生物样品中抗凝血类杀鼠剂分析研究进展

2015-06-01 12:30综述审校
复旦学报(医学版) 2015年5期
关键词:抗凝血头发质谱

朱 琳(综述) 向 平(审校)

(司法部司法鉴定科学技术研究所法医毒物化学研究室-上海市法医学重点实验室 上海 200063)

生物样品中抗凝血类杀鼠剂分析研究进展

朱 琳(综述) 向 平△(审校)

(司法部司法鉴定科学技术研究所法医毒物化学研究室-上海市法医学重点实验室 上海 200063)

在我国,抗凝血类杀鼠剂中毒频繁发生,易被误诊。检测生物样品中抗凝血类杀鼠剂对临床诊断、治疗和法医学调查有重要意义。本文综述了近年文献报道的生物样品中抗凝血类杀鼠剂的分析方法,着重比较各种生物样品选择、样品前处理、分离检测技术,并结合本课题组的应用案例给出血液、头发分析的结果参考。血液适用于抗凝血类杀鼠剂分析,其主要前处理方法有液-液提取、蛋白质沉淀等。头发可作为血液样品的重要补充,用于摄药历史的法医学调查。液相色谱-质谱(liquid chromatography-mass spectrometry,LC-MS)检测法兼顾灵敏度与选择性,是目前最常用的分析生物样品中抗凝血类杀鼠剂的技术。

抗凝血; 杀鼠剂; 中毒; 生物样品; 分析方法

随着急性剧毒杀鼠剂(如:毒鼠强)的禁用,抗凝血类杀鼠剂被广泛使用。人类抗凝血类杀鼠剂中毒在世界范围内屡有报道,最多见的摄入原因包括误食[1-3]、自行服毒[4]、甚至药物滥用者为达到更长欣快感而服用[2,5-6]。在美国,自2009—2013年发生抗凝血类杀鼠剂的接触报告共49702例[7-11]。抗凝血类杀鼠剂对人以外的非目标动物(家畜、宠物及野生动物哺乳动物、猛禽等)具有中毒和二次中毒威胁[12-16],在动物保护领域引起了重视。

在中国,抗凝血类杀鼠剂中毒频繁发生[17-23],甚至可能是公共安全事件,如2009年5月浙江乐清176人误食溴敌隆污染的炒饭中毒[24],2010—2011年福建某乡镇陆续出现不明原因的疑似大隆投毒[25]等。

抗凝血类杀鼠剂竞争性抑制肝脏中维生素K的转化,致凝血功能受损,其潜伏期为1~11天,中毒症状易与其他出血疾病混淆;加上服毒者隐瞒,或自认与症状无关而未提及服毒史,易导致误诊[20-21,26]。目前临床实践中多通过凝血参数提示诊断,但无法确诊。而检测生物样品中抗凝血类杀鼠剂,能直接为诊断和治疗[2]提供依据,也有助法医学专家调查案情[17]。

生物样品中的抗凝血类杀鼠剂含量多为ng/g级别,而薄层色谱[27]、液相色谱[28]、气相色谱[15]、气相色谱-质谱[29]检测法的灵敏度和专属性有限[30],曾是分析工作难点。近年来随着液相色谱-质谱检测技术的成熟,生物样品中的抗凝血类杀鼠剂检测方法得以进一步发展。

已有研究者综述了抗凝血类杀鼠剂分析研究进展[17,30-32],但尚缺对生物样品分析的重点讨论[32]。本文将简介抗凝血类杀鼠剂,综述近年来报道的分析方法,讨论生物样品选择、前处理、分析检测方法,并结合本实验室的案例应用,讨论人血液和头发的相关分析结果,供临床及法医毒物工作者参考。

抗凝血类杀鼠剂简介第1代抗凝血类杀鼠剂主要是华法林(warfarin,又名杀鼠灵)和杀鼠醚(coumatetralyl)。随着耐药鼠种群产生,开发出药效更强、作用更持久的第2代抗凝血类杀鼠剂(second generation anticoagulant rodenticides,SGARs),又被称为“超级华法林(superwarfarins)”、“长效杀鼠剂(long-acting rodenticides)”[1],包括4-羟基香豆素类的溴敌隆(bromadiolone,又名“乐万通”)、大隆(brodifacoum,又名“溴鼠灵”、“溴鼠隆”)、杀它仗(flocoumafen)等,以及茚满二酮类的敌鼠(diphacinone)、氯鼠(chlorophacinone)、杀鼠酮(valone)等。目前中国农业部登记的抗凝血类杀鼠剂有溴敌隆、大隆、敌鼠钠盐、氟鼠灵、杀鼠灵、杀鼠醚[33]。文献[34]及本课题组的案例显示,我国最常引起中毒的抗凝血类杀鼠剂是溴敌隆与大隆。

