Determination Of Barbiturates Research Articles

Ultrasound and surfactant-assisted dispersive liquid-liquid microextraction prior to poly(ethylene oxide)-mediated stacking in CE for highly sensitive determination of barbiturates in human fluids.

This study developed a facile, highly sensitive technique for extracting and quantifying barbiturates in serum samples. This method combined ultrasound and surfactant-assisted dispersive liquid-liquid microextraction with poly(ethylene oxide)-mediated stacking in capillary electrophoresis. Factors influencing the extraction and stacking performance, such as the type and volume of extraction solvents, the type and concentration of surfactant, extraction time, salt additives, sample matrix, solution pH, and composition of the background electrolyte, were carefully studied and optimized to achieve the optimal detection sensitivity. Under the optimized extraction (injecting 140μL C2 H4 Cl2 into 1mL of sample with pH 4 (5mM sodium phosphate containing 0.05mM Tween 20 and sonication for 1min) and separation conditions (150mM tris(hydroxymethyl)aminomethane-borate with pH 8.5 containing 0.5% (m/v) poly(ethylene oxide)), the limits of detection (signal-to-noise ratio=3) of five barbiturates ranged from 0.20 to 0.33ng/mL, and the calculated sensitivity improvement ranged from 868- to 1700-fold. The experimental results revealed excellent linearity (R2 >0.99), with relative standard deviations of 2.1%-3.4% for the migration time and 4.3%-5.7% for the peak area. The recoveries of the spiked serum samples were 97.1% -110.3%. Our proposed approach offers a rapid and practical method for quantifying barbiturates in biological fluids.

Determination of Barbiturates by Femtosecond Ionization Mass Spectrometry.

Barbiturates are highly susceptible to dissociation in mass spectrometry (MS) because of their long side chains combined with a nonaromatic ring consisting of several carbonyl and amine groups. As a result, they exhibit extensive α-cleavage and subsequent rearrangement, making the identification of these compounds difficult. Although a library of electron ionization MS (EIMS) is available, most barbiturates have very similar fragment patterns. Accordingly, it would be desirable to develop a technique for soft ionization, providing a molecular ion and large fragment ions as well. In this study, a molecular ion was clearly observed, in addition to large fragment ions, for a variety of barbiturates based on multiphoton ionization MS (MPIMS) using a tunable ultraviolet femtosecond laser as the ionization source (fs-LIMS). This favorable result was achieved when the optimal laser wavelength for minimizing the excess energy remaining in the ionic state was used. An examination of the photofragmentation pathways suggested that an H atom in the side chain was abstracted by an oxygen atom in the carbonyl group in the ring structure thus initiating fragmentation and subsequent rearrangement. Barbiturates that are substituted with alkyl groups (amobarbital and pentobarbital) had narrower spectral regions for optimal ionization than the other barbiturates with alkyl and alkenyl groups (butalbital and secobarbital) and more with alkyl and phenyl groups (phenobarbital). All of the barbiturates studied provided unique mass spectral patterns in fs-LIMS, which was useful for the reliable identification of these compounds in practical trace analysis.

Simultaneous determination of barbiturates, phenytoin and topiramate in hair by LC-MS/MS and application to real samples

IntroductionHair analysis is useful for monitoring exposure to drugs such as antiepileptics owing to long-term therapy and a high possibility of abuse of drugs, which could be fatal. An effective and rapid analytical method for the simultaneous determination of six barbiturates, as well as phenytoin and topiramate in hair samples was developed and validated by liquid-chromatography-tandem mass spectrometry (LC-MS/MS). MethodsThree different extraction methods were investigated for the development of an appropriate analytical method. Hair was finely cut and then extracted with methanol, methanol containing 1% hydrochloric acid, and liquid-liquid extraction in acidic condition. ResultsThere was no significant difference in the matrix effects among these three methods. Recoveries clearly declined in the extraction involving both acidic methanol extraction and a LLE in acidic condition. Methanol incubation was chosen as the appropriate extraction method with acceptable matrix effects and recoveries. After validating the methanol incubation, the limit of detection (LOD) and limit of quantification (LOQ) were determined as 0.01 and 0.02 ng/mg for topiramate and 0.25–0.5 and 0.5–1 ng/mg for the others in hair. The LC-MS/MS method was precise and accurate with a dynamic linear range of 0.02–5 ng/mg for topiramate and 0.5 or 1–50 ng/mg for others. This method was applied to authentic hair samples of two drug users. The hair concentrations of phenobarbital were 0.2–17.1 ng/mg in segmental analysis in one female subject and those of topiramate were 0.19–0.93 ng/mg in another female subject. DiscussionThe quantitative method was developed to determine 8 antiepileptics using LC-MS/MS. This method performed hair segmental analysis to provide useful informative and chronological data in both of the forensic and clinical toxicology fields.

