Chromatographic separation of the interconverting enantiomers of imidazo- and triazole-fused benzodiazepines.

The toolbox of medicinal chemists includes the 1,4-benzodiazepine scaffold as a "privileged scaffold" in drug discovery. Several biologically active small molecules containing a 1,4-benzodiazepine scaffold have been approved by the FDA for the treatment of various diseases, with most of them being used for their psychotropic effects. The therapeutic potential of 1,4-benzodiazepines has stimulated the interest of synthetic chemists in developing new synthetic strategies to a range of substituted analogues for biological evaluation. A structural variation of the classical benzodiazepine skeleton is observed e.g. in alprazolam, midazolam, and related benzodiazepines, which contain a 1,2,4-triazole or an imidazole ring fused to the benzodiazepine core. Irrespective of the presence of the fused heterocyclic ring, the seven-membered diazepine ring is far from planar, and its shape resembles a twist chair. Then, the unsymmetrical substitution pattern around the seven membered cycle renders these molecules chiral, as they lack any reflection-type symmetry element. However, chirality of this molecules is labile at room temperature, becausea simple ring flipping process converts one enantiomer into the other, and 1,4-benzodiazepines exist as a mixture of rapidly interconverting conformational enantiomers in solution at or near room temperature. Physical separation of the interconverting enantiomers of diazepam and of other related 1,4-benzodiazepin-2-ones can be accomplished by low temperature HPLC on chiral stationary phases (CSPs). If the HPLC column is cooled down to temperatures where the interconversion rate is sufficiently low, compared to the chromatographic separation rate, distinct separated peaks can be observed, provided the CSP is sufficiently enantioselctive. The apparent rate constants for the on-column enantiomerization and the corresponding free energy activation barriers were obtained by simulation of exchange-deformed HPLC profiles using a computer program based on the stochastic model. Here we report on the dynamic HPLC investigations carried out on a set of fused imidazo and triazolo-benzodiazepines (alprazolam, midazolam, triazolam and estazolam) The experimental dynamic chromatograms and the corresponding interconversion barriers reported in this paper show that the third fused heterocyclic ring increase the energy barrier by 2 kcal/mol.

Benzodiazepines in the oral fluid of Spanish drivers.

BACKGROUND: Driving under the influence of alcohol, illicit drugs and certain medicines is not allowed worldwide. Roadside drug testing is considered an important tool for determining such behavior. In Spain, mandatory roadside oral fluid drug testing is carried out regularly. The aim of this study was to determine the prevalence of benzodiazepines and benzodiazepines in combination with other drugs in drivers, examine benzodiazepine concentrations in drivers, and analyze the association of these factors with age and sex. METHODS: This study assessed data on Spanish drivers with confirmed drug-positive results recorded by the Spanish National Traffic Agency (Dirección General de Tráfico) between 2011 and 2016, accounting for 179,645 tests and 65,244 confirmed drug-positive tests. RESULTS: Benzodiazepines were confirmed in 4.3% of all positive roadside drug tests. In most of those cases (97.1%), other substances were also detected, particularly cocaine (75.3%) and cannabis (64.0%). The frequency of benzodiazepine-positive drivers (OR, 1.094; 95% CI, 1.088-1.100) increased with age, while the frequency of drivers who tested positive for benzodiazepines in conjunction with other substances, compared with drivers who tested positive for benzodiazepines alone, decreased with age (OR, 0.903; 95% CI, 0.825-0.988). Nordiazepam (54.8%) and alprazolam (46.9%) were the most common benzodiazepines detected. CONCLUSION: Concomitant use of benzodiazepines and other psychoactive substances was found to be a common behavior among drivers who tested positive on the road. It is important to raise awareness of all those involved in the consumption of driving-impairing substances (authorities, healthcare providers, patients and their families, etc.): roadside detection of driving-impairing substances is suggested, in addition to promoting the use of fewer driving-impairing medications and the provision of clear information to patients.

On-site rapid screening of benzodiazepines in dietary supplements using pipette-tip micro-solid phase extraction coupled to ion mobility spectrometry.

