Usual, unusual and unbelievable retention behavior in achiral supercritical fluid chromatography: Review and discussion.

Retention rules are well known in liquid chromatography. For the mobile phase composition, retention decreases when adding organic solvent to water for reversed-phase or increasing water proportion for hydrophilic interaction liquid chromatography, and a decrease in temperature usually increases retention. For supercritical fluids, the fluid density, which is related to temperature and column back-pressure, is significant for neat CO2 and with low percentages of organic modifiers, i.e. with compressible mobile phases. The increase in the modifier percentage reduces the fluid compressibility, leading to retention behaviors close to those observed with liquid mobile phases, for instance for temperature changes. Moreover, adsorption of carbon dioxide or modifiers modify the solutes/stationary phase interactions, further complicating the understanding of the observed retention changes, either with low amount of modifier, or with specific modifiers. Besides, the polar and nonpolar stationary phases (SPs) do not always behave identically, depending on physico-chemical properties. Silica, amino or ethyl-pyridine polar phases display mostly identical behavior for classical differences of compounds of different polarity, but can provide different retention order for more subtle differences, such as the position of polar groups. Moreover, the nature of the silica, inorganic or hybrid, or the additional charges onto the silica surface can also lead to different results. Even if the C18-bonded phases are not as popular as polar SPs, the non-polar SPs provide very high separation performances for suited compounds, i.e. for non-polar compounds, which are perfectly solubilized by supercritical fluids. Recently, unusual retention behaviors were observed with some specific C18-bonded phases, which display polar interactions in addition to dispersion interactions. Whatever the SPs used, supercritical fluids appear to favor specific effects that are not observed with liquid mobile phases that are more uniform in terms of physico-chemical properties. The objective of this paper is to describe different separation behaviors observed in SFC, to improve the general understanding of the specificities of the association of supercritical fluids and varied SPs.

Determination of Barbiturates in Biological Specimens by Flat Membrane-Based Liquid-Phase Microextraction and Liquid Chromatography-Mass Spectrometry.

The wide abuse of barbiturates has aroused extensive public concern. Therefore, the determination of such drugs is becoming essential in therapeutic drug monitoring and forensic science. Herein, a simple, efficient, and inexpensive sample preparation technique, namely, flat membrane-based liquid-phase microextraction (FM-LPME) followed by liquid chromatography-mass spectrometry (LC-MS), was used to determine barbiturates in biological specimens. Factors that may influence the efficiency including organic extraction solvent, pH, and composition of donor and acceptor phases, extraction time, and salt addition to the sample (donor phase) were investigated and optimized. Under the optimized extraction conditions, the linear ranges of the proposed FM-LPME/LC-MS method (with correlation coefficient factors ≥ 0.99) were 7.5-750 ng mL-1 for whole blood, 5.0-500 ng mL-1 for urine, and 25-2500 ng g-1 for liver. Repeatability between 5.0 and 13.7% was obtained and the limit of detection (LOD) values ranged from 1.5 to 3.1 ng mL-1, from 0.6 to 3.6 ng mL-1, and from 5.2 to 10.0 ng g-1 for whole blood, urine, and liver samples, respectively. This method was successfully applied for the analysis of barbiturates in blood and liver from rats treated with these drugs, and excellent sample cleanup was achieved.

Simultaneous analysis method for GHB, ketamine, norketamine, phenobarbital, thiopental, zolpidem, zopiclone and phenytoin in urine, using C18 poroshell column.

