Quantification of plasma carnitine and acylcarnitines by high-performance liquid chromatography-tandem mass spectrometry using online solid-phase extraction

Carnitine is an amino acid derivative that plays a key role in energy metabolism. Endogenous carnitine is found in its free form or esterified with acyl groups of several chain lengths. Quantification of carnitine and acylcarnitines is of particular interest for screening for research and metabolic disorders. We developed a method with online solid-phase extraction coupled to high-performance liquid chromatography and tandem mass spectrometry to quantify carnitine and three acylcarnitines with different polarity (acetylcarnitine, octanoylcarnitine, and palmitoylcarnitine). Plasma samples were deproteinized with methanol, loaded on a cation exchange trapping column and separated on a reversed-phase C8 column using heptafluorobutyric acid as an ion-pairing reagent. Considering the endogenous nature of the analytes, we quantified with the standard addition method and with external deuterated standards. Solid-phase extraction and separation were achieved within 8min. Recoveries of carnitine and acylcarnitines were between 98 and 105%. Both quantification methods were equally accurate (all values within 84 to 116% of target concentrations) and precise (day-to-day variation of less than 18%) for all carnitine species and concentrations analyzed. The method was used successfully for determination of carnitine and acylcarnitines in different human samples. In conclusion, we present a method for simultaneous quantification of carnitine and acylcarnitines with a rapid sample work-up. This approach requires small sample volumes and a short analysis time, and it can be applied for the determination of other acylcarnitines than the acylcarnitines tested. The method is useful for applications in research and clinical routine. Figure A method is presented for the analysis of carnitine and acylcarnitines in urine which includes a precipitation step, on-column extraction and LC-MS/MS. The run time is 8 minutes and the method was validated for carnitine, acetylcarnitine, octanoylcarnitine and palmitoylcarnitine. Analysis of a patient sample with medium-chain acyl-CoA dehydrogenase deficiency is show

Electrochemical Reduction and Oxidation of Ruddlesden–Popper-Type La2_{2}NiO3_{3}F2_{2} within Fluoride-Ion Batteries

Within this article, it is shown that an electrochemical defluorination and additional fluorination of Ruddlesden–Popper-type La$_2$NiO$_3$F$_2$ is possible within all-solid-state fluoride-ion batteries. Structural changes within the reduced and oxidized phases have been examined by X-ray diffraction studies at different states of charging and discharging. The synthesis of the oxidized phase La$_2$NiO$_3$F$_{2+x}$ proved to be successful by structural analysis using both X-ray powder diffraction and automated electron diffraction tomography techniques. The structural reversibility on re-fluorinating and re-defluorinating is also demonstrated. Moreover, the influence of different sequences of consecutive reduction and oxidation steps on the formed phases has been investigated. The observed structural changes have been compared to changes in phases obtained via other topochemical modification approaches such as hydride-based reduction and oxidative fluorination using F$_2$ gas, highlighting the potential of such electrochemical reactions as alternative synthesis routes. Furthermore, the electrochemical routes represent safe and controllable synthesis approaches for novel phases, which cannot be synthesized via other topochemical methods. Additionally, side reactions, occurring alongside the desired electrochemical reactions, have been addressed and the cycling performance has been studied

On the Surface Modification of LLZTO with LiF via a Gas-Phase Approach and the Characterization of the Interfaces of LiF with LLZTO as Well as PEO+LiTFSI

In this study we present gas-phase fluorination as a method to create a thin LiF layer on Li₆.₅La₃Zr₁.₅Ta₀.₅O₁₂ (LLZTO). We compared these fluorinated films with LiF films produced by RF-magnetron sputtering, where we investigated the interface between the LLZTO and the deposited LiF showing no formation of a reaction layer. Furthermore, we investigated the ability of this LiF layer as a protection layer against Li₂CO₃ formation in ambient air. By this, we show that Li₂CO₃ formation is absent at the LLZTO surface after 24 h in ambient air, supporting the protective character of the formed LiF films, and hence potentially enhancing the handling of LLZTO in air for battery production. With respect to the use within hybrid electrolytes consisting of LLZTO and a mixture of polyethylene oxide (PEO) and lithium bis(trifluoromethanesulfonyl)imide (LiTFSI), we also investigated the interface between the formed LiF films and a mixture of PEO+LiTFSI by X-ray photoelectron spectroscopy (XPS), showing decomposition of the LiTFSI at the interface

