Glutathione-stabilized Fe3 O4 nanoparticles as the sorbent for magnetic solid-phase extraction of diazepam and sertraline from urine samples through quantitation via high-performance liquid chromatography.

The synthesis and application of glutathione-coated magnetic nanocomposite were introduced with the purpose of developing a stable, cheap, operationally convenient, simple, fast, sensitive, and selective device for the microextraction of diazepam and sertraline for the first time. The prepared glutathione@Fe3 O4 nanocomposite was used as the sorbent in the form of magnetic solid-phase extraction. Afterward, the extracted analytes were desorbed by organic solvent and analyzed by high-performance liquid chromatography-ultraviolet detection. Several influential variables such as desorption time, desorption volume, sample pH, extraction time, and sorbent amount were screened through Plackett-Burman design and then optimized via Box-Behnken design. The obtained results showed that the above-mentioned method enjoys a good linear range (0.2-500 µg/L) with the coefficient of determination higher than 0.9927, low limits of determination (0.07-0.24 µg/L), acceptable limits of quantification (0.22-0.93 µg/L), good enrichment factors (128 and 153), and good spiking recoveries (95-105%) for diazepam and sertraline under the obtained optimized condition. Analyzing the real samples results in the confirmation of the presented method and it can be applied for the analysis of various organic compounds in biological samples.

Ultrasound-assisted low-density solvent dispersive liquid-liquid microextraction for the determination of 4 designer benzodiazepines in urine samples by gas chromatography-triple quadrupole mass spectrometry.

A novel microextraction technique based on ultrasound-assisted low-density solvent dispersive liquid-liquid microextraction (UA-LDS-DLLME) had been applied for the determination of 4 designer benzodiazepines (phenazepam, diclazepam, flubromazepam and etizolam) in urine samples by gas chromatography- triple quadrupole mass spectrometry (GC-QQQ-MS). Ethyl acetate (168µL) was added into the urine samples after adjusting pH to 11.3. The samples were sonicated in an ultrasonic bath for 5.5min to form a cloudy suspension. After centrifugation at 10000rpm for 3min, the supernatant extractant was withdrawn and injected into the GC-QQQ-MS for analysis. Parameters affecting the extraction efficiency have been investigated and optimized by means of single factor experiment and response surface methodology (Box-Behnken design). Under the optimum extraction conditions, a recovery of 73.8-85.5% were obtained for all analytes. The analytical method was linear for all analytes in the range from 0.003 to 10µg/mL with the correlation coefficient ranging from 0.9978 to 0.9990. The LODs were estimated to be 1-3ng/mL. The accuracy (expressed as mean relative error MRE) was within ±5.8% and the precision (expressed as relative standard error RSD) was less than 5.9%. UA-LDS-DLLME technique has the advantages of shorter extraction time and is suitable for simultaneous pretreatment of samples in batches. The combination of UA-LDS-DLLME with GC-QQQ-MS offers an alternative analytical approach for the sensitive detection of these designer benzodiazepines in urine matrix for clinical and medico-legal purposes.

Quantitation of benzodiazepines in blood and urine using gas chromatography-mass spectrometry (GC-MS).

The benzodiazepine assay utilizes gas chromatography-mass spectrometry (GC-MS) for the analysis of diazepam, nordiazepam, oxazepam, temazepam, lorazepam, alpha-hydroxyalprazolam, and alpha-hydroxytriazolam in blood and urine. A separate assay is employed for the analysis of alprazolam. Prior to solid phase extraction, urine specimens are subjected to enzyme hydrolysis. The specimens are fortified with deuterated internal standard and a five-point calibration curve is constructed. Specimens are extracted by mixed-mode solid phase extraction. The benzodiazepine extracts are derivatized with N-methyl-N-(tert-butyldimethylsilyl)trifluoroacetamide (MTBSFTA) producing tert-butyldimethyl silyl derivatives; the alprazolam extracts are reconstituted in methanol without derivatization. The final extracts are then analyzed using selected ion monitoring GC-MS.

A sensitive and selective method for the detection of diazepam and its main metabolites in urine by gas chromatography-tandem mass spectrometry.

A gas chromatography-tandem mass spectrometry method for detection of diazepam, nordazepam and oxazepam is presented. The method associates electron capture ionization and multiple reaction monitoring (MRM). No derivatization is performed; oxazepam undergoes thermal degradation during chromatographic injection and is thus quantified via its decomposition product. The negative molecular ions are so stable that they do not dissociate when collision is performed under "classical" conditions (i.e. with argon as collision gas). With xenon as collision gas, the energy transfer is sufficient to provide two product ions for diazepam and nordazepam and one product ion for the decomposition product of oxazepam. The sample preparation part involves liquid/liquid extraction with TOXI-TUBES A extraction tubes; it provides recovery yields between 68 and 95%, depending of the benzodiazepine considered, with coefficients of variation below 6% for 10 samples. The applicability of the method was demonstrated on urine extracts. From 1 mL of urine, the method provides quantitation limits of 0.15 ng/mL for diazepam, 1.0 ng/mL for nordazepam and 1.5 ng/mL for oxazepam. Mechanisms of dissociation of M*(-) ions of benzodiazepines are suggested.

