Plasma Diazepam Research Articles

Determination of diazepam in human plasma by developed and validated a high-performance liquid chromatographic ultraviolet method

Background and Aims: Diazepam is accepted as a safer drug to medicate in many serious cases, acting as an anticonvulsant, an anxiolytic and a treatment for many types of poisoning. Monitoring it is important in achieving successful treatment and reducing the risk of toxic effects. In this study, it is aimed to develop and validate a sensitive, repeatable, and reliable method based on high-performance liquid chromatographic analysis for the determination in human plasma. Methods: Separation was carried out using a reverse-phase C18 column (4.0 mm x 150 mm, 3 μm) at 30 °C. The solution was prepared with a 10 mM phosphate buffer and acetonitrile (1:1, v/v) was employed as a mobile phase at the isocratic flow with 0.5 mL/min rate. Quantification was applied at 230 nm. A solid-phase extraction method was established and optimized, which was then used in the preparation of the plasma (0.5 mL) samples to the analysis. Results: The method was found to be linear (r2 = 0.9805) between 100 and 1200 ng/mL. The analysis run was ≤12 min. Intraday and inter-day accuracy were found between -5.78 and 5.93 and precision was ≤1.82%. The limit of detection and quantification were calculated as 20.42 and 61.86 ng/mL, respectively. Recovery was found between the range of 95.12% and 106.83%. The method was determined to be robust according to changes in UV, mobile phase organic solvent content, mobile phase pH, column temperature, and operator. Conclusion: This simple, sensitive and reliable method is suggested for accredited-reference laboratories working on the therapeutic drug monitorization and/or overdose-toxicological quantified analysis of diazepam in human plasma.

Lack of clinically relevant differences in safety and pharmacokinetics after second‐dose administration of intranasal diazepam within 4 h for acute treatment of seizure clusters: A population analysis

ObjectiveCurrent diazepam nasal spray labeling requires waiting 4 h before administering a second dose. The objective of the current analyses was to examine safety and pharmacokinetic profiles of second doses of diazepam nasal spray given 0−4 h after the first dose.MethodsTwo datasets were analyzed. The first, a long‐term, repeat‐dose safety study of diazepam nasal spray, compared rates of treatment‐emergent adverse events (TEAEs), serious TEAEs, and treatment‐related TEAEs for patients receiving ≥1 second dose ≤4 h versus all second doses >4 h after the first. The second was a population pharmacokinetic analysis using data from three phase 1 studies to model drug exposure when a second dose of diazepam nasal spray was administered across multiple time points (1 min−4 h) following the first dose.ResultsIn the repeat‐dose safety study, a second dose of diazepam nasal spray was administered ≤24 h after the first to treat 485 seizure clusters in 79 patients. Rates of TEAEs were similar between patients receiving ≥1 second dose in ≤4 h (89.5%, n = 38) compared with >4–24 h only (80.5%, n = 41). The most common treatment‐related TEAEs were associated with nasal discomfort, which was mild or moderate and transient. There were no reports of respiratory or cardiac depression. The pharmacokinetic simulations of second doses predicted comparable elevations of plasma diazepam concentrations with administrations across a range of intervals after the first dose (1 min−4 h).SignificanceThese data indicate that the safety and pharmacokinetic profiles of a second dose of diazepam nasal spray administered within 4 h of the first dose are consistent with those associated with current labeling. This is potentially important for patients with seizure clusters who have a recurrent seizure within 4 h of first treatment and might benefit from immediate retreatment to reduce the risk of progression to status epilepticus.

Effects of CYP2C19*17 genetic polymorphisms on plasma and saliva concentrations of diazepam in patients with alcohol withdrawal syndrome.

