography-mass spectrometry

We describe a selective pressurized liquid extraction (SPLE) method, followed by gas chromatography–mass spectrometry (GC–MS), for the simultaneous extraction and clean-up of polybrominated diphenyl ethers (PBDEs) and polychlorinated biphenyls (PCBs) in sheep liver tissue samples. The on-line clean-up of liver tissue by SPLE was tested using differing amount of acid-modified silica (sulphuric acid:silica gel, 1:2, w/w), the most effective amount being 20 g. Different extraction solvents (iso-hexane and dichloromethane), either alone or in various combinations, were used to extract these target compounds from spiked liver samples. Variables affecting the SPLE extraction efficiency, including temperature, pressure, number of extraction cycles and static extraction time were studied; the optimum parameters were 80 °C, 10.3 MPa, 2 cycles and 5 min, respectively. The SPLE based method was compared with more traditional Soxhlet, off-line PLE, ultrasonic and heating extraction methods. Overall the mean percentage recoveries for all target chemicals using SPLE were 86–103% (n = 3, SD < 9%), and compared favourably with the Soxhlet (63–109%, n = 3, SD < 8%), off-line PLE (82–104%, n = 3, SD < 18%), ultrasonic (86–99%, n = 3, SD < 11%) and heating (72–102%, n = 3, SD < 21%) extraction methods. The limits of detection of the proposed method were 5–96 pg g⁻¹ and 2–29 pg g⁻¹ for the different PBDE and PCB chemicals studied, respectively. The outputs of the proposed method were linear over the range from 0.02 to 30 ng g⁻¹, for all PCB and PBDE congeners except for PBDE 100 and 153 (0.05–30 ng g⁻¹) and PBDE 183 (0.1–30 ng g⁻¹). The method was successfully applied to sheep liver samples for the determination of the target PBDE and PCB compounds.     

Development of an on-line matrix solid-phase dispersion/fast liquid chromatography/tandem mass spectrometry system for the rapid and simultaneous determination of 13 sulfonamides in grass carp tissues

A novel analytical protocol based on interfacing on-line matrix solid-phase dispersion (MSPD) with high-performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS) was developed for extraction and determination of 13 sulfonamide residues in grass carp tissues. The target analytes were separated on a fused-core C18-silica column with a period of 7 min and quantified by a triple–quadrupole linear ion-trap mass spectrometer in positive ion multiple-reaction monitoring (MRM) mode. The proposed method was optimized and validated according to Commission Decision 2002/657/EC. The matrix-matched calibration curves were performed at six concentration levels and good linear relationship (R² = 0.993–0.998) was observed within the range of 0.1–100 ng mL⁻¹. The mean values of relative standard deviation of intra- and inter-day ranging from 1.8% to 7.8% and from 2.8% to 10.3% were obtained, respectively. Moreover, satisfied recoveries (69.0–96.3%) of all studied sulfonamides were demonstrated in different spiked levels, with RSDs ≤ 13.2%. The proposed method has been applied successfully to the analysis of sulfonamides in several grass carp samples, and the results indicated that this novel instrumental coupling was fast, sensitive, reliable and environmental friendly with good prospects.     

Multiresidue analytical method for the determination of antimicrobials, preservatives, benzotriazole UV stabilizers, flame retardants and plasticizers in fish using ultra high performance liquid chromatography coupled with tandem mass spectrometry

A multiresidue analytical method for the determination of emerging pollutants belonging to personal care products (PCPs) (antimicrobials, preservatives), benzotriazole UV stabilizers (BUVSs) and organophosphorus compounds (OPCs) in fish has been developed using high speed solvent extraction (HSSE) followed by silica gel clean up and ultra fast liquid chromatography coupled with tandem mass spectrometry (UFLC–MS/MS) analysis. Developed extraction and clean up method yielded good recovery (>70%) for all the four groups of emerging pollutants, i.e. antimicrobials (78.5–85.6%), preservatives (85.0–89.4%), BUVSs (70.9–112%) and OPCs (81.6–114%; except for TEP – 68.9% and TPeP – 58.1%) with RSDs ranging from 0.7 to 15.4%. Intra- and inter-day repeatabilities were less than 19.8% and 19.0%, respectively at three spiked levels. The concentrations were given in lipid weight (lw) basis, and the method detection limits were achieved in the lowest range of 0.001–0.006 ng g⁻¹ for two antimicrobials, 0.001–0.015 ng g⁻¹ for four preservatives, 0.0002–0.009 ng g⁻¹ for eight BUVSs and 0.001–0.014 ng g⁻¹ for nine OPCs. Finally, the method was successfully validated as a simple and fast extraction method for the determination of 23 compounds belonging to PCPs, BUVSs and OPCs and applied to the analysis of three species of fish from Manila Bay, the Philippines. Concentrations ranged from 27 to 278 ng g⁻¹ for antimicrobials, 6.61 to 1580 ng g⁻¹ for paraben preservatives, −1 for BUVSs and ND (not detected) to 266 ng g⁻¹ for OPCs suggesting the ubiquitous contamination by these emerging pollutants in Manila Bay. This is the first method developed for the determination of triclocarban, four paraben preservatives and four BUVSs, in fish.     