Date-rape drugs have the potential to be used in drug-facilitated sexual assault, organ theft and property theft. Since they are colorless, tasteless and odorless, victims can drink without noticing, when added to the beverages. These drugs must be detected in time, before they are cleared up from the biofluids. A simultaneous extraction and determination method in urine for GHB, ketamine, norketamine, phenobarbital, thiopental, zolpidem, zopiclone and phenytoin (an anticonvulsant and antiepileptic drug) with LC-MS/MS was developed for the first time with analytically acceptable recoveries and validated. A 4 steps liquid-liquid extraction was applied, using only 1.000mL urine. A new age commercial C18 poroshell column with high column efficiency was used for LC-MS/MS analysis with a fast isocratic elution as 5.5min. A new MS transition were introduced for barbital. 222.7>179.8 with the effect of acetonitrile. Recoveries (%) were between 80.98-99.27 for all analytes, except for GHB which was 71.46. LOD and LOQ values were found in the ranges of 0.59-49.50 and 9.20-80.80ngmL(-1) for all the analytes (except for GHB:3.44 and 6.00µgmL(-1)). HorRat values calculated (between 0.25-1.21), revealed that the inter-day and interanalist precisions (RSD%≤14.54%) acceptable. The simultaneous extraction and determination of these 8 analytes in urine is challenging because of the difficulty arising from the different chemical properties of some. Since the procedure can extract drugs from a wide range of polarity and pKa, it increases the window of detection. Group representatives from barbiturates, z-drugs, ketamine, phenytoin and polar acidic drugs (GHB) have been successfully analyzed in this study with low detection limits. The method is important from the point of determining the combined or single use of these drugs in crimes and finding out the reasons of deaths related to these drugs.

Overcoming bioanalytical challenges associated with the separation and quantitation of GSK1278863, a HIF-prolyl hydroxylase inhibitor, and its 14 stereoisomeric metabolites.

GSK1278863 is an investigative drug under investigation for treatment of anemia associated with chronic kidney disease. Its metabolism is primarily metabolized by P450 enzymes where 19 unique metabolic species have been identified. These include multiple products of mono-, di-, and tri-oxygenation. Initially, two separate and complex ultra high performance liquid chromatography (UHPLC) reverse phase methodologies were developed, validated and applied to measure parent and various predominant and circulating metabolites in numerous clinical studies. However, 5 of the 6 oxidative metabolites may exist in different stereoisomeric forms, resulting in 14 separate species; therefore a chiral methodology was required to determine which stereoisomeric forms circulated in human. A variety of conventional approaches were explored, where in the end a supercritical fluid chromatography (SFC) method was required to separate this complex mixture of 14 stereoisomeric metabolites; data from these experiments provided important information on which species circulate in human. The details of these methodologies will be discussed herein.

Molecularly imprinted polymer with high-fidelity binding sites for the selective extraction of barbiturates from human urine.

In this paper we describe the synthesis of a molecularly imprinted polymer (MIP) by precipitation polymerisation, with barbital as the template molecule, and the application of the barbital MIP as a molecularly selective sorbent in the solid-phase extraction (SPE) of barbiturates from human urine samples. The MIP was synthesised by precipitation polymerisation using 2,6-bis-acrylamidopyridine as the functional monomer and DVB-80 as the cross-linking agent. The spherical MIP particles produced were 4.2 ± 0.4 µm in diameter; a non-imprinted control polymer (NIP) in bead form was 4.8 ± 0.4 µm (mean±standard deviation) in diameter. The particles were packed into a solid-phase extraction cartridge and employed as a novel sorbent in a molecularly imprinted solid-phase extraction (MISPE) protocol. The MIP showed high selectivity for the template molecule, barbital, a feature which can be ascribed to the high-fidelity binding sites present in the MIP which arose from the use of 2,6-bis-acrylamidopyridine as the functional monomer. However, the MIP also displayed useful cross-selectivity for other barbiturates besides barbital. For real samples, the MIP was applied for the extraction of four barbiturates from human urine. However, due to the high urea concentration in this sample which interfere the proper interaction of barbiturates onto the MIP, a tandem system using a commercially available sorbent was developed.

A high-throughput multivariate optimization for the simultaneous enantioseparation and detection of barbiturates in micellar electrokinetic chromatography-mass spectrometry.

