Hazards of secondary bromadiolone intoxications evaluated using high-performance liquid chromatography with electrochemical detection

Senso s 2007, 7, 1271-1286 senso s ISSN 1424-8220 © 2007 by MDPI www.mdpi.o g/senso s Full Resea ch Pape Haza ds o Seconda y B omadiolone In oxica ions E alua ed using High-pe o mance Liquid Ch oma og aphy wi h Elec ochemical De ec ion Sona K izko a 1 , Mi osla a Beklo a 2 , Ji i Pikula 2,* , Voj ech Adam 1 , Ales Ho na 3 and René Kizek 1 1 Depa men o Chemis y and Biochemis y, Facul y o Ag onomy, Mendel Uni e si y o Ag icul u e and Fo es y B no, Zemedelska 1, CZ-613 00 B no, Czech Republic 2 Depa men o Ve e ina y Ecology and En i onmen al P o ec ion, Ve e ina y and Pha maceu ical Uni e si y B no, Palackeho 1/3, CZ-612 42 B no, Czech Republic 3 Depa men o Food Enginee ing, Facul y o Technology, Tomas Ba a Uni e si y, T.G. Masa yka 275, CZ-762 72 Zlin, Czech Republic * Au ho o whom co espondence should be add essed; E-mail:[email p o ec ed] Recei ed: 28 June 2007 / Accep ed: 18 July 2007 / Published: 20 July 2007 Abs ac : This s udy epo ed on he possibili y o in oxica ions o non- a ge wild animals associa ed wi h use o b omadiolone as he ac i e componen o oden icides wi h an icoagula ion e ec s. A labo a o y es was done wi h ea hwo ms we e exposed o b omadiolone-con aining g anules unde he condi ions speci ied in he modi ied OECD 207 guideline. No mo ali y o ea hwo ms was obse ed du ing he ou een days long exposu e. When he ea hwo ms om he abo e es became a pa o he die o common oles in he ollowing expe imen , no mo ali y o consume s was obse ed oo. Howe e , elec ochemical analysis e ealed highe le els o b omadiolone in issues om ea hwo ms as well as common oles compa ed o con ol animals. The e we e de e mined compa able le els o b omadiolone in he li e issue o common oles a e p ima y (2.34±0.10 µg/g) and seconda y (2.20±0.53 µg/g) in oxica ion. The e o e, he isk o seconda y in oxica ion o small mammalian species eeding on b omadiolone-con aining Senso s 2007, 7 1272 ea hwo ms is he same as o p ima y in oxica ion h ough bai ed g anules. B omadiolone bio-accumula ion in he ood chain was moni o ed using he newly de eloped analy ical p ocedu e based on he use o a liquid ch oma og aphy coupled wi h elec ochemical de ec o (HPLC-ED). The HPLC-ED me hod allowed o de e mine he le els o b omadiolone in biological samples and is he e o e sui able o examining he en i onmen al haza ds o his subs ance. Keywo ds: an icoagulan oden icide; elec ochemis y, cyclic ol amme y; animal issues; non- a ge o ganisms; Eisenia e ida; Mic o us a alis; Lepus eu opaeus 1. In oduc ion Indus ial pollu an s come om a ious an h opogenic sou ces in o he en i onmen [1,2]. Ag icul u al pes icides including oden icides belong o such g oup o pollu an s [3-6]. Roden icides a e used o con ol pes oden s ha may be ha bou ing zoono ic in ec ious agen s [7-11]. Howe e , oden icides may ha e also di ec o indi ec e ec s on non- a ge o ganisms [12,13]. De imen al e ec s as well as mo ali y in bo h domes ic [14] and wild non- a ge animals [4,15] ha e been shown a e use o an icoagulan oden icides. One o he so-called second-gene a ion an icoagulan oden icide, b omadiolone, can be ound in li e s o p eda o s o small oden s such as he ox, ma en, and bi ds o p ey a suble hal concen a ions [16-18]. B omadiolone ac s as he an agonis o i amin K1 and inhibi s hus blood clo ing (Fig. 1). I may be cha ac e ized as ha ing a e y low LD50 alue and high s abili y [19]. The e o e b omadiolone may pe sis in he en i onmen (pa icula in he soil), bio-accumula e in an o