Determination of a novel antifibrotic small molecule GDC-3280 in human plasma and urine by liquid chromatography tandem mass spectrometry to support its first-in-human clinical trial.

A specific and robust LC-MS/MS method was developed and validated for the quantitative determination of GDC-3280 in human plasma and urine. The nonspecific binding associated with urine samples was overcome by the addition of CHAPS. The sample volume was 25 µL for either matrix, and supported liquid extraction was employed for analyte extraction. d6-GDC-3280 was used as the internal standard. Linear standard curves (R2 > 0.9956) were established from 5.00 to 5000 ng/mL in both matrices with quantitation extended to 50,000 ng/mL through dilution. In plasma matrix, the precision (RSD) ranged from 1.5 to 9.9% (intra-run) and from 2.4 to 7.2% (inter-run); the accuracy (RE) ranged from 96.1 to 107% (intra-run) and from 96.7 to 104% (inter-run). Similarly, in urine the precision was 1.5-6.2% (intra-run) and 1.9-6.1% (inter-run); the accuracy was 83.1-99.3% (intra-run) and 87.1-98.3% (inter-run). Good recovery (>94%) and negligible matrix effect were achieved in both matrices. Long-term matrix stability was established for at least 703 days in plasma and 477 days in urine. Bench-top stability of 25 h and five freeze-thaw cycles were also confirmed in both matrices. The method was successfully implemented in GDC-3280's first-in-human trial for assessing its pharmacokinetic profiles.

A validated surrogate analyte LC-MS/MS assay for quantitation of endogenous kynurenine and tryptophan in human plasma.

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.

A supported liquid extraction LC-MS/MS method for determination of concentrations of GDC-0425, a small molecule Checkpoint kinase 1 inhibitor, in human plasma.

A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the determination of GDC-0425 concentrations in human plasma has been developed and validated. Supported liquid extraction was used to extract plasma samples (50 µL) and the resulting samples were analyzed using reverse-phase chromatography and mass spectrometry coupled with a turbo-ionspray interface. The mass analysis of GDC-0425 was performed using multiple reaction monitoring transitions in positive ionization mode. The method was validated over the calibration curve range of 1.00-1000 ng/mL using linear regression and 1/x2 weighting. Within-run relative standard deviation ranged from 0.8 to 5.1%, while between-run RSD varied from 1.9 to 4.7% for QCs. The accuracy ranged from 90.0 to 101.0% of nominal for within-run and from 94.0 to 100.0% of nominal for between-run. Overall extraction recovery was 87.4% for GDC-0425 and 87.9% for GDC-0425-d9 . Stability of GDC-0425 was established in human plasma for 374 days at -20 and -70 °C and established in reconstituted sample extracts for 88 h when stored at 2-8 °C. Stable-labeled internal standard was used to minimize matrix effects. This assay was used to characterize the pharmacokinetics of GDC-0425 in cancer patients.

Simultaneous determination of itraconazole, hydroxy itraconazole, keto itraconazole and N-desalkyl itraconazole concentration in human plasma using liquid chromatography with tandem mass spectrometry.

A high-performance liquid chromatography tandem mass spectrometry (LC-MS/MS) assay was developed and validated for simultaneous determination of itraconazole (ITZ), hydroxy-itraconazole (OH-ITZ), keto-itraconazole (keto-ITZ) and N-desalkyl itraconazole (ND-ITZ) concentration in human plasma. One hundred and fifty microliters of human plasma were extracted using a solid-supported liquid extraction (SLE) method and the final extracts were analyzed using reverse-phase chromatography and positive electrospray ionization mass spectrometry. The standard curve range is 5-2500 ng/mL for ITZ and OH-ITZ and 0.4-200 ng/mL for keto-ITZ and ND-ITZ. The curve was fitted to a 1/x(2) weighted linear regression model for all analytes. The precision and accuracy of the LC-MS/MS assay based on the five analytical quality control (QC) levels were well within the acceptance criteria from both FDA and EMA guidance for bioanalytical method validation. Average extraction recovery was 97.4% for ITZ, 112.9% for OH-ITZ, 103.4% for keto-ITZ, and 102.3% for ND-ITZ across their respective curve range. Matrix factor was close to 1.0 at both high and low QC levels of all 4 analytes, which indicates minimal ion suppression or enhancement in our validated assay. Itraconazole and all three metabolites are stable in human plasma for 145 days stored at -70 °C freezers. The validated assay was successfully applied to a clinical study, which has a drug-drug interaction (DDI) arm using ITZ as a cytochrome P450, family 3, subfamily A (CYP3A) inhibitor.

Skill mix: in whose best interests?

The purpose of this article is to share the human resource experiences of a health visiting team. A historical attempt to recruit a part-time health visitor to the team failed, following the retirement of the previous post holder. A staff nurse was recruited into post to complement the 'skill mix' within the team. This paper highlights recruitment and retention issues and presents a working process for other teams to adopt. The article reflects the ever-changing workforce needs required to meet the challenges in primary care today.