Chromatographic separation of 2,3-benzodiazepines

A review of chromatographic methods for the determination of 2,3-benzodiazepines (2,3-BZs) is presented. The determinations are performed to investigate the presence of potential impurities in drug substances and to study their pharmacokinetic profile in biological samples, either in animals or in humans. Several methods dealt with a pretreatment of samples, i.e., liquid–liquid extraction by using a variety of solvents, solid-phase extraction, direct injection of specimens into the chromatographic apparatus. Different chromatographic techniques have been used. High-performance liquid chromatography allows optimal sensitivity and specificity by using ultraviolet or diode array detection methods. Gas chromatography-mass spectrometry and gas chromatography with nitrogen-phosphorous or electron-capture detectors have been also reported. Suitable methods for the separation of enantiomers of 2,3-BZs have been described. Thin-layer chromatography has been shown to be capable to isolate analytes from biological samples as urine or faeces. The reported chromatographic techniques are currently applied to define the metabolic pathways of 2,3-BZs in experimental and clinical studies.     

Application of solid-phase microextraction combined with derivatization to the determination of amphetamines by liquid chromatography

This work evaluates the utility of solid-phase microextraction (SPME) in the analysis of amphetamines by liquid chromatography (LC) after chemical derivatization of the analytes. Two approaches have been tested and compared, SPME followed by on-fiber derivatization of the extracted amphetamines, and solution derivatization followed by SPME of the derivatives formed. Both methods have been applied to measure amphetamine (AP), methamphetamine (MA), and 3,4-methylenedioxymethamphetamine (MDMA), using the fluorogenic reagent 9-fluorenylmethyl chloroformate (FMOC) and carbowax-templated resin (CW-TR)-coated fibers. Data on the application of the proposed methods for the analysis of different kind of samples are presented. When analyzing aqueous solutions of the analytes, both approaches gave similar analytical performance, but the sensitivity attainable with the solution derivatization/SPME method was better. The efficiencies observed when processing spiked urine samples by the SPME/on-fiber derivatization approach were very low. This was because the extraction of matrix components into the fiber coating prevented the extraction of the reagent. In contrast, the efficiencies obtained for spiked urine samples by the solution derivatization/SPME approach were similar to those obtained for aqueous samples. Therefore, the later method would be the method of choice for the quantification of amphetamines in urine.     

In vivo sampling with solid phase microextraction

This review discusses the most recent developments and future challenges in the application of solid phase microextraction (SPME) for sampling of live biological samples. The emphasis is placed on applications of fiber SPME for analysis of volatile emissions and drugs in biological fluids. The method development section highlights the main parameters that need to be considered in the case of in vivo experiments: extraction techniques, selection of extraction phases, calibration procedures, determination of free concentrations, and automation.     

Headspace solid-phase microextraction and gas chromatographic determination of dinitroaniline herbicides in human blood, urine and environmental water

Solid-phase microextraction (SPME) is a unique extraction and sampling technique, and it has been used for separation of volatile organics from water or other simple matrices. In this study, we have used SPME to separate dinitroaniline herbicides from complicated matrices of human urine and blood in order to broaden its application to biomedical analysis. The SPME conditions were optimized for water, urine and blood samples, in terms of pH, salt additives, extraction temperature, and fiber exposure time. Urine or water (1.0 ml) spiked with herbicides and 0.28 g of anhydrous sodium sulfate was preheated at 70°C for 10 min, and a polydimethylsiloxane-coated fiber for SPME was exposed to the headspace at 70°C for another 30 min; while spiked blood (0.5 ml) diluted with water (0.5 ml) was treated at 90°C in the same way. The herbicides were extractable under these conditions, and could be determined by gas chromatography–electron capture detector (GC–ECD). The recoveries of the herbicides, measured at the concentrations of 0.50 and 1.0 ng/ml urine or water, or 6.0 and 20 ng/0.5 ml blood, ranged from 35 to 64% for different herbicides from water or urine, and from 3.2 to 7.2% from blood. The headspace SPME yielded clean extracts of dinitroaniline herbicides from urine, blood or water, which could be directly analyzed by GC–ECD without further purification. The peak areas of the extracted herbicides were proportional to their concentrations in the range 0.1–10 ng/ml in water or urine, or 1–60 ng/0.5 ml in blood. The lowest detectable concentration of the herbicides lay in 0.1 ng/ml water or urine, or in 0.5 ng/0.5 ml blood. The intra- and inter-day coefficients of variation were within 14% for most of the analytes. Although the recoveries of the herbicides were rather low, the linearity of calibration curve and the precision were good. The developed method is more sensitive and much simpler in sample preparation than previously reported ones. With the established SPME method, a dosed herbicide was successfully separated and determined in rats' blood.     

