Polyoxomolybdate368 /polyaniline nanocomposite as a novel fiber for solid-phase microextraction of antidepressant drugs in biological samples.

A novel solid-phase microextraction fiber was synthesized by coating a stainless steel wire with polyoxomolybdate368 /polyaniline as a sorbent aimed at extraction of amitriptyline, nortriptyline, and doxepin as antidepressant drugs from urine and blood samples. The polyoxomolybdate368 /polyaniline composite coating was applied using electropolymerization process under constant potential. This composition leads to enhanced extraction efficiency of the fiber. Scanning electron microscopy images show that huge three-dimensional structures of polyoxomolybdate368 in composite induced more non-smooth and porous fiber. In order to optimize of the extraction process, a series of variables including concentration of the composite materials, coating thickness, pH, extraction time, salt addition, and stirring rate was investigated and optimum conditions were determined. Analysis of surface morphology and chemical composition was performed. High-performance liquid chromatography was used for separation and evaluation of mentioned antidepressant drugs from the matrixes. The experiments indicated a detection limits of <0.2 ng/L and a linear dynamic range of 0.3-100 ng/L (R2  > 0.994). The relative recovery values were found to be in the range of 92-98%. It was concluded that the purposed fiber is highly efficient in analyzing traces of antidepressant drugs in urine and blood.

Managing the column equilibration time in hydrophilic interaction chromatography.

For a wide variety of hydrophilic interaction chromatography stationary phases, a repeatable partial equilibration was demonstrated in gradient elution after purging with as little as 12 column volumes of mobile phase. Relative standard deviations of retention time of on average ~0.15% could be obtained after 1 or 2 conditioning (blank) runs. The equilibration period must be kept strictly constant, otherwise selectivity changes occur, but this is not problematic on modern instruments. Partial equilibration was largely independent of stationary phase or gradient slope. Alternatively, full column equilibration is favoured for stationary phases that do not trap extensive water layers, and for materials with a wider pore size that have a lower surface area. Temperatures somewhat above ambient also shorten the equilibration time. Some stationary phases under optimum conditions can achieve full column equilibration using purging with ~12 column volumes, which is useful for rapid set-up of isocratic separations or for conventional gradient analysis.

Injections from sub-µL sample volumes in commercial capillary electrophoresis.

A simple sample injection procedure compatible with commercial capillary electrophoresis (CE) instrumentation was developed, which enables handling sample volumes as little as 250nL for analytical applications where sample volume availability is of concern. Single-use micro-sampling inserts were prepared by thermal modification of polypropylene micropipette tips and the inserts were accommodated in standard CE vials in CE autosampler carousel. To ensure direct contact of separation capillary injection end with sample solution and to avoid possible damage to the capillary, a soft compression spring was placed at the bottom of the vial underneath the micro-sampling insert. Injections from sub-µL samples were carried out in conventional as well as in short-end injection mode, were compatible with standard i.d./o.d. (25-100µm/365µm) fused silica capillaries and with various background electrolyte solutions and detection modes. Excellent repeatability of replicate injections from 250nL to 3µL was achieved based on RSD values of quantitative analytical measures (peak heights ≤2.4% and peak areas ≤3.7%) for CE-UV-vis, CE-ESI-MS and CE-contactless conductivity detection of model basic drugs. The achieved RSD values were comparable with those for replicate injections of the drugs from standard CE vials. The reported concept of injections from micro-sampling inserts was further demonstrated useful in evaluation of micro-electromembrane extraction (µ-EME) of model basic drugs. Sub-µL volumes of operational solutions resulted in reduced lengths of µ-EME phases and improved extraction recoveries (66-91%) were achieved.

Dispersive solid-liquid phase microextraction based on nanomagnetic Preyssler heteropolyacid: A novel method for the preconcentration of nortriptyline.

In this study, a new, simple, rapid, and efficient method combined with ultraviolet visible spectrophotometry and high-performance liquid chromatography analysis was developed for the extraction and determination of nortriptyline. The tendency of the Preyssler tungsten heteropolyacid, H14 [NaP5 W30 O110 ], immobilized on the surface of mesoporous nanomagnetite to adsorb the drug from the solution has been investigated. This method involves the use of an appropriate mixture of nanosorbent that was homogenized in disperser solvent (1.0 mL, ethanol). At first, the mixture containing the nanomagnetic sorbent and disperser solvent was injected into the aqueous sample, and a cloudy solution was formed. Subsequently, separation of the two phases was carried out using a magnet. In the second stage, analyte was desorbed from the sorbent by methanol as the optimal desorption solvent using sonication method. The elution solvent containing enriched analyte was introduced to the instruments for further analysis. Optimization of experimental conditions with respect to the extraction efficiency was investigated. The method was linear in the range of 25-5000, while the detection limit (LOD = 3SB /m) was 7.9 ng/mL and the limit of quantification (LOQ = 10SB /m) was 26.4 ng/mL. The relative standard deviation was 4.66%. The method was successfully applied to human hair samples.