In this study, a reliable and novel coupling of pipette-tip micro-solid phase extraction (PT-µSPE) with corona discharge ionization-ion mobility spectrometry (CD-IMS) was developed for on-site fast detection of benzodiazepines (BZDs) in dietary supplements. The poly (styrene-co-divinylbenzene) (St-co-DVB) monoliths fabricated in pipette-tips through an in-situ polycondensation reaction. The extraction procedure of PT-µSPE for aqueous sample was performed for an appropriate number of aspirating/dispensing cycles by using a manual micropipettor. Then analytes retained in the polymeric monoliths were eluted with elution solvents by repeating the aspirating/dispensing cycles, and the eluates were analyzed by CD-IMS. In order to find the best extraction conditions, several parameters such as the number of aspirating/dispensing cycles for extraction and desorption, pH, elution solvent, amount of polymer and ionic strength were investigated. Under the optimal conditions, the LOD of this method was in the range of 5-15 ng mL-1. The spiked recovery values in real sample were obtained by PT-µSPE-IMS in the range of 84.2-112.1% and validated by ultraperformance liquid chromatography tandem mass spectrometry (UPLC-MS/MS). PT-µSPE as an on-site sampling method can be suitably combined to IMS which has great potential to achieve high throughput detection by using a multichannel micropipette. The satisfactory analytical results reveal that the developed method can be effectively applied for on-site screening BZDs in health products.

(±) Benzomalvins E isolated from Penicillium sp. SYPF 8411 in the rhizosphere soil of Codonopsis clematidea.

(+) Benzomalvins E (1) and (-) Benzomalvins E (2), a pair of epimeric derivatives, together with three known benzomalvins (3-5), were isolated from solid cultures of a interrhizospheric fungus Penicillium sp. SYPF 8411. The planar structure of (+) Benzomalvins E (1) has been previously reported. While, the absolute configuration of compound 1 was established by X-ray crystallographic analysis for the first time. The planar structure of the new compound 2 were elucidated by detailed interpretation of their HR ESI-TOF MS and NMR spectroscopic data. The absolute configuration of compound 2 was established by Rh2(OCOCF3)4-induced CD spectral data and the electronic circular dichroic (ECD) method. Furthermore, the epimerization induced by pH, temperature and H2O was revealed. Benzomalvins (1-3, 5), a type of indoximod, enhanced the cytotoxic capability of 5-fluorouracil against A549.

Voltammetric Electronic Tongue for the Simultaneous Determination of Three Benzodiazepines.

The presented manuscript reports the simultaneous detection of a ternary mixture of the benzodiazepines diazepam, lorazepam, and flunitrazepam using an array of voltammetric sensors and the electronic tongue principle. The electrodes used in the array were selected from a set of differently modified graphite epoxy composite electrodes; specifically, six electrodes were used incorporating metallic nanoparticles of Cu and Pt, oxide nanoparticles of CuO and WO3, plus pristine electrodes of epoxy-graphite and metallic Pt disk. Cyclic voltammetry was the technique used to obtain the voltammetric responses. Multivariate examination using Principal Component Analysis (PCA) justified the choice of sensors in order to get the proper discrimination of the benzodiazepines. Next, a quantitative model to predict the concentrations of mixtures of the three benzodiazepines was built employing the set of voltammograms, and was first processed with the Discrete Wavelet Transform, which fed an artificial neural network response model. The developed model successfully predicted the concentration of the three compounds with a normalized root mean square error (NRMSE) of 0.034 and 0.106 for the training and test subsets, respectively, and coefficient of correlation R ≥ 0.938 in the predicted vs. expected concentrations comparison graph.

New Brush-Type Chiral Stationary Phases for Enantioseparation of Pharmaceutical Drugs.

The importance of chirality in drug development is unquestionable, with chiral liquid chromatography (LC) being the most adequate technique for its analysis. Among the various types of chiral stationary phases (CSPs) for LC, brush-type CSPs provide the base for interaction analysis of CSPs and enantiomers, which provide valuable results that can be applied to interaction studies of other CSP types. In order to analyze the influence of aromatic interactions in chiral recognition, we designed a set of ten new brush-type CSPs based on (S)-N-(1-aryl-propyl)-3,5-dinitrobenzamides which differ in the aromatic unit directly linked to the chiral center. Thirty diverse racemates, including several nonsteroidal anti-inflammatory drugs and 3-hydroxybenzodiazepine drugs, were used to evaluate the prepared CSPs. Chromatographic analysis showed that the three new CSPs separate enantiomers of a wide range of compounds and their chromatographic behavior is comparable to the most versatile brush-type CSP-Whelk-O1. The critical role of the nonbonding interactions in positioning of the analyte (naproxen) in the cleft of CSP-6, as well as the analysis of interactions that make enantioseparation possible, were elucidated using computational methods. Furthermore, the influence of acetic acid as a mobile phase additive, on this enantiorecognition process was corroborated by calculations.