Ultraviolet derivatization of low-molecular-mass thiols for high performance liquid chromatography and capillary electrophoresis analysis

Thiols play an important role in metabolic processes of all living creatures and their analytical control is very important in order to understand their physiological and pathological function. Among a variety of methods available to measure thiol concentrations, chemical derivatization utilizing a suitable labeling reagent followed by liquid chromatographic or electrophoretic separation is the most reliable means for sensitive and specific determination of thiol compounds in real world samples. Ultraviolet detection is, for its simplicity, commonly used technique in liquid chromatography and capillary electrophoresis, and consequently many ultraviolet derivatization reagents are in used. This review summarizes HPLC and CE ultraviolet derivatization based methods, including pre-analytical considerations, procedures for sample reduction, derivatization, and separation of the primary biological aminothiols--cysteine, homocysteine, cysteinylglycine and glutathione, and most important thiol-drugs in pharmaceutical formulations and biological samples. Cognizance of the biochemistry involved in the formation of the analytes is taken.     

Rapid assay of cocaine, opiates and metabolites by gas chromatography—mass spectrometry

The simultaneous assay of cocaine, opiates and metabolites in small biological samples continues to be a difficult task. This report focuses upon tabulation of important techniques (extraction, derivatization, chromatographic conditions, detection mode, data acquisition) reported over the last decade that were used in the development of assays for these analytes. The most prevalent procedures for extraction of cocaine, opiates and metabolites were liquid—liquid and solid-phase extraction isolation methods. Following extraction analytes were derivatized and analyzed by gas chromatography—mass spectrometry. The technique most often used for chromatographic separation was fused-silica capillary column gas chromatography. Detection generally was performed by selected ion monitoring in the positive-ion electron-impact ionization mode, although full-scan acquisition and positive- and negative-ion chemical ionization methods have been used. It was apparent from the review that there is a continuing need for greater sensitivity and selectivity in the assay of highly potent opiates and for cocaine and metabolites.     

In vivo sampling with solid phase microextraction

This review discusses the most recent developments and future challenges in the application of solid phase microextraction (SPME) for sampling of live biological samples. The emphasis is placed on applications of fiber SPME for analysis of volatile emissions and drugs in biological fluids. The method development section highlights the main parameters that need to be considered in the case of in vivo experiments: extraction techniques, selection of extraction phases, calibration procedures, determination of free concentrations, and automation.     

Developments in multiple headspace extraction

This paper reviews new developments in multiple headspace extraction (MHE), especially its combination with two miniaturized extraction techniques, solid-phase microextraction (SPME) and single-drop microextraction (SDME). The combination of the techniques broadens the applicability of SPME and SDME to quantitative determination of analytes in complex liquid and solid matrixes. These new methods offer several advantages over traditional liquid–solid, liquid–liquid and headspace extraction techniques. The potential applications include extraction of volatiles and semivolatiles from environmental and physiological samples and from different polymer products such as medical and biomedical materials, food packaging and building materials. The theoretical principals of the techniques are also briefly reviewed.     

