Uncharged Compounds Research Articles

Use of dynamically coated capillaries for the determination of heroin, basic impurities and adulterants with capillary electrophoresis

Rapid, precise, accurate, and reproducible methodology using capillary electrophoresis (CE) with dynamically coated capillaries for the analysis of heroin and its basic impurities and adulterants is presented. Highly selective determination of the above solutes is obtained by analyzing the same sample preparation by two CE methods. For the determination of heroin, its basic impurities and basic adulterants, dynamic coating of the capillary surface is accomplished using a commercially available reagent kit with an added cyclodextrin ((CD) polycation coating followed by polyanion coating with dimethyl-β-cyclodextrin or hydroxypropyl-β-cyclodextrin). The addition of a cyclodextrin to the run buffer significantly improves the separation of these solutes. Neutral, acidic, and weakly basic adulterants which migrate near or after t 0 do not interfere with the more mobile basic solutes. The determination of neutral, acidic, and weakly basic adulterants in heroin is accomplished using a modification of the above commercially available reagent kit. After first coating with a polycation, a negative coating is obtained using a surfactant sodium dodecyl sulfate. Micellar electrokinetic chromatography (MEKC) with dynamically coated capillaries gives an excellent separation of the neutral, acidic, and weakly basic solutes, with considerably shorter run times compared to conventional MEKC. In addition for this system, most basic solutes in heroin have longer migration times than the uncharged and acidic compounds.

Chiral separation by capillary electrophoresis in nonaqueous medium.

In the last few years, nonaqueous background electrolyte (BGE) solutions have attracted increasing interest, not only in capillary zone electrophoresis (CZE) but also in other capillary electrophoretic techniques (CE) (). Nonaqueous capillary electrophoresis (NACE) exploits the vastly different physicochemical properties of organic solvents. With mere adjustment of BGE composition, parameters such as resolution, analysis time, and selectivity can be fine-tuned or even altered dramatically (, , , , , , , ). The solubility of the analytes and additives can be enhanced, and the possible adsorption of the analytes on the capillary wall can be reduced. Because solvation of the analytes differs considerably from that in aqueous solutions, a strong effect on the separation selectivity can be seen. In addition, low currents make the use of higher electric field strengths possible in separations. The interactions, which hardly take place in water, can be utilized in nonaqueous solvents permitting capillary electrophoretic separation of even uncharged compounds after the formation of complexes with ionic additives (,). Moreover, the solutes labile in aqueous solutions can be stable in nonaqueous media.

Enhancement of selectivity and resolution in the enantioseparation of uncharged compounds using mixtures of oppositely charged cyclodextrins in capillary electrophoresis.

The enantiomeric separation of some nonsteroidal anti-inflammatory drugs (NSAIDs) was investigated in capillary electrophoresis (CE) using dual systems with mixtures of charged cyclodextrin (CD) derivatives. A significant enhancement of selectivity and resolution could be achieved in the enantioseparation of these analytes in their uncharged form by the simultaneous addition of two oppositely charged CD derivatives to the background electrolyte. The combination of the single-isomer cationic CD, permethyl-6-monoamino-6-monodeoxy-beta-CD (PMMAbetaCD) and the single-isomer polyanionic CD, heptakis-6-sulfato-beta-cyclodextrin (HSbetaCD) in a pH 2.5 phosphoric acid-triethanolamine buffer, was designed and employed for the enantioseparation of profens. The improvement in selectivity and resolution can be attributed to the fact that the two CDs, which lead to independent and enantioselective complexation with the analyte enantiomers, have not only opposite effects on the electrophoretic mobility of these compounds but also opposite affinity patterns towards the enantiomers of these compounds. Binding constants for these enantiomers with each CD were determined using linear regression approach, in order to be able to predict the effect of the concentrations of the two CDs on enantiomeric selectivity and resolution in such dual systems.

In silico prediction of aqueous solubility, human plasma protein binding and volume of distribution of compounds from calculated pKa and AlogP98 values.