Selective extraction of alkaloids in human urine by on-line single drop microextraction coupled with sweeping micellar electrokinetic chromatography

A novel method of on-line single drop microextraction (SDME) coupled with sweeping micellar electrokinetic chromatography (MEKC) for the selective extraction and dual preconcentration of alkaloids was developed. In this technique, analytes of three alkaloids were firstly extracted from 4.0 mL basic aqueous sample solution (donor phase, 500 mM NaOH) into a layer of n-octanol at temperature 30 °C with the stirring rate of 1150 rpm, then back-extracted into the acidified aqueous acceptor (acceptor phase, 50 mM H₃PO₄) suspended at the tip of a capillary at 650 rpm. Then, the aqueous acceptor was introduced into capillary by hydrodynamic injection with a height difference of 15 cm between the inlet and outlet of capillary for 300 s, and analyzed directly by on-line sweeping MEKC. With the selective SDME, we were able to extract three alkaloids without any interfering components in human urine samples. Under the optimum conditions, the proposed method achieved limits of detections (LOD) of between 0.2 ng mL⁻¹ and 1.5 ng mL⁻¹ with 1583–3556-fold increases in detection sensitivity for three analytes, which indicated that it was a promising method for analysis of alkaloids in human urine.     

Electrosonic spray ionization--an ideal interface for high-flow liquid chromatography applications

Electrosonic spray ionization (ESSI) has been studied as an interface between high-performance liquid chromatography (HPLC) and mass spectrometry (MS), using sample flow rates up to 3.0 ml min⁻¹. This ionization interface was compared with pneumatically assisted electrospray ionization (ESI) using mass spectrometry for detection. For experiments that did not involve direct comparison of different flow rates, the ESI experiments were performed using post column splitting to work at optimal conditions. ESSI allows the interfacing of conventional or high-resolution liquid chromatography (LC) methods to mass spectrometry without post column splitting. High sample flow rates could be handled without a significant loss of signal intensity using a nebulization gas flow rate of 5.5 L min⁻¹. Since ESI needs to be operated with lower sample flow rates, it is limited to micro/nano LC systems, or post column splitting must be used. In particular, nano LC systems have to be treated with great care and require constant maintenance. When using post-column splitting, the increased diffusion can become a problem especially when using systems with very small void volumes. In all experiments ESSI showed better signal intensities than a commercially available, pneumatically assisted ESI source. ESSI does not require heating of the nebulizer gas, which should help to preserve the original structure of thermally unstable molecules. Therefore, ESSI is presented as an alternative to the commercially available heated ESI sources of AB SCIEX, Thermo Fischer, Agilent and Waters. The observed LC-ESSI-MS ion chromatograms are shown to be very stable even when using flow rates higher than 1.0 ml min⁻¹, which could be very suitable for ultra high performance LC, where sample flow rates up to 2.0 mL min⁻¹ with backpressures up to 1200 bar are used. Also, a difference in the relative intensities of singly and doubly protonated peptide monomers and dimers was observed between the two ionization methods. The coefficients of determination for the calibration of instrument response for Val–Tyr–Val and Met-Enkephalin showed excellent linearity over a wide concentration range (0.1–100 μM), while ESI results were only linear over a much smaller range (0.1–20 μM). The observed behavior is thought to be caused by insufficient ionization efficiency of solutions above ∼20 μM by ESI, exemplifying the robustness of ESSI as an interface between LC and MS.     