Electromembrane extraction of trace amounts of naltrexone and nalmefene from untreated biological fluids

Nalmefene and naltrexone are used to block the effects of narcotics and alcohol. In the present work, for the first time a microextraction technique was presented to reduce matrix interferences and improve detection limits of the drugs in urine and plasma samples. Electromembrane extraction (EME) followed by high performance liquid chromatography (HPLC) coupled with ultraviolet (UV) detection was optimized and validated for quantification of nalmefene and naltrexone from biological fluids. The membrane consists 85% of 2-nitrophenyl octyl ether (NPOE) and 15% di-(2-ethylhexyl) phosphate (DEHP) immobilized in the pores of a hollow fiber. A 100 V electrical field was applied to make the analytes migrate from sample solution with pH 2.0, through the supported liquid membrane (SLM) into an acidic acceptor solution with pH 1.0 which was located inside the lumen of hollow fiber. Extraction recoveries in the range of 54% and 75% were obtained in different biological matrices which resulted in preconcentration factors in the range of 109-149 and satisfactory repeatability (2.0相似文献   

Quantification of ceramide species in biological samples by liquid chromatography electrospray ionization tandem mass spectrometry

We present an optimized and validated liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) method for the simultaneous measurement of concentrations of different ceramide species in biological samples. The method of analysis of tissue samples is based on Bligh and Dyer extraction, reverse-phase high-performance liquid chromatography separation, and multiple reaction monitoring of ceramides. Preparation of plasma samples also requires isolation of sphingolipids by silica gel column chromatography prior to LC-ESI-MS/MS analysis. The limits of quantification were in a range of 0.01-0.50 ng/ml for distinct ceramides. The method was reliable for inter- and intraassay precision, accuracy, and linearity. Recoveries of ceramide subspecies from human plasma, rat liver, and muscle tissue were 78 to 91%, 70 to 99%, and 71 to 95%, respectively. The separation and quantification of several endogenous long-chain and very-long-chain ceramides using two nonphysiological odd chain ceramide (C17 and C25) internal standards was achieved within a single 21-min chromatographic run. The technique was applied to quantify distinct ceramide species in different rat tissues (muscle, liver, and heart) and in human plasma. Using this analytical technique, we demonstrated that a clinical exercise training intervention reduces the levels of ceramides in plasma of obese adults. This technique could be extended for quantification of other ceramides and sphingolipids with no significant modification.     

Detection of the isoflavone aglycones genistein and daidzein in urine using solid-phase microextraction–high-performance liquid chromatography–electrospray ionization mass spectrometry

An improved method of detection of the isoflavone aglycones, genistein and daidzein, is reported using solid-phase microextraction–high-performance liquid chromatography–electrospray ionization mass spectrometry (SPME–HPLC–ESI-MS). Extraction of the isoflavonoids from urine using SPME with a Carbowax–templated resin fiber coating allows rapid preconcentration of the analytes without the usual sample preparation required by other methods. Detection of the analytes is accomplished by HPLC–ESI-MS. Analysis of spiked samples of urine resulted in a linear range of 0.25 to 250 ng/ml for daidzein and 0.27 to 27.0 ng/ml for genistein. Limits of detection of daidzein and genistein were measured at 25.4 pg/ml for daidzein and 2.70 pg/ml for genistein. Daidzein and genistein were detected in urine following consumption of a soy drink.     