Determination of barbiturates in hair samples by using a validated UHPLC-HRMS method: application in investigation of drug-facilitated sexual assault

In recent years, benzodiazepines and benzodiazepine-like drugs are the most common substances associated with drug-facilitated sexual assaults (DFSA); however, barbiturates are also detected occasionally. Segmental hair analysis provides useful information on the historic pattern of drug use, enabling differentiation between single exposure in DFSA cases and chronic use. However, sensitive and specific methods for barbiturate analysis in hair samples are needed. Herein, we present an ultra-high-performance liquid chromatography coupled with high-resolution mass spectrometry (UHPLC-HRMS) method for qualitative and quantitative determination of seven barbiturates in hair samples. Firstly, a hair strand was decontaminated and then freeze-milled in liquid nitrogen. Next, 50 mg of powdered hair was extracted with methanol in an ultrasonic bath for 10 min in the presence of 10 ng phenobarbital-d5. The supernatant was dried under nitrogen gas, and the pellet was dissolved in 100 µL mobile phase. Afterwards, 10 µL of the suspension was injected into the UHPLC-HRMS system. The present method involved two UHPLC conditions for determination of barbiturates (I) and identification of the structural isomers amobarbital and pentobarbital (II). This method showed satisfactory linearity in a range of 0.02–20.00 ng/mg for UHPLC conditions I and II, both with a high determination coefficient (0.9991–0.9999). The selectivity, intra- and interday precision, accuracy and matrix effect of the method were acceptable. Next, the validated method was applied to investigate an authentic DFSA case. Hair samples (black, approximate 25 cm long) were collected 3 months after the assault, and the proximal segments (0–5 cm from the root; each segment was 1 cm long) were analyzed. Amobarbital was detected at a concentration of < LOQ (limit of quantification) and 0.09 ng/mg in the second and third 1 cm hair segment but not in the other segments. Thus, our method was successful in determining barbiturate concentration in human hair after a single-dose exposure, showing its potential for application in the investigation of DFSA cases.

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 Determination of Phenobarbital, Pentobarbital, Amobarbital and Secobarbital in Raw Milk via Liquid Chromatography with Electron Spray Ionization Tandem Mass Spectrometry.

A rapid, sensitive and specific liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the simultaneous determination of four barbiturates (phenobarbital, pentobarbital, amobarbital and secobarbital) in raw milk. The barbiturates were extracted using liquid-liquid extraction, ultrasonication and centrifugation, and purified on an SPE column. Analytes were separated by HPLC on a CSH C18 column eluted using an acetonitrile-water system with a linear gradient dilution programme, and detected by MS/MS. The recoveries of the barbiturates were 85.0-113.5%, and the intra- and inter-assay RSDs were less than 9.8% and 7.3%, respectively. The limit of detection was 5 ng/mL for all four of the barbiturates. The analytical method exhibited good linearity from 10 to 1000 ng/mL; the correlation coefficient (r2) was greater than 0.9950 for each barbiturate. This method was also applied to the determination of barbiturates in real milk samples and was found to be suitable for the determination of veterinary drug residues in raw milk.

Hollow fiber–liquid phase microextraction of barbiturates in liver samples

Barbiturates, especially phenobarbital, are involved in intoxication cases in Brazil and other countries. Liver tissue can be a useful alternate specimen when blood is not available (decomposition, severe fire, and exsanguination cases). Because the liver is a very complex matrix, it is essential is to eliminate potential interfering substances in this specimen before instrumental analysis of target compounds. In recent years, novel strategies for sample preparation have gained acceptance in the field of analytical toxicology. The objective of this work was to develop a gas chromatography–mass spectrometry (GC–MS) method using hollow fiber–liquid phase microextraction (HF–LPME) for determination of barbiturates (phenobarbital, secobarbital, pentobarbital, and butalbital) in liver samples. An aliquot of homogenized and acidified liver in aqueous solution (equivalent to 50 mg tissue) was submitted to extraction by the use of a 9-cm hollow fiber. The fiber was filled with eucalyptus oil in its pores and its lumen was filled with a NaOH solution (pH 13; acceptor phase). After extraction in an ultrasonic bath for 5 min, the acceptor phase was withdrawn and derivatized in the injector port of the GC–MS with trimethylanilinium hydroxide for flash methylation. A totally “green chemistry” approach of the sample extraction was obtained, because relatively large amounts of organic solvent generally used for extraction could be substituted by an essential oil used as a supported liquid membrane for HF–LPME. This method was validated and successfully applied to liver samples collected from five deceased persons with previous history of exposure to barbiturates. Phenobarbital concentrations in the liver found in these actual cases ranged from 1.3 to 16.7 μg/g.