Determination of primary and secondary aliphatic amines with high performance liquid chromatography based on the derivatization using 1,3,5,7-tetramethyl-8-(N-hydroxysuccinimidyl butyric ester)-difluoroboradiaza-s-indacene

In this article, the simultaneous determination of primary and secondary aliphatic amines including dimethylamine (DMA), diethylamine and eleven primary aliphatic amines by high performance liquid chromatography (HPLC) with fluorescence detection has been achieved using a BODIPY-based fluorescent derivatization reagent, 1,3,5,7-tetramethyl-8-(N-hydroxysuccinimidyl butyric ester)-difluoroboradiaza-s-indacene (TMBB-Su). The derivatization reaction of TMBB-Su with aliphatic amines was optimized with orthogonal design experiment and the derivatization reaction proceeded at 15 °C for 25 min. The baseline separation of these derivatives was carried out on a C₈ column with methanol-tetrahydrofuran-50 mM pH 6.50 HAc-NaAc buffer (55/5/40, v/v/v) as a mobile phase. Detected at the excitation and emission of 490 and 510 nm, respectively, the detection limits were obtained in the range of 0.01-0.04 nM (signal-to-noise ratio = 3). The proposed method has been applied to the determination of trace aliphatic amines in viscera samples from mice without complex pretreatment or enrichment method. The recoveries ranged from 95.1% to 106.8%, depending on the samples investigated.     

Determination of deoxynivalenol, T-2 and HT-2 toxins in a bread model food by liquid chromatography-high resolution-Orbitrap-mass spectrometry equipped with a high-energy collision dissociation cell

A sensitive and accurate method employing a single stage high resolution mass spectrometer equipped with a high-energy collision-dissociation cell (HCD) for the simultaneous determination of deoxynivalenol (DON), T-2 toxin (T-2) and HT-2 toxin (HT-2) in a processed bread model food has been developed. Two sample pre-treatment routes for the extraction of these mycotoxins were investigated, based on Mycosep^® column clean up or QuEChERS-like procedure, respectively. The former approach suffered less from matrix effects and allowed to achieve in bread samples LODs of 7, 12 and 17 ng/g for T-2, HT-2 and DON, respectively, with 0.5 ppm mass accuracy. Two acquisition modes, full scan MS and all ion fragmentation, exploiting the fragmentation features offered by an HCD chamber and integrated within the Orbitrap analyser, were compared for quantitative purposes. The method was applied to investigate the degradation of these mycotoxins during bread processing using a bread model food. Most T-2 hydrolyzed to HT-2 during dough preparation, and about 20–30% of HT-2 and DON was degraded during bread baking.     

Liquid chromatography/mass spectrometry compatible approaches for the quantitation of folic acid in fortified juices and cereals using aqueous normal phase conditions

Folic acid was separated under aqueous normal phase (ANP) conditions with Diamond Hydride™ columns and quantitated in fortified cereal and juice matrices using high performance liquid chromatography/ultraviolet absorption (HPLC-UV) and liquid chromatography/mass spectrometry (LC–MS) based methodologies. The folic acid was well-resolved from matrix components under the ANP conditions studied and allowed for the direct analyses of the fortified juices and cereals without the sample cleanup that is often required for other reported LC-based approaches. The calibration curve obtained from the LC–MS analyses demonstrated good linearity (R² = 0.9997) in the studied concentration range of 0.05–0.5 mg/L. The spiked flour percent recovery was 90% with HPLC-UV and 91% with LC–MS. Spiked juice percent recovery was 102% with LC–MS. However, analyses of juices were unsatisfactory either in terms of recovery or sensitivity. Therefore, analyses of juices will either need to be performed by LC–MS or, if by HPLC-UV, will require sample cleanup. Three cereals and one juice were analyzed with the methods.     

Direct aqueous determination of glyphosate and related compounds by liquid chromatography/tandem mass spectrometry using reversed-phase and weak anion-exchange mixed-mode column

