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APPLICATION Shimadzu News 1/2006 Acute poisoning with a rat bait LC-ESI-MS for simultaneous determination of antagonists in h uman

) are used therapeutically tralyl has therefore been rated as as . The same anti- extremely toxic (T+) in the Ger- coagulant effects are also being man Toxic Substance Act. The used effectively to combat toxicity data for rodents. Due to increasing resist- and coumatetralyl-containing rat ance of various rat species to poison baits in various forms of these compounds, second-genera- application are summarized in tion anticoagulants – the so-called Table 1. – have been devel- oped. These are highly effective, The most important diagnostic even at very low dosages and they parameters are the blood coagula- have long half-lives associated tion values (INR [International with long pharmacological action. Normalized Ratio] and the With increasing commercial dis- Quick-value). Immediate meas- tribution of superwarfarins, the ures in poisoning cases involving number of poisoning cases in anticoagulants are decontamina- humans and animals has also tion and, where necessary, admin- increased. In 2004 the poisoning istration of an antidote (vitamin emergency center in Berlin alone K1). When bleeding is acute and already registered 110 poisoning life threatening, the decreased lev- Figure 1: Bait preparation ingested by the patient cases involving el of factors must first ingestion. be replenished.

Dr. rer. nat. Thomas Grobosch, poison bait (Figure 1) was sent to Depending on the formulation, LC-MS analysis Boris Angelow, the BBGes Toxicology Depart- the active ingredient concentra- PD Dr. med. Dagmar Lampe ment’s laboratory. Analysis using tions of rat poison baits permitted For the detection and quantifica- Berlin Institution for Central Health LC-ESI-MS confirmed that poi- in Germany are between 0.0025 tion of coumatetralyl, an LC-MS Tasks (Berliner Betrieb für Zentrale soning was due to the rat poison’s and 0.79 %. Combination prepa- method was used that had been Gesundheitliche Aufgaben, BBGes), active compound coumatetralyl. rations, for instance Racumin® developed specifically for toxicol- Clinical Toxicology and Pharmacology Plus oat flake bait (coumatetralyl ogy analyses at the BBGes. This Vitamin K antagonists and , Bayer Crop- procedure is suitable for simulta- olving acute poisoning cases, Science) or Celaflor® Brumolin® neous identification and quantifi- as well as therapeutic drug 4-Hydroxycoumarin, a derivative (difethialon and sulfachinoxalin, cation of 10 indirect anticoagu- Smonitoring and drug testing of the sweet woodruff phyto- Scotts Celaflor GmbH & Co.) are lants in human serum. The are among the major tasks of the chemical , and 1.3- also available. method is based on an acidic Clinical Toxicology Department indandione belong to a group of (pH = 4.2) liquid-liquid extraction of the BBGes. This example indirect anticoagulants (Figure 2). Poisoning cases in humans and using 1-chlorobutane with subse- describes an LC-MS method for They are also known as vitamin K animals are indicated by an in- quent detection via LC-ESI-MS. the determination of rat poison antagonists as their action is based creased tendency towards bleed- This simple method for the deter- intoxications. on a functional disruption of the ing. Death occurs via internal mination of vitamin K antagonists synthesis of vitamin K-dependent bleeding or haemorrhagic shock. offers broad compound screening A 62-year old man was admitted coagulation factors. The active compound coumate- and high selectivities with short to the emergency station with

symptoms of nausea and vomit- Based on the half-life values of LD50 Rat (m) LD50 Rat (m) Name LD50 Rat (m) transdermal via inhalation ing. He admitted to having the coagulation factors already oral (mg/kg KG) ingested rat poison bait in a sui- circulating in the bloodstream, the (mg/kg KG) (mg/L) cide attempt. The patient’s medi- anti-coagulating effects of the rat Coumatetralyl 30.15 (f) > 100 0.05 cal history indicated an earlier poison are evident only after a < 500 (f) (dust) suicide attempt in 2003. No psy- certain time delay. Racumin powder 5000 > 5000 > 3.3 chiatric treatment had followed. Racumin > 5000 > 5000 – – A blood sample of the patient Some vitamin K antagonists baits together with a sample of blue rat (, , Table 1: Toxicity data for coumatetralyl: m = male, f = female

(From “Wirkstoffe in Pflanzenschutz und Schädlingsbekämpfungsmitteln: physikalisch-chemische und toxikologische Daten“, 3. Aufl., Industrieverband Agrar e.V. BLV, München, 2000)

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Shimadzu News 1/2006 APPLICATION