Sensitive Determination of Barbiturates in Biological Matrix by Capillary Electrophoresis Using Online Large‐Volume Sample Stacking*

In China, some forensic cases are caused by barbiturates. Thus, the determination of trace level barbiturates in body fluid is important for the poisoning investigation. In this study, an online large-volume sample stacking (LVSS) with polarity switching in capillary electrophoresis (CE) was applied for the sensitive determination of barbiturates. This technique involves injecting a large volume of sample into a capillary and removing the sample matrix plug out of the capillary by reversing the polarity. Quantitation limit obtained was 0.048, 0.057, 0.039, and 0.015 μg/mL for secobarbital, amobarbital, barbital, and phenobarbital (signal-to-noise ratio = 9). By using LVSS, the stacking was simply achieved at 171.7-, 169.7-, 202.7-, and 169.1-fold for the above four barbiturates. The relative standard deviation values of intraday and interday were <2.11% and 4.69%, respectively. Recoveries were ranged from 83.7 to 105.2%. Finally, the trace analysis method was applied to the analysis of real forensic specimens and has achieved satisfactory results.

Application of thermoresponsive HPLC to forensic toxicology: determination of barbiturates in human urine

A high-performance liquid chromatography (HPLC) method has been developed for assays of five barbiturates in human urine using a new thermoresponsive polymer separation column, which is composed of poly(N-isopropylacrylamide). By elevating the column temperature from 10°C to 50°C, the barbiturates metharbital, primidone, phenobarbital, mephobarbital, and pentobarbital became well separated by this method. The five barbiturates showed good linearity in the range of 0.2–10 μg/ml. Good accuracy, precision, and recoveries for these drugs were obtained at 1 and 5 μg/ml urine. The method with this new column type seems to have high potential for extensive use in forensic toxicology for analysis of many drugs and poisons by HPLC and HPLC-mass spectrometry.

Direct determination of barbiturates in urine by capillary electrophoresis using a capillary coated dynamically with polycationic polymers

Direct determination of barbiturates in urine by capillary electrophoresis using a capillary coated dynamically with polycationic polymers

Direct Determination of Barbiturates in Urine by Capillary Electrophoresis Using a Capillary Coated Dynamically with Polycationic Polymers

A new, simple and rapid capillary electrophoresis method, using hexadimethrine bromide (HDB) as electro-osmotic flow modifier, has been developed for the identification and determination of nine barbiturates, barbital acid, barbital, phenobarbital, pentobarbital, amobarbital, thiobarbituric acid, butobarbital, N-methyl-5-phenyl-ethyl barbital acid and 5-cyclohexenyl-5-ethyl barbital acid in urine with UV detection at 200 nm. The applied voltage was -25 kV and the capillary temperature was kept constant at 25 °C. The effects of buffer pH, the concentration of HDB and the concentration of α-cyclodextrin were studied systematically. Optimum separation was achieved with 20 mM borate buffer at pH 10.00 containing 0.04% (w/v) HDB and 2.06 mM α-cyclodextrin. Regression equations revealed good linear relationship between the peak area of each compound and its concentration. The correlation coefficients were from 0.9990 to 0.9997. The relative standard deviations of migration times and peak areas were <3.84 and 5.45% (intra-day). The nine barbiturates in urine were successfully determined within 7 min, without a prior preparation step and the method is useful for the investigation of intoxication.