抗凝血类杀鼠剂吸收途径包括口服、呼吸道[2,5-6]、皮肤和黏膜[35-41]吸收。抗凝血类杀鼠剂常在肝脏蓄积[42-43]。大鼠肝脏与血清中大隆浓度比值超过20[44]。华法林主要经肝脏的细胞色素P450羟基化代谢后,从尿液排泄。而第2代抗凝血类杀鼠剂溴敌隆[45]、大隆[44]和杀它仗[43]主要以原形从粪便排泄。

表1列举了一些中毒报道的血药浓度和消除半衰期。第2代抗凝血类杀鼠剂的生物半衰期可长至几天至几十天,大多显示两种消除时相的动力学[42,46]。亦有研究显示大隆为零级消除(取样时间至摄入后40天),但不排除到了低浓度时转为一级消除[47]。

目前治疗包括输注维生素K、凝血酶原复合物,重症时给予新鲜冷冻血浆或血液灌流等[2,48]。鉴于第2代抗凝血类杀鼠剂消除缓慢,且半衰期个体差异大(表1),在治疗过程中应测定患者血液中化合物浓度,设计给药方案,有利于合理治疗[46-47,49]。根据Lo等[46]的一个中毒案例,血液溴敌隆浓度降至10 ng/m L后,患者凝血功能恢复正常。而对于大隆,有研究者认为治疗终点可定在血清大隆浓度降至10 ng/m L[47,50]。

抗凝血类杀鼠剂分析使用的生物样品及前处理方法

血液 血液能直接反应循环系统中毒物浓度。抗凝血类杀鼠剂的水溶性普遍较差,易溶于有机溶剂。由于抗凝血类杀鼠剂呈弱酸性,液-液提取(liquid-liquid extraction,LLE)时可以考虑将水相调整为弱酸性[57]。

处理血浆可丙酮提取后乙醚沉淀蛋白质[58]或直接乙腈沉淀蛋白质[59]。有方法[45,60]使用了Toxi-B管(一种液-液提取装置,有机相包括30%~40%的二氯甲烷,10%~20%的庚烷,30%~40%的氯化锌)。

全血前处理常采用乙酸乙酯LLE,使用溶剂量

为2~8 m L[28,56,61-62]。Guan等[28]对比了乙酸乙酯、氯仿、乙醚和C18小柱对全血中5种抗凝血类杀鼠剂的回收率,乙酸乙酯的回收率最高。

表1 一些人类抗凝血类杀鼠剂中毒者的血液测量结果和半衰期Tab 1 Some anticoagulant rodenticide levels in human poisoning cases and the corresponding half-lives

血清的处理通常包括沉淀蛋白质和乙酸乙酯提取两个步骤[57]。

肝脏 第2代凝血类杀鼠剂在肝脏蓄积,因此肝脏常用于相关毒物动力学研究。最常用固相萃取法(solid phase extraction,SPE),且预先用乙腈[63]、丙酮[64]、或丙酮与二氯甲烷的混合物等[65]液-液提取。优点是样品净化充分,缺点是耗时较长。

Clelland[66]使用一次性吸管萃取(disposable pipette extraction,DPX)处理了肝脏样品,其原理同固相萃取,但无需活化和抽真空洗脱步骤,时间短;且每个样品使用的有机溶剂不超过1 m L,较传统固相萃取减少。

Vudathala等[67]开发了一个Qu ECh ERS (Quick、Easy、Cheap、Effective、Rugged、Safe,基于分散固相萃取原理)方法。与传统SPE对比,此方法便捷且节省溶剂,且可达到满意效果。

Hernández等[68]分析普通田鼠的肝脏、肠、肉时,将冻干的样品均一化后用甲醇(6 m L)提取。

毛发 抗凝血类杀鼠剂由于摄入隐蔽等原因,暴露来源往往难以明确[1],而头发检测时限长,根据头发生长速度约1 cm/月,通过分段分析,可大致推测某些外源化合物的摄入历史,并已在法医学等领域得以应用[10-11],头发还具有采样无侵犯性、易保存等优点。

目前报道的头发分析方法中,Röhrich等[69]将头发用甲醇超声提取(50℃,4 h),再用氯仿、丙酮提取,但未在实际应用中检出抗凝血类杀鼠剂。本文作者等[70]将头发冷冻研磨,加p H 6.8磷酸缓冲液超声后乙酸乙酯液-液提取,此法应用于实际中毒者的头发分段分析,检出了溴敌隆或大隆。