Diazepam is one of the most commonly prescribed tranquilizers for the therapy of alcohol withdrawal syndrome (AWS). However, diazepam therapy often turns out to be ineffective, and some patients experience dose-dependent adverse drug reactions. Previous studies have shown that the metabolism of diazepam involves the CYP2C19 isoenzyme, whose activity is highly dependent on polymorphism of the encoding gene. The study aimed to investigate the effects of CYP2C19*17 genetic polymorphisms on plasma and saliva concentrations of diazepam as well as its impact on the efficacy and safety rates of therapy in patients with AWS. The study was conducted on 100 Russian male patients suffering from the AWS who received diazepam injections at a dosage of 30.0 mg/day for 5 days. Genotyping was performed by real-time PCR with allele-specific hybridization. The efficacy and safety assessment was performed using psychometric scales. Based on the results of the study, we revealed differences in the efficacy and safety of therapy in patients with different CYP2C19 -806C>T genotypes. Therapeutic drug monitoring revealed the statistically significant difference in the levels of diazepam plasma concentration: (CC) 251.76 (214.43; 310.61) vs. (CT+TT) 89.74 (54.18; 179.13); P = 0.003, and diazepam saliva concentration: (CC) 3.86 (3.22; 5.12) vs. (CT+TT) 0.79 (0.44; 1.56); P = 0.003. Our study showed the effects of CYP2C19*17 genetic polymorphisms on the efficacy and safety rates of diazepam. Furthermore, we revealed the statistically significant differences in plasma and saliva concentration levels of diazepam in patients carrying different genotypes.

High-throughput and trace analysis of diazepam in plasma using DART-MS/MS and its pharmacokinetic application

A high-throughput quantitative analytical method based on Direct Analysis in Real Time tandem mass spectrometry (DART-MS/MS) has been developed and validated for the determination of diazepam in rat plasma, whereby analyzing of each sample needs merely 25 μL plasma, simple solid phase extraction sample preparation and 15 s acquisition time. The multiple reaction monitoring (MRM) transitions at m/z 285.2 → 193.1 and 316.0 → 270.0 were selected for the monitoring of diazepam and its internal standard clonazepam respectively. A good linearity within the range of 10–2000 ng/mL, an intra- and inter-day precisions within <7.78% as to an accuracy ranging from 1.04% to 7.92% have been achieved. The method has been successfully applied to the pharmacokinetic study of diazepam in rats’ plasma after a single intragastric administration at a dose of 10 mg/kg. The results indicate that this method fulfills the requirements of the bioanalysis in sensitivity and accuracy. It shows considerable promise for application of DART-MS to the quantitative investigation of other drugs.

Kinetic disposition of diazepam and its metabolites after intravenous administration of diazepam in the horse: Relevance for doping control.

In horses, the benzodiazepine diazepam (DIA) is used as sedative for pre-medication or as an anxiolytic to facilitate horse examinations. As the sedative effects can also be abused for doping purposes, DIA is prohibited in equine sports. DIA is extensively metabolized to several active metabolites such as nordazepam, temazepam and oxazepam (OXA). For veterinarians, taking into account the detection times of DIA and its active metabolites is needed for minimizing the risk of an anti-doping rule violation. Therefore, a pharmacokinetic study on 6horses was conducted using a single intravenous (IV) dose of 0.2mg/kg DIA Plasma and urine samples were collected at specified intervals until 16 and 26days post-administration, respectively. Samples were analysed by a sensitive liquid chromatography-electrospray ionization/tandem mass spectrometry method. DIA showed a triphasic elimination pattern in the horse. The mean plasma clearance of DIA was 5.9ml/min/kg, and the plasma elimination half-life in the terminal phase was 19.9h. Applying the Toutain model approach, an effective plasma concentration of DIA was estimated at 24ng/ml, and irrelevant plasma concentration (IPC) and irrelevant urine concentration (IUC) were computed to 0.047 and 0.1ng/ml, respectively. The detection time according to the European Horserace Scientific Liaison Committee (EHSLC), that is the time for which observed DIA plasma concentrations of all investigated horses were below the IPC was 10days. Using Monte Carlo Simulations, it was estimated that concentrations of DIA in plasma would fall below the IPC 18days after the DIA administration for 90% of horses. However, in the present study, a single administration of DIA could be detected for 24days in urine via the presence of OXA, its dominant metabolite.