Analysis of Benzodiazepines for Drug-Facilitated Assaults and Abuse Settings (Urine).

An overview of the detection of benzodiazepines and their respective metabolites and target analytes in urine by LC-MS/MS is described. This overview shows substantial differences in the approach to detection using this technique including optional use of ß-glucuronidase to hydrolyze conjugates present in urine. There are also significant variations in the extraction method employed from the use of direct injection, liquid-liquid extraction to solid-phase extraction options, with little apparent difference in limits of detection. Chromatography was largely based on the use of C18-bonded columns; however both C8- and phenyl-bonded columns were used to affect separation. Modern-day tandem mass spectrometers are capable of exceptional sensitivity enabling detection of sub-nanogram per milliliter amounts in urine, which provide for longer detection times in the urine of suspected drug-facilitated assaults. A method employed in the laboratory of the authors is provided by way of an example for readers wishing to establish a method in their own laboratory.

Rapid determination of designer benzodiazepines, benzodiazepines, and Z-hypnotics in whole blood using parallel artificial liquid membrane extraction and UHPLC-MS/MS.

Benzodiazepines (BZD) and Z-hypnotics are frequently analyzed in forensic laboratories, and in 2012, the designer benzodiazepines (DBZD) emerged on the illegal drug scene. DBZD represent a particular challenge demanding new analytical methods. In this work, parallel artificial liquid membrane extraction (PALME) is used for sample preparation of DBZD, BZD, and Z-hypnotics in whole blood prior to UHPLC-MS/MS analysis. PALME of BZD, DBZD, and Z-hypnotics was performed from whole blood samples, and the analytes were extracted across a supported liquid membrane (SLM) and into an acceptor solution of dimethyl sulfoxide and 200 mM formic acid (75:25, v/v). The method was validated according to EMA guidelines. The method was linear throughout the calibration range (R2 > 0.99). Intra- and inter-day accuracy and precision, as well as matrix effects, were within the guideline limit of ± 15%. LOD and LLOQ ranged from 0.10 to 5.0 ng mL-1 and 3.2 to 160 ng mL-1, respectively. Extraction recoveries were reproducible and above 52%. The method was specific, and the analytes were stable in the PALME extracts for 4 and 10 days at 10 and - 20 °C. No carry-over was observed within the calibration range. PALME and UHPLC-MS/MS for the determination of DBZD, BZD, and Z-hypnotics in whole blood are a green and low-cost alternative that provides high sample throughput (96-well format), extensive sample clean-up, good sensitivity, and high reproducibility. The presented method is also the first method incorporating analysis of DBZD, BZD, and Z-hypnotics in whole blood in one efficient analysis. Graphical abstract.

An insight into the determination of trace levels of benzodiazepines in biometric systems: Use of crab shell powder as an environmentally friendly biosorbent.

A vortex assisted dispersive solid phase extraction approach (VADSPE) based on crab shell powder as biodegradable and biocompatible µ-sorbent was developed for simultaneous analysis of three benzodiazepines (BZPs): Oxazepam, Flurazepamand Diazepam, in biological matrixes included blood, nail, hair and urine samples. The effective parameters in VADSPE process, including the volume of uptake solvent, the dosage of sorbent, extraction time and back extraction time, were optimized using response surface methodology(RSM) based on central composite design(CCD). The suggested technique allows successful trapping of BZPs in a single-step extraction. Under the optimized extraction conditions, the proposed approach was exhibited low limits of detection (0.003-1.2 µg·mL-1), an acceptable linearity (0.04-20 µg·mL-1). Method performance was assessed by recovery experiments at spiking levels of 10 µg·mL-1(n = 5) for BZPs in blood, nail, hair and urine samples. Relative recoveries were determined by HPLC, which were between 36%and 95.6%.

