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

Abstract

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.

Long-term delivery of ivermectin by use of poly(D,L-lactic-co-glycolic)acid microparticles in dogs

“…The assay for serum ivermectin concentrations was performed by use of HPLC with a fluorescence detection method. 11 After analytical method validation, 12 the limit of detection was determined to be 0.022 ng/mL, the limit of quantitation was 0.1 ng/mL, and the upper limit of linearity was 10 ng/mL. Correlation coefficients for all standard curves were ≥ 0.99.…”

Liquid chromatographic assay of ivermectin in human plasma for application to clinical pharmacokinetic studies

“…Most of the analytical methods for determining ivermectin and other avermectins in plasma are based upon conversion of these compounds to fluorescent products based upon a double dehydration reaction leading to aromatization followed by high-performance liquid chromatography (HPLC) with fluorometric detection. The fluorescent bioanalytical applications has been extensively reviewed by Fink et al [11]. The dehydration reaction uses trifluoracetic anhydride and Nmethylimidazole form a highly fluorescent six-membered aromatic ring in conjugation with a butadiene unit as shown in Fig.…”

Robotic Sample Preparation and HPLC Determination of the Major Component of Ivermectin in Human Plasma

“…Bioanalytical methods described for the determination of ivermectin in human and animal biological fluids involved enzyme-linked immunosorbent assay, [4] thin-layer chromatographic (TLC) [5] or high performance liquid chromatographic (HPLC) methods coupled to ultraviolet (UV) detection, [6][7][8][9][10][11] or fluorometric detection [12][13][14][15][16][17][18][19][20][21][22] of the derivatized product. The sample clean up and concentration was based on immunoaffinity columns, [11] liquid-liquid extraction [8,15,22] combined with, or solid-phase extraction (SPE), [10,[12][13][14][15][16][17]21] and on-line SPE.…”

“…Generally, TLC or HPLC methods with UV detection of underivatized ivermectin did not provide the required sensitivity (1 ng=mL or lower) and selectivity for clinical plasma analyses. 2) using the dehydration reagent [acetic or trifluoroacetic anhydride (TFAA)] and different organic base catalysts (pyridine, 1-methylimidazole or trialkylamines) has been extensively studied in our [23] and other laboratories [14,16,20] to establish the best reaction conditions for derivatization of ivermectin or its analogs at pg=mL concentration. Since ivermectin does not possess strong chromophores for UV or fluorescence detection, it has to be chemically modified to enhance its detectability and selectivity to achieve the lower limit of quantitation (LLOQ) of <1 ng=mL, using 1 mL or less volume of biological samples.…”

“…In most HPLC-UV detection methods, large sample volume (3-5 mL) and lengthy sample clean up procedures were required to achieve the sensitivity of low nanograms per milliliter. Automated on-line derivatization methods for ivermectin, or it analogs, have been reported [16,20,23] to eliminate the clean up procedure required after the derivatization, thus, increasing sample throughput and more constant derivatization yields. The conversion of ivermectin or its analogs to highly fluorescent derivatives (Fig.…”