Benzodiazepines In Plasma Research Articles

Quantification of 54 Benzodiazepines and Z-Drugs, Including 20 Designer Ones, in Plasma.

Benzodiazepines are widely used in the treatment of sleep and anxiety disorders, as well as epileptic seizures and alcohol withdrawal because of their broad therapeutic index and low cost. Due to their central nervous system depressant effects they are also often implicated in traffic accidents and drug-related intoxications. With an increasing number of designer benzodiazepines used in a recreational setting, there is a need for analytical methods to be able to quantify both the prescribed and designer benzodiazepines. A liquid chromatography-triple quadrupole mass spectrometry method was developed for the quantification of 34 prescribed and 20 designer benzodiazepines in plasma. Different sample preparation strategies, including protein precipitation, liquid-liquid extraction, solid-phase extraction and mini-QuEChERS, were tested. The best recoveries for all compounds of interest were obtained with a liquid-liquid extraction using methyl-tertiary-butyl-ether and 500μL plasma. The method was fully validated according to the European Medicines Agency guidelines for all compounds, except pivoxazepam, which is included for qualitative purposes only. In-sample stability issues were observed for cloxazolam, both at ambient temperature and during long-term storage at -20°C. Due to the large number of compounds included, the simple and time-efficient sample preparation and the relatively inexpensive instrumentation used, the presented method can be readily implemented in both therapeutic drug monitoring and forensic analyses.

Simultaneous analysis of eight benzodiazepines in blood and urine matrix by gas chromatography–mass spectrometry: Implications for air crash investigation

Introduction: Benzodiazepines are the most commonly prescribed class of drugs in India and are capable of impairing the performance of an aviator in therapeutic to subtherapeutic levels. Detection of benzodiazepines, particularly in blood, is not easy, since the concentrations present, especially following prescribed medical use, can be very low. Several publications have addressed estimation of benzodiazepines in plasma or serum; however, few have attempted their detection in whole blood. Urine, although a better specimen, benzodiazepines due to their extensive metabolism, its metabolites are excreted in urine instead of the parent compounds. Materials and Methods: In our laboratory, a method was developed for simultaneous detection and quantification of eight benzodiazepines in whole blood and urine matrix by gas chromatography–mass spectrometry selective ion monitoring (SIM) method. Chromatographic separation was optimized and achieved for separation of all 8 compounds using Agilent DB-5MS column. Retention time, selectivity and sensitivity were achieved by measuring each analyte in SIM mode. The developed method was tested and validated on actual biological samples for lorazepam, temazepam, diazepam, clonazepam, and nitrazepam. Conclusion: A single method was developed for the detection and quantification of eight benzodiazepines in whole blood and urine matrix by GC–MS SIM method. The method was also tested on limited number of actual biological samples for the lorazepam, temazepam, diazepam, clonazepam, and nitrazepam.

Quantification of eight benzodiazepines in human breastmilk and plasma by liquid-liquid extraction and liquid-chromatography tandem mass spectrometry: Application to evaluation of alprazolam transfer into breastmilk

Breastfeeding is strongly encouraged for infant and maternal health. Benzodiazepines (BZDs) are widely prescribed drugs for symptoms, such as anxiety and insomnia, which many women could experience during the postpartum period. However, limited information is currently available to evaluate the transfer of different BZDs into breastmilk. In order to assess the proprieties of this medication during breastfeeding, robust and sensitive analytical methods to quantify BZDs are required. For this purpose, we developed a method for quantification of BZDs, including alprazolam, bromazepam, clonazepam, clotiazepam, etizolam, flunitrazepam, lorazepam, and CM7116 (a metabolite of ethyl loflazepate), in human breastmilk and plasma using liquid chromatography/tandem mass spectrometry (LC/MS/MS). Sample preparation was performed by a simple liquid-liquid extraction (LLE) with ethyl acetate. For sample preparation of CM7116, the pretreatment process to completely obtain the metabolite was added before the LLE step. The BZDs were separated by a C18 column using a gradient elution of acetonitrile in aqueous ammonium acetate solution, and were detected in the positive ion electrospray mode with multiple reaction monitoring (MRM). Lower limits of quantification (LLOQs) in breastmilk ranged from 0.25 to 0.5 ng/mL, and those in plasma ranged from 0.5 to 1.0 ng/mL. The intra-day and inter-day precision, and accuracy of data were assessed and found to be acceptable. The developed method was successfully applied to measure the concentration of alprazolam in breastmilk and plasma, which were donated by a lactating woman who had been regularly treated with alprazolam. Milk to plasma (M/P) ratios were calculated as 0.52 (before oral administration) and 0.49 (2 h after administration) 3 days after delivery. The M/P ratio 1 month after delivery was calculated as 0.41 (2 h after administration). We estimated that the relative infant dose (RID) values of alprazolam ranged from 3.11 to 4.61%.