ganism and cause acu e o ch onic in oxica ions [20]. In addi ion he in e ac ion o b omadiolone and blood plasma p o eins is no clea ye . Recen ly, a s udy epo ing on he in e ac ion o b omadiolone and albumin was published [21]. Se e al p oduc s con aining b omadiolone ha e been app o ed o use as bai ed g anules in he Czech Republic. The g anules disin eg a e quickly in he ields o applica ion, hus he e is a isk o bo h p ima y and seconda y in oxica ions o non- a ge o ganisms, because he ca ying subs ances o g anules may a ac ea hwo ms. The insec i o ous and o he small mammalian species may possibly become in oxica ed by his way (Fig. 2). G ea a en ion has been paid o he su eillance o esidues o pes icides bo h in he en i onmen and oods [22-27]. Se e al analy ical app oaches such as gas ch oma og aphy mass spec oscopy, hin laye ch oma og aphy o immunochemis y [28,29] may be used o de e mine b omadiolone. Howe e , high pe o mance liquid ch oma og aphy (HPLC) coupled wi h a ious de ec o s ( luo escence o mass de ec o s) is he mos commonly used ins umen o de ec b omadiolone [30-34]. These ins umen s, howe e , su e om a ious eason, mos o all, high cos . Elec ochemical senso s and biosenso s ha e supe io p ope ies o e he o he exis ing measu emen sys ems because hey can p o ide quick, simple and low-cos on- ield de e mina ion o many biologically ac i e species and a numbe o dange ous pollu an s [35-57]. Senso s 2007, 7 1273 Figu e 1. Au opsy o pheasan s killed a he end o he expe imen e ealed no ma ked pa hological changes excep o occasional haemo hages wi hin he b eas muscles. The igu e shows a ypical inding in animals poisoned wi h oden icides, so i indica es ha he pa hological changes we e ound in opposi e o he ela i ely good heal h s a e o pheasan s [58]. Figu e 2. Scheme o he model expe imen simula ing he b omadiolone dis ibu ion wi hin he ood chain. THE RELATION BETWEEN BROMADIOLONE AND FOOD CHAIN – THE INFLUENCE OF THE RODENTICIDE ON NON-TARGET ORGANISMS Senso s 2007, 7 1274 Conside ing he abo e ac s, he aim o his wo k was o p opose a new me hod o b omadiolone de ec ion. We op imized high pe o mance liquid ch oma og aphy wi h elec ochemical de ec ion (HPLC-ED) o his pu pose and employed his app oach o de e mine he concen a ion o he a ge molecule in issues o animals om wo ophic le els. 2. Expe imen al Sec ion 2.1 Chemicals Ace ic acid, sodium ace a e, ace oni ile (HPLC g ade) and b omadiolone (Pes anal - analy ical s anda d) we e pu chased om Sigma Ald ich (S einheim, Ge many). Me hanol was pu chased om Fluka (S einheim, Ge many). Wa e o he p epa a ion o bu e s unde wen demine aliza ion by e e se osmosis using he ins umen s Aqua Osmo ic 02 (Aqua Osmo ic, Tisno , Czech Republic). Then, i was pu i ied using Millipo e RG (Millipo e Co p., USA, 18 MΏ). Wo king solu ions o he b omadiolone s anda d we e p epa ed daily by dilu ing he s ock solu ion o 1 mg/ml concen a ion wi h me hanol. 2.2 Roden icide-con aining p oduc The expe imen was ca ied ou wi h a comme cially a ailable oden icide (wi h 0.005% o b omadiolone; he eina e e e ed as PRODUCT), which is used commonly o he con ol o common oles in ga dens, o cha ds, ineya ds, and non-ag icul u al land. The PRODUCT is no app op ia e o la ge-scale use bu o he applica ion in he oles’ bu ows. The ecommended dose is 5-10 g anules pe open bu ow. 2.3 Bio es s 2.3.1 Toxici y o ea hwo ms – es in he a i icial soil subs a e The es was ca ied ou acco ding o he modi ied OECD 207 guideline [59]. Glass bo les (1,000 ml) we e illed wi h 500 g o he a i icial soil es subs a e consis ing o pea (10 %), kaolin clay (20 %), and silica sand (70 %). The subs a e was mixed wi h 10 g anules o he PRODUCT. Demine alised wa e was added a a le el co esponding o 35% o d y weigh o he subs a e. Fou bo les wi h he PRODUCT and ou con ol bo les we e used in he expe imen . Ten labo a o y ea ed indi iduals o Eisenia e ida we e weighed p io o he expe imen and placed on he su ace o he subs a e in each bo le. Only adul indi iduals wi h he cli ellum we e used; he minimum a e age weigh was 400 mg. The es was ca ied ou unde condi ions as ollows: empe a u e o 20±2°C and con inual ligh ning (450-600 lx). The e alua ion o he es was pe o med a e 14 days. One hal o con ol ea hwo ms and one hal o he ea hwo ms exposed o he PRODUCT we e ozen immedia ely a e collec ing. The emaining ea hwo ms we e used in he ollowing expe imen wi h common oles. Senso s 2007, 7 1275 2.3.2 P ima y and seconda y in oxica ion o common oles Nine common oles (Mic o us a alis) om he labo a o y ea ing we e used o expe imen ally s udy he possibili y o p ima y and seconda y in oxica ion by b omadiolone. Voles we e kep indi idually in 9 l glass aqua ia wi h wood sha ings as bedding. Th ee g oups o oles we e employed in he expe imen las ed 5 days. The i s g oup consis ed o con ol animals we e ed wi h common comme cially a ailable g anules o labo a o y mice. The second g oup o oles was ed wi h he PRODUCT g anules o which hey ecei ed 0.086 g daily. The hi d g oup was ed wi h ea hwo ms, which we e p e iously exposed o PRODUCT g anules o 14 days. D inking wa e was a ailable ad libi um. To ensu e adap a ion o oles o ea hwo ms as a eed, he oles used in he expe imen we e ed wi h ea hwo ms om he labo a o y ea ing o a pe iod o 7 days p io o he expe imen . A he s a o he expe imen he a e age weigh o oles om he i s , second and hi d g oup was 16.6 g, 15.5 g and 15.6 g, espec i ely. A he end o he 5-days long expe imen , he weigh o animals om he o e men ioned g oups was 17.3, 15.9 and 15.2 g, espec i ely. Voles we e weighed, eu hanized and hen subjec ed o au opsy. No g oss pa hological lesions we e ound. The li e was collec ed om each animal. The a e age weigh o he li e o animals om he o e men ioned g oups was 0.95, 0.93 and 0.94 g, espec i ely. 2.3.3 Ha es Th ee ha e cada e s om hun ing g ounds on ag icul u al p emises in dis ic s o he eas o he ci y o B no (Sou h Mo a ia, Czech Republic), whe e b omadiolone-con aining g anula bai s was used, we e collec ed in No embe 2005 and used in ou expe imen s. 2.4 P epa a ion o biological samples The samples o 0.5 g issue (ea hwo m, li e om he ea ed oles o li e and s omach con en o ha es) we e homogenized wi h 0.5 ml o me hanol using a homogenize (Ul a u ax) o 15 min. The homogena es was sonica ed a 150 W a 4 °C o 15 min. using K5 Sonica o (Slo akia) and subsequen ly o exed o 15 min. using Vo ex–2 Genie (Scien i ic Indus ies, New Yo k, USA). The mix u e was cen i uged o 20 minu es a 14,000 g a 4 °C using Uni e sal 32 R cen i uge (He ich- Zen i ugen GmbH, Tu lingen, Ge many). Subsequen ly, he supe na an liquo was collec ed and s o ed a –20 °C un il he nex analysis. P io o he analysis, he samples we e dilu ed 2 × wi h me hanol. 