Author Correction: Mutant p53 sustains serine-glycine synthesis and essential amino acids intake promoting breast cancer growth

Reprogramming of amino acid metabolism, sustained by oncogenic signaling, is crucial for cancer cell survival under nutrient limitation. Here we discovered that missense mutant p53 oncoproteins stimulate de novo serine/glycine synthesis and essential amino acids intake, promoting breast cancer growth. Mechanistically, mutant p53, unlike the wild-type counterpart, induces the expression of serine-synthesis-pathway enzymes and L-type amino acid transporter 1 (LAT1)/CD98 heavy chain heterodimer. This effect is exacerbated by amino acid shortage, representing a mutant p53-dependent metabolic adaptive response. When cells suffer amino acids scarcity, mutant p53 protein is stabilized and induces metabolic alterations and an amino acid transcriptional program that sustain cancer cell proliferation. In patient-derived tumor organoids, pharmacological targeting of either serine-synthesis-pathway and LAT1-mediated transport synergizes with amino acid shortage in blunting mutant p53-dependent growth. These findings reveal vulnerabilities potentially exploitable for tackling breast tumors bearing missense TP53 mutation

Mutant p53 sustains serine-glycine synthesis and essential amino acids intake promoting breast cancer growth

Reprogramming of amino acid metabolism, sustained by oncogenic signaling, is crucial for cancer cell survival under nutrient limitation. Here we discovered that missense mutant p53 oncoproteins stimulate de novo serine/glycine synthesis and essential amino acids intake, promoting breast cancer growth. Mechanistically, mutant p53, unlike the wild-type counterpart, induces the expression of serine-synthesis-pathway enzymes and L-type amino acid transporter 1 (LAT1)/CD98 heavy chain heterodimer. This effect is exacerbated by amino acid shortage, representing a mutant p53-dependent metabolic adaptive response. When cells suffer amino acids scarcity, mutant p53 protein is stabilized and induces metabolic alterations and an amino acid transcriptional program that sustain cancer cell proliferation. In patient-derived tumor organoids, pharmacological targeting of either serine-synthesis-pathway and LAT1-mediated transport synergizes with amino acid shortage in blunting mutant p53-dependent growth. These findings reveal vulnerabilities potentially exploitable for tackling breast tumors bearing missense TP53 mutations.Mutant p53 induces serine/glycine synthesis and essential amino acids intake. Under amino acid restriction, mutant p53 is stabilized and activates a transcriptional program that sustains a metabolic adaptive response promoting breast cancer cells growt

Topochemical Fluorination of La2NiO4+d::Unprecedented ordering of oxide and fluoride ions in La2NiO3F2

Synopsis La2NiO3F2 crystallizes in a new anion-ordered distortion variant of the n = 1 Ruddlesden−Popper-type structure. The unprecedented ordering of oxygen anions in the interlayer leads to an expansion of the lattice perpendicular to the stacking direction, accompanied by a strong tilting of NiO4F2 octahedra. A weakening of Ni−F−F−Ni superexchange interactions between the perovskite-type layers due to the reduced covalency of fluoride ions decreases the magnetic ordering temperature strongly. Abstract The Ruddlesden–Popper (K2NiF4) type phase La2NiO3F2 was prepared via a polymer-based fluorination of La2NiO4+d. The compound was found to crystallize in the orthorhombic space group Cccm (a = 12.8350(4) Å, b = 5.7935(2) Å, c = 5.4864(2) Å). This structural distortion results from an ordered half occupation of the interstitial anion layers and has not been observed previously for K2NiF4-type oxyfluoride compounds. From a combination of neutron and X-ray powder diffraction and 19F magic-angle spinning NMR spectroscopy, it was found that the fluoride ions are only located on the apical anion sites, whereas the oxide ions are located on the interstitial sites. This ordering results in a weakening of the magnetic Ni–F–F–Ni superexchange interactions between the perovskite layers and a reduction of the antiferromagnetic ordering temperature to 49 K. Below 30 K, a small ferromagnetic component was found, which may be the result of a magnetic canting within the antiferromagnetic arrangement and will be the subject of a future low-temperature neutron diffraction study. Additionally, density functional theory-based calculations were performed to further investigate different anion ordering scenarios