We have investigated whether three important ADME (absorption, distribution, metabolism, excretion) related properties (aqueous solubility, human plasma protein binding, and human volume of distribution at steady-state) can be predicted from chemical structure alone if only the predicted predominant ionisation state and lipophilicity (calculated logP [P = octanol-water partition coefficient]) are considered. A simple, fast method for the in silico prediction of aqueous solubility of predominantly uncharged compounds has been developed, while some potential is shown for the prediction of predominantly charged or zwitterionic compounds. Ten other known in silico prediction methods for aqueous solubility have also been evaluated. It has furthermore been demonstrated that the molecular weight (MW) profile of training sets for the development of aqueous solubility prediction methods can influence their predictive performance with regard to test sets of either matching or diverging profiles. The same property descriptors which have been found most relevant for the prediction of aqueous solubility have also proved useful for the prediction of human plasma protein binding and human volume of distribution at steady-state.

On-line automated sample preparation for liquid chromatography using parallel supported liquid membrane extraction and microporous membrane liquid–liquid extraction

An automated system was developed for analysis of non-polar and polar ionisable compounds at trace levels in natural water. Sample work-up was performed in a flow system using two parallel membrane extraction units. This system was connected on-line to a reversed-phase HPLC system for final determination. One of the membrane units was used for supported liquid membrane (SLM) extraction, which is suitable for ionisable or permanently charged compounds. The other unit was used for microporous membrane liquid–liquid extraction (MMLLE) suitable for uncharged compounds. The fungicide thiophanate methyl and its polar metabolites carbendazim and 2-aminobenzimidazole were used as model compounds. The whole system was controlled by means of four syringe pumps. While extracting one part of the sample using the SLM technique, the extract from the MMLLE extraction was analysed and vice versa. This gave a total analysis time of 63 min for each sample resulting in a sample throughput of 22 samples per 24 h.

Prediction of selectivity for enantiomeric separations of uncharged compounds by capillary electrophoresis involving dual cyclodextrin systems

The single-isomer polyanionic cyclodextrin (CD) derivative heptakis-6-sulfato-β-cyclodextrin (HSβCD) has been tested as chiral additive for the enantioseparation of non-steroidal anti-inflammatory drugs, such as fenoprofen, flurbiprofen, ibuprofen and ketoprofen, in capillary electrophoresis, using a pH 2.5 phosphoric acid–triethanolamine buffer in the reversed polarity mode. In most cases, the enantiomers of these acidic compounds, present in uncharged form at that pH, were only poorly resolved with HSβCD alone. However, the use of HSβCD in combination with the neutral CD derivative, heptakis-(2,3,6-tri- O-methyl)-β-cyclodextrin (TMβCD), which has a particularly high enantioselectivity towards these compounds, has led to complete enantioresolution in reasonably low migration times in most cases. Affinity constants for the enantiomers with the two cyclodextrins were determined, using linear regression in a two-step approach. Affinity constants with the charged HSβCD were first calculated in single systems while those with the neutral TMβCD were determined in dual systems. Selectivity for the enantiomeric separation of these compounds in dual CD systems could be predicted using recently developed mathematical models.

Separation of neutral compounds by microemulsion electrokinetic chromatography: fundamental studies on selectivity.

The selectivity of microemulsion electrokinetic chromatography (MEEKC) was studied utilizing some uncharged model compounds like aromatic amides, steroids, and esters of nicotinic acid. The cosurfactant of the microemulsion was found to be the most important factor affecting the selectivity, and alteration between 6.6% of 1-propanol, 1-butanol, tetrahydrofuran, and 2-ethoxyethanol caused several substantial changes in the migration order. In addition, the nature of the surfactant was found to significantly affect the selectivity. In this case, changes in order of migration was observed by replacement of half the content of sodium dodecyl sulfate (SDS) with either sodium dioctyl sulfosuccinate (SDOSS), 3-(N,N-dimethylmyristylammonio) propanesulfonate (MAPS), polyoxyethylene sorbitan monolaurate (Tween 21), and polyoxyethylene 23 lauryl ether (Brij 35). MEEKC was also accomplished with 3.3% of the anionic surfactant sodium cholate and with the cationic surfactant N-cetyl-N,N,N-trimethylammonium bromide (CTMA). Both provided substantial differences in selectivity as compared to the SDS-based systems. With SDS as surfacant, the concentration was varied within 1.0-4.5%. Minor selectivity changes were observed as the concentration of the surfacant was reduced, but the major effect was a reduction in the total migration time. The organic solvent of the microemulsion droplets was found only to have minor impact on the selectivity.