In vivo solid-phase microextraction for monitoring intravenous concentrations of drugs and metabolites

This protocol for in vivo solid-phase microextraction (SPME) can be used to monitor and quantify intravenous concentrations of drugs and metabolites without the need to withdraw a blood sample for analysis. The SPME probe is inserted directly into a peripheral vein of a living animal through a standard medical catheter, and extraction occurs typically over 2-5 min. After extraction, the analytes are removed from the sorbent and analyzed by, for example, liquid chromatography-tandem mass spectrometry. It has been validated in comparison with conventional blood analysis, and we describe here the in vitro experiments typically conducted during method development. The new-generation biocompatible SPME probes are designed specifically for extraction of semi-volatiles and nonvolatiles directly from aqueous samples and can be steam sterilized. Sorbents are coated on fine-gauge surgical steel wire (200-μm diameter), which is more rugged and biocompatible than conventional fibers (100-μm fused silica fiber). They incorporate a binding agent that resists fouling by the biological matrix and does not cause an immune response in the experimental animal. The sorbents used (coating thickness of ～50 μm) are selected for their affinity for the types of small molecules of interest. The procedure is illustrated by the analysis of benzodiazepines with polypyrrole-coated wires inserted into peripheral blood vessels of beagles, although it can be adapted for use in smaller animals. The in vivo sampling can require as little as 1 min, in which case the entire procedure from sampling to instrumental analysis can take as little as 30 min.     

Dispersive solid phase extraction combined with ion-pair ultra high-performance liquid chromatography tandem mass spectrometry for quantification of nucleotides in Lactococcus lactis

Analysis of intracellular metabolites in bacteria is of utmost importance for systems biology and at the same time analytically challenging due to the large difference in concentrations, multiple negative charges, and high polarity of these compounds. To challenge this, a method based on dispersive solid phase extraction with charcoal and subsequent analysis with ion-pair liquid chromatography coupled with electrospray ionization tandem mass spectrometry was established for quantification of intracellular pools of the 28 most important nucleotides. The method can handle extracts where cells leak during the quenching. Using a Phenyl-Hexyl column and tributylamine as volatile ion-pair reagent, sufficient retention and separation was achieved for mono-, di-, and triphosphorylated nucleotides. Stable isotope labeled nucleotides were used as internal standards for some analytes. The method was validated by determination of the recovery, matrix effects, accuracy, linearity, and limit of detection based on spiking of medium blank as well as standard addition to quenched Lactococcus lactis samples. For standard addition experiments, the isotope-labeled standards needed to be added in similar or higher concentrations as the analytes. L. lactis samples had an energy charge of 0.97 ± 0.001 which was consistent with literature, whereas some differences were observed compared with legacy data based on ³³P labeling.     

Developments in multiple headspace extraction

This paper reviews new developments in multiple headspace extraction (MHE), especially its combination with two miniaturized extraction techniques, solid-phase microextraction (SPME) and single-drop microextraction (SDME). The combination of the techniques broadens the applicability of SPME and SDME to quantitative determination of analytes in complex liquid and solid matrixes. These new methods offer several advantages over traditional liquid-solid, liquid-liquid and headspace extraction techniques. The potential applications include extraction of volatiles and semivolatiles from environmental and physiological samples and from different polymer products such as medical and biomedical materials, food packaging and building materials. The theoretical principals of the techniques are also briefly reviewed.     

Enantiomeric determination of amphetamines: exploring a novel one-step solid-phase microextraction-based approach

The recent advances in fiber manufacturing technology, solid-phase microextraction (SPME) is now widely studied for its effectiveness for the pretreatment of various categories of samples. This study explores a novel SPME approach for enantiomeric analysis of amphetamines, in which absorption/derivatization are accomplished in one step. Specifically, (S)-(-)-N-(Trifluoroacetyl)-prolyl chloride was adopted as the chiral derivatizing reagent and added directly into the sample matrix. Analytical parameters, such as temperature, absorption/desorption duration, and the amount of derivatizing reagent, were studied to determine their effects on the yield of analytes. The derivatization products resulting from this study show excellent desorption characteristics on the polydimethylsiloxane-coated fiber adopted in this study. Optimal operational parameters (absorption: 70 degrees C for 10 min; injection: 250 degrees C for 5 min) cause minimal negative impact on the fiber, allowing repeated use of the fiber for more than 30 times. For quantitation, data were collected under selected ion monitoring (SIM) mode using m/z 237 and 251 to designate derivatized amphetamine and methamphetamine. This method was evaluated and proved to be effective in (a) quantitative determination of the enantiomeric pairs of amphetamine and methamphetamine--in terms of repeatability, linearity, and limits of detection and quantitation; and (b) generating case-specimen data comparable to those derived from a conventional Liquid-liquid extraction approach. Good linearity for the calibration curves were established in the range of 1000-50 ng/mL for both analytes. The limits of detection for these analytes were 30 ng/mL.     