Experimental designs for the optimization of microextraction by packed sorbent (MEPS®) of benzodiazepines in human plasma

Experimental designs for the optimization of microextraction by packed sorbent (MEPS®) of benzodiazepines in human plasma

Development of Solid-Phase Extraction Coupled with Dispersive Liquid-Liquid Microextraction Method for the Simultaneous Determination of Three Benzodiazepines in Human Urine and Plasma.

A new pretreatment method termed solid-phase extraction (SPE) which is combined with dispersive liquid-liquid microextraction (SPE-DLLME) has been developed for the determination of oxazepam, alprazolam and diazepam in urine and plasma samples. The analytes were extracted from biological samples using SPE followed by DLLME. Various parameters that affect the efficiency of the two extraction techniques have been optimized. The calibration plots were linear in the range of 2.5-500 µg L(-1) for oxazepam, alprazolam and 1.0-500 µg L(-1) for diazepam with detection limits of 0.7 µg L(-1) for oxazepam, alprazolam and 0.4 µg L(-1) for diazepam in plasma sample. The results confirm the suitability of the SPE-DLLME-HPLC-UV as a sensitive method for the analysis of the targeted analytes in urine and plasma samples.

Hydrophilic interaction liquid chromatography/positive ion electrospray ionization mass spectrometry method for the quantification of alprazolam and α-hydroxy-alprazolam in human plasma

A hydrophilic interaction liquid chromatography/positive ion electrospray-mass spectrometry (HILIC-ESI/MS) has been developed and fully validated for the quantification of alprazolam and its main metabolite, α-hydroxy-alprazolam, in human plasma. The assay is based on 50μL plasma samples, following liquid-liquid extraction. All analytes and the internal standard (tiamulin) were separated by hydrophilic interaction liquid chromatography using an X-Bridge-HILIC analytical column (150.0mm×2.1mm i.d., particle size 3.5μm) under isoscratic elution. The mobile phase was composed of a 7% 10mM ammonium formate water solution in acetonitrile and pumped at a flow rate of 0.20mLmin(-1). Running in positive electrospray ionization and selected ion monitoring (SIM) the mass spectrometer was set to analyze the protonated molecules [M+H](+) at m/z 309, 325 and 494 for alprazolam, α-hydroxy-alprazolam and tiamulin (ISTD) respectively. The assay was linear over the concentration range of 2.5-250ngmL(-1) for alprazolam and 2.5-50ngmL(-1) for α-hydroxy alprazolam. Intermediate precision was less than 4.1% over the tested concentration ranges. The method is the first reported application of HILIC in the analysis benzodiazepines in human plasma. With a small sample size (50μL human plasma) and a run time less than 10.0min for each sample the method can be used to support a wide range of clinical studies concerning alprazolam quantification.

Spectrophotometric and chemometric studies on the simultaneous determination of two benzodiazepines in human plasma

A numerical simple, accurate and precise method based on spectrophotometric data coupled with multivariate calibration methods, PLS and MLR, combined with GA was developed for the simultaneous determination of two benzodiazepines, Clobazam and Flurazepam. A data set of absorption spectra obtained from a calibration set of mixtures containing the compounds was used to build GA-PLS and GA-MLR models. The models were tested using a dataset constructed from the compound synthetic solutions. The better model was also applied to plasma samples. The proposed method requires no preliminary separation steps and can be used for these drugs analysis in quality control laboratories.

Quantitation of drugs via molecularly imprinted polymer solid phase extraction and electrospray ionization mass spectrometry: benzodiazepines in human plasma

The association of solid phase extraction with molecularly imprinted polymers (MIP) and electrospray ionization mass spectrometry (ESI-MS) is applied to the direct extraction and quantitation of benzodiazepines in human plasma. The target analytes are sequestered by MIP and directly analyzed by ESI-MS. Due to the MIP highly selective extraction, ionic suppression during ESI is minimized; hence no separation is necessary prior to ESI-MS, which greatly increases analytical speed. Benzodiazepines (medazepam, nitrazepam, diazepam, chlordiazepoxide, clonazepam and midazolam) in human plasma were chosen as a proof-of-principle case of drug analyses by MIP-ESI-MS in a complex matrix. MIP-ESI-MS displayed good figures of merits for medazepam, nitrazepam, diazepam, chlordiazepoxide and midazolam, with analytical calibration curves ranging from 10 to 250 μg L(-1) (r > 0.98) with limit of quantification <10 μg L(-1) and acceptable within-day and between-day precision and accuracy.