Application of linear solvation energy relationships to polymeric pseudostationary phases in micellar electrokinetic chromatography.

Polymerized sodium 11-acrylamidoundecanoate (poly(Na 11-AAU)) was used as a pseudostationary phase (PSP) for micellar electrokinetic chromatography to separate uncharged compounds. The polymer PSP showed signifcantly different solute migration behaviors from conventional micelles including sodium dodecyl sulfate and poly (sodium 10-undecylenate), giving high separation efficiencies (>200000 theoretical plates/m). Linear solvation energy relationships were used to evaluate and characterize the chemical interactions that influence the retention behavior in the poly (Na 11-AAU) micellar system. It was found that the solute volume and solute hydrogen bond basicity mainly influenced the retention. The characteristic feature of the poly (Na 11-AAU) micellar system is that the micelle has a significantly higher capacity for dipole-dipole and dipole-induced dipole interactions as well as a slightly higher capacity for electron pair interactions than the aqueous phase. Due to its unique selectivity, the poly(Na 11-AAU) micellar system would become an attractive new option for selectivity optimization on methods development.

Sample clean-up, enrichment and determination of <i>s</i>-triazine herbicides from southern ethiopian lakes supported using liquid membrane extraction

(Received November 22, 1999; revised May 8, 2000) The liquid membrane extraction method has been employed for selectively extracting trace quantities of s-triazine herbicides in environmental waters collected from lakes Awassa, Chamo and Abbya, located in close proximity to the agricultural farms in Southern Ethiopia. In liquid membrane extraction, the uncharged triazine compounds from the flowing donor solution diffuse through a porous poly(tetrafluoroethylene) (PTFE) membrane, containing a water immiscible organic solvent. The s-triazine molecules are then irreversibly trapped in the stagnant acidic acceptor phase since they become protonated. Using both di-n-hexylether and n-undecane membrane solvents, s-traizine herbicides were extracted and low detection limits of about 1 ng/L have been obtained by extraction of three liters of sample solution spiked with 0.1 g/L of each triazine. Residues of atrazine and terbutryn ranging in concentration from 0.02 to 0.05 g/L have been successfully determined.Bull. Chem. Soc. Ethiop. 2000, 14(1), 9-24Bulletin of the Chemical Society of Ethiopia. ISSN: 1011-3924

Determination of critical micelle concentration of SDS in formamide by capillary electrophoresis

Using an organic solvent and a surfactant, the uncharged, hydrophobicp-arylacetophenones have been separated by micellar electrokinetic chromatography. As an example, the effect of formamide containing sodium n-dodecyl sulfate (SDS) on the separation has been studied. Separations of all selected uncharged hydrophobic compounds were shown to be strongly dependent on the concentration of surfactant. It is suggested that if there is no micelle formed in the formamide solvent, the uncharged hydrophobic compound cannot be separated even though in the presence of surfactant. The electro-migration of solutes was considered zero. After micelle formation, the uncharged compounds were partitioned between the micellar and the organic solvent phases. Neutral molecule partition in and out of micelles depends on the hydrophobicity of each analyte. Based on this suggestion the critical micelle concentrations of SDS, di-(ethylhexyl) sodium sulfosuccinate (AOT) and taurodexycholic acid sodium salt (STDC) in formamide were determined.

Characterisation of π–π interactions which determine retention of aromatic compounds in reversed-phase liquid chromatography

