Midazolam In Human Plasma Research Articles

Bioanalytical method to detect midazolam in plasma.

Midazolam (MDZ) is the first choice in palliative sedation, and commonly used in sleep induction in anesthesia, with rapid onset of action. However, monitoring of the level of sedation in patients is not accurate. We developed and validated a bioanalytical method to detect MDZ in plasma using high-performance liquid chromatography (HPLC) coupled to a photodiode array detector (PDA) for future monitoring of sedation. MDZ was extracted by solid-phase extraction (SPE). Analyses were performed on a C18 column, using 0.05% triethylamine and acetonitrile as mobile phase, analyzing at 220 nm. Recovery was evaluated by comparing extracted and nonextracted solutions. Precision, accuracy, linearity, limits of detection (LD) and quantification (LQ), specificity and selectivity were determined. The mean recovery obtained by SPE was 101.03%. The method was linear in the range 1.0-50.0 μg/mL. The LD and LQ were, respectively, 0.43 and 1.43 μg/mL. The specificity of the MDZ peak was adequate. The method was able to detect MDZ among other drugs. Plasma anticoagulants showed no interference with the drug detection. The bioanalytical method using HPLC-PDA and SPE was successfully validated and showed linearity, precision, accuracy, specificity and high sensitivity for detection of MDZ in human plasma.

An ultra-sensitive LC-MS/MS method to determine midazolam levels in human plasma: development, validation and application to a clinical study.

Midazolam is a commonly used marker substrate for the in vivo assessment of CYP3A activity. Reliable pharmacokinetic assessment at sub-pharmacological doses of midazolam requires an ultra-sensitive analytical method. A new, ultra-sensitive LC-MS/MS method for the determination of midazolam in human plasma using SPE was developed and fully validated. The lowest limit of quantitation is 0.1 pg/ml with a sample volume of 500 μl. The following parameters were validated: sensitivity, assay accuracy and precision, linearity, selectivity, and stability of midazolam at pertinent analytical and storage conditions. The validated method was utilized successfully for the sample assay during a midazolam microdosing study for the evaluation of CYP3A4 activity of a clinical candidate.

Development and Validation of Reverse Phase HPLC Method for the Determination of Midazolam in Human Plasma

:A simple, rapid, sensitive and specific high performance liquid chromatograpic (HPLC-UV) method was developed and fully validated for the quantification of midazolam in human plasma. Alprazolam was used as an internal standard. The analyte was extracted from human plasma samples by liquid-phase extraction technique. Analyte was chromatographed on a Zorbax XDB C18 column (3.5 µm, 4.6 x 100 mm i.d. from Agilent Technologies) by using a mixture of 0.05 M Na3PO4 (pH-4): toluene: acetonitrile (40:40:20, v/v/v) as the mobile phase at a flow-rate of 0.9 ml min−1 and the chromatogram was monitored with UV detection at 220 nm. The calibration curve obtained was linear (r2=0.99) over the concentration range of 2-200 ng ml−1. The results of the intra- and inter-day precision and accuracy studies were well within the acceptable limits. Extraction recoveries of MDZ from plasma were 85.73, 91.93 and 93.68 % at 5, 84 and 148 ng ml−1 respectively. The mean recovery of the internal standard was found to be 102.8 % at the spiked concentration of 1 µg ml−1. The method was validated and can be successfully applied for routine analyses.

Determination of Triazolam and Midazolam in Human Plasma Using Gas Chromatography with Microelectron Capture Detection for Clinical Application

A method for the simple and reliable determination of triazolam and midazolam in human plasma using gas chromatography with microelectron capture detection has been developed. Samples (0.5 mL of plasma) were prepared using a simple solvent extraction with 3% isoamyl alcohol/benzene in the presence of NaOH. Two microlitres of the extract were injected onto the capillary column ((5%-phenyl)-methylpolysiloxane). The method was found to be valid in terms of selectivity, linearity, precision, accuracy, and recovery over the concentration range of 0.2 to 20 ng/mL for triazolam, and from 0.5 to 200 ng/mL for midazolam, respectively. Intra- and inter-day precisions determined at three concentrations were from 4.1 to 9.3% for triazolam and from 2.9 to 13.0% for midazolam. The accuracies were within 17.7% for triazolam and within 13.0% for midazolam. This proposed method was successfully applied to a pharmacokinetic study of triazolam or midazolam in healthy volunteers.

