Biochemical evaluation and ligand binding studies on glycerophosphodiester phosphodiesterase from Staphylococcus aureus using STD-NMR spectroscopy and molecular docking analysis.

Glycerophosphodiester phosphodiesterase (GDPD) is a highly conserved enzyme in both prokaryotic and eukaryotic organisms. It catalyses the hydrolysis of various glycerophosphodiesters into glycerol-3-phosphate and corresponding alcohols, which serve as building blocks in several biosynthetic pathways. This enzyme is a well-known virulence factor in many pathogenic bacteria, including Staphylococcus aureus, and is thus considered a potential drug target. In this study, competent E. coli BL21(DE3)pLysS expression cells were used to express the GDPD enzyme from vancomycin-resistant Staphylococcus aureus (VRSA), which was then purified using size exclusion and anion exchange chromatography. The hydrolytic activity of GDPD was evaluated on the non-physiological substrate bis(p-nitrophenyl) phosphate (BpNPP), which indicated functional activity of the enzyme. 79 drugs were evaluated for their inhibitory potential against GDPD enzyme by the colorimetric assay. Out of 79 drugs, 13 drugs, including tenofovir (1), adenosine (2), clioquinol (11), bromazepam (12), lamotrigine (13), sulfadiazine (14), azathioprine (15), nicotine (16), sitagliptin PO4 (17), doxofylline (18), clindamycin phosphate (19), gentamycin sulphate (20), and ceftriaxone sodium (21) revealed varying degrees of inhibitory potential with IC50 values in the range of 400 ± 0.007-951 ± 0.016 µM. All drugs were also evaluated for their binding interactions with the target enzyme by saturation transfer difference (STD-NMR) spectroscopy. 10 drugs demonstrated STD interactions and hence, showed binding affinity with the enzyme. Exceptionally, tenofovir (1) was identified to be a better inhibitor with an IC50 value of 400 ± 0.007 µM, as compared to the standard EDTA (ethylenediaminetetraacetic acid) (IC50 = 470 ± 0.008 µM). Moreover, molecular docking studies have identified key interactions of the ligand (tenofovir) with the binding site residues of the enzyme.

A Comparative Study between Screen-Printed and Solid-Contact Electrodes for the Stability-Indicating Determination of Bromazepam.

Stability-indicating methods are awesome tools to ensure the safety and efficacy of active pharmaceutical ingredients (APIs). An accurate comparative study involving the use of potentiometric sensors for the determination of bromazepam (BRZ) in the presence of its main product of degradation and impurity was performed by the fabrication of two membrane electrodes. A screen-printed electrode (SPE) and a solid-contact glassy carbon electrode (SCE) were fabricated and their performance optimized. The fabricated sensors showed a linear electrochemical response in the concentration range 1.0 × 10-6 M to 1.0 × 10-2 M. The electrodes exhibited Nernstian slopes of 59.70 mV/decade and 58.10 mV/decade for the BRZ-SPE and BRZ-SCE membrane electrodes, respectively. The electrochemical performance was greatly affected by the medium pH. They showed an almost ideal electrochemical performance between pH 3.0 and pH 6.0. The fabricated membranes were applied successfully for the quantification of BRZ in the presence of up to 90% of its degradation product. Moreover, a successful application of the fabricated electrodes was performed for the sensitive and selective quantification of BRZ in its tablet form without any pretreatment procedure.

First evaluation of the anxiolytic-like effects of a bromazepam­palladium complex in mice.