Analysis of the broad-spectrum herbicide glyphosate and its related compounds is quite challenging. Tedious and time-consuming derivatization is often required for these substances due to their high polarity, high water solubility, low volatility and molecular structure which lacks either a chromophore or fluorophore. A novel liquid chromatography/tandem mass spectrometry (LC/MS–MS) method has been developed for the determination of glyphosate, aminomethylphosphonic acid (AMPA) and glufosinate using a reversed-phase and weak anion-exchange mixed-mode Acclaim^® WAX-1 column. Aqueous environmental samples are directly injected and analyzed in 12 min with no sample concentration or derivatization steps. Two multiple reaction monitoring (MRM) channels are monitored in the method for each target compound to achieve true positive identification, and ¹³C,¹⁵N-glyphosate is used as an internal standard to carry out isotope dilution mass spectrometric (IDMS) measurement for glyphosate. The instrument detection limits (IDLs) for glyphosate, AMPA and glufosinate are 1, 2 and 0.9 μg/L, respectively. Linearity of the detector response with a minimum coefficient of determination (R²) value (R² > 0.995) was demonstrated in the range of ∼10 to 10³ μg/L for each analytes. Spiked drinking water, surface water and groundwater samples were analyzed using this method and the average recoveries of analytes in three matrices ranged from 77.0 to 102%, 62.1 to 101%, 66.1 to 93.7% while relative standard deviation ranged from 6.3 to 10.2%, 2.7 to 14.8%, 2.9 to 10.7%, respectively. Factors that may affect method performance, such as metal ions, sample preservation, and storage time, are also discussed.     

Anion separations for liquid chromatography using propylpyridinium silica as the stationary phase

This work describes the characterization and potential applications of a silica-based anion-exchange phase prepared by a two-step modification process that incorporates a propylpyridinium group. The effects of pH and eluent concentration on anion separation were examined using 150 mm × 3.9 mm HPLC columns packed with the new phase. The mobile phase pH values ranged from 3.8 to 6.6 using phthalic acid/Tris solutions. The best separation was achieved using 2.5 mmol L⁻¹ phthalate/2.4 mmol L⁻¹ Tris solution at pH 4.2 as mobile phase with non-suppressed conductivity detection. The new stationary phase was used for the separation of some inorganic and organic anions showing good resolution. The stability of the silica-based anion exchange phase was also evaluated.Analytical curves, for concentrations ranging from 0.25 to 10 mg L⁻¹ for the inorganic anions chloride, nitrite, bromide and nitrate, showed good linear correlations (r > 0.998). The method was tested with certified rainwater samples. The measured and certified values were in good agreement, indicating that the new phase holds significant promise for the analysis of these anions in environmental samples.     

On the extra-column band-broadening contributions of modern, very high pressure liquid chromatographs using 2.1 mm I.D. columns packed with sub-2 μm particles

The efficiencies of two narrow bore columns (100 mm and 50 mm × 2.1 mm) packed with 1.7 μm totally porous BEH-C₁₈ particles were measured on two very high pressure liquid chromatographs (Acquity from Waters and 1290 Infinity HPLC System from Agilent) operating at maximum pressures of 1034 and 1200 bar, respectively. The probe compounds were a mixture of uracil, acetophenone, toluene, and naphthalene eluted in a 50/50 (v/v) solution of acetonitrile and water at 303 K with a flow rate of 0.40 mL/min. The apparent efficiencies of columns, which lumps the consequences of band broadening due to the column and the system contributions, may depend much on the extra-column volumes of the instruments used. Actually, it is known for a long time that the apparent column performance is strongly affected by the instrument characteristics, including the diameter of the connecting tubes, the injection technique (with or without needle seat capillary), and the detection cell volume. When the 1290 Infinity HPLC System is equipped with a needle seat, an inlet and an outlet connecting capillary tube with inner diameters around 115 μm, its extra-column variance for a 0.1 μL injection volume is 9.2 μL² while that of the Acquity instrument is 6.9 μL². Minor modifications suggested by their respective manufacturers allowed significant reductions of these variances, to 6.2 and 3.9 μL², respectively. Yet, in their optimized configurations and for weakly retained compounds (k ? 1), these modern, sophisticated instruments cannot provide more than 75% (1290 Infinity) and 85% (Acquity) of the maximum efficiency of a 2.1 mm × 50 mm BEH column. For more strongly retained compounds (k > 4), in contrast, they are both able to provide more than 95% of the maximum expected efficiency.     

Fast liquid chromatography-tandem mass spectrometry for the analysis of bisphenol A-diglycidyl ether, bisphenol F-diglycidyl ether and their derivatives in canned food and beverages