Simultaneous determination of alfentanil and midazolam in human plasma using liquid chromatography and tandem mass spectrometry

A fast, sensitive and selective liquid chromatography–tandem mass spectrometry (LC–MS/MS) method for the determination of alfentanil and midazolam in human plasma has been developed and validated. Alfentanil and midazolam were extracted from plasma using a mixed-mode cation exchange solid phase extraction method, with recoveries of both compounds greater than 80% at 3 different concentrations (1, 10 and 100 ng/ml). Compounds were analyzed on a C 18 column with a water and methanol mobile phase gradient with acetic acid as an additive, at a flow rate of 0.3 ml/min. The working assay range was linear from 0.25 to 100 ng/ml for each compound. The signal to noise ratio was 80 and 40 for alfentanil and midazolam, respectively, at the lowest concentration calibration standard, with less than 10% matrix suppression by human plasma at this concentration. Alfentanil and midazolam were stable in human plasma during storage at −80 °C, processing, and analysis. The procedure was validated and applied to the analysis of plasma samples from healthy human subjects administered oral and intravenous alfentanil and midazolam.

A sensitive, rapid and specific determination of midazolam in human plasma and saliva by liquid chromatography/electrospray mass spectrometry

A rapid, sensitive and selective liquid chromatography/electrospray mass spectrometry (LC/ES-MS) method was developed for the quantitative determination of the anaesthetic benzodiazepine midazolam (MID) in human saliva and plasma from patients undergoing anesthesia procedures. Biological samples spiked with diazepam-d5, the internal standard, were extracted into diethyl ether. Compounds were separated on a Xterra RP18 column using a mobile phase of acetonitrile/formic acid 0.1% at a flow rate of 0.25 mL/min under a linear gradient. Column effluents were analyzed using MS with an ES source in the positive ionization mode. Calibration curves were linear in the concentration ranges of 1-250 and 0.2-25 ng/mL in plasma and saliva, respectively. The limits of detection were 0.5 ng/mL in plasma and 0.1 ng/mL in saliva, using a 0.5-mL sample volume. The recoveries of the spiked samples were above 65%. The method was applied to ten real samples from patients undergoing midazolam treatment.

Determination of midazolam in human plasma by liquid chromatography with mass-spectrometric detection

A liquid chromatographic assay with mass-spectrometric detection was developed for the quantitative determination of the cytochrome P450 3A phenotyping probe midazolam in human plasma. Sample pretreatment involved a one-step extraction of 600 μl aliquots with ethyl acetate. Midazolam and the internal standard, lorazepam, were separated on a column ( 150 mm×4.6 mm , i.d.) packed with 5 μm Zorbax Eclipse XDB-C8 material, using a mobile phase composed of methanol and 10 mM aqueous ammonium acetate (60:40, v/v). Column effluents were analyzed using mass-spectrometry with an atmospheric pressure chemical ionization source. Calibration curves were linear in the concentration range of 1.00–200 ng/ml. The accuracy and precision ranged from 92.8 to 112% and 0.056 to 13.4%, respectively, for four different concentrations of quality control samples analyzed in triplicate on eight separate occasions. The developed method was subsequently applied to study the pharmacokinetics of midazolam in a group of 35 human subjects at a single dose of 25 μg/kg.

Quantitation of midazolam in human plasma by automated chip-based infusion nanoelectrospray tandem mass spectrometry.