A significant fraction of patients are affected by persistent fear and anxiety. Currently, there are several anxiolytic drug options, however their clinical outcomes do not fully manage the symptoms. Here, we evaluated the effects of a bromazepam­palladium derivative [2-{(7-bromo-2-oxo-1,3-dihydro-2H-1,4-benzodiazepin-5-il)pyridinyl-κ2-N,N}chloropalladium(II)], [(BMZ)PdCl2], on fear/anxiety and memory-related behavior in mice. For this, female Swiss mice were treated intraperitoneally (i.p.) with saline (NaCl 0.9%) or [(BMZ)PdCl2] (0.5, 5.0, or 50 µg/kg). After 30 min, different tests were performed to evaluate anxiety, locomotion, and memory. We also evaluated the acute toxicity of [(BMZ)PdCl2] using a cell viability assay (neutral red uptake assay), and whether the drugs mechanism of action involves the γ-aminobutyric acid type A (GABAA) receptor complex by pre-treating animals with flumazenil (1.0 mg/kg, i.p., a competitive antagonist of GABAA-binding site). Our results demonstrate that [(BMZ)PdCl2] induces an anxiolytic-like phenotype in the elevated plus-maze test and that this effect can be blocked by flumazenil. Furthermore, there were no behavioral alterations induced by [(BMZ)PdCl2], as evaluated in the light-dark box, open field, and step-down passive avoidance tests. In the acute toxicity assay, [(BMZ)PdCl2] presented IC50 and LD50 values of 218 ± 60 µg/mL and 780 ± 80 mg/kg, respectively, and GSH category 4. Taken together, our results show that the anxiolytic-like effect of acute treatment with [(BMZ)PdCl2] occurs through the modulation of the benzodiazepine site in the GABAA receptor complex. Moreover, we show indications that [(BMZ)PdCl2] does not promote sedation and amnesia and presents the same toxicity as the bromazepam prototype.

Voltage-clamp evidence of GABAA receptor subunit-specific effects: pharmacodynamic fingerprint of chlornordiazepam, the major active metabolite of mexazolam, as compared to alprazolam, bromazepam, and zolpidem.

BACKGROUND: Anxiolytic benzodiazepines, due to their clinical effectiveness, are one of the most prescribed drugs worldwide, despite being associated with sedative effects and impaired psychomotor and cognitive performance. Not every GABAA receptor functions in the same manner. Those containing α1 subunits are associated with sleep regulation and have a greater effect on the sedative-hypnotic benzodiazepines, whereas those containing α2 and/or α3 subunits are associated with anxiety phenomena and have a greater effect on the anxiolytic benzodiazepines. Therefore, characterization of the selectivity profile of anxiolytic drugs could translate into a significant clinical impact. METHODS: The present study pharmacodynamically evaluated chlornordiazepam, the main active metabolite of mexazolam, upon GABAA receptors containing α2 and/or α3, anxiety-related, and those containing an α1 subunit, associated with sleep modulation. RESULTS: As shown by whole-cell patch-clamp data, chlornordiazepam potentiated GABA-evoked current amplitude in α2 and α3 containing receptors without changing the current amplitude in α1 containing receptors. However, current decay time increased, particularly in GABAA receptors containing α1 subunits. In contrast, other anxiolytic benzodiazepines such as alprazolam, bromazepam, and zolpidem, all increased currents associated with GABAA receptors containing the α1 subunit. CONCLUSIONS: This novel evidence demonstrates that mexazolam (through its main metabolite chlornordiazepam) has a "pharmacodynamic fingerprint" that correlates better with an anxiolytic profile and fewer sedative effects, when compared to alprazolam, bromazepam and zolpidem, explaining clinical trial outcomes with these drugs. This also highlights the relevance of the pharmacological selectivity over GABAA receptor subtypes in the selection of benzodiazepines, in addition to their clinical performance and pharmacokinetic characteristics.

A case of schizophrenia with congenital color vision deficiency: From the perspective of color universal design to promote medication adherence.

Color-blindness, or more accurately, color vision deficiency (CVD), which is the inability or decreased ability to distinguish different colors, is one of the commonest visual disorders. Patients with schizophrenia usually have multiple types of visual processing impairments, including color vision impairments. Here, we present a case of schizophrenia with congenital CVD. The patient was aware of his color deficiency since elementary school. We assessed his ability to distinguish medicines based on their color, including those that he had been previously prescribed. Although he could distinguish all of the tablets, he could not distinguish the color of the blister packs, specifically that of the bromazepam 2 mg pack (green) from the 1 mg pack (red). This case suggests that CVD patients might misunderstand the color of medications, which might lead to medication errors, or poor drug adherence. The color universal design principle should be considered when designing tablets and their blister packs, in order to improve medication adherence.