2.5 Elec ochemical measu emen s in he s a iona y sys em Elec ochemical cyclic ol amme ic measu emen was ca ied ou using he AUTOLAB Analyse (EcoChemie, The Ne he lands) connec ed o VA-S and 663 (Me ohm, Swi ze land). The h ee- elec ode sys em was used; i consis ed o he ca bon pas e elec ode (pas e composi ion: 70% ca bon powde and 30% mine al oil, w/w; he e lon body in which he diame e o he ac i e elec ode su ace was 2.5 mm; he su ace o he elec ode was polished p io o each measu emen using a ine il a ion pape acco ding o [60-62]); e e ence elec ode (Ag/AgCl/3 mol l -1 KCl), and he auxilia y elec ode om glassy ca bon. GPES so wa e (EcoChemie, The Ne he lands) was employed o p ocess aw da a Senso s 2007, 7 1276 using Le el 4 Sa i zky and Golay il e . Expe imen s we e ca ied ou a oom empe a u e. The measu emen was pe o med in he po en ial ange om 0.6 o 1 V using he ollowing pa ame e s: he po en ial s ep o 2.4 mV, he scan a e o 0.3 V.s -1 . Ace a e bu e (0.2 M, pH 4.0) was used as a suppo ing elec oly e. 2.6 Elec ochemical measu emen s in he low sys em The low sys em consis ed o he ch oma og aphic pump (Model 583 ESA Inc., Chelms o d, MA, USA), Gua d cell (Model 5020 ESA, USA), eac ion loop (1m) o Flow Injec ion Analysis (FIA), and he ch oma og aphic column Pola is (C18-A, 150 × 4.6 mm, wi h pa icle size o 3 µm; Va ian, Inc.) and he elec ochemical de ec o . The de ec o consis ed o pa s as ollows: low ampe ome ic analy ical cell (Model 5040, ESA, USA) con aining he e e ence (hyd ogen-palladium), auxilia y ca bon and he wo king elec ode made o glassy ca bon, he Coulochem III con ol module. The sample (5 µl) was injec ed manually using a 5 µl injec ion loop (Supelco, Belle on e, PA, USA). The da a om he de ec o we e p ocessed using he CSW 32 so wa e. The isoc a ic mobile phase consis ed o 0.2 M ace a e bu e (pH 4.0/ace oni ile, 40:60, / ). The de ec ion po en ial was op imized; see in “Resul s” sec ion. 3. Resul s 3.1 De ec ion o b omadiolone using cyclic ol amme y To employ HPLC wi h elec ochemical de ec ion o measu emen o b omadiolone we aimed on s udy o he basic elec ochemical beha iou o he a ge molecule using cyclic ol amme y a he ca bon pas e elec ode. The ol ammog ams wi h a dis inc oxida ion signal a po en ial o 836 mV due o b omadiolone a e shown in Fig. 3A. Dependence o he peak heigh on he scan a e was loga i hmic (y = 44.413 ln(x) - 78.08). In addi ion, he po en ial o he peak shi ed g adually o posi i e po en ials (Fig. 3B). The dependence o he peak heigh on he concen a ion o b omadiolone was also de e mined. The signal enhanced sha ply wi h he concen a ion in he gi en concen a ion ange up o a concen a ion o 1 µg/ml, hen mo e g adually (Fig. 3C). In spi e o he ac ha he peak heigh o b omadiolone enhanced wi h i s inc easing concen a ion, bu he dependence measu ed is no s ic ly linea wi hin he in e al. Fo analy ical pu poses his dependence can be spli in o wo s ic ly linea lines a a concen a ion o b omadiolone anging om 0.625 o 10 µg/ml (y = 600.53x + 67.114, R 2 = 0.9912); and a a concen a ion o b omadiolone anging om 20 o 300 ng/ml (y = 62.576x + 259.78, R 2 = 0.9927, inse in Fig. 3C). The limi o de ec ion o b omadiolone measu ed on he ca bon pas e elec ode by cyclic ol amme y was e alua ed as 5 ng/ml. I can be concluded ha CV is a me hod sui able o undamen al s udying o elec ochemical beha iou o a a ge molecule; howe e , i can no be used o analysis o eal sample due o ela i e low selec i i y. The e o e he expe imen al esul s ob ained we e used in an op imiza ion p ocedu e o he de ec ion o b omadiolone using low analysis. Senso s 2007, 7 1277 Figu e 3. The elec ochemical analysis o b omadiolone using cyclic ol amme y. (A) Cyclic ol ammog ams o b omadiolone (1 µg/ml) a scan a e o 5, 50, 150, and 300 mV/s. (B) Dependence o he heigh and posi ion o po en ial o he b omadiolone signal on he scan a e. (C) Va ia ion in he heigh o he b omadiolone signal on he concen a ion a a scan a e o 300 mV/s. Pa ame e s o measu emen : 0.2 M ace a e bu e (pH 4.0), in he po en ial ange om 0.6 o 1 