Analysis of Multiple Anticoagulant Rodenticides in Animal Blood and Liver Tissue Using Principles of QuEChERS Method

Abstract

A quick and easy method for the analysis of anticoagulant rodenticides in blood or tissue using principles of dispersive solid-phase extraction (dSPE), commonly known as QuEChERS (short for quick, easy, cheap, effective, rugged, and safe), was developed. Briefly, a combination of magnesium sulfate, PSA, florisil, and basic alumina was used to cleanup blood samples. Further, to cleanup liver tissue samples, C(18) sorbent was included along with the previously mentioned. The samples were analyzed using high-performance liquid chromatography equipped with a reversed-phase C(18) column (150 x 4.6 mm, 5-microm particle size) and a UV and fluorescence detector. The mobile phase consisted of 0.03 M tetrabutylammonium hydroxide (TBA) adjusted to pH 7/methanol (1:1, v/v) as solvent A and methanol as solvent B in a gradient run. The method detection limit was as low as 10 ng/mL for brodifacoum and difenacoum in blood and 10 ng/g in liver; 50 ng/mL for bromadiolone, difethialone, and chlorphacinone in blood and similarly 50 ng/g in liver; and 100 ng/mL for coumafuryl, pindone, warfarin, and diphacinone in blood and 100 ng/g in liver samples. A number of clinical samples of both blood and liver were analyzed; the comparison of this modified QuEChERS and traditional solid-phase extraction data was found to be in close agreement. This method resulted in drastic reduction in processing time and solvent cost both in terms of consumption and disposal, thus making it an attractive alternative to the traditional solid-phase extraction.

Analytical methods for determination of anticoagulant rodenticides in biological samples

“…This quick, easy, cheap, effective, rugged, and safe (QuEChERS) method was employed for extraction of anticoagulant rodenticides using acetonitrile, and the cleanup step was carried out by mixing this extract with primary-secondary amine (PSA), magnesium sulfate and C18 sorbent [13].…”

“…In other methods, isocratic elution was used for indanediones while gradient elution was employed for hydroxycoumarins analysis [9,11]. Vudathala et al [13] successively developed a methodology of gradient elution for simultaneous analysis of indanediones and hydroxycoumarins from blood and liver tissues. HPLC was used by employing mobile phase composed of 0.03 M tetrabutyl ammonium hydroxide (TBA) buffer (pH 7) and methanol (25:75) (solvent A) and methanol (solvent B) at a flow rate of 0.6 mL/min.…”

“…HPLC-UV detection has been used for analysis of diphacinone, chlorophacinone in biological fluids and tissues [9][10][11]13,30], difethialone, pindone, coumafuryl and warfarin in blood, plasma and liver [13,15,16], and warfarin, coumatetralyl, crimidine and bromadiolone in blood and urine [17]. However these methods have low detection sensitivity mostly ranging from 10 to 100 ng/ mL.…”

“…However these methods have low detection sensitivity mostly ranging from 10 to 100 ng/ mL. Similarly HPLC-FLD has also been employed for rodenticides detection [6][7][8][9][10][11][12][13][14]30]. Although fluorescence detection offers enhanced sensitivity for hydroxycoumarin based rodenticides, but it still requires an extensive sample clean-up.…”

Epidemiology and symptomatology of long acting anticoagulant rodenticide poisoning

“…[3,4] Due to their high-lipid solubility, increased affinity for hepatic enzymes, and significant increase in potency as well as their ability to overcome resistance, LAARs earned the moniker "superwarfarin", which continues in use today. [5][6][7][8] LAARs induce anticoagulation through inhibition of the Vitamin K Epoxide Reductase enzyme. [1] This inhibition leads to reduced ability to gamma-carboxylate terminal glutamine residues of Vitamin K Dependent coagulation factors.…”