An automated chip-based infusion nanoelectrospray ionization (nanoESI) platform was used to demonstrate reproducible quantitation of drug molecules from biological matrices. Three sample preparation strategies were explored including protein precipitation of plasma with acetonitrile, de-salting of the plasma, and a combination of protein precipitation with subsequent de-salting of the dried and reconstituted extract. The best results were obtained when fortified human plasma samples containing midazolam were precipitated with acetonitrile containing alprazolam as the internal standard (IS). The supernatant was concentrated to dryness, reconstituted in aqueous acid, and de-salted by automated reversed-phase solid-phase extraction (SPE) prior to infusion nanoESI-MS/MS. Analyses employed a triple quadrupole mass spectrometer operated in selected reaction monitoring (SRM) mode. Each sample was infused for approximately 10 s and the resulting ion current profiles were integrated. Area ratios were used for regression analysis of standard samples (1.5-500 ng/mL). Quality control samples (3, 250, and 400 ng/mL) in five replicates from three different analysis days demonstrated intra-assay precision (< or =16%), inter-assay precision (< or =5%), and overall accuracy (+/-9% deviation). Infusion reproducibility of the assay was established by analyzing extracts after storage for 24 h at ambient temperature. Control plasma samples from six different sources probed the potential utility of this technique for the analysis of clinical samples. At the lower limit of quantitation (LLQ), variability and mean overall accuracy were < or =13% CV and +/-3% deviation, respectively, while at the upper limit of quantitation (ULQ) variability and mean overall accuracy were < or =9% CV and +/-9% deviation, respectively. Inter-chip variability was established by determining standard sample extracts across five different chips (< or =12% CV). Throughput for the assay was 55 s per sample, although this time may be shortened to 40 s per sample with recent improvements in the automated nanoESI system. No contamination or carryover was observed using this promising automated nanoESI-MS/MS platform.

Determination of midazolam in human plasma by solid-phase microextraction and gas chromatography/mass spectrometry.

A new method is described for the qualitative and quantitative analysis of midazolam, a short-acting 1,4-imidazole benzodiazepine, in human plasma. It involves a plasma deproteinization step, solid-phase microextraction (SPME) of midazolam using an 85-microm polyacrylate fiber, and its detection by gas chromatography/mass spectrometry (GC/MS) in selected ion monitoring (SIM) mode, using pinazepam as internal standard. The assay is linear over a midazolam plasma range of 1.5-300 ng/mL, relative intra- and inter-assay standard deviations at 5 ng/mL are below 7%, and the limit of detection is 1 ng/mL. The method is simple, fast and sufficiently sensitive to be applied in clinical and forensic toxicology as well as for purposes of therapeutic drug monitoring.

Capillary GC Determination of Fentanyl and Midazolam in Human Plasma

A capillary GC method is described for simultaneously measuring fentanyl and midazolam in human plasma. Plasma was extracted at basic pH with cyclohexane–isopentanol (197:3) with Ro21-2212 added as internal standard and fentanyl derivative as carrier. The drug was back-extracted in H2SO4, and then the extract was made basic and reextracted with hexane. The organic phase was evaporated at 40°C under N2and the residue was dissolved in ethanol. A portion was analyzed on a wide-bore capillary column (10 m × 0.53 mm) of HP-1 (2.65 μm), operated at 255°C with N2as carrier gas and N–P detector. The calibration graph for fentanyl and midazolam was linear within the respective ranges of 0.5–100 and 5–1000 ng/ml with a correlation coefficient of 0.9998. The recovery of fentanyl and midazolam was 99.5 ± 4.5 and 99.9 ± 3.2%. The coefficient of variation within a run and between runs was less than 7%. The detection limit for fentanyl was 0.2 ng/ml plasma, and for midazolam was 0.5 ng/ml plasma. The method was applied to therapeutic drug monitoring and pharmacokinetic studies.

