A versatile high-performance LC-MS/MS assay for the quantification of voriconazole and its N-oxide metabolite in small sample volumes of multiple human matrices for biomedical applications.

Voriconazole (VRC) pharmacokinetics, in particular its complex metabolism, is still not fully understood which challenges its optimal therapeutic use. To increase knowledge on the pharmacokinetics of this antifungal drug, it is essential to broaden the perspective and expand in vitro and clinical in vivo investigations in particular to aspects such as unbound plasma, target-site and metabolite concentrations. Innovative sampling approaches such as microdialysis, a minimally-invasive technique for the analysis of compound concentrations in target-site human tissue fluids, are associated with bioanalytical challenges, i.e. small sample volumes and low concentrations. Thus, a bioanalytical LC-MS/MS assay for the simultaneous quantification of VRC and its main N-oxide (NO) metabolite in human plasma, ultrafiltrate and microdialysate was developed and validated according to the European Medicines Agency guideline. Quantification was rapid, simple and feasible for clinically relevant concentrations from 5 to 5000 ng/mL in plasma and ultrafiltrate as well as from 4 to 4000 ng/mL in microdialysate. Due to the high sensitivity of the assay, only 20 µL of plasma or ultrafiltrate and 5 µL of microdialysate were required. For VRC and NO in all matrices, between-run accuracy was high with a maximum mean deviation of 7.0% from the nominal value and between-run precision was demonstrated by ≤ 11.8% coefficient of variation. Both compounds proved stable under various conditions. The assay suitability was demonstrated by the application to a clinical study quantifying simultaneously VRC and NO concentrations in plasma, ultrafiltrate and microdialysate. Additionally, the assay was successfully adapted for pharmacokinetic analyses in human tissue-derived in vitro experiments. Overall, by reducing the required sample volume, the bioanalytical method allows for an increased number of plasma samples in vulnerable populations, e.g. infants, and enables the generation of concentration-time profiles with a higher temporal resolution in microdialysis studies. Consequently, the developed assay is apt to elucidate the complex pharmacokinetics of VRC in clinical settings as prerequisite for therapy optimisation.

Automated Real-Time Tumor Pharmacokinetic Profiling in 3D Models: A Novel Approach for Personalized Medicine.

Cancer treatment often lacks individual dose adaptation, contributing to insufficient efficacy and severe side effects. Thus, personalized approaches are highly desired. Although various analytical techniques are established to determine drug levels in preclinical models, they are limited in the automated real-time acquisition of pharmacokinetic profiles. Therefore, an online UHPLC-MS/MS system for quantitation of drug concentrations within 3D tumor oral mucosa models was generated. The integration of sampling ports into the 3D tumor models and their culture inside the autosampler allowed for real-time pharmacokinetic profiling without additional sample preparation. Docetaxel quantitation was validated according to EMA guidelines. The tumor models recapitulated the morphology of head-and-neck cancer and the dose-dependent tumor reduction following docetaxel treatment. The administration of four different docetaxel concentrations resulted in comparable courses of concentration versus time curves for 96 h. In conclusion, this proof-of-concept study demonstrated the feasibility of real-time monitoring of drug levels in 3D tumor models without any sample preparation. The inclusion of patient-derived tumor cells into our models may further optimize the pharmacotherapy of cancer patients by efficiently delivering personalized data of the target tissue.

A rapid, simple and sensitive liquid chromatography tandem mass spectrometry assay to determine amoxicillin concentrations in biological matrix of little volume.

To assess pharmacokinetics of amoxicillin (AMX) in mice, limitations such as a small sampling volume and low drug concentrations have to be addressed. Similar challenges are faced in a clinical framework, e.g. for therapeutic drug monitoring in neonates or small-scale in vitro investigations. An assay enabling quantification of small sample volumes but still at very low concentrations covering a broad concentration range is thus needed. A simple, rapid and highly sensitive liquid chromatography tandem mass spectrometry (LC-MS/MS) method was developed and successfully validated for quantification of AMX in mouse serum according to European Medicines Agency guidelines. Sample preparation enabled the use of only 10 µL of serum, which is 5-fold less than comparable assays and allows to reduce the number of mice used in pharmacokinetic studies. After protein precipitation with 40 µL chilled methanol and dilution of the supernatant with water, the sample was injected into the LC system on a Poroshell 120 Phenyl Hexyl column (2.1 × 100 mm, 2.7 µm). Chromatographic separation was achieved using a gradient method consisting of acetonitrile and ultra-pure water, both with 0.1% (V/V) formic acid. Positive electrospray ionisation in multiple reaction monitoring mode was used for detection and quantification of AMX. Application to murine study samples demonstrated the reliability of the developed method being accurate and precise with a quantification range from 0.01 to 10 µg/mL. The assay is easily transferable due to a simple sample preparation and confirmed stability of AMX under various applied conditions.

SFC for chiral separations in bioanalysis.

This review covers literature investigating methods for enantioselective chromatography using supercritical fluids as mobile phase constituents (SFC) in the field of bioanalysis. It provides an overview on method development and screening approaches published in scientific literature 2014-2018. Chiral stationary phases are used to create a chiral environment that allows for discrimination of enantiomers. Especially polysaccharide-based stationary phases are used in methods of recent investigations. In comparison to HPLC chiral SFC separation provides more selective cavity effects of inclusion-type chiral separation phases. Modifier and additive choices as well as further operating conditions like backpressure, temperature and flow rate are summarized and critically discussed. Further on, observed sample pretreatment and possible detection techniques are presented. SFC hyphenated to mass detection was found of major relevance and is therefore further discussed. Coupling of SFC with different detectors allows for straightforward use in bioanalysis. Interfacing MS detectors is generally performed including a make-up pump. Thus, applied make-up conditions were also reviewed. While most of the chiral separations in HPLC are performed in normal phase mode, and thus, challenge MS hyphenation, SFC-MS hyphenation can be easily achieved. This allows for convenient application in chiral trace analyses, often required in bioanalysis. Even worse in enantioseparation than in achiral chromatography, method development in SFC suffers from a lack of knowledge in separation mechanisms and thus approaches are often quite unique and most often achieved by screening using a One-Factor-at-a-Time (OFAT) design. Broad screening experiments with methodical approaches still appear as method of choice for now.

