A silica fiber coated with a ZnO-graphene oxide nanocomposite with high specific surface for use in solid phase microextraction of the antiepileptic drugs diazepam and oxazepam.

A novel ZnO-graphene oxide nanocomposite was prepared and is shown to be a viable coating on fused silica fibers for use in solid phase microextraction (SPME) of diazepam and oxazepam from urine, this followed by thermal desorption and gas chromatographic quantitation using a flame ionization detector. A central composite design was used to optimize extraction time, salt percentage, sample pH and desorption time. Limits of detection are 0.5 µg·L-1 for diazepam and 1.0 µg·L-1 for oxazepam. Repeatability and reproducibility for one fiber (n = 4), expressed as the relative standard deviation at a concentration of 50 µg·L-1, are 8.3 and 11.3% for diazepam, and 6.7 and 10.1% for oxazepam. The fiber-to-fiber reproducibility is <17.6%. The calibration plots are linear in the 5.0-1000 µg·L-1 diazepam concentration range, and from 1.0-1000 µg·L-1 in case of oxazepam. The fiber for SPME has high chemical and thermal stability (even at 280 °C) after 50 extractions, and does not suffer from a reduction in the sorption capacity. Graphical abstract A hydrothermal method was introduced for preparation of ZnO- GO nano composite on a fused silica fiber as solid phase microextraction with high mechanical, chemical stability and long service life.

Applying cloud point extraction technique for the extraction of oxazepam from human urine as a colour or fluorescent derivative prior to spectroscopic analysis methods.

Two new methods based on cloud point extraction (CPE) technique were developed and optimized for the extraction and preconcentration of oxazepam from human urine, as an azo or fluorescent derivative. The first method is a spectrophotometric one, which is based on the acid hydrolysis of the oxazepam to a benzophenone, diazotization of the benzophenone, and then the coupling with oxine to form an azo dye. The second method is a spectrofluorimetric one, which involves reduction of the target compound using Zn°/HCl at room temperature with the formation of a highly fluorescent derivative. The main factors affecting the chemical reactions and CPE were investigated and optimized systematically. Under optimum experimental conditions, the calibration graphs were linear in the range of 0.1 to 1.5 (0.05 to 2.0) µg/ml with correlation coefficients of 0.9989 (0.9985), for the CPE-spectrophotometric (CPE-spectrofluorimetric) method. The limit of detection was found to be 0.034 (0.018) µg/ml and the relative standard deviation was calculated to be 1.35 (2.52)%. Recoveries in the spiked samples ranged from 87 to 94%. Finally, the proposed methods were applied to the determination of oxazepam in human urine.

Application of a novel solid-phase-extraction sampler and ultra-performance liquid chromatography quadrupole-time-of-flight mass spectrometry for determination of pharmaceutical residues in surface sea water.

In the present study, a multi-residue method based on a bag-solid phase extraction (bag-SPE) technique was evaluated for determination of 10 pharmaceuticals in surface water close to the effluent of a sewage treatment plant (STP) and along a coastal gradient from a STP effluent. The 10 compounds selected were caffeine, atenolol, metoprolol, oxazepam, carbamazepine, ketoprofen, naproxen, ibuprofen, diclofenac and gemfibrozil. All analyses were performed using ultra-performance liquid chromatography (UPLC) combined with quadrupole time-of-flight (QTOF) mass spectrometry. The detection limits (LOD) ranged from 1.0 to 13 ng L(-1). The method showed linear concentration ranges from 25 to 800 ng L(-1) with regression coefficients (R(2)) better than 0.9801. The recoveries of the selected analytes ranged from 11 to 65% with relative standard deviations (RSD) of <16% and inter-day variations of less than 18%. Isotopically labeled surrogate standards were used to compensate for sampling losses and matrix effects. Four of the selected 10 pharmaceuticals (caffeine, metoprolol, oxazepam and carbamazepine) were quantified, at concentrations ranging from 4 to 210 ng L(-1).

Simultaneous analysis of diazepam and its metabolites in rat plasma and brain tissue by HPLC-UV and SPE.