This paper presents the influence of π–π interactions on the retention behaviour of aromate-rich and aromate-poor analytes separated on various chromatographic sorbents. The presence of π–π interactions between the analytes and a polymer based sorbent can be very useful for separating compounds with similar retention behaviour on silica based reversed-phase sorbents (where this type of interaction is absent). Various C 4 and C 18 modified silica sorbents (5 μm and 15 μm) are compared with some poly(styrene-divinyl benzene) (PS-DVB) sorbents. For this purpose five aromates and four non-aromates were selected. The retention times of the nine uncharged test compounds (hydrocortisone, hydrocortisone acetate, testosterone, testosterone propionate, nitrobenzene, anisole, toluene, valerophenone, cumene) increase with increasing hydrophobicity (log P). On silica based reversed-phase sorbents this relationship is the same for aromates and non-aromates. On PS-DVB sorbents this relationship is different: the aromatic compounds need more acetonitrile to elute from the column than the non-aromatic compounds with a similar hydrophobicity: at log P 2.7 the difference in acetonitrile concentration is approximately 18–20% for all tested polymer matrices which have no groups covalently bound to their PS-DVB moieties (Vydac™, Polymer Laboratories, Amersham Pharmacia Biotech). This difference is probably due to the presence of an π–π interaction between the sorbent and the analyte. The importance of the π–π interaction decreases when more acetonitrile is needed to elute an aromatic analyte from the column. The retention of these mono-aromates largely depends on hydrophobic interactions and π–π interactions. The higher the log P of the analyte (containing an aromatic group), the more important the hydrophobic interaction becomes. Although covalent coupling of functional groups to the PS-DVB sorbent (with a phenyl, isopropyl or ether linked via a –CH 2CHOHCH 2O– spacer) leads to a more hydrophilic sorbent, it does not lead to a decrease in π–π interaction since the surface still accessible for the compounds tested. A comparison of the retention behaviour of the aromatic test compounds with the non-aromatic test compounds on silica based reversed-phase sorbents with that on PS-DVB based sorbents shows the importance of determining π-π interactions.

Electrophoretic stereoisomer separation of aspartyl dipeptides and tripeptides in untreated fused-silica and polyacrylamide-coated capillaries using charged cyclodextrins

The separation of diastereomeric and enantiomeric aspartyl model dipeptides and tripeptides including the isomeric iso-Asp containing ß-peptides was studied in untreated fused-silica capillaries and polyacrylamide-coated capillaries by capillary electrophoresis using negatively charged cyclodextrins as chiral selectors. While most diastereomers could be separated in the acidic pH range two dipeptide diastereomers were also resolved at alkaline pH. The separation of the enantiomers using carboxymethyl-ß-cyclodextrin or sulfobutyl-ß-cyclodextrins could be performed either between pH 2 and 3 when the peptides are positively charged or at pH 5.25 where the peptides migrate as uncharged compounds with the electroosmotic flow. Exploiting the self-mobility of the negatively charged cyclodextrins and reversing the polarity of the applied voltage, a reversal of the migration order of the enantiomers was achieved in polyacrylamide-coated capillaries.

Enantioseparation of uncharged compounds by capillary electrophoresis using mixtures of anionic and neutral β-cyclodextrin derivatives

The use of the polyanionic sulfobutyl-β-cyclodextrin in combination with a neutral cyclodextrin derivative such as trimethyl-β-cyclodextrin (TMCD) or dimethyl-β-cyclodextrin (DMCD) in a pH 3 phosphoric acid–triethanolamine buffer has proved to be very suited to the enantioseparation of acidic compounds such as non-steroidal anti-inflammatory drugs. In this paper, the usefulness of such dual cyclodextrin systems was evaluated for the enantioseparation of weakly acidic and neutral compounds. Fairly good results with respect to chiral resolution were obtained at pH 3 for weak acids in these dual systems. However, no complete enantioseparation could be achieved under these conditions for the neutral drug chlormezanone. Another ionizable derivative, carboxymethyl-β-cyclodextrin, was then investigated together with TMCD or DMCD at pH 3 and 5. After optimisation of the concentration of the neutral cyclodextrin derivative, high enantioresolution could be obtained at pH 5 for chlormezanone as well as for all the other compounds tested.

Characterisation of retention in micellar high-performance liquid chromatography and in micellar electrokinetic chromatography using lipophilicity and polarity indices