其他生物样品 死亡案件的法医毒物分析中,除了血液、肝、肾等,有报道胆汁检出抗凝血类杀鼠剂[71],肝脏的甲醛保存液也可检出[71]。

在野生动物的抗凝血类杀鼠剂暴露监测中,动物尸体检查常用肝脏[58]。采样无侵犯性的生物样品则有粪便[45]以及猛禽食团[14]。毛发分析或将给野生动物的保护性监测提供了新选择[70]。

虽然已建立以尿液为样品的方法[59,72-73],但极少从尿液检出抗凝血类杀鼠剂的报道。如某女,因出血入院20天后,血液中检出大隆(422 ng/m L),尿液无检出[62]。有报道一危重患者的尿液(血尿)中检出溴敌隆为73 ng/m L,同时其血液中溴敌隆为1 150 ng/m L[48],很可能源于尿液混杂的血液。如前述,溴敌隆、大隆、杀它仗主要随粪便排出,尿液排泄很少[43-45];即使对通过经尿液排出的化合物,如敌鼠,检测时距摄入已久,其排泄速率已变慢[74],因此尿液不是抗凝血类杀鼠剂分析的理想样品。

分析方法

液相色谱-紫外检测和液相色谱-荧光检测法

抗凝血类杀鼠剂不易挥发,应用气相色谱法时需衍生化,且灵敏度有限[30],如Sato等[29]开发的气相色谱-质谱(GC-MS)法,检测限为10~30 ng/m L。而液相色谱法不需要衍生化步骤。高效液相色谱的分离,多为以C18为固定相的反向色谱。就灵敏度而言,荧光检测器(FLD)检测限可达小于10 ng/m L[57,65,75],优于紫外(UV)通常在20~75 ng/m L范围的检测限[28,76-77]。

液相色谱-质谱检测和液相色谱-串联质谱检测法 相较紫外和荧光检测器,质谱有极佳的灵敏度和选择性。Hernández等[68]测量4种抗凝血类杀鼠剂,用UV或FLD,检出限是9~89 ng/g,而用质谱检出限为0.6~1.5 ng/g。液相色谱-质谱(LC-MS)是近年新开发方法中的主流,其主要缺点是价格较高。

电喷雾电离(electrospray ionization,ESI)是目前检测生物样品中抗凝血类杀鼠剂最常用的离子源[45,58,61-62,66,68,70,78]。金米聪等[79]用大气压化学电离(atmospheric pressure chemical ionization,APCI)和ESI分析杀鼠醚,APCI正模式响应最好。Röhrich等[69]考察了ESI、APCI、大气压光电离(atmospheric pressure photoionization,APPI)3种离子源分析抗凝血类物质,在APCI、ESI负模式下鼠得克和大隆有最好响应,溴敌隆则只在此两种模式下有响应。蔡欣欣等[59]考察了不同离子源,敌鼠、氯鼠只在ESI负模式离子化。因此,ESI是同时检测抗凝血类杀鼠剂的合适之选。

基质效应指色谱分离时共同流出的物质使待测物的离子化效率降低或增高,是LC-MS方法必须考察的问题。已报道的抗凝血类杀鼠剂分析方法中,待测物离子化皆受抑制[62,68-70,78,80],抑制率为6.5%[78]~75%[70]。为了减轻基质效应,可调整色谱条件或优化样品处理,如Clelland为减轻脂肪酸引起的基质效应,在DPX后加入了碱性铝[66]。

目前LC-MS和LC-MS/MS分析抗凝血类杀鼠剂的方法最常采用四极杆质谱检测器,其中三重四级杆的多反应监测(MRM)模式检测限可达到0.1 ng/m L[62]。目前报道的两个测量头发中抗凝血类杀鼠剂的LC-MS/MS方法均为使用此检测法[69-70]。Fourel等[60]用离子阱质谱分析13种抗凝血类物质,检出限为5~25 ng/m L。Schaff等[80]用轨道阱高分辨质谱分析血液中的抗凝血类杀鼠剂,同时用全扫描质谱和串联质谱采集数据,该方法检测限为2~10 ng/m L。

其他分析方法 除高效液相色谱,亦有使用离子色谱法的分析[53,78,81],如Chen等[78]使用离子色谱-离子阱质谱分析四种茚满二酮类杀鼠剂,定量下限为0.2~0.5 ng/m L。

