Innovative methodology to transfer conventional GC-MS heroin profiling to UHPLC-MS/MS

Nowadays, in forensic laboratories, heroin profiling is frequently carried out by gas chromatography coupled with mass spectrometry (GC-MS). This analytical technique is well established, provides good sensitivity and reproducibility, and allows the use of large databases. Despite those benefits, recently introduced analytical techniques, such as ultra-high-pressure liquid chromatography (UHPLC), could offer better chromatographic performance, which needs to be considered to increase the analysis throughput for heroin profiling. With the latter, chromatographic conditions were optimized through commercial modeling software and two atmospheric pressure ionization sources were evaluated. Data obtained from UHPLC-MS/MS were thus transferred, thanks to mathematical models to mimic GC-MS data. A calibration and a validation set of representative heroin samples were selected among the database to establish a transfer methodology and assess the models' abilities to transfer using principal component analysis and hierarchical classification analysis. These abilities were evaluated by computing the frequency of successful classification of UHPLC-MS/MS data among GC-MS database. Seven mathematical models were tested to adjust UHPLC-MS/MS data to GC-MS data. A simplified mathematical model was finally selected and offered a frequency of successful transfer equal to 95%. Figur

Enantiomeric methadone quantitation on real post-mortem dried matrix spots samples: Comparison of liquid chromatography and supercritical fluid chromatography coupled to mass spectrometry.

This study describes two bioanalytical methods for the quantitation of the two methadone enantiomers in dried matrix spots using high performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS) and high performance supercritical chromatography tandem mass spectrometry (HPSFC-MS/MS). Dried matrix spots were obtained by spotting 10 µL of each sample fluid on a Whatman paper. Methadone and its main metabolite, EDDP, were extracted with 100 µL methanol and subsequently injected into the LC-MS/MS and SFC-MS/MS systems. Enantiomeric separation was achieved with AGP-column for the LC conditions and with Chiralpak IH-3 in SFC. The two methods were fully validated and 93 post-mortem samples were analysed with both analytical methods. Results from validation parameters and results obtained for all post-mortem samples were compared with a significant spearman correlation of r <sub>s</sub> = 0.9978 for R-methadone and r <sub>s</sub> = 0.9981 for S-methadone. The LC method provided better results in terms of uncertainty, retention factor and resolution, whereas SFC provides better sensitivity, with lower LOD. Median R-/S-methadone ratio in peripheral blood was found equal to 1.60 (N = 32), varying from 0.79 to 4.23. The reported values were in good agreement with previously published results. Based on the results obtained here, SFC-MS/MS can be considered a reliable alternative to the widely used LC-MS/MS for the quantitation of methadone enantiomers in bioanalysis and should be evaluated for other bioanalytical methods. Both methods can be easily and quickly used in toxicological routine analysis for the methadone quantitation in human fluids matrices, even if considering that the polysaccharide coated column IH-3 used in SFC does not allow the enantiomeric EDDP separation

A novel ultra-high-performance supercritical fluid chromatography hyphenated to tandem mass spectrometry method for the analysis of urinary endogenous steroids in the anti-doping context.