The relative importance of the adsorption and partitioning mechanisms in hydrophilic interaction liquid chromatography.

We propose an original model of effective diffusion along packed beds of mesoporous particles for HILIC developed by combining Torquatos model for heterogeneous beds (external eluent+particles), Landauers model for porous particles (solid skeleton+internal eluent), and the time-averaged model for the internal eluent (bulk phase+diffuse water (W) layer+rigid W layer). The new model allows to determine the analyte concentration in rigid and diffuse W layer from the experimentally determined retention factor and intra-particle diffusivity and thus to distinguish the retentive contributions from adsorption and partitioning. We apply the model to investigate the separation of toluene (TO, as a non-retained compound), nortriptyline (NT), cytosine (CYT), and niacin (NA) on an organic ethyl/inorganic silica hybrid adsorbent. Elution conditions are varied through the choice of a third solvent (W, ethanol, tetrahydrofuran (THF), acetonitrile (ACN), or n-hexane) in a mobile phase (MP) of ACN/aqueous acetate buffer (pH 5)/third solvent (90/5/5, v/v/v). Whereas NA and CYT retention factors increase monotonously from W to n-hexane as third solvent, NT retention reaches its maximum with polar aprotic third solvents. The involved equilibrium constants for adsorption and partitioning, however, do not follow the same trends as the overall retention factors. NT retention is dominated by partitioning and NA retention by adsorption, while CYT retention is controlled by adsorption rather than partitioning. Our results reveal that the relative importance of adsorption and partitioning mechanisms depends in a complex way from analyte properties and experimental parameters and cannot be predicted generally.

Nano-electromembrane extraction.

The present work has for the first time described nano-electromembrane extraction (nano-EME). In nano-EME, five basic drugs substances were extracted as model analytes from 200 µL acidified sample solution, through a supported liquid membrane (SLM) of 2-nitrophenyl octyl ether (NPOE), and into approximately 8 nL phosphate buffer (pH 2.7) as acceptor phase. The driving force for the extraction was an electrical potential sustained over the SLM. The acceptor phase was located inside a fused silica capillary, and this capillary was also used for the final analysis of the acceptor phase by capillary electrophoresis (CE). In that way the sample preparation performed by nano-EME was coupled directly with a CE separation. Separation performance of 42,000-193,000 theoretical plates could easily be obtained by this direct sample preparation and injection technique that both provided enrichment as well as extraction selectivity. Compared with conventional EME, the acceptor phase volume in nano-EME was down-scaled by a factor of more than 1000. This resulted in a very high enrichment capacity. With loperamide as an example, an enrichment factor exceeding 500 was obtained in only 5 min of extraction. This corresponded to 100-times enrichment per minute of nano-EME. Nano-EME was found to be a very soft extraction technique, and about 99.2-99.9% of the analytes remained in the sample volume of 200 µL. The SLM could be reused for more than 200 nano-EME extractions, and memory effects in the membrane were avoided by effective electro-assisted cleaning, where the electrical potential was actively used to clean the membrane.

Implementation of droplet-membrane-droplet liquid-phase microextraction under stagnant conditions for lab-on-a-chip applications.

In the current work, droplet-membrane-droplet liquid-phase microextraction (LPME) under totally stagnant conditions was presented for the first time. Subsequently, implementation of this concept on a microchip was demonstrated as a miniaturized, on-line sample preparation method. The performance level of the lab-on-a-chip system with integrated microextraction, capillary electrophoresis (CE) and laser-induced fluorescence (LIF) detection in a single miniaturized device was preliminarily investigated and characterized. Extractions under stagnant conditions were performed from 3.5 to 15 microL sample droplets, through a supported liquid membrane (SLM) sustained in the pores of a small piece of a flat polypropylene membrane, and into 3.5-15 microL of acceptor droplet. The basic model analytes pethidine, nortriptyline, methadone, haloperidol, and loperamide were extracted from alkaline sample droplets (pH 12), through 1-octanol as SLM, and into acidified acceptor droplets (pH 2) with recoveries ranging between 13 and 66% after 5 min of operation. For the acidic model analytes Bodipy FL C(5) and Oregon Green 488, the pH conditions were reversed, utilizing an acidic sample droplet and an alkaline acceptor droplet, and 1-octanol as SLM. As a result, recoveries for Bodipy FL C(5) and Oregon Green 488 from human urine were 15 and 25%, respectively.

Separation and determination of amitriptyline and nortriptyline by dispersive liquid-liquid microextraction combined with gas chromatography flame ionization detection.