Electromembrane extraction of substances with weakly basic properties: a fundamental study with benzodiazepines.

AIM: Electromembrane extraction (EME) of weakly basic benzodiazepines was investigated (-1.47 < pKa < 5.01). MATERIALS & METHODS: 96-well EME was performed with strongly acidic conditions in the acceptor solution using 250-mM trifluoroacetic acid to maximize ionization. RESULTS & CONCLUSION: Recoveries more than 80% were obtained for analytes with pKa > 2, whereas EME was less efficient for substances with pKa < 2. The latter was trapped in the supported liquid membrane due to less acidic pH conditions in the acceptor solution close to the supported liquid membrane. EME followed by UHPLC-MS/MS analysis was evaluated from human plasma, and the results were in compliance with EMA guidelines. Both electrokinetic migration and passive diffusion contributed to mass transfer when performing EME of weakly basic analytes.

Quantitative determination of risperidone, paliperidone and olanzapine in human serum by liquid chromatography-tandem mass spectrometry coupled with on-line solid-phase extraction.

A recent guideline recommends therapeutic drug monitoring for risperidone, paliperidone and olanzapine, which are frequently used second-generation antipsychotics. We developed a simple high-performance liquid chromatography-tandem mass spectrometry coupled with an online solid-phase extraction method that can be used to measure risperidone, paliperidone and olanzapine using small (40 µL) samples. The analytes were extracted from serum samples automatically pre-concentrated and purified by C8 (5 µm, 2.1 × 30 mm) solid-phase extraction cartridges, then chromatographed on an Xbidge™ C18 column (3.5 µm, 100 × 2.1 mm) thermostatted at 30°C with a mobile phase consisting of 70% acetonitrile and 30% ammonium hydroxide 1% solution at an isocratic flow rate of 0.3 mL/min, and detected with tandem mass spectrometry. The assay was validated in the concentration range from 2.5 to 160 ng/mL. Intra- and inter-day precision for all analytes was between 1.1 and 8.2%; method accuracy was between 6.6 and 7.6%. The risperidone and paliperidone assay was compared with a high-performance liquid chromatography-ultraviolet assay currently used in our hospital for risperidone and paliperidone therapeutic drug monitoring, and the results of weighted Deming regression analysis showed good agreement. For the olanzapine assay, we compared 20 samples in separate re-assays on different days; all the relative errors were within the 20% recommended limit.

Isolation, identification and characterization of two novel process-related impurities in olanzapine.

Olanzapine is a marketed antipsychotic agent for treatment of both positive and negative symptoms of schizophrenia. The chemical synthesis of olanzapine drug substance involved the reaction of thienobenzodiazepine hydrochloride with N-methylpiperazine. During the manufacture of olanzapine, two unknown impurities were present in pilot batches in the range of 0.08-0.22% based upon HPLC analysis. These unknown impurities were enriched from the mother liquor of reaction by preparative HPLC, and characterized by UV, FT-IR, LC-MS/TOF, 1D-NMR (1H, 13C, DEPT), 2D-NMR (1H-1H COSY, HSQC, HMBC, ROESY) and single-crystal X-ray diffraction analysis. Based on the complete spectral analysis and knowledge of the synthetic route of olanzapine, these two new impurities were identified as 2-methyl-4-(4-methyl piperazin-1-yl)-10-((methylthio)methyl)-thieno[2,3-b][1,5] benzodiazepine (impurity-I) and 10-(3-(1H-benzo[d]imidazol-2-yl)-5-methylthiophen-2-yl)-2-methyl-4-(4-methyl piperazin-1-yl)-thieno[2,3-b][1,5]benzodiazepine (impurity-II). Finally, prospects to the formation and controlling of impurity-I and II were discussed in detail.

Fabrication of magnetic zinc adeninate metal-organic frameworks for the extraction of benzodiazepines from urine and wastewater.

