Charakterisierung der Mikroflüssigkeitschromatographie – Ableitung theoretischer Leistungsgrenzen und praktische Aspekte für die Routineanalytik am Beispiel der Zytostatika

Miniaturized liquid chromatography (LC) is ideally suited to reduce the resource consumption without compromising data quality in the analytical laboratory. However, the applicability and handling of miniaturized LC in routine analysis is often being questioned. In addition, only little information on the theoretical performance limits of this separation technique is available. Therefore, the aim of this study was the characterization of micro-liquid chromatography (micro-LC) from the theoretical and practical point of view. For the evaluation the development of a fast method for the analysis of antineoplastic drugs from wipe samples using micro-LC-MS/MS was applied. The investigated analytes comprise polar as well as non-polar compounds. In addition, three critical peak pairs were included that either cannot be differentiated by mass spectrometry or are affected by ion suppression at co-elution. Therefore, a chromatographic separation was mandatory. Micro-LC was used as separation dimension using a column with an inner diameter of 300 µm due to the advantages of reduced resource consumption and high linear velocity leading to increased sample throughput. To achieve a chromatographic separation, a suitable chromatographic phase system was identified by principal component analysis (PCA). Afterwards, the column efficiency was investigated using van Deemter and kinetic plot analysis for isocratic and gradient elution. The results indicate a higher packing quality of the sub-2 µm fully porous particle packed column compared to larger particle diameters of various morphology. In addition, similar values for the reduced plate height were found in micro-LC compared to conventional column inner diameters (≥ 2.1 mm). The investigation of peak capacity also demonstrates the benefit of sub-2 µm fully porous particles for gradient times between 5 s and 5 min. Further optimization of the extraction efficiency, ion source parameters and system design was done to establish the method on basis of micro-LC-MS/MS. This hyphenation technique enables to separate the target analytes within 2.5 min at a flow rate of only 25 µL min-1. Thereby the sample throughput can be increased by a factor of 2 reducing the resource consumption by 98% compared to the previously used conventional LC-MS/MS method. Method validation was accomplished to demonstrate that sensitivity and robustness of the developed method is given in a routine environment. Furthermore, it was shown that the method can be used to analyze real samples from hospital pharmacies in order to verify the reference value of 0.1 ng cm-2 for workplace contamination of antineoplastic drugs. In summary, it was exemplarily demonstrated that micro-LC can successfully replace conventional LC-MS in targeted ultrasensitive MS analysis.Verfahren wie die miniaturisierte Hochleistungsflüssigkeitschromatographie (LC) sind ideal geeignet, um den Verbrauch an Ressourcen zu verringern ohne dabei die Datenqualität im analytischen Labor negativ zu beeinflussen. Nichtsdestotrotz wird die Anwendbarkeit dieser Technik in der Routineanalytik aufgrund der Handhabbarkeit oftmals kritisch hinterfragt. Des Weiteren sind nur wenige Informationen bezüglich der theoretischen Leistungsfähigkeit dieser Trenndimension verfügbar. Aus diesem Grund ist das Ziel dieser Arbeit die Charakterisierung der Mikro-Flüssigkeitschromatographie (Mikro-LC) aus Sicht der Theorie und Praxis. Zur Bewertung wurde die Entwicklung einer schnellen Analysenmethode ausgewählter Zytostatika von Wischproben mittels Mikro-LC-MS/MS verwendet. Die untersuchten Analyten umfassen dabei sowohl polare als auch unpolare Substanzen. Weiterhin sind drei kritische Peakpaare enthalten, bei denen entweder eine Ionensuppression bei Koelution beobachtet wird oder eine Unterscheidung mittels Massenspektrometrie nicht möglich ist. Aus den genannten Gründen ist eine chromatographische Trennung notwendig. Um dies zu erreichen, wurden Mikro-LC-Säulen mit einem Innendurchmesser von 300 µm eingesetzt. Dies bietet neben dem reduzierten Verbrauch von Ressourcen den Vorteil, dass durch hohe lineare Fließgeschwindigkeiten ein erhöhter Probendurchsatz generiert werden kann. Um eine chromatographische Trennung zu erreichen, wurde ein geeignetes Phasensystem durch den Einsatz der Hauptkomponentenanalyse (PCA) identifiziert. Anschließend wurde die Effizienz miniaturisierter Trennsäulen mittels van Deemter und Kinetic Plot Analyse sowohl für die isokratische Arbeitsweise als auch für die Gradientenelution untersucht. Dabei stellte sich heraus, dass vollporöse Partikel mit einem Partikeldurchmesser von kleiner 2 µm (sub-2 µm) eine verbesserte Packungsqualität gegenüber größeren Partikeln unterschiedlicher Morphologie aufweisen. Weiterhin konnte festgestellt werden, dass im Vergleich zu konventionellen Innendurchmessern vergleichbare Packungsqualitäten erreicht werden können. Die Untersuchung der Peakkapazität für Gradientenlaufzeiten zwischen 5 s und 5 min ergab, dass mit vollporösen sub-2 µm Partikeln eine höhere Peakkapazität pro Zeiteinheit generiert werden konnte. Des Weiteren wurden neben dem Systemaufbau, die Extraktionseffizienz sowie die Ionenquellenparameter optimiert, um die Methode auf Basis der Mikro-LC-MS/MS zu etablieren. Durch diese Kopplung konnten die Komponenten innerhalb von 2.5 min bei einer Flussrate von lediglich 25 µL min-1 getrennt werden. Dadurch konnte der Probendurchsatz um den Faktor 2 bei gleichzeitiger Reduzierung der Ressourcen um 98% im Vergleich zur zuvor etablierten konventionellen LC-MS/MS gesteigert werden. Anschließend wurden Validierungsdaten erhoben, um die Sensitivität und Robustheit der entwickelten Methode im Routineumfeld zu demonstrieren. Anhand von Realproben aus Krankenhausapotheken wurde gezeigt, dass der Referenzwert für die Arbeitsplatzkontamination von Zytostatika von 0,1 ng cm-2 mittels der Methode überprüft werden kann. Damit konnte exemplarisch gezeigt werden, dass die Mikro-LC qualifiziert ist, die konventionelle LC-MS im Bereich der ultra-sensitiven Targetanalytik zu ersetzen

