A twin purification/enrichment procedure based on two versatile solid/liquid extracting agents for efficient uptake of ultra-trace levels of lorazepam and clonazepam from complex bio-matrices.

In this research work, two consecutive dispersive solid/liquid phase microextractions based on efficient extraction media were developed for the influential and clean pre-concentration of clonazepam and lorazepam from complicated bio-samples. The magnetism nature of the proposed nanoadsorbent proceeded the clean-up step conveniently and swiftly (∼5min), pursued by a further enrichment via a highly effective and rapid emulsification microextraction process (∼4min) based on a deep eutectic solvent (DES). Finally, the instrumental analysis step was practicable via high performance liquid chromatography-ultraviolet detection. The solid phase used was an adequate magnetic nanocomposite termed as polythiophene-sodium dodecyl benzene sulfonate/iron oxide (PTh-DBSNa/Fe3O4), easily and cost-effectively prepared by the impressive co-precipitation method followed by the efficient in situ sonochemical oxidative polymerization approach. The identification techniques viz. FESEM, XRD, and EDX certified the supreme physico-chemical properties of this effective nanosorbent. Also the powerful liquid extraction agent, DES, based on bio-degradable choline chloride, possessed a high efficiency, tolerable safety, low cost, and facile and mild synthesis route. The parameters involved in this versatile hyphenated procedure, efficiently evaluated via the central composite design (CCD), showed that the best extraction conditions consisted of an initial pH value of 7.2, 17mg of the PTh-DBSNa/Fe3O4 nanocomposite, 20 air-agitation cycles (first step), 245µL of methanol, 250µL of DES, 440µL of THF, and 8 air-agitation cycles (second step). Under the optimal conditions, the understudied drugs could be accurately determined in the wide linear dynamic ranges (LDRs) of 4.0-3000ngmL-1 and 2.0-2000ngmL-1 for clonazepam and lorazepam, respectively, with low limits of detection (LODs) ranged from 0.7 to 1.0ngmL-1. The enrichment factor (EF) and percentage extraction recovery (%ER) values were found to be 75 and 57% for clonazepam and 56 and 42% for lorazepam at the spiked level of 75.0ngmL-1, possessing proper repeatabilities (relative standard deviation values (RSDs) below 5.9%, n=3). These valid analytical features provided quite accurate drug analyses at therapeutically low spans and levels below potentially toxic domains, implying a proper purification/enrichment of the proposed microextraction procedure.

Enhanced selectivity in CZE multi-chiral selector enantioseparation systems: proposed separation mechanism.

It has been reported many times that the commercial mixtures of chiral selectors (CS), namely highly sulfated beta-CDs (HS-beta-CDs), provide remarkable enantioselectivity in CZE when compared with single-isomer CDs, even single-isomer HS-beta-CDs. This enhanced enantioselectivity of multi-CS enantioseparative CZE is discussed in the light of multi-CS model that we have introduced earlier. It is proposed on a theoretical basis and verified experimentally that the two enantiomers of a chiral analyte under interaction with a mixture of CSs are very likely to differ in their limit mobilities, which is opposite to single-CS systems where the two limit mobilities are likely to be the same. Thus while the enantioseparation is usually controlled by different distribution constants between the two enantiomers and CS used in single-CS systems, an additional, electrophoretic, enantioselective mechanism resulting from different limit mobilities may play a significant role in multi-CS systems. This additional mechanism generally makes the multi-CS systems more selective than the single-CS systems. The possible inequality of limit mobilities is also significant for optimization of separation conditions using mixtures of CSs. A practical example supporting our considerations is shown on enantioseparation of lorazepam in the presence of a commercial mixture of HS-beta-CDs and a single-isomer HS-beta-CD, heptakis(6-O-sulfo)-beta-CD.

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.

Preparation and characterization of poly(methyltetradecylsiloxane) stationary phases immobilized by gamma radiation onto zirconized silica.

The preparation of stationary phases with enhanced chemical stability in alkaline eluents has been the principal objective of many chromatographers. New and improved silica substrates and advanced chemical modification methods are among the possibilities being investigated to reach this objective. The present work has evaluated these two possibilities for new stationary phases. First, the silica surface was modified by reaction with zirconium tetrabutoxide to produce zirconized silica particles having about 21% (w/w) of zirconium. Then poly(methyltetradecylsiloxane) (PMTDS) was immobilized onto this surface using different doses (50-120 kGy) of gamma radiation. These new phases were characterized using elemental analysis and infrared and solid-state (29)Si-nuclear magnetic resonance (NMR) spectroscopies. These new stationary phases presented column efficiencies of about 68,000 plates m(-1), symmetric peaks for apolar compounds and retention factors that depend on the irradiation dose and show improved stability in high pH mobile phases. The separation of several pharmaceuticals at pH 11 is presented.

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.

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.

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.

Bonded cellulose-derived high-performance liquid chromatography chiral stationary phases. I. Influence of the degree of fixation on selectivity.

Four mixed 10-undecenoyl-3,5-dimethylphenylaminocarbonyl derivatives of cellulose, with an increasing proportion of alkenoyl groups, were synthesized and chemically bonded on allylsilica gel. The influence of the degree of fixation of the cellulose derivative on the matrix for the four resulting chiral stationary phases on their selectivity is discussed.

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.

Estimation of plasma lorazepam by gas-liquid chromatography and a benzene extraction.

Plasma lorazepam can be easily and reliably estimated using a simple single stage benzene extraction and gas-lizuid chromatography with flunitrazepam as the internal standard. Reproducible results within the clinical range of plasma levels were obtained. This method permits the extraction and chromatographing of 40 samples in approximately 7 1/2 hours.