NDMA impurity in valsartan and other pharmaceutical products: Analytical methods for the determination of N-nitrosamines.

Batch recalls for valsartan containing pharmaceutical products in July 2018 initiated a discussion on possible contaminations with N-nitrosodimethylamine (NDMA). It appeared that NDMA was generated during synthesis of the active pharmaceutical ingredient (API) from the solvent dimethylformamide (DMF) and the reagent nitrite. Discussion on NDMA as API impurity is extended to other drugs since then. Already several years before scientific literature reported NDMA as impurity of several other drugs, thus underlining the apparent risk. At present none of the pharmacopoeias tests for NDMA and only very limited publications of methods for its determination in pharmaceuticals are published so far. This review summarizes aspects for the analyses of nitrosamines (NAs) with special focus on NDMA and discusses their potential applicability for drug analyses. The majority of recent publications utilize GC-MS or GC-MS/MS due to its high selectivity and low detection levels. GC-TEA also provides high selectivity for nitrosamines. However, current availability of this combination is very limited. Alternatively, LC-MS/MS is also performed in NA analysis. An integration of a general test in future pharmacopoeias is suggested due to the toxicological relevance and broader spectrum of possible APIs that may be affected.

Plant derived triterpenes from Gypsophila elegans M.Bieb. enable non-toxic delivery of gene loaded nanoplexes.

To this date, a number of different Gypsophila species from the family of Caryophyllaceae were phytochemically characterized and tested for diverse pharmacological effects. With Gypsophila elegans M. Bieb., we investigated a scarcely explored Gypsophila species, providing a number of potential transfection enhancing triterpene saponins, and so-called sapofection agents. So far triterpene saponins have not been isolated in Gypsophila elegans M.Bieb. Crude extracts from roots and seeds, as well as each purification step were tested for delivery modulation of gene-loaded nanoplexes into neuroblastoma cells. The application of the bioassay guided isolation strategy enabled the assessment of the most active Gypsophila compound, the bisdesmosidic triterpene saponin gypsophilosid A. Gypsophilosid A was isolated by chromatographic techniques, and characterized by electrospray mass spectrometry and intense NMR-spectroscopy, using a variety of 1D and 2D-NMR experiments such as HSQC, HMBC, HQQC, TOCSY and NOESY. In neuroblastoma cells, gypsophilosid A increased the transfection efficiency of gene-nanoplexes up to 80% compared to 2% in the control group without saponin. Our results proved the successful applicability of the implemented methods to detect, isolate and identify saponins, which are biochemically active in terms of transfection.

SFC-MS/MS as an orthogonal technique for improved screening of polar analytes in anti-doping control.

HPLC is considered the method of choice for the separation of various classes of drugs. However, some analytes are still challenging as HPLC shows limited resolution capabilities for highly polar analytes as they interact insufficiently on conventional reversed-phase (RP) columns. Especially in combination with mass spectrometric detection, limitations apply for alterations of stationary phases. Some highly polar sympathomimetic drugs and their metabolites showed almost no retention on different RP columns. Their retention remains poor even on phenylhexyl phases that show different selectivity due to π-π interactions. Supercritical fluid chromatography (SFC) as an orthogonal separation technique to HPLC may help to overcome these issues. Selected polar drugs and metabolites were analyzed utilizing SFC separation. All compounds showed sharp peaks and good retention even for the very polar analytes, such as sulfoconjugates. Retention times and elution orders in SFC are different to both RP and HILIC separations as a result of the orthogonality. Short cycle times could be realized. As temperature and pressure strongly influence the polarity of supercritical fluids, precise regulation of temperature and backpressure is required for the stability of the retention times. As CO2 is the main constituent of the mobile phase in SFC, solvent consumption and solvent waste are considerably reduced. Graphical Abstract SFC-MS/MS vs. LC-MS/MS.

The influence of chronic L-carnitine supplementation on the formation of preneoplastic and atherosclerotic lesions in the colon and aorta of male F344 rats.

L-Carnitine, a key component of fatty acid oxidation, is nowadays being extensively used as a nutritional supplement with allegedly "fat burning" and performance-enhancing properties, although to date there are no conclusive data supporting these claims. Furthermore, there is an inverse relationship between exogenous supplementation and bioavailability, i.e., fairly high oral doses are not fully absorbed and thus a significant amount of carnitine remains in the gut. Human and rat enterobacteria can degrade unabsorbed L-carnitine to trimethylamine or trimethylamine-N-oxide, which, under certain conditions, may be transformed to the known carcinogen N-nitrosodimethylamine. Recent findings indicate that trimethylamine-N-oxide might also be involved in the development of atherosclerotic lesions. We therefore investigated whether a 1-year administration of different L-carnitine concentrations (0, 1, 2 and 5 g/l) via drinking water leads to an increased incidence of preneoplastic lesions (so-called aberrant crypt foci) in the colon of Fischer 344 rats as well as to the appearance of atherosclerotic lesions in the aorta of these animals. No significant difference between the test groups regarding the formation of lesions in the colon and aorta of the rats was observed, suggesting that, under the given experimental conditions, L-carnitine up to a concentration of 5 g/l in the drinking water does not have adverse effects on the gastrointestinal and vascular system of Fischer 344 rats.