Automated determination of midazolam in human plasma by high-performance liquid chromatography using column switching

An automated gradient high-performance liquid chromatographic method using a column-switching technique was developed in order to determine and quantify midazolam (separated from the metabolite α-hydroximidazolam) in human plasma. After dilution with an internal standard (flurazepam) solution, containing 20% acetonitrile, 400 μl of the plasma samples were injected onto a precolumn (17 × 4.6 mm I.D., C 18 Corasil 37–53 μm) and retained. Proteins and polar plasma components were washed out using a 0.1 M sodium hydroxide solution, followed by an equilibration with a phosphate buffer of pH 8.0. After column-switching midazolam and flurazepam were eluted and transferred to the analytical column (RP-select B) in the backflush mode, separated by gradient elution and detected at 230 nm by ultraviolet detection. Precision of replicate analyses on the same day was 1.5% for midazolam and 0.7% for flurazepam. Recovery of midazolam was in the range 80–89% and the detection limit was 2 ng/ml plasma.

Sensitive determination of midazolam in human plasma by capillary gas chromatography with surface ionization detection

Sensitive determination of midazolam in human plasma by capillary gas chromatography with surface ionization detection

The determination of total and unbound midazolam in human plasma. A comparison of high performance liquid chromatography, gas chromatography and gas chromatography/mass spectrometry

Midazolam concentrations in patients' plasma was determined after extraction with high performance liquid chromatography (HPLC), gas chromatography (GC) and gas chromatography/mass spectrometry (GC/MS). GC was selected for routine plasma assays in terms of selectivity, simplicity, precision, accuracy and sensitivity (0.02 microgram/mL); HPLC analysis was less sensitive (0.1 microgram/mL) than GC; GC/MS was used for analysis validation. Plasma protein binding of midazolam was determined by GC in patients' plasma after in vitro incubation with midazolam, ultrafiltration and extraction; 5% of the drug was unbound to plasma proteins. Midazolam distribution in lipoprotein fractions separated by ultracentrifugation of plasma obtained from patients on prolonged midazolam treatment was also assayed by GC.

Quantitation by gas chromatography of the 1- and 4-hydroxy metabolites of midazolam in human plasma.

The imidazobenzodiazepine midazolam (MDZ), along with its 1-hydroxy and 4-hydroxy metabolites (1-OH-MDZ, 4-OH-MDZ) can be simultaneously quantitated by electron capture gas-liquid chromatography. After addition of diazepam as internal standard, alkalinized plasma samples are extracted into benzene-isoamyl alcohol. The organic extract is separated, evaporated to dryness, reconstituted, and chromatographed using 3% SP-2250 as the liquid phase. The identity of 4-OH-MDZ, not previously detected in human plasma in unconjugated form, was verified by negative-ion chemical ionization mass spectroscopy. After single oral doses of MDZ administered to humans, concentrations of MDZ and 1-OH-MDZ (alternatively named 1-hydroxymethyl midazolam) were similar, and both compounds were eliminated from plasma in parallel. Concentrations of 4-OH-MDZ were considerably lower, but this metabolite was also eliminated in parallel with the parent compound.

Determination of midazolam and two metabolites of midazolam in human plasma by gas chromatography—negative chemical-ionization mass spectrometry

A method is described for measuring midazolam, a new anesthesia induction agent and hypnotic, and its hydroxymethyl and desmethyl metabolites in human plasma. Deuterated analogues of each compound are added to plasma as internal standards. The compounds are extracted from plasma with benzene containing 20% 1, 2-dichloroethane and after removal of the extracting solvent are dissolved in a solution of bis-(trimethylsilyl)acetamide and acetonitrile. An aliquot of this solution is analyzed by gas chromatography—mass spectrometry with the mass spectrometer set to monitor in the gas chromatographic effluent the M$$$ ions of drug, metabolites and internal standards generated by methane electron-capture negative chemical ionization. For all three compounds, the limit of quantitation is 1 ng ml −1, and the precision (relative standard deviation) at a concentration of 5 ng ml −1 is less than 6%. Measurable amounts of the hydroxymethyl, but not the desmethyl, metabolite of midazolam could be found in the plasma of humans given either an intravenous or an oral dose of midazolam maleate.