Determination of cyanide and volatile alkylnitriles in whole blood by headspace solid-phase microextraction and gas chromatography with nitrogen phosphorus detection

Simultaneous determination of cyanide and volatile alkylnitriles such as acetonitrile, cis- and trans-crotononitrile, allylnitrile and butyronitrile at low ppb concentration on whole blood (rat and mice) by headspace solid-phase microextraction (HS-SPME) followed by gas chromatography (GC) with nitrogen phosphorus detection has been achieved for the first time. SPME extraction time and temperature were optimized using a star experimental design. Optimum conditions for cyanide extraction were chosen to analyze unspiked blood samples containing alkylnitriles as that analyte occurs at the lowest concentrations. For all analytes, the developed methodology yielded good quality parameters. In all cases, good reproducibility (relative standard deviation < or =12%), detection limits (<3ng mL(-1)) and quantification limits (<4 ng mL(-1)) were recorded.     

Application of solid-phase microextraction and gas chromatography–mass spectrometry for measuring chemicals in saliva of synthetic leather workers

Saliva is of interest as a diagnostic aid for oral and systemic diseases, to monitor therapeutic drugs, and detect illicit drug abuse. It is also attractive for biological monitoring of exposure to hazardous solvents. The major advantage of this indicator over other biological monitoring targets is that the saliva is noninvasive and less confidential in comparison with blood and urine. Salivary analysis is generally acceptable by study subjects and can be applied to investigation of a wide variety of compounds. However, very few studies have been conducted on the saliva matrix to monitor exposure to hazardous solvents. The aim of this study is to establish an analytical method, headspace solid-phase microextraction (HS-SPME) followed by gas chromatography–mass spectrometry (GC–MS), by which the saliva matrix can be monitored for multiple compounds with various polarities, such as methyl ethyl ketone (MEK), isopropyl alcohol (IPA), and N,N-dimethyl formamide (DMF) (common solvents used in synthetic leather manufacture), as well as acetone (ACE) and N-methyl formamide (NMF) (metabolites of IPA and DMF, respectively). We studied this technique as an alternative biological monitoring method for investigating exposure to hazardous solvents. A Carboxen/Polydimethylsiloxane (CAR/PDMS 75 μm) fiber coating was employed for this study, and various extraction and desorption parameters were evaluated. The extraction efficiency and reproducibility of analyses was improved by pre-incubation. The limits of detection were 0.004, 0.003, 0.006, 0.05, and 0.10 μg/mL for ACE, MEK, IPA, DMF, and NMF, respectively. Method validation was performed on standards spiked in blank saliva, and a correlation was made between HS-SPME and traditional solvent pretreatment methods. It was found that correlation coefficients (r) were greater than 0.996 for each analyte, with no significant differences (p > 0.05) between two methods. However, the SPME method achieved lower limits of detection, with good accuracy (recovery 95.3–109.2%) and precision (1.17–8.22% CV) for both intra- and inter-assay, when quality control samples were analyzed for all five compounds. The partition coefficient for each compound between the headspace of the saliva sample and the CAR/PDMS fiber coating was 90.9, 170.1, 36.4, 3.70 and 0.92 for ACE, MEK, IPA, DMF and NMF, respectively. Real sample analyses were performed on workers in a synthetic leather factory. In summary, the SPME method is a highly versatile and flexible technique for chemical measurement, and we demonstrate its application for monitoring biological exposure to hazardous solvents. Saliva monitoring using sensitive SPME approaches for determining workplace exposure should prove useful as an alternative exposure monitoring method.     