Krizkova等[82]用液相色谱-循环伏安法测定田鼠肝脏的溴敌隆含量,该电化学检测器能达到5 ng/m L的检测限。Beklova等[83]使用碳糊电极,以微分脉冲伏安法(differential pulse voltammetry,DPV)测定了动物组织中的溴敌隆,检出限为0.5 ng/m L。然而,这两种电化学分析方法选择性较低,只适合单一目标物的分析[82]。

应用案例关于中国人群中接受治疗的抗凝血类杀鼠剂中毒者体内含量的报道尚不多。作者所在的研究团队近年检测了多例疑似抗凝血类杀鼠剂中毒患者的血液[62]和头发[70]。现列举部分案例如下。

血液检测 应用案例1:男,39岁,与妻子争执后服下家中鼠药(数量不明),当天被送诊,无明显症状。血液中含溴敌隆12.1 ng/m L。

案例2:男,29岁,因不明原因的出血就诊,纤维蛋白原含量(Fg)为4.7 g/L(正常值1.8~3.5 g/L),活化部分凝血酶时间(APPT)为123 s(正常值27~41 s),凝血酶原时间60 s(正常值0~16 s)。2天后血样送实验室检测,含溴敌隆89.5 ng/m L。

案例3:男,47岁,口腔出血、胃十二指肠出血、血尿、腰痛一年半。口服维生素K使其凝血指标回复正常,数月后出血复发。血样送本实验室检测,含溴敌隆10.8 ng/m L。

头发检测 血液检出抗凝血类杀鼠剂的5例患者(全血中溴敌隆或大隆含量为1.1~422 ng/m L)处,征得同意后采集头发,分段分析。出现症状后9 ~25天采集头发的患者,其近发根1 cm或0.5 cm的头发检出,含量为检出限(溴敌隆10 pg/mg,大隆25 pg/mg)至78 pg/mg,而远端头发无检出。推测摄入时间,与患者描述的症状出现时间大致相符。

1例55岁女性患者的头发采集于出现症状6个月后,近头皮0~8 cm的发段均可检出大隆,各段含量从检出限到54 pg/mg,难以发现明显分布规律。漫长的半衰期使抗凝血类杀鼠剂在摄入后很长时间都能从血液循环进入头发,目前尚难从头发分析判断单次或重复摄药。

结语生物样品中抗凝血类杀鼠剂的分析对中毒的诊断、治疗以及法医调查很重要。已建立的生物样品中抗凝血类杀鼠剂的分析方法中,液相-质谱兼顾选择性和灵敏度,是目前最常用的分析技术。进一步研究方向可能有:(1)步骤更少、更环境友好型的前处理及自动化,如针对复杂样品的QuEChERS方法等;(2)用量更少、更易采集和保存的样品种类,如唾液、干血点(dry blood spot)等;(3)考察头发中的各段含量分布及其随时间的变化规律,美发处理等因素对其影响,头发能否判断反复或单次摄入等问题。

[1]Watt BE,Proudfoot AT,Bradberry SM,et al. Anticoagulant rodenticides[J].Toxicol Rev,2005,24(4):259-269.

[2]Spahr JE,Maul JS,Rodgers GM.Superwarfarin poisoning:a report of two cases and review of the literature[J]. Am J Hematol,2007,82(7):656-660.

[3]Olmos V,Lopez CM.Brodifacoum poisoning with toxicokinetic data[J].Clin Toxicol(Phila),2007,45(5):487-489.

[4]Iglesias Lepine ML,Epelde Gonzalo F,Casanas Ferrer F,et al.Superwarfarin rodenticide intoxication in adults:an update on bromadiolone,brodifacoum,and difethialone [J].Emergencias,2013,25(3):201-203.

[5]Waien SA,Hayes D,Leonardo JM.Severe Coagulopathy as a consequence of smoking crack cocaine laced with rodenticide[J].N Engl J Med,2001,345(9):700-701.

[6]La Rosa FG,Clarke SH,Lefkowitz JB.Brodifacoum intoxication with marijuanking[J].Arch Pathol Lab Med,1997,121(1):67-69.

[7]Bronstein AC,Spyker DA,Cantilena LR,et al.2009 Annual report of the American Association of Poison Control Centers'National Poison Data System(NPDS):27th annual report[J].Clin Toxicol(Phila),2010,48 (10):979-1178.

[8]Bronstein AC,Spyker DA,Cantilena LR,et al.2010 Annual report of the American Association of Poison Control Centers′National Poison Data System(NPDS):28th annual report[J].Clin Toxicol(Phila),2011,49 (10):910-941.