The R- and S-configurations of barbiturates display differences in potency and biological activity. In this study, multivariate micellar electrokinetic chromatography-mass spectrometry (MEKC-MS) approach for the simultaneous analysis of three chiral barbiturates (mephobarbital, pentobarbital, and secobarbital) is developed using a polymeric chiral surfactant. After screening 11 amino acid polymeric surfactants, polysodium N-undecenoxycarbonyl-L-isoleucinate (poly-L-SUCIL) was found to be the best chiral selector. The multivariate central composite design (CCD) is used to optimize the chiral resolution, decrease the total analysis time, and improve the ESI-MS signal-to-noise (S/N) ratio. In the preliminary set of experiments, the ranges of the factors investigated in the multivariate approaches are determined. Next, the CCD design is conducted to determine the best overall chiral resolution with shortest possible run times. This optimization resulted in simultaneous enantioseparation in less than 32 min of all three barbiturates with 3-5 fold higher sensitivity by MS compared to UV detection. The adequacy of the multivariate model is validated by three replicate experimental runs at the predicted optimum conditions. The predicted results of MEKC-MS are found to be in good agreement with the experimental data for migration times, resolution, and S/N ratio. The optimized method provided good results in terms of linearity and recovery values of chiral barbiturates spiked in human serum after solid-phase extraction procedure.

[The use of enzymatic hydrolysis for isolation of barbituric acid derivatives from blood (as exemplified by phenobarbital and barbamyl)].

Modern isolation techniques by direct extraction with organic solvents or after protein precipitation by various sedimenting or salting-out agents are characterized by low efficiency and do not permit to liberate derivatives of barbituric acid from their complexes with blood proteins. The use of enzymatic hydrolysis makes it possible to break bonds between barbiturates and protein and thereby improve the efficiency of isolation. We performed enzymatic hydrolysis of the model phenobarbital-blood and barbamyl-blood complexes with the use of trypsin, pepsin, chymotrypsin, and papain. The degree of phenobarbital extraction with trypsin and barbamyl was estimated at 62.1 +/- 1.2% and 75.1 +/- 1.6% respectively; in other words, it was 32.7 +/- 1.0% and 51.1 +/- 1.0% higher than that achieved by traditional methods. Certain validation characteristics of the new method are presented.

A convenient method for the preparation of barbituric and thiobarbituric acid transition metal complexes.

A convenient method for the preparation of barbiturate transition metal complexes: (i) Cr(3+), Mn(2+), Fe(3+), Zn(2+) and Cd(2+) ions with barbituric acid (H(2)L) and (ii) Cr(3+) and Mo(5+) with 2-thiobarbituric acid (H(2)L') was reported and this has enabled seven complexes to be formulated as: [Cr(HL)(2)(OH)(H(2)O)].H(2)O, [Mn(HL)(2)(H(2)O)(2)], [Fe(2)(L)(OH)(3)(H(2)O)(4)].2H(2)O, [Zn(HL)(2)], [Cd(HL)(2)], [Cr(HL')(OH)(2)(H(2)O)].H(2)O and [Mo(HL')(2)]Cl. These new barbiturate complexes were synthesized and characterized by elemental analysis, molar conductivity, magnetic measurements, spectral methods (mid infrared, (1)H NMR, mass, X-ray powder diffraction and UV/vis spectra) and simultaneous thermal analysis (TG and DTG) techniques. The molar conductance measurements proved that, all complexes of barbituric and 2-thiobarbituric acids are non-electrolytes except for [Mo(HL')(2)]Cl. The electronic spectra and magnetic susceptibility measurements were used to infer the structures. The IR spectra of the ligands and their complexes are used to identify the mode of coordination. Kinetic and thermodynamic parameters such as: E, DeltaH, DeltaS and DeltaG are estimated according to the DTG curves. The two ligands and their complexes have been studied for their possible biological antifungal activity.

Chiral recognition ability and solvent versatility of bonded amylose tris(3,5-dimethylphenylcarbamate) chiral stationary phase: enantioselective liquid chromatographic resolution of racemic N-alkylated barbiturates and thalidomide analogs.

