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1 4 3 Vol. 4 No Journal of Food Safety and Quality Jun., 张秀尧 1*, 蔡欣欣 1, 林学尧 2, 陈端秀 (1., ; 2., ) 2 摘要 : 目的 - 方法, - (9:1, v/v), 5.0 mmol/l, BEH C 18,, 结果 %~108% 89.7%~109%, 1.3%~11% 1.1% 18% (n=6), (S/N =10) 0.05 μg/l 0.1 μg/l 结论, 关键词 : - ; ; ; ; ; Determination of 3 kinds of Aconitum alkaloids in human urine and whole blood using ultra-pressure liquid chromatography-mass spectrometry ZHANG Xiu-Yao 1*, CAI Xin-Xin 1, LIN Xue-Yao 2, CHEN Duan-Xiu 2 (1. Wenzhou Center for Disease Control and Prevention, Wenzhou , China; 2. Ruian Center for Disease Control and Prevention, Ruian , China) ABSTRACT: Objective To establish a rapid ultra-pressure liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for detection of aconitine, mesaconitine and hypaconitine in human urine and whole blood. Methods Urine sample was directly injected into the separation system and plasma sample was initially prepared by precipitation of proteins with acetonitrile methanol (9:1, v/v). The analysis of the alkaloids was performed on an ACQUITY UPLC BEH C 18 column using gradient elution with methanol and 5.0 mmol/l ammonium bicarbonate in water, and then detected by the positive electrospray ionization-ms/ms (ESI-MS/MS) method under MRM mode, and quantified by matrix internal standard method using noscapine as internal standard. Results The average recoveries were 93.9%~108% and 89.7%~109% for the three alkaloids in urine and whole blood, with relative standard deviation (RSD) of 1.3%~11% and 1.1%~18% (n=6), respectively. The quantitative limits (S/N=10) of the alkaloids in both of urine and whole blood were 0.05 μg/l and 0.1 μg/l. Conclusion This method is sensitive and accurate, and has been successfully applied to determine the 3 alkaloids in one case of food poisoning. KEY WORDS: ultra-pressure liquid chromatography-tandem mass spectrometry (UPLC-MS/MS); aconitine; mesaconitine; hypaconitine; urine; whole blood * 通讯作者 :,,, [email protected] *Corresponding author: ZHANG Xiu-Yao, Senior Technologist, Wenzhou Center for Disease Control and Prevention, No.41, Xinchengdadao Road, Wenzhou , China. [email protected]

2 引言,, ( 1),, [1], 0.2 mg, 1~2 mg [2] ; [3],, [4], 2 材料与方法 2.1 仪器与试剂 ACQUITY UPLC-Quattro Premier XE - ( Masslynx 4.1, Waters ); MR32i ( Jouan ); MS3 ( IKA ); N-EVAP (24, Organomation ); Gradient A10 Milli-Q ( Millipore ) (HPLC, Merck ); (HPLC, Tedia ); (1 ml, ); Eppendorf (1.5mL, Eppendorf ) ( ), 1000 µg/ml, 35, ( kg) 2.2 样品前处理方法 1 3 Fig. 1 Structures of three kinds of Aconitum alkaloids, 液相色谱 (HPLC) [5] (GC/MS) [6] [7-12] HPLC, ; GC/MS - ( ) (TMCS-BSTFA),,, BEH C 18,,,, 尿液样品前处理 1.0 ml Eppendorf, 40 μl (, 100 μg/l), r/min 10 min, 6 Eppendorf, 1.0 ml, μg/l 50 μg/l, 40 μl (100 μg/l),, 全血样品前处理 5 ml, 100 μl, 700 μl - (9: 1, v/v), 3 min, 1000 r/min 3 min, 50, 200 μl 10%, Eppendorf, r/min 10 min, 6, 100 μl, μg/l 50 μg/l, 4 μg/l,,,

3 3, : 色谱与质谱条件 色谱条件 : ACQUITY UPLC BEH C 18 (50 mm 2.1 mm, 1.7 µm, Waters ), ; A, B 5.0 mmol/l ; : 3.5 min 45%A 65%A, 0.1 min 95%A, 1.9 min, 0.1 min 45%A, 1.4 min; : ml/min; : 50 ; 10 μl 质谱条件 ; ESI 1.0 kv; 120 ; 400 ; 50 L/hr, 500 L/hr, Pa 1, 2.2 min, 3.5 min, 3 结果与分析 3.1 质谱条件的优化 MRM,, 4, 2, (dwell time) 15~20, 色谱条件的优化 ACQUITY UPLC BEH C 18 (50 mm 2.1 mm, 1.7 µm), -0.1%, 3,,, RPLC-MS/MS [13-16],,,,,, ACQUITY UPLC BEH,, 5 mmol/l 0.1%, 45%, 3, R s 0.8, 3.35 min,,,, C 18, 3 A, B 2 mmol/l 5 mmol/l 7 min Table 1 表 1 质谱的 MRM 参数 MS parameters for multiple reaction monitoring (m/z) (V) (ev) (min) * > aconitine 646.4> >572.4 * mesaconitine 632.3> >556.4 * hypaconitine 616.3> (IS) noscapine 414.1>353.0 * : * (quantitative ion pair)