In this work a fast liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS) method using a C18 Fused Core™ column, was developed for the simultaneous analysis of bisphenol A diglycidyl ether (BADGE), bisphenol A (2,3-dihydroxypropyl) glycidyl ether (BADGE·H₂O), bisphenol A bis(2,3-dihydroxypropyl) ether (BADGE·2H₂O), bisphenol A (3-chloro-2-hydroxypropyl) glycidyl ether (BADGE·HCl), bisphenol A bis(3-chloro-2-hydroxypropyl) ether (BADGE·2HCl) and bisphenol A (3-chloro-2-hydroxypropyl)(2,3-dihydroxypropyl ether) (BADGE·HCl·H₂O) and bisphenol F diglycidyl ether (BFDGE), bisphenol F bis(2,3-dihydroxypropyl) ether (BFDGE·2H₂O), bisphenol F bis(3-chloro-2-hydroxypropyl) ether (BFDGE·2HCl). The LC method was coupled with a triple quadrupole mass spectrometer, using an ESI source in positive mode and using the [M+NH₄]⁺ adduct as precursor ion for tandem mass spectrometry experiments. The method developed was applied to the determination of these compounds in canned soft drinks and canned food. OASIS HLB solid phase extraction (SPE) cartridges were used for the analysis of soft drinks, while solid canned food was extracted with ethyl acetate. Method limits of quantitation ranged from 0.13 μg L⁻¹ to 1.6 μg L⁻¹ in soft drinks and 1.0 μg kg⁻¹ to 4.0 μg kg⁻¹ in food samples. BADGE·2H₂O was detected in all the analyzed samples, while other BADGEs such as BADGE·H₂O, BADGE·HCl·H₂O, BADGE·HCl and BADGE·2HCl were also detected in canned foods.     

New method for development of carbon clad silica phases for liquid chromatography: Part I. Preparation of carbon phases

Owing to its combination of unique selectivity and mechanical strength, commercial carbon clad zirconia (C/ZrO₂) has been widely used for many applications, including fast two-dimensional liquid chromatography (2DLC). However, the low surface area available (only 20–30 m²/g for commercial porous ZrO₂) limits its retentivity. We have recently addressed this limitation by developing a carbon phase coated on the high surface area of HPLC grade alumina (C/Al₂O₃). This material provides higher retentivity and comparable selectivity, but its use is still limited by how few HPLC quality types of alumina particles (e.g., particle size, surface area, and pore size) are available. In this work, we have developed useful carbon phases on silica particles, which are available in various particle sizes, pore sizes and forms of HPLC grade. To make the carbon phase on silica, we first treat the silica surface with a monolayer or less of metal cations that bind to deprotonated silanols to provide catalytic sites for carbon deposition. After Al (III) treatment, a carbon phase is formed on the silica surface by chemical vapor deposition at 700 °C using hexane as the carbon source. The amount of Al (III) on the surface was varied to assess its effect on carbon deposition, and the carbon loading was varied at different Al (III) levels to assess its effect on the chromatographic properties of the various carbon adsorbents. We observed that use of a concentration of Al (III) corresponding to a full monolayer leads to the most uniform carbon coating. A carbon coating sufficient to cover all the Al (III) sites, required about 4–5 monolayers in this work, provided the best chromatographic performance. The resulting carbon phases behave as reversed phases with reasonable efficiency (50,000–79,000 plates/m) for non-aromatic test species.     

Enantioseparation of phenylsuccinic acid by high speed counter-current chromatography using hydroxypropyl-β-cyclodextrin as chiral selector

High speed counter-current chromatography (HSCCC) was successfully applied to resolution of phenylsuccinic acid (PSA) with hydroxypropyl-β-cyclodextrin (HP-β-CD) as chiral selector (CS). The two-phase solvent system composed of n-hexane–methyl tert-butyl ether–0.1 mol L⁻¹ phosphate buffer solution with pH = 2.51 (0.5:1.5:2, v/v/v) was selected. Influence factors involved in the chiral separation were investigated, including the concentration of chiral selector, pH value of the aqueous phase, the separation temperature, and the thermodynamic parameters of inclusion complex were calculated. The complex formation constants were determined using analytical instrument. Two HSCCC elution modes were studied and peak resolution equation was discussed. Under optimum separation conditions, 712 mg of PSA racemate was separated using preparative apparatus. The purities of both of the fractions including (+)-PSA and (−)-PSA from the preparative CCC separation were over 98.5% determined by HPLC and enantiomeric excess of (+)-PSA and (−)-PSA reached 97.6% and 98.6%, respectively. Recovery for the target compounds from the CCC fractions reached 80–82% yielding 285 mg of (+)-PSA and 292 mg of (−)-PSA.     