In this study, an alternative method for synthesizing magnetic cobalt adeninate metal-organic frameworks was developed, and the synthesized materials were examined for their potential application for separating and enriching benzodiazepines from complex samples. Benzodiazepines, widely used as hypnotics, muscle relaxants, sedatives, and anxiolytics, are a class of drugs that require accurate detection and monitoring. Results showed that Fe3 O4 nanoparticles could be well anchored onto the external surface of cobalt adeninate metal-organic frameworks by using amino-silane as a linkage. Their adsorption of benzodiazepines was mainly promoted by intermolecular hydrogen binding, π-π interactions and electrostatic attraction. Their potential application was evaluated by extraction of benzodiazepines in urine and wastewater samples prior to liquid chromatography with mass spectrometry. Under optimum conditions, the calibration curves were linear with a correlation coefficient of ≥0.9928 in the concentration range of 10-5000 ng/L for lorazepam and 5-5000 ng/L for estazolam, chlordiazepoxide, alprazolam, midazolam and triazolam. The limits of detection were in the range of 0.71-2.49 ng/L. The percent of extraction recoveries were 80.2-94.5% for urine and 84.1-94.4% for wastewater, respectively. Results suggested that magnetic cobalt adeninate metal-organic frameworks could potentially be a promising material for enriching benzodiazepines from urine and wastewater with high accuracy and precision.

Superparamagnetic Fe3 O4 @SiO2 core-shell composite nanoparticles for the mixed hemimicelle solid-phase extraction of benzodiazepines from hair and wastewater samples before high-performance liquid chromatography analysis.

Magnetic Fe3 O4 /SiO2 composite core-shell nanoparticles were synthesized, characterized, and applied for the surfactant-assisted solid-phase extraction of five benzodiazepines diazepam, oxazepam, clonazepam, alprazolam, and midazolam, from human hair and wastewater samples before high-performance liquid chromatography with diode array detection. The nanocomposite was synthesized in two steps. First, Fe3 O4 nanoparticles were prepared by the chemical co-precipitation method of Fe(III) and Fe(II) as reaction substrates and NH3 /H2 O as precipitant. Second, the surface of Fe3 O4 nanoparticles was modified with shell silica by Stober method using tetraethylorthosilicate. The Fe3 O4 /SiO2 composite were characterized by X-ray diffraction, scanning electron microscopy, Fourier transform infrared spectroscopy, and vibrating sample magnetometry. To enhance their adsorptive tendency toward benzodiazepines, cetyltrimethylammonium bromide was added, which was adsorbed on the surface of the Fe3 O4 /SiO2 nanoparticles and formed mixed hemimicelles. The main parameters affecting the efficiency of the method were thoroughly investigated. Under optimum conditions, the calibration curves were linear in the range of 0.10-15 µgmL(-1) . The relative standard deviations ranged from 2.73 to 7.07%. The correlation coefficients varied from 0.9930 to 0.9996.

High performance liquid chromatographic enantioseparation development and analytical method characterization of the carboxylate ester of evacetrapib using an immobilized chiral stationary phase with a non-conventional eluent system.

Using multiple HPLC chromatographic modes and various chiral columns in the context of an automated screening system, a potential separation was initially identified for the methyl ester of evacetrapib and its stereoisomers using an immobilized polysaccharide-based HPLC column. The bonded nature of this column, the Chiralpak(®) IC, allows for enhanced separation development with a diverse solvent range not amenable to standard coated chiral stationary phases. The ternary eluent system ultimately identified provided isomer resolutions not obtainable via the more established hexane/alcohol or polar organic chromatographic modes. A systematic separation development process is described, first for the resolution of the isomers, and later incorporating five potential impurities. A robust separation system was eventually developed that effectively resolves all compounds within a reasonable analysis time.

Comparison of the performance of different silicon-based SALDI substrates for illicit drug detection.

Surface-assisted laser desorption ionization mass spectrometry (SALDI-MS) is an emerging technique used for the detection of small molecules (<700 Da) such as illicit drugs. In recent times, this technique has been employed for the detection of illicit drugs in various body fluids including saliva. Three common SALDI techniques, desorption ionization on porous silicon (DIOS), nanostructure-initiator mass spectrometry (NIMS) and nanostructured laser desorption ionization (NALDI(™)) are compared for the detection of four drug classes, amphetamines, benzodiazepines, opiates and tropane alkaloids. We focus in our comparison on structural and chemical characteristics, as well as analytical performance and longevity.