DEVELOPMENT AND VALIDATION OF A GC-NPD/micro-ECD METHOD USING DUAL COLUMN FOR THE DETERMINATION OF BENZODIAZEPINE IN HUMAN WHOLE BLOOD AND PLASMA

Four benzodiazepines flunitrazepam, nitrazepam, clonazepam, and alprazolam, have been analyzed from biological matrices by dual column gas chromatography using a nitrogen-phosphorus detector (NPD) and a micro-electron capture detector (m-ECD). The recoveries of the four benzodiazepines spiked into human whole blood and plasma were 88.8 - 97.9 and 90.7 - 97.5%, respectively. The regression equations for the four benzodiazepines showed excellent linearity. The detection limits (LODs) were 0.14 - 0.95 ng/mL for whole blood and 0.13 - 0.93 ng/mL for plasma. The method is simple and sensitive for the determination of benzodiazepines in human whole blood and plasma, and may be useful in forensic science practice.

Development of a Liquid Chromatographic Method for the Simultaneous Determination of Six Benzodiazepines in Human Plasma After Solid-Phase Extraction

A simple and fast procedure is presented for the simultaneous determination of alprazolam, bromazepam, diazepam, lorazepam, lormetazepam, and tetrazepam in human plasma. The drugs were isolated by solid-phase extraction, and the subsequent analysis was carried out by high-performance liquid chromatography coupled with a diode-array detector (HPLC-DAD). This allowed recoveries of around 90% to be obtained with good sensitivity, selectivity, and resolution of the eluting peaks. The detector response at 230 nm was linear within the benzodiazepine concentration range of 20–4000 ng/mL in human plasma, with values of less than 6.7% and 5.4% for precision and accuracy, respectively.

Experimental design for optimization of microwave-assisted extraction of benzodiazepines in human plasma

A simple and fast microwave-assisted-extraction (MAE) method has been evaluated as an alternative to solid-phase extraction (SPE) for the determination of six benzodiazepines widely prescribed in European countries (alprazolam, bromazepam, diazepam, lorazepam, lormetazepam and tetrazepam) in human plasma. For MAE optimization a Doehlert experimental design was used with extraction time, temperature and solvent volume as influential parameters. A desirability function was employed in addition to the simultaneous optimization of the MAE conditions. The analysis of variance showed that the solvent volume had a positive influence on the extraction of all the analytes tested, achieving a statistically significant effect. Also, the extraction time had a statistically significant effect on the extraction of four benzodiazepines. The selected MAE conditions-89 degrees C, 13 min and 8 mL of chloroform/2-propanol (4:1, v/v)-led to recoveries between 89.8 +/- 0.3 and 102.1 +/- 5.2% for benzodiazepines using a high performance liquid chromatography method coupled with diode-array detection. The comparison of MAE and SPE shows better results for MAE, with a lower number of steps in handling the sample and greater efficiency. The applicability of MAE was successfully tested in 27 plasma samples from benzodiazepine users.

Quantitation of Benzodiazepines in Urine, Serum, Plasma, and Meconium by LC-MS-MS

A single method for confirmation and quantitation of a panel of commonly prescribed benzodiazepines and metabolites, alpha-hydroxyalprazolam, alpha-hydroxyethylflurazepam, alpha-hydroxytriazolam, alprazolam, desalkylflurazepam, diazepam, lorazepam, midazolam, nordiazepam, oxazepam, temazepam, clonazepam, and 7-aminoclonazepam, was developed for three specimen types, urine, serum/plasma, and meconium. Quantitation was by liquid chromatography tandem-mass spectrometry (LC-MS-MS) using a Waters Alliance-Quattro Micro system. The instrument was operated in multiple reaction monitoring mode with an electrospray ionization source in positive ionization mode. The method was evaluated for recovery, imprecision, linearity, analytical measurement range, specificity, and carryover. Average recovery and imprecision (within-run, between-run, and total % CV) were within +/- 15% of the target concentrations for urine (10 to 5000 ng/mL) and serum/plasma (10 to 2500 ng/mL) and within +/- 20% for meconium (10 to 5000 ng/g). In all, 205 patient specimens were analyzed, and the results compared to a previous in-house gas chromatography-MS method or LC-MS-MS results from an outside laboratory. Oxazepam glucuronide was evaluated as a hydrolysis control for the urine and meconium specimens.