The chiral recognition ability and solvent versatility of a new chiral stationary phase containing amylose 3,5-dimethylphenylcarabamate immobilized onto silica gel (CHIRALPAK IA) is investigated. Thus, the direct enantioselective separation of a set of racemic N-alkylated barbiturates and 3-alkylated analogs of thalidomide was conducted using different nonstandard solvents as eluent and diluent, respectively in high-performance liquid chromatography (HPLC). The separation, resolution, and elution order of the investigated compounds were compared on both immobilized and coated amylose tris(3,5-dimethylphenylcarbamate) chiral stationary phases (Chiralpak IA and Chiralpak AD, respectively) using a mixture of n-hexane/2-propanol (90:10 v/v) as mobile phase with different flow-rates and fixed UV detection at 254 nm. The effect of the immobilization of the amylose tris(3,5-dimethylphenylcarbamate) chiral stationary phase on silica (Chiralpak IA) on the chiral recognition ability was noted as the bonded phase (Chiralpak IA) was superior in chiral recognition and possesses a higher resolving power in most of the reported cases than the coated one (Chiralpak AD). A few racemates were not or poorly resolved on the immobilized Chiralpak IA or the coated Chiralpak AD when using standard solvents were most efficiently resolved on the immobilized Chiralpak IA upon using nonstandard solvents. Furthermore, the immobilized phase withstands the nonstandard (prohibited) HPLC solvents such as dichloromethane, ethyl acetate, tetrahydrofuran, methyl-tert-butyl ether, and others when used as eluents or as a dissolving agent for the analyte itself. The direct analysis of a real sample extracted from plasma using DCM on Chiralpak IA is also shown.

True and false reversal of the elution order of barbiturates on a bonded cellulose-based chiral stationary phase.

A set of racemic N-alkylated barbiturates were investigated for their direct enantioselective HPLC separation on a new chiral stationary phase (CSP) containing cellulose tris(3,5-dimethylphenylcarabamate) immobilized onto silica gel (Chiralpak IB) and its coated version (Chiralcel OD). They were online detected by UV and optical rotation detectors to trace the elution order of their enantiomers. Surprisingly, examples of false and true reversal of the elution order of enantiomers of barbiturates were observed and reported.

Investigation of the novel mixed-mode stationary phase for capillary electrochromatography. I. Preparation and characterization of sulfonated naphthalimido-modified silyl silica gel.

A novel packing material, 3-(4-sulfo-1,8-naphthalimido)propyl-modified silyl silica gel (SNAIP), was prepared for the use as a stationary phase of capillary electrochromatography (CEC). The sulfonic acid groups on SNAIP stationary phase contributed to the generation of electroosmotic flow (EOF) at low pH and served as a strong cation-exchanger. In CEC with SNAIP, a mixed-mode separation was predicted, comprising hydrophobic and electrostatic interactions as well as electrophoretic migration process. In order to understand the retention mechanism on SNAIP, effects of buffer pH, concentration, and mobile phase composition on EOF mobility and the retention factors of barbiturates and benzodiazepines were systematically investigated. Moreover, the retention behavior of barbiturates on SNAIP was investigated and compared with those on octadecyl silica (ODS), phenyl-bonded silica, and 3-(1,8-naphthalimido)propyl-modified silyl silica gel to confirm the presence of pi-pi interaction on its retention mechanism. It was observed that a column efficiency was more than 85,000 N/m for retained compounds and the relative standard deviations for the retention times of EOF marker, thiourea, and five barbiturates were below 2.5% (n = 4). Under an applied voltage of 20 kV and a mobile phase consisted of 5 mM phosphate (pH 3.8) and 40% methanol, the baseline separation of five barbiturates was achieved within 3 min.

Rapid separation of barbiturates and benzodiazepines by capillary electrochromatography with 3-(1,8-naphthalimido)propyl-modified silyl silica gel.