Dynamic high performance liquid chromatography on chiral stationary phases. Low temperature separation of the interconverting enantiomers of diazepam, flunitrazepam, prazepam and tetrazepam.

Diazepam and the structurally related 1,4-benzodiazepin-2-ones tetrazepam, prazepam and flunitrazepam are chiral molecules because they adopt a ground state conformation featuring a non-planar seven membered ring devoid of any reflection-symmetry element. The two conformational enantiomers of this class of benzodiazepines interconvert rapidly at room temperature by a simple ring flipping process. Low temperature HPLC on the Whelk-O1 chiral stationary phase allowed us to separate the conformational enantiomers of diazepam and of the related 1,4-benzodiazepin-2-ones, under conditions where the interconversion rate is sufficiently low, compared to the chromatographic separation rate. Diazepam, tetrazepam and prazepam showed temperature dependent dynamic HPLC profiles with interconversion plateaus indicative of on-column enantiomer interconversion (enantiomerization) in the temperature range between -10 °C and -35 °C, whereas for flunitrazepam on-column interconversion was observed at temperatures between -40 °C and -66 °C. Simulation of exchange-deformed HPLC profiles using a computer program based on the stochastic model yielded the apparent rate constants for the on-column enantiomerization and the corresponding free energy activation barriers. At -20 °C the enantiomerization barriers, ΔG(≠), for diazepam, prazepam and tetrazepam were determined to be in the range 17.6-18.7 kcal/mol. At -55 °C ΔG(≠) for flunitrazepam was determined to be in the 15.6-15.7 kcal/mol range. The experimental dynamic chromatograms and the corresponding interconversion barriers reported in this paper call for a reinterpretation of previously published results on the HPLC behavior of diazepam on chiral stationary phases.

Dispersive micro-solid-phase extraction of benzodiazepines from biological fluids based on polyaniline/magnetic nanoparticles composite.

In this study, diverse types of Fe3O4 nanocomposites modified by polyaniline, polypyrrole, and aniline-pyrrole copolymer were synthesized through chemical oxidative polymerization process for dispersive-µ-solid phase extraction (D-µ-SPE) in the presence of various dopants. The results showed that the nanocomposite modified by polyaniline with p-toluene sulfonic acid as a dopant demonstrated higher extraction efficiency for lorazepam (LRZ) and nitrazepam (NRZ). Also the synthesized magnetic sorbents were characterized. The nanocomposite sorbent in combination with high performance liquid chromatography-UV detection was applied for the extraction, preconcentration and determination of lorazepam and nitrazepam in urine and plasma samples. Different parameters influencing the extraction efficiency including: sample pH, amount of sorbent, sorption time, elution solvent and its volume, salt content, and elution time were optimized. The obtained optimal conditions were: sample pH, 6; amount of sorbent, 5 mg; sorption time, 5.0 min; elution solvent and its volume, 0.5 mM cethyltrimethyl ammonium bromide in acetonitrile, 150 µL; elution time, 2.0 min and without addition of NaCl. The calibration curves were linear in the concentration range of 1-2000 µg L(-1). The limits of detection (LODs) were achieved in the range of 0.5-1.8 µg L(-1) for NRZ and 0.2-2.0 µg L(-1) for LRZ, respectively. The percent of extraction recoveries and relative standard deviations (n=5) were in the range of 84.0-99.0, 6.1-7.8 for NRZ and 90.0-99.0, 4.1-7.0 for LRZ, respectively. Ultimately, the applicability of the method was successfully confirmed by the extraction and determination of NRZ and LRZ in human urine and plasma samples.

Boseongazepines A-C, pyrrolobenzodiazepine derivatives from a Streptomyces sp. 11A057.

Three new pyrrolobenzodiazepine derivatives, boseongazepines A-C (1-3), were isolated from a culture broth of Streptomyces sp. 11A057, together with the known compound usabamycin B (4). The structures of 1-4 were determined through the analysis of spectroscopic data including extensive 1D-, 2D-NMR, and MS techniques. Cell growth inhibition effects of these compounds were evaluated against Jurkat, K-562, HL-60, and HepG2 cell lines.