Bromazepam increases the error of the time interval judgments and modulates the EEG alpha asymmetry during time estimation.

AIM: This study investigated the bromazepam effects in male subjects during the time estimation performance and EEG alpha asymmetry in electrodes associated with the frontal and motor cortex. MATERIAL AND METHODS: This is a double-blind, crossover study with a sample of 32 healthy adults under control (placebo) vs. experimental (bromazepam) during visual time-estimation task in combination with electroencephalographic analysis. RESULTS: The results demonstrated that the bromazepam increased the relative error in the 4 s, 7 s, and 9 s intervals (p = 0.001). In addition, oral bromazepam modulated the EEG alpha asymmetry in cortical areas during the time judgment (p ≤ 0.025). CONCLUSION: The bromazepam decreases the precision of time estimation judgments and modulates the EEG alpha asymmetry, with greater left hemispheric dominance during time perception. Our findings suggest that bromazepam influences internal clock synchronization via the modulation of GABAergic receptors, strongly relating to attention, conscious perception, and behavioral performance.

[Potentially inappropriate psychotropic drug prescription in elderly people in West Occitanie area]. / Prescription de psychotropes potentiellement inappropriés chez les sujets âgés en Occitanie Ouest.

AIM: Psychotropic drugs remain frequently prescribed in elderly people. Some of them are classified as « inappropriate ¼ because they could increase the risk of adverse drug reactions. The aim of this study is to evaluate the prevalence of exposure to potentialy inappropriate psychotropic drugs (PIPs) in the elderly living in nursing home residents (EHPAD) in West Occitanie area. METHODS: We carried out a retrospective study on the cohort of PAAPI program (Personne âgée et amélioration des prescriptions inappropriées) participating in PAAPI program set up in West Occitanie in 2016 including 3095 patients during 2 years. PIPs were identified by the list EU(7)PIM and completed with the guidelines mailed by the National Drug Safety Agency. We measured the prevalence of exposure to PIPs in this population. RESULTS: The majority of the residents (n=2301, 74.4%) were female of the average age was 87.1±8.1 years and. The prevalence of exposure to psychotropic drugs was about 77.5% with an average of 1.6 prescription lines per patient (±1,1). We found antidepressants (36.5% of PIP) with mainly paroxetine (37.3% of antidepressants PIP), followed by venlafaxine (32.8%), hypnotics and sedatives (26.6% of PIP) with zopiclone with inappropriate dose (59.4% of hypnotics PIP), anxiolytics (25.8% of PIP) with alprazolam (37.9% of anxiolytics PIP), followed by bromazepam (16%) and antipsychotics (11.2% of PIP) with cyamemazine (100% of inappropriate prescription) followed by aripiprazole and haloperidol (respectively 100% and 14.7% of inappropriate prescription). CONCLUSION: According to our data, third of elderly people in Nursing Homes were exposed to a PIP suggesting that guidelines about psychotropic drugs prescription are not well followed. Then, information campaign of healthcare professionnals could be useful to improve psychotropic drug prescription in the elderly population.

Prevalence and concentrations of sedative-hypnotic drugs in blood of drivers involved in road traffic crashes in the Padova region of Italy - not so easy to interpret.