Quantification of plasma carnitine and acylcarnitines by high-performance liquid chromatography-tandem mass spectrometry using online solid-phase extraction

Carnitine is an amino acid derivative that plays a key role in energy metabolism. Endogenous carnitine is found in its free form or esterified with acyl groups of several chain lengths. Quantification of carnitine and acylcarnitines is of particular interest for screening for research and metabolic disorders. We developed a method with online solid-phase extraction coupled to high-performance liquid chromatography and tandem mass spectrometry to quantify carnitine and three acylcarnitines with different polarity (acetylcarnitine, octanoylcarnitine, and palmitoylcarnitine). Plasma samples were deproteinized with methanol, loaded on a cation exchange trapping column and separated on a reversed-phase C8 column using heptafluorobutyric acid as an ion-pairing reagent. Considering the endogenous nature of the analytes, we quantified with the standard addition method and with external deuterated standards. Solid-phase extraction and separation were achieved within 8 min. Recoveries of carnitine and acylcarnitines were between 98 and 105 %. Both quantification methods were equally accurate (all values within 84 to 116 % of target concentrations) and precise (day-to-day variation of less than 18 %) for all carnitine species and concentrations analyzed. The method was used successfully for determination of carnitine and acylcarnitines in different human samples. In conclusion, we present a method for simultaneous quantification of carnitine and acylcarnitines with a rapid sample work-up. This approach requires small sample volumes and a short analysis time, and it can be applied for the determination of other acylcarnitines than the acylcarnitines tested. The method is useful for applications in research and clinical routine

Comparison Of Liver Cell Models Using The Basel Phenotyping Cocktail

Currently used hepatocyte cell systems for in vitro assessment of drug metabolism include hepatoma cell lines and primary human hepatocyte (PHH) cultures. We investigated the suit-ability of the validated in vivo Basel phenotyping cocktail (caffeine [CYP1A2], efavirenz [CYP2B6], losartan [CYP2C9], omeprazole [CYP2C19], metoprolol [CYP2D6], midazolam [CYP3A4]) in vitro and characterized four hepatocyte cell systems (HepG2 cells, HepaRG cells, and primary cryopreserved human hepatocytes in 2-dimensional [2D] culture or in 3D-spheroid co-culture) regarding basal metabolism and CYP inducibility. Under non-induced conditions, all CYP activities could be determined in 3D-PHH, CYP2B6, CYP2C19, CYP2D6 and CYP3A4 in 2D-PHH and HepaRG, and CYP2C19 and CYP3A4 in HepG2 cells. The highest non-induced CYP activities were observed in 3D-PHH and HepaRG cells. mRNA expression was at least 4-fold higher for all CYPs in 3D-PHH compared to the other cell systems. After treatment with 20µM rifampicin, mRNA increased 3 to 50-fold for all CYPs except CYP1A2 and 2D6 for HepaRG and 3D-PHH, 4-fold (CYP2B6) and 17-fold (CYP3A4) for 2D-PHH and 4-fold (CYP3A4) for HepG2. In 3D-PHH at least a 2-fold in-crease in CYP activity was observed for all inducible CYP isoforms while CYP1A2 and CYP2C9 activity did not increase in 2D-PHH and HepaRG. CYP inducibility assessed in vivo using the same phenotyping probes was also best reflected by the 3D-PHH model.Our studies show that 3D-PHH and (with some limitations) HepaRG are suitable cell systems for assessing drug metabolism and CYP induction in vitro. HepG2 cells are less suited to as-sess CYP induction of the 2C and 3A family. The Basel phenotyping cocktail is suitable for the assessment of CYP activity and induction also in vitro

The basel cocktail for simultaneous phenotyping of human cytochrome P450 isoforms in plasma, saliva and dried blood spots.