Diazepam is frequently used as an adjuvant during antidepressant therapy. Recently, some studies have suggested that the treatment with benzodiazepines could have different efficacy in depressed patients as opposed to non-depressed ones. To clarify the matter, a study is currently underway, regarding the drug metabolism in rats. In order to obtain a more complete and significant set of data, the main diazepam metabolites have also been considered, namely: nordiazepam, temazepam and oxazepam. A feasible and reliable HPLC method has been developed for the simultaneous determination of these compounds in plasma and brain tissue of rats. The method has been applied to "normal" rats and to genetic rat models of depression in order to estimate drug metabolism in different breeds. Analyte separation was achieved on a C8 reversed phase column using an acidic phosphate buffer/acetonitrile mixture as the mobile phase. The detection wavelength was 238 nm. An original sample pre-treatment, based on solid-phase extraction (SPE) was developed in order to eliminate endogenous interference, using only 250 microL of matrix (brain homogenate or plasma) for a complete analysis. The method has been validated with good results in terms of precision, extraction yield, sensitivity, selectivity and accuracy on both matrices and has been successfully applied to samples from some rats subjected to the preliminary study. The obtained data will hopefully contribute to the clarification of possible differences between depressed and non-depressed subjects with respect to benzodiazepine biotransformation.

Optimization of 12 chiral analytes with 8 polymeric surfactants.

This manuscript discusses the results of studies that were performed to determine optimum capillary electrophoresis (CE) conditions for the enantiomeric resolution of twelve chiral analytes with eight amino acid based polymeric surfactants. The parameters that were optimized include pH, buffer type, and concentration of surfactant. The results indicated that the optimum conditions for enantiomeric separations with the amino acid based polymeric surfactants examined in this study using CE were analyte dependent, not surfactant dependent. In other words, the optimum conditions for a particular analyte were the same for all the amino acid based polymeric surfactants examined in this study. The results of these studies indicate that when using a large group of related amino acid based polymeric surfactants only a few surfactants need to be optimized for each analyte under study. These studies were limited to anionic surfactants that contain the amino acids glycine, L-alanine, L-valine, and L-leucine only. No inference can be necessarily drawn about surfactants containing other types of amino acids such as threonine and serine, which contain extra heteroatoms, or phenylalanine that has an aromatic moiety.

Application of polymeric surfactants in micellar electrokinetic chromatography-electrospray ionization mass spectrometry of benzodiazepines and benzoxazocine chiral drugs.

Chiral micellar EKC (CMEKC) coupled to ESI-MS using polymeric surfactants as pseudostationary phases is investigated for simultaneous enantioseparation of two benzodiazepines, (+/-)-oxazepam ((+/-)-OXA) and (+/-)-lorazepam ((+/-)-LOR), and one benzoxazocine, (+/-)-nefopam ((+/-)-NEF). First, enantioselectivity and electrospray sensitivity of six chiral polymeric surfactants for all three chiral compounds are compared. Second, using poly(sodium N-undecenoyl-L-leucinate) as pseudostationary phase, the organic modifiers (methanol (MeOH), isopropanol, and ACN) are added into the running buffer to further improve chiral resolution (RS). Next, a CMEKC-ESI-MS method for the simultaneous enantioseparation of two benzodiazepines is further developed by using a dipeptide polymeric surfactant, poly(sodium N-undecenoxy carbonyl-L,L-leucyl-valinate) (poly-L,L-SUCLV). The CMEKC conditions including nebulizer pressure, capillary length, ammonium acetate concentration, pH, poly-L,L-SUCLV concentration, and capillary temperature were optimized to achieve maximum chiral RS and highest sensitivity of MS detection. The spray chamber parameters (drying gas temperature and drying gas flow rate) as well as sheath liquid conditions (MeOH content, pH, flow rate, and ionic strength) were found to significantly influence MS S/N of both (+/-)-OXA and (+/-)-LOR. Finally, a comparative study between simultaneous UV and MS detection showed high plate numbers, better chiral RS, and enhanced detectability with CMEKC-MS. However, speed of analysis was faster using CMEKC-UV.

Enantioseparations by capillary electro-chromatography: differences exhibited by normal- and reversed-phase versions of polysaccharide stationary phases.

The influence of using normal-phase and reversed-phase versions of four commercial polysaccharide stationary phases on chiral separations was investigated with capillary electrochromatography (CEC). Both versions of the stationary phases, Chiralcel OD, OJ, and Chiralpak AD, AS were tested for the separation of two basic, two acidic, a bifunctional, and a neutral compound. Different background electrolytes were used, two at low pH for the acid, bifunctional and neutral substances, and three at high pH for the basic, bifunctional and neutral ones. This setup allowed evaluating differences between both stationary-phase versions and between mobile-phase compositions on a chiral separation. Duplicate CEC columns of each stationary phase were in-house prepared and tested, giving information about the intercolumn reproducibility. In general, reversed-phase versions of the current commercial polysaccharide stationary phases are found to be best for reversed-phase CEC, even though at high pH no significant differences were seen between both versions. Most differences were observed at low pH. For acidic compounds, it was seen that an ammonium formate electrolyte performed best, which is also an excellent electrolyte if coupling with mass spectrometry is desired. For basic, bifunctional and neutral compounds, no significant differences between the three tested electrolytes were observed at high pH. Here, a phosphate buffer is preferred as electrolyte because of its buffering capacities. However, if coupling to mass spectrometry is wanted, the more volatile ammonium bicarbonate electrolyte can be used as an alternative.