Relations of CYP2C19*2 genetic polymorphisms to plasma and saliva concentrations of diazepam in patients hospitalized for alcohol withdrawal

Diazepam is one of the most widely prescribed tranquilizers for the therapy of alcohol withdrawal syndrome (AWS). However, diazepam therapy often turns out to be ineffective, and some patients experience dose-dependent adverse drug reactions. Previous studies have shown that the metabolism of diazepam involves the CYP2C19 isoenzyme, whose activity is highly dependent on polymorphism of the encoding gene. The objective of our study was to investigate the effects of CYP2C19*2 genetic polymorphisms on plasma and saliva concentrations of diazepam as well as its impact on the efficacy and safety rates of therapy in patients with AWS. The study was conducted on 100 Russian male patients with AWS who received diazepam in injections at a dosage of 30.0 mg/day for 5 days. Genotyping was performed by real-time polymerase chain reaction. The efficacy and safety assessment was performed using psychometric scales. We revealed differences in the efficacy and safety of therapy in patients with different CYP2C19 681G&gt;A genotypes. Therapeutic drug monitoring (TDM) revealed the statistically significant differences in the levels of diazepam plasma concentration: (GG) 199.83 [82.92; 250.58] vs (GA+AA) 313.47 [288.99; 468.33], p=0.040, and diazepam saliva concentration: (GG) 2.80 [0.73; 3.80] vs (GA+AA) 5.33 [5.14; 6.00], p=0.003).

Mathematical Modeling of In Vitro‐In Vivo Atypical Kinetics of Diazepam

Cytochrome P450 (CYP) and uridine 5'‐diphospho‐glucuronosyltransferase (UGT) enzymes are responsible for metabolism of most marketed drugs. Due to the large binding sites of certain CYPs and UGTs, CYP and UGT substrates tend to show non ‐ Michaelis‐Menten kinetics (atypical kinetics). Although several in vitro data provide evidence for the existence of atypical kinetics, this characteristic is seldom observed with in vivo data. The purpose of this project is to characterize the atypical kinetics of diazepam and its sequential metabolism in in vitro and in vivo systems. Numerical methods will be used to obtain kinetic parameters, which will be used for in vitro‐in vivo correlations (IVIVC).Diazepam, which exhibits atypical kinetics in vitro, and its primary metabolites (temazepam, nordiazepam), and secondary metabolite (oxazepam) were characterized with in vitro studies with pooled male Sprague Dawley rat liver microsome (MRLM), pooled female Sprague Dawley (FRLM), and purified rat cyp isoforms (cyp3a1, cyp3a2, and cyp2c11) separately. Rat microsomal partition and rat plasma protein binding assays were conducted via equilibrium dialysis to determine the free fraction of the drug. Two substrate binding (ESS) models provided better fits than one substrate (ES) models, as determined from the resulting AICc values. Preliminary in vitro data from MRLM and cyp3a1 showed obvious sigmoidal saturation curves for temazepam formation. Only cyp3a1 show significant sigmoidal curves for nordiazepam formation. For cyp3a1, Km and Vmax values for temazepam and nordiazepam calculated with the EES model are significantly different from those with the ES model. The intrinsic clearance (CLint) values calculated from the MRLM and cyp3a1 ESS models is not constant within the observed range of diazepam plasma levels of rats. No obvious sigmoidicity of diazepam saturation curves was observed for cyp3a2 (male‐specific) and cyp2c11 (male‐specific) isoforms, which are other cyp isoforms responsible for diazepam metabolism in male rats. In order to characterize in vitro‐in vivo correlations for diazepam, in vivo PK studies with female and male rats are currently underway.Support or Funding InformationThis research was supported by NIGMS grants: R01GM114369 and R01GM104178.

Determination of minimal steady-state plasma level of diazepam causing seizure threshold elevation in rats.

