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Background: Volumetric absorptive microsampling may reduce the blood collection burden associated with therapeutic drug monitoring of immunosuppression to prevent organ transplant rejection. This work describes the development of a laboratory and analytical technique for quantifying tacrolimus and mycophenolic acid (MPA) from the Tasso-M20™ in human whole blood using bead-based impact-assisted extraction. Results: The sampled blood volume was accurate with estimated volumes within

While the rates of new cases and deaths for high grade serous ovarian cancer (HGSC) have declined steadily in the past decade, HGSC remains the fifth leading cause of cancer death in women. As many patients succumb to chemoresistance, there is a need to uncover novel targeted drugs. One way to do so is by drug repurposing. A computational screen, DrugPredict, led to the discovery that amiodarone, an antiarrhythmic, is a potent apoptosis-inducer and Myc-degrader in numerous patient-derived ovarian cancer cell lines. In search of a more selective, but less toxic compound, structure-activity relationship was applied to identify DL78. Due to its structural differences, DL78 is considered to be independent of amiodarone. DL78 lacks hERG activity, retains Myc regulation, and gains selective anticancer properties. This compound produces significant cell death selectively in patient-derived HGSC cells, including isogenic cisplatin-resistant lines, but does not harm non-transformed fallopian tube cells. Cancer cells treated with DL78 arrest prior to anaphase and showcase microtubule abnormalities and upregulation of mitotic regulators. The prolonged arrest leads to loss of Myc, mitotic catastrophe, and apoptosis selectively in the HGSC cells, while fallopian tube cells can cycle normally. DL78’s activity is confirmed to be dependent on Myc expression because targeted inhibition of Myc decreases its potency and prevents the DL78-induced cell cycle arrest. Finally, pharmacokinetic analysis shows that DL78 saturates the tumor following intraperitoneal injection, yet drug concentrations in the blood and liver present at normal levels. Overall, these phenotypes do not show with amiodarone treatment, suggesting that structurally different DL78 performs through a different mechanism. Further classification of this tumor-selective compound may reveal targeted ovarian cancer vulnerabilities. Citation Format: Jessica Teitel, Michele Cusato, Margaret Farah, Agharnan Gandhi, Pil Lee, Andrew White, John Takyi-Williams, Bo Wen, Andre Monterio Da Rocha, Analisa DiFeo. Novel Myc-dependent compound DL78 selectively induces mitotic catastrophe and cell death in high grade serous ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1570.

Paper Spray (PS) ionization has been demonstrated extensively for rapid quantitation of pharmaceuticals in dried matrix spots. In this study, experimental and validation parameters of paper spray mass spectrometry were explored. Firstly, the effect on analyte signal response by angular positioning of the paper/spray tip to the mass spectrometer (MS) inlet was studied and found that, the analyte signal response depends on the flow rate of the paper substrate, when the spray tip is positioned in angle to the MS inlet. Secondly, the extent of carryover on the PS system due to the reuse of the paper substrate metal clip holder was evaluated and found to be compound dependent and could be minimal for some compounds even when the metal clip holder is reused. Thirdly, the blood matrix effects in different paper substrates on analyte signal was evaluated and found that, in the absence/presence of a matrix, the absolute signal response/suppression of the analytes varied significantly among the papers. Moreover, the precision value of standard line slopes constructed in six different lots of rat blood was 2.2%, less than the cut-off value of