The retention of various barbiturates, phenylurea and triazine herbicides was measured in high-performance liquid chromatography (HPLC) on a C 8 column in aqueous micellar and non-micellar mobile phases containing anionic and cationic surfactants. The retention was characterised using the lipophilicity and polarity indices suggested earlier. The migration of the compounds tested was measured in the same mobile phases using micellar electrokinetic chromatography (MEKC) in fused-silica capillaries. The effects of the surfactants on the separation of uncharged compounds were less in the non-micellar than in the micellar region, but were still significant. The lipophilicity and polarity indices can be applied here, too, to characterise and predict the retention behaviour as a function of the concentration of the organic modifier and of the surfactant in the mobile phase or in the working electrolyte. Unlike the situation in micellar HPLC, the effects of the concentration of the surfactant on the lipophilicity selectivity in an homologous series are negligible in MEKC, where the polarity parameters may be used to characterise the contribution of the electrophoretic migration to the retention. The behaviour observed may possibly be explained by the lipophilic part of the surfactant molecules and of the organic solvent in aqueous–organic mobile phases having similar effects on the separation. The present approach offers the possibility of determining the critical micellar concentration from the migration times obtained using MEKC.

Electronic absorption spectroscopy for the investigation of the solution binding of gold, palladium, mercury and silver salts to the lower rim substituted calix[4]arenes 25,26,27,28-(2- N,N-dimethyldithiocarbamoylethoxy)calix[4]arene and 25,26,27,28-(2-methylthioethoxy)calix[4]arene

The two uncharged compounds 25,26,27,28-(2- N,N-di methyldithiocarbamoylethoxy)calix[4]arene ( 1) and 25,26,27,28- (2-methylthioethoxy)calix[4]arene ( 2) are effective extractants for transferring Hg(II), Ag(I), Pd(II) and Au(III) from aqueous solution into chloroform. The electronic absorption spectra of 1 and 2 show additional bands at long wavelength upon complexation with AuCl 4 −, PdCl 4 2− and PdBr 4 2−, and analogous bands for Hg 2+ and Ag + with 1. For 1 these new bands are considered to be either of the charge transfer type or transitions within the C=S moiety. These new bands for the complexes with 2 are assigned to LMCT transitions of the S → M type. These spectral features are used to obtain information about the solution structures of the complexes that are formed between these metal ions and both 1 and 2.

Effect of molecular size and charge on biofilm sorption of organic matter

Packed-bed reactors were used to examine the effects of molecular size and charge on biofilm sorption of organic compounds selected for their similarity to the size and charge ranges of aquatic natural organic matter (NOM). In addition, the sorbate compounds were chosen for nonbiodegradability, which was verified by BOD tests. In experiments using uncharged NOM-surrogate compounds it was found that as sorbate molecular size increased, biofilm sorption decreased. Also, negatively charged NOM-surrogate compounds were found to be less well sorbed to biofilm than neutral molecules. Apparently biofilm sorption of small molecules is enhanced by diffusion of the sorbate into the porous biofilm matrix; whereas larger sorbate molecules probably accumulate at the biofilm-water interface because they are excluded from the biofilm pores. The observed decrease in biofilm sorption of anionic organic molecules compared to removal of neutral sorbates was thought to be the result of electrostatic repulsion between the negatively-charged sorbate and the negatively-charged biofilm particularly slowing diffusion of the sorbate molecules within the biofilm.

Cyclodextrin Solubilization of ETH-615, a Zwitterionic Drug

Therapeutic usefulness of many zwitterionic drugs is hampered by their very low aqueous solubility. The purpose of this work was to investigate the effects of cyclodextrins on the solubility of the zwitterionic drug ETH-615, the role that charge might play in the cyclodextrins complexation, and the influence of polymers and ion-pairing agents on the cyclodextrin solubilization. The effects of five different β-cyclodextrin derivatives were evaluated, i.e., the anionic β-cyclodextrin sulfobutyl ether sodium salt and carboxymethyl-β-cyclodextrin sodium salt, the uncharged 2-hydroxypropyl-β-cyclodextrin and randomly methylated β-cyclodextrin, and the cationic 2-hydroxy-3-trimethyl-ammoniopropyl-β-cyclodextrin. The uncharged cyclodextrins had much larger solubilizing effect on ETH-615 than the charged ones. However, due to the highly polar zwitterionic structure of ETH-615 the stability constants of its cyclodextrin complexes were several orders of magnitude smaller than those commonly observed for uncharged lipophilic compounds. Cyclodextrin solubilization of ETH-615 was enhanced by water-soluble polymers and ion-pairing agents.