Diazepam, administered by the intravenous, oral, or rectal routes, is widely used for the management of acute seizures. Dosage forms for delivery of diazepam by other routes of administration, including intranasal, intramuscular, and transbuccal, are under investigation. In predicting what dosages are necessary to terminate seizures, the minimal exposure required to confer seizure protection must be known. Here we administered diazepam by continuous intravenous infusion to obtain near-steady-state levels, which allowed an assessment of the minimal levels that elevate seizure threshold. The thresholds for various behavioral seizure signs (myoclonic jerk, clonus, and tonus) were determined with the timed intravenous pentylenetetrazol seizure threshold test in rats. Diazepam was administered to freely moving animals by continuous intravenous infusion via an indwelling jugular vein cannula. Blood samples for assay of plasma levels of diazepam and metabolites were recovered via an indwelling cannula in the contralateral jugular vein. The pharmacokinetic parameters of diazepam following a single 80-μg/kg intravenous bolus injection were determined using a noncompartmental pharmacokinetic approach. The derived parameters Vd , CL, t1/2α (distribution half-life) and t1/2β (terminal half-life) for diazepam were, respectively, 608mL, 22.1mL/min, 13.7 minutes, and 76.8minutes, respectively. Various doses of diazepam were continuously infused without or with an initial loading dose. At the end of the infusions, the thresholds for various behavioral seizure signs were determined. The minimal plasma diazepam concentration associated with threshold elevations was estimated at approximately 70ng/mL. The active metabolites nordiazepam, oxazepam, and temazepam achieved levels that are expected to make only minor contributions to the threshold elevations. Diazepam elevates seizure threshold at steady-state plasma concentrations lower than previously recognized. The minimally effective plasma concentration provides a reference that may be considered when estimating the diazepam exposure required for acute seizure treatment.

Biomimetic synthesis of proline-derivative templated mesoporous silica for increasing the brain distribution of diazepam and improving the pharmacodynamics of nimesulide

Herein a new kind of proline-derivative templated mesoporous silica with curved channels (CMS) was biomimetically synthesized and applied as carrier to improve the drug dissolution and bioavailability of hydrophobic diazepam (DZP) and nimesulide (NMS). Drugs can be incorporated into CMS with high efficiency; during this process, they successfully transformed to amorphous phase. As a result, the dissolution rate of DZP and NMS was significantly improved. Biodistribution study confirmed that CMS converted DZP distribution in mice with the tendency of lung targeting and brain targeting. At 45 min postadministration, the concentrations of DZP in plasma, lung and brain were 8.57-fold, 124.94-fold and 19.55-fold higher from 1:3 DZP/CMS sample than that of pure DZP sample, respectively. At 90 min postadministration, the content of DZP in brain was 62.31-fold higher for 1:3 DZP/CMS sample than that of pure DZP. Besides, the anti-inflammatory and analgesic effects of 1:3 NMS/CMS were systematic evaluated using mouse ankle swelling test (MAST), mouse ear swelling test (MEST) and mouse writhing test (MWT). The results indicated that after incorporating into CMS, the therapeutic effects of NMS were obviously improved, and the inhibition rates of 1:3 NMS/CMS in all pharmacodynamics tests varied from 102.2% to 904.3%.

Development of a simple and sensitive HPLC-MS/MS method for determination of diazepam in human plasma and its application to a bioequivalence study.

We developed a simple, sensitive, and effective ultra-performance liquid chromatography/tandem mass spectrometry (HPLC-MS/MS) method with an electrospray ionization (ESI) interface in multiple reaction monitoring (MRM) and positive ion modes to determine diazepam concentrations in human plasma using voriconazole as an internal standard (IS). Diazepam and IS were detected at transition 285.2→193.1 and 350.2→127.1, respectively. After liquid-liquid extraction (LLE) using 1.2 ml of ethyl acetate:n-hexane (80:20, v/v), diazepam and IS were eluted on a Phenomenex Cadenza CD-C18 column (150 × 3.0 mm, 3 µm) with an isocratic mobile phase (10 mM ammonium acetate in water:methanol [5:95, v/v]) at a flow rate of 0.4 mL/min. The peak retention time was 2.32 min for diazepam and 2.01 min for IS, respectively. The lower limit of quantitation (LLOQ) was 0.5 ng/mL (S/N > 10) using 50 µL of plasma, and no interferences were observed in chromatograms. Our analytical method was fully validated and successfully applied to a bioequivalence study of two formulations of diazepam in healthy Korean volunteers.

Omeprazole inhibits oxidative drug metabolism. Studies with diazepam and phenytoin in vivo and 7-ethoxycoumarin in vitro.