LC-MS conditions HPLC

Binary pump system with membrane degasser, autosampler, oven, UV detector (Shimadzu) s in h uman serum Mobile phase: A: methanol, B: methanol/0.1 % HCOOH (10/90, v/v) · Flowrate: 0.60 mL/min · Gradient: 0 – 0.7 min: 95 % B; 0.7 – 1.1 min: 50 % B linear; 1.1 – 3.2 min: 6 % B linear; 3.2 – 3.8 min: 6 % B; 3.8 – 4.2 min: 95 % linear · Separation column: Atlantis C18 (2.1 x 20 mm, 3 µm, Waters) · Oven temperature: 40 °C · Gradient for the determination of cholecalciferol (vitamin D3): 0 – 0.7 min: 20 % B; 0.7 – 3.5 min: 6 % B linear; 3.5 – 3.7 min: 6 % B; 3.7 – 4.1 min: 20 % B linear; 4.1 – 4.2 min: 20 %. analysis times using two masses for identification/quantification. MS Figure 3 shows the compounds identified using this method. LCMS-2010 system (Shimadzu) Ion source: ESI positive & negative · Nebulizer gas: nitrogen; 4,5 L/min · Block- and CDL temperature: 300 °C · Detector voltage: 1.9 kV Based on suspected poisoning SIM-masses: acenocoumarol ESI (-): 353 & 352; ESI (-): 523 & 521; bromadiolon ESI (-): 527 & 525; coumachlor ESI (-): with a coumarin derivative, serum 343 & 341; coumatetralyl ESI (-): 292 & 291; ESI (-): 445 & 443; difethialon ESI (-) 539 & 537; ESI (-) 543 & 542; samples as well as bait samples phenprocoumon ESI (-): 280 & 279; warfarin ESI (-) 308 & 307, internal standard ESI (+): 432 were subjected to liquid-liquid extraction and subsequently ana- lyzed according to the LC-MS method described below. In addi- tion, a systematic toxicological analysis (STA) was carried out using HPLC-DAD. The bait samples were first extracted using methanol, the resulting solution was then filtered after centrifuga- tion and 5 µL was injected direct- ly into the LC-MS system (for LC-MS conditions see insert). Figure 2: Structural similarity between vitamin K and its antagonists

For quantification, a 6-point cali- bration was carried out. The cali- bration was linear for all calibra- tion points in the range from 10 up to 250 µg/L (r2 > 0.995). The determination limit was 5 µg/L (S/N 10). For the precision the following coefficients of variation were determined based on quality control samples (day-to-day, n = 6, 100 µg/L): acenocoumarol (5.2 %), coumachlor (5.9 %), coumatetralyl (4.8 %), phenpro- coumon (6.0 %), warfarin (6.0 %), brodifacoum (14.9 %), bro- madiolon (8.2 %), difenacoum (8.4 %), difethialon (11.7 %), flo- coumafen (14.2 %).

Results

The selected analytical conditions enable fast elution of the listed compounds within 5 min at a determination limit of 10 µg/L. The experimental conditions (mobile phase, analytical column, ESI interface) conform to the usual LC-MS system configura- tions at the institute laboratory

Figure 3: Structures of the vitamin K antagonists and the internal standard

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APPLICATION Shimadzu News 1/2006

and therefore do not require long The active compound could be identified via the retention time eleven days later (LOQ = 10 reconfiguration times. identified unequivocally from the and the two characteristic masses. µg/L). This is due either to a dis- chromatogram (Figure 5) via the There were no interfering peaks. tinctive distribution behavior or a Figure 4 shows SIM chromato- retention time (2.0 min) as well as Both results pointed to a cou- half-life of less than two to three grams of the 10 established vita- the two masses (m/z 292 and matetralyl-containing days. min K antagonists (standard each 291). Quantification resulted in a bait. In order to test whether the 100 µg/L). The symmetry of the coumarin concentration of 121 rodenticide sample was the com- Summary peaks is acceptable. To safeguard µg/L. The control sample (expect- bination preparation Racumin® the validity of the analytical ed value 100 µg/L) showed a cou- Plus oat flake bait (Bayer AG), a This example of a suicide attempt result, two masses were used. In matetralyl concentration of 94.7 methanol extract of the rodenti- with a coumatetralyl-containing marked contrast with the identifi- µg/L which was within the cide sample was analyzed via LC- rat poison was presented to cation after fragmentation in a required range of 80 – 120 µg/L APCI-MS to detect cholecalcifer- demonstrate a sensitive and reli- first run and quantification after a (± 20 % of the expected value). ol. However, no indication on the able analytical method for the second run, our analytical strate- In addition, the sample under- presence of cholecalciferol was simultaneous determination of 10 gy enabled a simultaneous identi- went screening for basic com- found. vitamin K antagonists (among fication and quantification within pounds (HPLC-DAD) whereby them 5 superwarfarins) in human one run due to the high sensitivi- no further compounds were iden- Although a coumatetralyl concen- serum/plasma using LC-ESI-MS. ty, whereby a higher sensitivity tified. tration of 121 µg/L was measured The method is based on a simple was obtained for mass [M-1 amu]. when the patient was admitted, and fast liquid-liquid extraction Within the scope of method vali- In the bait sample the active com- coumatetralyl could no longer be with subsequent LC-MS analysis. dation, 12 tested blank samples pound coumatetralyl was also detected in a control sample taken obtained via 6 different sampling methods were free from the above-mentioned vitamin K antagonists. 80e3 70e3 The upper determination limit of 60e3 250 µg/L (3 decades) is not suffi- cient for the determination of 50e3 phenprocoumon and warfarin 40e3 Intensity (therapeutic range up to 3000 30e3 µg/L). Therefore for the investi- gation of these compounds, corre- 20e3

spondingly less material should 10e3 be used or the sample should be diluted with coumarin-free blank 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 4.00 4.25 4.50 4.75 plasma/serum. The precision of Minutes the method is suitable for obtain- ing acceptable results at low anal- Figure 4: SIM chromatograms of the 10 vitamin K antagonists (each 100 µg/L, ESI negative) ysis frequency when time is essential.

Based on the suspected coumarin 60,000 intoxication, the patient sample 55,000 was investigated using the above- 50,000 described analytical procedure. IS 45,000

40,000

35,000 Coumatetralyl c = 121 µg/L

Intensity 30,000

25,000

20,000

15,000

10,000

5000

0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 4.00 4.25 4.50 4.75

Minutes

Figure 5: Serum sample of the patient with internal standard (SIM chromatogram)

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