Rethinking Drug Analysis in Health Care: High-Throughput Analysis of 71 Drugs of Abuse in Oral Fluid Using Ion Mobility--High-Resolution Mass Spectrometry.

We have identified a clinical need for a sensitive, specific, flexible, comprehensive and affordable analytical technology to efficiently detect polydrug use. In addition, the current standard practice of surveilled urine sampling is uncomfortable for the patient; hence, more patient-friendly sample collection methods are requested. To fill these needs, we have developed and validated a high-throughput liquid chromatography-high-resolution mass spectrometry (LC--HRMS) method for the analysis of drugs of abuse (DoA) in oral fluid (OF). The method covers a panel of 71 substances including traditional DoA, prescription narcotics and new psychoactive substances (NPS), with a guaranteed limit of identification of <3 µg/L for 87% of the analytes. Method validation showed high accuracy (>99.7%), sensitivity (>99.7%) and specificity (100%). Most analytes had a high process efficiency during the salting-out liquid-liquid extraction sample preparation and no or only a minor matrix effect during the analysis. We have implemented this method in clinical routine and present data from 18,579 OF samples collected during routine patient treatment in mainly psychiatric and addiction clinics in West Sweden between September 2020 and June 2021. Seventy-one percent of the samples were positive and a total of 41,472 DoA findings were detected. Amphetamine (27%), buprenorphine (25%), nordiazepam (18%) and alprazolam (16%) were most prevalent. New psychoactive substances were detected in 189 samples (1.0%). The occurrence of polydrug use was common; 34% of the positive samples contained three analytes or more and 12% six or more. To the best of our knowledge, this is the first method for comprehensive analysis of DoA in OF using LC--HRMS and the largest dataset published on the detection of DoA in OF. With the current complex and variable drug use pattern, this broad, cost-effective and reliable method has largely replaced immunoassay screening in urine in our laboratory.

Osmoprotective effect of ubiquinone in lipid vesicles modelling the E. coli plasma membrane.

Bacteria need to be able to adapt to sudden changes in their environment, including drastic changes in the surrounding osmolarity. As part of this adaptation, the cells adjust the composition of their cytoplasmic membrane. Recent studies have shown that ubiquinones, lipid soluble molecules involved in cell respiration, are overproduced by bacteria grown in hyperosmotic conditions and it is thus believed that these molecules can provide with osmoprotection. Hereby we explore the mechanisms behind these observations. Liposomes with a lipid headgroup composition mimicking that of the cytoplasmic membrane of E. coli are used as suitable models. The effect of ubiquinone-10 (Q10) on water transport across the membranes is characterized using a custom developed fluorescence-based experimental approach to simultaneously determine the membrane permeability coefficient and estimate the elastic resistance of the membrane towards deformation. It is shown that both parameters are affected by the presence of ubiquinone-10. Solanesol, a molecule similar to Q10 but lacking the quinone headgroup, also provides with osmoprotection although it only improves the resistance of the membrane against deformation. The fluorescence experiments are complemented by cryogenic transmission electron microscopy studies showing that the E. coli membrane mimics tend to flatten into spheroid oblate structures when osmotically stressed, suggesting the possibility of lipid segregation. In agreement with its proposed osmoprotective role, the flattening process is hindered by the presence of Q10.

Choice of cuvette material can influence spectroscopic leakage and permeability experiments with liposomes.

Liposome solute permeability experiments are widely performed to gain information about lipid membrane characteristics. Spectroscopic methods are often used for this purpose, usually monitoring the leakage of a self-quenching fluorescent dye (e.g., carboxyfluorescein, CF) from the liposomes. Hereby, we investigate the effect of liposome-cuvette interactions, a seldom considered detail, on the results obtained from liposomal permeability experiments. The spontaneous leakage of CF from liposomes with different surface properties and phase states is followed using quartz and polystyrene cuvettes, and the results are compared. It is shown that for most lipid compositions the leakage profiles vary notably between different cuvette materials. Reproducibility of the measurements also varies depending on the cuvettes used, with polystyrene providing with more robust results. To explain these observations, the interaction of liposomes with polystyrene and quartz-like surfaces was characterized with the help of the quartz crystal microbalance with dissipation monitoring (QCM-D). Our results show that, while liposomes seldom interact with polystyrene, quartz-liposome interactions are almost unavoidable and have a large impact on the leakage experiments mainly via two mechanisms: i) the rupturing of liposomes on the cuvette surface causing a fast release of encapsulated CF, and ii) the disruption of adsorbed liposomes caused by magnetic stirring. Depending on their composition, the liposomes interact in different ways with quartz, affecting thus the extent of each proposed mechanism. The experiments demonstrate the importance of considering the cuvette material when planning and conducting spectroscopic experiments with liposomes.

Effect of ubiquinone-10 on the stability of biomimetic membranes of relevance for the inner mitochondrial membrane.

Ubiquinone-10 (Q10) plays a pivotal role as electron-carrier in the mitochondrial respiratory chain, and is also well known for its powerful antioxidant properties. Recent findings suggest moreover that Q10 could have an important membrane stabilizing function. In line with this, we showed in a previous study that Q10 decreases the permeability to carboxyfluorescein (CF) and increases the mechanical strength of 1-palmitoyl-2-oleyl-sn-glycero-phosphocholine (POPC) membranes. In the current study we report on the effects exerted by Q10 in membranes having a more complex lipid composition designed to mimic that of the inner mitochondrial membrane (IMM). Results from DPH fluorescence anisotropy and permeability measurements, as well as investigations probing the interaction of liposomes with silica surfaces, corroborate a membrane stabilizing effect of Q10 also in the IMM-mimicking membranes. Comparative investigations examining the effect of Q10 and the polyisoprenoid alcohol solanesol on the IMM model and on membranes composed of individual IMM components suggest, moreover, that Q10 improves the membrane barrier properties via different mechanisms depending on the lipid composition of the membrane. Thus, whereas Q10's inhibitory effect on CF release from pure POPC membranes appears to be directly and solely related to Q10's lipid ordering and condensing effect, a mechanism linked to Q10's ability to amplify intrinsic curvature elastic stress dominates in case of membranes containing high proportions of palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE).

Ubiquinone-10 alters mechanical properties and increases stability of phospholipid membranes.

Ubiquinone-10 is mostly known for its role as an electron and proton carrier in aerobic cellular respiration and its function as a powerful antioxidant. Accumulating evidence suggest, however, that this well studied membrane component could have several other important functions in living cells. The current study reports on a previously undocumented ability of ubiquinone-10 to modulate the mechanical strength and permeability of lipid membranes. Investigations of DPH fluorescence anisotropy, spontaneous and surfactant induced leakage of carboxyfluorescein, and interactions with hydrophobic and hydrophilic surfaces were used to probe the effects caused by inclusion of ubiquinone-10 in the membrane of phospholipid liposomes. The results show that ubiquinone in concentrations as low as 2 mol% increases the lipid packing order and condenses the membrane. The altered physicochemical properties result in a slower rate of release of hydrophilic components, and render the membrane more resistant towards rupture. As judged from comparative experiments using the polyisoprenoid alcohol solanesol, the quinone moiety is essential for the membrane stabilizing effects to occur. Our findings imply that the influence of ubiquinone-10 on the permeability and mechanical properties of phospholipid membranes is similar to that of cholesterol. The reported data indicate, however, that the molecular mechanisms are different in the two cases.