A capillary electrochromatographic (CEC) method was applied to the simultaneous separation of barbiturates (barbital, phenobarbital, secobarbital and thiopental) and benzodiazepines (nitrazepam, diazepam and triazolam). The separation was performed in a 75 microm i.d. capillary, packed with 3-(1,8-naphthalimido)propyl-modified silyl silica gel (NAIP), studying the effects of buffer pH and mobile phase composition. Using an applied voltage of 20 kV and the short-end injection method (9 cm capillary effective length), the mobile phase of 1.0 mM citrate buffer (pH 5.0) containing 45% methanol provided the baseline separation of seven toxic drugs in less than 9 min. In CEC with NAIP, the benzodiazepines were separated by the combination of hydrophobic and pi-pi interactions, whereas the separation of the barbiturates was based on the hydrophobic interaction.

Capillary electrochromatographic analysis of barbiturates in serum.

A capillary electrochromatographic method was developed for the separation of barbiturates. The separation was optimized in a 75 microm ID capillary, packed with 3-(1,8-naphthalimido)propyl-modified silyl silica gel (NAIP), studying the effect of buffer pH, buffer concentration, and mobile phase composition. Using an applied voltage of 20 kV and the short-end injection method (9 cm capillary effective length), the mobile phase of 1.0 mM citrate buffer (pH 5.0) containing 40% methanol provided the baseline separation of barbital, phenobarbital, secobarbital, and thiopental (internal standard) in less than 4.5 min. The method was successfully applied to the analysis of barbiturates in human serum. Under the optimal conditions, good repeatability and linearity were obtained in the range of 2.90-43.29 microg/mL for barbital, phenobarbital, and secobarbital.

On-line coupling of packed capillary electrochromatography with coordination ion spray-mass spectrometry for the separation of enantiomers.

Pressure-supported packed capillary electrochromatography (CEC) and packed capillary high-performance liquid chromatography (pHPLC) have been coupled on-line to electrospray ionization-mass spectrometry (ESI-MS) and coordination ion spray-mass spectrometry (CIS-MS). Separation of enantiomers of barbiturates and chlorinated alkyl phenoxypropanoates were performed on a permethylated beta-cyclodextrin stationary phase by pressure-supported CEC. For on-line detection with ESI- and CIS-MS, a modified sheath-liquid interface was used. CIS-MS is a universal, novel ionization technique which improves the selectivity as well as the sensitivity. Charged complexes were formed through the addition of central complexing ions such as silver(I), cobalt(II), copper(II), and lithium(I) to the sheath flow. Advantages of CIS-MS detection compared to the ESI-MS mode are discussed. In the CIS-MS mode, increased sensitivity and high selectivity was attained through different possibilities of complexation. The superiority of pressure-supported CEC compared to pHPLC in the hyphenation with CIS-MS is demonstrated.

Steady-state concentration distribution of artificial receptor and target analyte in plasticized PVC membrane between solutions differing in target analyte concentration.

A barbiturate receptor has proven effective in improving selectivity in solid-phase microextraction of barbiturates when doped into plasticized poly(vinyl chloride) (PVC). It would be beneficial to have selective extractions for any given organic species; however, the receptors do not exist. They will be found by screening of libraries of potential receptors; thus, a screening method is needed. It is important to screen the receptors in the medium in which they will work: plasticized PVC. We hypothesize that we can make receptors move in solution in response to the presence of a solute to which they bind. This work examines whether we can establish a sufficient free energy gradient for a good receptor to move to a predetermined place in space. A difference in the barbiturate solute (substrate or guest) concentration in solutions bathing the two sides of a plasticized PVC membrane containing the barbiturate receptor (or host) creates a spatial concentration gradient of the substrate in the membrane. This causes the receptor's chemical potential to vary across the membrane. Upon binding to the analyte, the receptor undergoes a local activity drop, which decreases its free energy. This process produces a flux of receptor to accumulate at place where there is a high substrate concentration. A concentration gradient of substrate can be maintained across the membrane at steady state. In membranes for which the formation of the complex is favored, the receptor responds to the gradient of substrate. In membranes for which binding is not favored, a gradient of substrate is completely ignored by the receptor. Thus, the receptor does respond to the gradient but only if the concentration gradient of guest corresponds to a chemical potential gradient.