BACKGROUND & OBJECTIVES: This study reports the prevalence and concentrations of sedative-hypnotic drugs as exemplified by benzodiazepines (BZD) and zolpidem (Z-hypnotic) in blood samples from drivers involved in road traffic accidents (RTA) in the Padova region of Italy. Another aim of the study was to estimate the prevalence of these drugs with concentrations in blood above the therapeutic intervals and above specific per se limits. METHODS: A total of 4066 blood samples collected from drivers involved in RTA were analysed for the presence of alcohol, drugs of abuse and medicinal drugs with sedative-hypnotic properties. Prevalence of drivers positive for BZDs and zolpidem were reported according to the reporting limit of our laboratory (1 ng/mL) in a sort of zero tolerance approach and compared with the prevalence according to analytical cut-offs used in the "European Union's research project on Driving Under the Influence of Drugs, Alcohol and Medicines" (DRUID). The impairment-based, per se limits adopted in Norway and in England and Wales and the values used to define "therapeutic ranges" in blood and in plasma/serum were also applied to the case study. RESULTS: 175 blood samples were positive for sedative-hypnotics above 1 ng/mL, with the following prevalence: diazepam 44%, nordazepam 41.8%, lorazepam 32.6%, zolpidem 28%, oxazepam 25.6%, alprazolam 16%, delorazepam 11,6%, lormetazepam 11,6%, temazepam 11.6%, clonazepam 11.6%, triazolam 6.9%, N-desalkylflurazepam 4.6%, bromazepam 2.3%. When applying DRUID analytical cut-offs, the prevalence of BZDs and zolpidem sharply decreases. Applying the impairing cut-offs used in Norway, 56% of positive samples were above the limits equivalent to a BAC of 0.2 g/L, 39% above the limits corresponding to 0.5 g/L, and 23% above the cut-off corresponding to 1.2 g/L. Only 1% of the drivers had drug concentrations above the per se concentration limits adopted in England and Wales [26]. When comparing blood levels with therapeutic ranges in plasma, bromazepam, lormetazepam and delorazepam were often found above the highest limits. The adjustment of the concentrations with the plasma-to-blood ratios causes a significant increase of cases above the therapeutic ranges in plasma. CONCLUSIONS: Sedative-hypnotic drugs are medicinal substances frequently identified in drivers involved in RTA, commonly in concentrations associated with driving impairment. Besides the concentrations of drugs in blood, several factors have to be considered to conclude that a driver was impaired. The frequent association with alcohol, cocaine and other BZDs, confirms the abuse potential of these medications.

Development and validation of a stability indicating RP-HPLC-DAD method for the determination of bromazepam.

A reliable, selective and sensitive stability-indicating RP-HPLC assay was established for the quantitation of bromazepam (BMZ) and one of the degradant and stated potential impurities; 2-(2-amino-5-bromobenzoyl) pyridine (ABP). The assay was accomplished on a C18 column (250 mm × 4.6 mm i.d., 5 µm particle size), and utilizing methanol-water (70: 30, v/v) as the mobile phase, at a flow rate of 1.0 ml min-1. HPLC detection of elute was obtained by a photodiode array detector (DAD) which was set at 230 nm. ICH guidelines were adhered for validation of proposed method regarding specificity, sensitivity, precision, linearity, accuracy, system suitability and robustness. Calibration curves of BMZ and ABP were created in the range of 1-16 µg mL-1 with mean recovery percentage of 100.02 ± 1.245 and 99.74 ± 1.124, and detection limit of 0.20 µg mL-1 and 0.24 µg mL-1 respectively. BMZ stability was inspected under various ICH forced degradation conditions and it was found to be easily degraded in acidic and alkaline conditions. The results revealed the suitability of the described methodology for the quantitation of the impurity (ABP) in a BMZ pure sample. The determination of BMZ in pharmaceutical dosage forms was conducted with the described method and showed mean percentage recovery of 99.39 ± 1.401 and 98.72 ± 1.795 (n = 6), respectively. When comparing the described procedure to a reference HPLC method statistically, no significant differences between the two methods in regard to both accuracy and precision were found.

Information about benzodiazepines: what does the internet offer us? / Informação sobre benzodiazepínicos: o que a internet nos oferece?

This study analysed the quality of information published on the internet regarding 4 benzodiazepines that are widely used in Brazil: alprazolam, bromazepam, clonazepam and diazepam. This choice is justified by the fact that these drugs are widely used and can generate chemical dependency, and the internet is an important source of information about them. We analysed 20 sites for each drug. More than half (56.3%) of the sites were classified as deficient or very deficient. The most frequent problems with the sites were the absence of a description of the person responsible for the site (60%), incomplete information (62.5%), the absence of a contact for additional information (45%) and the absence of the last date the site was updated (82%). These results reinforce concerns regarding the quality of the health information published on the internet, which has already been noted in the literature, and the need to adopt minimum quality criteria for this information.