[9]Bronstein AC,Spyker DA,Cantilena LR,et al.2011 Annual report of the American Association of Poison Control Centers′National Poison Data System(NPDS):29th annual report[J].Clin Toxicol(Phila),2012,50 (10):911-1164.

[10]Mowry JB,Spyker DA,Cantilena LR,et al.2012 Annual report of the American Association of Poison Control Centers′National Poison Data System(NPDS):30th annual report[J].Clin Toxicol(Phila),2013,51(10):949 -1229.

[11]Mowry JB,Spyker DA,Cantilena LR,et al.2013 Annual report of the American Association of Poison Control Centers′National Poison Data System(NPDS):31st annual report[J].Clin Toxicol(Phila),2014,52(10):1032-1283.

[12]Berny P,Caloni F,Croubels S,et al.Animal poisoning in Europe.Part 2:Companion animals[J].Vet J,2010,183 (3):255-259.

[13]Sanchez-Barbudo IS,Camarero PR,Mateo R.Primary and secondary poisoning by anticoagulant rodenticides of nontarget animals in Spain[J].Sci Total Environ,2012,420:280-288.

[14]Elliott JE,Hindmarch S,Albert CA,et al.Exposure pathways of anticoagulant rodenticides to nontarget wildlife[J].Environ Monit Assess,2013,186(2):895 -906.

[15]Duvall MD,Murphy MJ,Ray AC,et al.Case studies on second-generation anticoagulant rodenticide toxicities in nontarget species[J].J Vet Diagn Invest,1989,1(1):66 -68.

[16]Norström K,Kaj L,Brorström-Lundén E.Screening 2012,Rodenticides[R].Stockholm:IVL Swedish Environmental Research Institute Ltd,2013.

[17]严慧,沈敏.抗凝血杀鼠药的分析研究进展[J].复旦学报:医学版,2011,38(4):361-366.

[18]王臻,刘忠文,张茵,等.抗凝血灭鼠剂中毒导致获得性维生素K依赖性凝血因子缺乏症——46例临床分析[J].血栓与止血学,2011,17(4):166-168.

[19]刘厚鹏.维生素K1治疗抗凝血鼠药中毒疗效观察[J].基层医学论坛,2014,14(28):3877-3878.

[20]刘存学,程海波,刘晋豫.抗凝血灭鼠药中毒误诊3例分析[J].临床急诊杂志,2013,14(4):184-185.

[21]肖礼民.成人重度溴敌隆中毒27例临床分析[J].中国乡村医药,2014,21(1):38.

[22]刘大洪,邓端英,彭伟,等.抗凝血灭鼠药中毒32例救治体会[J].基层医学论坛,2014,18(25):3437-3438.

[23]陈锋.抗凝血治疗杀鼠药中毒的临床观察[J].现代诊断与治疗,2014,25(2):392-393.

[24]汪晓华,陈晓军.溴敌隆食物中毒176例诊治分析[J].中国全科医学,2010,13(15):1674-1675.

[25]黄国,闫磊,张翠媚.2010—2011年福建省某乡镇不明原因出血性疾病调查分析[J].疾病预防控制通报,2012,27 (4):21-22.

[26]金宝灿,广跃乾,王天昌,等.抗凝血杀鼠剂中毒21例误诊分析[J].贵州医药,2012,36(7):641-642.

[27]Rengel I,Friedrich A.Detection of anticoagulant rodenticides(4-hydroxycoumarins)by thin-layer chromatography and reversed-phase high-performance liquid chromatography with fluorescence detection[J]. Vet Res Commun,1993,17(6):421-427.

[28]Guan F,Ishii A,Seno H,et al.A method for simultaneous determination of five anticoagulant rodenticides in whole blood by high-performance liquid chromatography[J].J Pharm Biomed Anal,1999,21(1):179-185.

[29]Sato S.Coumarin rodenticides[M]∥Suzuki O,Watanabe K.Drugs and poisons in humans.Berlin Heidelberg:Springer,2005:599-608.

[30]董颖,王炯,杜鸿雁.抗凝血类杀鼠剂检测技术研究进展[J].中国法医学杂志,2013,28(3):221-223.

[31]刘波.常见鼠药分类及其检测方法研究进展[J].职业与健康,2015,31(2):276-279.

[32]陈海燕.茚满二酮类鼠药的分析进展[J].中国卫生检验杂志,2008,18(12):2843-2845.

[33]中华人民共和国农业部农药检定所.中国农药信息网农药综合查询系统[EB/OL].(2008-09-25)[2015-04- 02].http://www.chinapesticide.gov.cn/service/zhcx/ shuoming.html

[34]韩奇杰,李凯平,赖跃,等.溴敌隆中毒研究进展[J].中国法医学杂志,2012,27(6):471-473.