Separation and HPLC analysis of 15 benzodiazepines in human plasma

Benzodiazepines (BZDs) are often prescribed to schizophrenic or depressed patients, as a part of polypharmacy regimens. An HPLC method has been developed for the simultaneous determination of 15 BZDs in human plasma. Separation was obtained by using a C8 RP column and a mobile phase composed of 65% aqueous phosphate buffer at pH 3.0 and 35% ACN. The UV detector was set at 220 nm and clomipramine was used as the internal standard. A careful pretreatment procedure of plasma samples was developed, using SPE with C1 cartridges, which gives high extraction yields (> 97%). The LOQs were always lower than 7.6 ng/mL and the LODs always lower than 2.6 ng/mL for all analytes. The method was successfully applied to plasma samples from depressed and schizophrenic patients undergoing polypharmacy with one or more BZDs. Precision data, as well as accuracy results, were satisfactory and no interference from other drugs was found. Hence, the method seems to be suitable for the therapeutic drug monitoring (TDM) of patients undergoing therapy with one or more BZDs.

Liquid chromatography–electrospray ionisation mass spectrometry for the determination of nine selected benzodiazepines in human plasma and oral fluid

A new simple and rapid liquid chromatographic–mass spectrometric technique was designed for the determination of nine benzodiazepines in plasma and oral fluid. Benzodiazepines were extracted from alkalinised spiked and clinical plasma and oral fluid samples using a single step, liquid–liquid extraction procedure with diethyl ether. The chromatographic separation was performed with a Xterra ® RP 18, 5 μm (150 × 2.1 mm i.d.) reversed-phase column using deuterated analogues of the analytes as internal standard. The recovery ranged from 70.3 to 86.9% for plasma and 63.9 to 77.2% for oral fluid. The limits of detection ranged from 0.5 to 1 ng/ml in plasma and 0.1 to 0.2 ng/ml for oral fluid. The method was validated for all the compounds, including linearity and the main precision parameters. The procedure, showed to be sensitive and specific, was applied to real plasma and oral fluid samples. The method is especially useful to analyse saliva samples from drivers undergoing roadside drug controls.

Clinically relevant anterograde amnesia and its relationship with blood levels of benzodiazepines in suicide attempters who took an overdose

The relationship between anterograde amnesia, sedation and plasma levels of benzodiazepines was studied prospectively in a group of 24 patients who took an overdose of benzodiazepines. Patients were tested on two sequential days after having taken an overdose. Anterograde amnesia was tested by using a verbal recall test and a photo recognition test. Sedation was scored on a visual analogue scale (VAS) by the patient and the interviewer. The concentration of benzodiazepines in plasma was measured by using a radioreceptor assay that adds benzodiazepines and their active metabolites. The cumulative amount of benzodiazepines was expressed as diazepam equivalents (DZE). Diazepam equivalents determined by this radioreceptor assay were significantly higher on the first day than on the second day. Ratings on the verbal recall test were significantly lower on the first day than on the second day. There was a significant relation between decrease of diazepam equivalents and increase of verbal recall: more than 30% of increase of verbal recall was explained by decrease of diazepam equivalents. There was not a strong relation between decrease of diazepam equivalents and reduction of level of sedation as scored by the patients. There was almost no relation between decrease of diazepam equivalents and reduction of level of sedation as scored by the interviewer. No relation was found between verbal recall, sedation and diazepam equivalents. There was no relation between diazepam equivalents and photo recognition. It was concluded that anterograde amnesia was strongly associated with benzodiazepines in patients who take benzodiazepines in an overdose. Sedation does not predict the degree of anterograde amnesia.

Determination of the Benzodiazepine Plasma Concentrations in Suicidal Patients Using a Radioreceptor Assay