Este trabalho analisou a qualidade da informação veiculada na internet sobre 4 benzodiazepínicos amplamente utilizados no Brasil: alprazolam, bromazepam, clonazepam e diazepam. Essa escolha se justifica pelo fato desses medicamentos serem amplamente utilizados, poderem gerar dependência química e a internet ser importante fonte de informação sobre eles. Foram analisados 20 sites para cada medicamento. Mais da metade (56,3%) dos sites foram classificados como deficientes ou muito deficientes. Os problemas mais frequentes foram a ausência da descrição do responsável pelo sítio (60%), informação incompleta (62,5%), ausência de contato para informação adicional (45%) e da última data da atualização (82%). Os resultados reforçam a preocupação com a qualidade da informação em saúde veiculada na internet, já apontada pela literatura, e a necessidade de adoção de critérios mínimos de qualidade para esta informação.

Stability indicating spectrophotometric methods for quantitative determination of bromazepam and its degradation product.

Four simple, sensitive and selective stability indicating spectrophotometric methods are presented for quantitative determination of the benzodiazepine drug; bromazepam (BMZ) and one of its reported potential impurities and degradation product; 2-(2-amino-5-bromobenzoyl) pyridine (ABP) in methanol. Method A, is isoabsorptive point coupled with D0 method, where good linearity was obtained by measuring the absorbance of BMZ at 264 nm (Aiso) in the concentration range of 2-25 µg mL-1, and the absorbance of ABP at its λmax 396 nm in concentration range of 0.5-24 µg mL-1. Method B, is ratio subtraction; the absorbance was measured at 233 nm for BMZ using 20 µg mL-1 of ABP, while ABP was determined directly at its λmax 396 nm using methanol as a solvent. Method C, was based on measuring the total peak amplitude of the first derivative of the ratio spectra (DD1) of BMZ from 301 to 326 nm using 10 µg mL-1 of ABP as a divisor and determination of ABP at peak amplitude of 293 nm using 5 µg mL-1 of BMZ as a divisor. In method D, ratio difference method, good linearity was achieved for determination of BMZ and ABP by measuring the differences between the amplitudes of ratio spectra at 312 nm and 274 nm and differences between the amplitudes of ratio spectra at 274 nm and 312 nm, respectively. The stability of BMZ was investigated under different ICH recommended forced degradation conditions. The suggested methods were then successfully applied for determination of BMZ in its pharmaceutical formulations.

Kinking of Bilateral Internal Carotid Arteries as Cause of Cognitive Dysfunction.

INTRODUCTION: Dolichoarteriopathies of the internal carotid artery (DICAs) is divided into three forms: tortuous, coiling and kinking. In case of kinking, internal carotid artery forms a sharp angle of <90 degrees, while in the background there is metaplasia of a tunica media with unknown etiology. The association with stroke is still questionable, but it is believed that it can be associated with cerebral ischemia and with clinical symptomatology that accompanies cerebral ischemia. AIM: Aim of article was to present diagnostic and therapeutic modality of patient with verified internal carotid artery kinking. CASE REPORT: The 55-year-old male patient was admitted to the Department of Neurology, General Hospital «Prim.dr. Abdulah Nakas¼, due to dizziness and instability while walking, forgetfulness, memory loss and low mood. He has previously been reported to be hypertensive and with diagnosis of diabetes mellitus and dyslipidemia. Doppler sonography also suspects on distal subocclusion of the internal carotid artery (low flow rates were observed). Diagnostic transcranial Doppler (TCD) of vertebrobasilar artery showed decreased blood flow velocities in both vertebral and basilar artery and indicated atherosclerotic altered blood vessels of the brain. CTA findings indicate bilateral kinking of internal carotid artery with right duplex Kinking. SPECT with 15 mCi 99mTc-hexamethylpropyleneamineoxime (99mTc-HMPAO) verified global cortex hypoperfusion, indicating chronic vascular failure. The patient was treated with acetylsalic acid, clopidogrel, atorvastatin, donepezil, memantine, escitalopram, bromazepam, along with antihypertensive and antidiabetic therapy (per os). CONCLUSION: A severe degree of kinking can cause neurological symptomatology, especially if it is bilateral. Symptoms of cerebrovascular disease are more pronounced when autoregulation of cerebral hemodynamics is impaired. Bilateral severe degree of kinking possibly can cause cognitive impairment. Diagnosis, analysis of the existence of possible risk factors for the onset, and the existence of genetic predisposition are a prerequisite for better understanding of the disease and optimal treatment.

