High-performance liquid chromatographic assay for the determination of a semisynthetic avermectin analog (eprinomectin) in bovine milk at parts per billion levels--method development and validation.

A sensitive and automated method has been developed and validated to determine marker residue eprinomectin B(1a) in bovine milk. Extraction of eprinomectin B(1a) from milk is accomplished with acetonitrile after the addition of an internal standard. The extract containing the analytes is evaporated to dryness and reconstituted in a solution containing 30% 1-N-methylimidazole in acetonitrile. Online derivatization is carried out with trifluoroacetic anhydride. Determination of eprinomectin B(1a) and its internal standard is carried out by HPLC using a reversed-phase C(18) column with a mobile phase consisting of methanol, acetonitrile, water, triethylamine and phosphoric acid. The overall extraction recovery of eprinomectin B(1a) is 94% with milk supplemented between 2 and 50 ng/ml eprinomectin B(1a). Precision RSD averaged 3.0% in Laboratory 1 (n=25) compared to 4.3% in Laboratory 2 (n=35). The limit of quantitation is approximately 2 ng/ml eprinomectin B(1a), the limit of detection is approximately 0.25 ng/ml using this method.

Synthesis of nodulisporic acid 2' '-oxazoles and 2' '-thiazoles.

[reaction--see text] The semisynthetic conversion of nodulisporic acid A (1) into a set of three heterocyclic side chain derivatives provided compounds, highlighted by 6, with an improved spectrum of ectoparasiticidal activity and pharmacokinetic profile relative to the natural product.

An automated method for the determination of subnanogram concentrations of eprinomectin in bovine plasma.

Eprinomectin is a potent anthelmintic compound that kills certain parasitic nematodes and arthropods of cattle. A sensitive and automated bioanalytical assay was developed for quantitation of eprinomectin in bovine plasma in support of clinical development of eprinomectin for use in all classes of cattle. This assay determined the concentration of eprinomectin in plasma by reversed-phase high performance liquid chromatography (HPLC) with fluorometric detection. Plasma sample preparation included liquid extraction performed by the Packard MultiPROBE robotics workstation, followed by solid phase extraction performed by the Gilson ASPEC XL automated workstation. The HPLC assay included automated pre-column derivatization with a fluorogenic reagent system which included trifluoroacetic anhydride and N-methylimidazole as the catalyst. This reversed-phase chromatographic analysis was based on the fluorescence detection of derivatized eprinomectin and an internal standard, L-648 548, which was similarly derivatized by the fluorogenic reagents. The assay was automated and validated for two concentration ranges of 0.05-10 and 0.5-200 ng ml-1. The lower limit of quantitation of eprinomectin in plasma was 0.05 ng ml-1. The %RSD of the assay was 10% or better at all concentrations. This automated analysis of eprinomectin was used for high-throughput clinical assays with acceptable accuracy and precision.

Stability studies and high-performance liquid chromatographic procedures for L-648,548 and its major degradates in an animal health formulation.

L-648,548 is a new avermectin which was evaluated for the development of an animal health formulation. A stability-indicating method for the essay of 5% (w/v) L-648,548 in an animal health formulation has been developed using reversed-phase high-performance liquid chromatography (HPLC) with UV detection (245 nm). The procedure to determine L-648,548 is linear and accurate over the range 80-120% of the target concentration with a limit of quantitation of 0.2%. Validation data are presented. Also two related degradates of this compound were observed during the stability studies of the L-648,548 formulation. These degradates were determined to be the 2-epimer formed in the presence of base and the 8a-oxo degradate formed by oxidation. Identification of these compounds following direct chemical synthesis was based on mass, UV and NMR spectroscopy. The mechanistic pathways for the formation of these degradates are discussed. The 8a-oxo degradate has a modified chromophore, thus requiring a second HPLC method with detection at 280 nm that was also validated.

Evolution of a specific fluorogenic derivatization of ivermectin for bioanalytical applications. A review.

