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 illicit and medicinal drugs and their metabolites in oral fluid and preserved oral fluid by liquid chromatography–tandem mass spectrometry

An LC-MS/MS method using 0.5 ml of oral fluid was developed for the determination of morphine, codeine, 6-monoacetylmorphine, methadone, amphetamine, methamphetamine, 3,4-methylenedioxyamphetamine, 3,4-methylenedioxymethamphetamine, 3,4-methylenedioxy-N-ethylamphetamine, benzoylecgonine, cocaine, delta-9-tetrahydrocannabinol, zolpidem, zopiclone, alprazolam, clonazepam, oxazepam, nordiazepam, lorazepam, flunitrazepam, diazepam, diphenhydramine and amitriptyline. The method was fully validated in terms of linearity (the method was linear between 1–5 μg/L and 100–200 μg/L) recoveries (7.5–82.6%), within-day and between-day precisions and accuracies (CV and MRE, both <15%), limits of detection (0.5 μ g/L) and quantitation (the lowest point on the calibration curve), relative ion intensities, freeze-and-thaw stability and matrix effect. The method was applied to preserved oral fluid collected by a special commercial device, the StatSure Saliva Sampler™.     

Hydrophilic interaction liquid chromatography–tandem mass spectrometry (HILIC-MS/MS) determination of cocaine and its metabolites benzoylecgonine, ecgonine methyl ester, and cocaethylene in hair samples

This study reports the development and validation of a method using hydrophilic interaction liquid chromatography–tandem mass spectrometry (HILIC-MS/MS) for the analysis of cocaine and its metabolites benzoylecgonine (BE), ecgonine methyl ester (EME), and cocaethylene (CE) in hair samples. Decontamination was performed as follows: Firstly, the aliquot of hair was briefly rinsed with 2 mL dichloromethane, then was washed three times with 10 mL 0.01 M phosphate buffer, pH 6, for 15 min, followed by 2 mL 2-propanol for less than 2 min, and, finally, a last rinse with 2 mL dichloromethane was again done. Cocaine compounds were extracted from 10 mg of hair by incubation with 2 mL 0.1 M HCl at 50 °C for 12 h and purified by solid phase extraction with Oasis MCX cartridges. Analysis was performed by LC-MS/MS using an Atlantis HILIC silica chromatographic column. The method was fully validated. Linearity was established over the concentration range 0.020–10.0 ng/mg for cocaine (COC), 0.010–10.0 ng/mg for BE and CE, and 0.005–2.0 ng/mg for EME, and the correlation coefficients were all >0.99. Extraction efficiency was >70% for all analytes. Limits of detection were 0.0005 ng/mg for CE and 0.001 ng/mg for the other analytes (COC, BE, and EME). Lower limits of quantification were the lowest points of the calibration curves with acceptable accuracy and precision (coefficient of variation ≤20%). Intra- and inter-day imprecision ranged between 1.5% and 9.5% and 0.7% and 12.6%, respectively. Intra- and inter-day inaccuracy ranged from 0.5% to 12.3% and from 0.7% to 7.1%, respectively. With regard to matrix effects, suppression was <27.5% in all cases. The method was applied to the analysis of several samples derived from forensic cases.     

Comparison of liposomes formed by sonication and extrusion: rotational and translational diffusion of an embedded chromophore

We report on the physical and optical characterization of liposomes formed by extrusion and sonication, two widely used methods for vesicle preparation. We also address the issue of whether the properties of bilayers formed from liposomes prepared by the two techniques differ at the molecular and mesoscopic levels. We used the phospholipid 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), with and without cholesterol, to form liposomes, incorporating 1-oleoyl-2-[12-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]dodecanoyl]-sn-glycero-3-phosphocholine (18:1-12:0 NBD-PC) as an optical probe of dynamics. We measured the physical morphology of liposomes by transmission electron microscopy (TEM) and dynamic light scattering (DLS), and the rotational and translational diffusion of 18:1-12:0 NBD-PC by time correlated single photon counting (TCSPC) and fluorescence recovery after pattern photobleaching (FRAPP), respectively. We find that, despite apparent differences in average size and size distribution, both methods of preparation produced liposomes that exhibit the same molecular scale environment. The translational diffusion behavior of the tethered chromophore in planar bilayer lipid membranes formed from the two types of liposomes also yielded similar results.     

Simultaneous determination of opiates, methadone, amphetamines, cocaine, and metabolites in human placenta and umbilical cord by LC-MS/MS

LC-MS/MS methods for the quantification of morphine, morphine-3-glucuronide, morphine-6-glucuronide, codeine, 6-acetylmorphine, cocaine, benzoylecgonine, ecgonine methyl ester, hydroxybenzoylecgonine, cocaethylene, amphetamine, methamphetamine, 3,4-methylenedioxyamphetamine, 3,4-methylenedioxymethamphetamine (MDMA), methadone, and 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine in human placenta and umbilical cord were developed and validated. Specimens (1?±?0.02 g) were homogenized with the Ultra-Turrax T8 disperser and centrifuged, and the supernatant was submitted to solid-phase extraction with Oasis MCX cartridges. Chromatographic separation was performed using an Atlantis T3 analytical column (100?×?2.1 mm, 3 μm) and a gradient of 0.1 % formic acid and acetonitrile. Selectivity was verified in 10 different blank specimens. The method was linear from 1–5 to 100–500 ng/g, depending on the analyte. Limits of detection and quantification ranged from 0.5 to 2.5 ng/g and 1 to 5 ng/g, respectively. Method imprecision was ≤15.3 %, except for MDMA at low quality control (18.1 %); accuracy, 87.1 to 114 %; extraction efficiency, 16.3 to 154.0 % (%CV?=?1.8-39.4 %); matrix effect, ?75.7 to 449.9 % (%CV?=?3.5–50 %); and process efficiency, 8.7 to 316.0 %. The method was applied to authentic placenta and umbilical cord specimens from drug-user pregnant women.