[35]Spiller HA,Gallenstein GL,Murphy MJ.Dermal absorption of a liquid diphacinone rodenticide causing coagulaopathy[J].Vet Human Toxicol,2003,45(6):313 -314.

[36]Martin-Bouyer G,Khanh NB,Linh PD,et al.An epidemic of a"new"haemorrhagic disease in infants attributable to talcum powder contaminated with warfarin in Ho-Chi-Minh Ville(Vietnam)[J].Arch Toxicol Suppl,1984,7:494-498.

[37]Abell TL,Merigian KS,Lee JM,et al.Cutaneous exposure to warfarin-like anticoagulant causing an intracerebral hemorrhage:a case report[J].J Toxicol Clin Toxicol,1994,32(1):69-73.

[38]Park BK,Choonara IA,Haynes BP,et al.Abnormal vitamin K metabolism in the presence of normal clotting factor activity in factory workers exposed to 4-hydroxycoumarins[J].Br J Clin Pharmacol,1986,21 (3):289-293.

[39]Svendsen SW,Kolstad HA,Steesby E.Bleeding problems associated with occupational exposure to anticoagulant rodenticides[J].Int Arch Occup Environ Health,2002,75(7):515-517.

[40]Hong J,Yhim HY,Bang SM,et al.Korean patients with superwarfarin intoxication and their outcome[J].J Korean Med Sci,2010,25(12):1754-1758.

[41]虞孝里,李思惠,王招兄,等.职业性亚急性抗凝血杀鼠剂中毒4例临床分析[J].中国工业医学杂志,2000,13 (1):33-34.

[42]Vandenbroucke V,Bousquet-Melou A,De Backer P,et al. Pharmacokinetics of eight anticoagulant rodenticides in mice after single oral administration[J].J Vet Pharmacol Ther,2008,31(5):437-445.

[43]Huckle KR,Hutson DH,Warburton PA.Elimination and accumulation of the rodenticide flocoumafen in rats following repeated oral administration[J].Xenobiotica,1988,18(12):1465-1479.

[44]Bachmann KA,Sullivan TJ.Dispositional and pharmacodynamic characteristics of brodifacoum in warfarin-sensitive rats[J].Pharmacology,1983,27(5):281-288.

[45]Sage M,Fourel I,Cœurdassier M,et al.Determination of bromadiolone residues in fox faeces by LC/ESI-MS in relationship with toxicological data and clinical signs after repeated exposure[J].Environ Res,2010,110(7):664 -674.

[46]Lo VM,Ching C,Chan A YW,et al.Bromadiolone toxicokinetics:diagnosis and treatment implications[J]. Clin Toxicol(Phila),2008,46(8):703-710.

[47]Bruno GR,Howland MA,Mcmeeking A,et al.Longacting anticoagulant overdose:brodifacoum kinetics and optimal vitamin K dosing[J].Ann Emerg Med,2000,36 (3):262-267.

[48]高丽丹,杨红军,陈芝.血液灌流救治一例危重溴敌隆中毒患者报告[J].中国疗养医学,2011,20(2):169-170.

[49]Boettcher S,Wacker A,Moerike K,et al.Acquired coagulopathy caused by intoxication with the superwarfarin-type anticoagulant rodenticide flocoumafen [J].Eur J Haematol,2011,86(2):173-175.

[50]Gunja N,Coggins A,Bidny S.Management of intentional superwarfarin poisoning with long-term vitamin K and brodifacoum levels[J].Clin Toxicol(Phila),2011,49 (5):385-390.

[51]Weitzel JN,Sadowski JA,Furie BC,et al.Surreptitious ingestion of a long-acting vitamin K antagonist/ rodenticide,brodifacoum:clinical and metabolic studies of three cases[J].Blood,1990,76(12):2555-2559.

[52]Pavlu J,Harrington DJ,Voong K,et al.Superwarfarin poisoning[J].Lancet,2005,365(9459):628.

[53]Cai M,Dong X,Chen X,et al.An assay for identification and determination of toxic rodenticide valone in serum by ion chromatography-electrospray ionization tandem mass spectrometry with ion trap detector[J].Talanta,2009,78 (1):242-247.

[54]Grobosch T,Angelow B,Sch,et al.Acute bromadiolone intoxication[J].J Ana Toxicol,2006,30(4):281-286.

[55]Vindenes V,Karinen R,Hasvold I,et al.Bromadiolone poisoning:LC-MS method and pharmacokinetic data[J].J Forensic Sci,2008,53(4):993-996.