High-performance liquid chromatographic methods for the determination of topoisomerase II inhibitors

Various methods for separating eleven different types of topoisomerase II (TOPO-2) inhibitors, including epipodophyllotoxins, anthracyclines, anthracenediones, anthrapyrazoles, anthracenebishydrazones, indole derivatives, aminoacridines, benzisoquinolinediones, isoflavones, bisdioxopiperazines and thiobarbituric acids, are summarized. Proper sample preparation and storage is critical to the successful analysis of some TOPO-2 inhibitors due to difficulties associated with adsorption, instability and complex biological components. Solid-phase and liquid–liquid extractions are widely used to separate TOPO-2 inhibitors from biological samples, although simple deproteinization followed by direct analysis of the supernatant is preferable to extraction based on its speed and simplicity. High-performance liquid chromatography (HPLC) is the favored method for the bioanalysis of TOPO-2 inhibitors. UV or diode array detection is generally employed for early pharmacokinetic studies, while fluorescence or electrochemical detection is used more frequently for analytes with fluorescent or oxidative–reductive properties. For analyses requiring highly sensitive and/or specific detection, electrospray mass spectrometry (ESI-MS or ESI-MS–MS) provides a suitable alternative. A comprehensive compilation of the HPLC techniques currently used to separate TOPO-2 inhibitors will aid the future development of analytical methods for new TOPO-2 inhibitors.     

Determination of amphetamines in human urine by headspace solid-phase microextraction and gas chromatography

Solid-phase microextraction (SPME) is under investigation for its usefulness in the determination of a widening variety of volatile and semivolatile analytes in biological fluids and materials. Semivolatiles are increasingly under study as analytical targets, and difficulties with small partition coefficients and long equilibration times have been identified. Amphetamines were selected as semivolatiles exhibiting these limitations and methods to optimize their determination were investigated. A 100- micro m polydimethylsiloxane (PDMS)-coated SPME fiber was used for the extraction of the amphetamines from human urine. Amphetamine determination was made using gas chromatography (GC) with flame-ionization detection (FID). Temperature, time and salt saturation were optimized to obtain consistent extraction. A simple procedure for the analysis of amphetamine (AMP) and methamphetamine (MA) in urine was developed and another for 3,4-methylenedioxyamphetamine (MDA), 3,4-methylenedioxy-N-methamphetamine (MDMA) and 3,4-methylenedioxy-N-ethylamphetamine (MDEA) using headspace solid-phase microextraction (HS-SPME) and GC-FID. Higher recoveries were obtained for amphetamine (19.5-47%) and methamphetamine (20-38.1%) than MDA (5.1-6.6%), MDMA (7-9.6%) and MDEA (5.4-9.6%).     

Determination of acrolein by headspace solid-phase microextraction gas chromatography and mass spectrometry

We developed a headspace solid-phase microextraction (headspace SPME) method to measure acrolein in human urine. This new technique resolves some problems with the headspace gas chromatography and mass spectrometry (GC–MS) method which we developed previously. With the original method, a column and a filament were damaged by the injection of air. A 0.5-ml urine (or phosphate-buffered saline) sample in a glass vial containing propionaldehyde as an internal standard was heated for 5 min. The SPME fiber (65 μm carbonwax–divinylbenzene fiber) was exposed to the headspace and then inserted into a GC–MS instrument in which a DB-WAX capillary column (30 m×0.32 mm, film thickness 0.5 μm) was installed. The total analysis time was 15 min. The inter-assay and intra-assay coefficients of variation were 10.07 and 5.79%, respectively. The calibration curve demonstrated good linearity throughout concentrations ranging from 1 to 10 000 nM. The headspace SPME method exhibits high sensitivity and requires a short analysis time as well as the previous method. We conclude that this method is useful to measure urinary acrolein.