4 样品前处理方法的优化 -, - (3: 2, v/v) [8] [9] [10] [11] [12],, 5 μg/l 3, 8%~17%,,,,,,, 3 12%~20%, [17] 53%~67%, 3, 60 V, m/z 184>m/z 184, 4 ev, m/z 184, m/z 184, m/z 184, m/z184 > m/z 184,, 3, ,,,,,,,, [18-19] 3.4 内标物的选择, 3, [8] [10] [11] [12], [9], 3,, 3.5 线性范围和检出限 6, 2.2, Masslynx 4.1 Targetlynx ( 1/x), 0.1~50 μg/l 0.999, 3,, 2 3 (LOD), 10 (LOQ) μg/l, 0.05 μg/l; μg/l, 0.1 μg/l,, 3.6 方法的回收率和精密度,, 30 min,,, %~108% 89.7%~109%, 1.3%~11% 1.1%~18%, 实际样品的测定 ,, 6, 18 19, ,, 22 5, 3,,,,

5 3, : Fig. 2 2 (0.1 μg/l) UPLC-MS/MS chromatograms of a spiked urine sample (0.1 μg/l) 表 2 加标回收率和精密度 (n=6) Table 2 Recoveries and RSDs of the Aconitum alkaloids in urine and whole blood (n=6) (μg/l) (%) (%) (μg/l) (%) (%)

6 720 4 Table 3 表 3 患者尿液 全血和洗胃液中 3 种乌头生物碱的浓度 Concentrations of Aconitum alkaloids in urine, blood and gastric lavage fluid in their poisoning cases 病例年龄性别 尿液 (μg/l) 全血 (μg/l) 洗胃液 (μg/l) 1 48 男 男 女 男 , 4 2, 3,, mg/l 1.8 mg/l 3 3, 3, 3, 9, 12 h,, 2 min [1], 1 d, 7 [20], 4 结论 - 3,,, 参考文献 [1]. [J]., 2007, 18(5): [2] Zhang F. Review of the methods of detection of Aconitum alkaloids in human body fluids [J]. Li Shizhen Med Mater Med Res, 2007, 18(5): [3],. 132 [J]., 2009, 35(4): 290. [4] Han W, Xiong G. Diagnosis and treatment of aconitine poisoning: a report of 132 cases [J]. J Mod Clin Med, 2009, 35(4): 290. [5]. [M]. :, [6] Guo JY. Practical forensic medicine [M]. Changchun: Changchun Press, [7]. 78 [J]., 2008, 5: [8] Li B. Clinical analysis of acute poisoning aconitine-78 cases report [J]. West China Med J, 2008, 5: [9],,,. [J]., 2005, 20 (4): [10] Jin M, Li HH, Wang S, et al. A method for rapid separation and determination of aconitine in plasma by reversed-phase high performance liquid chromatography [J]. Chin J Forensic Med, 2005, 20 (4): [11] Ito K, Ohyama Y, Konishi Y, et al. Method for the simultaneous determination of Aconitum alkaloids and their hydrolysis products by gas chromatography-mass spectrometry in human serum [J]. Planta Med, 1997, 63: [12] Hayashida M, Hayakawa H, Wada K, et al. A column-switch LC/MS/ESI method for detecting tetrodotoxin and Aconitum alkaloids in serum [J]. Legal Med, 2003, 5(5): S101 S104. [13],. LC-MS/MS [J]., 2010, 27(8): [14] Gao K, Xing JJ. A rapid method for determination of aconitine in biology samples by LC-MS/MS [J]. Chin J Mod Appl Pharm, 2010, 27(8): [15],,. LC-MS/MS [J]., 2010, 45(8): [16] Qiu K, Wu H, Wang HY. Simultaneous determination of aconitine and hypaconitine in plasma and urine by LC-MS/MS [J]. Chin Pharm J, 2010, 45(8): [17],,,. LC-MS/MS [J]., 2012, 17(4): [18] Wang ZQ, Jing X, Zeng R, et al. Determination of aconitine concentration in rats plasma by LC-MS/MS [J]. Chin J Clin Pharmcol Ther, 2012, 17(4): [19],,,. - [J].

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