A Method for the Simultaneous Identification and Quantitation of Five Superwarfarin Rodenticides in Human Serum

Abstract

A high-performance liquid chromatographic method with ultraviolet (UV) and fluorescence detection was developed for the analysis of one indandione and four hydroxycoumarin anticoagulant rodenticides in human serum. The superwarfarin rodenticides, chlorophacinone, bromadiolone, difenacoum, brodifacoum, and difethialone, can be identified and quantitated simultaneously with this method. After adding a buffer (pH 5.5), the anticoagulants were extracted from serum with chloroform-acetone. The organic phase was separated and evaporated to dryness, and the residue was subjected to chromatographic analysis. The anticoagulants were separated by reversed-phase chromatography and detected by UV absorption at 285 nm and by fluorescence at an excitation wavelength of 265 nm and an emission wavelength of 400 nm. Extraction efficiencies from 55 to 131% were obtained. The within-run precision ranged from 2.0 to 7.1% for UV detection and from 0.0 to 4.8% for fluorescence detection. Between-run precision ranged from 1.3 to 16.0% for UV detection and from 1.8 to 9.0% for fluorescence detection. The anticoagulants can be quantitated at serum concentrations down to 3-12 ng/mL for fluorescence detection and down to 20-75 ng/mL for UV detection. No interferences were observed with the related compounds warfarin and vitamin K1.

Superwarfarins

“…Another HPLC method for detecting brodifacoum in serum and liver using difenacoum as the internal standard has been reported (O'Bryan and Constable, 1991). There is also a method for the simultaneous detection of five superwarfarin rodenticides in human serum (Kuijpers et al, 1995).…”

“…Other serum methods have been reported; for example, detection limits of 3-12 ng/mL for fluorescence and 20-75 ng/mL for UV detection (Mura et al, 1992;Kuijpers et al, 1995;McCarthy et al, 1997;Feng et al, 1999). Tissue methods include a solid phase cartridge extraction from liver, with recoveries ranging from 52% for difenacoum to 78% for warfarin.…”

Analytical methods for determination of anticoagulant rodenticides in biological samples

“…Various organic solvents and their mixtures such as acetonitrileethylether [8], chloroform-acetone [10], acetone-diethyletherchloroform [11], acetonitrile-acetone [12], dichloromethaneacetone [14], 1-chlorobutane [18], ethyl acetate [19][20][21]28,32], acetone [22,23], acetonitrile [24][25][26]29] and methanol [30,33] were used to extract anticoagulant rodenticides from biological samples. Among all organic solvents used in LLE, acetonitrile-ethyl ether (9:1) [8] produced the highest recovery (92-109%) in plasma and liver tissues.…”

“…butylamine and borate buffer (pH 10.4), to maximize the native responses. Isocratic elution has also been used by other authors for rodenticides determinations [8,10,[15][16][17]. In other methods, isocratic elution was used for indanediones while gradient elution was employed for hydroxycoumarins analysis [9,11].…”

Superwarfarin intoxication via skin exposure causing high INR levels and vitamin K deficiency: A case report

“…Tarımsal alanlarda ilaç-lamalarda kullanılmakta ve özellikle fare zehirlerinde yüksek miktarlarda bulunmaktadır. Superwarfarinli rodentisitler; brodifacoum, bromadiolone, chlorophacinone, difenacoum, ve difethiacone gibi gruplara ayrılmaktadır (5) . Olgumuzda kullanılan superwarfarinli rodentisitte brodifacoum etken maddesi vardır.…”