[56]Jin MC,Ren YP,Xu XM,et al.Determination of bromadiolone in whole blood by high-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry[J].Forensic Sci Int,2007,171 (1):52-56.

[57]Felice LJ,Chalermchaikit T,Murphy MJ.Multicomponent determination of 4-hydroxycoumarin anticoagulant rodenticides in blood-serum by liquid-chromatography with fluorescence detection[J].J Anal Toxicol,1991,15 (3):126-129.

[58]Vandenbroucke V,Desmet N,De Backer P,et al.Multiresidue analysis of eight anticoagulant rodenticides in animal plasma and liver using liquid chromatography combined with heated electrospray ionization tandem mass spectrometry[J].J Chromatogr B Analyt Technol Biomed Life Sci,2008,869(1-2):101-110.

[59]蔡欣欣,张秀尧.超高效液相色谱三重四极杆质谱法同时快速测定血浆和尿液中11种杀鼠剂[J].分析化学,2010,38(10):1411-1416.

[60]Fourel I,Hugnet C,Goy-Thollot I,et al.Validation of a new liquid chromatography-tandem mass spectrometry ion-trap technique for the simultaneous determination ofthirteen anticoagulant rodenticides,drugs,or natural products[J].J Ana Toxicol,2010,34(2):95-102.

[61]Jin M,Chen X.Rapid determination of three anticoagulant rodenticides in whole blood by liquid chromatography coupled with electrospray ionization mass spectrometry [J].Rapid Commun Mass Spectrom,2006,20(18):2741-2746.

[62]Yan H,Xiang P,Zhu L,et al.Determination of bromadiolone and brodifacoum in human blood using LCESI/MS/MS and its application in four superwarfarin poisoning cases[J].Forensic Sci Int,2012,222(1-3):313-317.

[63]Albert CA,Wilson LK,Mineau P,et al.Anticoagulant rodenticides in three owl species from western Canada,1988-2003[J].Arch Environ Contam Toxicol,2009,58 (2):451-459.

[64]Gallocchio F,Basilicata L,Benetti C,et al.Multi-residue determination of eleven anticoagulant rodenticides by high-performance liquid chromatography with diode array/fluorimetric detection:investigation of suspected animal poisoning in the period 2012-2013 in northeastern Italy[J].Forensic Sci Int,2014,244:63-69.

[65]Shore RF,Birks JDS,Afsar A,et al.Spatial and temporal analysis of second-generation anticoagulant rodenticide residues in polecats(Mustela putorius)from throughout their range in Britain,1992-1999[J].Environ Pollut (Oxford,UK),2003,122(2):183-193.

[66]Clelland BL.Forensic applications of Raman microspectroscopy,capillary electrophoresis,chromatography,and mass spectrometry for the analysis of textile fibers,dyes,illicit drugs,and anticoagulant rodenticides[D]. South Carolina:University of South Carolina,2006.

[67]Vudathala D,Cummings M,Murphy L.Analysis of multiple anticoagulant rodenticides in animal blood and liver tissue using principles of Qu EChERS method[J].J Anal Toxicol,2010,34(5):273-279.

[68]Hernández AM,Bernal J,Bernal JL,et al.Analysis of anticoagulant rodenticide residues in Microtus arvalis tissues by liquid chromatography with diode array,fluorescence and mass spectrometry detection[J].J Chromatogr B,2013,925:76-85.

[69]Röhrich J,Zörntlein S,Becker J,et al.LC/MS/MSanalysis of anticoagulant rodenticides in hair.[EB/OL].(2009-04-30)[2015-03-09].http://www.chem-agilent.com/cimg/ ASMS2009-04_LC/MS/MS_Analysis.pdf.

[70]Zhu L,Yan H,Shen B,et al.Determination of bromadiolone and brodifacoum in human hair by liquid chromatography/tandem mass spectrometry and its application to poisoning cases[J].Rapid Commun Mass Spectrom,2013,27(4):513-520.

[71]Palmer RB,Alakija P,De Baca JE,et al.Fatal brodifacoum rodenticide poisoning:autopsy and toxicologic findings[J].J Forensic Sci,1999,44(4):851-855.

[72]Guan F,Ishii A,Seno H,et al.Use of an ion-pairing reagent for high-performance liquid chromatographyatmospheric pressure chemical ionization mass spectrometry determination of anionic anticoagulant rodenticides in body fluids[J].J Chromatogr B Analyt Technol Biomed Life Sci,1999,731(1):155-165.