The effect of treatment with omeprazole, a substituted benzimidazole, on the elimination of diazepam and phenytoin was studied in two groups of 8 subjects. Omeprazole given orally in a daily dose of 40 mg over 7 days decreased diazepam plasma clearance from 22.4 +/- 2.8 to 10.1 +/- 1.5 ml/kg X h and prolonged the half-life of diazepam from 36.9 +/- 4.1 to 85.0 +/- 14.7 h. Plasma concentrations of desmethyldiazepam, a diazepam metabolite, were reduced after omeprazole treatment. Omeprazole also reduced the plasma clearance of phenytoin from 25.1 +/- 2.0 to 21.4 +/- 1.8 ml/kg X h and prolonged its half-life from 20.7 +/- 1.9 to 26.3 +/- 2.7 h. Renal excretion of the major metabolite of phenytoin (p-hydroxyphenyl-phenyl-hydantoin) was not changed. Omeprazole did not affect the volume of distribution and the plasma protein binding of either diazepam or phenytoin. In vitro studies with human liver microsomes showed that omeprazole in equimolar concentrations (0.5 mM) was a stronger inhibitor than cimetidine of 7-ethoxycoumarin deethylase activity. These data confirm that omeprazole interferes with the elimination of other drugs by an inhibition of the drug-metabolizing monooxygenase system of the human liver.

Positive Allosteric Modulator of GABA Lowers BOLD Responses in the Cingulate Cortex.

Knowledge about the neural underpinnings of the negative blood oxygen level dependent (BOLD) responses in functional magnetic resonance imaging (fMRI) is still limited. We hypothesized that pharmacological GABAergic modulation attenuates BOLD responses, and that blood concentrations of a positive allosteric modulator of GABA correlate inversely with BOLD responses in the cingulate cortex. We investigated whether or not pure task-related negative BOLD responses were co-localized with pharmacologically modulated BOLD responses. Twenty healthy adults received either 5 mg diazepam or placebo in a double blind, randomized design. During fMRI the subjects performed a working memory task. Results showed that BOLD responses in the cingulate cortex were inversely correlated with diazepam blood concentrations; that is, the higher the blood diazepam concentration, the lower the BOLD response. This inverse correlation was most pronounced in the pregenual anterior cingulate cortex and the anterior mid-cingulate cortex. For subjects with diazepam plasma concentration > 0.1 mg/L we observed negative BOLD responses with respect to fixation baseline. There was minor overlap between cingulate regions with task-related negative BOLD responses and regions where the BOLD responses were inversely correlated with diazepam concentration. We interpret that the inverse correlation between the BOLD response and diazepam was caused by GABA-related neural inhibition. Thus, this study supports the hypothesis that GABA attenuates BOLD responses in fMRI. The minimal overlap between task-related negative BOLD responses and responses attenuated by diazepam suggests that these responses might be caused by different mechanisms.

Preconcentration and determination of chlordiazepoxide and diazepam drugs using dispersive nanomaterial-ultrasound assisted microextraction method followed by high performance liquid chromatography.

Benzodiazepines (BDs) are used widely in clinical practice, due to their multiple pharmacological functions. In this study a dispersive nanomaterial-ultrasound assisted- microextraction (DNUM) method followed by high performance liquid chromatography (HPLC) was used for the preconcentration and determination of chlordiazepoxide and diazepam drugs from urine and plasma samples. Various parameters such as amount of adsorbent (mg: ZnS-AC), pH and ionic strength of sample solution, vortex and ultrasonic time (min), and desorption volume (mL) were investigated by fractional factorial design (FFD) and central composite design (CCD). Regression models and desirability functions (DF) were applied to find the best experimental conditions for providing the maximum extraction recovery (ER). Under the optimal conditions a linear calibration curve were obtained in the range of 0.005-10μgmL(-1) and 0.006-10μgmL(-1) for chlordiazepoxide and diazepam, respectively. To demonstrate the analytical performance, figures of merits of the proposed method in urine and plasma spiked with chlordiazepoxide and diazepam were investigated. The limits of detection of chlordiazepoxide and diazepam in urine and plasma were ranged from 0.0012 to 0.0015μgmL(-1), respectively.