Impairments in memory and psychomotor function appear to be induced by benzodiazepines not only after long-term use, but also after administration of a single dose. Because it is known on which neurotransmitter system the benzodiazepines exert their action, the use of a quantitative radioreceptor assay (RRA) can be a useful tool in studying the interrelationship between the neurochemical and memory processes. The RRA measures the sum of the main compound(s) and all active metabolites present, where it relates the biological activity to the pharmacodynamic effect instead of relating it to the plasma levels of the individual compounds. To correlate the loss of memory with the benzodiazepine concentration, plasma concentrations were determined in suicidal patients. From suicidal patients (n = 84), the benzodiazepines in plasma were measured with a direct radioreceptor assay using tritiated flunitrazepam as the labelled ligand. The receptor material was a lyophilized preparation from calf cortex. Furthermore, the samples were subjected to high-performance liquid chromatographic (HPLC) analysis, and the HPLC data were converted to diazepam equivalents using cross-reactivities of the individual compounds. Patients who had ethanol residues in their plasma were excluded from this correlation experiment. The data (n = 40) obtained with the two analytical techniques were compared and correlated to assess the validity of the radioreceptor assay in establishing the relationship between the loss of memory and the total amount of benzodiazepines present. The cumulative amount of diazepam determined with the RRA and the sum of compounds determined with the HPLC method, after correction using the cross-reactivities, were plotted and correlated using regression analysis. Regression analysis showed an x variable of 0.75 and a correlation coefficient of 0.67. The intercept was not significantly different from zero (P = 0.49, t-test), whereas the slope was significantly different from zero (P < 0.01). Benzodiazepines can be directly determined in plasma using this radioreceptor assay. The data obtained from HPLC analysis were easily converted to diazepam equivalents using the cross-reactivities. A discrepancy between the data obtained from the two analytical techniques, however, indicates that certain metabolites are present, which were not quantitated in the HPLC analysis, but were measured in the radioreceptor assay. Therefore, the radioreceptor assay proved to be a valuable tool for the assessment of clinical effects, such as the demonstration of the loss of memory in suicidal patients after a benzodiazepine overdose.

Direct injection HPLC method for the determination of selected benzodiazepines in plasma using a Hisep column

A direct plasma injection HPLC method has been developed for the determination of selected benzodiazepines (nitrazepam, clobazam, oxazepam, lorazepam). The method uses an analytical hydrophobic shielded phase (Hisep) column equipped with a Hisep guard column, are easy to perform and requires 20 ul of a filtered plasma sample. The chromatographic run time is less than 15 min using a mobile phase of 15:85 v/v acetonitrile–0.18 M ammonium acetate pH 2.5. The method is good for 175 injections before replacement of the guard column. The method was linear in the range 0.5–18 ug ml −1 (r>0.99, n=6) for the analytes with R.S.D. less than 10.82%. Interday and intraday variability were found to be less than 14%. The limits of detection and quantitation were 0.16 (s/n>3) and 0.5 ug ml −1 (s/n>10), respectively, for each of the four benzodiazepines.

Simple method for the determination of benzodiazepines in human body fluids by high-performance liquid chromatography–mass spectrometry

Three benzodiazepines, etizolam, brotizolam and lorazepam, have been analyzed from human plasma and urine samples by high-performance liquid chromatography–mass spectrometry (HPLC–MS) with a new polymer column (MSpak GF), which enabled direct injection of crude biological samples. The recoveries of the three benzodiazepines spiked into plasma and urine were 81.7–90.7 and 78.7–91.5%, respectively. The regression equations for the three benzodiazepines showed excellent linearity in the range 10–500 ng ml −1 of plasma or urine. The detection limits were 2 ng ml −1 for etizolam and brotizolam, 5 ng ml −1 for lorazepam in both samples. The intra- and inter-day precisions for plasma and urine were not greater than 12.7%. The data obtained from determination of the benzodiazepines in rat plasma after oral administration of the drugs are also presented.

Direct LC analysis of five benzodiazepines in human urine and plasma using an ADS restricted access extraction column.

An alkyl-diol-silica (ADS) precolumn was used for the direct and on-line extraction of several benzodiazepines from serum and urine. The protein component of the biological sample was flushed through the ADS column, while simultaneously extracting the benzodiazepine compounds in the pores of the ADS stationary phase. The role of hydrophobic interactions in the extraction mechanism was confirmed. Column switching was employed to elute the extracted analytes from the ADS column into a high-performance liquid chromatography reverse-phase C18 column for the isocratic separation and UV detection of the benzodiazepines. Sample preconcentration via large volume injections was possible, improving the limits of detection. The calculated clonazepam, oxazepam, temazepam, nordazepam and diazepam detection limits were 38.8, 24.2, 31.7, 31.3, 45.0 ng/ml in serum, respectively, and 48.4, 24.5, 31.7, 33.1, 52.9 ng/ml for urine, respectively. The method was linear over the range of 50-10000 ng/ml in both matrices with an average linear coefficient (R(2)) value of 0.9918. The injection repeatability and intra-assay precision of the method were evaluated over ten injections, resulting in a percent relative standard deviation <5%. The ADS extraction column was robust, providing many direct injections of biological fluids for the extraction and subsequent determination of benzodiazepines.

Benzodiazepines and hip fractures in elderly people: case-control study

Objective: To determine whether benzodiazepines are associated with an increased risk of hip fracture.Design: Case-control study.Participants: All incident cases of hip fracture not related to traffic accidents or cancer in...