Fritless Packed Columns for Capillary Electrochromatography: Separation of Uncharged Compounds on Hydrophobic Hydrogels

A novel column is described that does not require frits to keep packing material within a capillary. A continuous bed is prepared in situ in aqueous solution by radical copolymerization of N-isopropylacrylamide and 2-acrylamido-2-methylpropanesulfonic acid (the resultant gel is denoted poly(AMPS-co-IPAAm). N,N'-Methylenebisacrylamide is used for cross-linking. On the application of an electrical field, electroosmotic flow (EOF) is developed in the bed along the capillary, where fluid propulsion would be otherwise difficult to achieve. The resultant EOF transports neutral compounds through the column without forcing the gel out of the capillary. Examination of the fluid motion in the continuous bed using a video microscope system and an image processor shows a relatively flat flow profile of EOF. The bed functions as the stationary phase for reversed-phase capillary electrochromatography (CEC). This new approach is an alternative to packed capillary columns which have been used previously in CEC. A high efficiency is obtained for a steroid which is separated on a 4.0% total monomer concentration (T), 10.0% degree of cross-linking (C), and 10.0% mole fraction of AMPS in the total monomer (S), poly(AMPS-co-IPAAm) column. A mixture of polyaromatic hydrocarbons is separated on a 6.9% T, 5.8% C, and 5.5% S poly(AMPS-co-IPAAm) column. The capacity factor of benzo[a]pyrene increases from 0.63 to 1.91 as the acetonitrile content in a Tris-boric acid buffer is decreased from 45 to 30% (v/v). The run-to-run RSD of analyte migration time is less than 0.73%, and the day-to-day RSD is acceptable. Potential benefits of this approach are also mentioned.

Stereoselective interaction of uncharged esters at four muscarinic receptor subtypes

We investigated the binding and pharmacological properties of the esters of 3,3-dimethylbutan-1-ol (the carbon analogue of choline) with either diphenylglycolic acid, (R)-phenylcyclohexylglycolic acid, or (S)-phenylcyclohexylglycolic acid [BS-6181, (R)-BS-7826 and (S)-BS-7826, respectively] at muscarinic M1, M2, M3 (Hm3) and M4 receptors. The three uncharged compounds were muscarinic receptor antagonists, with pA2 or pKi values between 7.9 5.6. The achiral ester BS-6181 displayed highest affinity for M1, M3 (Hm3) and M4 receptors (pA2 or pKi = 7.2−7.6) and lower affinity for M2 receptors (pA2 or pKi = 6.7and6.8). The four muscarinic receptor subtypes were able to distinguish between the two enantiomers of the cyclohexyl derivative of BS-6181 [(R)- and (S)-BS-7826], with a preference for the (R)-isomer (up to 79-fold). Interestingly, the (S)-enantiomer of BS-7826, being the distomer, was found to be M4 selective (pKi/M4= 6.9; pA2 or pKi/M1-M3 (Hm3) = 5.6−6.2). These results indicate that uncharged compounds may (stereo)selectively bind to muscarinic receptors via hydrophobic interactions. Thus, an ionic bond between muscarinic ligands and an anionic site of the receptor is not absolutely necessary for recognition of muscarinic receptors.

Transport properties of the multidrug resistance-associated protein (MRP) in human tumour cells

In this paper we demonstrate that the expression of the multidrug resistance-associated protein (MRP) in a variety of intact human tumour cells results in the ATP-dependent, mutually exclusive extrusion of both the acetoxymethyl ester and the free anion forms of the fluorescent dye calcein, as well as that of a fluorescent pyrenemaleimide-glutathione conjugate. The MRP-dependent transport of all these three model compounds closely correlates with the expression level of MRP and is cross-inhibited by hydrophobic anticancer drugs, by reversing agents for MDR1, and also by compounds not influencing MDR1, such as hydrophobic anions, alkylating agents, and inhibitors of organic anion transporters. Cellular glutathione depletion affects neither the MRP-dependent extrusion of calcein AM or free calcein, nor its modulation by most hydrophobic or anionic compounds, although eliminating the cross-inhibitory effect of glutathione conjugates. These results suggest that the outward pumping of both hydrophobic uncharged and water-soluble anionic compounds, including glutathione conjugates, is an inherent property of MRP, and offer sensitive methods for the functional diagnostics of this transport protein as well as for the rapid screening of drug-resistance modulating agents.