[Selected psychoactive drugs--clinical problems and medical-legal aspects]. / Wybrane srodki odurzajace--problemy kliniczne i medyczno-sadowe.

The multifarious aspects of psychoactive drug use present a significant challenge to the contemporary analyst. During the first stage of the present experiment, the recovery from human serum and urine of some psychoactive drugs with acidic or basic properties was studied. The efficiency of this process was determined using solutions of drug standards added to serum or urine. Classic liquid-liquid extraction, as well as solid phase extraction methods were compared. The efficiency of recovery was checked using high-performance liquid chromatography (HPLC). The results of this study confirm the usefulness of RP-18 sorbent from Merck and the importance in terms of quantitative analysis of the technique selected for isolation of the xenobiotic from the biological material. The second stage of the experiment was aimed at qualitative determination of some narcotics using thin-layer chromatography (TLC). By stepwise comparison and elimination it was possible to develop an optimal system of chromatographic separation using laminar staining. The proposed system and the conditions for separation ofxenobiotics with six selected elution systems and laminar visualization confirm the feasibility of separating 22 psychoactive drugs. The practical use of the system is limited mainly to screening. Conditions for quantitative analysis of diazepam, tramadol, and pethidine in biological material (serum, urine) using high-performance liquid chromatography, as well as morphine in serum using an immunoenzyme assay have been developed. The procedures have been applied to analysis of narcotics and psychoactive drugs administered prior to anesthesia (morphine, diazepam, pethidine) or for suppression of post-operative pain (morphine, tramadol) in 31 patients of an intensive care unit. 10 ml of blood was drawn at fixed times: 30 minutes prior to surgery (S1), at start of surgery (S2), 60 minutes later (S3), 30 minutes after administration of analgesic (S4), and 60 minutes after administration of analgesic (S5). Urine samples were also collected: immediately after surgery (M1) and 90 minutes after administration of analgesic (M2).

Liquid-phase microextraction as a sample preparation technique prior to capillary gas chromatographic-determination of benzodiazepines in biological matrices.

Liquid-phase microextraction (LPME) and gas chromatography were applied to determine diazepam and the main metabolite N-desmethyldiazepam in human urine and plasma. The analytes were extracted from 3.0-3.5 ml sample volumes directly into 25 microl of extraction solvent. The microextraction device consisted of a porous hollow fiber of polypropylene attached to two guiding needles inserted through a septum and a 4 ml vial. The hollow fiber filled with extraction solvent was immersed in sample solution. The extraction device was continuously vibrated at 600 rpm for 50 min. An aliquot (1 microl) of the extraction solvent with preconcentrated analytes was injected directly into the capillary gas chromatograph. Thirty samples were extracted simultaneously on the vibrator, providing a high sample capacity. The limits of detection were from 0.020 to 0.115 nmol/ml for diazepam and N-desmethyldiazepam in plasma and urine using a nitrogen-phosphorus detector (NPD).

Determination of benzodiazepines in human urine and plasma with solvent modified solid phase micro extraction and gas chromatography; rationalisation of method development using experimental design strategies.

Solid phase micro extraction (SPME) and gas chromatographic analysis was used for the analysis of several benzodiazepines (oxazepam, diazepam, nordiazepam, flunitrazepam and alprazolam) in human urine and plasma. Several factors likely to affect the analyte recovery were screened in a fractional factorial design in order to examine their effect on the extraction recovery. Parameters found significant in the screening were further investigated with the use of response surface methodology. The final conditions for extraction of benzodiazepines were as follows: Octanol was immobilised on a polyacrylate fibre for 4 min. The fibre was placed in the sample and extraction took place at pH 6.0 for 15 min. Urine samples were added to 0.3 g ml(-1) sodium chloride. In plasma, the extraction recovery was less than in urine and releasing the benzodiazepines from plasma proteins followed by protein precipitation was found necessary prior to sampling. The method was validated and found linear over the range of samples. The limits of detection in urine were determined to be in the range 0.01-0.45 micromol l(-1). The corresponding limits of detection in plasma were in the range 0.01-0.48 micromol l(-1). Finally, the method developed was applied to determine some benzodiazepines after administration of a single dose. This method offers sufficient enrichment for bioanalysis after a single dose of high dose benzodiazepines as diazepam, but for low dose benzodiazepines as flunitrazepam, further sensitivity is needed.

Identification of parent benzodiazepines by gas chromotography/mass spectroscopy (GC/MS) from urinary extracts treated with B-glucuronidase.