V, po en ial s ep: 2.4 mV, expe imen s we e ca ied ou a oom empe a u e. 3.2 De ec ion o b omadiolone using low injec ion analysis wi h glassy ca bon elec ode To make he analysis o b omadiolone-con aining samples apid and sui able o ou ine, a new app oach o he de ec ion o b omadiolone using Flow Injec ion Analysis (FIA) was p oposed. The elec ochemical esponse o b omadiolone was s udied a a cons an low a e o he mobile phase (0.5 ml/min) and a a ying wo king elec ode po en ial. The de ec ion po en ial o 840 mV was selec ed based on he hyd odynamic ol ammog am (no shown). To ob ain he maximum esolu ion o he b omadiolone peak and he maximum in ensi y o he signal, he e ec o an o ganic componen o he mobile phase (ace oni ile) was s udied (Fig. 4A). The inc eased po ion o he o ganic phase was ound bene icial o he de ec ion o b omadiolone. This ac is likely o be associa ed wi h he physicochemical p ope ies o b omadiolone as an non-pola compound (mo e soluble in o ganic sol en s) al hough ou p e ious s udies indica ed a signi ican dec ease in he elec ochemical signal in he p esence o he inc eased po ion o he o ganic componen in he mobile phase [58,63-66]. The low a e o he mobile phase also a ec s he elec ochemical signal. Va ia ions in he b omadiolone 0 100 200 300 400 500 600 700 800 900 0 2 4 6 8 10 12 y = 44.413Ln(x) - 78.08 R 2 = 0.9801 0 50 100 150 200 0 100 200 300 400 500 0.72 0.76 0.80 0.84 0 100 200 300 400 500 Scan a e (mV/s) Po en ial (V) 0.6 0.7 0.8 0.9 1.0 500 nA Po en ial (V) Cyclic ol ammog ams 5 mV/s 50 mV/s 150 mV/s 300 mV/s Scan a e B omadiolone signal Peak heigh (nA) Scan a e (mV/s) A B C O OH O CH CH 2 CH B OH y = 600.53x + 67.114 R 2 = 0.9912 0 100 200 300 0 0.1 0.2 0.3 0.4 B omadiolone concen a ion (µ µµ µg/ml) Peak heigh (nA) B omadiolone concen a ion (µ µµ µg/ml) Peak heigh (nA) Senso s 2007, 7 1278 signal wi h he low a e anging om 0.1 ml/min o 2.0 ml/min we e de e mined. The b omadiolone signal inc eased a a low a e up o 0.8 ml/min. Then i did no change (no shown). Figu e 4. The analysis o b omadiolone using high pe o mance liquid ch oma og aphy wi h elec ochemical de ec ion. (A) The e ec o he o ganic componen in he mobile phase (ace oni ile) on he b omadiolone peak heigh . (B) Ch oma og am o b omadiolone a a concen a ion o 50 µg/ml, e en ion ime o he peak o 9.5min. (C) Dependence o he heigh o he signal on he b omadiolone concen a ion. Expe imen al condi ions we e as ollows: ch oma og aphic column Pola is C18-A, 150 × 4.6 mm, diame e : 3 µm (Va ian, Inc.). The sample was injec ed manually. Isoc a ic mobile phase consis ed o 0.2 M ace a e bu e (pH 4.0)/ace oni ile (40:60, / ) and he de ec ion po en ial o 840 mV we e used. The low a e o he mobile phase was 0.8 ml/min. 3.3 High pe o mance liquid ch oma og aphic analysis o b omadiolone Unde he expe imen al condi ions op imized using FIA (wo king elec ode po en ial 840 mV, mobile phase 0.2 M ace a e bu e (pH 4.0)/ace oni ile (40:60, / ), he low a e o he mobile phase 0.8 ml/min) high pe o mance liquid ch oma og aphy coupled wi h elec ochemical de ec o was used o de e mine he con en o b omadiolone in eal samples. The signal o b omadiolone was obse ed wi h he e en ion ime o 9.5 min (Fig. 4B). In addi ion, he calib a ion dependence o b omadiolone a a concen a ion ange om 0.3 o 50 µg/ml was de e mined. The s ic ly linea calib a ion cu e was ob ained: y = 3.5x + 0.9; R 2 = 0.9997, wi h a ela i e s anda d de ia ion o abou 4 % (Fig. 4C). When we p oposed he echnique o de e mina ion o b omadiolone, he oxic e ec s o he a ge molecule on a ious expe imen al models we e in es iga ed. 