Using Mitra sampling to support first-in-human pharmacokinetic evaluations for PF-07059013.

Aim: A sensitive and selective method for the determination of PF-07059013 in dried blood collected by Mitra™ tips was developed and qualified from 50 to 50,000 ng/ml. Materials & methods: PF-07059013 is isolated from 10 µl of human dried blood by extraction with methanol and analyzed by HPLC-MS/MS. Results & conclusions: In addition to routine validation elements, impact of hematocrit and Mitra tip's lot-to-lot variation on assay accuracy were evaluated. The qualified method was used in one clinical study with excellent performance. Correlation coefficient between blood concentrations obtained from liquid-incurred blood samples and dried-incurred blood samples is 0.95. Clinical Trial Registration: NCT04323124 (ClinicalTrials.gov).

Bioanalytical method validation and sample analysis for nirmatrelvir in dried blood collected using the Tasso-M20 device.

Aim: A sensitive and selective method for the determination of nirmatrelvir in dried human blood collected by Tasso-M20 was developed and validated from 20.0 to 20,000 ng/ml. Materials & methods: Nirmatrelvir and its stable-labeled internal standard were isolated from approximately 20 µl of blood dried on one volumetric absorptive pad inside the Tasso-M20 device by extraction with methanol, followed by dilution of the supernatant. The extracts were analyzed by high-performance liquid chromatography coupled with tandem mass spectrometric detection. Results & conclusion: The method was fully validated. Hematocrit levels do not impact assay accuracy. Stabilities to cover sample drying and storage at a variety of conditions were conducted. The validated method was used in multiple clinical studies with excellent performance.

Toxicokinetic evaluation of atrasentan in mice utilizing serial microsampling: validation and sample analysis in GLP study.

BACKGROUND: A semi-automated 96-well protein precipitation followed by HPLC-MS/MS method for the determination of atrasentan (2R-[4-methoxyphenyl]-4S-[1,3-benzodioxol-5-yl]-1-[N,N-di-(N-butyl)-aminocarbonyl-methyl]-pyrrolidine-3R-carboxylic acid) in mouse whole blood was developed, validated and utilized in GLP toxicokinetic evaluations. Six 40-µl whole blood samples were collected from a single mouse over the course of a 12 h blood collection window. To avoid sample volume losses, whole blood was selected as the matrix in place of the more typically used plasma. A 10-µl assay volume was used to ensure sufficient volumes are available for dilutions, repeats and incurred sample reanalysis. The samples (10-µl aliquot) were fortified with stable-labeled internal standard (d18-atrasentan) and lysed thoroughly prior to protein precipitation. The chromatographic separation was performed on a Zorbax(®) SB-C18 (50 x 2.1 mm; 5 µm) HPLC column with a mobile phase consisting of 25 mM ammonium acetate and 0.25% (v/v) acetic acid in 50/50 (v/v) acetonitrile/water. The MS measurement was conducted under positive ion mode using multiple-reaction monitoring of m/z 511→354 for analyte and 529→354 for stable-labeled internal standard. The peak area ratio (analyte:stable-labeled internal standard) was used to quantitate atrasentan. RESULTS: A dynamic range of 5-1400 ng/ml was established after validation. The challenges associated with a small-volume whole-blood assay involved anticoagulant overloading with commercial blood collection tubes, managing phospholipids to ensure a robust assay and automation. In-depth discussions are provided in this article. The validated method was then used for GLP toxicokinetic evaluations. To demonstrate the method reproducibility, approximately 10% of the incurred samples from the study were repeated in singlet. Excellent assay reproducibility was demonstrated where 100% of samples met incurred sample reanalysis acceptance criteria. CONCLUSION: Good quality exposure data were obtained from every serial sampled mouse in the study.

Comparing similar spectra: from similarity index to spectral contrast angle.

We investigated a spectral-contrast-angle (theta) method to determine whether mass spectra of structural isomers are the same or significantly different. This method represents collisionally activated dissociation (CAD) spectra as vectors in space. Mass spectra of different isomers are represented as different vectors, having characteristic lengths and direction. The derived spectral contrast angle, which is a measure of the angle between two vectors corresponding to two closely related spectra, is a measure of whether the mass spectra are the same or significantly different. We compare this method with the similarity index (SI) method and show that the spectral contrast angle method is superior and can differentiate between very similar spectra in cases where the SI cannot. Both methods can be implemented simply in situations where the analyst is called on to decide, on the basis of mass or product-ion spectra, whether reference and unknown compounds are the same or to evaluate the reproducibility of spectra comprised of many peaks.