Urinary glucuronide metabolites of the benzodiazepines were converted back to the parent molecules after treatment with B-glucuronidase. The benzodiazepines were extracted by a one-step liquid/liquid extraction from urine or by a liquid/solid phase extraction. For the limit of detection (LOD), a standard solution of diazepam and oxazepam was serially diluted and analyzed to the point at which a reproducible analytical result was no longer obtained. Using a temperature program and a splitless mode of injection, excellent quantitation was achieved within an 8-min run time. Based upon specimens obtained from patients under a physician's care, we have determined that urinary concentrations of the benzodiazepines > 200 ng/ml are most likely due to abuse rather than to a prescribed ingestion under strict medical surveillance. Therefore, the calibration standard and cutoff concentration for a positive result was set at 200 ng/ml.

Selective detection of chlorine-containing compounds by gas chromatography/chemical reaction interface mass spectrometry.

Chemical reaction interface mass spectrometry (CRIMS) was studied as a gas chromatographic detection technique for chlorine-containing compounds. Both SO2 and HBr were tested as reactant gases. With SO2, a detection limit of 50 pg of diazepam and a linear range of 4 orders of magnitude were achieved, and the experimental data were reproducible. With HBr, the detection limit was 10 ng of diazepam and the linear dynamic range was only 2 orders of magnitude. The possible pharmacological application of CRIMS was studied using urine spiked with diazepam and several of its metabolites, and the results show CRIMS to be a simple but potentially powerful method in drug metabolism studies.

Evaluation of an enzyme immunoassay for determining diazepam and nordiazepam in serum and urine.

We evaluated a commercially available enzyme immunoassay method for determining the concentration of total diazepam plus its active metabolite, nordiazepam, in small amounts of serum and urine. The coefficients of variation were 5% within assay and 8% between assay. Concentrations so measured for patients' sera correlated well with those obtained both by gas chromatography with electron-capture detection and by liquid chromatography with ultraviolet detection. For urine samples, the correlation between values obtained by the enzyme immunoassay method and those for total diazepam/nordiazepam/oxazepam obtained by liquid chromatography was adequate for routine clinical toxicological use. The ability of the immunoassay to determine other benzodiazepines was also evaluated.

Analysis of therapeutic and commonly abused drugs in serum and urine by gas-liquid chromatography using a photoionization detector.

A simple, rapid and sensitive gas chromatographic procedure using the photoionization detector (PID) was developed for the detection and quantitation of several drugs in serum and urine. In order to evaluate the performance of the PID, the results were compared with those of the flame-ionization detector (FID). The data indicate that the PID is 8-16 times more sensitive than the FID for the drugs studied in the barbiturate group. Excellent reproducibility was found for samples quantitated with the PID on a routine basis. The PID and FID produced statistically similar results on extracted serum samples. The correlation coefficient was 0.99. The PID also produced chromatograms with less background than those obtained with the FID for many extracted serum samples. The advantages of the PID for drug analysis in biological fluids include simplicity of operation, lack of solvent response, universal drug response, non-destructive character and stability.

Frequency of use of diazepam in individuals on probation and in methadone maintenance programs.

During the spring of 1977 a survey was taken of drug analysis results for over 17,500 urine samples from Los Angeles County probationers and over 8,500 urine samples from Los Angeles County methadone clinic enrollees to determine the frequency of diazepam (Valium) use. The probation department specimens were found to contain diazepan less frequently than the methadone clinic specimens as a group. There were, however, wide variations in use frequency between the seven methadone clinics.

Thin-layer detection of diazepam and/or chilordiazepoxide alone or in combination with major drugs of abuse in drug abuse urine screening programs.

Three extraction procedures for the detection of diazepam, oxazepam, chlorazepate and/or chlordiazepoxide in human urines are presented. All three procedures are based on the acid hydrolysis of benzodiazepines and/or their conjugated metabolites to give the corresponding benzophenones. Procedure I involves the direct acid hydrolysis of raw urine and is recommended when the aim is to test the abuse of benzodiazepine derivatives only. Procedure II Is a two-step extraction method in which a wide variety of drugs of abuse including cocaine (test based on the detection of benzoylecgonine) are extracted by the first step using paper loaded with cation-exchange resin and the benzodiazepines are tested in the second step by the acid hydrolysis of the spent urine left after removing the ion-exchange paper. Procedure III involves the use of inert fibrous matrix and then its acid hydrolysis. The detection procedure is based on the identification of methylaminochlorobenzophenone (MACB) and aminochlorobenzophenone (ACB). MACB is detected as a yellow-colored compound while ACB is detected by spraying with Bratton-Marshall reagent. Specificity of detection of ACB has been achieved by the selection of a thin-layer developing solvent system in which sulfonamides with primary aromatic amino groups remain at the origin.

[Gas chromatographic analysis of benzodiazepines. 2. Diazepam and its metabolites]. / Zur gaschromatographischen Analytik der Benzodiazepine. 2. Mitteilung: Diazepam und seine Metabolite

A gas-chromatographic method is reported which completely resolves diazepam and its major metabolites and thus enables the specific quantitation of these compounds after extraction from serum and urine. The sensitivity limits are about 3 ng/ml if 4 ml serum or urine are extracted.