The resurgence of Cannabis therapeutic discoveries have led to the need for sensitive and selective analytical methods for the detection of cannabinoids and their metabolites in biological matrices. High resolution mass spectrometry (HRMS) enables good sensitivity and provides more selectivity due to its accurate mass measurement of the targeted compounds. The aim of this study was to develop and validate a sensitive liquid chromatography high resolution mass spectrometry (LC-HRMS) method for the quantitative analysis of cannabidiol (CBD), cannabinol (CBN), Δ9-tetrahydrocannabinol (Δ9-THC) and its major metabolites 11-Hydroxy-Δ9-THC and 11-Nor-9-carboxy-Δ9-THC in human plasma. The method utilized a simple liquid-liquid extraction of the cannabinoids from plasma samples followed by an isocratic chromatographic separation and detection by ESI-HRMS Q-Exactive plus platform. The lower limit of quantification (LLOQ) was 0.2 ng/ mL for the targeted cannabinoids and its metabolites with sample volume of 0.5 mL plasma. The method was linear from 0.2 to 100.0 ng/mL with an average correlation coefficient of >0.995 using weighted (1/x) linear least-squares regression. No significant carry-over was noticed for all analytes and the extraction recovery ranged from 60.4 % to 85.4 %. Dilution results indicated no influence on the accuracy of analysis. The method's intra-day and inter-day precision (CV %) ranged from 2.90 to 10.80 % and accuracy within -0.9 to 7.0 from nominal. Matrix effect ranged from 1.1 % to 49.8 %. The analytes were stable in the autosampler for 6 and 12 h, respectively. This method was sensitive and can be applicable to cannabinoids pharmacokinetics study.

The purpose of this reported study was to develop and validate an LC-MS/MS method for the quantification of goserelin in a Pheroid (R) formulation simulated intestinal fluid. Biopharmaceuticals are formulated in drug delivery systems to improve their gastrointestinal stability. Goserelin, a peptide drug was formulated in Pheroid (R) delivery system and its gastrointestinal stability assessed using simulated intestinal fluid, which required an assay to determine the varying amounts of goserelin remaining after a specific time. Several extraction methods and solvents investigated to extract goserelin from complex matrix led to either poor recovery, peak shape or high background interference. A rapid gradient reversed-phase method coupled to tandem mass spectrometry detection was optimized for the separation and quantification of the extracted peptide. A simple, reproducible and good recovery extraction procedure for goserelin quantification was achieved through simultaneous acetonitrile protein precipitation and water-saturated n-butanol liquid-liquid extraction with water dilution. The method was found to be rapid, specific, precise and accurate, and successfully applied to determine goserelin remaining content in a simulated intestinal fluid, with potential use in other lipid-based formulation evaluated in simulated intestinal fluids. (C) 2019 Elsevier B.V. All rights reserved

Here we report a new site-specific conjugation strategy to modify proteins via thiazolidine ligation. Proteins harbouring a 1,2-aminothiol moiety introduced by amber codon suppression technology could be modified chemoselectively with aldehyde-functionalized reagents, such as a biotin-labeled peptide or ubiquitin, under mild conditions to yield homogeneous biotinylated or ubiquitinated products.

Aim: Current bioanalytical methods applied in nonclinical PK studies for screening drug candidates demand significant amount of time and resources, hence, the need to develop alternative methods. Results: A proof-of-concept paper spray–MS method for the detection and quantitation of small molecules in plasma has been developed and validated using sunitinib and benzethonium as model compounds. The method includes single oral or intravenous administration of sunitinib to mice and serial micro-volume (20 µl) blood collection at different time intervals. The method is rapid with overall analysis time of 1 min and a full PK profile of sunitinib was obtained from a single mouse. Conclusion: The paper spray–MS approach is simple, sensitive and can potentially enable significant reduction of animal use and cost.

