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Aim: Indoleamine 2,3-dioxygenase (IDO) and tryptophan 2,3-dioxygenase (TDO) catalyze the initial and rate-controlling step of tryptophan metabolism through the kynurenine pathway, which plays an important role in mediating immune response. Accurate measurement of tryptophan and kynurenine is critical for monitoring the activity of IDO/TDO. Experimental: Surrogate analytes ([15N2]-Tryptophan and [13C6]-Kynurenine) were used for preparation of calibration standard and quality control. A fit-for-purpose validation using an approach of surrogate analyte and authentic matrix was carried out. Results: Acid precipitation was used in sample preparation, which yielded good recovery without significant matrix effect. Precision and accuracy results were well within the acceptance criteria. The assay demonstrated successful application to a clinical study to confirm a transient depletion of kynurenine upon IDO inhibition. Conclusion: A robust, specific and simple LC–MS/MS method was developed and validated with a fit-for-purpose style for measuring tryptophan and kynurenine in human plasma samples.

In human respiratory disease studies, liquid samples such as nasal secretion (NS), lung epithelial lining fluid (ELF), or upper airway mucosal lining fluid (MLF) are frequently collected, but their volumes often remain unknown. The lack of volume information makes it hard to estimate the actual concentration of recovered active pharmaceutical ingredient or biomarkers. Urea has been proposed to serve as a sample volume marker because it can freely diffuse through most body compartments and is less affected by disease states. Here, we report an easy and reliable LC-MS/MS method for cross-matrix measurement of urea in serum, plasma, universal transfer medium (UTM), synthetic absorptive matrix elution buffer 1 (SAMe1) and synthetic absorptive matrix elution buffer 2 (SAMe2) which are commonly sampled in human respiratory disease studies. The method uses two stable-isotope-labeled urea isotopologues, [15N2]-urea and [13C,15N2]-urea, as the surrogate analyte and the internal standard, respectively. This approach provides the best measurement consistency across different matrices. The analyte extraction was individually optimized in each matrix. Specifically in UTM, SAMe1 and SAMe2, the unique salting-out assisted liquid-liquid extraction (SALLE) not only dramatically reduces the matrix interferences but also improves the assay recovery. The use of an HILIC column largely increases the analyte retention. The typical run time is 3.6min which allows for high throughput analysis.

A liquid chromatographic-tandem mass spectrometry (LC-MS/MS) method for the determination of taselisib (GDC-0032, RO5537381) concentrations in human plasma has been developed and validated to support bioanalysis of clinical samples. Solid phase extraction (SPE) was used to extract plasma samples (50μL) and the resulting samples were analyzed using reversed phase chromatography and mass spectrometry coupled with an atmospheric pressure chemical ionization interface. The mass analysis of taselisib was performed using multiple reaction monitoring transitions in positive ionization mode. The method was validated over the calibration curve range 0.400-400ng/mL using linear regression and 1/x(2) weighting. The within-run relative standard deviation (%RSD) ranged from 1.3 to 5.6%, while the between-run %RSD varied from 2.0 to 4.5% for LLOQ, low, medium, medium high and high QCs. The accuracy ranged from 94.7 to 100.3% of nominal for within-run and 96.0-99.0% of nominal for between-run for the same QCs. Extraction recovery of taselisib was between 83.8% and 92.9%. Stability of taselisib was established in human plasma for 977days at -20°C and -70°C and established in sample extracts for 96h when stored at 2 - 8°C. Stable-labeled internal standard was used to minimize matrix effects. Mean single dose pharmacokinetic parameters determined using this method for a phase I/II clinical trial were: Cmax=35.2ng/mL, AUC0-inf=1570ngh/mL, and T1/2=39.3h.