Analysis of Complexation Interactions between Metal Ions and Drugs under Pseudo-physiological pH Conditions by a High-throughput Screening Method Using a Solid-phase Extraction Cartridge.

A high-throughput screening method for the complexation between metal ions and drugs was established by combining solid-phase extraction (SPE) using a nitrilotriacetic acid (NTA) modified silica spin cartridge with subsequent HPLC analysis. First, a test metal ion solution was passed through the NTA cartridge, then a test drug solution diluted in phosphate buffered saline (pH 7.4) was passed through the metal-chelated NTA cartridge. The complexation behavior between the metal and the drug on the NTA cartridge was evaluated by HPLC quantification of the drug in the SPE eluate. Comprehensive analysis of the complexation behavior between 11 different metal ions and 55 drugs showed that Cu2+, Ni2+, Co2+, Zn2+, Cr3+ and Fe3+ formed complexes with 12, 5, 4, 2, 1 and 1 kinds of drugs, respectively. Bromazepam selectively formed complexes with Cu2+, Ni2+ and Co2+.

Development and Validation of an Analytical Method for the Detection and Quantification of Bromazepam, Clonazepam and Diazepam by UPLC-MS/MS in Surface Water.

The development of analytical methods capable of determining micropollutants is essential for quality control of drinking water. Benzodiazepines, a class of pharmaceuticals with anxiolytic properties, have received increasing attention as micropollutants. The purpose of this study was to develop an analytical method for determination of three benzodiazepine drugs (bromazepam, clonazepam and diazepam) in surface water. For the extraction of the matrix analytes, SPE cartridges (C18, 500 mg/3 mL) were used. The method was validated according to the quality criteria of the USEPA 8000D Validation Guide. The developed and validated method showed recovery values between 57 and 100%, RSD < 20% and R2 > 0.9949. LD ranged between 2.70 and 5.00 ng L-1 for bromazepam and clonazepam respectively whereas LQ was 0.01 µg L-1 for all analytes. The matrix affected the signal intensity of clonazepam thus evidencing the matrix effect by analysis statistic (F test).

Effect of Evening Bromazepam Administration on Blood Pressure and Heart Rate in Mild Hypertensive Patients.

AIM: To assess the effects of chronic evening oral administration of bromazepam alone or in combination with propranolol on ambulatory blood pressure (BP) and heart rate (HR) in mild hypertensive subjects. METHODS: Thirty-seven mild hypertensive patients after a 2-week placebo period were randomized to bromazepam 3 mg, propranolol 40 mg, bromazepam 3 mg plus propranolol 40 mg or placebo for 2 weeks according to a double-blind, double dummy, cross-over design. After each treatment period, 24-h BP and HR ambulatory monitoring was performed by using a non-invasive device. RESULTS: Ambulatory monitoring showed that during night-time SBP and DBP values were unaffected by bromazepam as compared to placebo, whereas SBP was significantly reduced by propranolol both when taken alone and in combination with bromazepam. HR nocturnal values were significantly reduced by propranolol, whereas they were significantly increased by bromazepan both when taken alone (+11.5%, p < 0.05 vs. placebo) and in combination with propranolol (+12.8%, p < 0.05 vs. propranolol). No significant difference in day-time values of SBP, DBP and HR was observed among the 4 treatment groups. CONCLUSIONS: In mild hypertensive patients, evening consumption of bromazepam for a 2-week period did not affect BP, while it increased nocturnal HR. Such an increase was observed both when bromazepam was taken alone and in combination with propranolol, which suggests that it depends on a bromazepam mediated decrease in vagal tone. Whatever the mechanism, the HR nocturnal increase might be of clinical relevance, due to the role of high HR as cardiovascular risk factor, particularly in already at risk hypertensive subjects.