Determination of imipramine and trimipramine by capillary electrophoresis with electrochemiluminescence detection

The tricyclic antidepressants (TCA) imipramine (Imi) and trimipramine (Tri) were successfully analyzed by capillary electrophoresis (CE) coupling with Tris(2,2-bipyridyl) ruthenium(II)-based (Ru(bpy)₃²⁺) end-column electrochemiluminescence (ECL) detection. The addition of β-CD to the running buffer was found to enable baseline separation of the two analytes and the addition of acetonitrile (ACN) as an organic additive to improve the repeatability and sensitivity of the CE method. Under the optimized separation and detection conditions (50 mM PBS (pH = 7.0) and 2 mM Ru(bpy)₃²⁺ in the ECL detection cell, 20 mM Tris (pH = 2.0), 0.2 mM β-CD and 20% ACN (v/v) as running buffer), wide linear ranges of 0.1-5 μM and 0.1-5 μM were achieved, with the correlation coefficients of 0.9990 (n = 8) and 0.9980 (n = 8) for Imi and Tri, respectively. Detection limits 5 nM and 1 nM (S/N = 3) were obtained for Imi and Tri, respectively. The method was also successfully applied for the determination of Imi in pharmaceutical dosage form.     

CdSe quantum dots capped PAMAM dendrimer nanocomposites for sensing nitroaromatic compounds

The detection of nitroaromatic compounds, best known as raw materials in explosives preparations, is important in many fields including environmental science, public security and forensics. CdSe quantum dots capped with PAMAM-G₄ dendrimer were synthetized in water and used for the detection of trace amounts of three nitroaromatic compounds: 4-methoxy-2-nitrophenol (MNP), 2-amine-5-chloro-1,3-dinitrobenzene (ACNB) and 3-methoxy-4-nitrobenzoic acid (MNB). To increase the apparent water solubility of these compounds α-cyclodextrin (α-CD) was used to promote the formation of inclusion complexes. The studied nitroaromatic compounds (plus α-CD) significantly quenched the fluorescence intensity of the nanocomposite with linear Stern-Volmer plots. The Stern-Volmer constants (standard deviation in parenthesis) were: MNB, K_SV = 65(5) × 10⁴ M⁻¹; ACNB, K_SV = 19(2) × 10⁴ M⁻¹; and, MNP, K_SV = 33(1) × 10² M⁻¹. These constants suggest the formation of a ground state complex between the nitroaromatric compounds and the sensor which confers a relatively high analytical sensitivity. The detection sensibilities are about 0.01 mg L⁻¹ for MNB and ACNB and about 0.1 mg L⁻¹ for MNP. No interferences or small interferences are observed for trinitrotoluene [K_SV = 10(2) × 10² × M⁻¹], 2,4-dinitrotoluene [K_SV = 20(3) × 10 M⁻¹], 2,6-dinitrotoluene [K_SV = 11(4) × 10 M⁻¹] and nitrobenzene [K_SV = 2(1) × 10³ × M⁻¹].     

Simultaneous determination of clenbuterol, salbutamol and ractopamine in milk by reversed-phase liquid chromatography tandem mass spectrometry with isotope dilution

A simple, sensitive and reliable analytical method was developed for the simultaneous determination of clenbuterol (CLB), salbutamol (SAL) and ractopamine (RAC) in milk by ultra high performance liquid chromatography–positive electrospray ionization tandem mass spectrometry (UHPLC–ESI-MS/MS) with isotope dilution. Samples were directly purified through HLB cartridge. Then the eluate was dried under nitrogen and residues were redissolved in mobile phase. Samples were analyzed by LC–MS/MS on an Acquity UPLC^® BEH C₁₈ column with gradient elution. The samples were quantified using clenbuterol-D₉, salbutamol-D₃ and ractopamine-D₆ as internal standards. The proposed method was validated according to the European Commission Decision 2002/657/EC determining specificity, decision limit (CCα), detection capability (CCβ), recovery, precision, linearity, robustness and stability. CCα values were 0.054, 0.006 and 0.008 μg/kg for CLB, SAL and RAC, respectively. CCβ values were 0.058, 0.007 and 0.009 μg/kg for CLB, SAL and RAC, respectively. The mean recoveries, repeatability (expressed as coefficient of variation, CV_r), and reproducibility (CV_R) varied from 95.8 to 106.2%, from 3.60 to 6.44% (CVr), and from 4.77 to 7.53% (CV_R), respectively. The method is demonstrated to be suitable for the determination of clenbuterol, salbutamol and ractopamine in milk. The total time required for the analysis of one sample, including sample preparation, was about 45 min.