用HPLC法同时测定比格犬血浆中地西泮、去甲西泮和氟马西尼的浓度

用HPLC法同时测定比格犬血浆中地西泮、去甲西泮和氟马西尼的浓度

Assessment of pharmacokinetics and tolerability of intranasal diazepam relative to rectal gel in healthy adults

Diazepam rectal gel (RG) is currently the only approved rescue therapy for outpatient management of seizure clusters in the United States. There is an unmet medical need for an alternative rescue therapy for seizure clusters that is effective, and more convenient to administer with a socially acceptable method of delivery. An intranasal diazepam formulation has been developed, and this study evaluates the tolerability and bioavailability of diazepam nasal spray (NS) relative to an equivalent dose of diazepam-RG in healthy adults. Twenty-four healthy adults were enrolled in a phase 1, open-label, 3-period crossover study. Plasma diazepam and metabolite concentrations were measured by serial sampling. Dose proportionality for 5- and 20-mg intranasal doses and the bioavailability of 20mg diazepam-NS relative to 20mg diazepam-RG were assessed by maximum plasma concentration (Cmax) and systemic exposure parameters (AUC0-∞ and AUC0-24). The mean Cmax values for 20mg diazepam-NS and 20mg diazepam-RG were 378 ± 106 and 328 ± 152 ng/mL, achieved at 1.0 and 1.5h, respectively. Subjects administered intranasal and rectal gel formulations experienced nasal and rectal leakage, respectively. Diazepam absorption following intranasal administration was consistent but 3 subjects with diazepam-RG had low plasma drug levels at the earliest assessment of 5 min, due to poor retention, and were excluded from analysis. Excluding them, the treatment ratios (20mg diazepam-NS:20mg diazepam-RG) and 90% confidence intervals for diazepam Cmax and AUC0-24 were 0.98 (0.85-1.14) and 0.89 (0.80-0.98), respectively, suggesting that the bioavailability was comparable between the two formulations. Dose proportionality was observed between the lowest and highest dose-strengths of intranasal formulation. Both intranasal and rectal treatments were well tolerated with mild to moderate adverse events. Results suggest that a single-dose of 20mg diazepam-NS is tolerable and comparable in bioavailability to that of diazepam-RG. The intranasal formulation may provide caregivers and patients with a more socially acceptable and convenient alternative rescue therapy in the acute treatment of seizure clusters.

Diabetes and hypertension increase the placental and transcellular permeation of the lipophilic drug diazepam in pregnant women

BackgroundPrevious studies carried out in our laboratories have demonstrated impaired drug permeation in diabetic animals. In this study the permeation of diazepam (after a single dose of 5 mg/day, administered intramuscularly) will be investigated in diabetic and hypertensive pregnant women.MethodsA total 75 pregnant women were divided into three groups: group 1 (healthy control, n = 31), group 2 (diabetic, n = 14) and group 3 (hypertensive, n = 30). Two sets of diazepam plasma concentrations were collected and measured (after the administration of the same dose of diazepam), before, during and after delivery. The first set of blood samples was taken from the mother (maternal venous plasma). The second set of samples was taken from the fetus (fetal umbilical venous and arterial plasma). In order to assess the effect of diabetes and hypertension on diazepam placental-permeation, the ratios of fetal to maternal blood concentrations were determined. Differences were considered statistically significant if p ≤ 0.05.ResultsThe diabetes and hypertension groups have 2-fold increase in the fetal umbilical-venous concentrations, compared to the maternal venous concentrations. Feto: maternal plasma-concentrations ratios were higher in diabetes (2.01 ± 1.10) and hypertension (2.26 ± 1.23) groups compared with control (1.30 ± 0.48) while, there was no difference in ratios between the diabetes and hypertension groups. Umbilical-cord arterial: venous ratios (within each group) were similar among all groups (control: 0.97 ± 0.32; hypertension: 1.08 ± 0.60 and diabetes: 1.02 ± 0.77).ConclusionsOn line with our previous findings which demonstrate disturbed transcellular trafficking of lipophilic drugs in diabetes, this study shows significant increase in diazepam placental-permeation in diabetic and hypertensive pregnant women suggesting poor transcellular control of drug permeation and flux, and bigger exposure of the fetus to drug-placental transport.