Activation of Anxiogenic Circuits Instigates Resistance to Diet-Induced Obesity via Increased Energy Expenditure.

Anxiety disorders are associated with body weight changes in humans. However, the mechanisms underlying anxiety-induced weight changes remain poorly understood. Using Emx1Cre/+ mice, we deleted the gene for brain-derived neurotrophic factor (BDNF) in the cortex, hippocampus, and some amygdalar subregions. The resulting mutant mice displayed impaired GABAergic transmission and elevated anxiety. They were leaner when fed either a chow diet or a high-fat diet, owing to higher sympathetic activity, basal metabolic rate, brown adipocyte thermogenesis, and beige adipocyte formation, compared to control mice. BDNF re-expression in the amygdala rescued the anxiety and metabolic phenotypes in mutant mice. Conversely, anxiety induced by amygdala-specific Bdnf deletion or administration of an inverse GABAA receptor agonist increased energy expenditure. These results reveal that increased activities in anxiogenic circuits can reduce body weight by promoting adaptive thermogenesis and basal metabolism via the sympathetic nervous system and suggest that amygdalar GABAergic neurons are a link between anxiety and metabolic dysfunction.

Performance Comparison Between Monolithic, Core-Shell, and Totally Porous Particulate Columns for Application in Greener and Faster Chromatography.

Background: The introduction of monolithic rods and core-shell particles as new morphologies of packing materials different from the conventional totally porous particles resulted in a leap forward for performance in LC. Meanwhile, environmental safety has become increasingly important in many areas, especially in industry and research laboratories. Objective: This study compared the efficiencies of commercially available columns of different lengths and diameters when greener chromatographic conditions were utilized. The main purpose of this study is to help practitioners select the most appropriate stationary phase for faster and greener analysis. Methods: The three types of stationary phases were compared in terms of separation efficiency, number of theoretical plates, peak shape, selectivity, resolution, analysis time, mobile phase consideration, and permeability using six drug molecules. Results: Results indicated that core-shell and monolithic stationary phases had superiority over the conventional totally porous particles in terms of efficiency and speed of analysis. Monolithic rods had lower column backpressure and higher permeability, so they are more suitable for higher mobile phase flow rates and viscosities. However, core-shell particles provided enhanced peak shapes and number of theoretical plates. Conclusions: The choice will depend on the main purpose of analysis and the composition of the mobile phase. Compromise must be made to obtain the best trade-off between separation efficiency and analysis speed. Highlights: This study is the first to consider green chromatography concepts for the selection of the best stationary phase of new morphologies.

Evaluation of benzodiazepines and zolpidem in nails and their stability after prolonged exposure to chlorinated water.

The study aims the development and validation of a LC-MS/MS method for the identification and quantification of benzodiazepines and zolpidem in nails as alternative keratinized matrix to hair in long-term monitoring of anxiolytic and hypnotic drugs. Both fingernail and toenail samples (1-2 mm) were collected by clipping the excess overhang of the nail from volunteers and from postmortem cases. They were washed twice with organic solvents, dried under nitrogen stream, pulverized, immersed in a methanol solution (internal standard: diazepam-D5) and sonicated up to two hours. The solution was then direct injected in the LC-MS/MS system. Mass spectrometry was set in MRM mode, selecting two transitions for each substance. 32 analytes among benzodiazepines, metabolites and hypnotics were included in the list. The method fulfilled the internationally required criteria for validation. Limits of detection ranged from 0.03 pg/mg (zolpidem) to 13.1 pg/mg (bromazepam). 9 subjects under therapy were positive at 7 different benzodiazepines and/or metabolites (lorazepam, desalkylflurazepam, bromazepam, diazepam, alprazolam, lormetazepam and prazepam), while 5 molecules were measured in 4 postmortem cases (diazepam, desmethyldiazepam, delorazepam, 7-aminoclonazepam and zolpidem). In vitro experiments on eight authentic samples suggested that benzodiazepines in nails are influenced by the prolonged exposure to chlorinated water.