The evolution of the fluorogenic derivatization of ivermectin is traced through a series of continual modifications that have resulted in improvements in speed and sensitivity. Since the original development of this selective analytical technique, the reaction time has been shortened from 24 h at 100 degrees C to < 30 s at room temperature and, through modifications of the derivatization reagent and catalyst, the sensitivity has also been increased 50-fold to 20 pg of analyte with no significant decrease in precision. A procedure is reported, based on the use of fluorescence derivatization, which eliminates the use of solid-phase columns for sample preparation and fluorophore isolation, and is faster and less cumbersome than previous methods. The method was evaluated with cattle and canine plasma samples over the concentration range 1.0-40 ng ml-1 of ivermectin. It has an accuracy of 1.9% (mean relative error) over this concentration range and a precision of 5.6% (RSD) at the 1 ng ml-1 ivermectin concentration level in a 1 ml plasma sample.

Efficacy in sheep and pharmacokinetics in cattle that led to the selection of eprinomectin as a topical endectocide for cattle.

Eprinomectin (MK-397 or 4"-epi-acetylamino-4"-deoxy-avermectin B1) is a novel avermectin selected for development as a topical endectocide for all cattle, including lactating cows. The initial efficacy assessments were made in sheep to identify subclasses of the avermectin/milbemycins that possessed inherent activity against a spectrum of nematode parasites. This included examination of several hundred analogs each given orally to a single sheep experimentally infected with a range of parasitic nematodes. Representatives of several subclasses, most notably the 4"-epi-amino avermectin B1 subclass, were identified as possessing potent, broad-spectrum activity against the endoparasites, whereas subclasses such as those with a variety of synthetic substitutions at C-4a or oximes at C-5 were among the least potent. Eprinomectin, a member of the 4"-epi-amino subclass, possessed potent activity against the range of nematodes when tested at 0.025 mg kg-1 per os. Milk and plasma concentration profiles were also made for these and other selected avermectin/milbemycins following topical administration to lactating dairy cattle. The molecular structure of each compound had a significant effect on the milk to plasma ratio, but the ratio of each was constant over time, implying an equilibrium between the 2 compartments. Compounds that were saturated at the C-22,23 bond had milk to plasma ratios > or = 1.0, whereas those unsaturated at this bond were generally < or = 1.0. The milk to plasma ratio of eprinomectin was < or = 0.2. Therefore, not only is eprinomectin the most potent broad-spectrum avermectin/milbemycin identified to date, but it also possesses one of the lowest milk partitioning coefficients in this class of antiparasitics.

Identification of the secondary degradates of L-648,548 in an animal health formulation.

L-648,548 is a semisynthetic analog of avermectin. During stability investigations of this compound in an animal health formulation, two new degradates were discovered. These degradates (L-648,548 phenol and its 8,9-Z isomer) were identified as the reaction products of 5-oxo-L-648,548 formed by oxidation of L-648,548. Addition of base to the reaction medium containing 5-oxo-L-648,548 was found to catalyze the formation of L-648,548 phenol via a postulated dehydration by an E1cb elimination followed by the rapid tautomerization of the C5 carbonyl. Photolysis of L-648,548 phenol with visible light (including ambient laboratory lighting) was found to readily produce 8,9-Z-L-648,548 phenol. This transformation was confirmed to be exclusively a photoinduced process.

Analytical method development and preformulation stability studies of L-665,871 (a beta-agonist) in swine feed.

An analytical procedure for the determination of the beta-agonist L-665,871 by LC with fluorescence detection has been developed. The mobile phase consists of acetonitrile-phosphate buffer (pH 3.0) with ion-pairing reagent hexanesulphonic acid sodium salt for the analysis of swine feed samples with 1 and 20 microgram g-1 L-665,871. Storage of feed samples at room temperature, however, indicated that the stability L-665,871 and its analogue L-674,239 were greatly compromised by the presence of moisture in the feed. Acidification with maleic acid and/or freezing the feeds were found to significantly improve drug stability.