Dispersive liquid-liquid microextraction (DLLME) coupled with gas chromatography-flame ionization detection (GC-FID) was applied for the determination of two tricyclic antidepressant drugs (TCAs), amitriptyline and nortriptyline, from water samples. This method is a very simple and rapid method for the extraction and preconcentration of these drugs from environmental sample solutions. In this method, the appropriate mixture of extraction solvent (18 microL Carbon tetrachloride) and disperser solvent (1 mL methanol) are injected rapidly into the aqueous sample (5.0 mL) by syringe. Therefore, cloudy solution is formed. In fact, it is consisted of fine particles of extraction solvent which is dispersed entirely into aqueous phase. The mixture was centrifuged and the extraction solvent is sedimented on the bottom of the conical test tube. 2.0 microL of the sedimented phase is injected into the GC for separation and determination of TCAs. Some important parameters, such as kind of extraction and disperser solvent and volume of them, extraction time, pH and ionic strength of the aqueous feed solution were optimized. Under the optimal conditions, the enrichment factors and extraction recoveries were between 740.04-1000.25 and 54.76-74.02%, respectively. The linear range was (0.005-16 microg mL(-1)) and limits of detection were between 0.005 and 0.01 microg mL(-1) for each of the analytes. The relative standard deviations (R.S.D.) for 4 microg mL(-1) of TCAs in water were in the range of 5.6-6.4 (n=6). The performance of the proposed technique was evaluated for determination of TCAs in blood plasma.

Study of overload for basic compounds in reversed-phase high performance liquid chromatography as a function of mobile phase pH.

The retention and overloading properties for eight basic solutes and two quaternary ammonium compounds were studied over the pH range 2.7-10.0 using phosphate and carbonate buffers. At low pH, a hybrid inorganic-organic silica-ODS phase (XTerra RP-18, 15 cm x 0.46 cm) showed substantial loss in efficiency when sample masses exceeded about 0.5 microg; these results were similar to those obtained previously on pure silica ODS and wholly polymeric phases, suggesting a common overloading mechanism. At pH 7-8.5, substantial improvements in loading capacity were obtained on XTerra due apparently to the unexpectedly strong influence of small decreases in solute ionisation. Data from the quaternary compounds suggested that silanol ionisation on this phase was still small even at intermediate pH. For many bases, loading capacity continued to improve as the pH was raised to 10, in line with the decrease in the proportion of ionised solute. However, for the highest pK(a) solutes, peak shape worsened at high pH, possibly due to the negative influence of increasing column silanol ionisation.

The adsorption mechanism of nortryptiline on C18-bonded Discovery.

The adsorption isotherms of an ionizable compound, nortriptyline, were accurately measured by frontal analysis (FA) on a C(18)-Discovery column, first without buffer (in an aqueous solution of acetonitrile at 15%, v/v of ACN), then with a buffer (in 28%, v/v ACN solution). The buffers were aqueous solutions containing 20 mM of formic acid or a phosphate buffer at pH 2.70. The linear range of the isotherm could not be reached with the non-buffered mobile phase using a dynamic range larger than 40,000 (from 1.2 x 10(-3) g/L to 50 g/L). With a 20 mM buffer in the liquid phase, the isotherm is linear for concentrations of nortriptyline inferior to 10(-3) g/L (or 3 micromol/L). The adsorption energy distribution (AED) was calculated to determine the heterogeneity of the adsorption process. AED and FA were consistent and lead to a trimodal distribution. A tri-Moreau and a tri-Langmuir isotherm models accounted the best for the adsorption of nortriptyline without and with buffer, respectively. The nature of the buffer affects significantly the middle-energy sites while the properties of the lowest and highest of the three types of energy sites are almost unchanged. The desorption profiles of nortriptyline show some anomalies in relation with the formation of a complex multilayer adsorbed phase of acetonitrile whose excess isotherm was measured by the minor disturbance method. The C(18)-Discovery column has about the same total saturation capacity, around 200 g of nortriptyline per liter of adsorbent (or 116 mg/g), with or without buffer. About 98-99% of the available surface consists in low energy sites. The coexistence of these different types of sites on the surface solves the McCalley's enigma, that the column efficiency begins to drop rapidly when the analyte concentration reaches values that are almost one hundred times lower than those that could be predicted from the isotherm data acquired under the same experimental conditions. Due to the presence of some relatively rare high energy sites, the largest part of the saturation capacity is not practically useful.

Separation mechanism of nortriptyline and amytriptyline in RPLC.