15 y = 3.5319x + 0.9243 R 2 = 0.9997 0 40 80 120 160 200 0 20 40 B omadiolone concen a ion (µ µµ µg/ml) Peak heigh (µA) 0 10 20 30 40 50 60 70 80 90 100 30% 35% 40% 60% Ace oni ile con en in mobile phase (%, / ) Peak heigh (%) 7 9 11 13 50 µA b omadiolone 50 µg/ml Re en ion ime (min) RT 9.5 min B AC Re en ion ime (min) 8.5 9.5 10.5 con ol b omadiolone posi i e 300 nA Re en ion ime (min) 8.5 9.5 10.5 con ol b omadiolone posi i e 300 nA300 nA 50 µA Senso s 2007, 7 1279 3.4 B omadiolone exposu e 3.4.1 Ea hwo ms Du ing he 14-days long expe imen , all ea hwo ms exposed o b omadiolone su i ed. A he beginning o he expe imen hei a e age weigh was 430 mg. Howe e , he weigh o con ol ea hwo ms was 293 mg while ea hwo ms exposed o he PRODUCT g anules weighed 344 mg a he end o he expe imen ,. The dec ease in he weigh o con ol ea hwo ms (mo e han 30 %) was caused by s a a ion since he subs a e con ained no sui able nu ien s. Ea hwo ms exposed o he PRODUCT g anules los only 20 % o hei o iginal weigh . They we e assumed o ha e ecei ed g anules con aining b omadiolone. Using he p oposed app oach o homogeniza ion and ex ac ion, he samples o ea hwo ms (Eisenia e ida) we e p epa ed and subsequen ly analyzed by HPLC-ED. The a e age le el o b omadiolone was 1.7 ± 0.2 µg pe ea hwo m, which co esponds o 3.3 ± 0.9 µg pe g o ea hwo m weigh (Fig. 5). No signal due o b omadiolone was de e mined in samples om con ol ea hwo ms. Figu e 5. The con en o b omadiolone in he issues om expe imen al animals. No b omadiolone signal was de e mined in samples om he con ols ( oles, ea hwo ms). The le els o b omadiolone in ea hwo ms we e ecalcula ed ela i e o he weigh o ea hwo ms. The le els o b omadiolone in oles we e ecalcula ed o he weigh o he li e . Fo o he de ails see in Fig. 3 and “Expe imen al sec ion”. 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 B omadiolone con en (µg/g) Con ol Eisenia e ida Mic o us a alis (p ima y exposu e) Mic o us a alis (seconda y exposu e) Biological samples Lepus eu opaeus (li e ) Lepus eu opaeus (s omach con en ) 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 B omadiolone con en (µg/g) Con ol Eisenia e ida Mic o us a alis (p ima y exposu e) Mic o us a alis (seconda y exposu e) Biological samples Lepus eu opaeus (li e ) Lepus eu opaeus (s omach con en ) Senso s 2007, 7 1286 69. Paole i, M.G.; Sommaggio, D.; Fa e o, M.R.; Pe uzzelli, G.; Pezza ossa, B.; Ba ba ie i, M. Ea hwo ms as use ul bioindica o s o ag oecosys em sus ainabili y in o cha ds and ineya ds wi h di e en inpu s. Appl. Soil Ecol. 1998, 10, 137-150. 70. Dell'Omo, G.; Tu k, A.; Sho e, R.E. Seconda y poisoning in he common sh ew (So ex a aneus) ed ea hwo ms exposed o an o ganophospha e pes icide. En i on. Toxicol. Chem. 1999, 18, 237- 240. 71. B own, P.; Cha l on, A.; Cu hbe , M.; Ba ne , L.; Ross, L.; G een, M.; Gillies, L.; Shaw, K.; Fle che , M. Iden i ica ion o pes icide poisoning in wildli e. J. Ch oma og . A 1996, 754, 463- 478. 72. Guan, F.; Ishii, A.; Seno, H.; Wa anabe, K.; Kumazawa, T.; Suzuki, O. A me hod o simul aneous de e mina ion o i e an icoagulan oden icides in whole blood by high- pe o mance liquid ch oma og aphy. J. Pha m. Biomed. Anal. 1999, 21, 179-185. 73. Guan, F.Y.; Ishii, A.; Seno, H.; Wa anabe-Suzuki, K.; Kumazawa, T.; Suzuki, O. Use o an ion- pai ing eagen o high-pe o mance liquid ch oma og aphy-a mosphe ic p essu e chemical ioniza ion mass spec ome y de e mina ion o anionic an icoagulan oden icides in body luids. J. Ch oma og . B 1999, 731, 155-165. 74. Ob yan, S.M.; Cons able, D.J.C. 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