BACKGROUND AND OBJECTIVE Phenotyping cocktails use a combination of cytochrome P450 (CYP)-specific probe drugs to simultaneously assess the activity of different CYP isoforms. To improve the clinical applicability of CYP phenotyping, the main objectives of this study were to develop a new cocktail based on probe drugs that are widely used in clinical practice and to test whether alternative sampling methods such as collection of dried blood spots (DBS) or saliva could be used to simplify the sampling process. METHODS In a randomized crossover study, a new combination of commercially available probe drugs (the Basel cocktail) was tested for simultaneous phenotyping of CYP1A2, CYP2B6, CYP2C9, CYP2C19, CYP2D6 and CYP3A4. Sixteen subjects received low doses of caffeine, efavirenz, losartan, omeprazole, metoprolol and midazolam in different combinations. All subjects were genotyped, and full pharmacokinetic profiles of the probe drugs and their main metabolites were determined in plasma, dried blood spots and saliva samples. RESULTS The Basel cocktail was well tolerated, and bioequivalence tests showed no evidence of mutual interactions between the probe drugs. In plasma, single timepoint metabolic ratios at 2 h (for CYP2C19 and CYP3A4) or at 8 h (for the other isoforms) after dosing showed high correlations with corresponding area under the concentration-time curve (AUC) ratios (AUC0-24h parent/AUC0-24h metabolite) and are proposed as simple phenotyping metrics. Metabolic ratios in dried blood spots (for CYP1A2 and CYP2C19) or in saliva samples (for CYP1A2) were comparable to plasma ratios and offer the option of minimally invasive or non-invasive phenotyping of these isoforms. CONCLUSIONS This new combination of phenotyping probe drugs can be used without mutual interactions. The proposed sampling timepoints have the potential to facilitate clinical application of phenotyping but require further validation in conditions of altered CYP activity. The use of DBS or saliva samples seems feasible for phenotyping of the selected CYP isoforms

Using dried blood spots to facilitate therapeutic drug monitoring of antiretroviral drugs in resource-poor regions

Abstract Objectives We evaluated whether dried blood spots (DBS) are suitable to monitor combined ART when samples are collected in rural Tanzania and transported over a long distance to a specialized bioanalytical laboratory. Methods Plasma and DBS samples were collected in Tanzania from study patients treated with nevirapine, efavirenz or lopinavir. In addition, plasma, whole blood and DBS samples were obtained from a cohort of HIV patients at the site of the bioanalytical laboratory in Switzerland. DBS samples were analysed using a fully automated LC-MS/MS method. Results Comparison of DBS versus plasma concentrations of samples obtained from the bridging study in Switzerland indicated an acceptable bias only for nevirapine (18.4%), whereas for efavirenz and lopinavir a pronounced difference of −47.4% and −48.1% was found, respectively. Adjusting the DBS concentrations by the haematocrit and the fraction of drug bound to plasma proteins removed this bias [efavirenz +9.4% (−6.9% to +25.7%), lopinavir +2.2% (−20.0% to +24.2%)]. Storage and transportation of samples from Tanzania to Switzerland did not affect the good agreement between plasma and DBS for nevirapine [-2.9% (−34.7% to +29.0%)] and efavirenz [-9.6% (−42.9% to +23.8%)]. For lopinavir, however, adjusted DBS concentrations remained considerably below [-32.8% (−70.4% to +4.8%)] corresponding plasma concentrations due to decay of lopinavir in DBS obtained under field conditions. Conclusions Our field study shows that the DBS technique is a suitable tool for therapeutic drug monitoring in resource-poor regions; however, sample stability remains an issue for certain analytes and therefore needs special consideration