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.

[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.

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.

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.

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.

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.

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.

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.

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.

Flash alkylation for the identification of barbiturates in biological material.

The gas chromatographic identification of barbiturates isolated from biological material is not optimal, and due to their polar nature, it is difficult to obtain good resolution on most columns. To improve resolution, flash alkylation appeared to be a reasonable alternative. Methyl, ethyl, and butyl derivatives were prepared for various drugs found in the "acid-neutral" extract and the Kovats retention indices (Ir) were determined on SE-30, OV-17, and SP-2250. The Ir of the alkylated derivatives of the barbiturates are highly reproducible. One column and two alkylated derivatives can be used for the qualitative analysis of barbiturates in extracts of biological material, even when the Stas-Otto extract is used.

Isolation of drugs from post mortem specimens with the automated sample processor. Part I. Barbiturate derivatives.

Employing an automated sample processor, barbiturate positive tissue homogenates were analyzed to yield gas and liquid chromatographic data. Excellent quality extracts were produced that could be assayed by either GC/NPD or HPLC. Good precision and accuracy was found (CV 2-5%) with approximately 200 mg of tissue sample. The technique has been applied to authentic post mortem tissues and fluids.

Extraction of acidic drugs from water and plasma: study of recovery with five different solvents.

The recovery of 28 acidic drugs from water and plasma was investigated spectrophotometrically using five different solvents for extraction: hexane, diethyl ether, toluene, n-butyl chloride, and chloroform. Nine barbiturates, three sulfonamides, four diuretics, and twelve other acidic drugs were studied. As classes, the barbiturates, the sulfonamides, and the diuretics were optimally extracted with diethyl ether from both water and plasma, while considerable variation in recovery was noted among solvents for the other drugs. In general, recoveries from water were higher than those from plasma with most solvents, except for the sulfonamides for which recoveries were often better from plasma. Extraction recoveries for the individual drugs are tabulated and compared.

The Microspheres based Detoxification System (MDS). A new extracorporeal blood purification technology based on recirculated microspherical adsorbent particles.

Plasma sorption processes have so far been performed with filters and appropriate adsorption columns. In this paper, we introduce a newly developed plasma sorption system, which is based on the high adsorption capacity of microspheres in a recirculation system. The technology has been applied successfully in vitro to eliminate endotoxins, low density lipoproteins (LDL) and barbiturates from human plasma.

Application of Empore C-8 extraction disks for screening urine in systematic toxicological analysis.

Solid-phase extraction (SPE) by means of disposable columns has become a widely accepted technique for sample pretreatment in toxicology, both for directed analyses and for screening analyses. However, the sample capacity in SPE is usually limited to a few millilitres. Therefore, we have investigated to what extent these problems can be overcome by using Empore extraction disks, consisting of chemically modified C-8 reversed-phase silica, embedded in an inert polytetrafluoroethylene (PTFE) matrix. Human urine was selected as the matrix and dexetimide and mepyramine were initially used as test drugs because these drugs were available in tritiated form. Additional drugs investigated included codeine, hexobarbital, imipramine, methamphetamine, and nitrazepam. In these investigations, the sample capacity for untreated urine was at least 25 mL, and analyte quantities up to 250 micrograms could be retained by these filters. Washing with water/methanol mixtures was successful in removing substantial amounts of endogenous interferences, and methanol proved to be an acceptable eluent. Thus, these disks seem to have interesting potential for toxicological analysis in that sample concentration and cleanup can be achieved at the same time.

Rapid isolation with Sep-Pak C18 cartridges and wide-bore capillary gas chromatography of some barbiturates.

A simple and rapid method for isolation of nine barbiturates with Sep-Pak C18 cartridges from human urine, plasma and whole blood, is presented; the detection of drugs was made by wide-bore capillary gas chromatography (GC) with flame ionization. The drug-containing samples, after mixing with dilute acid solution, were directly applied to the cartridges and eluted with either chloroform/methanol (9:1) or acetonitrile. Separation of the nine drugs was satisfactory with use of an intermediately polar HP-17 capillary column. Recoveries of most compounds were excellent for both chloroform/methanol and acetonitrile as elution solvents. However, backgrounds were cleaner and evaporation time was much shorter for the chloroform/methanol system. The present isolation method with use of Sep-Pak C18 cartridges and wide-bore capillary GC seem very useful in the fields of forensic chemistry, clinical toxicology and clinical pharmacology.