The first step in the detection of testosterone (T) doping is to measure the urinary steroid profile for the athlete biological passport (ABP). To harmonise the analysis between anti-doping laboratories, urinary steroid profiling is parametrised in deep detail and shall be performed by gas chromatography hyphenated to mass spectrometry (GC-MS). However, due to its requirement for extensive sample preparation, alternatives to GC-MS are being actively pursued. The aim of this study was the evaluation of Ultra-High-Performance Supercritical Fluid Chromatography hyphenated to tandem Mass Spectrometry (UHPSFC-MS/MS) as an alternative for the quantification of endogenous urinary steroids. In this context, we developed a high throughput sample extraction method, followed by a novel UHPSFC-MS/MS method for the analysis of 10 endogenous urinary steroids which are relevant for doping control analysis. Depending on the steroid, the herein presented method is capable of quantification from 0.5 ng/mL up to 10 µg/mL. After validation, the applicability of the method was evaluated by analysing 132 authentic urine samples, which demonstrated results similar to classical GC-MS analysis. Steroid concentrations determined by UHPSFC-MS/MS were slightly overestimated in comparison with GC-MS, but the ratios had <10 % difference between the two methods. As the ABP considers the steroid ratios for passport evaluation, the herein presented method could be used for steroid profiling without reducing the sensitivity of the ABP. Thus, we would propose to consider UHPSFC-MS/MS as an alternative to GC-MS after more tests would have been performed to support our findings. Furthermore, we have also investigated the potential of this technology for sample purification prior to Isotope Ratio Mass Spectrometry (IRMS) for the differentiation between exogenous and endogenous origin of T and its metabolites. While the achieved separation was sufficient to purify urine samples for IRMS analysis in our proof-of-concept study, the instrumental parameters should be further refined for future use

Assessment of chemical stability of monoclonal antibody and antibody drug conjugate administered by pressurized intraperitoneal aerosol chemotherapy.

Pressurized intraperitoneal aerosol chemotherapy (PIPAC) is a new therapeutic approach for patients with peritoneal cancer. So far, most published studies investigated the administration of established cytostatic agents through PIPAC. This study aimed to evaluate the effect of PIPAC on two breakthrough anti-cancer agents, specifically anti-PD1 pembrolizumab, and anti-HER2 antibody-drug conjugate (ADC) - trastuzumab-deruxtecan. We conducted systematic analyses on samples of pembrolizumab and trastuzumab-deruxtecan at clinically relevant concentrations before and after PIPAC administration using an experimental setup of a hermetic container system, mimicking the abdominal cavity and using identical features as in clinical use. We utilized a range of chromatographic and spectroscopic techniques to explore potential alterations in the primary, secondary, and tertiary structures of the drugs, focusing on post-translational modifications resulting from the aerosolization. Our findings indicate that PIPAC did not compromise the integrity of tested biopharmaceuticals. The size variants of both drugs, assessed by size exclusion chromatography (SEC), remained unchanged. Reversed-phase liquid chromatography (RPLC) and hydrophobic interaction chromatography (HIC) revealed no significant differences in hydrophobicity variants, the average drug-to-antibody ratio (DAR), or DAR distribution before and after PIPAC treatment. Circular dichroism (CD) spectroscopy confirmed that the secondary and tertiary structures were preserved. While pembrolizumab showed no change in charge variants post-PIPAC, trastuzumab-deruxtecan exhibited a non-negligible change in the quantity of charge variants on the monoclonal antibody itself, while the payload remained unchanged. This shift could possibly be related to the metallic composition of the CapnoPen® device (made of nickel and chromium) used in PIPAC and for these experiments. Together, our results suggest that PIPAC does not alter the structure of pembrolizumab and trastuzumab-deruxtecan, paving the way for future clinical trials

Comparative study of the LC-MS/MS and UPLC-MS/MS for the multi-residue analysis of quinolones, penicillins and cephalosporins in cow milk, and validation according to the regulation 2002/657/EC

The aim of this study was to develop and validate an analytical method to simultaneously determine European Union-regulated beta-lactams (penicillins and cephalosporins) and quinolones in cow milk. The procedure involves a new solid phase extraction (SPE) to clean-up and pre-concentrate the three series of antibiotics before analysis by liquid chromatography¿tandem mass spectrometry (LC-MS/MS) and ultra-high-performance liquid chromatography¿tandem mass spectrometry (UPLC-MS/MS). LC-MS/MS and UPLC-MS/MS techniques were also compared. The method was validated according to the Directive 2002/657/EC and subsequently applied to 56 samples of raw cow milk supplied by the Laboratori Interprofessional Lleter de Catalunya (ALLIC) (Laboratori Interprofessional Lleter de Catalunya, Control Laboratory Interprofessional of Milk of Catalunya)