[Purification of extracts from biological material using aluminum oxide]. / Ochistka vytiazhek iz biologicheskogo materiala s ispol'zovaniem okisi aliuminiia.

An effective, rapid, and safe operation is suggested for purification of extracts from biological material from co-extractive substances during testing for barbiturates, benzophenones, and nichlofolane. The purification is carried out on a column packed with aluminum oxide.

Enantiomer separation by capillary electrochromatography on a cyclodextrin-modified monolith.

A chiral monolithic stationary phase was prepared by packing a capillary with bare porous silica and sintering the silica bed at high temperature. The resulting silica monolith was polymer-coated with Chirasil-Dex, a permethylated beta-cyclodextrin covalently linked via an octamethylene spacer to dimethylpolysiloxane. Subsequently, Chirasil-Dex was thermally immobilized on the silica support and a chiral monolith of very high stability (30 kV, more than 400 bar pressure) was obtained. The enantiomer separation of various chiral compounds by monolithic (rod) capillary electrochromatography (rod-CEC) was feasible. This method was compared with capillary liquid chromatography (LC) in a single-column mode using unified equipment. About two to three times higher efficiency was found in the rod-CEC mode as compared to rod-LC. The influence of pressure-driven flow support on efficiency, resolution, elution time and baseline stability was investigated. The amount and nature of organic modifier strongly influences efficiency and resolution.

Preparation of barbiturate optical isomers and their effects on GABA(A) receptors.

BACKGROUND: Barbiturate anesthetics are optically active and usually exist in two mirror-image enantiomeric forms. Their stereoselective effects in mammals are well known, but remarkably few data are available concerning their effects on anesthetic targets in vitro. This is in part because of the lack of availability of pure barbiturate enantiomers. Such in vitro data could be used to test the relevance of putative molecular targets. METHODS: A high-performance liquid chromatography technique using a permethylated beta-cyclodextrin column was used to separate the optical isomers of three barbiturates in preparative quantities. The effects of the isomers on GABA-induced currents in stably transfected mouse fibroblast cells were investigated using the whole-cell patch-clamp technique. RESULTS: Highly purified optical isomers of hexobarbital, pentobarbital, and thiopental were prepared, and their effects were studied on a gamma-aminobutyric acid type A receptor of defined subunit composition. For each of the three barbiturates, both enantiomers potentiated gamma-aminobutyric acid-induced currents at pharmacologically relevant concentrations, with the S-enantiomer being more potent than the R-enantiomer by a factor of between 1.7 and 3.5. The degree of stereoselectivity did not vary greatly with anesthetic concentration. CONCLUSIONS: The rank order and degree of stereoselectivity that we have observed for the enantiomers of hexobarbital, pentobarbital, and thiopental acting on the gamma-aminobutyric acid type A receptor are entirely consistent with this receptor playing a central role in the anesthetic actions of barbiturates.

Development of an assay for the extraction and quantification of nine 5-n-alkyl-5-ethyl barbituric acids in various rat tissues.

Methods were developed to quantify a series of nine homologous 5-n-alkyl-5-ethyl barbituric acids in 15 rat tissues. Tissue homogenates were spiked with one of four multicomponent mixtures (methyl to n-propyl, n-propyl to n-pentyl, n-pentyl to n-heptyl and n-pentyl to n-nonyl). Liquid-liquid extraction was used to extract the homologues from the rat tissues. Reverse phase HPLC with UV detection at 214 nm was used to separate and quantify the individual barbiturates. The limit of detection for each respective homologue was 1 microg x g(-1) except skin and bone (2 microg x g(-1)). The methodology developed reduced a potential 135 individual assays to a more manageable 16.