Simultaneous determination of barbiturates in human biological fluids by direct immersion solid-phase microextraction and gas chromatography?mass spectrometry

Simultaneous determination of barbiturates in human biological fluids by direct immersion solid-phase microextraction and gas chromatography?mass spectrometry

Simultaneous determination of barbiturates in human biological fluids by direct immersion solid-phase microextraction and gas chromatography–mass spectrometry

Simultaneous determination of seven barbiturates in human whole blood and urine by combining direct immersion solid-phase microextraction (DI–SPME) with gas chromatography-mass spectrometry (GC–MS) is presented. The main parameters affecting the DI–SPME process, such as SPME fibers, salt additives, pHs, extraction temperatures and immersion times were optimized for simultaneous determination of the drugs. The extraction efficiencies were 0.0180–0.988 and 0.0156–2.76% for whole blood and urine, respectively. The regression equations of the drugs showed excellent linearity for both samples; the correlation coefficients ( r 2) were 0.994–0.999. The detection limits for whole blood were 0.05–1 μg ml −1, and those for urine 0.01–0.6 μg ml −1. Actual quantitation could be made for pentobarbital in whole blood and urine obtained from volunteers, who had been orally administered a therapeutic dose of the drug. The DI–SPME/GC–MS procedure for barbiturates established in this study is simple and sensitive enough to be adopted in the fields of clinical and forensic toxicology.

Determination in serum of some barbiturates using micellar liquid chromatography with direct injection

A procedure was developed for the determination of several barbiturates, amobarbital, barbital, hexobarbital, and secobarbital, using a C18 column ( 120×4.6 mm ) and micellar liquid chromatography (MLC) mobile phases containing sodium dodecyl sulfate (SDS) and propanol, butanol, or pentanol as a modifier, with UV detection at 230 nm. After the application of an interpretative strategy of optimization, the four barbiturates can be resolved and determined in serum samples, allowing the direct injection in 0.10 M SDS–4% (v/v) butanol, pH 7, with an analysis time below 8 min. In the proposed MLC procedure, linearities ( r>0.999), limits of detection ( ng mL −1) in the 30–70 range, repeatabilities, and intermediate precision below 1.8% are adequate for the quantification. The proposed method could be applied to the determination of barbiturates in serum samples with recoveries that agreed with the concentration added.

Determination of barbiturates in urine by micellar liquid chromatography and direct injection of sample

A liquid chromatographic procedure for the determination of six barbiturates (barbital, diallyl barbituric acid, phenobarbital, butabarbital, amobarbital and pentobarbital) in urine samples is described. The proposed system uses a Spherisorb octadecyl-silane ODS-2 C 18 analytical column and a guard column of similar characteristics. The UV detector was set at 240 nm. A study to select adequate composition of the micellar mobile phase for the separation of these compounds in urine samples is performed. Maximum resolution was achieved with a 0.07 M sodium dodecylsulphate-0.3% propanol at pH 7.4 eluent. Limits of detection at 240 nm were ranged between 0.13 μg ml −1 for diallyl barbituric acid and 2.7 μg ml −1 for amobarbital. The procedure allows for the determination of these compounds in 20 minutes, it does not require prior a sample preparation step and it can be very useful to the investigation of intoxication.

Automated procedure for determination of barbiturates in serum using the combined system of PrepStation and gas chromatography–mass spectrometry

A system of an automatic sample preparation procedure followed by on-line injection of the sample extract into a gas chromatograph-mass spectrometer (GC–MS) was developed for the simultaneous analysis of seven barbiturates in human serum. A sample clean-up was performed by a solid-phase extraction (SPE) on a C 18 disposable cartridge. A SPE cartridge was preconditioned with methanol and 0.1 M phosphate buffer. After loading 1.5 ml of a diluted serum sample into the SPE cartridge, the cartridge was washed with 2.5 ml of methanol–water (1:9, v/v). Barbiturates were eluted with 1.0 ml of chloroform–isopropanol (3:1, v/v) from the cartridge. The eluate (1 μl) was injected into the GC–MS. The calibration curves, using an internal standard method, demonstrated a good linearity throughout the concentration range from 0.1 to 10 μg ml −1 for all barbiturates extracted. The proposed method was applied to 27 clinical serum samples from three patients who were administrated secobarbital.

Determination of barbiturates by solid-phase microextraction (SPME) and ion trap gas chromatography–mass spectrometry

Solid-phase microextraction (SPME) in conjunction with quadrupole ion trap GC–MS was applied to the determination of a series of barbiturates. A 65 μm Carbowax–divinylbenzene (DVB) SPME fiber was used to successfully extract a series of eight barbiturates from aqueous solution. Absorption kinetics and distribution coefficients for the 65 μm Carbowax–DVB SPME fiber were determined for the compounds. In addition the method was evaluated with respect to linearity, limit of detection, precision, desorption time, and the effect of salt. Limits of detection reached 1 ng/ml for the barbiturates. Linearity was established for the barbiturates over a concentration range of 10–1000 ng/ml, with coefficients of correlation 0.99. Overall, the precision of the method fell between 2.2%–6.5%, depending on the barbiturate. SPME was applied to the identification and quantitation of the barbiturates in a urine matrix. The method was validated by analyzing a reference standard pentobarbital-spiked urine sample. Both standard addition and internal standard with [2H5]-pentobarbital techniques were evaluated, with recoveries found to be 93% and 104%, respectively. SPME was then used to rapidly screen a urine specimen tested positive for barbiturates, and butalbital was detected and quantified.