Tackling Issues Observed during the Development of a Liquid Chromatography Method for Small Molecule Quantification in Antibody-Chelator Conjugate

In the context of targeted radionuclide therapy, antibody-chelator conjugates (ACCs) are an evolving class of antibody-related drugs with promising applications as tumor-targeted pharmaceuticals. Generally, a typical ACC consists of a recombinant monoclonal antibody (mAb) coupled to radionuclide via a chelating agent. Characterizing the ACC structure represents an analytical challenge since various impurities must be constantly monitored in the presence of formulation components during the quality control (QC) process. In this contribution, a reliable method devoted to the monitoring of an ACC sample, and its small molecule-related synthesis impurities, has been developed via liquid chromatography (LC). A problem-solving approach of common analytical issues was used to highlight some major issues encountered during method development. This included separation of poorly retained impurities (issue #1); interferences from the formulation components (issue #2); analysis of impurities in presence of ACC at high concentration (issue #3); and recovery of impurities during the whole analytical procedure (issue #4). To the best of our knowledge, this is the first time that a chromatographic method for the analysis of ACC synthesis impurities is presented. In addition, the developed approach has the potential to be more widely applied to the characterization of similar ACCs and other antibody-related drugs.</p

Pharmacodynamics, pharmacokinetics and CYP3A4 interaction potential of the selective P2X3 receptor antagonist filapixant: A randomized multiple ascending-dose study in healthy young men

Aims: We report on investigations exploring the P2X3-receptor antagonist filapixant's effect on taste perception and cough-reflex sensitivity and describe its pharmacokinetics, including its CYP3A4-interaction potential. Methods: In a randomized, placebo-controlled, double-blind study, 3 × 12 healthy men (18-45 years) were assigned (3:1) to filapixant (20, 80 or 250 mg by mouth) or placebo twice daily over 2 weeks. A single dose of midazolam (1 mg), a CYP3A4 substrate, was administered with and without filapixant. Assessments included a taste-strips test, a taste questionnaire, cough challenge with adenosine triphosphate, adverse event reports and standard safety assessments. Results: Taste disturbances were observed mainly in the 250-mg group: six of nine participants (67%) in this group reported hypo- or dysgeusia in the questionnaire; eight participants (89%) reported taste-related adverse events. Five participants (56%) had a decrease in overall taste-strips-test scores ≥2 points (point estimate −1.1 points, 90% confidence interval [−3.3; 1.1]). Cough counts increased with adenosine triphosphate concentration but without major differences between treatments. Filapixant exposure increased proportionally to dose. Co-administration of filapixant had no clinically relevant effect on midazolam pharmacokinetics. Area under the concentration-time curve ratios and 90% confidence intervals were within 80-125%. No serious or severe adverse events were reported. Conclusions: Overall, filapixant was safe and well tolerated, apart from mild, transient taste disturbances. Such disturbances occurred more frequently than expected based on (in vitro) receptor-selectivity data, suggesting that other factors than P2X3:P2X2/3 selectivity might also play an important role in this context. The cough-challenge test showed no clear treatment effect. Filapixant has no clinically relevant CYP3A4 interaction potential

Online and Splitless NanoLC × CapillaryLC with Quadrupole/Time-of-Flight Mass Spectrometric Detection for Comprehensive Screening Analysis of Complex Samples

A novel multidimensional separation system based on online comprehensive two-dimensional liquid chromatography and hybrid high-resolution mass spectrometry has been developed for the qualitative screening analysis and characterization of complex samples. The core of the system is a consistently miniaturized two-dimensional liquid chromatography that makes the rapid second dimension compatible with mass spectrometry without the need for any flow split. Elevated temperature, ultrahigh pressure, and a superficially porous sub-3-μm stationary phase provide a fast second dimension separation and a sufficient sampling frequency without a first dimension flow stop. A highly loadable porous graphitic carbon stationary phase is employed in the first dimension to implement large volume injections that help countervailing dilution caused by the sampling process between the two dimensions. Exemplarily, separations of a 99-component standard mixture and a complex wastewater sample were used to demonstrate the performance of the dual-gradient system. In the second dimension, 30 s gradients at a cycle time of 1 min were employed. One multidimensional separation took 80–90 min (∼120 min including extended hold and re-equilibration in the first dimension). This approach represents a cost-efficient alternative to online LC × LC strategies working with conventionally sized columns in the rapid second dimension, as solvent consumption is drastically decreased and analytes still are detectable at environmentally relevant concentrations

Feedback from the Science Caf\ue9 from the Sixth European Bioanalysis Forum Young Scientist Symposium

The 6th Young Scientist Symposium, a meeting organized by young scientists for young scientists under the umbrella of the European Bioanalysis Forum vzw and in collaboration with the Universities of Bologna and Ghent, included a variety of interesting presentations on cutting edge bioanalytical science and processes. Integrated in the meeting, an interactive round table session, the Science Cafe, discussed the challenges related to sustainability for bioanalytical lab activities. This manuscript reflects conclusions from these discussions. They can provide our community a compass for future business practices to embrace more sustainable laboratory activities considerate of smarter use of a wide array of resources and laboratory tools, resulting in increased wellbeing for our next generations and our planet