[73]Hao Y,Dong X,Liang S,et al.Analysis of four anticoagulants rodenticides in foodstuff and body fluids by high-performance liquid chromatography with photodiodearray detector[J].Anal Methods,2014,6(7):2356-2362.

[74]Rattner BA,Horak KE,Lazarus RS,et al.Toxicokinetics and coagulopathy threshold of the rodenticide diphacinone in eastern screech-owls(Megascops asio)[J].Environ Toxicol Chem,2014,33(1):74-81.

[75]Kuijpers EA,Den Hartigh J,Savelkoul TJ,et al.A method for the simultaneous identification and quantitation of five superwarfarin rodenticides in human serum[J].J Anal Toxicol,1995,19(7):557-562.

[76]Lotfi H,Dreyfuss MF,Marquet P,et al.A screening procedure for the determination of 13 oral anticoagulants and rodenticides[J].J Anal Toxicol,1996,20(2):93-100.

[77]Chalermchaikit T,Felice LJ,Murphy MJ.Simultaneous determination of eight anticoagulant rodenticides in blood serum and liver[J].J Anal Toxicol,1993,17(1):56-61.

[78]Chen X,Cai M,Ouyang X,et al.Ion chromatography tandem mass spectrometry for simultaneous confirmation and determination of indandione rodenticides in serum[J]. Biomed Chromatogr,2009,23(11):1217-1226.

[79]金米聪,符展明,王立.HPLC-APCIMS联用法测定全血中杀鼠迷[J].分析测试学报,2006,25(2):97-99

[80]Schaff JE,Montgomery MA.An HPLC-HR-MS-MS method for identification of anticoagulant rodenticides in blood[J].J Anal Toxicol,2013,37(6):321-325.

[81]Jin M,Chen X,Ye M,et al.Analysis of indandione anticoagulant rodenticides in animal liver by eluent generator reagent free ion chromatography coupled with electrospray mass spectrometry[J].J Chromatogr A,2008,1213(1):77-82.

[82]Krizkova S,Beklova M,Pikula J,et al.Hazards of secondary bromadiolone intoxications evaluated using high-performance liquid chromatography with electrochemical detection[J].Sensors,2007,7(7):1271-1286.

[83]Beklova M,Krizkova S,Supalkova V,et al.Determination of bromadiolone in pheasants and foxes by differential pulse voltammetry[J].Int J Environ Anal Chem,2007,87(6):459-469.

Review of analysis on anticoagulant rodenticides in biological specimens

ZHU Lin,XIANG Ping△
(Department of Forensic Toxicology,Institute of Forensic Science,Ministry of Justice-Shanghai Key Laboratory of Forensic Medicine,Shanghai 200063,China)

In China,poisoning of anticoagulant rodenticides is common,and the diagnosis is difficult. Analysis of anticoagulant rodenticides in biological samples is very important to clinical diagnosis,treatment,and forensic investigation.The present article reviews the recently reported analytical methods for anticoagulant rodenticides in biological specimens,focusing on the choice of biological specimen,sample preparation,separation and detection technologies,along with the application to poisoning cases using blood or scalp hair by the the authors′study group.The conclusions:blood is appropriate for analysis of anticoagulant rodenticides,and the sample preparation mainly includes liquidliquid extraction or protein precipitation.Hair could serve as implementation to blood in the forensic investigation of exposure history.To date,liquid chromatography-mass spectrometry(LC-MS)is the commonest technology for analyzing anticoagulant rodenticides in biological specimens for its sensitivity and selectivity.

anticoagulants; rodenticides; poisoning; biological specimen; analytical methods

R 595

B

10.3969/j.issn.1672-8467.2015.05.018

2015-03-10;编辑:张秀峰)

中央级公益性科研院所基本科研业务费专项资金项目(2013G-9);上海市法医学重点实验室资助项目(14DZ2270800)

△Corresponding author E-mail:xiangping2630@163.com

*This work was supported by the Central Public-Interest Scientific Institution Basal Research Fund(2013G-9)and the Research Fund of Shanghai Key Forensic Medicine Laboratory(14DZ2270800).

猜你喜欢
抗凝血头发质谱
我的头发『断了』
气相色谱质谱联用仪在农残检测中的应用及维护
头发飘起来了
头发剪坏的你可以这么办
头发
吹扫捕集-气相色谱质谱联用测定水中18种挥发性有机物
抗凝血类灭鼠剂中毒的诊治分析
肝素抗凝血浆用于急诊生化检验的价值分析
藏北草原第一代抗凝血灭鼠剂应用技术研究
枣霜化学成分的色谱质谱分析