Mercurimetric Potentiometric Determination of Barbiturates Using a Solid-State Iodide Ion-Selective Electrode

A simple potentiometric method is described for mercurimetric titration of barbiturates using a solid-state iodide ion-selective electrode. The optimum titration conditions involve the use of a borate buffer of pH 9–10 as a medium and mercury(II) perchlorate of pH 1.7–1.9 as a titrant. Under these conditions, titration curves with two sharp consecutive inflection breaks are obtained. The first inflection corresponds to quantitative and stoichiometric reaction of barbiturates with mercury(II) and the second break is due to the reaction of the buffer with the titrant. No interferences are caused by Cl−, Br−, PO4 3- and many excipients and diluents commonly used in the drug formulations. Determination of barbiturates in various pharmaceutical preparations gives reproducible results with an average recovery of 99.1% of the nominal (st.dev. 0.3 %) and the method offers significant advantages over the titrimetric method of the British Pharmacopoeia.

Determination of Barbiturates by Solid-Phase Microextraction and Capillary Electrophoresis

The solid phase microextraction device is based on plasticized poly(vinyl chloride) (PVC) as an extraction solvent, which is coated on a primed steel rod. The plasticizer is a phosphate triester. A simple device for back-extraction of analytes into a few microliters of back-extraction solution is described. A Teflon tube, with an inside diameter just larger than the PVC-coated extraction rod, is terminated at one end by a syringe. A few microliters of back-extraction solution is placed into the open end of the tube. Because the Teflon does not wet, the drop remains together. After exposure to sample, the PVC-coated extraction rod containing the analytes is inserted into the back-extraction solution-containing tube. The aqueous back-extraction solvent spreads out in the narrow annular space thus formed. After a suitable period, the rod is removed, and the droplet re-forms. It can be ejected into a sample vial with the syringe. The device has been used in a CE-based determination of barbiturates. Extraction, back-extraction, and separation of 10 barbiturates takes less than 30 min. Extraction and preconcentration of benzoates is also briefly considered.

Application of 3-(1,8-Naphthalimido) Propyl-Modified Silyl Silica Gel as a Stationary Phase In High Performanc Liquid Chromatography of Barbiturates and Diastereomeric Compounds

Abstract The application of a column packing material, 3-(l,8-naphthalimido)propyl-modified silyl silica gel (NAIP) developed by us, for high performance liquid chromatography (HPLC) of barbiturates and diastereomeric compounds is described. The separation of barbiturates by using an NAIP column as a stationary phase was at first investigated. Six commercially available barbiturates (Barbital, Phenobarbital, Amobarbital, Pentobarbital, Secobarbital and Thiopental) were well separated on an NAIP column by an isocratic elution with borate buffer (pH 7.0)-acetonitrile (9:1, v/v). Further, the applicability of the HPLC system including an NAIP column to the determination of barbiturates in human blood plasma was examined. Barbiturates extracted from plasma sample using a solvent extraction (hexane-diethyl ether, 3:7. v/v) were subjected to the HPLC system and determined by an internal standard method with UV detection at 220 nm. The recoveries of barbiturates spiked to human plasma were obtained in the range of 88.6 to 100.0%. The lower detection limits of barbiturates spiked ranged from 0.05 to 0.33 fig/mL, and the within-day and day-to-day precisions for plasma sample (n=5) gave relative standard deviations of less than 9 07% and 18.68%, respectively. Next, the applicability of an NAIP column to the separation of diastereomeric derivatives was studied. Epinephrine (EP), norephedrine (NE) and α-phenylethylamine (PA) carrying an aromatic ring were used as representative targets for derivatization with three kinds of chiral reagents. Resolution behavior on NAIP was compared with that on a conventional reverse phase ODS column. Diastereomeric derivatives of targets were satisfactorily separated by an NAIP column as well as an ODS column. The best separation of S(+)-1-(1-naphthyl)ethyl isocyanate derivatives with PA was obtained on an NAIP column and the calibration curves showed good linearity in the concentration range examined.