Effect of temperature on enantiomer separation of oxzepam and lorazepam by high-performance liquid chromatography on a beta-cyclodextrin derivatized bonded chiral stationary phase.

A reversed-phase high-performance liquid chromatography (HPLC) method with beta-cyclodextrin (beta-CD) derivatized as chiral stationary phase is used to directly separate oxazepam (Oxa) and lorazepam (Lor) enantiomers. The effect of temperature on the direct HPLC separation of Oxa and Lor enantiomers is studied for the commercially available beta-CD derivatized bonded chiral stationary phase. Chromatographic peak coalescence, appearing as a plateau between the resolved peaks, is observed at column temperatures of above 13 degrees C. Peak coalescence on the beta-CD derivatized bonded column is attributable to racemization of the Oxa enantiomer. By reducing the column temperature to 13 degrees C, the enantiomeric composition of Oxa and Lor could be determined on the chiral column. This method is expected to be useful for the resolution of 3-hydroxybenzodiazepines. At the same time, the separation mechanism is studied by calculating the thermodynamic parameters. The results reveal that the separation of Oxa and Lor enantiomer is a case of enthalpy-controlled separation, inclusion mechanism does not control the separation. The interaction between Oxa and beta-CD is an additionally strong pi-pi interaction or hydrogen bonding, but that between Lor or beta-CD derivatized is a weak pi-pi interaction or hydrogen bonding.

Monolithic silica columns with chemically bonded beta-cyclodextrin as a stationary phase for enantiomer separations of chiral pharmaceuticals.

An enantioselective silica rod type chiral stationary phase (CSP) is presented; a novel combination of the well known enantiomer separation properties of beta-cyclodextrin and the unique properties concerning the flow behavior of silica monoliths. Two different synthesis routes are described, and it was found that the in situ modification of a plain silica rod column turned out to be the best. The chromatographic behaviour of the beta-cyclodextrin silica rod was studied and compared with a very similar commercially available beta-cyclodextrin bonded particulate material (ChiraDex). Even if the amount of beta-cyclodextrin bound to the silica rod was only about half of the amount of beta-cyclodextrin bound to ChiraDex) particles, good resolutions were achieved for a set of chiral test components like Chromakalin, Prominal, Oxazepam, Methadone and some other drugs. By taking advantage of the unique features of the silica rods relating to their flat H/u (Van Deemter) curves, fast enantiomer separations could be demonstrated.

Separation of oxazepam, lorazepam, and temazepam enantiomers by HPLC on a derivatized cyclodextrin-bonded phase: application to the determination of oxazepam in plasma.

The enantioselective high-performance liquid chromatography (HPLC) of three racemic 3-hydroxybenzodiazepines, oxazepam (Oxa), lorazepam (Lor), and temazepam (Tem), is a difficult operation because of the spontaneous chiral inversion in polar solvent. To solve this problem, we have developed an HPLC method based on a chiral Cyclobond I-2000 RSP column, maintained at 12 degrees C, and a reversed mobile phase (acetonitrile in 1% triethylamine acetate buffer, TEAA) at a flow rate of 0.4 ml/min. Peaks were detected by a photodiode-array detector at 230 nm for quantification and by an optical rotation detector for identification of (+) and (-) enantiomers. The results showed that peak resolutions of Oxa, Lor, and Tem enantiomers, analyzed under the same conditions, were 3.2, 2.0, and 1.8, respectively. For the determination of Oxa enantiomers in plasma of rabbits, extraction with diethyl ether at pH 1.5, a polar organic mobile phase, and a Cyclobond I-2000 SP column were used. Other analytical conditions were the same as previously described. Blood samples were immediately cooled at 4 degrees C and centrifuged at 0 degrees C for the collection of plasma. The results showed a difference in plasma S(+)- and R(-)-oxazepam concentrations in rabbits. No racemization of S(+)- or R(-)-Oxa enantiomers, added alone to blank plasma, was observed after extraction and enantioselective HPLC analysis.

Direct calculation and computer simulation of the enantiomerization barrier of oxazepam in dynamic HPLC experiments--a comparative study.

