Interlaboratory assessment of cryomilling sample preparation for residue analysis.

The effectiveness of the comminution approach used for bulk field samples limits the size of the subsample that must be extracted and analyzed to ensure an adequately representative and reproducible measurement. In many cases this subsample size restricts the residue method to the use of larger vessel formats, limiting downstream throughput. The introduction of a secondary fine-milling step to this process using a subsample size already known to be representative can further improve sample homogeneity and allow direct method scaling to small high-throughput formats. Dramatic increases in method throughput can then be achieved through the simultaneous processing of numerous samples in parallel. This approach was evaluated across a diverse grouping of crop matrices using two substantially different pesticide types. Both fortified and field-collected samples demonstrated a high degree of precision and reproducibility across laboratories. Additional benefits of this approach include significant reductions in cost and solvent waste generation, as well as improvements in assay quality and transferability.

Supramolecular behavior of the amphiphilic drug (2R)-2-ethylchromane-2-carboxylic acid arginine salt (a novel PPARalpha/gamma dual agonist).

PURPOSE: This study was conducted to evaluate the aggregation properties of an amphiphilic drug. METHODS: Aggregation of the drug was studied by various methods including phase-contrast and polarized microscopy, spectrophotometry, surface tensiometry, atomic force microscopy, and dynamic light scattering. Lymph-cannulated rats were used to assess fractions of drug that were absorbed into lymphatics. RESULTS: During the pharmaceutical development of an alpha/gamma dual PPAR agonist, a derivative of a chromane-2-carboxylic acid (compound 1), it was discovered that the compound was able to form various aggregates in aqueous media from pH 6.5 to 7.1, whereas aggregating predominantly into micelles at higher pH values. Critical micelle concentrations seemed to be quite low, about 0.25 mM (0.17 mg/mL) in deionized water as determined by spectrophotometric (dye) and surface tensiometry (du Nuoy) methods. Aggregation of compound 1 into large supramolecular aggregates was visualized via phase-contrast microscopy and atomic force microscopy. The observed aggregates ranged from 250 nm to greater than 10 microm in size. Formation of liquid crystalline phases was observed by polarized microscopy as the material was gradually hydrated with water. Lymph studies in rats indicated that up to 6.9% of the orally administered dose of compound 1 in pH 6.5 buffer appeared in lymph, suggesting that supramolecular aggregation may also occur in vivo leading to partitioning between the portal and the lymph routes. CONCLUSIONS: The aforementioned supramolecular aggregation was found to have a profound effect on the pharmaceutical development of the drug and potentially on in vivo absorption of the drug.

The role of biopharmaceutics in the development of a clinical nanoparticle formulation of MK-0869: a Beagle dog model predicts improved bioavailability and diminished food effect on absorption in human.

MK-0869 (aprepitant), a potent substance P antagonist, is the active ingredient of EMEND which has recently been approved by the FDA for the prevention of chemotherapy-induced nausea and vomiting. Early clinical tablet formulations of MK-0869 showed significant food effects on absorption, suggesting that formulation could have a significant role in improving bioavailability. A Beagle dog model was developed in an effort to guide novel formulation development. Using the suspension of the micronized bulk drug used for the tablet formulations, the food effect on absorption was confirmed in the dog at a similar magnitude to that observed in humans. Further dog studies demonstrated a clear correlation between particle size and in vivo exposures, with the nanoparticle (NanoCrystal) colloidal dispersion formulation providing the highest exposure, suggesting dissolution-limited absorption. The NanoCrystal dispersion also eliminated the food effect on oral absorption in the dog at a dose of 2mg/kg. Regional absorption studies using triport dogs indicated that the absorption of MK-0869 was limited to the upper gastrointestinal tract. These results provided strong evidence that the large increase in surface areas of the drug nanoparticles could overcome the narrow absorption window and lead to rapid in vivo dissolution, fast absorption, and increased bioavailability. In addition, the dog model was used for optimizing formulation processes in which the nanoparticles were incorporated into solid dosage forms, and for selecting excipients to effectively re-disperse the nanoparticles from the dosage units. The human pharmacokinetic data using the nanoparticle formulation showed excellent correlations with those generated in the dog.

The importance of chromatographic separation in LC/MS/MS quantitation of drugs in biological fluids: detection, characterization, and synthesis of a previously unknown low-level nitrone metabolite of a substance P antagonist.

The observation of an interference peak in plasma samples from dogs dosed with compound I led to the discovery of an unidentified metabolite. The unknown metabolite had the same molecular weight as the parent drug, and their fragmentation profiles were also quite similar. LC/MS/ MS analysis of the plasma extracts of dogs and rats dosed with I and its deuterium-labeled analogue suggested a nitrone structure for the unknown metabolite. Synthesized nitrone matched the unknown metabolite with identical retention time and nearly identical fragmentation profile. The nitrone slowly decomposed in acidic aqueous solution at ambient temperature and also underwent in-source, thermal-induced hydrolysis during electrospray ionization mass spectrometric analysis. The reaction of the nitrone with diethyl acetylenedicarboxylate readily generated a [2 + 3] cycloaddition product. The example shown here clearly demonstrates that precautions must be taken when LC/MS/MS quantitation is conducted in the selected reaction monitoring mode.