Metabolism of difebarbamate in man.

The metabolism of 1,3-bis(3-butoxy-2-carbamoyloxypropyl)-5-ethyl-5-phenyl- (1H,3H,5H)-pyrimidine-2,4,6-trione (difebarbamate) in man was studied. Human volunteers received a single oral dose of 25 mg/kg difebarbamate. Urine was extracted with Amberlite XAD-2 resin and the extracts were separated by preparative HPLC after enzymatic hydrolysis. Four major metabolites were isolated and their structures were determined using NMR and mass spectrometry. The oxygen dealkylation led to the formation of two metabolites: 1-(3-butoxy-2-carbamoyloxypropyl)-3-(2-carbamoyloxy-3-hydrox ypropyl)-5-ethyl-5- phenyl-(1H, 3H, 5H)-pyrimidine-2,4,6,-trione and 1,3-bis(2-carbamoyloxy-3-hydroxypropyl)-5-ethyl-5-phenyl-(1H,3H,5H )- pyrimidine-2,4,6,-trione. The hydrolysis of the carbamoyloxy group with the oxygen dealkylation led to the formation of 1-(2-carbamoyloxy-3-hydroxypropyl)-3-(2,3-dihydroxypropyl)-5-ethyl - 5-phenyl-(1H,3H,5H)-pyrimidine-2,4,6,-tione, whereas the 4-hydroxylation of the benzene ring together with the oxygen dealkylation led to the formation of 1,3-bis(2-carbamoyloxy-3-hydroxypropyl)-5-ethyl-5-(4-hydroxyphenyl )-(1H,3H,5H)- pyrimidine-2,4,6,-trione. No traces of the parent drug were found.

[Characteristics of barbiturate adsorption on activated charcoal]. / Zakonomernosti adsorbtsii barbituratov na aktivirovannykh ugliakh.

Equilibration patterns of barbiturates adsorption on activated charcoals were studied using aqueous solutions of barbital and sodium barbital and commercially available charcoals BAU, AR-3, AR-B, AR-A, SKT-6A. The rate of barbiturates adsorption was described as an additive function of concentrations of amide, ionized and non-ionized imidol forms calculated considering the reactions of dissociation and hydrolysis. Relatively low equilibrium concentrations of barbiturates in the solid phase and simultaneous sorption of the barbital anion as well as of its non-ionized form were related to the considerable rate of the substance ionization due to both tautomeric transformation and dissociation of the acid formed. The procedure for calculation of equilibrium described assumes that the rate of barbital and sodium barbital adsorption on activated charcoals of various brands might be estimated at various pH values of a solution, based on dissociation and hydrolysis reactions, which is of importance under conditions of the barbiturates enterosorption.

[Use of temperature coefficients of Kovach indexes in the identification of barbiturates]. / Primenenie temperaturnykh koéffitsientov indeksov Kovacha dlia identifikatsii barbituratov.

The conditions of direct gas chromatographic assessment of barbiturates in reference solutions and cadaveric material extracts were analyzed with the aim of their unification. Along with the Kovach indexes, their temperature coefficients seem promising for the identification of barbiturates. Critical parameters of the evaporator temperature regimen were established, that may be used for barbiturate identification as well. The threshold values of gas chromatographic detection of phenobarbital, barbital, etaminal, hexenal, quietal, and hexobarbital were determined.

[The use of high-pressure liquid chromatography for identifying barbiturates in cadaveric material]. / Ispol'zovanie vysokoéffektivnoi khromatografii dlia identifikatsii barbituratov v trupnom materiale.

A method for chemical toxic analysis of barbiturates has been developed, making use of acetone as an extracting agent and high-pressure liquid chromatography for the identification of the isolated substances. Analysis with the use of this method is preceded by extraction and chromatographic purification. The co-extractive substances do not interfere with the identification of barbiturates.

[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.