Dynamic chromatographic methods constitute a versatile approach to the rapid and precise determination of enantiomerization barriers of stereolabile drugs. In the present study enantioselective dynamic high-performance liquid chromatography (DHPLC) was employed to determine the enantiomerization barrier of oxazepam. Dynamic elution profiles, exhibiting plateau formation and/or peak broadening between 20 and 60 degrees C at pH 2.6 and pH 8 were obtained in the presence of the chiral stationary phase (CSP) Nucleodex-beta-PM (permethylated beta-cyclodextrin chemically bonded to silica) using a 6:4 mixture of phosphate buffer and methanol as mobile phase. Evaluation of the experimental chromatograms was performed by the novel approximation function (AF) (without computer simulation), and by the stochastic model implemented in the ChromWin simulation software (with computer simulation) furnishing the respective apparent forward rate constants, k(1)(app)(T). From the rate constants, k(1)(app)(T), measured at variable temperatures, the kinetic Eyring activation parameters, deltaG(T)(#), deltaH(#) and deltaS(#), of the enantiomerization of oxazepam were obtained. By variation of the flow rate of the mobile phase, the expected independence of the enantiomerization barrier from the chromatographic time scale was demonstrated for the first time.

Influence of specific albumin ligand markers used as modifiers on the separation of benzodiazepine enantiomers by chiral liquid chromatography on a human serum albumin column.

Specific ligand markers for the various binding sites of human serum albumin (HSA) have been described in the literature. Some of these markers (medium chain fatty acids, warfarin, digoxin, and bilirubin) were used as mobile phase modifiers. Using a high performance liquid chromatographic (HPLC) column containing HSA as stationary phase, their influence was investigated on the separation in this phase of the enantiomers of three benzodiazepines (temazepam, oxazepam, and lorazepam). Displacement effects were observed with medium chain fatty acids. This influence was proportional to the chain length and to the concentration of acid. Allosteric cooperative effects were noted with digoxin for the three benzodiazepines. Both displacement and cooperative effects were observed with warfarin. Stereoselectivity was decreased for temazepam and oxazepam and increased for lorazepam.

Resolution of several racemic 3-hydroxy-1,4-benzodiazepin-2-ones by high-performance liquid chromatography on a chiral silica-bonded stationary phase.

The resolution of four racemic 3-hydroxy-1,4-benzodiazepin-2-ones, widely used in therapeutics, by means of a chiral stationary phase is described. The chiral selector used is (S)-N-(3,5-dinitrobenzoyl)phenylalanine. This chiral stationary phase showed both good enantioselectivity and efficiency for the compounds. Elution times were in all cases shorter than those previously reported for such compounds on different stationary phases. Racemic oxazepam was used to evaluate the loading capacity of the chiral stationary phase.

Rapid extraction of oxazepam from greyhound urine for high performance liquid chromatography analysis.

A rapid and efficient procedure is described for the extraction and analysis of oxazepam, the major urinary metabolite of diazepam in greyhounds. Urine was extracted by passing through a bonded silica column (Bond Elut) following enzyme hydrolysis. The adsorbed drug was eluted and then detected and measured by high performance liquid chromatography (HPLC). Recoveries were in excess of 85% at 50 ng/ml concentrations. Detection was possible up to 30 h after a single oral dose of diazepam (5 mg).

Diastereoisomeric glucuronides of oxazepam. Isolation and stereoselective enzymic hydrolysis.

Oxazepam glucuronide isolated from swine urine by previously published methods was separated into its diastereoisomers by ion-exchange chromatography on a preparative scale. Quantitative high-performance liquid chromatography was used to monitor the separation. The two isomers were obtained in analytically pure form and then characterized by elemental analysis, oxazepam content, mass spectrometry, ultraviolet spectroscopy, optical rotation and optical rotatory dispersion-circular dichroism. The latter permitted the assignment of the dextrorotatory and the levorotatory isomers to the (S)- and (R)- configurations, respectively. Rates of enzymic hydrolysis depend on the configuration of the substrate as well as on the enzyme preparation used. Rate of cleavage was highest with the (S)-(+)-glucuronide and beta-glucuronidase from Escherichia coli. This enzyme possesses the highest degree of stereoselectivity; it hydrolyzes the (S)-(+)-isomer more than 400 times faster than the (R)-(-)-form. Bovine liver glucuronidase is less stereoselective, whereas glucuronidase preparations of molluscan origin exhibit little stereoselectivity. The ready hydrolysis of one of the glucuronides by an enzyme from an intestinal microorganism may play a role in the enterohepatic circulation of oxazepam.