A significant limitation in performing preclinical studies using small animals is the need for macrovolume blood sampling, a similar situation encountered with newborn or neonates screening. Dried blood spot (DBS) method which requires small sample volume has overcome this limitation. However, the required extensive sample pre-treatment prior to chromatographic separation and detection is a major bottleneck. High throughput analysis is a priority in preclinical drug discovery and clinical settings. Paper spray mass spectrometry which is ideally suited to direct DBS analysis without sample pre-treatment and chromatographic separation prior to mass analysis at ambient environment could provide the required throughput. At present there is no report on the application of paper spray mass spectrometry for preclinical studies or biomarker analysis. The studies described in this thesis were undertaken to evaluate the application of paper spray mass spectrometry for direct analysis of small molecule drugs and biomarker in dried matrix spot samples. In the first part of the thesis, paper spray mass spectrometry (PS-MS) method was developed and validated. Potential analytical liabilities, such as carryover and ion suppression were systematically evaluated using structurally diverse test compounds. It was observed that ionization suppression was prevalent for the test compounds, but it could be effectively managed by using a stable isotope labeled internal standard (SIL-IS). Carryover was found to be minimal for some compounds and variable for others and could generally be overcome by cleaning the metal clip and inserting matrix blanks in-between samples. The results from the validation study indicated that the PS-MS assay was precise and accurate, obtaining a LLOQ of 1 and 5 ng/mL for sunitinib and benzethonium respectively in dried plasma spots (DPS). The test compound, sunitinib, was stable in all conditions studied and analyte extraction and detection sensitivity could be improved with sufficient off-line or on-line pre-moistening of the dried spot prior to PS-MS analysis after longer storage period. For quantitative bioanalysis, SIL-IS coated PCR tube sampler prepared by evaporation and concentration technique was used to sample microvolume (5 µL) of plasma, serum and whole blood and the results compared to that of conventional pre-mix method. The tube sampler results were found to correlate well with those from a conventional laboratory pre-mixed procedure and comparable accuracy and precision were achieved. Hence, < 10 µL blood could be sampled directly from small rodents during preclinical studies. Evaluation of the PS-MS assay to ascertain that it will provide comparable data to LC-MS/MS assay when used for in vivo bioanalytical analysis showed that PS-MS assay results correlate well with those from conventional LC-MS system, and comparable accuracy, precision, linearity and sensitivity (LLOQ of 1 ng/mL of sunitinib in DBS) were achieved with analysis speed of about 48-fold higher with sample volume of 5 µL. In addition, the presence of stable metabolite in the samples showed no impact on the parent quantitation for the test compound in PS-MS assay. Further application of the PS-MS assay to preclinical pharmacokinetic (PK) study found that, the assay was simple and rapid with overall analysis time of 1 min and a full plasma concentration vs. time profile of sunitinib could be obtained from a single mouse. The assay required small sample volume, thereby reducing the overall cost and the use of animals in the preclinical study. Therefore, PS-MS assay could be a potential valuable tool for high throughput in vivo screening of drug candidates. In the second part of the thesis, DBS method was developed as an alternative sampling method for the analysis of biomarkers in DBS samples and further investigated the direct detection of biomarkers from DBS samples using paper spray method. Hemoglobin Constant Spring (Hb CS) was the selected disease model used for this study, which was among a group of inherited blood diseases that were previously studied in our lab and the potential peptide markers identified using peptidomic approach. A DBS prepared by spotting 10 µL packed red cell on Schleicher & Schuell (S&S) 903 filter paper, dried for 4 h and punched-out spots of eight taken with subsequent solvent extraction and precipitation with 0.1 % formic acid in acetonitrile was sufficient to detect all Hb CS specific peptide markers using MALDI-MS and nanoLC-MS/MS analysis with comparable results to liquid packed red cell samples of 100 µL. Stability results also showed that peptide markers were stable in the DBS at 4 ℃ and room temperature for a period up to 12 months and at 37 ℃ and 40 ℃ for 1 month period. Direct analysis of the peptide markers from DBS using PS-MS method proved unsuccessful probably due to the nature of the paper substrate used or low percentage of Hb CS specific peptide markers present in the peripheral blood. However, this sampling method can potentially be used to screen Hb CS disease in newborns, critically ill patients or individuals from remote/resource-limited areas or be coupled to the discovery and validation study. DOCTOR OF PHILOSOPHY (SBS)

Ambient ionization MS has become very popular in analytical science and has now evolved as an effective analytical tool in metabolomics, biological tissue imaging, protein and small molecule drug analysis, where biological samples are probed in a rapid and direct fashion with minimal sample preparation at ambient conditions. However, certain inherent challenges continue to hinder the vibrant prospects of these methods for in situ analyses or to replace conventional methods in bioanalysis. This review provides an introduction to the field and its application in bioanalysis, with an emphasis on the most recent developments and applications. Furthermore, ongoing challenges or limitations related to quantitation, sensitivity, selectivity, instrumentation and mass range of these ambient methods will also be discussed.