Drugs and Poisons in Humans - A Handbook of Practical Analysis (Part 65)

Introduction:As coumarin rodenticides, warfarin, coumatetralyl, coumafuryl, coumachlor and bromadiolone are commercially available in Japan. The coumarin rodenticides do not show direct anticoagulant action causing bleeding, but inhibit the metabolic cycle of vitamin K; the inhibition causes the interference with protein biosynthesis of vitamin K-dependent coagulant factors (II, VII, IX and X factors) in the liver, which are very important for the blood coagulation system. The lowered coagulant factors cause the bleeding deaths of the rodents [1]. Warfarin, coumatetralyl or coumafuryl is not effective with single administration, but becomes effective by repeated intakes of a small amount of each poison...
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Drugs and Poisons in Humans - A Handbook of Practical Analysis (Part 65) 7.8II.7.8 Coumarin rodenticides by Shouichi SatoIntroductionAs coumarin rodenticides, warfarin, coumatetralyl, coumafuryl, coumachlor and bromadi-olone are commercially available in Japan. The coumarin rodenticides do not show direct anti-coagulant action causing bleeding, but inhibit the metabolic cycle of vitamin K; the inhibitioncauses the interference with protein biosynthesis of vitamin K-dependent coagulant factors(II, VII, IX and X factors) in the liver, which are very important for the blood coagulationsystem. The lowered coagulant factors cause the bleeding deaths of the rodents [1]. Warfarin,coumatetralyl or coumafuryl is not effective with single administration, but becomes effectiveby repeated intakes of a small amount of each poison for 4–5 days successively. Coumachlorand bromadiolone are much more potent and long-lasting rodenticides with long biologicalhalf-lives; they provoke poisoning signs and symptoms, which last for a long time, only withtheir single administration [2]. Such a potent rodenticide is called “super-warfarin”. Warfarin is also very popular as an oral anticoagulant drug for treatment and prevention ofthromboembolism. Although the analysis of coumarin rodenticides and anticoagulants is carried out largely byHPLC [3, 4], a GC/MS method for analysis of 4 rodenticides is presented in this chapter.Reagents and their preparation• Coumarin rodenticides can be obtained in the forms of crystals or powder. They are slightly water-soluble and almost stable under storage at room temperature [4].• Standard compounds: warfarin and coumachlor can be purchased from Sigma (St. Louis, MO, USA); coumatetralyl and bromadiolone from Wako Pure Chemical Ind., Ltd. (Osaka, Japan). A 100-mg aliquot each is dissolved in 100 mL methanol (1 mg/mL) as a stock solu- tion. To use one of them as internal standard (IS), the above solution is diluted 10-fold with 50 % methanol aqueous solution (100 µg/mL). The above solutions should be stored at 4 °C under light-shading conditions.• Mixed standard solution for calibration curves: 1-mL aliquots of the above 4 stock solu- tions (1 mg/mL) is mixed with 9 mL of 50 % methanol aqueous solution (final volume 10 mL, 100 µg/mL for each compound).• Spiked serum solutions for the calibration curves [5]: 50-, 10-, 1- and 0.3-µL volumes of the above mixed standard solution (100 µg/mL) are spiked into 1-mL volume blank serum specimens (final concentration, 5, 1, 0.1 and 0.03 µg/mL, respectively).• 30 % Methanol buffer solution: 70 mL of 0.1 M citrate buffer solution (pH 6.0) is mixed with 30 mL methanol.• Extraction solvent: chloroform/isopropanol (9:1, v/v).• Derivatization reagents: trimethylsilyldiazomethane (TMS-DAM, 10 %, v/v in hexane, GL Sciences, Tokyo, Japan), N-methyl-N-(tert-butyldimethylsilyl)trifluoroacetamide (MTBSTFA)© Springer-Verlag Berlin Heidelberg 2005600 Coumarin rodenticides and N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) (both from Pierce, Rockford, IL, USA, and other manufacturers). • Serum: pooled serum obtained from healthy subjects. GC/MS conditions GC column: a DB-5MS methylsilicone medium-bore capillary column (30 m × 0.25 mm i.d., film thickness 0.25 µm, J & W Scientific, Folsom, CA, USA). Conditions; GC/MS instrument: Shimadzu GCMS-QP5050A (Shimadzu Corp., Kyoto, Japan); column (oven) temperature: 210 °C (1 min, splitless) → 10 °C/min → 330 °C (3 min); in- jection temperature: 250 °C; carrier gas: He; flow rate: 0.9 mL/min (sampling time, 2 min); inter- face temperature: 250 °C; detector temperature: 250 °C; ion source: EI; electron energy: 70 eV. Ions selected for quantitation: those shown in > Table 8.1. Procedure i. A 0.5-mL volume of a specimena is mixed with 1 mL of 0.1 M citrate buffer solutionb (pH 6.0) and 20 µL IS solutionc. ii. The solution is poured into an activated Oasis®HLB cartridgesd,e,f (Waters, Milford, MA, USA). iii. The cartridge is washed with 3 mL purified water and 3 mL of 30 % methanol buffer so- lutiong. ⊡ Table 8.1 Molecular formulae and mass spectral ions for coumarin rodenticides (anticoagulants) Common name (IUPAC) Molecular M.W. DI* Principal mass spectral ions (m/z) DP** formula ME TMS TBDMS derivative derivative derivative Warfarin (3-(α-acetonylbenzyl)- C19H16O4 308.4 265 ...