The single and the competitive equilibrium isotherms of nortriptyline and amytriptyline were acquired by frontal analysis (FA) on the C18- bonded discovery column, using a 28/72 (v/v) mixture of acetonitrile and water buffered with phosphate (20 mM, pH 2.70). The adsorption energy distributions (AED) of each compound were calculated from the raw adsorption data. Both the fitting of the adsorption data using multi-linear regression analysis and the AEDs are consistent with a trimodal isotherm model. The single-component isotherm data fit well to the tri-Langmuir isotherm model. The extension to a competitive two-component tri-Langmuir isotherm model based on the best parameters of the single-component isotherms does not account well for the breakthrough curves nor for the overloaded band profiles measured for mixtures of nortriptyline and amytriptyline. However, it was possible to derive adjusted parameters of a competitive tri-Langmuir model based on the fitting of the adsorption data obtained for these mixtures. A very good agreement was then found between the calculated and the experimental overloaded band profiles of all the mixtures injected.

Effect of anionic additive type on ion pair formation constants of basic pharmaceuticals.

Due to their beneficial effect on selectivity, peak shape, and sample loading, the use of mobile phase anionic additives, such as formate (HCOO-), chloride (Cl-), and trifluoroacetate (CF3COO-), is increasing in both reversed-phase chromatography (RPLC) and liquid chromatography-mass spectrometry (LC/MS). Similarly, perchlorate is a common "ion pair" agent in reversed-phase separation of peptides. Although many studies have suggested that anions effect in chromatography is due to the formation of ion pairs in the mobile phase between the anions and cationic analytes, there has been no independent verification that ion pairs are, in fact, responsible for these observations. In order to understand the mechanisms by which anionic additives influence retention in chromatography and ionization efficiency in electrospray mass spectrometry, we studied the formation of ion pairs between a number of prototypical basic drugs and various additives by measuring the effect of anionic additives on the electrophoretic mobility of the probe drugs under solvent conditions commonly used in chromatography. For the first time, ion pair formation between basic drugs and anionic additives under conditions commonly used in reversed-phase liquid chromatography has been confirmed independently with all anions (i.e. hexafluorophosphate, perchlorate, trifluoroacetate, and chloride) used in this study. We measured ion pair formation constants (Kip) for different anionic additives using capillary electrophoresis (CE) and obtained quantitative estimates for the extent of ion pairing in buffered acetonitrile-water. The data clearly indicate that different anionic additives ion pair with cationic drugs to quite different extents. The ion pair formation constants show a clear trend with the order being: PF6- > ClO4- > CF3COO- > Cl-. However, the extent of ion pairing is not large. At a typical RPLC mobile phase additive concentration of 20mM, the percentages of the analytes that are present as ion pairs are about 15%, 6%, and 3% for hexafluorophosphate, perchlorate, and trifluoroacetate, respectively. The fraction of the analytes present as a chloride pair is even smaller.

Affinity screening by packed capillary high performance liquid chromatography using molecular imprinted sorbents. II. Covalent imprinted polymers.

This study concentrates on the production of covalent molecular imprint polymers (MIPs) as highly selective sorbents for nortriptyline (NOR), a representative tricyclic antidepressant (TCA). The functionalized template contains a polymerizable 4-vinylphenyl carbamate moiety used to bind the template molecule to the polymer matrix. Polymerization with a cross-linker followed by hydrolytic cleavage of the labile carbamate functionality leaves an MIP with selective binding sites capable of binding template through hydrogen bonding interactions. Demonstrated chromatographically through a "selection index", these MIPs showed high selectivity for the template molecule (NOR) among a library of structurally similar compounds. The recognition was found to correlate with structural similarity to the template compound. A direct comparison between covalent and non-covalent molecular imprinting strategies reveals a great deal of improvement in the peak shape of the retained compound resulting from covalent imprinting (evidenced by peak asymmetry factors A.).

Capillary electrochromatography of hydrophobic amines on continuous beds.

The capillary electrochromatographic separation performance of hydrophobic amines and a related quaternary ammonium compound on continuous beds based on polymers of acrylamide has been studied. The chromatographic bed is polymerized in situ and the character of the polymers with regard to hydrophobicity and charge has been systematically changed by regulating its content of isopropyl and sulfonate ligands, respectively. The best performance was obtained for columns with a molar ratio of 1:80 for the sulfonate and isopropyl groups, and resulted in efficiencies up to 200000 plates per meter. The effects on retention, resolution and elution order by ionic strength, pH, and content of acetonitrile in the mobile phase have been investigated. The quaternary ammonium compound was always the least retained irrespective of pH. By increasing the pH, a reversal of the migration order between the tertiary and secondary amine was obtained. The results indicate a complex migration/retention mechanism where ion-exchange, adsorption and electrophoretic mobilities play a role. The concentration limit of detection could be lowered from 1.3 microg/mL to 50 pg/mL by using a high content of 2-propanol (96%) in the sample compared to dissolving the analytes in the mobile phase.