Effect of Pluronic F68 on plasma and brain diazepam concentrations in mice

Effect of Pluronic F68 on plasma and brain diazepam concentrations in mice

A RAPID HILIC METHOD FOR ANALYSIS OF DIAZEPAM AND ESTAZOLAM IN HUMAN PLASMA

A rapid method based on hydrophilic interaction chromatography (HILIC) using UV detection was developed for analysis of diazepam and estazolam in human plasma. The method utilized a Kromasil KR100-5SIL column (250 mm × 4.6 mm I.D., 5 µm) with a mobile phase consisting of acetonitrile-sodium formate buffer (10 mM, pH = 5.5) (90:10, v/v) at a flow rate of 1.0 mL/min. The detection wavelength was 254 nm. The chromatographic run time was less than 5 min per injection. All the experiments were performed at room temperature. The isolation of the analytes from plasma samples was achieved by solid phase extraction (SPE) using Oasis HLB cartridges. The standard curve range was 25–5000 ng/mL (r2 > 0.99) for diazepam and estazolam using 0.2 mL of human plasma. The limit of quantification (LOQ) and limit of detection (LOD) for both compounds were 25 and 10 ng/mL, respectively. Recoveries of the analytes ranging from 70.7% to 88.5% with intra- and inter-day precision (RSD) less than 11% were obtained at the concentrations above the LOQ. The proposed method is rapid, simple, accurate, and its mobile phase is compatible for mass spectrometry, proving an alternative method for benzodiazepine drug monitoring in forensic and clinical toxicology as well as pharmacokinetic studies.

Evaluation of plasma diazepam and nordiazepam concentrations following administration of diazepam intravenously or via suppository per rectum in dogs

To evaluate the pharmacokinetics of diazepam administered per rectum via compounded (ie, not commercially available) suppositories and determine whether a dose of 2 mg/kg in this formulation would result in plasma concentrations shown to be effective for control of status epilepticus or cluster seizures (ie, 150 to 300 ng/mL) in dogs within a clinically useful interval (10 to 15 minutes). 6 healthy mixed-breed dogs. Dogs were randomly assigned to 2 groups of 3 dogs each in a crossover-design study. Diazepam (2 mg/kg) was administered IV or via suppository per rectum, and blood samples were collected at predetermined time points. Following a 6- or 7-day washout period, each group received the alternate treatment. Plasma concentrations of diazepam and nordiazepam were analyzed via reversed phase high-performance liquid chromatography. Plasma concentrations of diazepam and nordiazepam exceeded the targeted range ≤ 3 minutes after IV administration in all dogs. After suppository administration, targeted concentrations of diazepam were not detected in any dogs, and targeted concentrations of nordiazepam were detected after 90 minutes (n = 2 dogs) or 120 minutes (3) or were not achieved (1). On the basis of these results, administration of 2 mg of diazepam/kg via the compounded suppositories used in the present study cannot be recommended for emergency treatment of seizures in dogs.

Restricted access magnetic core-mesoporous shell microspheres with C8-modified interior pore-walls for the determination of diazepam in rat plasma by LC-MS

In this study, a novel enrichment technique based on magnetic core-mesoporous shell microspheres with C8-modified interior pore-walls (C8-Fe3O4@mSiO2) was successfully developed for the determination of diazepam in rat plasma by LC-MS. Due to the unique properties of the synthesized C8-Fe3O4@mSiO2 microspheres (C8-modified magnetic mesoporous microsphere), small drug molecules like diazepam can enter the mesopore channels and be efficiently absorbed through hydrophobic interaction by interior C8-groups (Octyl functional groups). Large molecules like proteins are excluded from the mesopore channels as a result of size exclusion effect, leading to direct extraction of drug molecules from protein-rich biosmaples such as plasma without any other pretreatment procedure. Moreover, diazepam adsorbed C8-Fe3O4@mSiO2 microspheres could be simply and rapidly isolated through placing a magnet on the bottom of container, and then diazepam could be easily eluted from C8-Fe3O4@mSiO2 microspheres for further LC-MS analysis. Extraction conditions such as amounts of C8-Fe3O4@mSiO2 microspheres added, adsorption time, elution solvent and elution time were investigated. Method validations including linear range, the limit of detection, precision, and recovery were also studied. The results indicated that the proposed method based on C8-Fe3O4@mSiO2 microspheres was simple and accurate for the analysis of diazepam in the rat plasma. And it will provide new ideas for analyzing plasma concentration and pharmacokinetics of similar drugs.